Table of Contents Meeting Participants .................................................................................................................... ix Steering Committee .................................................................................................................. xvii Sustaining Members .................................................................................................................. xix Honorary Lifetime Members .................................................................................................... xxi Symposia I: Advances in Fundamental and Commercial ABA Research BIOSYNTHESIS AND CATABOLISM OF ABSCISIC ACID ..................................................... 1 J.A.D. Zeevaart* ABSCISIC ACID ANALOGS - TOWARD DEVELOPMENT OF ABA-BASED PLANT GROWTH REGULATION ............................................................................................................. 6 S. Abrams*, K. Nelson, Y. Gai, I Zaharia, P. Galka and I Alarcon CHALLENGES FOR THE COMMERCIAL DEVELOPMENT OF S-ABSCISIC ACID (ABA) ....................................................................................................... 7 P. D. Petracek*, D. Woolard, R. lJ1enendez, and P. Warrior ABSCISIC ACID SIGNALING NETWORKS IN ARABIDOPSIS ............................................ 1 0 R. Finkelstein*, T Lynch, I Brocard-Gifford, ME. Garcia, TL. Thomas Session I: Biotic and Abiotic Stress SUGAR MOVER ENHANCES CROP PERFORMANCE, AND BIOTIC/ABIOTIC TOLERANCE OF PLANTS .......................................................................................................... 19 J.H Stoller, R. Salzman and A. Liptay* THE ROLE OF ROOT TO SHOOT SIGNALING IN COORDINATING RESPONSES TO SOIL COMPACTION ................................................................................................................... 20 S.L. Aphale*, T.S. Stokes, C.R. Black, lB. Taylor and J.A. Roberts ROOT FEED TM FOR ENHANCED CROP PERFORMANCE AND BIOTIC/ABIOTIC CROP PLANT TOLERANCE .................................................................................................................. 24 R. Salzman * and A. Liptay Session II: PGRS in Turfgrass and Vegetable Production ANNUAL BLUEGRASS SEEDHEAD CONTROL IN OVERSEEDED PERENNIAL RYEGRASS WITH TURF GROWTH REGULATORS .............................................................. 27 A. G. Estes * and L. B. McCarty iii
Transcript
Table of Contents
Meeting Participants .................................................................................................................... ix
Steering Committee .................................................................................................................. xvii
Sustaining Members .................................................................................................................. xix
Honorary Lifetime Members .................................................................................................... xxi
Symposia I: Advances in Fundamental and Commercial ABA Research
BIOSYNTHESIS AND CATABOLISM OF ABSCISIC ACID ..................................................... 1 J.A.D. Zeevaart*
ABSCISIC ACID ANALOGS - TOWARD DEVELOPMENT OF ABA-BASED PLANT GROWTH REGULATION ............................................................................................................. 6 S. Abrams*, K. Nelson, Y. Gai, I Zaharia, P. Galka and I Alarcon
CHALLENGES FOR THE COMMERCIAL DEVELOPMENT OF S-ABSCISIC ACID (ABA) ....................................................................................................... 7 P. D. Petracek*, D. Woolard, R. lJ1enendez, and P. Warrior
ABSCISIC ACID SIGNALING NETWORKS IN ARABIDOPSIS ............................................ 1 0 R. Finkelstein*, T Lynch, I Brocard-Gifford, ME. Garcia, TL. Thomas
Session I: Biotic and Abiotic Stress
SUGAR MOVER ENHANCES CROP PERFORMANCE, AND BIOTIC/ABIOTIC TOLERANCE OF PLANTS .......................................................................................................... 19 J.H Stoller, R. Salzman and A. Liptay*
THE ROLE OF ROOT TO SHOOT SIGNALING IN COORDINATING RESPONSES TO SOIL COMPACTION ................................................................................................................... 20 S.L. Aphale*, T.S. Stokes, C.R. Black, lB. Taylor and J.A. Roberts
ROOT FEED TM FOR ENHANCED CROP PERFORMANCE AND BIOTIC/ABIOTIC CROP PLANT TOLERANCE .................................................................................................................. 24 R. Salzman * and A. Liptay
Session II: PGRS in Turfgrass and Vegetable Production
ANNUAL BLUEGRASS SEEDHEAD CONTROL IN OVERSEEDED PERENNIAL RYEGRASS WITH TURF GROWTH REGULATORS .............................................................. 27 A. G. Estes * and L. B. McCarty
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t'TIFEAGLE' BERMUDAGRASS RESPONSE TO THREE PLANT GROWTH REGULATORS ........................................................................................................... 28 T. G. Willis *, H Liu, T. Whitwell, JE. Toler, L.B. McCarty
MICROBIAL QUALITY AND SHELF LIFE OF HARPIN TREATED HEAD LETTUCE .......................................................................................................................... 29 J Fonseca*, W Kline, C. Wyenandt, M Hoque, H Ajwa and N French
t'TIFWAY' BERMUDAGRASS RESPONSE TO PRIMO AND CUTLESS ........................... .30 F. W Totten *, JE. Toler, and L.B. McCarty
Symposium II: PGRS in Tree and Vine Crops
USES OF PGRS IN CITRICULTURE .......................................................................................... 33 J Burns*
DO HORMONES PLAY A ROLE IN ALTERNATE BEARING IN CITRUS? ......................... 34 S. Verreynne and C. Lovatt*
BENEFITS OF PRESTIGE (CPPU, Forchlorfenuron) USE IN CALIFORNIA TABLE GRAPES ......................................................................................................................... 35 R. Hopkins*, R. Beach, and R. Menendez
USES OF PGRS IN TREE NUT CROPS ..................................................................................... 36 B. Beede*
Symposium III: Molecular and Morphological Aspects of Plant Hormones and Reproductive Development
MORPHOLOGY AND REGULATION OF FLOWERING IN APPLE. .................................... .41 S. McArtney * and E. Hoover
GENETIC DISSECTION OF AUXIN BIOSYNTHESIS IN ARABIDOPSIS ........................... .42 Y. Zhao*
HORMONAL REGULATION OF FRUIT DEVELOPMENT ................................................... .43 J Ozga*
GENES CONTROLLING FRUIT DEVELOPMENT IN ARABIDOPSIS ................................. .44 J Dinneny, C. Ferrcmdiz, K. Gremski, S. Liljegren, A. Roeder, and M Yanofsky*
Symposium IV: Ornamental Plant Growth Regulation
CHEMICAL REGULATION OF SENESCENCE IN ORNAMENTALS .................................. .47 M Reid*
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ORNAMENTAL GROWTH RESPONSES FROM PROHEXADIONE-CA APPLICATIONS ........................................................................................................................... 48 D. Barcel*
EFFICACY AND PHYTOTOXICITY OF FASCINATION (6-BENZYLADENINE AND GA4+GA7) ON A VARIETY OF ORNAMENTAL PLANTS .................................................... 52 H Lieth*
GROWTH REGULATION OF ORNAMENTALS IN EUROPE-FOCUS ON ALTERNATIVE METHODS ....................................................................................................... 53 C. Wang Hansen *
Session III: Regulation of Growth and Development/Analytical Methods
COMPARISON OF COTTON VARIETAL RESPONSES TO APPLICATION OF AUXI-GRO® WP PLUS CAL-MAX ............................................................................................ 57 M D. Rethwisch *, M Reay, J. Grudovich, J. Wellman and D. M Ramos
SALICYLATE ACTIVITY. 4 ROLE OF ETHYLENE IN PARAQUAT DAMAGE ................. 58 F. P. Silverman, P. D. Petracek*, Z. Ju, D.F. Heiman, and P. Warrior
ETHEPHON DEFOLIATION OF PLUMERIA Plumeria rubra for WINTER FLOWERING ............................................................................................................... 65 R. Criley*
GIBBERELLIN SYNTHESIS INHIBITOR AFFECTS ANNUAL XYLEM PRODUCTION AND VESSEL ELEMENT ANATOMY IN SOME TREES ........................................................ 69 WR. Chaney*, D.M Mickey, HA. Holt
IMMUNOSENSOR ASSAY: A NOVEL METHOD TO ANALYZE PHYTOHORMONES .................................................................................................................... 74 L. Xiao *, R. Wang, J. Li, G. Sheng
Session IV: Flowering/Seed and Fruit Development
POST ANTHESIS PGR APPLICATION AND FIRST AND SECOND CROP PRODUCTION IN DRILL-SEEDED RICE ............................................................................................................ 79 R. Dunand*
REGISTRATION OF 2,4-D FOR INCREASING FRUIT SIZE OF MANDARINS AND MANDARIN HYBRIDS IN CALIFORNIA ................................................................................. 80 C. Thomas Chao *, L. Ferguson, and c.J. Lovatt
PEACH FLOWER BUD THINNING BY DORMANT SEASON APPLICATIONS OF VEGETOIL™ ................................................................................................................................ 81 G. L. Reighard*, D. R. Ouellette, and K. H Brock
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FLORIGENIC PROMOTER OF L YCHEE (Litchi chinensis, Sonn) SYNTHESIZED INLEAVES ................................................................................................................................... 87 TL. Davenport* and Z. Ying
Contributed Papers
EFFECTS OF LIQUID FERTILIZER CONTAINING 5-AMINOLEVULINIC ACID ON THICKENING GROWTH IN TULIP BULBS .................... 91 R. Yoshida *, E.Ohta, K.Iwai, T Tanaka and H Okada
tDEVELOPING IMPROVED NURSERY CULTURE FOR THE PRODUCTION OF ROOTED CUTTINGS OF CANADA YEW (Taxus canadensis) ................................................ 95 L. Webster*
NEW INNOVATIONS WITH FLURPRIMIDOL USE ON TURFGRASS, CONTAINERIZED ORNAMENTALS, AND LANDSCAPE ORNAMENTALS .................... 101 B. T Bunnell* and S.D. Coclu'eham
STUDIES ON PLANT-ASSOCIATED ACTINOMYCETES AND THEIR SECONDARY METABOLITES ................................................................................................ 102 Y. Igarashi*, S. Miura, M Azumi, T Furumai and R. Yoshida
DEVELOPMENTAL REGULATION OF THE GA BIOSYNTHESIS GENES, GA20ox, GA30x, AND GA20x DURING GERMINATION AND YOUNG SEEDLING GROWTH OF PEA (Pisum sativum L.) ................................................................ .106 B. Ayele, J Ozga*, and D. Reinecke
GIBBERELLIN AND AUXIN LEVELS IN MATURE EMBRYOS AND YOUNG SEEDLINGS OF Pisum sativum L. ............................................................................................. 107 B. Ayele, J Ozga*, and D. Reinecke
IMPROVEMENT OF YIELD IN GREENHOUSE GROWN DETERMINATE MULTIFLOWERED PEAS WITH GIBBERELLIN TREATMENTS ...................................... 1 08
* Sonja L. MaId, H Mullen, R. Pharis and S. Singer
ETHYLENE SENSITIVITY OF CUT RACEMES OF ADVANCED BREEDING LINES OF PINK FLOWERED BLUEBONNET ....................................................................... 121
W.A. Mackay *, N Sankhla and T.D. Davis
EFFECT OF NITRIC OXIDE GENERATING COMPOUNDS ON FLOWER SENESCENCE IN CUT RACEMES OF PINK FLOWERED LUPINUS HAVARDIIWATS . ...................................................................................................................... 126
N Sankhla, W.A. Mackay* and T.D. Davis
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COMPATIBILITY OF SALAD CROPS GROWN IN MIXED CROP \HYDROPONIC SYSTEMS ....................................................................................................... 133 SL. Edney, J T. Richards, MD. Sisko, NC. Yorio, G. W. Stutte*, and R. M Wheeler
SENSITIVITY SCREENING OF RADISH SEEDLINGS TO SPACECRAFT VOCs ............. 141 I Eraso*, G. W. Stutte, 0. Monje, S Anderson and R. D. Hickey
ROOTSTOCK EFFECTS ON GROWTH OF APPLE SCION WITH DIFFERENT GROWTH HABITS ..................................................................................................................... 142 T. Tworkoski* and S Miller
tEMBRYOGENESIS INDUCTION WITH IAA AND IAA CONmGATES IN CARROTS ................................................................................................................................... 143 K. Tworkoski*, A. Newton, and E. Shea
Author Index .............................................................................................................................. 151
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PARTICIPANT LIST
Suzanne Abrams Plant Biotechnology Institute National Research Council Canada 110 Gymnasium Place Saskatchewan, Canada S7N OW9 phone: 306-975-5333 fax: 306-975-4839 e-mail: [email protected]
Marcus Adair Valent BioSciences 214 Cross Breeze Drive Memphis, TN 38018 USA phone: 701-756-4676 e-mail: [email protected]
Shanta Aphale University of Nottingham Plant Sciences Division Bonigton Campus Sutton Bonington Leicestershire, UK LE125RO United Kingdom e-mail: [email protected]
David Barce1 Chemtura 13393 Jackson Street Salinas, CA 93906 USA phone: 831-449-7266 fax: 831-449-7123 e-mail: [email protected]
Robert Beede University of California Cooperative Extension - Kings County 680 N. Campus Drive, Suite A Hanford, CA 93230 USA phone: 559-384-6534 fax: 559-582-5166 e-mail: [email protected]
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Bruce Brown Dormex Company USA, LLC 5151 North Palm Ave., Ste. 620 Fresno, CA 93704 USA phone: 559-229-8183 fax: 559-229-0893 e-mail: [email protected]
Todd Bunnell SEPRO Corporation 11550 North Meridian Street Suite 600 Carmel, IN 46032 USA phone: 317-216-5667 fax: 317-580-8295 e-mail: [email protected]
J ache Burns Citrus Research and Education Center University of Florida 700 Experiment Station Road Lake Alfred, FL 33850 USA phone: 863-956-4631 fax: e-mail: [email protected]
Betty Carson Chemical Dynamics 4206 Business Lane Plant City, Florida 33566 USA
William R. Chaney Professor Emeritus of Tree Physiology 3727 118th Avenue Allegan, MI 49010 USA Phone: 269-673-3099 email: [email protected]
C. Thomas Chao University of California-Riverside Department of Botany and Plant Sciences 4106 Batchelor Hall Extension Riverside, CA 92521-0124 USA phone: 909-787-3441 fax: 909-787-4437 e-mail: ctchao(tllcitrus.ucr.edu
Charles W. Coggins University of California Department of Botany and Plant Sciences 1870 Cape Court Riverside, CA 92506-4600 USA phone: 951-789-0895 fax: 951-827-4437 e-mail: [email protected]
Pamela J. Coker Public Health Foundation 223 Corral Court Fishers, IN 46038 USA phone: 864-710-6936 e-mail: [email protected]
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Kyle Coleman AMV AC Chemical 110 S. Nevada Street Kennewick, WA 99336 USA phone: 509-430-2299 fax: 509-783-5909 email: [email protected]
Richard A. Criley Dept. of Tropical Plant & Soil Sciences University of Hawaii 3190 Maile Way, Rm. 102 Honolulu, Hawaii 96822-2232 USA phone: 808-956-8492 fax: 808-956-3894 e-mail: [email protected]
Gary Custis PBI Gordon Corporation Field Research & Development Dept. 1217 W. 12th Street Kansas City, MO 64101-0090 USA phone: 816-460-6215 fax: 816-460-3715 e-mail: [email protected]
Thomas Davenport University of Florida - IFAS Tropical Research & Education Center 18905 SW 280 Street Homestead, Florida 33031 USA phone; 305-246-7001 x215 fax: 305-246-7003 e-mail: [email protected]
Jeffrey H. Dobbs Olympic Horticultural Products 1095 Applecross Dr. Roswell, GA 30075 USA phone: 770-992-0121
Richard T. Dunand LSU Agricultural Center Rice Research Station 1373 Caffey Road Rayne, Louisiana 70578 USA phone: 337-788-7531 fax: 337-788-7553 e-mail: [email protected]
Bryan W. Ellsworth L T BioSyn, Inc. 3406 Pomona Blvd. Pomona, CA 91768 USA phone: 909-348-5133 fax: 909-348-5135 e-mail: [email protected]
Alan G. Estes Clemson University E-142 P & A Bldg. Horticulture Department Clemson, SC 29634-0319 USA phone: 864-656-4959 fax: 864-656-4960 e-mail: [email protected]
Louise Ferguson University of California-Davis Plant Sciences Department Kearney Agricultural Center 9240 S. Riverbend Ave. Parlier, CA 93648 USA phone: 559-646-6541 fax: 559-646-6593 e-mail: [email protected]
Ignacio Eraso Dynamac Corporation DYN-3
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Kennedy Space Center, FL 32899 USA phone: 321-861-2939 fax: 321-861-2922 email: [email protected]
Alan Estes Horticulture Department Clemson University E-142 P&A Building Clemson, SC 29634-0319 USA phone: fax: 864-656-4960 e-mail: [email protected]
Louise Ferguson University of California-Davis Pomo1ogy Department Kearney Agricultural Center 9240 S. Riverbend Avenue Parlier, CA 93648 USA phone: 559-646-6541 fax: 559-646-6593 e-mail: [email protected]
Ruth Finklestein University of California-Santa Barbara MCD Biology Department Santa Barbara, California 93106 USA phone: 805-893-4800 fax: 805-893-4724 e-mail: [email protected]
Jorge M. Fonseca The University of Arizona Yuma Agricultural Center 6425 8th St. Yuma, Arizona 85364 USA phone: 928-782-3836 fax: 928-782-1940 e-mail: [email protected]
Kevin D. Forney Fine Americas, Inc. 15602 Marty Ave. Bakersfield, CA 93314 USA phone: 661-588-7137 fax: 661-588-6863 e-mail: [email protected]
Phil Grau Phil Grau Consulting 3064 W. Wellington Lane Fresno, CA 93711 USA phone: 559-431-4827 fax: 559-431-4879 e-mail: [email protected]
Conny Wang Hansen Danish Institute of Agricultural Sciences Department of Horticulture P.O. Box 102 Aarslev, Denmark DK-5792 phone: 0045-89993312 fax: 45-89993490 e-mail: [email protected]
James R. Hansen Valent Biosciences Corporation 870 Technology Way Libertyville, IL 60048 USA phone: 847-968-4799 e-mail: [email protected]
Marcus Heisler Caltech 1200 E. California Blvd. Pasadena, CA 91125 USA e-mail: [email protected]
Yasuhiro Igarashi Biotechnology Research Center Toyama Prefectural University 5180 Kurokawa, Kosugi, Imizu Toyama, Japan 939-0398 Japan phone: fax: 81-766-56-2498 e-mail: [email protected]
Greg Johnson Fine Americas, Inc. 1850 Mt. Diablo Blvd. Suite 405 Walnut Creek, CA 94596 USA phone: 925-932-8800 fax: 925-932-8892 e-mail: [email protected]
Bruce L. Kirkpatrick Valent Biosciences Corporation 870 Technology Way Libertyville, IL 60060 USA
Heiner Lieth Plant Sciences Department Environmental Horticulture - Mailstop 6 One Shields Ave. University of California-Davis Davis, CA 95616-8587 USA phone: 530-752-7198 fax: 530-752-1819 email: [email protected]
Albert Liptay Stoller Enterprises, Inc. 4001 W. Sam Houston Pkwy. N. Suite 100 Houston, TX 77043-1226 USA phone: 713-461-1493, ext. 126 fax: 713-461-4467 e-mail: [email protected]
Carol J. Lovatt University of California-Riverside Department of Botany and Plant Sciences Riverside, CA 92521-0124 USA phone: 951-827-4663 fax: 951-827-4437
Wayne A. Mackay Texas A & M University-TAES Dallas 17360 Coit Road Dallas, TX 75252-6599 USA phone: 972-952-9251 fax: 972-952-9216 e-mail: [email protected]
Sonja Maki Biology Department Carleton College One North College Street Northfield, MN 55057 USA phone: 507-646-4544 fax: 507-646-5757 [email protected]
Stephen R. Malone Syngenta Crop Protection Seed Treatment Technology Platform 317 330th Street Stanton, MN 55018 USA phone: 507-663-7658 fax: 507-645-7519 email: [email protected]
Jerry V. Mayeux Plant BioTech, Inc. HC 66 Box 74 Deming, New Mexico 88030 USA phone: 505-894-4900 fax: 505-212-0034 [email protected]
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Ricardo Menendez Valent Biosciences Corporation 870 Technology Way Libertyville, IL 60048 USA phone: 847-968-4715 fax: 847-968-4801 e-mail: [email protected]
AlanR. Mick Dormex Company USA, LLC. 5151 N. Palm, Suite 620 Fresno, CA 93704 USA phone: 559-229-8183 fax: 559-229-0893 e-mail: [email protected]
Steven McArtney NC State University Mountain Horticultural Crops Research Center 455 Research Drive Fletcher, NC 28732 USA phone: 828-684-3562 fax: 828-684-8715 Steve _ [email protected]
Alan Mick Donnex Company USA, LLC 5151 North Palm Avenue, Suite 620 Fresno, CA 93704 USA phone: 828-684-3562 fax: 828-684-8715
Steven L. Morrison Answers for Agriculture P.O. Box 178029 San Diego, CA 92117 USA phone: 619-507-0600 e-mail: [email protected]
Dean K. Mosdell Syngenta 501-1 S. Reino Rd. #183 Newbury Park, CA 91320 USA phone: 805-400-0514 e-mail: [email protected]
Jeffrey P. Norrie Acadian Seaplants Ltd. R&D Division 30 Brown Ave. Dartmouth, Nova Scotia B3B lX8 Canada phone: 902-468-2840 fax: 902-468-3474 e-mail: [email protected]
Jocelyn Ozga University of Alberta 4-10 AgricultureIForestry Centre Dept. of Ag, Food & Nutritional Sci. Edmonton, Alberta T6G 295 Canada phone: 780-492-2653 fax: 780-492-4265 email: [email protected]
Peter Petrocek Valent BioSciences Corporation 6131 Oakwood Road Long Grove, Illinois 60047 USA e-mail: [email protected]
Richard Pharis Biological Sciences Department University of Calgary 2500 University Drive NW Calgary, Alberta T2N IN4 Canada phone: 403-220-5015 fax: 403-289-9311 e-mail: [email protected]
Jerome Pier Western Farm Services 509 Webber Ave., Suite 201 Stockton, CA 95203 USA phone: 209-610-0565 fax: 209-464-4652 e-mail: [email protected]
Michael Reid Department of Plant Sciences University of California, Davis 1 Shields Ave. Davis, CA 95616 USA phone: 530-754-6751 fax: 530-754-6753 e-mail: [email protected]
Gregory Reighard Department of Horticulture Clemson University Box 340319 Clemson, SC 29634-0375 USA phone: 864-656-4962 fax: 864-656-4960 e-mail: [email protected]
Michael Rethwisch University of California Cooperative Extension 290 N. Broadway Blythe, CA 92225-1649 phone: 760-921-5060 fax: 760-921-5059 e-mail: [email protected]
Ron Salzman Stoller Enterprises, Inc. 4001 W. Sam Houston Parkway Houston, TX 77043 USA phone: 713-461-1493 fax: 713-461-4467 e-mail: [email protected]
Brady Smith Genetics Graduate Group University of California-Davis 634 Lessley Place Davis, CA 95616 USA e-mail: [email protected]
Ron Smith 12 Chateau Drive McLeod Hill, New Brunswick CANADA E3A 5X2 phone: 506-453-1792 e-mail: rsmith0225(mrogers.com
Jerry Stoller Stoller Enterprises, Inc. 4001 West Sam Houston Parkway North Suite 100 Houston, TX 77043-1226 phone: 713-461-1493 fax: 713-461-4467 e-mail: [email protected]
Thomas Stopyra Chemical Dynamics, Inc. Research & Development 4206 Business Lane
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Plant City, Florida 33566 USA phone: 813-752-4950 fax: 813-752-8639 e-mail: [email protected]
Michael Straumietis Advanced Nutrients # 109-31063 Wheel Avenue Abbotsford, BC V2T 6HI Canada phone: 604-854-6793 fax: 604-854-4371 e-mail: [email protected]
Gary Stutte Dynamac Corporation Space Life Sciences Laboratory Mail Code DYN-3 Kennedy Space Center, FL 32899 USA phone: 321-861-3493 fax: 321-861-2925 e-mail: [email protected]
. Frederick Totten Clemson University Horticulture Department E-143 P&A Building Clemson, SC 29634-0319 phone: fax: 864-656-4960
Kathryn Tworkoski Loyola College of Maryland MS 2967 4501 N. Charles St. Baltimore, MD 21210 e-mail: [email protected]
Tom Tworkoski USDA-ARS Appalachian Fruit Research Station 2217 Wiltshire Road Kearneysville, WV 25430 USA
PeterT. Yu LTBiosyn 1842 E.Workman #323 West Covina, CA 91791 USA phone: 619-300-5092
YuanjiZhou Chinese Society of Plant Physiology 319 Yue Yang Rd. Shanghai,China 200031 China phone: 0086-21-54922853 fax: 0086-21-43922857 e-mail: [email protected]
Ryuji Yoshida Toyama Prefectural University College of Technolgy Kurokawa 5180 Kosugi-machi, Toyama 939-0398 Japan phone: 81-766-56-7500 fax: 81-766-56-0396 e-mail: [email protected]
Jan Zeevaart Michigan State University MSU-DOE Plant Research Lab East Lansing, MI48824-13112 USA phone: 517-353-3230 fax: 517-353-9168 e-mail: [email protected]
YhundeZhao University of California San Diego, Biology 9500 Gilman Dr MC 0116 La Jolla, CA 92093-0116 USA phone: fax: 858-534-7108 e-mail: [email protected]
Mr. S. Gary Custis (2"d Vice President) PBI/Gordon Corporation Field Research & development Dept. 1217 W. 12th Street Kansas City, MO 64101-0090 816-460-6215 (phone) 1816-460-3715 (fax)
Dr. Steven J. McArtney (Secretary) North Carolina State University Mountain Horticultural Crops Research Center 455 Research Drive Fletcher, NC 28732 828-684-3562 828-684-8715 (fax)
Dr. Ronald F. Smith (Member at Large - 3) See Editor of PGRSA Quarterlv
Dr. C. Thomas Chao (Member at Large - 2) University of California-Riverside Department of Botany and Plant Sciences 2137 Batchelor Hall Riverside, CA 92521-0124
Dr. Jose Pablo Morales-Payan (Member at Large -1) University of Puerto Rico - Mayaguez Horticulture Department AP303 P.O. Box 9030 Mayaguez, PR 00681-9030 787-832-4040 ext. 2088
Dr. Richard T. Dunand (Business Manager) LSU Agricultural Center Rice Research Station 1373 Caffey Road Rayne, LA 70578 337-788-7531 337-788-7553 (fax)
Acadian Seaplants Ltd. AMV AC Chemical Corporation
BASF Corporation Bayer CropScience
Chemtura Dormex Company USA, LLC
Dynamac Corporation Fine Americas, Inc.
Olympic Horticultural Products PBI Gordon Corporation
Plant Biotech, Inc. Syngenta Professional Products
SePRO Corporation Stoller Enterprises
Valent BioSciences Corporation
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Plant Growth Regulation Society of America
Honorary Lifetime Members
Anson Coole
Horace G. Cutler
C. Dave Fritz
Masayuki Katsumi (JSCRP)
Edward F. Sullivan
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SYMPOSIA I
ADVANCES IN FUNDAMENTAL AND COMMERCIAL ABA
RESEARCH
lABSCISIC ACID METABOLISM
Jan A.D. Zeevaart1* and Seung-Hwan Yang1
ABSTRACT Abscisic acid (ABA) is necessary for seed development and dormancy, and also plays a
role in adaptation to abiotic stresses. To understand how the levels of ABA are regulated, it is essential to understand the pathways of ABA synthesis and degradation. In green plants, ABA is a cleavage product of 9-cis-epoxycarotenoids, yielding the CIS product xanthoxin, which is further converted to ABA via AB-aldehyde. The cleavage of carotenoids is catalyzed by ninef.is-~poxycarotenoid gioxygenase (NCED) and is a major limiting step in ABA biosynthesis. The main catabolic step is 8' -hydroxylation of ABA, resulting in phaseic acid (P A), which is further converted to dihydrophaseic acid (DPA). ABA 8'-hydroxylase is encoded by a small gene family ofP450 monooxygenases, the CYP70 7As. Using mutants and molecular-genetic approaches, biosynthetic and catabolic enzymes have been characterized. NCEDs and CYP707 As play major roles in regulating ABA levels through biosynthesis and catabolism, respectively.
INTRODUCTION Abscisic acid (ABA) is a plant hormone that is involved in seed development and
adaptation to various stresses. It also plays an important role in seed dormancy. When ABA levels increase during water stress, stomates close and changes in gene expression lead to an increase in stress tolerance. Likewise, in developing seeds ABA is needed for gene expression for storage materials and tolerance of desiccation.
Levels of ABA can change rapidly by as much as 100-fold due to changes in water status of the tissue. Conversely, ABA content can decrease quickly when the tissue is rehydrated. Regulation of physiological processes by ABA is mainly due to changes in the ABA content. Thus, in order to understand how ABA levels are regulated, it is important to know how ABA is synthesized and catabolized, and how these processes are regulated. There are several recent reviews discussing ABA metabolism (Nambara and Marion-Poll, 2005; Schwartz et aI., 2003; Schwartz and Zeevaart, 2004; Xiong et aI., 2003) and the reader is referred to these articles for original references.
ABSCISIC ACID BIOSYNTHESIS ABA is a sesquiterpenoid (CIS), which means that it is built up of three isoprenoid units
(3xCs). However, efforts to label ABA with the terpenoid precursor mevalonic acid proved unsuccessful. Exposing stressed leaves to an atmosphere containing 180 2 resulted in the production of ABA that had one 180 atom in the carboxyl group, but was not labeled in the ring positions, indicating that ABA is derived by cleavage from a larger molecule, presumably a carotenoid, in which the oxygen atoms on the ring are already present. It is now well established that ABA in green plants is a breakdown product of the 9-cis-epoxycarotenoid xanthophylls, 9-cis-violaxanthin and 9' -cis-neoxanthin, as shown in Figure 1. The first mutant in the pathway, aha}, results in accumulation of zeaxanthin and a lack of xanthophylls. The aha2, aha3, and aao3 mutants all block steps in the conversion ofxanthoxin via AB-aldehyde to ABA. However, mutagenesis in Arabidopsis with selection for mutants lacking seed dormancy andlor with a wilty phenotype did not yield any mutants for the proposed cleavage step of carotenoids. The
1
breakthrough came with the viviparous mutant, vp14, in maize. Recombinant VP14 protein specifically cleaved 9-cis-epoxycarotenoids (C40) to give xanthoxin (CIS) and C2S-apocarotenoids. Since this seminal discovery, many more genes encoding carotenoid cleavage enzymes have been isolated. VP 14 is the founding member of a large family of carotenoid cleavage enzymes that are able to cleave carotenoids at different positions. For example, central cleavage of p-carotene in animals results in retinal (C20, vitamin A). Nine-fis-~oxycarotenoid gioxygenase (NCED) cleaves the 11,12 double bonds of9-cis-violaxantin and 9'-cis-neoxanthin to give xanthoxin, and ultimately ABA. Enzymes that cleave carotenoids at other positions are called Qarotenoid Qleavage gioxygenases (CCD). NCEDs are encoded by small gene families (5 in Arabidopsis), so that functional redundancy may preclude isolation of need mutants in selection schemes.
Af'!!Mt'lli<S!f'lthf§l
J,o;!: .. ~1 :1" . :tU .... Nlolll:>:amll1in
Figure 1. The biosynthetic pathway of ABA from the xanthophylls 9-cis-violaxanthin and 9' -cisneoxanthin. The first part of the pathway involves synthesis of carotenoids. The first committed step in ABA biosynthesis is the cleavage step. The mutants abal-aba3, and aao3 in Arabidopsis for different steps in the pathway are indicated. ABA3 encodes a molybdenum cofactor which is required by abscisic aldehyde oxidase, AA03. The mutant vp14 is in maize.
2
The cleavage step in ABA biosynthesis is strongly upregulated by water stress and thus a major regulatory step in the pathway. As expected, overexpression ofNCEDs in transgenic plants results in elevated ABA levels, increased seed dormancy, reduced transpiration rates, and increased tolerance of drought. There are no reports of transgenic plants overexpressing other ABA biosynthetic genes that cause similar phenotypes. This supports the notion that the cleavage reaction is the main step controlling ABA biosynthesis in green tissues, in which carotenoid substrates are non-limiting.
ABSCISIC ACID CATABOLISM The two main pathways of ABA catabolism are conjugation and oxidation. Conjugation
of ABA with glucose gives the ABA glucose ester (ABA-GE) and is catalyzed by ABA glucosyl transferase. Oxidation can take place at the 7' -, 8' -, and 9' -carbon atoms of the ABA molecule, but occurs predominantly at the 8' -position and is catalyzed by ABA 8' -hydroxylase (Figure 2). Oxidation at the 8' -position gives rise to the unstable intermediate 8' -hydroxy ABA, which is converted to phaseic acid (P A), which is in tum converted to dihydrophaseic acid (DP A). This end product, as well as DP A glucoside, can accumulate to very high levels in certain organs, such as seeds.
Figure 2. Catabolism of ABA takes place via conjugation to ABA-GE and oxidation at the 7'-, 8' -, and 9' -carbons ofthe ABA molecule. Oxidation at the 8' -position is the predominant pathway in most species. GE = glucose ester; GS = glucoside.
3
Although it was known since 1976 that 8' -hydroxylase is a P 450 monooxygenase (CYP), it was not until last year that the genes encoding ABA 8' -hydroxylase were isolated (Kushiro et aI., 2004; Saito et aI., 2004). In Arabidoposis, four genes, CYP707Al-4, encode 8'-hydroxylase. Expression of CYP707 A2 is specifically upregulated when Arabidopsis seeds are imbibed, thus rapidly depleting the ABA pool and releasing the seeds from dormancy. As expected, seeds of the mutant cyp 70 7a2 lack seed dormancy. Expression of all four CYP707A3 genes was increased when stressed leaves were rehydrated. Thus, it is clear that expression of the CYP707A genes plays a key role in regulating ABA levels. Recombinant CYP707 A protein oxidized ABA exclusively to 8' -hydroxy ABA and P A. Thus, it appears that 7' - and 9' -hydroxylation of ABA are catalyzed by different enzymes, presumably also P450s.
REGULATION OF ABA METABOLISM The current status of our knowledge of ABA metabolism indicates that NCEDs and
CYP707 As are the two main groups of enzymes that regulate the levels of ABA through biosynthesis and catabolism, respectively. Nevertheless, there are reports that either stress or application of ABA upregulates expression of all genes in the pathway, except ABA2. It has not been shown, however, that increased expression of genes other than NCEDs also results in increased ABA production. In particular, application of ABA also induces expression of CYP707As, thus enhancing ABA degradation. The possibility exists that increased catabolism leads to increased biosynthesis through feedback regulation, although there is no evidence at present for such a mechanism.
Recent work has shown that the final step in ABA biosynthesis in turgid plants is localized in vascular bundles, specifically in companion cells and xylem parenchyma cells (Koiwia et aI., 2004; Christmann et aI., 2005). However, the first committed step in ABA biosynthesis, the cleavage of carotenoids, takes place in chloroplasts, mainly in the mesophyll. Thus, movement of intemlediates appears to be involved in ABA biosynthesis.
Very little is known about the mechanism by which water stress triggers ABA biosynthesis, except that loss of turgor appears to be the signal. Most likely, this initial signal is sensed at the plasma membrane, causing increased transcription of NeED genes in the nucleus, import ofthe NCED protein into the chloroplasts and cleavage of carotenoids. Most of this signaling pathway remains to be elucidated.
ACKNOWLEDGEMENT Our work on abscisic acid metabolism is supported by the National Science Foundation
and the U.S. Department of Energy.
LITERATURE CITED
Christmann A, T Hoffinann, I Teplova, E Grill, and A Muller. 2005. Generation of active pools of abscisic acid revealed by in vivo imaging of water-stressed Arabidopsis. Plant Physiol. 137:209-219.
Nambara E, and A Marion-Poll. 2005. Abscisic acid biosynthesis and catabolism. Annu. Rev. Plant BioI. 56:165-185.
4
Koiwai H, K Nakaminami, M Seo, W Mitsuhashi, T Toyomasu, and T Koshiba. 2004. Tissuespecific localization of an abscisic acid biosynthetic enzyme, AA03, in Arabidopsis. Plant Physio1. 134:1697-1707.
Kushiro T, M Okamoto, K Nakabayashi, K Yamagishi, S Kitamura, T Asami, N Hirai, T Koshiba, Y Kamiya, and E Nambara. 2004 The Arabidopsis cytochrome P450 CYP707 A encodes ABA 8' -hydroxylases: key enzymes in ABA catabolism. EMBO 1. 23: 1647 -1656.
Saito S, N Hirai, C Matsumoto, H Ohigashi, D. Ohta, K Sakata, and M Mizutani. 2004. Arabidopsis CYP707As encode (+)-abscisic acid 8'-hydroxylase, a key enzyme in the oxidative catabolism of abscisic acid. Plant Physio1. 134: 1439-1449.
Schwartz SH, X Qin, and JAD Zeevaart. 2003. Elucidation of the pathway of abscisic acid biosynthesis by mutants, genes, and enzymes. Plant Physio1. 131:1591-1601.
Schwartz SH, and JAD Zeevaart. 2004. Abscisic acid biosynthesis and metabolism. In PJ Davies, ed, Plant Hormones. Biosynthesis, Signal Transduction, Action. Kluwer, Dordrecht, The Netherlands, pp. 137-155.
5
ABSCISIC ACID ANALOGS - TOWARD DEVELOPMENT OF ABA-BASED PLANT GROWTH REGULATORS
S. Abrams1*, K. Nelson, Y. Gai, I. Zaharia, M. Galka, P. Galka, I. Alarcon
ABSTRACT
We are developing chiral ABA analogs to probe the structural requirements of binding pockets of proteins that recognize ABA and ABA catabolites. Our overall objectives are to establish the biological activity of each compound in the pathway and to design analogs to target specific proteins, in a rational design of ABA-based plant growth regulators. These analogs are being used in physiological and biochemical studies on enzymes that metabolize ABA such as glucosyl transferases and P450 monooxygenases that hydroxylate ABA. Affinity probes with the essential structural features of ABA preserved have been developed to identify novel ABA-binding proteins. Recent progress in these studies will be reported.
IPlant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK S7H 3A6 Canada
6
CHALLENGES FOR THE COMMERCIAL DEVELOPMENT OF S-ABSCISIC ACID (ABA)
PD Petracek1 * , D Woolard, R Menendez, and P Warrior
ABSTRACT ABA is one of the natural hormones found ubiquitously in plants. ABA is involved in
many major events of plant growth and development including dormancy, germination, bud break, flowering, fruit set, general growth and development, stress tolerance, ripening, abscission, and senescence. One of the most well known roles of ABA is the regulation of water relations in plants through the control of stomata opening and closure. Over 5000 scientific papers and patents on the molecular biology, biochemistry, physiology, and applied efficacy of ABA have been published since Okhuma et al. proposed its chemical structure in 1965. Despite this breadth of information, a commercial use for ABA in agriculture has not yet been identified or pursued. Like other PGRs, commercialization of ABA must meet several critical challenges. First, an ABA use must solve a commercial problem with a favorable return on investment for the user. Second, ABA must be manufactured at a cost that is commensurate with the value of its agronomic applications. Third, ABA must be registered and approved for conm1ercial use. Overall, the commercialization of ABA requires a substantial investment in production optimization, laboratory research, field development, and regulatory support.
The commercial development of a PGR follows both sequential and parallel paths. These paths include research and development on the active ingredient (discovery of activity, laboratory/greenhouse research, field research, field development, and commercial development), production of the active ingredient (initial production through synthesis, fermentation, or extraction, and optimization of production) and registration of the active ingredient (physical and chemical properties testing, safety testing, and environmental safety testing).
For agrochemicals such as herbicides, insecticides, and fungicides, the question is often known and the answer is found. For example: How do I kill this weed, but not my crop? For PGRs, the answer is often known and the question is found. For example: Here is ABA, now find a use. The primary challenge for commercial development of ABA is to find the question that ABA can answer. The steps to meeting that challenge include the following:
1. Review the background information. In the published literature there are more than 5000 articles on abscisic acid based on a search through Agricola. Most studies on ABA pertain to biochemistry or basic physiology. Few of them pertain to exogenous application. The lack of applied ABA studies is due in part to the cost of ABA. Field studies require gram quantities of active ingredients. For example, S-ABA in the 2004-2005 Sigma catalog cost $533/mg. One liter of 1000 ppm S-ABA would cost $533,000.
lValent BioSciences Corporation, 6131 Oakwood Road, Long Grove, Illinois 60047
7
Among patents there are more than 60 patents on abscisic acid based on a search through the US Patent Office. Many of the patents pertain to conifer embryogenesis. Additionally academic researchers with ABA experience have been consulted.
2. List the potential targets. This may be done by first identifying targets based on a review of the background information, second organizing targets based on chronology of plant life, and third prioritizing targets based on most credible evidence and potential utility.
3. Develop the methodology. This requires, first determining how to evaluate the targeted effects (e.g. lab/greenhouse studies vs. field trials and internal research vs. external cooperator), second developing a usable formulation prototype that maintains a soluble and stable active ingredient, and third developing protocol to evaluate effects of active concentration, application timing, adjuvant requirements, and application method (e.g. spray, drench, or sprench).
4. Execute the studies, review the data, and reprioritize. Examples ABA effects include the increase in grape coloration, reduction in sweet com chilling injury, extension in shelf-life of ornamentals, reduction of transpiration (Figure 1, top) and reduction in growth (Figure 1, bottom).
Conclusion The critical challenges for the cOlmnercial development of S-abscisic acid include: 1. Identifying commercial opportunities with favorable returns on investment for the user. 2. Reducing in the cost of manufacturing to a level that is commensurate with the value of its agronomic application. 3. Registering the product with the US EPA and obtaining approval from state agencies for commercial use.
8
~ Ze o'E -0 1-1.) «-0::0 -c a.!\! (J).s::. z ... ~! 1-
0
~
120
100
80
60
40
20
0
24
20
4
_ Water control
__ Ethanol control
-'- ABA ('10 ppm) ~ ABA (100 ppm)
-1 0 1 2 3 4 5 6 7 8 9 10 DAYS AFTER TREATMENT
_ Water control __ Ethanol control
~ ABJ:\(10ppm)
~ ABA ('100 ppm) ""4·"'" {>J.3!\ CWOO NJm)
0L-~_1~~0--L1~2~~3~~4--75~6~~7~~8--79~1~0
DAYS AFTER TREATMENT
Figure 1. Rutgers Tomato seeds were planted in Promix PGX and seedlings were grown for in a 25C growth chamber with a 16-hour light cycle for the first 10 days. The seedlings were transplanted into standard 6-inch pots with Promix BX and held in a 25/18C greenhouse. Fourteen days after planting (n = 10 plants/treatment), plants were treated by pipetting 10 ml of treatment solution (water control, ethanol control, 10, 100, or 1000 ppm ABA) onto moist soil of each pot. The seedlings were watered daily. Transpiration was measured daily with a LiCor Li-1600 steady state porometer attached to the newest leaf large enough to span the orifice of the porometer (top figure). Height to the tallest node was measured daily (bottom figure).
9
ABA SIGNALING NETWORKS IN ARABIDOPSIS
R.Finkelstein1*, T. Lynch1, I. Brocard-Gifford1
, M.E. Garcia\ T.L. Thomas2.
ABSTRACT Abscisic acid (ABA) regulates both seed maturation and germination, and several ABA
insensitive (ABI) loci encoding transcription factors are required for normal seed maturation and inhibition of gern1ination and seedling growth by ABA. We are expanding our knowledge of this signaling network by detennining which of these factors interact with each other, identifying additional factors that interact directly with the ABI gene products, and identifying genes that are directly regulated by the ABI factors. These studies have revealed extensive cross-regulation among these factors, and identified a novel plant-specific family of proteins that modifies ABA and abiotic stress sensitivity. Regulatory targets of the ABI factors constitute less than 10% of all ABA-regulated genes, including presumed desiccation protectants and additional regulators, but these may be core components of ABA response because ABI over-expression confers hypersensitivity to ABA and other stresses. Our results may lead to strategies for manipulation of seed nutritional, germination or storage qualities.
INTRODUCTION The plant hormone abscisic acid (ABA) affects many agronomically important features of
plant growth including embryo development, seed and bud dormancy, water movement and retention, tolerance of a variety of abiotic stresses (e.g. drought, salinity, and cold), and senescence. Many of these effects involve changes in gene expression, and transcriptional profiling studies in Arabidopsis have shown that as much as 5% of the genome is regulated by ABA (Hoth et al., 2002; Seki et al., 2002; Suzuki et al., 2003). However, it is likely that distinct subsets of these genes are ABA-responsive within any given organ or tissue type. The specificities of these responses depend on the local ABA concentration and the available signal transduction components. Consequently, one strategy to modify only subsets of ABA responses requires that we identify specific regulators with limited effects.
Attempts to understand ABA signaling have employed biochemical, pharmacological and genetic approaches to identify possible signal transduction components. To date, over 50 loci have been shown to affect some aspect of ABA signaling; many of these also regulate responses to other signals, such as other hormones or environmental conditions, providing a mechanism for integrating response to multiple signals (Finkelstein et al., 2002). Many ofthe mutants affecting these loci have no obvious phenotypic defects in the absence of ABA or stress treatments and, even under stress conditions, some phenotypes are detectable only by molecular analysis. The subtleties and complexities of these phenotypes are consistent with a high degree of genetic redundancy and many points of interaction or "cross-talk" among signaling pathways. The products of these loci include transcription factors, protein kinases and phosphatases, GTPbinding proteins, enzymes involved in phospholipid metabolism or RNA processing, and many unknowns. A current challenge is to discern which combinations of these components interact to form a signaling network and to identify regulators that can enhance stress tolerance without severely stunting growth.
Among these ABA effects, control of seed maturation and the subsequent commitment to seedling growth are major determinants of a plant's reproductive success. Several loci affecting sensitivity to ABA at gennination have been identified as the genes disrupted in the ABA insensitive (abi) mutants of Arabidopsis (Finkelstein et al., 2002). Three ofthese loci (ABI3, ABI4 andABI5) encode transcription factors. We are broadening our studies of the network by identifying additional genes that interact with or regulate these loci and investigating their function in ABA and/or stress signaling in seeds and seedlings.
IMolecular, Cellular and Developmental Biology Department, UC Santa Barbara, Santa Barbara, CA 93117 USA 2Dept. of Biology, Texas A&MUniversity, College Station, TX 77843 USA
10
MATERIALS AND METHODS Plant Genetic Materials Single and double mutant lines with defects inABI4, ABI5, FUS3, LEel, ABF3 andABFl were isolated as described in (Brocard-Gifford et aI., 2003; Finkelstein et aI., 2005). T -DNA insertion lines for the A5IPs were obtained from the SIGnAL collection (Alonso et aI., 2003), distributed by the ABRC. Yeast Two-Hybrid Screen
A translational fusion of the DNA binding domain (BD) ofGAL4 and all but the first eight amino acids of ABI5 was constructed as described in (Nakamura et aI., 2001), and transformed into yeast (Saccharomyces cerevisiae) strain PJ69 (James et aI., 1996) to be used as "bait" in a two-hybrid screen. An activation domain (AD) fusion cDNA library derived from 3-day-old etiolated tissue (Kim et aI., 1997), distributed by the ABRC, was transformed into the yeast and cells containing potentially interacting fusions were selected by complementation of defects in histidine and adenine biosynthesis. Plasmids encoding the activation domain fusions were isolated and their fusion genes sequenced to identify the predicted interacting protein. Additional interactions were tested by transforming the AD fusion plasmids back into yeast, in combination with other BD fusions. Interactions were assayed qualitatively by complementation of the auxotrophies, and quantitatively by activation of a beta-galactosidase reporter gene, as described in (Nakamura et aI., 2001). Plant Material For Microarray Analyses
In order to emphasize the effects of each specific ABI regulator, we chose to compare ABA-responsive gene expression in ABI over-expression (35S:ABl) vs. mutant (abi) lines. Plants (11-12 days old) were treated with 50 flM ABA and 20 flM cycloheximide (CHX) for 4 hrs prior to harvest and RNA extraction; the short induction period and inclusion of CHX was intended to limit inductions to those that are primary effects of the constitutively over-expressed ABI factor. RNA was prepared by hot phenol extraction, followed by serial precipitations, as described in (Finkelstein et aI., 1985). Prior to use in micro array experiments, effectiveness of the transgene expression and the ABA and CHX treatments were confinned by Northern blot analyses of genes whose expression patterns were predictable based on previous studies. Microarray Analyses
Affymetrix ATHI Gene Chips were used to characterize transcriptomes associated with specific genomes and treatments. All procedures for generating labeled targets and hybridization of these targets to elements on the Gene Chips were carried out as recommended by Affymetrix. Briefly, cDNA was derived from total RNA and subsequently used to generate biotin labeled cRNA which was fragmented to lengths between 35 and 200 bp. Hybridization ofthe fragmented cRNA to the Gene Chip Array was carried out in a Affymetrix GeneChip Hybridization Oven 640. Appropriate washing and staining of the arrays was then conducted on an Affymetrix GeneChip Fluidics Station 400 under the control of Affymetrix Microarray Suite 5.0 software. The stained and washed array was finally scanned with an Agilent GeneArray Scamler.
The output information from the scanner, including intensity data and hybridization quality flags, and annotation information on the genes represented on the ATHI Genechip were then used as input to GeneSpring 7 (Silicon Genetics/Agilent) for analysis and data mining. The program was used to normalize the intensity data. All values below 0.01 were set to 0.01. For purposes of comparison, the data from each chip were compared to the control chip which was the chip hybridized with targets derived from seedlings over expressing the ABI4 gene in the absence of added ABA. Each measurement for each gene on each chip was divided by the median of that gene's measurements on the control chip. Only genes flagged as present or marginal in the experimental chips were considered for determining those that were up-regulated relative to the control. Similarly, only genes flagged as present or marginal in the control hybridization were considered for determining genes down-regulated with respect to the control.
RESULTS AND DISCUSSION We have used three approaches to elucidating the network surrounding the ABI
transcription factors: testing for potential interactions with known loci encoding additional
11
transcription factors, screening for proteins that can interact directly with the ABI proteins in a yeast two-hybrid system, and identifying regulatory targets of the ABI factors by micro array analyses. Tests of Interactions Among Known Loci
ABI3, ABI4, and ABI5 encode transcription factors that are members of three distinct families that playa major role in the seed/seedling transition and ABA response: the B3-, AP2-, and bZIP domain families (Finkelstein et aI., 2002) (Figure 1). Other loci regulating embryonic identity were identified as defective in leafy cotyledon (lee) class mutants, including those encoding additional B3 family members (e.g. FUSCA3 and LEAFY COTYLEDON2). Other bZIPs mediating ABA or stressresponsive gene expression (ABFs, AREBs, and AtDPBFs) were identified based on binding to cis-acting ABAresponse elements, but very little genetic information was available for the loci encoding these factors until recently (Bensmihen et aI., 2005;Bensmihen et aI., 2002;Kim et aI., 2004). Consequently, both the LEAFY COTYLEDON (LEC) class loci and the other members of the ABI51ABFIAREBIAtDPBF clade of bZIPs were good candidates for interactors with the ABI transcription factor loci.
Figure 1. Factors regulating the seed/seedling transition
Furthermore, genetic interactions between ABI3 and the LEC class loci had already been demonstrated (parcy et aI., 1997). To test for genetic interactions between theLEC class loci and ABI4 or ABI5, or among potentially redundantABI5-related bZIPs, we constructed and characterized double mutants. The potential for direct interactions was tested in a yeast twohybrid assay, and by detennining whether the various combinations of factors were coexpressed.
The genetic studies demonstrated both synergistic and antagonistic interactions among the ABI and LEC class factors, the nature of the interaction varying with the specific response (Brocard-Gifford et aI., 2003). Although such interactions are most consistent with a complex combinatorial control network, no direct interactions were observed by two-hybrid assays (Brocard-Gifford et aI., 2003). However, detailed expression analyses showed substantial crossregulation among these loci, and distinct temporal and spatial regulation. Although all of these factors are expressed throughout embryo development, LEC1 is most abundant earliest (Harada, 2001) and is required for a subsequent increase in FUS3 expression (Brocard-Gifford et aI., 2003). FUS3 inhibits GA synthesis and promotes ABA synthesis, which in tum promotes FUS3 stability, thereby enhancing FUS3 activity in mid-embryogeny (Gazzarrini et aI., 2004). LEC1 and FUS3 also regulate expression and/or stability of ABI3 (Parcy et aI., 1997), ABI5, and several related bZIPs (Brocard-Gifford et aI., 2003). It is not known whether any of these reflect direct cross-regulation, but ABI5 at least is auto-regulatory (Brocard et aI., 2002).
In contrast to the dramatic defects of the lee class mutants, loss of function for most of the ABI5 related clade ofbZIPs have almost no discernible phenotype (Bensmihen et aI., 2005; Bensmihen et aI., 2002; Finkelstein et aI., 2005; Kim et aI., 2004). However, ABI5 andABF3 appear to function redundantly, such that double mutants are more resistant to ABA than the monogenic parents (Finkelstein et aI., 2005). Surprisingly, loci with similar effects antagonistically regulate each other's expression (Finkelstein et aI., 2005), possibly providing a
12
mechanism to emphasize regulation by distinct family members in different tissues or stages of development.
Screens for inter actors Relatively few direct interactions with the ABI factors were observed by two-hybrid
assays. Therefore, we screened for additional factors that might participate in complexes affecting ABI-dependent signaling. The strongest interactions for ABI5 were observed with a family of four proteins containing 3 highly conserved (70-86% identical) domains, none of which have any known function. We have 100
designated these A5IPs, for ABI~-interacting nroteins. To determine whether these proteins (/) participate in ABA signaling in plants, we ~ 80
obtained T -DNA insertion lines for all ofthe '<t
loci and constructed over-expression lines for ~ 60
most. Comparison of ABA sensitivity of A5IP c
loss- and gain-of-function lines confirms a ~ 40
--Col -e a5ip2 ---b 85ip3 ---~ a5ip4 ~ws role for all in ABA response (Figure 2). .~
However, despite the high degree of sequence aJ ,,·,·,,$v· 3f.iSJUf('!
conservation, different family members have ~ 20 -+- 35S:A5IP2/+
., ... ~ \~~ af.ijp1
. f'C' t ABA 't"ty ~ 35S:A51P3 opposmg e lec s on senSllVl ',., --"E'~ l3illi6
Expression analyses and additional yeast two- 0 L_-II-__ liJ::.-=...:::..'Ii~~!!!!!I!!!!!!!!!IIIII!!!!tP"-" hybrid assays indicate that the A5IPs may interact 0 1 10
with multiple members of the ABI5-related bZIP clade in different tissues or developmental stages. Although the genetic data confirm the importance of the A5IPs in regulating ABA response, they do not address mechanism, which might involve effects on stability, activity or localization of ABI5 and related bZIPs (Figure 3).
Embryo Drought, osmotic or maturation ~ /' nutrient stress
ABA
~
ABA-regulated gene expression (including ABI5)
-< ~
[ABA] (iJM)
Figure 2. ABA sensitivity of A5IP mutant and over-expression lines, listed beneath their respective wildtype backgrounds.
ABI5 degradation Germination Seedling growth
Embryo maturation Desiccation tolerano
Identifying regulatory targets of ABI transcription factors Comparison of either loss- or gain-offunction lines for either ABI4 or ABI5 suggests that these factors have similar roles in ABA and stress response (Brocard et aI., 2002; Finkelstein, 1994; Soderman et aI., 2000). Furthermore, the ABA and stress hypersensitivity of the overexpression lines suggests that the subset of genes that are regulated by these loci are central components of ABA response. To identify specific regulatory targets of the ABI factors, we used micro arrays to compare ABA-inducible expression in overexpression vs. mutant lines.
Figure 3. Hypothetical model of interactions among A5IPs, ABI5 and possibly related bZIPs affecting the commitment to seedling growth. The biochemical functions, and hence the mechanism of action, of the A5IPs are unknown.
13
Cycloheximide was included to limit transcript accumulation to those genes that do not require additional protein synthesis to promote their expression. These studies test the hypotheses that the ABls have similar effects due to
the overlap in their targets, and that these targets include some regulators as well as genes encoding mediators of desiccation tolerance and other protective functions. In contrast to abil-l, which perturbs regulation of greater than 90% of ABA-regulated genes (Hoth et aI., 2002), each ABI appeared to regulate less than 100 ABA-inducible genes (~1O% of the total), with only 20-30% of their targets in common. The largest class of shared targets is late embryogenesis abundant and related genes, which encode extremely hydrophilic proteins and are correlated with desiccation tolerance in many contexts (Wise and Tunnacliffe, 2004). Interestingly, additional targets include transcription factors, receptor-like kinases, protein phosphatases, and enzymes of phospholipid metabolism, suggesting that these ABIs may act in part by activating components of other signaling pathways. Reverse genetic studies are in progress to test the relevance of some of these other regulators to ABA or other stress responses.
CONCLUSIONS These studies have shown that some of the ABI genes are members of gene families with
overlapping functions that may be either synergistic or antagonistic, and that there is substantial cross-regulation of expression among some ABI genes and their homologs. We have also identified strong interactions with another novel family of proteins that affect ABI function in early seedling growth, the A5IPs. Finally, our micro array studies identified 15 potential ABIregulated regulators that may contribute to ABI-dependent gene expression, as well as provide a mechanism to integrate responses to ABA and other stress signals. By integrating molecular, genetic and physiological data, we hope to develop a coherent model of ABA action that could have applications in modifying seed quality and yield or stress tolerance of plants.
ACKNOWLEDGEMENTS We thank the ABRC team at Ohio State University for efficient distribution of the T-DNA
insertion lines from the SALK SIGnAL collection. This work was supported by grants from the National Science Foundation (IBN-9982779) and USDAINRIlCGP (03-35304-13201) to RRF, and USDAlNRI/CGP 01-35304-10940 to TLT. Funding for the SIGnAL indexed insertion mutant collections was provided by the National Science Foundation.
LITERATURE CITED
Alonso, 1M, AN Stepanova, TJ Leisse, CJ Kim, H Chen, P Shinn, DK Stevenson, J Zimmerman, P Barajas, R Cheuk, et aI. 2003. Genome-Wide Insertional Mutagenesis of Arabidopsis thaliana. Science 301: 653-657.
Bensmihen, S, S Rippa, G Lambert, D Jublot, V Pautot, F Granier, J Giraudat and F Parcy. 2002. The Homologous ABI5 and EEL Transcription Factors Function Antagonistically to Fine-Tune Gene Expression during Late Embryogenesis. Plant Cell 14: 1391-1403.
Bensmihen, S, J Giraudat and F Parcy. 2005. Characterization ofthree homologous basic leucine zipper transcription factors (bZIP) of the ABI5 family during Arabidopsis thaliana embryo maturation. J. Exp. Bot 56: 597-603.
BrocaI'd, I, T Lynch and R Finkelstein. 2002. Regulation and role of the Arabidopsis ABAinsensitive (ABI)5 gene in ABA, sugar and stress response. Plant PhysioI. 129: 1533-1543.
Brocard-Gifford, I, T Lynch and R Finkelstein. 2003. Regulatory networks in seeds integrating developmental, ABA, sugar and light signaling. Plant Physio1. 131: 78-92.
Finkelstein, R, K Tenbarge, J Shumway and M Crouch. 1985. Role of abscisic acid in maturation of rapeseed embryos. Plant Physiol 78: 630-636.
14
Finkelstein, R, S Gampala and CRock. 2002. Abscisic acid signaling in seeds and seedlings. Plant Cell 14: SI5-S45.
Finkelstein, R, SSL Gampala, TJ Lynch, TL Thomas and CD Rock. 2005. Redundant and distinct functions of the ABA response loci ABA-INSENSITIVE(ABI) 5 andABRE-BINDING FACTOR (ABF)3. Plant Mol. BioI. in press.
Finkelstein, RR. 1994. Mutations at two new Arabidopsis ABA response loci are similar to the abi3 mutations. Plant J. 5: 765-771.
Gazzarrini, S, Y Tsuchiya, S Lumba, M Okamoto and P McCourt. 2004. The transcription factor FUSCA3 controls developmental timing in Arabidopsis through the hormones gibberellin and abscisic acid. DeveI. Cell 7: 373-385.
Harada, J. 2001. Role of Arabidopsis LEAFY COTYLEDON genes in seed development. J. Plant PhysioI. 158: 405-409.
Hoth, S, M Morgante, J-P Sanchez, M Hanafey, S Tingey and N-H Chua. 2002. Genome-wide gene expression profiling in Arabidopsis thaliana reveals new targets of abscisic acid and largely impaired gene regulation in the abil-l mutant. J. Cell Sci. 115: 4891-4900.
James, P, J Halladay and E Craig. 1996. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144: 1425-1436.
Kim, J, K Harter and A Theologis. 1997. Protein-protein interactions among the Aux/IAA proteins. Proc. NatI. Acad. Sci. USA 94: 11786-11791.
Kim, S, J-Y Kang, D-I Cho, JH Park and SY Kim. 2004. ABF2, an ABRE-binding bZIP factor, is an essential component of glucose signaling and its overexpression affects multiple stress tolerance. Plant J. 40: 75-87.
Nakamura, S, T Lynch and R Finkelstein. 2001. Physical interactions between ABA response loci of Arabi do psis. Plant J. 26: 627-635.
Parcy, F, C Valon, A Kohara, S Misera and J Giraudat. 1997. The ABSCISIC ACIDINSENSITIVE3, FUSCA3, and LEAFY COTYLEDONlloci act in concert to control multiple aspects of Arabidopsis seed development. Plant Cell 9: 1265-1277.
Seki, M, J Ishida, M Narusaka, M Fujita, T Nanjo, T Umezawa, A Kamiya, M Nakajima, A Enju, T Sakurai, et aI. 2002. Monitoring the expression pattern of around 7,000 Arabidopsis genes under ABA treatments using a full-length cDNA microarray. Funct. Integr. Genomics 2: 282-291.
Soderman, E, I Brocard, T Lynch and R Finkelstein. 2000. Regulation and function ofthe Arabidopsis ABA-insensitive4 (ABI4) gene in seed and ABA response signaling networks. Plant Physio!. 124: 1752-1765.
Suzuki, M, MG Ketterling, Q-B Li and D McCarty. 2003. Viviparous 1 alters global gene expression patterns through regulation of abscisic acid signaling. Plant PhysioI. 132: 1664-1677.
Wise, M and A Tunnac1iffe. 2004. POPP the question: What do LEA proteins do? Trends Plant Sci 9: 13-17.
15
SESSION I
BIOTIC AND ABIOTIC STRESS
17
SUGAR MOVER ENHANCES CROP PERFORMANCE, AND BIOTIC/ABIOTIC TOLERANCE OF PLANTS
Jerry H. Stoller\ Ron Salzman* and Albert Liptay
ABSTRACT
Sugar Mover is a commercial product for enhancing photosynthate partitioning and acquisition in the plant. Results indicate an increase in carbon fixation and an increase in sugar transport from leaves to the sink: tissues. Application of Sugar Mover can also adjust the hormone balance during the various stages of growth, resulting in a more desirable architecture of the crop plant. Evidence suggests that Sugar Mover may influence hormone synthesis downstream ofthe MEP pathway, resulting in altered ABA:GA ratios.
I Stoller Enterprises Inc, 4001 W Sam Houston Parkway N, Houston TX 77043 USA
19
THE ROLE OF ROOT TO SHOOT SIGNALLING IN COORDINATING RESPONSES TO SOIL COMPACTION
S.L. Aphale1*, T.S. Stokes, C.R. Black, LB. Taylor and J.A. Roberts
ABSTRACT Soil compaction imposes physical and hypoxic stresses on roots, promoting consequent
reductions in stomatal conductance and shoot growth. Ethylene and ABA are recognized long distance signals, mediating responses to various stresses including soil drying, flooding and compaction. Wildtype and mutant genotypes of tomato with a reduced capacity to produce ABA or ethylene were grown in a novel growth system in which ballotini beads were used to simulate sub-critical compaction stress. The objective was to establish the role of ethylene and ABA in mediating plant growth under compacted conditions. Their role as long-distance messengers mediating responses to soil compaction at the gene and whole plant level is discussed.
INTRODUCTION The pressure which roots must exert to grow through soil is determined largely by soil
water content and bulk density; as the soil dries, its resistance to root penetration rises, increasing the stress imposed on the root tip (Passioura, 1988). Soil compaction affects root and shoot growth and may reduce crop yield (Clark et al., 2003). Decreases in stomatal conductance and leaf growth may occur in the absence of any change in foliar water status, suggesting that root-sourced signals may be responsible for eliciting these responses, as occurs when roots are subjected to drought (Davies and Zhang, 1991).
Innovative split-pot systems developed to investigate responses to compaction in sensitive species such as tomato (Mulholland et al., 1996) have shown that reductions in stomatal conductance are correlated with increases in xylem sap ABA concentration (Hussain et al., 1999). In a detailed review of the evidence obtained from split-pot and root excision studies using wildtype and ABA-deficient mutants of tomato, Roberts et a1. (2002) concluded that root-sourced ABA has a central role in mediating stomatal responses to compaction.
Similar approaches have been used to investigate the role of ethylene or its biosynthetic precursors, particularly l-aminoacyclopropane-l-carboxylic acid (ACC), in mediating responses to compaction (He et a1., 1996). Hussain et al. (1999) noted that reductions in leaf growth were closely correlated with increased ethylene evolution from leaf tissue, while Roberts et a1. (2002) reported that leaf growth was suppressed when wildtype plants were grown on compacted soil, but not in the ACO lAS mutant, which has a reduced ability to produce ethylene. This observation suggests a role for ethylene in mediating reductions in leaf growth. The role of crosstalk between ABA and ethylene in mediating compaction responses has also been investigated. In experiments in which wildtype, ethylene-deficient and AC01AS and ABA-deficient mutant notabilis genotypes of tomato were grown in a splitpot system, Hussain et al. (2000) showed that wild-type levels of ABA restricted the increase in ethylene production which normally occurs when plants experience sub-critical soil compaction.
In the present study, an aerated hydroponic culture system containing ballotini beads placed was used to examine the impact of mechanical impedance to root growth on wildtype (Ailsa Craig) and ABA-deficient mutant. (no tab ilis) genotypes of tomato. Effects on stomatal conductance and shoot and root growth were examined in the context of temporal and spatial
IPlant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, LEICS, LE12 5RD, U.K
20
changes in the expression of genes involved in the biosynthesis of ABA (9' -cisepoxycarotenoid dioxygenase; NCED) and ethylene (ACC synthase; AC01).
MATERIALS AND METHODS Germination and seedling establishment
Seeds of Ailsa Craig and notabilis were germinated and propagated until 22 days after emergence; they were then transferred to an aerated hydroponic system containing modified Hoagland's nutrient solution. Sub-critical mechanical impedance was simulated by adding glass ballotini beads (2.85 mm diameter) to the nutrient solution. The plants were arranged in a randomised block design; data were analysed using Genstat version 5.
Plant growth measuremeuts Leaf area and leaf and root dry weights were determined at each harvest. Daily
increments in leaf area were calculated from non-destructive measurements of leaf length using an allometric equation; leaf expansion rate was calculated for each treatment by linear regression analysis.
Gas exchange measurements A portable CIRAS-2 infrared gas analyser (PP Systems, Hitchin, UK) was used to make
daily instantaneous gas exchange measurements (stomatal conductance and net photosynthetic rate) for the youngest fully expanded leaf (terrninalleaflet of leaf 3).
Leaf and root tissue RNA extraction and two step RT-PCR At each harvest, leaves and roots from plants grown on nutrient solution containing
ballotini (compacted treatment) or without ballotini (uncompacted) were snap-frozen using liquid nitrogen. RNA extracted using the Qiagen RNeasy® plant mini kit was treated with DNase using RQ 1 RNase-free DNase (Promega) and cleaned up for downstream RT -PCR using the RNA cleanup protocol described in the Qiagen RNeasy® plant mini kit. First strand synthesis was carried using the ABgene Reverse-iITh1 1 st strand synthesis kit to generate high yields of full-length cDNA. Oligo-dT primers were used in the RT step and gene-specific primers for NCED and ACOI for the amplification. 35 cycles ofPCR were carried out before analysing a 10 pI aliquot of the RT-PCR reaction medium using agarose gel electrophoresis.
Experimental treatments Two experiments were carried out. In Experiment 1, the effects of sub-critical compaction
on stomatal conductance, leaf area and dry weight were examined for the wildtype Ailsa Craig and ABA-deficient mutant notabilis of tomato over a 12 day period. In Experiment 2, changes in the expression of key genes regulating ABA and ethylene biosynthesis were examined by harvesting root and leaf tissue of Ailsa Craig at defmed times during the experimental period; these samples were analyzed using RT-PCR to identify effects on gene expression. These were correlated with changes in gas exchange and leaf growth.
RESULTS In Experiment 1, stomatal conductance decreased significantly after one day of compaction
stress in both the wildtype Ailsa Craig and ABA-deficient notabilis (Fig. la) relative to uncompacted control plants, although the effect was greater extent in the wildtype (P<O.OOI). Net C02 assimilation (Fig. 1 b) declined relative to uncompacted controls in Ailsa Craig (P<O.OOI but not in notabilis. Leaf area and dry weight (data not shown) were reduced by
21
compaction in both genotypes (P<0.001, but to a greater extent in the wildtype plants (P<0.001).
" II) 900 1: 800 (5 E 700, .s 600 Q)
" 500 c
il 400 :J
300 "0 c 0 200 " ~ 100 E 0 0
U5 0 1 2345678 9 10 1112
lirre (days) after start of treatrrent
9 -", 8 1: 7 (5
6 E .s 5 c
4 :§ 3 E
'iii 2 II) til 1 d" 0 0
0 1 2 3 4 5 6 789101112
Tirre (days) after start of treatment
__ Ailsa
Craig (contro I)
____ Ailsa
Craig (beads)
- • -0· •• notabilis (control)
_. -0-' - notabilis (beads)
Figure 1. (a) Stomatal conductance and (b) net CO2 assimilation rate for Ailsa Craig and notabilis plants subjected to control and sub-critical compaction stress. Values are the mean ± double standard error for five plants per treatment
In Experiment 2, stomatal conductance declined in the compacted treatment within two days of imposing compaction stress (Fig. 2a; (P<O.OOl), but recovered to the control level by the end of the experimental period. Leaf expansion rate (Fig. 2b) was greatly reduced within one day of imposing compaction stress (P<0.001), an effect which was maintained throughout the experimental period. RT -PCR analysis (data not shown) revealed that NCED 1 expression in the roots increased within one day of imposing stress but decreased again on the following day. Further analysis is required to elucidate changes in AC01 expression in the leaves and roots.
-",
~600~:r::I o E 500 2-~ 400
~ 300 :J "0 § 200
" 1ii 100 15 E 0 ~~-L~~~~~-L~~~~ V5 o 1 2 3 4 5 6 7 8 9 101112131415
lime (days)
N E .s. t1l Q) ... t1l
to .3
30
25
20
15
10
5
0
0123456789101112131415
lime (days)
Figure 2. (a) Timecourses of stomatal conductance and (b) leaf area in Ailsa Craig plants subj ected to compaction stress. Vertical bars in (a) show standard errors of the difference between means for comparing treatments at specific times and changes with time.
DISCUSSION The initial reduction in stomatal conductance induced by compaction was greater in the
wildtype Ailsa Craig than in the ABA-deficient mutant notabilis (Fig. 1; (P<0.001», probably because its inability to produce wildtype ABA concentrations limits its stomatal responses (Roberts et al., 2002). The reduced stomatal conductance in the wildtype was accompanied by increased NCEDl expression in the roots, suggesting that ABA biosynthesis increased following exposure to compaction. However, this increase might have resulted from handling stress rather than de novo synthesis induced by compaction. The observed
22
stomatal closure in plants exposed to impeded rooting conditions may have resulted from increased root to shoot transpOli of ABA, or altematively from redistribution between intemal ABA pools (Wilkinson and Davies, 2002).
The observation that the reduction in leaf area induced by compaction was smaller in notabilis than in Ailsa Craig suggests that this effect may have resulted from its lower endogenous ABA concentration. By contrast, Roberts et al. (2002) reported that the reduction in leaf growth was greater in notabilis than in Ailsa Craig and concluded that wildtype ABA levels were necessary to limit the increase in ethylene concentrations in plants grown on compacted soil and prevent the ethylene-induced reduction in leaf growth seen in notabilis. FUliher genetically-based studies are required to elucidate whether differences in ethylene biosynthesis between notabilis and Ailsa Craig are causally linked with observed differences in ABA biosynthesis and leaf growth.
LITERATURE CITED
Clark LJ, WR Whalley and PB Barraclough 2003. How do roots penetrate strong soil? Plant and Soil 255: 93-104.
Davies WJ and J Zhang 1991. Root signals and the regulation of growth and development of plants in dtying soil. Annual Review of Plant Physiology and Plant Molecular Biology 42: 55-76.
He CJ and SA Finlayson, MC Drew, WR Jordan and PW Morgan 1996. Ethylene biosynthesis during aerenchyma formation in roots of maize subjected to mechanical impedance and hypoxia. Plant Physiology 112: 1679-1685.
Hussain A, CR Black, IE Taylor and JA Roberts 2000. Does an antagonistic relationship between ABA and ethylene mediate shoot growth when tomato (Lycopersicon esculentum Mill.) plants encounter compacted soil? Plant, Cell and Environment 23: 1217-1226.
Mulholland BJ, CR Black, IE Taylor, JA Robelis and JR Lenton 1996. Effect of soil compaction on barley (Hordeum vulgare L.) growth. 1. Possible role for ABA as a rootsourced chemical signal. Joumal of Experimental Botany 47: 539-549.
Hussain A, BJ Mulholland, CR Black, IE Taylor and JA Roberts 1999. Novel approaches for examining the effects of differential soil compaction on xylem sap ABA concentration, stomatal conductance and growth in barley (Hordeum vulgare L.). Plant, Cell and Environment 22: 1377-1388.
Passioura JB 1988. Root signals control leaf expansion in wheat seedlings growing in dtying soil. Australian Joumal of Plant Physiology 15: 687-697.
Robelis JA, A Hussain, IE Taylor and CR Black 2002. Use of mutants to study longdistance signalling in response to compacted soil. Journal of Experimental Botany 53: 45-50.
Wilkinson, Sand WJ Davies 2002. ABA-based chemical signalling and the coordination of responses to stress in plants. Plant, Cell and Environment 25: 195-210.
23
ROOT FEEDTM FOR ENHANCED, CROP PERFORMANCE AND BIOTIC/ABIOTIC CROP PLANT TOLERANCE
Jerry H. Stoller1, Ron Salzman and Albert Liptay*
ABSTRACT
This presentation is about the application of a commercial product which will allow a crop plant to regulate its hormone balance and enhance its tolerance to biotic and abiotic stress. The objective is to help the plant achieve a greater percentage of its genetic potential. Experiments indicate improved fruit quality, greater percentage of large fruits, higher yields, minimized physiological disorders and enhanced tolerance to insects and diseases. Root Feed is applied directly to the crop roots through drip irrigation on a weekly basis.
1 Stoller Enterprises Inc, 4001 W Sam Houston Parkway N, Houston TX 77043 USA
24
SESSION II
PGRS IN TURFGRASS AND VEGETABLE PRODUCTION
25
ANNUAL BLUEGRASS SEEDHEAD CONTROL IN OVERSEEDED PERENNIAL RYEGRASS WITH TURF GROWTH REGULATORS
A.G. EstesI* and L. B. McCarty
ABSTRACT
A study was conducted looking at annual bluegrass seedhead control in overseeded perennial ryegrass. Proxy (ethephon) 2L at 6.8 lb ai A-I, Primo Maxx (trinexapac-ethyl) 1L at 0.086 lb ai A-I, Cutless (flurprimidol) 50 WP at 1.5 lb ai A-I, Trimmit (paclobutrazol) 2 SC at 0.125 lb ai A-I and Embark T &0 (mefluidide) 0.2 L at 0.1225 lb ai A-I were evaluated alone and in tank mixes at half rates with one another. Applications were made in mid-February prior to annual bluegrass seedhead production and again four weeks later in mid-March. Annual bluegrass seedhead control was evaluated on a 0 -100% scale where 0 = no control and 100 = complete seedhead control throughout the study. The study also evaluated perennial ryegrass injury, quality and height.
'TIFEAGLE' BERMUDAGRASS RESPONSE TO THREE PLANT GROWTH REGULATORS
T.G. Willisl*, H. Liu1, T. Whitwell\ J.E. Toler2, and L.B. McCartyl
ABSTRACT
The study was performed for 20 weeks on 'TifEagle' bermudagrass [Cynodon dactylon (L.) Pers. x C. trasvaalensis Burtt-Davey]. Primo MAXX 1EC (trinexapacethyl), Cutless 50WP (flurprimidol), and Proxy 2L (ethephon) were evaluated alone and as a tank mix. Initial PGR applications were made on 17 May 2004 with sequential applications made biweekly. PGR rates included: Primo MAXX at 2 oz A-I (0.0175 kg ai ha-I
), Cutless at 2 oz A-I (0.07 kg ai ha-I), and Proxy at 2.5 oz 1000 ft2 (1.91 kg ai ha-I
).
Measurements included: turfgrass quality, turfgrass density, clipping yield reduction, root length density, root biomass, ball roll distances, shoot chlorophyll, root carbohydrates, overseeding establishment and spring transition from Poa trivialis L. to hybrid bermudagrass.
IDepartment of Horticulture, Clemson University, E-143 P&A Bldg., Clemson SC 29634-0319 USA 2Department of Applied Economics and Statistics, Clemson University, F-148 P&A Bldg., Clemson, SC 29634-0319 USA
28
MICROBIAL QUALITY AND SHELF LIFE OF HARPIN-TREATED HEAD LETTUCE
Jorge M. Fonseca1*, Wesley L. Kline2, Christian A. Wyenande, Murshidul Hoque3
, Husein Ajwa3
and Ned French4
ABSTRACT
The effect of pre-harvest application of Extend®, a newly developed second generation harpin product, on shelf life of fresh-cut lettuce was investigated. The lettuces were grown in locations A: Watsonville, CA; B: Cedarville, NJ; and C: Yuma, AZ, and treated five days before harvest at 140,280, and 420 glha. Lettuce processed and bagged was stored at 2-4°C and evaluated for quality during 20 days at 5-day intervals. Experimental conditions and subsequent results from the three trials varied. Lettuce from trial A treated at 280-420 glha consistently had a higher overall quality and lower microbial population than the control. In trial B, where wet conditions prevailed immediately after treatment application and during harvest, few differences among treatments were observed. In trial C, lettuce treated at 280 glha had consistently higher quality and lower microbial counts during early stages of storage (data not shown).
Although overall results are mixed, data from evaluations conducted 5 days after processing showed differences in overall quality between control and treated lettuce at all three locations. Differences in microbial population were also observed in trial A and C. In trial B, the control showed the lowest microbial count but no statistical difference was recorded among treatments (Table 1). This study revealed that a field application of harp in improves quality of fresh-cut lettuce under conditions to be elucidated. Our results encourage further studies to determine the influence of environmental conditions on the plant's response to harp in and, demonstrate that agents that elicit hypersensitive response may be used to improve quality of fresh-cut products.
Table 1: The effect of a field application of Extend®, on overall visual quality and mesophilic bacteria count of fresh-cut head lettuce grown in three different locations, 5-6 days after process1l1g.
Overall Visual Quality Microbial Population (9-1 scale) (LOglO CFU/g)
Locations -+ A B C A B l1S C
Control 7.8 b 5.83 b 7.61 b 5.98 a 6.86 6.81 a EBC-351 7.9 b 5.89b 7.86b 5.35 ab 6.36 6.64 ab 140 g/ha EBC-351 8.4 a 6.39 a 8.28 a 5.15 bc 6.26 6.57b 280 g/ha EBC-351 8.5 a 6.28 a 8.33 a 5.11 c 6.18 6.78 ab 420 g/ha
l1S mdlCates no slgmficant differences among values m the same column; *Values followed by different letters in the same column are significantly different (P<0.05).
IThe University of Arizona - Yuma Agricultural Center, Yuma AZ 85364 USA; 2Rutgers Cooperative ExtensionBridgeton, NJ 08302 USA; 3University of Cali fomi a, Davis /USDA-ARS, Salinas CA 93905 USA; 4 Eden Bioscience Corporation, Little Rock, AR 72223 USA
29
'TIFWAY' BERMUDAGRASS RESPONSE TO PRIMO AND CUTLESS
1* 2 1 F.W. Totten ,IE. Toler, and L.B. McCarty .
ABSTRACT
The study was perfonned for 12 weeks from 10 July to 4 October, 2004 on registered Tifway bennudagrass [Cynodon dactylon (L.) xc. transvalensis Burtt-Davy]. Primo MAXX lEC (trinexapac-ethyl) and Cutless 50WP (flurprimidol) were evaluated alone and as a tank mix at three rates applied every three weeks. The rates for Primo were 0,6, and 12 oz A-I (0,0.052, and 0.105 kg ai ha-1
) and for Cutless, 0, 4, and 8 oz A-I (0, 0.14, and 0.28 kg ai ha-1
). Applications were made every three weeks for the duration of the study. In summary, acceptable visible injury was recorded 1 and 2 WAIT, and greatest clipping reduction was recorded 8 WAlT. In both cases, the highest recorded values were for the high rates of both Primo and Cutless. Percent lateral regrowth was reduced by Cutless up to 26% 2 WAIT.
1 Department of Horticulture, Clemson University, E-143 P&A Bldg, Clemson, SC 29634-0319 USA. 2 Department of Applied Economics and Statistics, Clemson University, F-148 P&A Bldg, Clemson, SC 29634-0319 USA
30
SYMPOSIUM II
PGRS IN TREE AND VINE CROPS
31
USES OF PGRS IN CITRICULTURE
Jackie Bums l
ABSTRACT
Plant growth regulators (PGRs) are used in fresh and processed citrus fruit production to maintain or increase grower and/or industry profitability. Preharvest application of auxins such as NAA have been used to thin fruitlets and improve size of certain fresh citrus types, whereas 2,4-D can be used to reduce preharvest drop of mature fruit. Gibberellins such as GA3 delay
rind aging and extend the harvest period, thereby improving fruit size. Some uses for GA 3 in
improving fruit set and juice yield have been reported. Postharvest applications of PGRs in citrus include applications of2,4-D for 'button' retention in lemons, GA3 for delaying rind
senescence, and ethylene for degreening. Recent work has demonstrated the potential for the abscission agent 5-chloro-3-methyl-4-nitro-lH-pyrazole to improve mature fruit removal when used with mechanical harvesting, and to maintain the following year's yield when mechanically harvesting late season Valencia. In all cases, an understanding of environmental, timing and application effects are crucial to successful management of PGRs and to maximize economic benefit.
lCitrus Research and Education Center, University of Florida, Institute of Food and Agricultural Sciences, 700 Experiment Station Road, Lake Alfred, FL 33850, USA
33
DO HORMONES PLAY A ROLE IN ALTERNATE BEARING IN CITRUS?
J. Verreynne1,2 and C. Lovatt1*
ABSTRACT
Whereas climatic events initiate alternate bearing, a heavy on-crop followed by a light off-crop, it is perpetuated by endogenous tree factors. The mechanism and underlying physiology by which fruit influence return bloom of citrus was unresolved. Fruit were removed from individual shoots monthly or from entire on-crop 'Pixie' mandarin trees during periods critical to shoot initiation (summer) and phase transition (winter). Fruit removal provided clear evidence that the inhibitory effect of fruit on return bloom was greatest during the summer. The on-crop reduced summer-fall shoot growth and spring bud break on the one-year-old wood of fruitbearing parent shoots. Buds collected during the summer from on-crop 'Pixie' mandarin trees were characterized by high indoleacetic acid and low isopentenyladenosine concentrations compared to buds from off-crop trees. The. starch level of the buds was not affected. Inhibition of summer-fall vegetative shoot growth of on-crop trees appears to be due to a mechanism similar to apical dominance, not a lack of available carbohydrate.
IDepartment of Botany and Plant Sciences, University of Cali fomi a, Riverside, CA 92521-0124 USA 2Current address: Department of Horticultural Science, University of Stellenbosch, Private Bag Xl, Matieland, 7602 South Africa
34
BENEFITS OF PRESTIGE (CPPU, Forchlorfenuron) USE IN CALIFORNIA TABLE GRAPES
R. Hopkins1*, R. Beach2, and R. Menendez3
ABSTRACT
CPPU was developed and patented in Japan by Kyowa Hakko in 1984. CPPU is a phenylurea type synthetic type compound with cytokinin-like activity. CPPU has been shown to have many marked physiological effects in plants such as increase in fruit size, fruit set and delay senescence among others. In 2005, CPPU became commercially available for the California table grape and kiwifruit markets under the trade name Prestige. Prestige is used in table grapes to increase berry size and quality, to improve pack-out and reduce berry shatter. Prestige can also help growers manage harvest and improve fruit quality after cold storage and during shelf life. This presentation will report on field research conducted with Prestige in California table grapes for the last five consecutive seasons.
lField R&D Specialist, Valent BioSciences Corp., 2Global Field Development Manager, Valent BioSciences Corp., 3Global Business Manager, Valent BioSciences Corp., 870 Technology Way, Libertyville, IL 60048 USA
35
USES OF PLANT GROWTH REGULATORS IN TREE NUT CROPS
Robert H. Beede l
ABSTRACT
Plant growth regulator (PGR) use in tree nut crops targets maximizing total yield and improving harvest efficiency more than increasing visual appeal. The effect of the PGR must be substantial in order to justify the development, registration, and application costs. This paper will review PGR research known to the author in walnuts, almonds, pistachios, and pecans, and it will also identify some of the horticultural challenges faced in each of these crops that might be mitigated by PGR use.
Other than limited acreage in Oregon, California is the only state in North America where walnuts are commercially grown. Ethephon is used frequently as a preharvest aid. Applied at packing tissue brown (the final stage of nut maturity where the tissue surrounding the kernel turns from white to a uniformly tan to brown color), ethephon provides exogenous ethylene to increase the rate of hull dehiscence. The result is an accelerated harvest by seven to ten days and improved nut quality from lighter colored kernels. Evidence will be presented to suggest that walnut cultivars are not equally responsive to ethephon, a phenomenon which could be the more likely cause for poor response than inadequate coverage, high temperature, or incorrect timing. Research will also be shown to suggest that stress tolerant cultivars, such as Serr, resist defoliation from ethephon treatment, even when applied under low soil moisture conditions. Severe defoliation results when ethephon is applied to pecan. This affects next year's buds.
Results from current work with ReTain®, an AVG-based, ethylene synthesis inhibitor compound from Valent BioSciences, will be presented as a practical solution to walnut pistillate flower abortion (PFA). ReTain® appears to sufficiently reduce ethylene production from multiple pollen tube growth. This significantly increases fruit set and commercial yield of the Serr variety, which can have PFA above 50%.
Walnuts are also characterized as having poor branch angle and attachment of laterals arising from current season growth. This prevents growers from developing their primary scaffold branches in the same year as the trunk and thus, delays commercial harvest to the fifth or sixth year. An unsuccessful attempt with Promalin (BA plus G~-7) will be presented.
Pistachios have been shown to be responsive to the rest breaking agents hydrogen cyanamide (Dormex) and horticultural mineral oil, with the latter now widely used commercially due to cost and convenience. Research presented will show the beneficial effects of oil on advancing bud break, maturity, yield, and percent split nuts. Benzyladenine (6 BA) in combination with urea applied in June and July has also been shown to mitigate alternate bearing by significantly increasing yield in the off-year. The amount of 6 BA and urea directly effect response. Efforts to improve nut size with gibberellic acid (GA) and fruit removal with abscisic acid (ABA) will also be presented.
lCooperative Extension, Kings and Tulare Counties, University of California, Hanford, CA 93230
36
Attempts to improve almond nut removal, advance maturity, and achieve better cross pollination between cultivars with Alar®, ethephon, and Cycoce1® had some success but the side effects of smaller kernel size and phytotoxicity stopped further investigation in the 1970's. The author is unaware of any research with PGR's to control tree size once full canopy is attained.
37
SYMPOSIUM III
MOLECULAR AND MORPHOLOGICAL ASPECTS OF
PLANT HORMONES AND REPRODUCTIVE DEVELOPMENT
39
MORPHOLOGY AND REGULATION OF FLOWERING IN APPLE
S. McArtneyl* and E. Hoover2
ABSTRACT
The formation of bud scales, transition leaves, true leaves, bracts and flower primordia were observed in buds removed from non-flowering spurs on one-year old woody shoots of apple (Malus x domestica Borkh. cv. 'Royal Gala') over six consecutive years. Doming of the apical meristem, which represents the first easily observable transition to floral development, occurred from 72 d to 99 d after full bloom depending on the year. The period when doming was observed was poorly synchronized within the populations of sampled buds each year, lasting from 22 d in duration in the shortest year to 50 d in duration in the longest year. There was significant variation between years in the minimum number of appendages observed within buds at the time of doming suggesting the concept of a cultivar-specific "critical appendage number" may not be valid.
An investigation of the effect of cultivar on appendage formation, doming and flower morphogenesis revealed that each of the four cultivars studied exhibited a unique pattern of floral development over time, determined by fitting response probabilities to each of five ordinal stages of development. The rate of appendage formation during the first 60 d after bloom was highest in 'Fuji' and 'Pacific Rose' and lowest in 'Braeburn'. Although all cultivars exhibited a high level of flower bud fornlation (>75% of buds floral) the probability of observing doming was never greater than 0.13, indicating that flower morphogenesis proceeded rapidly once buds were committed to floral development. The highest probability of observing doming occurred sooner after bloom of 'Fuji' (86 d) than 'Royal Gala', 'Braeburn' or 'Pacific Rose' (104-112 d). These differences were not related to either bloom date or harvest date of the cultivar.
Exogenous application of GA3, G~+7 or GA7 at rates ranging from 100-400 ppm in the on year of a biennial bearing cycle can reduce the severity of the cycle by inhibiting flower bud formation. Application of gibberellins at bloom inhibited flower bud formation of spurs whereas later applications (42-84 d after bloom) inhibited flower bud formation on current season shoots only. Flower bud formation was promoted by application(s) of 5 ppm NAA or 400-700 ppm Ethrel during the period from 35-95 d after bloom.
Homologues to genes that regulate flowering in other species are being discovered in apple, including floral meristem identity genes (MdTFL1), genes involved in floral induction (AFL, MdMADS5), flower differentiation (AFL1, AFL2) and floral organ identity (MdMADS12, MdMADS12, MdMADS14, MdMADS15).
I Department of Horticultural Science, North Carolina State University, Fletcher, NC 28732 2 Department of Horticultural Science, University of Minnesota, St. Paul, MN 55108
41
GENETIC DISSECTION OF AUXIN BIOSYNTHESIS IN ARABIDOPSIS
Yunde Zhao1
ABSTRACT
Although indole-3-actetic acid (IAA) , the main auxin in plants, has a relative simple structure and can be easily synthesized in a test tube, the auxin biosynthesis pathways have not been well defined. We previously identified a dominant auxin overproduction mutant yucca that involves in the hydroxylation of tryptamine, a rate-limiting step in auxin biosynthesis. YUCCA encodes a flavin monooxygenase and belongs to a family of 11 members in Arabidopsis. We have systematically investigated the role of each YUCCA homolog in auxin biosynthesis and plant development by analyzing the T-DNA insertion lines of the YUCCA genes, phenotypes of YUCCA overexpression lines, and expression patterns of each YUCCA gene. Most YUCCA genes have distinct, yet overlapping expression patterns and single YUCCA knockout lines did not display any obvious developmental phenotypes. However, Arabidopsis plants with mUltiple YUCCA genes deactivated by T-DNA insertion showed dramatic phenotypes, namely decreased apical dominance and fertility and defected flower development. Characterization of both gainof-function and loss-of-function mutants of the YUCCA family genes provides not only insights in auxin biosynthesis, but also provides tools for analyzing auxin-regulated plant development.
1 Section of Cell and Developmental Biology, Division of Biological Sciences, University of California San Diego, 9500 Gilman Drive, La loIIa, CA 92093-0116
42
HORMONAL REGULATION OF FRUIT DEVELOPMENT
Jocelyn Ozga1* and Dennis Reinecke1
ABSTRACT
Fruit development involves a complex interplay of cell division, differentiation and expansion of sporophytic and gametophytic tissues that is carefully coordinated over time. Plant hormones are signals that regulate many processes of plant development including fruit development leading to mature fruit and viable inature seed. Auxins and gibberellins (GAs) have been implicated in the coordination of development between the fruit and the seeds. In the past hormone application studies and hormone analysis studies have supported that fruit development is in part regulated by hormonal interaction. More recently, biochemical and molecular studies are showing how hormones effect fruit development. This talk will focus on understanding the interaction between auxin and gibberellin in pea fruit and seed development using physiological, biochemical, and molecular approaches.
lPlant Physiology and Molecular Biology Research Group, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5
43
GENES CONTROLLING FRUIT DEVELOPMENT IN ARABIDOPSIS
Jose Dinneny, Cristina Femindiz, Kristina Gremski, Sarah Liljegren, Adrienne Roeder, and Martin Y anofskyl *
ABSTRACT
The Arabidopsis fruit is typical of several thousand members of the Brassicaceae family and consists of two valves that are separated by a thin structure called the rep1um. A narrow stripe of valve margin cells differentiates at the va1ve/rep1um boundary and is necessary for fruit opening at maturity. We have used molecular and genetic approaches to identify the genes that control valve margin specification to understand how the valve margin cells are precisely positioned at the va1ve/rep1um boundary during fruit development. The SHATTERPROOF (SHP), INDEHISCENT (IND) and ALCATRAZ (ALC) genes encoded transcription factors that are expressed in the valve margin where they act to promote valve margin differentiation, and mutants lacking the activities of these gene fail to disperse their seeds at maturity. The FRUITFULL (FUL) gene encodes a transcription factor that is expressed in valve cells and functions to negatively regulate expression of the valve margin identity genes in the valves. The REPLUMLESS (RPL) gene encodes a transcription factor that is expressed in the rep1um where it functions to negatively regulate valve margin identity gene expression. Together, negative regulation by FUL and RPL leads to a narrow stripe of expression of SHP, IND and ALC, thus positioning valve margin formation precisely at the va1ve/rep1um border. Two additional genes, FILAAJENTOUS FLOWER (FIL) and YABBY3 (YAB3), previously shown to encode transcription factors that pattern lateral organs, function to positively regulate expression of the FUL and SHP genes. Together, these studies have led to a model for the regulatory interactions among genes that pattern the Arabidopsis fruit.
REFERENCES
Ferrandiz, C., Liljegren, S.l, and M.F. Yanofsky. (2000). Negative regulation of the SHATTERPROOF genes by FRUITFULL during Arabidopsis fruit development. Science 289:436-438.
Liljegren, S.l, Ditta, G.S., Eshed, Y., Savidge, B., Bowman, l, and M.F. Yanofsky. (2000). SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis. Nature 404:766-770.
Liljegren, S.l, Roeder, A.H.K., Kempin, S.A., Gremski, K, 0stergaard, L., Guimil, S., Khammungkhune, D. and Yanofsky, M.F. (2004). Control of fruit patterning in Arabidopsis by INDEHISCENT. Cell, 116:843-853.
Rajani, S., and Sundaresan, V. (2001). The Arabidopsis myc/bHLH gene ALCATRAZ enables cell separation in fruit dehiscence. Current Biology 11: 1914-22.
Roeder, A.H.K., Ferrandiz, C., and Yanofsky, M.F. (2003). The role of the REPLUMLESS homeodomain protein in patterning the Arabidopsis fruit. Current Biology 13:630-635.
IDivision ofBio1ogica1 Sciences, University of California at San Diego, La Jolla, CA 92093-0116
44
SYMPOSIUM IV
ORNAMENTAL PLANT GROWTH REGULATION
45
CHEMICAL REGULATION OF SENESCENCE IN ORNAMENTALS
Michael S. Reid1*
ABSTRACT
The life of ornamentals - cut flowers and potted foliage and flowering plants - usually ends with leaf yellowing, petal wilting andlor abscission ofleaves petals, and flowers. A range of plant growth regulators have been developed to prevent or at least delay these symptoms during marketing and display. Premature senescence resulting from exposure to exogenous or endogenous ethylene can effectively be controlled with inhibitors of ethylene action, including silver ion and I-methylcyclopropene. These inhibitors may also delay petal and leaf abscission, although manipulation of auxin gradients can also be effective. The anti-senescence effects of gibberellins and cytokinins are also employed commercially for preventing premature leaf yellowing. Thidiazuron, a non-metabolized cytokinin, is very effective in extending the life of some potted plants.
IDepartment of Plant Sciences, University of Cali fomi a, Davis, 1 Shields Ave., Davis, CA 95616, USA
47
ORNAMENT AL GROWTH RESPONSES FROM PROHEXADIONE-CA APPLICATIONS.
D. Barce1 1
ABSTRACT Prohexadione-Ca was spray applied to various red flowering herbaceous plants
(argyranthemum, coleus, double impatiens and vegetative petunia) at 500, 1000 or 2000 PPM. The plants used in the trial were split into three groups at time of application, depending on growth stage -1) open blooms, 2) Buds only, and 3) pre-bloom (all flower buds & blooms removed). Plants were evaluated for growth reduction, phytotoxicity and bloom effect. Height reduction at 2000 PPM was most significant on petunia (74%), followed by argyranthemum (50%) and impatiens (30%), with only a slight reduction seen on coleus. Bloom effect was observed as a loss of red pigment in leaves (coleus) or flowers, and was relative to the dose applied. At 2000 PPM, most flowers were white to light pink. Level of flower color dismption was similar for all three growth stages. Apart from height control or the loss of anthocyanin production, no leaf phytotoxicity was observed.
INTRODUCTION
With the ever increasing diversity of annual and perelll1ial herbaceous plants, growers are requiling the use of plant growth regulator's (PGR's) for desirable crop growth. Commercially acceptable growth involves height and width control, leaf color, number of blooms, bloom timing and overall crop quality. These factors allow growers to provide for a high quality and shippable plants (Keever, 2003).
CUlTent PGR's on the market range in activity from slight to extremely active; however, the various PGR's can often result in marked differences in growth responses. Most plants respond with reduced growth, some have increased bloom counts and others may respond with reduced bloom count and or a delay in bloom. Some plants have little to no response. It is the purpose of this trial to evaluate prohexadione-Ca for PGR efficacy and final crop quality on red-pigmented floweling herbaceous plants.
Prohexadione-CA is an acylcyclohexanedione and acts on blocking 3Bhydroxylation (Rademacher 2000) and prevents the biosynthesis of active GA's. This process is understood to happen late in the bio-synthesis pathway of gib berellins.
MATERIALS AND METHODS
Four plant types were tested; Petunia 'Cascadia Lavender', Argyranthemum 'Red', Coleus 'Red/Copper' and Double Impatiens 'Rose Pink'. Plants were ii'om vegetatively produced cuttings provided by The Flower Fields® Ecke Ranch. The rooted plugs were potted into four-inch plastic pots and allowed to establish for two weeks.
1 Chemtura Crop Protection (fonnerly Crompton Corp.) Salinas, CA. 93906.
48
Each of the four crop blocks were divided into three sub-blocks of differing growth stages: 1) plants in bloom; 2) plants having flower buds only; 3) plants having no flowers or flower buds. The sub-blocks were produced by pinching out the flowers and/or flower buds. Plants were maintained using typical growing practices for commercial greenhouse crops.
Test matelials (PGR's) consisted of 1) Prohexadione-Ca DF formulation applied at 500,1000 or 2000 PPM; 2) Prohexadione-Ca SC , fonnu1ation applied at 1000 PPM; and 3) a water check for all crop plots and all three sub-blocks. Spray volume was 2 qts. /l00 ft2 or a nice wet spray to the foliage / flowers. The experimental design was a randomized complete block (RCB) for the split plots, consisting of 10 plants (replicates) per treatment per sub-block, resulting in 150 plants per crop used in the trial. Data was collected and analyzed at P=0.05 using the Agricultural Research Management computer software.
RESULTS AND DISCUSSION
Plants were evaluated for height, growth area [height x width, for petunia only] phytotoxicity and bloom effect. The petunia growth habit was spreading and sprawling, thus a need to include both height and width measurements. Bloom effect was a measure of color change (check vs. treated) in the flowers. Phytotoxicity was a measure ofleaf and stem distortion. Both bloom effect and phytotoxicity was rated on a scale of 1 to 4, where 1 =none and 4=severe. The phytotoxicity rating also included a 5, where 5=dead. Data was taken at 7, 20, and 34 DAT (days after treatment).
Height Control: Overall, argyranthemum and petunia [Fig. 1] were the most responsive followed by the impatiens. The coleus plots had a degree ofvaliability resulting in little significant difference in height control compared to the untreated control. The peliod of7 to 20 DAT was the most active, i.e., argyranthemum height was reduced on average 18% to 43%, respectively for applications made pre-bloom. Applications made to budded or blooming plants were less responsive, providing 36% and 24% control respectively at 20 DAT for argyranthemum. This may be more of a function ofplalltS having been pinch of bud or blooms vs. the non pinched blooming plants.
Petunia was not significantly affected by treatments at 7 DAT; however, at 20 DAT there was dramatic growth reduction, i.e., 65% for pre-bloom, 50% for budded and 54% for blooming plants. These responses were similar for all three rates tested, i.e. 500, 1000 and 2000 PPM. The SC fonnulation was slightly less active than the WP formulation.
On impatiens, only the 2000 PPM rate was consistently active in reducing height, about a 33% reduction overall.
Fig. 1. Average overall height and growth reduction from Prohexadione-Ca spray applications.
49
Bloom Effect: Earlier testing with Prohexadione-Ca resulted in the reduction of red pigments from flowers (Barce12004, Morrison 2004, Hartley 2004) Based on these observations the rating used for bloom effect was based on a scale of 1 to 4, 1 =no change in flower color from untreated check 2=slightly less color, 3=moderate color loss, 4=severe color loss. The color progression went from red flowers to near white flowers. Because of previous tlial results showing the loss of red flower pigments (open flowers having been sprayed with Prohexadione-Ca) it was the goal of this tlial to evaluate potential color loss of flowers where applications were made at the three growth peliods, i.e., pre-bloom, budded plants and open bloom.
Pre-Bloom Stage: Plants had no flowers or flower buds at time of application, and no open blooms had developed on any of the plants by 7 DAT. At this time, however, the coleus showed moderate to severe loss of red color in the foliage at all rates tested. At 20 DAT, petunia, coleus and argyranthemum showed significant color loss (2.3 for petunia, 3.5 for both coleus and argyranthemum). Treatments delayed the flowering of impatiens, as the untreated check did have blooms while the treatments did not. By 34 DAT, the effects of color loss had begun to subside. The petunia blooms were back to nonnal color, the coleus foliage was normal except for the 2000 PPM dose, having only a slight loss (2.0). The impatiens had developed flowers by 34 DAT, with the 1000 or 2000 PPM rates ofthe WP resulting in a significant loss of color, 2.8 and 3.8 respectively. The SC fonnulation, however, had only slight color loss on the impatiens and would have been considered acceptable.
Budded Stage: At 7 DAT, significant color loss was noted on petunia, coleus and argyranthemum, all being rated as moderate to severe. The impatiens had not yet developed open blooms. At 20 DAT, petunia had slight to moderate color loss, coleus and argyranthemum [Fig. 2.] had moderate to severe loss, and the impatiens had slight to severe color loss, showing a good dose response. By 34 DAT the petunia was normal, while the coleus, argyranthemum and impatiens had slight to moderate loss at 1000 or 2000 PPM respectively.
Open Bloom Stage: This stage was the most dismptive to foliage and flower color, with all plants showing moderate to severe loss at 7 DAT. This was also tme at 20 DAT [Fig. 2.], however, the petunia was more tolerant showing only a slight loss at 500 or 1000 PPM, and moderate loss at 2000 PPM. By 34 DAT only the impatiens showed slight to moderate loss while the other plants were of acceptable color.
Water Check 1000 PPM OB 1000 PPMBI
Fig.2. Argyranthemum bloom effect at 20 DAT for Check, open bloom treatment (OB) or bud initiation treatment (B1)
50
Phytotoxicity: None of the treatments resulted in damaged or defonned leaves, i.e., leaf curl or crinkling ofleafmargins. The coleus leaves did have color loss but no damage to leaf shape or stmcture.
hl smmnmy, Prohexadione-Ca spray applications did result in reduced plant height, showing a clear dose response that peaked in activity at about 20 DAT and diminishing by 34 DAT. Red flower and foliage pigments were significantly affected and reduced. This color loss was similar for all plants, regardless of whether they were sprayed before bloom or in full bloom. The color loss was dose responsive, pealdng about 20 DAT and diminishing by 34 DAT.
LITERATURE CITED
Barcel DJ, 2004. Prohexadione-CA efficacy trials on zinnia and geranium plants. Crompton Cmp, Salinas, CA.
Hartley D, 2004 Prohexadione-CA efficacy tl'ials on various herbaceous flowering plants, Colorado State University, Fort Collins, CO.
Monison S, 2004 Prohexadione-CA efficacy trials on flowering Geranium and poinsettia plants, University of San Diego, San Diego, CA.
Rademacher W, 2000. Annual Review of Plant Physiology and Plant Molecular Biology. Vol. 51: 501-53l.
51
EFFICACY AND PHYTOTOXICITY OF FASCINATION (6-BENZYLADENINE AND GA4+GA7) ON A VARIETY OF ORNAMENTAL PLANTS
Dr. Heiner Lieth1* and L.L. Dodge1
ABSTRACT
The plant growth regulator product "Fascination" is a combination of 6-Benzyl Adenine (at 1.8% by weight) and Gibberellic Acid (G~+GA7 at 1.8% by weight). During 2004 and 2005 we tested the effect of Fascination on a 37 distinct ornamental crops with the goal of developing independent efficacy and phytotoxicity data for new product label registration. The rate proposed for the product label consisted of 250 ppm a.i. so that this was the IX rate used in all experiments. In addition to this rate and a control (OX) we also applied the product at half (0.5X, 125 ppm) and double (2X, 500 ppm) strength. In general, for each of the tested species and varieties, 36 plants were randomly chosen and individually tagged for treatment. The plants were grown in greenhouse and nursery settings where the experimentation was carried out using the standardized IR4 Fascination protocol (June 2004) which called for a 6-week time-frame for each experiment. The plants always received 2 foliar spray applications, one on day 0 and another on day 21. Phytotoxicity and efficacy measurements were taken at day 0, 21 and 42. Phytotoxicity evaluations were based on a numerical rating scale of 0 (no injury) to 10 (complete kill). The measurements also included plant height, width and a count of the numbers of shoots or branches per pot.
Fascination was not effective at inducing branching and also caused no phytotoxicity on the following plants: Alchemilla mollis 'Auslese', Campanula persicifolia 'Telham Beauty', Centaurea montana, Coreopsis grandiflora 'Baby Sun', Gaura lindheimeri 'Siskiyou Pink', Gazania linearis 'Colorado Gold', Gypsophila elegans 'Covent Garden', Hibiscus moscheutos 'Disco Belle Pink', Lamium maculatum 'Shell Pink', Lavandula angustifolia 'Munstead', Leucanthemum x superbum 'Snowlady', Monarda didyma 'Jacob Kline', Nepeta cataria, Penstemon sp. 'Red Rocks', Perovslda atriplicifolia, Rudbeckia fulgida 'Goldstrum', Sedum spurium, Solidago rugosa 'Fireworks', and Vinca 'Tall Rosea Mix'. Ofthese, Fascination was effective at generating longer flower stems (which could be beneficial in cut flower production): Rudbeckia, Penstemon, Campanula, Leucanthemum, Solidago, and Gaura. In the case of the last two, Fascination might also be useful to control flowering.
On the following plants Fascination caused no phytotoxicity and was effective at increasing branching at the IX rate (250 ppm): Artemisia lactiflora 'Guizho', Hedera helix, Lobelia cardinalis, Phlox divaricata ssp. laphamii, Salvia leucantha, and Stachys byzantina 'Silver Carpet'. Some plant showed benefit from Fascination without phytotoxicity at rates other than IX: Hypericum calycinum responded well at 125 ppm while Calyopteris clandonensis 'Longwood Blue', Salvia splendens, and Verbena canadensis 'Homestead Purple' resulted in improved branching at a rate of 500 ppm.
The following showed significant phytotoxicity when treated with Fascination: Aquilegia vulgaris plena 'Black Barlow' , Astilbe taquetii, Gaillardia x grandiflora 'Summer Kiss' , Iberis sempervirens 'Snowflake', Physostegia virginiana 'Vivid', Aster novae angliae 'Purple Dome', and Calendula officinalis 'Orange Mix.'
lEnvironmental Horticulture IR4 Center, Plant Sciences Department, Mailstop 6, University of California, Davis, CA 95616
52
GROWTH REGULATION OF ORNAMENTALS IN EUROPE - FOCUS ON ALTERNATIVE METHODS
1". 1 Hansen, C.W .. and Petersen, K.K.
In Europe the largest amount of pesticides used in horticulture is for chemical growth regulation in ornamental plant production. The intensive use of chemical growth retardants is of environmental concern. In recent years, restrictions on the use of chemical PGRs have been introduced in Europe, and reflect a need for developing efficient non-chemical methods for plant growth regulation. Experiments with a range of genetically and ecologically widely differing plant species have shown that chemical growth regulation can be significantly reduced by using a low phosphorus (P) buffer technique (Compalox®-P) as a single factor or combined with drought stress or reduced nitrogen (N) availability. In some plant species chemical growth regulation can be completely avoided by using the low P buffer technique, whereas in other species combinations with other alternative methods or reduced amounts of chemical growth regulators are needed to obtain sufficient growth regulation. Results from 94 tests using the Compalox®-P buffer technique are available in a low P database (http://www.agrsci.org/ahp/cwh).
In a study with Hibiscus rosa-sinensis drought stress was applied as cyclic drought, alternating between container capacity and drought to visible wilt (-650 to -800 hPa as measured with tensiometers placed in the growth substrate). Also in this study we used the Compalox®-P buffer technique to maintain a predetermined and stable P concentration in the growth substrate (Hansen & Nielsen, 2001). Reduced N was either provided as a constant low N fertigation or as a dynamic supply where N availability varied throughout production according to the plant demand. Drought stress as a single factor reduced plant height by 30%, and low P by 15% when compared with the control. Combining reduced P with drought resulted in an additive growth regulating effect (36% reduction). Continuous low N availability reduced plant height by 20%, but resulted in severe N deficiency symptoms. There was no clear growth regulating effect of dynamic N availability. The desired plant height was obtained by a combination of drought stress and reduced amounts (17%) and applications (3 compared with 7) ofthe chemical growth regulator Cycocel (Hansen & Petersen, 2004; Hansen & Petersen, 2005).
Post-production evaluations showed that chemically growth regulated plants had by far the highest percentage of damaged and wilted flower buds throughout the post-production evaluation compared with plants from all other treatments (Figure 1). Besides the growth regulation effects, reduced P in particularly, but also reduced N availability during production also improved the post-production quality by significantly reducing the number of senescent flower buds compared with chemically growth-regulated plants (Figure 1). Several cycles of drought did not influence post-production stress tolerance.
1 Danish Institute of Agricultural Sciences, Department of Horticulture, P.O.Box 102, DK-5792 Aarslev, Denmark. E-mail: [email protected]
53
Improving plant tolerance to post-production stress by delaying floral senescence and reducing root dieback (Hansen & Nielsen, 2001; Hansen & Petersen, 2004) may have considerable importance to the horticultural industry and to the consumer, since most cultivated flowering plants have inadequate keeping quality when growth regulated chemically. The presentation at the PGRSA meeting 2005 provides you with an update on promising alternative methods for plant growth regulation, the potential benefits of using these methods for growth regulation, and what attempts were made in Europe to implement the results to the horticultural industry.
Control DynamicN LowP
Chemical PGR Drought
III Fresh flower buds
50% ~ Opened flowers
D Senescent flower buds
Figure 1. Post-production evaluations showed that chemically growth regulated plants had the highest percentage of senescent flower buds throughout the 28 days post-production evaluation in interior room compared with control plants (not growth regulated) and with plants grown at reduced nutrient availability.
LITERATURE CITED Hansen, C.W. and Nielsen, K.L. (2001). Reduced phosphorus availability as a method to
reduce chemical growth regulation and to improve plant quality. In: Horst, WIJ. Et al. (eds.). Plant Nutrition: Food security and sustainability of agro-ecosystems through basic and applied research. Kluwer Academic Publishers, 314-315.
Hansen, C.W. and Petersen, K.K (2004). Reduced nutrient and water availability to Hibiscus rosa-sinensis 'Cairo Red'as a method to regulate growth and improve postproduction quality. Europ.J.Hort.Sci., 69 (4) 159-166.
Hansen, C.W. and Petersen, K.K (2005). Effects of reduced nutrient and water availability on plant growth and post-production of Hibiscus rosa-sinensis. Acta Horticulturae 669:269-273.
For more information about the Compalox®-P buffer technique, please see http://www. martinswerk. de and http://www. agrsci.org/ahP/cwh
54
SESSION III
REGULATION OF GROWTH AND DEVELOPMENT/ANALYTICAL
METHODS
55
COMPARISON OF COTTON VARIETAL RESPONSES TO APPLICATION OF AUXI-GRO® WP PLUS CAL-MAX
M. D. Rethwischl*, M. Reayl, J. Grudovichl, J. Wellmanl and D. M Ramosl
ABSTRACT
AuxiGroa WP (active ingredients = 29.2% gamma aminobutyric acid and 29.2% glutamic acid) was applied at the rate of 4 oz'!acre in combination of one qUacre of CalMax (a fertilizer containing 11 % calcium) to three cotton varieties at second boll stage of development. Data documented a very highly significant (p<0.000 1) increase in leaf chlorophyll of 25-33% at 2.5 weeks after application. Treatments resulted in three additional nodes of growth at 17 days post treatment, but also resulted in approximately 10% less fruiting structure retention. Varieties differed in their lint responses to the treatment. Yield increases were noted in DPL 449BR and Phytogen 71 OR, but a large yield reduction occurred in FiberMax 991BR. Fiber lengths were reduced in all three varieties, with greatest reduction (0.04-0.05 inch) noted in FiberMax 991BR and Phytogen 710R. Micronaire was reduced slightly (0.1), did not change, or increased (0.58) in DPL 449BR, FiberMax 991BR and Phytogen 710R respectively.
lUniversity of California Cooperative Extension - Riverside County, 290 N. Broadway, Blythe, CA 92225-1649 USA
57
SALICYLATE ACTIVITY. 4 ROLE OF ETHYLENE IN PARAQUAT DAMAGE
FP Silvennan\ PD Petracek1*, Z JU1,2, DF Heiman\ and P Warrior1
ABSTRACT Paraquat is a free radical-generating herbicide that inhibits electron transport
through photo system I. We have found that simultaneous application of sodium salicylate (NaSA) with paraquat decrease herbicidal activity. Because NaSA is an inhibitor of ethylene biosynthesis, we wanted to detennine if ethylene is causal in salicylate protection from paraquat. Tobacco plants were treated with paraquat amended with inhibitors of ethylene biosynthesis, inhibitors of ethylene action, or ethephon, an ethylene-releasing agent. Inhibition of ethylene biosynthesis by aminoethoxyvinylglycine (AVG) did not significantly affect paraquat activity, while aminooxyacetic acid (AOA), a general inhibitor of pyridoxal phosphate-dependent enzymes, slowed but did not stop paraquat activity. Inhibitors of ethylene perception (1-methylcyclopropane, I-MCP; silver thiosulphate, STS) had no effect on paraquat activity. Moreover, NaSA protected both Arabidopsis wild type and the ethylene-insensitive mutant ein2-1 from paraquat. Only NaSA and the ethylene generator ethephon significantly protected tobacco from paraquat. We conclude that although ethylene may modulate paraquat activity, its perception is not essential for paraquat damage or NaSA protection.
INTRODUCTION Paraquat is a non-selective contact herbicide. Paraquat inhibits photosysnthesis by
accepting electrons from photo system I, which in tum generates reactive oxygen species (ROS) in the light. The ROS generated, which include superoxide anion, hydrogen peroxide, and the hydroxyl radical, cause lipid peroxidation and membrane damage. Paraquat has been used experimentally to induce plant stress.
Salicylic acid is a phenolic that is commonly found in plants. It has been shown to be a signal molecule in thennogenesis of certain Arum lilies, and to be a signal in plant defense induction (Raskin, 1992). Applied SA has been shown to reduce the effects of abiotic stresses, including chilling and heat stress (Dat et aI., 1998; Janda et aI., 1999). Additionally, SA is an inhibitor of the terminal step of ethylene biosynthesis, the oxidation of l-aminocyclopropane carboxylic acid to ethylene catalyzed by ACC oxidase (Leslie and Romani, 1988). Ethylene is a plant hormone involved in plant growth and senescence. Ethylene is produced in response to herbicide treatment, and it may be one of the causes of herbicide damage. However, we have demonstrated that ethylene production is a consequence of herbicide treatment, but not a necessary component of paraquat action.
The protection of tobacco from paraquat by salicylate provided us an opportunity to test the role of ethylene in paraquat activity. The objectives of these studies were 1) to test to role of ethylene modulators in paraquat activity, and 2) to define any interaction between ethylene and salicylate in protection of plants from paraquat.
1 Valent BioSciences Corporation, 6131 Oakwood Road, Long Grove, Illinois, USA 60047 2 Present Address: Shandong University of Technology, Zibo, Shandong, People's Republic of China
58
MATERIALS AND METHODS Plant Material
Xanthi-nc tobacco was grown in an environmentally-controlled growth chamber in a 16 h light/8 h dark photoperiod at 25°C for 4-5 weeks after sowing. Plants typically had 5 to 7 fully expanded leaves when treated (Silverman et aI., 2004).
Cotton (SG-105, Delta Pine Land Company, Scott MS) was sown and grown for 10 d in a growth chamber in a 16 h light/8 h dark photoperiod at 25 EC, at 250 moles m-2
S-1. At the time of treatment, the cotyledons were fully-expanded and the first true leaves had not expanded. Cotyledons were removed 24 hours after spray applications and sealed in disposable 50 mL polypropylene tubes. Ethylene determinations were made 5-6 hours later as described previously (Greenberg et aI., 2000).
Seed for the Arabidopsis thaliana (L.) Heyn. ein2-l mutant was obtained fl.-om the Arabidopsis Biological Resource Center (The Ohio State University, Columbus, OH). The corresponding wild type (Columbia) seed was obtained from Lehle Seed (Round Rock, TX). Arabidopsis plants were grown in Pro-Mix PGX under cool white fluorescent lamps at 150 J.!moles m-2
S-1 under a 16 h light/8 h dark photoperiod at 25°C and treated at maturity.
Herbicide treatments Herbicides were foliar-applied with sufficient volume to insure good coverage.
Plants were sprayed with either the active ingredient (paraquat, methyl viologen; Aldrich) or the commercial product (Gramoxone Max®; Syngenta) in a solution containing either 0.25% (v/v) crop oil concentrate (CO C) (cotton and tobacco), or 0.1 % COC (Arabidopsis). Plants were rated for herbicidal damage as percent leaf area affected at selected times following application.
Ethylene modulators The ethylene biosynthesis inhibitors used were aminoethoxyvinylglycine (A VG;
ReTain®; Valent BioSciences Corporation), aminooxyacetic acid (AOA; SigmaAldrich), and sodium salicylate (NaSA; Sigma-Aldrich). The ethylene action inhibitors used were 1-methylcyc1opropene (l-MCP, EthyIBloc®; Floralife® Inc.), silver thiosulfate (STS, Silgard®; Gard Inc.). The ethylene generator used was 2-chloroethylphosphonic acid (Ethephon, Flore1®; Southern Agricultural Industries).
With the exception of 1-MCP, all ethylene modulators were dissolved in water for application. STS and AOA were applied by soil drench, while ethephon, A VG and NaSA were applied either alone or in combination sprays with the herbicide. The ethylene action inhibitor 1-MCP was applied by sealing tobacco plants in an airtight 115 liter drum overnight with 200 ppm 1-MCP. Following gas exposure, plants were equilibrated for 2 hours before being spray-treated.
Statistics Data were subjected to analysis of variance, and means were separated by
Duncan's new multiple range test (p=0.05) using PlotIT software (Scientific Programming Enterprises, Haslett, MI).
59
RESULTS AND DISCUSSION The conversion of S-adenosylmethionine (SAM) to aminocyclopropane (ACC) is
the rate-limiting step in ethylene biosynthesis (McKeon et aI., 1995). This step, catalyzed by ACC synthase, is inhibited by either AVO or AOA. The next step, oxidative conversion of ACC to ethylene, is inhibited by salicylate (Leslie and Romani, 1988). NaSA inhibition of the conversion of ACC to ethylene is dose-dependent (Figure 1). The perception of ethylene is blocked by STS or I-MCP. Finally, the perception of ethylene is blocked in ein2-1, which has a lesion in the pathway of ethylene-mediated signal transduction (Alonso et aI., 1999).
E
e -c: o ()
A
E Q. Q. co s::!.. 0
~
E Q. Q.
C'! e c:(
~ z + 0
~
E E E Q. Q. Q. Q. Q. Q.
co """ 0
!e. co ~ ~
~ c:( c:( en C\l en z C\l z z + + + 0 0 0
~ ~ ~
Figure 1. Application of sodium salicylate (NaSA) inhibited conversion of aminocyclopropane to ethylene in mung bean hypocotyls is dose dependent manner.
Tobacco plants sprayed with paraquat alone showed signs of leaf damage and dessication within the first 24 hours. The simultaneous application of sodium salicylate (NaSA) with paraquat inhibited paraquat damage (Figure 2). At 3 days after spray application, a 50% reduction in damage was observed when the combination treatment was used. NaSA alone caused no significant leaf damage.
To better understand the mechanism of action of salicylate-mediated protection from paraquat, the effect of paraquat on ethylene generation of cotton seedlings was determined. Treatment with paraquat alone (500 /lM) increased ethylene evolution of cotton cotyledons from 1.4 pmole/gram/hr to 467.4 pmole/gramlhr. The combination of NaSA (5 mM) with paraquat (0.5 mM) reduced ethylene generation by 9-fold to 51.3 pMoles/gramlhr and resulted in less herbicidal damage.
To better understand the role of ethylene biosynthesis inhibition in protection of tobacco from paraquat, both AVO and AOA were examined (Figure 3). AVO alone (100 ppm) induced a slight amount of chlorosis on the new growth oftobacco. AVO was unable to protect tobacco from paraquat (Figure 3A) and showed no interactions with NaSA protection of paraquat (not shown). In contrast, AOA slowed paraquat damage in
60
a dose-dependent manner 2 days after application (Figure 3B), but had no effect 4 days after application.
"C 1 00 II)
1:) oS! ::;: 75 n:s
«~ 50 'tti II) ..J
C 25 II)
!:! II) c..
El coe 8 NaSA, 10 mM • Paraquat, 38 ppm • NaSA+Paraquat
2 4 6 8 10
Day Post Spraying
Figure 2. Simultaneous application of sodium salicylate (NaSA) protects tobacco from paraquat damage (COC: 0.25% v/v crop oil concentrate).
The role of ethylene perception was tested with two inhibitors of ethylene action: I-MCP and silver thiosulfate. Neither inhibitor affected paraquat activity. A further test of the role of ethylene perception was made using the ein2-1 mutant of Arabidopsis, which is unable to perceive ethylene. Both paraquat damage and salicylate protection were equal in ein2-1 and in WT Columbia (Table 1).
To determine if ethylene could result in increased protection, the ethylene generator ethephon was used. Simultaneous foliar application of ethephon with paraquat significantly protected tobacco from herbicidal damage (Figure 4). Ethylene is an important endogenous regulator of plant growth and development. In addition to its role in fruit ripening, ethylene is integral to other developmental processes. Ethylene is produced in response to all forms of plant stress and is often considered to be a consequence of stress and not essential for plant response. The salicylate-mediated protection from paraquat provided an opportunity to examine the role of ethylene in the response of plants to herbicide stress.
Salicylic acid is an endogenous growth regulator in plants and has been shown to be involved in thermogenesis and plant defense. We have shown that NaSA protects against paraquat (Silvelman et aI., 2004). Several workers have previously shown that SA pretreatment can protect plants against against paraquat and oxidative stresses (see Strobel and Kuc, 1995). However, we have shown that simultaneous treatment also protects against paraquat.
61
A 0 3 days • 6 days
Figure 3. The ethylene biosynthesis inhibitors showed differential affects on paraquat herbicidal activity. A. Aminoethoxyvinylglycine (A VG) did not significantly affect paraquat (PQ) activity (Treatments: COC, 0.25% v/v crop oil concentrate; A VG, 100 ppm A VG+COC; PQ, 500 ppm PQ+COC; PQ+A VG, PQ+AVG+COC). B. Aminooxyacetic acid (AOA) delays herbicidal activity of paraquat (Treatments: PQ, 187 ppm PQ + 0.25% v/v COC; PQ+AOAI0, PQ+COC+ 10 ppm AOA pretreatment; PQ+AOAI00, PQ+COC+ 100 ppm AOA pretreatment; PQ+AOA500, PQ+COC+500 ppm AOA pretreatment).
Of the compounds tested here, only NaSA and ethephon reduced paraquat activity. NaSA is an ethylene biosynthesis inhibitor (Leslie and Romani, 1988). The only other ethylene biosynthesis inhibitor that showed any modulation of paraquat activity is AOA. AOA is a general inhibitor ofPLP-dependent enzymes, and it is likely to be acting on other targets. Ethephon is used as a rip~ning and synchronizing agent in many crops. The mode of action of ethephon protection may be through induction of lignification (reviewed in Enyedi et aI., 1992), or through some other mechanism.
Ethylene perception does not appear to be necessary for either paraquat activity or NaSA protection from paraquat. Neither STS nor I-MCP treatment resulted in paraquat protection. Moreover, NaSA protected both WT (Columbia) and ethylene insensitive ein-2-1 Arabidopsis from paraquat damage (Table 1).
Table 1. Sodium salicylate safens both Columbia WT and ein2-1 Arabidopsis from paraquata.
Columbia WT Treatments Control Sodium salicylate Paraquat Sodium salicylate + paraquat ein2-1 (Columbia) Treatments Control Sodium salicylate Paraquat Sodium salicylate + paraquat
48 h o.o±o.o 2.0±0.5 83.8 ± 1.5 13.8 ±2.3
48 h 0.0 ±O.O 2.5 ± 0.1
76.3 ± 2.3 8.8 ±2.5
Hours post spray application 96h
O.O±O.O 2.5 ± 0.1 92.8 ± 2.3 21.3 ± 1.5
Hours post spray application 96h
O.O±O.O 2.5 ± 0.1 85.8 ± 3.7 13.8 ± 3.1
168 h O.O±O.O 2.5 ± 0.1 95.3 ± 2.2 30.0 ± 2.3
168 h O.O±O.O 2.0±0.5 85.0 ± 7.6 23.8 ± 3.6
a All data are expressed as the mean ± the standard error of the mean of the percent leaf area affected.
Taken together, these data suggest that neither ethylene biosynthesis nor perception is necessary for paraquat activity or salicylate protection from paraquat damage. We suggest that sodium salicylate functions in paraquat protection through an ethylene-independent pathway.
LITERATURE CITED
Alonso, 1M, T Hirayama, GRoman, S Nourizadeh, and JR Ecker. 1999. EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis. Science. 284:2148-2152.
Dat, JF, H Lopez-Delgado, CH Foyer, and 1M Scott. 1998. Parallel changes in H20 2 and catalase during thermotolerance induced by salicylic acid or heat acclimation in mustard seedlings. PlantPhysioI. 116:1351-1357.
63
Enyedi, AJ, N Yalpani, FP Silverman, and I Raskin. 1992. Signal molecules in systemic plant resistance to pathogens and pests. Cell 70:879-886.
Greenberg, J, FP Silverman, and H Liang. 2000. Uncoupling salicylic acid-dependent cell death and defense-related responses from disease resistance in the Arabidopsis mutant acd5. Genetics 156:341-350.
Janda, T, G Szalai, I Tari, and E Paldi. 1999. Hydroponic treatment with salicylic acid decreases the effects of chilling injury in maize (Zea mays) plants. Planta 208: 175-180.
Leslie, C and R Romani. 1988. Inhibition of ethylene biosynthesis by salicylic acid. Plant PhysioI. 88:833-837.
McKeon, T, J Fernandez-Maculet, and SF Yang. 1995. Biosynthesis and metabolism of ethylene, p. 118-139. In: P. Davies (ed.). Plant Hormones: Physiology, Biochemistry, and Molecular Biology. Kluwer Academic Publishers, Boston.
Raskin, I. 1992. Role of salicylic acid in plants. Ann. Rev. Plant Physiol. Plant Mol. BioI. 43:439-463.
Silverman, FP, PD Petracek, Z Ju, DF Heiman, and P Warrior, 2004. Enhanced herbicide composition. US Patent Appl. No. 2004/0009876 Al.
Strobel, Nand J Kuc. 1995. Chemical and biological inducers of systemic resistance to pathogens protect cucumber and tobacco plants from damage caused by paraquat and cupric chloride. Phytopathol. 85:1306-1310.
64
ETHEPHON DEFOLIATION OF PLUMERIA RUBRA FOR WINTER FLOWERING
Richard A. Crileyl
ABSTRACT
A popular lei flower, the plumeria (Plumeria rubra) is dormant during the winter tourist season in Hawaii and is unavailable for its use as a greeting to visitors. Foliar sprays of ethephon cause defoliation and result in the inability of the plant to perceive short photoperiods. Plumeria trees were treated at different times in fall 2003 and fall 2004 with 800 ppm a.i. ethephon, and shoot tips were tagged to follow inflorescence development. Results from the 2004-2005 period are reported. Trees treated 10/23/04 averaged 78.4 days to flower with a range of 44 to 126 days. The average fell on 1/9/05 with 50% of tagged shoots producing their first flower by 1/5/05. Trees treated 11/30/04 averaged 81 days to flower with a range of 48 to 113 days. The average bloom date for this treatment was 2/18/05, with 50% of the tagged shoots producing their first flowers by 2/24/05. Tagged shoots on untreated plants were timed from the 10/23/04 date of first treatment and averaged 123.7 days to flower with a range of 53 to 167 days. The average date for flowering was 2/23/05, but the 50% flowering date was 3/3/05. Despite the poor data in 2003-2004, fall ethephon treatments were responsible for some earlier flowering of plumeria. These results are useful for commercial growers of plumeria and the winter tourism trade in Hawaii.
INTRODUCTION
A plumeria lei is a traditional greeting among both residents and tourists in Hawaii. As a lei flower, plumeria is much underestimated in tenns of its value in the floriculture industry because many flowers are gathered from backyards and roadside plantings and thus are not counted in the annual census conducted by the USDAlHawaii Agricultural Statistics Service, which has reported on average 13 commercial producers during the past 5 years. In 2004, 13 producers reported farm gate sales of $513,000 from some 16 million blooms (RASS, 2005). The "image value" of plumeria flowers to Hawaii's tourist industry is probably many times that amount.
During winter, plumeria flower production nearly ceases at a time when visitor counts are high. Other flowers are imported to meet the needs for lei flowers with pre strung dendrobium orchids from Thailand being among the most prominent. Plumeria growers have tried different strategies in an effort to produce flowers for the winter market, ranging from hand defoliation to planting many acres so that a least a few flowers can be gathered, or choosing warm parts ofthe state in which to grow plumeria trees.
Inflorescences are largely produced in the spring and may continue to bear flowers for six months, although the last flowers are small and infrequent. Murashige (1966) reported that leaf retention and abscission were controlled by daylength. Lawton
1 Department of Tropical Plant and Soil Sciences, University of Hawaii, 310 Maile Way, Rm 102, Honolulu, HI 96822
65
and Akpan (1968) reached the same conclusion and added that stem growth and leaf production continued under long days. Sheehan and Murashige (1962) reported that plants treated with long days or gibberellic acid continued to produce leaves and did not go dormant until natural short days were imposed. Criley (1995) reported that ethephon applications to plumeria prior to September caused little defoliation and the inflorescence stalk developed on July and August treatments as a nub with no flowers. Later treatments resulted in earlier flowering. These results suggested that inflorescences were initiated during the long days of summer and that development ceased under the influence of shortening day lengths in the fall. Since winter night temperatures at sea level in Hawaii seldom drop much below 16 C, growth can resume with elongation of the terminal inflorescence given periods of warmth.
MATERIALS AND METHODS
In fall 2003, an experiment was initiated on a block of 'Celadine' plumeria trees at the University of Hawaii Waimanalo Research Farm to determine if enhanced temperatures could speed the development of inflorescences following treatment with ethephon. The ethephon concentration used was 800 ppm, a concentration previously shown effective in defoliation studies (Stevens and Criley, 1985). Ethephon was applied on 9/30103 and 10/24/03 with good defoliation occun-ing within two weeks. At three weeks after treatment, two-thirds of the inflorescences were covered with plastic bags to raise the temperature sun-ounding the terminal, a treatment suggested by Canadian grape growers (Eddy, 2003). Half of these bags were removed after 4 weeks and the remainder after 8 weeks. One-third of ethephon treated terminals were not covered, and a set of trees that were not sprayed were included as controls. For each treatment, 15 inflorescences were" tagged for a total of 147 branches (September) and 137 branches (October); some branch tips were lost to rot. The date of first open flower on an inflorescence was recorded.
In fall of 2004, 800 ppm ethephon sprays were applied to foliage of a plumeria cultivar identified only as 'Graveyard Yellow" (its original source was a graveyard) on 10/23/04 and 11/30104. Fifty temlinals were tagged on nine trees at each spray date and their date of first open flower was recorded. An unsprayed set of nine trees served as controls with 50 tagged terminals. Some terminals were accidentally broken off the 11/30104 trees, leaving 45 tagged terminals.
RESULTS
Heavy rains during fall and winter 2003-2004 caused the plastic bags covering the terminals to fill with water. Cool, overcast weather negated much of the influence of the treatments, and little or no difference was noted between the two bagged treatments and controls. Even the few flowers that opened during the rainy weather were deformed as a result of Botrytis infection. A few September-treated terminals did flower in late December and early January, but on average, approximately 146 days were required following defoliation for first bloom (about 2/24/04) for the September treatment and 129 days for the October treatment (about 3/1/04), with controls performing about the same as
66
the treated terminals (2/20104). In general, the results were disappointing and details are not presented.
The 2004-2005 experiment met with better weather. Fifty percent of the Octobertreated terminals produced their fIrst flowers by January 5, 2005, while fIfty percent of the November treatment had come into bloom by February 14, 2005 (Table 1). Untreated controls reached fIfty percent bloom by March 3, 2005 (Table 1). The earliest Octobertreated terminals flowered before Christmas, approximately 50 days after treatment and fully 40% had presented their first flowers by the end of the year. Because terminal development was uneven, flowering was not concentrated, but ethephon-treated plumeria generally required fewer days to reach 100% fIrst bloom (Figure 1).
DISCUSSION
The 2003-2004 results showed the dependence of plumeria on warm, sunny weather to continue the development of previously-initiated inflorescences. Treatment with ethephon to defoliate plumeria for early flowering would be most successful on the drier, sunnier leeward sides of the islands. The bagging treatment proved to be more work than it was worth, and since the bags fIlled with water from the rainy weather, their effect in raising temperatures was not recorded. The bags had to be secured tightly or they would blow off in the wind.
This research demonstrated that plumeria trees with well-developed inflorescence structures can be forced into flower in time to meet the needs of the lei flower industry during a season when the availability of plumeria flowers is normally low. My hypothesis is that with ethephon-induced defoliation, leaves are not present to sense photoperiod and send the terminal into a dormant state. Under suitable conditions of warmth, active growth resumes and the terminal inflorescence elongates and produces flowers.
Ethephon has received a registration for defoliating and defruiting a number of ornamentals, including plumeria. Alternative defoliation materials have not been evaluated for plumeria.
LITERATURE CITED
Criley, R. A. 1995. Enhanced winter flowering of plume ria with ethephon. Acta Hortic. 394:325-330
Eddy, D. 2003. Sun trapping. Amer. 1 West. Fruit Grower June 2003:6-7. Hawaii Agricultural Statistics Service (RASS) 2005. Hawaii flowers & nursery products
Arumal summary. USDAlHASS, Honolulu, HI 96814 Lawton, J. R. S. and E. E. J. Akpan. 1968. Periodicity inplumeria. Nature 218:384-386. Murashige, T. 1966. The deciduous behavior of the tropical plant, Plumeria accuminata.
Physiol. Plant. 19:348-355. Sheehan, t. J. and T. Murashige. 1963. Growth responses of plumeria to photoperiod
and gibberellic acid. Proc. Fla. State Hort. Soc. 76:477-479. Stevens, G. and R. A. Criley. 1985. Induced fruit abscission of tropical ornamental
trees with ethephon and chorflurenol. HortScience 20:382-383.
67
Table 1. Flowering dates (first open flower) for 10% percentiles of 'Graveyard Yellow'plumeria defoliated with ethephon on October 23,2004 (N=50) or November 30, 2004 (N = 45) or untreated (N=50).
Figure 1. Average days from treatment to first open flower for each ten percent increment of flowering for ethephon-treated 'Graveyard Yellow' plumeria. Days to flower for untreated controls were determined from October 23,2004.
68
GIBBERELLIN SYNTHESIS INIDBITOR AFFECTS ANNUAL XYLEM PRODUCTION AND VESSEL ELEMENT ANATOMY IN SOME TREES
William R. Chaneyl*, Denise M. Mickeyl and Harvey A. Hoitl
ABSTRACT Red oak (Quercus rubra L.), white oak (Quercus alba L.), sweetgum
(Liquidambar styraciflua L.), and yellow poplar (Liriodendron tulipifera L.) were treated with paclobutrazol using the soil drench or soil injection method at a dose rate of9.6 grams a.i. per tree. Five or six growing seasons after treatment, trees were harvested and cross-sections of the main stem were removed for analysis. Total tree height, diameter growth of the trunk, the width of annual rings of xylem, and the size and number of vessels in the earlywood were compared in paclobutrazol treated and untreated trees. Tree height was reduced in all four species, whereas diameter growth at 137 cm above ground-line (DBH) and arumal ring width for five or six growing seasons were reduced by 33 and 36 percent, respectively, only in sweetgum and white oak trees treated with paclobutrazol. The cross-sectional area of individual vessel elements also was reduced by paclobutrazol treatment only in white oak and sweetgum. The number of vessels per unit area of xylem tissue was not affected by paclobutrazol in any of the four species.
INTRODUCTION The growth regulator paclobutrazol (PBZ) used by arborists to reduce shoot growth has
been shown to have additional benefits for trees and shrubs including improved resistance to drought stress, darker green leaves, protection against some fungal and bacterial pathogens, and enhanced development of fibrous roots (Chaney 2003; Chaney et al. 1996; Fletcher et al. 2000; Rademacher 2000). Cambial growth, as well as shoot growth, has also been shown to be reduced for up to three years after treatment in some tree species (Bai et al. 2004). The objective of this experiment was to determine total tree height, diameter growth of the trunk, the width of annual rings of xylem, and the size and number of vessels in the earlywood of several trees species five or six years after they were treated with the growth retardant paclobutrazol or left untreated.
MATERIALS AND METHODS Experimental trees were located at Martell Experimental Forest Farm near the Purdue
University campus in Tippecanoe County, Indiana. Red oak (Quercus rubra L.) and white oak (Quercus alba L.) ranging from 8-10 cm basal diameter were treated in April 1995 with paclobutrazol (PBZ) using the soil drench method at a dose rate of 9.6 grams a.i. per tree. The following April, sweetgum (Liquidambar styraciflua L.) and yellow poplar (Liriodendron tulipifera L.) (4-8 cm basal diameter) were treated with paclobutrazol using the soil injection method at the same dose rate. At the end of the growing season in 2000, three control and three treated trees of each species were randomly selected and harvested at ground level. Total tree
Department Forestry and Natural Resources, 715 W. State Street, Purdue University, West Lafayette, IN 47907
69
height and diameter 137 cm above ground line (DBH) were measured before a 2.5 to 5.0 cm thick cross-section was removed approximately 137 cm from the base of each tree.
The stem disks were sanded smooth using a rotary sander to enable better viewing of the xylem rings at low magnification. Annual xylem ring widths were measured for the 1995-2000 growing seasons for the red and white oak and for the 1996-2000 growing seasons for the sweetgum and tuliptree with an Acu-Rite III digital readout system (Acu-Rite, Jamestown, New York). The samples were measured by placing them on the movable stage of the instrument and viewing the annual rings of xylem through a lOX magnifying glass with a cross-hair. As the stage was moved by a hand crank, the digital readout gave a measurement of ring width to the nearest 0.001 mm. Measurements were taken at four points at right angles on each sample.
To determine the cross-sectional area of vessels in the earlywood of the 2000 annual growth ring, a small section of wood was cut from each disk. The pieces were boiled for approximately one hour to soften the wood for sectioning. Twenty-um-thick cross-sections were made using an A.a. Spencer Sliding Microtome, model 860 (American Optical Corporation, Buffalo, New York). The microtome slices were stained with a 1 % solution of safranin and mounted on glass slides. Ten vessel cells were randomly selected in the earlywood and two diameters of each cell at right-angles were measured at 20X magnification using a microscope with an ocular eyepiece. Cross-sectional area of the cells was determined using the equation Area = 1t X length 1 x length 2.
The number of vessel cells was also counted for each sample. Samples were viewed at lOX magnification and all the vessels in ten replications of an area of 1 mm2 were counted.
Data were analyzed using analysis of variance and comparison of means with Tukey's Studentized Test (p :S0.05).
RESULTS AND DISCUSSION Total tree height was reduced six years after treatment with pac1obutrazol in white
and red oak and five years after treatment in sweetgum and tuliptree (Table 1). Although no measurements of height were made at the time of treatment in 1995 or 1996, the height of the white oak and red oak were measured in 1991 for another study using the same trees and no difference in height was found within each species (Chaney and Byrnes 1993).
Table 1. Comparison of total height of trees untreated or treated with paclobutrazol.
Treatment
Control PBZ-Treated
White Oak Red Oak ............................... m
8.4 a* 8.9 a 5.8 b 7.1 b
Sweetgum Tuliptree
9.9a 11.1 a 9.3 b 6.6 b
*Means in columns followed by the same letter are not statistically different (p :::;0.05).
The width of the annual xylem ring increment was reduced in white oak and sweetgum beginning the year these trees were treated and continued to be reduced thoughout the study (Table 2). Annual accumulation of xylem was not affected in red oak until the fifth year after treatment, whereas tulip tree was not affected by treatment with pac1obutrazol. This is the same pattern of response for the same tree species reported in an earlier study but over a shorter time period (Bai et al. 2004).
70
Table 2. Comparison of annual xylem ring increment (mm) of red oak and white oak for six years after treated with pac1obutrazol (PBZ) and sweetgum and tuliptree five years after treatment with pac1obutrazol.
Treatment Species Year
Red oak 1995 1996 1997 1998 1999 2000 Control 2.77 a* 2.13 a 2.09 a 2.69 a 2.74 a 3.33 a PBZ-Treated 2.81 a 2.57 a 2.42 a 2.11 a 1.92 a 2.08 b
White oak Control 3.04 a 3.08 a 2.08 a 2.79 a 1.51 a 2.00 a PBZ-Treated 1.55 b 0.70b 0.54 b 0.50b 0.46b 0.58b
Sweetgum Control 4.04 a 3.17 a 3.85 a 3.10 a 2.99 a PBZ-Treated 3.29b 2.39 a 1.55 b 1.68 b 1.34 b
Tuliptree Control 2.89 a 2.95 a 2.34 a 2.60 a 3.71 a PBZ-Treated 2.72 a 2.13 a 2.01 a 2.24 a 2.88 a
*Means in columns for each species followed by the same letter are not statistically different (p :::;0.05).
The number of vessel elements per square millimeter of cross sectional area of the xylem did not varied between untreated and PBZ-treated trees for any of the four species investigated (Table 3). The number of vessels in white and red oak is small because these tree species are ring-porous and typically have fewer but larger vessels in the earltwood, whereas sweetgum and tuliptree are diffuse-porous in anatomical structure and have a large number of small vessels evenly distributed across annual rings of xylem.
Table 3. Number of vessel elements per mm2 of cross sectional area.
Treatment
Control PBZ-Treated
White Oak Red Oak ........................... mm2
9.52 a* 8.64 a 10.10 a 9.46 a
Sweetgum
60.87 a 59.67 a
Tuliptree
59.20 a 54.19 a
*Means in columns followed by the same letter are not statistically different (p :::;0.05).
Cross-sectional areas of vessels in white oak and sweetgum were significantly reduced in PBZ-treated trees compared to those in untreated trees, whereas the area of vessels in red oak and tuliptree was unaffected (Table 4).
The data reported here substantiate well established responses of reduction in height growth following treatment of trees with paclobutrazol (Fletcher et al. 2000; Rademacher 2000). Reductions in cambial growth in some tree species, but not others, have also been reported (Bai et al. 2004; Estabrooks 1993; Lehman et al. 1990), but the data reported here show that the effect ofpaclobutrazol can extent for at least 5-6
71
Table 4. Average cross-sectional area (mm2) of individual vessels.
Treatment
Control PBZ-Treated
White Oak Red Oak Sweetgum ........................... mm2
0.20 a* 0.16 a 0.14b 0.16a
0.0073 a 0.0033 b
Tuliptree
0.0086 a 0.0077 a
*Means in columns followed by the same letter are not statistically different (p :S0.05).
years after treatment of trees. In marked contrast to most published reports, Costa et aI. 1995 showed an increase in trunk diameter of 'Blanquilla' pear trees when they were treated with paclobutrazoI. The reduction in cross-sectional area of vessels found for white oak and sweetgum has not to our knowledge been reported before. Change in xylem anatomy could increase the resistance to water flow in transpiration and smaller vessels could reduce the potential for movement of disease organisms in xylem.
Generally, the larger springwood vessels of ring porous tree species are more conducive to rapid distribution of oak wilt (Ceratocystis fagacearum) conidia. In addition, tylose formation is much more extensive in large earlywood vessels as compared to smaller late vessels (Drake 1956; Nair 1964; Parmeter et aI. 1954;). Larger vessels are also more susceptible to embolisms. Trees infected late in the summer or fall of the previous year often do not show wilt symptoms until the formation of earlywood begins the following year. This has been noted in numerous cases where trees inoculated on various dates late in the season, all show symptoms on nearly the same date the following year (Nair 1964; Skelly and Merrill 1968; Skelly and Wood 1974).
There have been several reports of growth regulators being used to alter host anatomy and to reduce susceptibility to oak wilt (Kuntz et aI. 1968). Northern pin oaks treated with 2,3,6-trichlorophenyl acetic acid formed wood without xylem vessels, but these trees were able to conduct adequate water for growth and transpiration through xylem parenchyma, fibers, and tracheids. When trees infected with oak wilt (c. fagacearum) were treated with this compound, the pathogen and vascular plugging was confined to vessels formed prior to treatment (Geary and Kuntz 1962; Venn et aI., 1968).
LITERATURE CITED
Bai, S., W. Chaney, and Y. Qi. 2004. Response of cambial and shoot growth in trees treated with paclobutrazoI. Jour. Arboric. 30:137-145.
Chaney, W.R. 2003. Tree growth retardants: Arborists discovering new uses for an old tool. Tree Care Indust. 14(3):54-59.
Chaney, W.R., G.S. Premachandra and H.A. Holt. 1996. Physiological basis for benefits of tree growth regulators, pp. 8-18. In: Proceedings Western Plant Growth Regulator Society, Sacramento, CA, January 24-25, 1996.
Chaney, W.R. and W.R. Byrnes. 1993. Effect of seedling age and taproot length on performance of oak. Northern Jour. Applied For. 10: 175-178.
72
Costa, l, M. Bosch, and A Blanco. 1995. Growth and cropping of 'Blanquilla' pear trees treated with paclobutrazol. Jour. Hort. Sci. 70:433-443.
Drake, C.R 1956. The spread and control of oak wilt. Ph.D. Thesis. University of Wisconsin, Madison, WI.
Estabrooks, E.N. 1993. Paclobutrazol sprays reduce vegetative growth and increase fruit production in young McIntosh apple trees. Canadian Jour. Plant Sci. 73:1127-1135.
Fletcher, RA., A Gilley, N. Sankhla, and T.D. Davis. 2000. Triazoles as plant growth regulators and stress protectants. Hortic. Rev. 24:55-138.
Geary, T.F. and lE. Kuntz. 1962. The effect of growth regulators on oak wilt development. Phytopathology 52:733.
Kuntz, lE., V.M.G. Nair and K.O. Venn. 1968. A new approach to oak wilt control. National Center for Weed Control Conference Proceedings, pp. 36-37.
Lehman, L.J., C.R. Unrath and E. Young. 1990. Mature 'Starkrimson Delicious' apple tree response to paclobutrazol application method. Hortscience 25:429-430.
Nair, V.M.G. 1964. Pathogenesis of oak wilt in bur oaks. Ph.D. Thesis. The University of Wisconsin, Madison, WI.
Parameter, J.R, lE. Kuntz and Al Riker. 1954. Oak wilt development in bur oaks. Wisconsin College of Agriculture Forestry Research Notes 16: 1-2.
Rademacher, W. 2000. Growth retardants: Effects on gibberellin biosynthesis and other metabolic pathways. Ann. Rev. Plant Physiol. Plant Mol. BioI. 51:501-531.
Skelly, lM. and W. Merrill. 1968. Susceptibility of red oaks to infection by Ceratocystis fagacearum during the dormant season in Pennsylvania. Phytopathological Notes 58: 1425-1426.
Skelly, lM. and F.A. Wood. 1974. Longevity of Ceratocystis fagacearum in ammate treated and nontreated root systems. Phytopathology 64: 1483-1485.
Venn, K.O., V.M.G. Nair and lE. Kuntz. 1968. Effects of TCPA on oak sapwood formation and the incidence and development of oak wilt. Phytopathology 58: 1071.
73
IMMUNOSENSOR ASSAY: A NOVEL METHOD TO ANALYZE
PHYTOHORMONES
Langtao Xiao 1, Ruozhong Wang1, Jin Li2
, Guoli Sheng2
ABSTRACT
Phytohonnones play very important roles at almost all developmental stages of plants. Because of their extremely low concentrations in plant tissues and they are highly sensitive to environmental factors such as light, heat and oxygen, sensitive assay is a limiting factor for phytohonnonal research. All traditional phytohonnonal assays including bioassay, GC, HPLC, ELISA and RIA have their disadvantages. Mainly based on radioimmunoassay and biosensor technology, immunosensor assay is a new technology for phytohonnonal analysis proposed by Hunan Provincial Key Laboratory ofPhytohonnones and Growth Development and State Key Laboratory of Chemo/Biosensing and Chemometrics at Hunan University in 2002. After construction of the first indole acetic acid (IAA) immunosensor, several kinds of immunosensors for phytohonnones were successfully developed.
IAA immunisensors: Cunently we developed the two types of immunosensors for IAA. (1) Piezoelecttric immunosensor. The detection was based on competitive immunoreactions between IAA and an bound to the anti-IAA antibody immobilized on a quartz microbalance, the frequency change of sensor caused by antigen was linearly related to logarithm of the concentration ofIAA in the range from O.Sng / mL to Sllg / mL. (2) Amperometric immunosensor. The determination was based on an enzyme-linked competitive immunoreaction between free IAA and IAA labeled with HRP to bind on the anti-IAA antibody immobilized on the soI-gel-alginate-carbon composite electrode(SACE) surface. The response signal expressed as percentage cunent reduction (CR%) was linearly related to the logarithm of the concentration ofIAA in range ofSllg / mL to SOOllg / mL with a regression equation of the fOlm y = 37.80x-22.47 and conelation coefficient of 0.9922.
Cytokinin immunosensor: The immunosensor based on a multilayer-coated glassy carbon electrode was designed to detennine isopentenyl adenosine (iP A) in plants. The multilayer consists ofpolypynole and poly(m-phenylenediamine) with ~Fe(CN)6 and horseradish peroxidase (HRP) entrapped during electropolymerization. The fenocyanide doped in polypynole functions as the mediator. The glucose oxidase bound on the immunosensor by the competitive immunoreaction involving iP A catalyzed the oxidation of the added glucose with the fonnation of H20 2, which was in turn reduced in the presence of HRP entrapped in poly(m-phenylenediamine ).
1 Hunan Provincial Key Laboratory of Phytohormones and Growth Development, Hunan Agricultural University, Changsha,
410128, China
2 State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, China
Supported by Hunan Provincial Natural Science Foundation OIJJYl003, Hunan Provincial Department of Education, 99B05
74
The current of the oxidized production of ferrocyanide reduced at -50m V was inversely proportional to the concentration of iP A in the competitive immunoreaction. The percentage of current response reduction (CR%) was linearly related to the logarithm of the concentration
of iP A in the 51lg / mL to 300 Ilg / mL range, with a regression equation of the form y = 42.13x -27.79 and a correlation coefficient of 0.9861.
Immunosensors for other phytohormones: Similar work to develop immunosensors for other phytohormones such as gibbereUins and abscisic acid is also in progress. FUlther study is needed to improve the sensitivity and reuse time of immunosensors. Immunosensor assay has been used to analyze hybrid rice samples and the results were in satisfactory agreement to those obtained by high-performance liquid chromatography method, indicating its high application potentiaL
75
SESSION IV
FLOWERING/SEED AND FRUIT DEVELOPMENT
77
POST ANTHESIS PGR APPLICATION AND FIRST AND SECOND CROP PRODUCTION IN DRILL-SEEDED RICE
R. T. Dunand 1
ABSTRACT
Second crop growth originates from axillary buds located at nodes along the culm of the rice plant. These buds begin actively growing after the crop matures and is harvested. This second crop growth is limited by declining temperature and day length during the fall. Initiating growth of a second crop early has the potential to pennit heading and grain filing to occur under more optimum conditions. Evaluations of plant growth regulators applied to the first crop to enhance second crop production were conducted.
Cocodrie, a variety that matures early and can produce a second crop, was drill-seeded on 7-in row spacings on March 26,2004. Plot size was 8.75 (15 rows) x 23 ft. Customary agricultural practices were followed to provide adequate pest control and water management for the first and second crops. Plant growth regulators (PGRs) were applied during the dough stage of the first crop (July 30). Rates of2,4-dichlorophenoxy acetic acid, 2,4-D, (Amine 2,4-D Weed Killer, Platte Chemical Co., Fremont, NE); N-(2-chloro-4-peridinyl)-N'-phenyl urea, CPPU, (Prestige, Valent U.S.A., Walnut Creek, CA); trinexapac-ethyl, TE, (Palisade, Syngenta Crop Protection, Inc., Greensboro, NC); mefluidide, MF, (Embark, PBI/Gordon Corp., Kansas City, MO); and maleic hydrazide, MH, (Royal MH-30 SG, Chemtura, Middlebury, CT) were variable and as follows: 2,4-D: 1.13, 11.3, and 113 g/A; CPPU: 0.25,2.5, and 25 g/A; TE: 14 g/A; MF: 0.25 and 0.5Ib/A; and MH: 1.5 and 3 lb/A. Crop growth and production were unaffected by rates.
First crop growth and maturity were unaffected by the PGRs, and yield was decreased by the mitotic inhibitors. Mature plant height ranged between 105 and 107 cm, and grain moisture at harvest ranged between 18.4 and 19.0% for the PGRs and control. Grain yield ranged between 7395 and 7408 lb/A for 2,4-D, CPPU, TE, and the control. Grain yields with MF (6682Ib/A) and MH (6458Ib/A) were significantly reduced.
Early second crop growth was noticeably affected by the PGRs that had affected first crop yield. Leaf density (leaves above the stubble of the first crop) at 12 days after harvest (DAR) was significantly higher following MF and MH (10 and Illeaves/ft2) compared with the other PGRs and control (1 to 5 leaves/ft2). Similarly, at 20 DAR, panicle densities with MF and MH were 4 and 7 panicles/ft2 compared with 3 panicles/ft2 or fewer for the other PGRs and control. At maturity, the effects were less. Plant height ranged between 68 and 71 cm, grain moisture between 15.3 and 16 %, and grain yield between 2356 and 2430 lb/A for the PGRs and control.
Total yield (first plus second crop) was influenced just as first crop grain yield. Grain yield ranged between 9753 and 98141b/A for 2,4-D, CPPU, TE, and control. Grain yield with MF (9112Ib/A) and MH (8881Ib/A) were significantly lower.
Plant growth regulators applied to the first crop in rice can impact the second crop. Mitotic inhibitors like mefluidide and maleic hydrazide can be injurious to the first crop and enhance second crop growth. Further studies are needed to define the influence of plant growth regulators on second crop production.
lLSU Agricultural Center, Rice Research Station, 1373 Caffey Road, Rayne, LA 70578 USA
79
REGISTRATION OF 2,4-D FOR INCREASING FRUIT SIZE OF MANDARINS AND MANDARIN HYBRIDS IN CALIFORNIA
1* 2 1 C.T. Chao , L. Ferguson, and C.l Lovatt
ABSTRACT
A 24C registration of2,4-D (ALCO CitrusFix) for fruit size increase of mandarins and mandarin hybrids was granted by the Department of Pesticide Regulation, California Environment Protection Agency in January 2005. This registration was based on the efficacy data generated from experiments on 'Fina Sodea' Clementine mandarin conducted in 2002 and 2003. 24 ppm of 2,4-D was able to increase the yield and commercially desirable large sized fruit significantly in both "ON" (2002) and "OFF" (2003) years. The average yield per tree was increased from 61.58 kg of control (nonspray) to 77.84 kg (I 26%) in 2002 and from 31.81 kg to 45.16 kg (t 42%) in 2003. The 24 ppm 2,4-D treatment was able to increase the large-jumbo-mammoth sized fruit from 47.05 kg to 66.30 kg (t 41 %) in 2002 and from 16.75 kg to 23.66 kg (t 41 %) in 2003. Additional experiments of 2,4-D with 12, 24, and 48 ppm and more timing on 'Afourer' mandarin and 'Minneola' tangelo in 2003 and 2004 showed that 2,4-D treatments could increase yield and large sized fruit in an "ON" year (2004). This application of 2,4-D should offer growers an additional tool to enhance their return on mandarins and mandarin hybrids.
1 Department of Botany and Plant Sciences, University of California-Riverside, Riverside, CA 92521 2Department of Plant Sciences, University of California-Davis, Davis, CA 95616
80
PEACH FLOWER BUD THINNING BY DORMANT SEASON APPLICATIONS OF VEGETOIL™
. 1 * 1 1 G. L. Reighard ,D. Ouellette and K. Brock
lClemson University, Department of Horticulture, Clemson, SC 29634 USA
ABSTRACT Removal of flower buds, flowers or young developing fruit in early spring increases
potential peach fruit size by reducing competition for stored carbohydrates. Peach growers often wait ~ 30 days after full bloom to start hand-thinning fruit, which can limit potential :fruit size. Experiments were conducted in commercial orchards in South Carolina to determine the efficacy of Vegetoil™ (Va), an emulsified soybean oil adjuvant, for pre-bloom thinning of peach cultivars. Cultivars were sprayed in January or February of 2003 and 2004 using rates of 8 or 10% by volume of va (a.i., 93% soybean oil). Dormant oil at 2 or 3% was the control treatment. Bloom was either delayed or advanced by va depending on cultivar and year. va significantly reduced the number of live flower buds at bloom in some cultivars in some years. va also significantly decreased hand-thinning costs and for some cultivars improved fruit size and commercial pack-out. Although pre-bloom va treatments were variable in efficacy, in every year that treatments were applied in grower orchards, one or more of the donnant Vegetoil™ applications significantly increased net returns for the grower in that orchard. va performed similarly but not always the same as vegetable grade soybean oil (SO) mixed with an emulsifier.
INTRODUCTION Thinning is necessary to adjust the number of fruits (i.e., peaches) on a fruit tree so that
they will adequately size for commercial acceptance. Thinning practices that achieve a marketable :fruit size that maximizes pack-out yield per tree increase orchard production efficiency for growers. An important component of maximizing fruit size and yield is time of thinning. Peach thinning can be done pre-bloom (e.g., floral buds), during bloom (e.g., flowers) or post-bloom (e.g., fruits/fruitlets).
Thinning at the pre-bloom stage increases the available stored carbohydrates for cell growth in the remaining flower buds. Soybean oil (SO), when applied pre-bloom, has thinned peach [prunus persica (L.) Batsch] flower buds (Deyton et aI., 1992; Myers et aI., 1996; Moran et aI., 2000). Flower bud death due to SO is concentration dependent (Moran et aI., 2000; Myers et aI., 1996; Deyton et aI., 1992) and increases with an increase in SO concentration. The efficacy of thinning with SO from year to year is not known (Moran et aI., 2000). Variability in bud thinning and bloom delay using SO can be attributed to cold injury (Moran et aI., 2000) and management factors (pendergrass et aI., 2000). The objectives of this research were to determine the efficacy of both soybean vegetable oil plus an emulsifier and the commercially available soybean oil adjuvant Vegetoil™ to thin flower buds, reduce thinning expenses, and increase :fruit size and improve pack-out of peach :fruit in South Carolina peach orchards.
MATERIALS AND METHODS Two and three peach cultivars at Titan Peach Farms (Ridge Spring, SC) were selected in
2003 and 2004, respectively. All trees were trained to an open-center system and were spaced
81
4.6 to 5.2 m apart in rows 6.1 m apart. Each treatment was applied to a minimum of 2 and a maximum 4 hectares of orchard per cultivar to obtain packinghouse data. Ten l-year-old shoots on each of 10 trees in each treatment block were randomly selected. The total number of flower buds and number of open flowers were recorded twice for each of the six blocks between March 11 and March 19, 2003 to determine differences in timing of bloom. Fruit from these same 10 trees were commercially hand-thinned in late April. Fruits thinned from each tree were counted and weighed. The total time required for a crew to thin each treatment block was recorded to determine hand-thinning costs. For each harvest, data included percent pack-out (grade 1 fruit), weight (yield), and number of fruit in each of 5 size classes (5,4, 5.7, 6,4, 7.0 and 7.6 cm). Handthinning and spray application costs were subtracted from returns from fruit sales to determine gross returns per hectare minus thinning costs. Data for flower counts and fruitlets removed were replicated, but treatment thinning times, yield, and pack-out data were non-replicated and only non-statistical numbers are rep011ed.
An airblast sprayer was calibrated to deliver l328 Llha for the dormant and soybean oil applications. Latron B-1956 was pre-mixed with soybean oil at 10% of its concentration (e.g., 3.785 L Latron per 37.85 L soybean oil). Latron was not mixed with Vegetoil™ (Drexel Chemical Co.), since it is a pre-mixed formulation of soybean vegetable oil and emulsifier.
An orchard of five-year-old 'Blazeprince' peach trees were sprayed either twice with 3% DO (Jan. 28 & Feb. 8, 2003), with 8% Vegetoil™ (VO) on Jan. 31, 2003, or with 8% soybean oil (SO) on Feb. 7, 2003. Four-year-old 'Fireprince' peach trees were sprayed with either 3% DO on Jan. 5,2003, or with 8% VO on Jan. 31,2003. The 8% VO treatment was applied at either 935 or 1328 L/ha. Fruit from each treatment block from the first 4 harvests (of 7 total) were not packed separately. Therefore, harvest totals for 'Fireprince' include data from only the last 3 harvests. Total returns from fruit sales were estimated by averaging market price for fruit in size classes 6,4 and 7.0 cm.
Ten-year-old 'Summerprince' peach trees were sprayed with either 10% VegetoiFM (VO) (a.i. 93% soybean oil) or with 9% soybean oil (SO) on February 3, 2004. Control trees were sprayed with 2% dormant oil (DO) in early January 2004. All flower buds were recounted and the number of open flowers recorded March 11 and March 16 to determine flower bud mortality and timing of bloom. Fruit were hand-thinned April 22-25, and again on May 5. Five-year-old 'Fireprince' peach trees were sprayed with either 8% soybean oil (SO) on January 29,2004 or with 9% Vegetoil™ (VO) on February 3. Control trees were sprayed with 2% dormant oil (DO) in early January 2004. Flower bud numbers were re-counted and the number of open flowers recorded March 11 and March 16 to determine flower bud mortality and timing of bloom. Fruit were hand-thinned on May 6, and again May 19-21. Eight-yearold 'Flameprince' peach trees were sprayed either with 8% Vegetoil™ on February 4, 2004 or with 1 % Tergitol on March 19 at 80-90% full bloom. Control and Tergitol-treated trees were sprayed with 2% dormant oil (DO) in early Januruy 2004. Number of fruit on each of the selected shoots was recorded on April 12. Fruit in the control and Tergitol-treated blocks were hand-thinned on May 12.
RESULTS AND DISCUSSION For the 'Blazeprince', bloom period was similar between treatments (data not shown).
The 8% VO and 8% SO treatments reduced the amount of hand-thinned fruitlets by 15% and 35%, and thinning costs by 10% and 25%, respectively, compared to the DO control (Table 1). Both oil treatments slightly advanced fruit maturity. The percent pack-out was highest in the VO block (Table 2). Fruit yield was slightly lower with the oil treatments, but fruit size was about
82
10% larger than the control fruit. Vegetoil™ produced lower gross revenue due to a reduction in fruit yield.
For 'Fireprince', 8% VegetoiFM sprayed 1328 L/ha advanced bloom and also had the greatest thinning effect (Table 1). Thirty-five percent fewer fruitlets were hand-thinned compared to the DO control. However, hand-thinning costs were similar. VegetoifM sprayed at 935 L/ha resulted in the highest fruit yield, twice that of the DO control, but fruit size was smaller compared with the other treatments (Table 2). However, yield data were collected only from the last 3 of 7 harvests, which may have biased the earlier ripening va treatments. Gross dollar return was positively correlated with fruit yield.
In 2004, 9% SO slightly advanced bloom of 'Summerprince', with 45% more flowers open on March 16, compared to the DO control (data not shown). SO and va also slightly reduced the number of surviving flower buds. This difference did not translate into a reduced number of hand-thinned fruitlets (Table 3), since 1 % Tergitol was sprayed during bloom on all trees, including control trees. Slightly fewer fruit were thinned from control trees following the Tergitol application, resulting in the control treatment being the least expensive to hand-thin. Fruit maturity was slightly delayed with 10% va as 25% of the total fruit in the 10% va orchard was picked over the first two harvest dates, compared to approximately 35% of the fruit in the other orchards. Yield per hectare was 10% lower with va, compared to the SO and control treatments (Table 4). Pack-out percent and mean fruit weight were higher with the va and SO treatments compared to the control. Trees sprayed with 9% SO had twice as many fruit in the largest size class (7.0 cm) as other trees. Gross dollar return per hectare was highest with SO-treated trees, ~ $4000 more than with other treatments (Table 5). Despite producing similar yields, SO-treated trees returned more than control trees due to higher percent pack-out and a higher percentage of fruit in the 6.4 and 7.0 cm size classes.
Table 1. Fruit thinning 2003 data for 5 peach cultivars at Titan Peach Fanns, Ridge Spring, SC
Fruit thinning Fruitlet weight Cost @7.501hr
Count/treeZ Total/tree Mean per tree per ha Cultivar Field Treatment (kg) (g) ($) ($) Blazeprince Davis A 8% Vegetoil 662 b 1.96 a 2.96 a 0.89 318.75
DavisB 8% Soybean oil 489 c 1.34 b 2.76 a 0.74 265.03 Davis C 3% Dormant oil '780 a 2.09 a 2.65 a 0.98 350.99
Fireprince Be114-2A 8% Vegetoil- 1328Uha 307 b 2.73 a 8.61 a 0.71 254.29 Be114-2B 8% Vegetoil- 935Uha 409 ab 3.48 a 8.50 a 0.79 282.94 Be114-2C 3% Dormant oil 479 a 3.91 a 8.16 a 0.70 250.71
Z Mean separation within columns and cultivar by LSD, p< 0.05.
Table 2. Harvest data in 2003 for 2 2each cuItivars at Titan Peach Farms, Ridge S2ring, South Carolina.
Costs/ha CuItivar Yield Mean Gross Hand- Spray Net
Packout per tree perha fruit wt returnlha Thinning application return/ha Blazepril7ce-Davis (%} (kg} (kg} (g) ($) ($) ($) ($)
Table 3. Fruit thinning 2004 data for 3 peach cultivars at Titan Peach Farms, Ridge Spring, SC.
Fruit Fruitlet weight Thinning thinned Total/tree Mean cost/hax
Cultivar TreatmentZ per treeY (kg) (g) ($)
SummerprinclI0% Vegetoil (2/3/04)+ 1 % Tergitol (3/18/0" 6031 a 11.8 a 2.0 b 2079 9% Soybean oil (2/3/04)+ 1% Tergitol (3/18/ 5192 a 16.2 a 3.0 a 2200 2% Dormant oil (1/04) + 1 % Tergitol (3/18/0. 4947 a 13.2 a 2.6 a 1992
Fireprince 8% Soybean oil (1/29/04) 696 a 11.4 a 17.0 a 548 9% Vegetoil (2/3/04) 266 b 5.0 b 19.6 a 398 2% Dormant oil (1/04) 715 a 12.6 a 18.6 a 405
Flameprince 8% Vegetoil (2/4/04) 991 b 19.0 b 19.5 a 1092 2% Dormant oil (1/04)+ 1 % Tergitol (3/19/0. 1199 b 21.8 b 18.3 ab 659 2% Dormant oil (1/04) 1856 a 32.5 a 17.7 b N/A
z Control- 2% dormant oil application Y Mean separation within columns by Duncan's multiple range test, P < 0.05. x Tree number in Flameprince control blocks insufficient for cost analysis.
Fireprince' bloom in 2004 was delayed slightly by 9% Vegetoil™ (data not shown). Significantly fewer flowers (e.g., 35%) in the VO treatment were open on March 16, compared to the SO and control treatments. The 9% VO treatment reduced the amount of hand-thinned fruitlets by 60% compared to the DO control, but thinning costs were not reduced (Table 3). Fruit maturity was delayed with 8% SO. Only 10% of the total fruit in the SO orchard was picked on the first two harvest dates, compared to 20% of the fruit in the other orchards. Fruit yield was noticeably lower with trees sprayed with 9% VO. However, mean fruit weight was highest with va, with almost 60% of fruit 7.6 em or greater in size (Table 4). Percent pack-out was similar with all treatments. The low-yielding Va-treated trees reduced gross dollar return per hectare by ~ $3900-4400, compared to SO-treated and control trees (Table 5).
'Flameprince' flower bud density and fruit set were not recorded for the 8% VO treatment. The fruit hand-thinned from VO trees were much less compared to the control trees (Table 3). Hand-thinning was more expensive with VO-treated trees, which did not receive the added Tergitol (i.e., chemical bloom thinner) treatment. VO slightly delayed fruit maturity compared to the DO+ Tergitol treatment. va also slightly improved pack-out, and increased fruit yield by more than 20% compared to trees sprayed with Tergitol (Table 4). va produced a gross dollar return per hectare that was ~$5000 higher than a Tergitol application (Table 5).
84
Table 4. Harvest data in 2004 for 3 ,Qeach cultivars at Titan Peach Farms, Ridge S,Qring, South Carolina.
Cultivar/treatment Yield Mean Size class (% of fruit) per tree perha Packout fruit wt 5.4 cm 5.7 cm 6.4 cm 7.0cm 7.6 cm
CONCLUSIONS Pre-bloom thinning treatments were variable in efficacy but did increase grower gross
returns as much as $4944/ha in one Vegetoil1M application. However, environment and cultivar interactions resulted in a gross return loss of $3924 for another Vegetoil™ treatment. These data show that timing, peach cultivar, and likely other factors affect the efficacy of using soybean oil as a flower bud thinner, but the potential to reduce labor costs and increase fruit size is possible.
85
ACKNOWLEDGEMENTS Funding for this project was provided by the South Carolina Ag Experiment Station
project SC-170005 and the South Carolina Peach Council. The investigators thank Titan Peach Farms, Inc., Jason Rodgers and Chalmers Can for their generous assistance and material support.
LITERATURE CITED Deyton, D.E., Sams, C.E. and Cummins, lC. 1992. Application of dormant oil to peach trees
modifies bud-twig internal atmosphere. HortScience 27: 1304-1305. Moran, R.E., Deyton, D.E., Sams, e.E. and Cummins, J.e. 2000. Applying soybean oil to
dormant peach trees thins flower buds. HortScience 35: 615-619. Myers, R.E., Deyton, D.E. and Sams, C.E. 1996. Applying soybean oil to dormant peach trees
alters internal atmosphere, reduces respiration, delays boom, and thins flower buds. J. Amer. Soc. Hort. Sci. 121: 96-100.
Pendergrass, R., Roberts, R.K., Deyton, D.E. and Sams, C.E. 2000. Economics of using soybean oil to reduce peach freeze damage and thin fruit. HortTechnology 10: 211-217.
86
FLORIGENIC PROMOTER OF L YCHEE (Litchi chinensis, Sonn) SYNTHESIZED IN LEAVES .
T.L. Davenpore* and Z. Ying1
ABSTRACT
In mango, the florigenic promoter is synthesized in leaves and is translocated to buds in the phloem whereas in citrus, it is synthesized in stem tips. Because lychee trees appear to have the same phenology as mango, and appear regulated by similar environmental cues, we needed to determine the source of the putative florigenic promoter of this crop to further understand the flowering mechanisms. Replicate branches from five trees each of two cultivars, 'Brewster' and 'Mauritius' were isolated from the rest of the canopy by girdling. All of the stem terminals on each branch were tip pruned to stimulate uniform bud break during cool, floral inductive conditions. One branch on each tree was defoliated to remove the potential source of the floral promoter and another was left with the full complement of leaves. Another set of replicate branches was left undisturbed to document normal flowering behavior. Virtually 100% of the lateral shoots initiating in the branches with leaves formed flowering shoots. In contrast, branches in which all of the leaves were removed formed only vegetative shoots. These results indicate that the floral promoter of lychee is synthesized in leaves.
I University of Florida, IFAS, Trop. Res. & Ed. Ctr., Homestead, FL 33031
87
CONTRIBUTED PAPERS
89
EFFECTS OF LIQUID FERTILIZER CONTAINING 5-AMINOLEVULINIC ACID
ON THICKENING GROWTH IN TULIP BULBS
R. Yoshida1', E. Ohta1, K. Iwai2 , T. Tanaka and H. Okadaa
ABSTRACT
The promotive effect of Pentakeep-V (PKV) on thickening growth of tulip bulbs was
studied. PKV markedly increased the number of tulip bulbs, especially of commercial
bulbs. This effect of PKV was found in both cvs. Ballerina and Leen-van-der-Mark. Thus,
this PKV dilution was very useful for the production of commercial bulbs in tulip plants.
INTRODUCTION Tulip plants are a main crop in winter cropping on drained paddy field, especially in
Toyama Prefecture, Japan. The commercial bulbs are used for flower bulbs in gardening
and cut flower. However, the thickening growth from seeds to seed bulbs is very slow,
approximately 5 to 7 years need to obtain commercial bulbs from original seeds.
Therefore, the promotion of thickening growth of tulip seed bulbs using plant growth
regulator is very useful for increasing actual income of farmer. In this experiment, we
examined whether or not liquid fertilizer containing 5-aminolevulinic acid (5-ALA) and
micronutrients (PKV) affects the thickening growth of seed bulb (circumference of bulb,
5 and 6 cm) in tulip plants.
MATERIALS AND METHODS The cultivars of tulip plants used were Ballerina and Leen-van-der-Mark. Both
cultivars were cultivated under field condition. The seed bulbs of 5cm (cv. Ballerina) and
6cm (cv. Leen-van-der-Mark) were transplanted in field on October 23, 2004. At
flowering time on May 5, 2005, each testing PKV dilution (5000-fold and 2000-fold) was
sprayed three times onto leaves and stem after flower picking. The water solution was
also applied to control plants. The spraying volume per 10 are was Ca. 100 liter. These
testing solutions contained 0.1 % of Approach as a wetting agent. The liquid of PKV used
was composed of N 9.5%, MgO 5.7%, MnO 0.3% and B203 0.45% as macro-and micro
nutrients, and also DTPA-Fe, ZnS04, Na2Mo04 and 5-aminolevulinic acid (5-ALA) as
l.Toyama Prefectural University, Kosugi, Toyama 939-0311, Japan
2.Seiwa Co., Ltd., Hachobori 1-6-1, Chuo-ku, Tokyo 104-0032, Japan
3.Cosmo oil Co., Ltd,.Shibaura 1-1-1, Minatoku, Japan
91
growth promoters.
At harvesting time on June 20, 2005, the total yield of bulbs and the number in each
bulbs size were determined. The bulb size was classified to the circumference (cm) of
bulb (>10cm, >9cm, >8cm, >7cm, >6cm, >5cm and <5cm).
RESULTS AND DISCUSSION
The promotive effect of PKV on the yield in each bulb size, in case of cv. Ballerina, is
shown in Table l. As shown in table, the foliar spray of PKV markedly increased the
total bulb number per 6 m2• The rate increased was 106% for PKV 5000-fold dilution
and 120% for PKV 2000-fold one. However, no the promotive effect of PKV on the bulb
weight in each bulb size was observed (Table 2).
On the other hand, in case of cv. Leen-van-der-Mark, the promotive effect of PKV on
the thickening growth of each bulb size is shown in Table 3. The total bulb number in
plants treated with PKV 5000-fold and 2000-fold dilutions were increased by 102% and
III % compared with that of control, respectively. Both the PKV dilutions tested
increased the yield of commercial bulbs (>10cm). But PKV treatments had no the
promotive effect on the bulb weight in each bulb size (Table 4). Thus, high yielding for
commercial bulbs was found in tulip plants treated with PKV 2000-fold dilution.
Yoshida el al. (2) have been reported that at low concentration 5-ALA has promotive
effect on growth of welsh onion, garlic and rakkyo. Yoshida et al. (3) also found that
5-ALA in the presence of microelements increased the fresh and dry yield of Komatsuna,
Brassica camprestlis var. perviridis under alkaline soil conditions. Hotta et al. (1) have
reported that the foliar application of 5-ALA at low concentration (0.18 to l.6 mM)
increased fixation in light and decreased release of C02 in darkness.
From our results and these findings, we wish to emphasize that PKV (5-ALA in the
presence of macro-and micro-nutrients) and 5-ALA alone behave as a stimulant for the
thickening growth of tulip bulbs, especially for high yielding of commercial bulbs in
tulip plants.
LITERATURE CITED
l. Hotta Y., T. Tanaka, H. Takaoka, Y. Takeuchi and M. Konnai (1997). Promotive effects
of 5-aminolevulinic acid on the yield of several crops. Plant Growth Regulation
22:109-114.
2. Yoshida R., T. Tanaka and Y. Hotta (1996). Regulation of fuructan accumulation in
rakkyo (Allium baken) and shallot (Allium ascalonicum) by 5-aminolevulinic acid.
92
Proceedings of PGRSA. 177-182.
3. Yoshida R., Y. Fukuda, K. Shimotsubo, K. Iwai, S. Watanabe and T. Tanaka (2004).
Growth promotive effects of 5-aminolevulinic acid in the presence of micro elements on
yield in Komatsuna, Brassica campestris var. perviridis under alkaline soil conditions.
4th International Crop Science Congress, CD-Rom.
Table 1. Effect ofPKV on the yield in each bulb size
DEVELOPING IMPROVED NURSERY CULTURE FOR THE PRODUCTION OF ROOTED CUTTINGS OF CANADA YEW (Taxus canadensis Marsh.)
L. Webster1*, R. F. Smith2, and S.I. Cameron2
ABSTRACT Since the early 1990s, when the ftrst anti-cancer drug containing paclitaxel was marketed,
yew species (Taxus spp.) around the world have been threatened by unsustainable harvesting practices. Canada yew (Taxus canadensis Marsh.) a species native to eastern Canada and the north eastern United States is now facing similar pressures. In response to an increasing demand for biomass, in 1997, a domestication program was started. In 2003, a series of nursery trials was initiated to evaluate the potential of several plant growth regulators and cultural treatments for improving rooting of recalcitrant clones. This poster reports initial results from these trials.
INTRODUCTION In 1958 the National Cancer Institute (NCI) initiated a search for chemotherapeutic cancer
drugs. A plant screening program was developed to find natural compounds for the use in the fight against cancer. In 1972 the first species was found that contained the anti-cancer chemical paclitaxel (Taxol®): Paciftc yew (Taxus brevifalia Nutt.), found in mountainous areas of the northwestern United States (Hansen 1999) and western Canada (Farrar 1995). Subsequently, paclitaxel has been found in the stem bark, roots and needles of several other yew species, including Canada yew (Taxus canadensis Marsh.) (Nandi, S. K. et al 1996, Senneville et al 2001). While all Taxus species contain paclitaxel, other specific 'taxanes' and related compounds and their concentrations vary greatly among species.
In the 1990's the NCI designated Taxol as an "emergency priority", since it was a very promising anti-cancer drug and already in short supply. The sustainability and survival of Taxus spp. around the world continues to be of great concern. The relatively high commercial value of taxane-yielding biomass has resulted in signiftcant reductions in native populations of virtually all of the yew species throughout the world (Smith and Cameron 2002). Consequently, there is concern that similar overharvesting could threaten Canada yew populations (c.f. Senneville et al 2001). A sustainable harvest system is being developed; but insufftcient research has been conducted to date to quantify the impact of different harvesting levels on the regrowth and abundance of the species (Smith and Cameron 2002).
As an alternative response to an increasing demand for biomass, in 1997 a domestication program was started, in which rooting cuttings are an integral component of field crop production. Canada yew shoots are moderately amenable to rooting, but cuttings are slow to root, and recalcitrant clones still cannot be rooted effectively. In 2003, a series of nursery trials was initiated to evaluate the potential of several plant growth regulators and cultural treatments for improving rooting of Canada yew cuttings.
1 Faculty of Forestry and Environmental Management, University of New Brunswick, P.O. Box 4400, Fredericton, New Brunswick, Canada, E3B SA3 2 Natural Resources Canada, Canadian Forest Service, P.O. Box 4000, Fredericton, New Brunswick, Canada, E3B SP7
95
Research objectives The objectives of this research are: (i) to develop a method to increase rooting in recalcitrant
clones of Canada yew, (ii) to evaluate methods to decrease time to root, cuttings and iii) to test alternative rooting methods such as rooting directly into field crop beds. Improvement(s) in any ofthese areas would increase the efficacy, and decrease the cost, of producing rooted cuttings.
MATERIALS AND METHODS Cuttings between 2 and 7 inches in length were collected in the fall of 2003. Shoots were cut
just below the annual bud scar and comprised a maximum of two years of growth. All cuttings (with the exception of the no hormone treatment in the horn lone trial) were dipped in a commercial rooting hormone mixture (Stirn-Root No.3, containing 0.8% indole-3-butyric acid). Excess rooting powder was tapped off. All cuttings were struck into 67 cell multi-pot trays with a standard mix of 2:1 peat moss: vermiculite. A 0.2" diameter hole with a depth of 0.75" was pre drilled in each cell prior to striking the cutting. The soil was pinched around the cutting after sticking to hold it in place.
Three experiments were initiated: a terminal bud removal trial, a hormone trial and a cold storage trial. The number of cuttings struck per tray per clone varied from 30 to 67, based upon the total number of cuttings available within the specified size range from the parent plant. For each clone, the total number of cuttings was divided equally among treatments. The trays were arranged in the greenhouse by trial, clone, and treatment in a randomized block layout.
Terminal Bud trial The terminal bud trial consisted of twenty-one clones. There were two trays per clone for a
total of 42 trays. One tray per clone had the terminal bud removed and the other tray the terminal bud was left intact.
Hormone trial The hormone trial consisted of eight clones. There were two trays per clone for a total of 16
trays. One tray per clone had IBA (indole-3-butyric acid) hormone applied, and the other tray cuttings did not have hormone applied.
Storage trial The storage trial consisted of twenty-eight clones. There were two trays per clone for a total
of 56 trays. Material for one tray was struck on the day of collection and the material for the second tray was stored in a cooler at 4°C. Ten clones were stored for one week, nine clones were stored for four weeks and nine clones were stored for six weeks.
Cuttings were grown in a greenhouse at the Canadian Forest Service in Fredericton, New Brunswick. Post-striking, trays containing the cuttings were placed in the greenhouse with misting to maintain the relative humidity at 70%. The day/night temperature was 22/18 °C. Ambient light levels ranged between 37 and 146 micromole/sec/nr for the rooting period. Once callus and (or) roots formed, the temperature, frequency of watering and light in the greenhouse were reduced to provide a cold period of eight weeks where the greenhouse temperature was at maintained at 5°C. Greenhouse growing resumed in the spring with 22/18 °C day/night temperatures, relative humidity of 60% and a 16-18 hour photo period extended through
96
supplementary lighting (high pressure sodium). Cuttings were maintained at these conditions throughout the summer and harvested during the fall of 2004.
Measurements Cuttings were harvested individually, placed into plastic bags, and put into frozen storage
until processed. Cuttings that did not have roots were discarded. At the time of processing, roots were washed and the following measurements taken:
Terminal bud trial: The length of the initial cutting, length of new growth, root collar diameter, and tenninal shoot diameter were measured and then the samples dried at 65°C for 48-72 hours. Dry weights were measured separately for roots, the original cutting needles and stem, terminal shoot (current-year) needles and stem, and lateral shoot(s) (current-year) needles and stem(s).
Hormone trial and storage trial: length of cutting with new growth. Dry weights were measured separately for roots, cutting, and CU11'ent year growth.
RESULTS AND DISCUSSION
Terminal Bud trial The production and basipetal translocation of auxins from terminal buds is important in
stimulating adventitious rooting (Cooper 1936). Removing the tenninal bud reduced sugar:starch concentration ratios in the basal stem of Pinus banksiana cuttings, which may have caused a reduction in rooting success (Haissig 1989). The effect of tenninal bud removal has not been studied on Taxus spp. so it was unknown how cuttings would be affected.
Operationally, there is an advantage if removal of the terminal bud did not does not decrease rooting success, since an increased number of cuttings can be taken from each parent plant. However the removal of Canada yew tenninal buds caused a decrease in mean survival for all clones from 39% to 22%. Survival rate was typically higher for all clones with terminal bud attached. With the exception of two clones (designated Band R) which had a minimal increase in survival with tenninal bud removed (Fig 1). Therefore, there may be auxins or other phytohormones within the tenninal bud that are translocated at time of rooting to the basal stem (Haissig 1989). For cuttings that rooted, the amount of new growth produced by cutting was positively correlated with the initial cutting length (P< 0.0001). There was no significant difference in growth between cuttings with terminal bud removed and without tenninal bud removed (p=0.59).
97
ABCDEFGH JKLMNOPQRSTU
Clone
[o,.} ... Without Terminal Bud ..... .. With Termin~
Figure 1. Survival between clones with and without a terminal bud.
Hormone trial Mean survival for all clones with and without hormone treatments was 49% and 36%
respectively. This is similar to previous results from IBA hormone application to Pacific Yew cuttings where rooting success increased from 30.6% to 50.0% (Mitchell 1997). Hormone application had no significant effect on survival within clones (Fig. 2). Cuttings receiving honnone produced significantly more top (p<0.005) and root (P<0.009) growth than did the untreated controls. Many clones rooted but produced no current shoot growth. Hormone treatment did not increase rooting success in recalcitrant clones.
~ Horm~-=_~ No" Hormone .... ,~, .. Hormone .... g . .. ' No Hormone I
80
70 60 ~ 50 C. 40 ~ 30 .~
::J 20 IIJ
10 o
Figure 2. Survival (lines with points) and Average root weight (bars) between clones with and without hormone treatment.
Several studies have shown that cold storage of dormant conifer cuttings prior to striking is an effective treatment for improving rooting success (Behrens 1988). This was also true for Canada yew cuttings. The average survival of cuttings that had cold storage treatment was about 10% higher at any of the three storage times compared to cuttings that were immediately struck at the time of collection (Fig. 3). This implies that rooting success is not enhanced by increasing the
98
cold storage times. Operationally, however, cold storage does allow for material to be collected and stored for striking at a later time, while actually increasing rooting potential without degrading the plant material.
...
80
75 -~ -iij 70 .~
~ :I t/J
65
60 . Week 1 Week4 Week6
.. {, ... Control .s; .. Treatedl
Figure 3. Average survival of cuttings struck at time of Collection versus those stored for one, four, or six weeks (n = 10, 9, 9 clones respectively).
CONCLUSION
I
Clonal differences were significant for all treatments and experiments. For the clones that had poor survival, the cuttings that did root typically did not exhibit bud flush and subsequent top growth. The standard practice of using a honnone dip to root yew cuttings should also be used for Canada yew, but it does not appear to enhance rooting in recalcitrant clones. Pretiminmy findings indicate that storing cuttings for as little as one week prior to striking can achieve modest gains in rooting efficiency.
ACKNOWLEDGEMENTS We would like to expresses thanks John Letourneau, Dan Flemming, Fiona McBain-Hogg
and Paula Stewart-Leblanc, for technical support, Laurie Yeates and Terry Hay for greenhouse support, and Dr. Marek Krasowski for project guidance and other students; Maureen Cameron, Robert Eveleigh, and Katherine Smith, who helped with processing of samples. Funding from the New Brunswick Innovation Foundation, the Plant Growth Regulation Society (Student Travel Award), Chatham Biotec Ltd. in collaboration with the Canadian Forest Service, and the University of New Brunswick, School of Graduate Studies and the Faculty of Forestry (Travel Grant) is gratefully acknowledged.
LITERATURE CITED
Behrens, V. 1988. Storage ofunrooted cuttings. In: Davis, T. D., Haissig, B. E., and Sankhla, N. eds. Adventitious Root Fonnation in Cuttings. Portland, Oregon: Dioscorides Press, 235-247.
99
Cooper, W. C. 1936. Transport of root-forming hormone in woody cuttings. Plant. Physiol. 11: 779-793.
Farrar, J. L.1995. Trees in Canada. Fitzhenry and Whiteside Limited and the Canadian Forest Service. Markham, Canada.
Haissig, B. E. 1989. Removal of the stern terminal and application of auxin change carbohydrates in Pinus banksiana cuttings during propagation. Physiol. Plant. 77: 179-184.
Hansen, R. C. 1999. from "preface" to Taxus and Taxol - A compilation of research findings. Special circular 150. Ohio Agriculture Research and Development Center. The Ohio State University.
Mitchell, A. K 1997. Propagation and growth of pacific yew (Taxus brevifolia Nutt.) cuttings. Northwest Sci. 71:56-63.
Nandi, S. K, Palni, L. S., and Rikhari, H. C. 1996. Chemical induction of adventitious root fonnation in Taxus baccata cuttings. Plant Growth Reg. 19: 117-122.
Selmeville, S., Beaulieu, J., Daoust, G., Deslauriers, M., and Bousquet, J. 2001. Evidence for low genetic diversity and metapopulation structure in Canada yew (Taxus canadensis): considerations for conservation. Can. J. For. Res. 31: 110-116.
Smith, R. F., and Cameron, S. I. 2002. Domesticating ground hemlock (Taxus canadensis) for producing taxanes: a case study In: Proc. 29th Annual Mtg. Plant Growth Reg. Soc. Arner. Halifax N.S. July 28-Aug 1,2002. pp 40-45.
100
NEW INNOVATIONS WITH FLURPRIMIDOL USE ON TURFGRASS, CONTAINERIZED ORNAMENTALS, AND LANDSCAPE ORNAMENTALS.
B. T. Bunnell1* and S. D. Cockreham1
ABSTRACT
Flurprimidol (FP) is a nitrogen-containing heterocycle Type II, class B plant growth regulator (PGR) in the pyrimidine class of chemistry. Specifically, flurprimidol inhibits the enzyme cytochrome P450 monooxygenase blocking the formation of ent-kaurenoic acid, a precursor to active GA' s. PGRs with similar chemistries and modes of action include: ancymidol and the triazoles, paclobutrazol (PB) and uniconazole. Registered uses for FP in the US include turfgrass and established landscape ornamentals. Registration is pending for FP application to containerized ornamentals and trees. Extensive research trials have been performed in these use sites to evaluate plant responses to flurprimidol. Discussions will include: plant site ofFP uptake and resulting height control in containerized ornamentals, lateral recovery of turfgrass following FP applications, and FP activity on established perennial landscape ornamentals.
In FP tissue absorption trials performed at North Carolina State University in Spring 2002, investigators applied equal amounts ofFP and PB (119 ppm, or 1.25 mg) to leaves, stems and roots of containerized potted sunflower (Helianthus), cultivar 'Pacino'. Above-ground applications were made by painting stems and leaves, while a concentrated dose was drenched to roots. The objective was to determine differential absorption ofFP among plant tissues and compare with absorption of PB. Results indicate that FP is more active through the stem and substrate for root uptake than through foliar application. Furthennore, FP elicits greater activity through the stem compared to PB. With the greatest activity ofFP through the stem, active sites on whole plants can be characterized as: stems » roots » leaves.
In the lateral recovery studies on turfgrass, FP and PB were applied twice, 4 weeks apart at equal active ingredient per acre rates (0.28 and 0.56 kg ai/ha) on a creeping bentgrass [Agrostis stoloniferous L. val'. palustris (Huds.)] fairway in central Indiana. The objective was to evaluate the lateral regrowth (RG) of creeping bentgrass following sequential applications of FP and PB. In order to evaluate lateral RG of creeping bentgrass, soil cores were extracted from each replicate plot prior to application and backfilled with topdressing sand. In order to measure lateral RG, a wire mesh grid was constructed equal to the dimension ofthe original extracted core. Digital images were taken every 2 weeks with the wire mesh grid overlaying the backfilled soil core to calculate lateral regrowth. By 4 weeks after initial treatment (WAIT), PB at 0.56 kg ai/ha reduced lateral RG by 11 to 13% compared to FP at 0.28 and 0.56 kg ai/ha. At the final rating date, at 8 WAIT, plots receiving sequential applications ofPB at 0.56 kg ai/ha reached 82% total RG, whereas sequential applications ofFP at 0.56 kg ai/ha reached 98%.
Current and future investigations will evaluate a granular formulation of FP for application to established perennial landscape ornamentals. Expected responses to FP to landscape ornamentals include: shoot growth regulation resulting in less pruning frequency and trim biomass, darker green foliage, more compact growth habit, and the potential for improved plant health.
ISePRO Corporation, 11550 N Meridian St., Suite 600, Cannel, IN 46032 USA
101
STUDIES ON PLANT-ASSOCIATED ACTINOMYCETES AND THEIR SECONDARY
METABOLITES (3)
Y Igarashi 1 *, S. Miura 1, M. Azumi 1, T. Furumai 1 and R. Yoshida2
ABSTRACT
Effect of endophytic actinomycetes on plant growth was investigated. Crop seeds were
bacterized with the spores of endophytic actinomycetes and grown in a green house. Of the tested
microorganisms, Streptomyces hygroscopicus S-17 induced the significant growth promotion of tomato
cd 2 times in height and ca 8 times in fresh weight compared to the control. One of the secondary
metabolites, pteridic acid A showed growth promotion in the root formation test of kidney bean
hypocotyls and the tobacco BY-2 cell culture.
INTRODUCTION
Actinomycetes are widely distributed in association with plant in natural environments. These
plant-associated (endophytic) microorganisms are opportunistic and/or saprophytic but do not show
pathogenicity in general. Endophytic actinomycete is the new entity for the screening of novel bioactive
compounds. We have isolated several new bioactive compounds from endophytic actinomycetes in
search for lead molecules for pharmaceutical and agrochemical usages. Although the endophytic
actionomycetes produce various secondaty metabolites, biological significance of such metabolites in
plant-microbe community is open to question. Previously, we identified that endophytic actinomycetes
are producing plant hormone-like substances; toyocamycin is a potent cytokinin-like callus growth
promoter, and pteridic acid is an auxin-like adventitious roots formation promoter. In this study, effects
of endophytic actinomycetes on plant growth was investigated in view of agricultural application.
MATARIALS AND METHODS
Bacterial strains. Endophytic actinomycetes were isolated from plant samples according to
the procedure previously reported (Igarashi, 2002). Actionomycete strains used in this study are as
follows: Streptomyces hygroscopicus S-17 (isolated from Pteridium aqui!inum), S. hygroscopicus S-346
(isolated from Clethra barbinervis), S. sp. S-231 (isolated from Leucothe granaya), S. sp. (isolated from
Allium chinese).
1 Biotechnology Research Center, Toyama Prefectural University, Kosugi, Toyama 939-0398, Japan 2 College of Technology, Toyama Prefectural University, Kosugi, Toyama 939-0398, Japan
102
Seed bacterization. To a mature slant culture of an actinomycete was added a solution of
10% DMSO/I0% glycerol. The slant tube was sonicated for 5 min and the spore suspension was taken
into a sterilized tube. Crop seeds were soaked in the suspension for 30 min at room temperature. Then,
the seeds were planted in a plastic pot filled with commercially available culture soil, and grown in a
green house.
Plant growth promotion activity of secondary metabolites. Isolation of pteridic acid and its
root fonnation activity has been described previously (Igarashi, 2002). Tobacco BY-2 cells were cultured
in LS medium in dark at 25°C. After the addition of pteridic acid or benzyladenine, the cells were
cultured for 7 days and the fresh weight of the cells was measured. Isolation of 6-prenylindole has been
reported previously (Sasaki, 2002). Five mm length slices of barley sprouts were incubated with
6-prenylindole in a buffer solution (PH 7) for 48 hr in dark, and the length of the slices was measured.
RESULTS AND DISCUSSION
Previously we reported that the crop growth is promoted by seed bacterization with
actinomycetes that are isolated from a wide variety of plants (Igarashi, 2003; Igarashi, 2004). Although
it is uncertain whether these actinomycetes are plant-specific or not, they are considered endophytes
because they can be reisolated from the crops inoculated to.
Seed bacterization of crops was carried out by immersing the seeds in the spore suspension.
The seeds were planted in a pot and grown in a green house. After four weeks of cultivation, growth of
bacterized crops was compared with that of untreated ones. Among the tested microorganisms, the
actinomycete Streptomyces hygroscopicus S-17 showed significant growth promotion of tomato ca 2
times in height and ca 8 times in fresh weight compared to the control (Fig 1, Fig. 2). Several factors are
possibly involved in this growth activating effect: firstly, the microorganism induces the systemic
acquired resistance or phytoalexin production; secondly, the actinomycete produces antimicrobial
substances effective against plant-deleterious soil microorganisms; thirdly, the actinomycete produces
bioactive compounds that have plant growth promoting activity.
Cont S-17 S-231 S-328 S-346
Fig. 1. Growth of tomato seedlings after bacterization.
103
cont S-17 S-231 S-328 S-346
Fig. 2. Growth of tomato in fresh
weight (g/plant).
Although actinomycetes produce numerous kinds of secondary metabolites, their plant
bioactivity is known very little. We investigated the plant growth promoting activity of the secondary
metabolites of strain S-17 which induces significant growth promotion by seed bacterization. By using
HPLC, MS and NMR, we have identified that at least 10 chemically different classes of secondary
metabolites are produced by strain S-17. Of these compounds, pteridic acid A induced the adventitious
root formation of kidney bean hypocotyls (Table 1) and growth promotion of tobacco BY-2 cells (Table
2), suggesting the possible involvement of secondary metabolites in plant growth promotion. Further
analysis of the mechanism of plant growth promotion by seed bacterization is currently investigated.
by pteridic acid A. cells induced by pteridic acid A.
Number of roots/hypocotyl Cell fresh weight (mg)/flask
Conc (!-lM) Pteridic acid A
0 30.5±5.9
0.001 39.7±4.8
0.01 42.3±I2.9
0.1 60.0±8.5
1 66.3±8.5
LITERATURE CITED
IAA Initial 55±5
34.7±4.8
42.3±4.6
51.0±5.1
86.7±1.1
Control
0.1 !-lM Pteridic acid A
3.0 !-lM Pteridic acid A
1.0 !-lM Benzyladenine
447±4
936±88
702±48
820±33
Igarashi Y, T. Iida, S. Miura, R. Yoshida, and T. Furumai. 2003. Secondary metabolites of endophytic
actinomycetes with plant growth promoting activity. The abstract of 13th International Symposium on
the Biology of Actinomycetes. p.20.
Igarashi Y, R. Yoshida, S. Miura, and T. Furumai. 2004. Plant growth promoting activity of secondary
metabolites of endophytic actinomycetes. The abstract of 18th International Conference on Plant Growth
Substances. p. 59.
Igarashi Y, T. Iida, T. Sasaki, N. Saito, R. Yoshida, and T. Furumai. 2002. Isolation of actinomycetes
from live plants and evaluation of antiphytopathogenic activity of their secondary metabolites.
Actinomyceto!. 16: 9-13.
104
Igarashi Y, T. Iida, R. Yoshida, and T. Furumai. 2002. Pteridic acids A and B, novel plant growth
promoters with auxin-like activity from Streptomyces hygroscopicus TP-A0451. J. Antibiotics. 55:
764-767.
Sasaki T, Y Igarashi, M. Ogawa, and T. Furumai. 2002. Identification of 6-prenylindole as an antifungal
metabolite of Streptomyces sp. TP-A0595 and synthesis and bioactivity of 6-substituted indoles. J.
Antibiotics. 55: 1009-1012.
105
DEVELOPMENTAL REGULATION OF THE GA BIOSYNTHESIS GENES, GA20ox, GA30x, and GA200x DURING GERMINATION AND YOUNG SEEDLING GROWTH OF PEA (Pisum sativum L.)
Belay Ayele 1 , Jocelyn Ozga1 *, and Dennis Reinecke 1
ABSTRACT
To understand the role of gibberellins (GAs) during gennination and early postgenninative stages of large-seeded dicotyledonous plants, we profiled the expression pattern of genes encoding three regulatory GA biosynthesis enzymes (PsGA20oxl, PsGA30xl and PsGA2oxl) in pea (Pisum sativum L.) using real-time RT-PCR. To broaden our inferences on the role of GAs in these processes, we compared the GA biosynthesis gene expression patterns in two distinctly different genotypes of pea (,Alaska' a model cultivar for vining pea containing the wild-type internode length gene LE and 'Carneval' a model cultivar for semi-leafless field pea containing the Ie mutation producing shorter internodes), both of which germinate readily on imbibition under nonnal environmental conditions. Residual amounts of PsGA20oxl, PsGA30xl, and PsGA20xl transcripts were detected in the mature embryos (0 days after imbibition; DAI) of both genotypes. Transcription of PsGA20oxl, PsGA30xl, and PsGA20xl mRNAs occurred in all tissues examined (cotyledons, embryo axis, shoots and roots from 0.5 to 6 DAI) and was developmentally regulated within each tissue. Cotyledonary GA biosynthesis gene transcript patterns suggest that a signal from the axis triggers GA biosynthesis in the cotyledon. The high levels of PsGA200xl and PsGA30xl mRNA in the embryonic axis at 1 DAI suggests that the embryo axis is a major site for GA biosynthesis for stiinulation of axis expansion. GA biosynthesis gene expression in 2 to 6 DAI shoots and roots (when their growth in fresh weight and length increased linearly) indicates a key role for de novo GA biosynthesis in early growth of seedlings. Supported in part by NSERC grant #138166.
1 Plant Physiology and Molecular Biology Research Group, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5
106
GIBBERELLIN AND AUXIN LEVELS IN MATURE EMBRYOS AND YOUNG SEEDLINGS OF Pisum sativum L.
Belay Ayelel, Jocelyn Ozgal*, and Dennis Reineckel
ABSTRACT
Gibberellins (GAs) and auxins are two classes of hormones that influence processes during seed germination and early seedling growth. This study examines how auxin and GA levels change during these developmental phases in two different genotypes of pea (pisum sativum L.), 'Alaska' (LE) and 'Carneval' (Ie), both which germinate readily upon imbibition.. The endogenous levels of gibberellins (GA19, GA2o, GAl, GAs, GA29, and GA3) and auxins (IAA and 4-CI-lAA) in mature embryos and component tissues (root, shoot and cotyledon) of 4-d-old seedlings were determined by gas chromatography-mass spectrometry. Relative to their level in the mature embryos, there was an increase ofIAA and a decrease of 4-CI-lAA in the cotyledons 4 days after imbibition, suggesting a difference in the roles of these two auxins in the germination process. 4-d-old roots of both cultivars contained higher levels of both lAA and 4-CIlAA than the corresponding shoots. IAA and 4-CI-lAA levels of 4-d-old 'Alaska' shoot were elevated in comparison to that in 'Cameval' shoots. There was an elevated level of GA20 in the mature embryo of both genotypes which decreased markedly by 4 days after imbibition, suggesting the role ofGA2o as a substrate to be 3P-hydroxylated to GAl upon germination. Some GAl was detected in mature embryos of both genotypes; however, no endogenous GAl was detected in the three organs of 'Cameval'(le) 4 days after imbibition. The three tissues of 'Alaska' seedlings contained GAl, and its level was highest in the root, the most actively growing organ in 4 days after imbibition seedlings. This together with the higher level of the two auxins found in the root suppOli the theory that biologically active GA (GAl) and auxins are mainly localized in actively growing tissue. The inactive 2P-hydroxylated GAs (GA29 and GAs) were much higher in the mature embryo and the three tissues of 4-d-old seedlings of 'Alaska' than that of 'Cameval'. Supported in part by NSERC grant # 138166.
1 Plant Physiology and Molecular Biology Research Group, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5
107
IMPROVEMENT OF YIELD IN GREENHOUSE GROWN DETERMINATE MULTIFLOWERED PEAS WITH GIBBERELLIN TREATMENTS
Sonja L. Makil *, H. Mullen l, R. Pharis2 and S. Singerl
ABSTRACT
The detenninate (det) line of pea displays a more synchronous flower development than wildtype (WT) due to the early cessation of growth of the terminal meristem. However, yield may be reduced in determinate lines compared to WT indeterminate lines. We have previously analyzed the effect of different gibberellins (GAs) on the growth and development ofa determinate, multiflowered line of pea (det multi) in growth room growing conditions. In the current study, we evaluate the effects of GA4 and two ring-D modified GAs, 16,17-dicholoromethano dihydro GAs (Die) or the exo-enriched isomer of 16, 17-dihydro GAs (DiHGAs) on growth and flowering of the det multi line under greenhouse growing conditions. DiHGAs is known to be a competitive substrate inhibitor of 3~-hydroxylation of 3-deoxy GAs. The det multi plants were treated once with 5 or 25 !!g of G~ or each of the two ring D-modified GAs when plants had six expanded leaves. Gibberellin treatments resulted in an increase in seed yield primarily due to enhanced development of lower floral, axillary nodes.
INTRODUCTION
Gibberellins (GAs) play critical roles in plant reproductive development (Pharis and King, 1985 and King and Evans, 2003). Utilization of dwarf varieties of crops has been an important consideration in designing crop production systems. For example, the 'Super Dwarf rice plant is deficient in the 3~-hydroxylase that catalyzes the conversion ofGA2o (inactive) to GAl (active) (Mitsunaga, et aI., 1994, Itoh, et aI., 2001). The two predominant GA biosynthetic pathways for vegetative tissue of higher plants have been extensively studied and experiments with GA mutants have identified a feedback phenomenon where bioactive GAs can downregulate their own biosynthesis (Hedden and Phillips, 2000).
Gibberellins are also developmentally regulated and genetic dissection of GA signaling pathways is uncovering some intriguing links between plant hormones and developmental genes, especially in the reproductive phase of plant development. In rice, for example, expression of the catabolic GA20x gene is located in a ring around vegetative shoot apices and this expression decreases drastically after the phase transition from vegetative to reproductive growth (Tomoaki, et aI, 2001, see also discussion in King and Evans, 2003). Agamous-like 15 (AGL15) in Arabidopsis directly controls the GA catabolic gene, AtGA2ox6 (Wang, et aI., 2004). Interaction between the KNOX class of transcriptional regulators and GA biosynthesis genes has emerged as a link between developmental genes and hormones (see review by Hay, et aI., 2004).
lBiology Department, Carleton College, One North College Street, Northfield, MN 55057 USA, 2Biological Sciences Department, University of Calgary, Calgary, Alberta, T2N IN4, Canada
108
Recently, floral homeotic genes have been shown to be targets of DELLA protein nuclear repressors and GAs may regulate floral development by opposing DELLA repressors (Yu, et aI., 2004). Mutations with DELLA protein changes have been used by plant breeders to create highyielding semi-dwarf varieties of wheat and it has been shown recently that flexible control of plant architecture in Arabidopsis can be achieved through switchable expression of a DELLA gene (Ait-ali, et aI, 2003). Hence, a highly flexible control of inflorescence architecture may be feasible once we better understand the genetic regulation of inflorescence architecture in pea.
There is considerable interest in genes which affect plant architecture. Pea belongs to a small number of plant geme in which a number of genes involved in branching, flowering, and photoperiod responsiveness have been identified (Beveridge, et aI., 2003). We have previously reported on some of the interesting changes seen in shoot architecture after various pea genotypes were treated with specific GAs or ring D-modified GAs (Maki, et aI., 2004). Our work with determinate, semi-dwarf, multiflowered pea lines indicates that yield in these lines can be improved by treatments with ring D-modified GAs derivatives which also very likely function as competitive substrates for the enzyme that effects 3B-hydroxylation of3-deoxy GAs. For example, outgrowth of pre-inflorescence buds was increased in determinate lines following treatment with 25 /lg of GAt (Malci, et aI., 2004; Fig. 2a). While basal branching is common in pea it usually results in vegetative growth, not in increased yield. Outgrowth of upper axillary buds is far less common during development and treatments which result in enhanced floral axillary branching may be beneficial in some lines.
MATERIALS AND METHODS
Seeds of the det multi line (Fig. 1) were originally provided to S. Singer by the late Dr. Gerry Marx (Geneva Experiment Station, Ithaca, NY). The det multi line is in a gibberellin deficient background (Ie). Seeds were sown in soil-less potting mix (Prime-Gro7, Thenn-O-Rock East, New Eagle, PA) in 15 cm pots (1 per pot) and placed in a greenhouse. Long day conditions were maintained in the Spring 2005 study by HID lighting from 0300 until 0700 followed by incandescent lighting from 1700 until 2100. Long days were provided in the Summer 2005 study using natural daylength. The apical bud was treated with a single application of either GAt or two ring-D modified GAs, 16,17-dicholoromethano dihydro GAs (Die) or the exo-emiched isomer of 16, 17-dihydro GAs (DiHGAs) in 5 !J,L (25 !J,g application) or 1 !J,L (5 !J,g application) of 50% ethanol when plants had 6 expanded leaves (3 plants per treatment in the Spring 2005 study and 10 plants per treatment in the Summer 2005 study).
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exo - C -16,17 - dihydro GI't;
CL
C -16,17 - dichloromethano dihydro GI't;
Figure 1. Schematic of the det multi line in a dwarf background (Ie). First order inflorescence meristem terminating in a stub (S 1), second order inflorescence meristem terminating as a stub (S2), second order floral meristem (F2), third order floral meristem (F3). DETERlI1INATE (DETIPsTFLl a) is a TERlI1INAL FLOWERIICENTRORADIALIS homologue in pea (Foucher, et aI., 2003).
H
endo - C - 16,17 - dihydro GI't;
110
Figure 2. Structures of the gibberellins used in this study.
60,------------------------------------,
+" C CIl
_ control
50 1'0',,\,',01 diCGA5 tif,\jftl exo-DiHGA5
40
Q; C/l 30
"C Ql Ql
(J)
20
10
o -'---------------'
Gibberellin Treatment
Figure 3. Effect of single 25 f!g applications of 16,17 -dicholoromethano dihydro GAs (diCGA5) or exo-enriched isomer of 16,17-dihydro GAs (DiHGAs) on seed yield in the Spring 2005 study (average ± S.E.).
Figure 4. Spring 2005 Study. det multi line grown during January-April, 2005 under greenhouse conditions with optimal lighting. Left, control; center, exo-enriched-16,17-dihydro GAs (25f!g) treated; right, 16,17 -dichloromethano dihydro GAs (25 f!g) treated.
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Effect of 5 ug GA Applications on Plant Height
120,------------------------------------,
-e- control 100 '-i1!o-' DiCGA5-5ug
E 80 ~ .... .<= .~ 60 J: C ctl 0:: 40
20
" .J;. ... exo-DiHGA5-5ug -e- GM-5ug
o+----.-----.----.----.----~----,_--~
o 2 3 4 5 6 7
Week
Figure 5. Effect of single 5 ~g GA applications of 16,17-dichloromethano dihydro GA5 (DiCGAs), exo-enriched isomer of 16,17 -dihydro GAS (DiHGAs), or G~ on plant height in the Summer 2005 study (average ± S.B., n=lO).
Effect of 25 ug Applications on Plant Height
140
-e- control .. ~.~ ... 120 --3\-- DiCGA5-25ug
... /!; ....•. exo-DiHGA5-25ug °L. :"$:.
-e- GM-25ug /".l..
100 .i'·
E ~ .. '
1: 80 Ol 'iii J:
60 C ctl .'
0:: 40
20 ,~
0 0 2 3 4 5 6 7
Week
Figure 6. Effect of25 ~g GA applications of 16,17-dichloromethano dihydro GA5 (DiCGAs), exo-enriched isomer of 16, 17-dihydro GA5 (DiHGAs), or G~ on plant height in the Summer 2005 study (average ± S.B., n=10).
112
Table 1. Effect of Gibberellin Treatments on the Node of Flower Initiation, Flowers per node and Total Flowers and PodslPlant.
No. of Flowers Total Lateral
on Axillary Length! Axillary Branches Main Stem Pods! Seeds!
G~-25 ug 1B.B.±.0.2 2. 9.±.0. 2 2.5.±.0.3 1.5.±.0.2 3.6.±.1.0 1.4.±.0.5 0.45.±.0.09 7.6.±.0.B 32.0.±.2.7 NFl = node of floral initiation NFIIF = number of flowers at the node of floral initiation NFI+ IfF = number of flowers at the node of floral initiation + 1 NFI+2fF = number of flowers at the node of floral initiation +2
(ii) Metabolism of C-16, 17 -dihydro GAs to C-16, 17 dihydro GA3 and C-16, 17 -dihydro
GA6 may (speculatively) occur in dicots such as pea, but not in monocots (dihydro
GA3 is known to be growth-active, see Mander et al1995 and Evans et al1994). However, such conversions by pea 3-oxidase preparations (King et aI., 2004) or by the Arabidopsis GA30x fusion protein (Zhou et a12004) have not been seen. That said, the GA30x fusion protein was able to convert C-16, 17 -dihydro GA20 to C-
16,17 -dihydro GAl (Zhou et al 2004), so it is not impossible that the intact plant
system, perhaps with essential co-factors present, can convert dihydro GAs to dihydro
GA3 or dihydro GA6 (the GA30x fusion protein could convert GAs to GA6 (Zhou et
aI., 2004) and intact pea shoots can convert GAs to GA3 (Durley et al1972).
(iii) Inhibition of catabolic GA biosynthetic steps, for example 2-oxidation of GA20 to
GA29, GA9 to GAs!' or even GAl to GAs and G~ to GA34 by ring D-modified GAs derivatives could yield a net effect of more "growth-effector" GAl or G~ being present, even though 3 ~-hydroxylation was coincidentally also being inhibited. However, King et aI., 2004, found no evidence of competitive inhibition of pea GA20x-l or GA20x-2 by a wide range of ring D-modified GAs derivatives, even
though GAs per se showed good inhibitory activity on GA20x-l. Hence, this "avenue" for yielding growth promotion in dicots by application of ring D-modified GAs derivatives is ruled out.
(iv) inhibition of catabolic oxidation at C-16, i.e. prevention of the formation of C-16, 17-dihydrodihydroxy GAs (C-16 diols) by ring D-modified GAs derivatives is, as noted
for (iii) above, another possible way to obtain more "effector" GAl or G~. For
example, there was coincidental inhibition of C-16-diol fonnation for GA20 when C-
16,17-dihydro GAs application was used to inhibit 3~-hydroxylation ofGA2o to GAl in rice (Takagi, Pearce, Pharis, unpublished results based on work by Takagi et aI., 1994). Such coincidental inhibition ofGA2o catabolism (to GA2o-16-diol) by dihydro
GAs could yield a net increase in GAl production in these pea plants by
"overloading" the 3~-hydroxylase with substrate GA2o.
Since C-16-diols (and their -17 -O-glucosides) of a range of GA structures are common in higher plants (Santes et aI., 1995 and references cited therein, Hasegawa et aI., 1995), the possibility that C-16,17-dihydro GAs and other ring D-modified GA5 derivatives act as competitive inhibitors of C-16 oxidation of GAs during catabolic GA metabolism is well worth exploring.
To summarize, then, while the mechanism for growth inhibition by ring D-modified GAs derivatives is readily explained, i.e. competitive inhibition ofGA 3~-hydroxylation, their ability to promote growth, especially in dicots, remains "speculative", although potential mechanisms (ii) and (iv) above need to be researched more extensively.
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Promotion of Axillary Growth and Subsequent Flowering of those Axillaries in Pea
The mechanism of the release and subsequent growth of the upper axillary buds in the det multi line of pea could be due in part to the reduction of apical dominance in this line as the apical meristem ceases growth abruptly. Axillary buds are released in the det multi plant without gibberellin treatment, however their growth is minimal. The ratio of total lateral length to main shoot length was increased in both of the ring-D modified GA treatments, relative to GA4-treated plants or to control plants (Fig. 7). This was caused primarily by an increase in the number of upper axillary buds which elongated (Table I).
All treatments, including G~, slightly reduced the number of flowers at the node of flower initiation (NFl) and at each of the two nodes above the NFL However, the number of flowers produced on axillary branches below the NFl was increased in all treatments. Plants treated with DiCGAs and exo-DiHGAs produced more flowers on axillary branches than GA4 treatments. In fact, the greatest number of axillary branch flowers were produced on plants treated with 25 ug of exo-DiHGAs. The exo-DiHGAs treatment also resulted in the greatest number of axillary branches greater than 10 cm.
The increase in the number of flowers produced from ring-D modified GA treatment raises the possibility that the ring D-modified GAs derivatives are acting as per se florigens. Evans et al (1990) concluded that "if there is a GA receptor involved in the floral induction of Lalium, it has different structural specificities from the GA receptor involved in stem elongation". In the latter case, the retardive effect of the ring D-modified GAs on the growth of the Lalium stem (underlying the potential floral apex) can be presumed to be a result of "competitive substrate inhibition" of GA30x activity.
In the current study, we determined the number of flowers which developed fully. Examination of axillary branches on a microscopic level would be necessary to detennine whether the ring-D modified GAs were increasing the number of flowers initiated. Never-the-Iess, the increased production of fully developed flowers, and subsequent pods, in ring-D modified GA-treated plants suggests that these treatments may be beneficial for increased pod and seed yield in determinate lines of pea. Future studies will focus on determining whether expression of three pea floral genes (UNl, DET, and PlU) in pea are altered following ring-D modified gibberellin treatment. We thank Prof. L.N. Mander, Res. School of Chemistry, Australian National University, for the gift of Ring-D modified GAs. Research supported by NSF 0422840.
LITERATURE CITED
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Arumingtyas, E.L., Floyd, R.S., Gregory, M.J. and Murfet, I.e. 1992. Pisum Genetics 24:17-31.
Brian, P.W., Grove, J.F., Mulholland, T.P.C. 1967. Relationships between structure and growthpromoting activity of the gibberellins and some allied compounds, in four test systems. Phytochemistry 6:1475-1499.
Crozier, A., Kuo, C.C., Durley, RC., Pharis, RP. 1970. The biological activities of 26 gibberellins in nine plant bioassays. Canadian Journal of Botany 48:867-877.
Durley, RC., Railton, I.C., Pharis, RP. 1972. Interconversion of gibberellin A5 to gibberellin
A3 in seedlings of dwarf pea, cv Meteor. Phytochemistry 12:1609-1612.
Evans, L.T., King, RW., Mander, L.N., Pearce, D.W., Pharis, RP. 1996. Method of treating plants or plant tissues with C-16,17-dihydro gibberellins. PCT/AU92/00426 and U.S. Patent No. 5,532,206.
Evans, L. T., Chu, A., King, R.W., Mander, L.N., Pharis, RP. 1990. Gibberellin structure and florigenic activity in Lolium temulentum, a long-day plant. Planta 182:97-106.
Evans, L.T., King, RW., Mander, L.N., Pharis, R.P. 1994. The differential effects ofC-16,17 dihydro GAs and related compounds on stem elongation and flowering in Lolium temulentum L. Planta 193: 107 -114.
Foster, K.R., In-jung Lee, Pharis, RP., Morgan, P.W .. 1997. Effects of Ring D-modified gibberellins on development and endogenous gibberellins in selected Sorghum bicolor maturity genotypes. Jouranl of Plant Growth Regulation 16:79-87.
Foucher, F., Morin, J., Courtiade, J" Cadioux, S., Ellid, N., Banfield, M., Rameau, C. 2003. DETERMINATE and LATE FLOWERING are two TERA1INAL FLOWERlICENTRORADIALIS homologs that control two distinct phases of flowering initiation and development in pea. Plant Cell 15:2742-2754.
Hasegawa, M., Nakajima, M., Takeda, K., Yamaguchi, I., Murofushi, N. 1995. Endogenous levels of gibberellins in normal and male sterile anthers of rice (Oryza sativa cv. Nihonmasari). BioSci. Biotech. Biochem. 59:1716-1720.
Hay, A., Craft, J., Tsiantis, M. 2004. Plant hormones and homeoboxes: bridging the gap? BioEssays 26:395-404.
Hedden, P. and Phillips, A.L. 2000. Gibberellin metabolism: new insights revealed by the genes. Trends in Plant Science 5:523-530.
Huang, J., Pray, C., Rozelle, S. 2002. Enhancing the crops to feed the poor. Nature. 418:678-684.
Itoh, H., Ueguchi-Tanaka, M., Sentoku, N., Kitano, H., Matsuoka, M., Kobayashi, M. 2001. Cloning and functional analysis of two gibberellin 3 beta-hydroxylase genes that are differently
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expressed during the growth of rice. Proceedings of the Nationall Acadamy of Science 98(15):8909-14.
King, RW. and Evans, L.T. 2003. Gibberellins and flowering of grasses and cereals: prizing open the lid of the "florigen" black box. Annual Reviews of Plant Biology 54: 12.1-12.22.
King, RW., Evans, L.T., Mander, L.N., Moritz, T., Pharis, RP., Twitchin, B. 2003. Synthesis of gibberellin GA6 and its role in flowering of Lalium temulentum. Phytochemistry 62:77-82.
King, RW., Seto, H., Sachs, RM. 2000. Response to Gibberellin Structural Variants Shows that Ability to Inhibit Flowering Correlates with Effectiveness for Promoting Stem Elongation of Some Plant Species. Journal of Plant Growth Regulation 19:437-444.
Maki, S.L., Mullen, H., Pharis, R and Singer, S. 2004. Effects of a range of gibberellin structures on growth of Pisum sativum genotypes differing in shoot architecture. Proceedings of the 31 st Annual Meeting of the Plant Growth Regulation Society of America. Charleston, SC.
Mander, L.N., Camp, D., Evans, L.T., King, R.W., Pharis, RP., Sherburn, M., Twitchin, B. 1995. Designer gibberellins: The quest for specific activity. Acta Horticulturae 394:45-55.
Martin D.N., Proebsting W.M., Hedden P. 1997. Mendel's dwarfing gene: cDNAs fi'om the Le alleles and function of the expressed proteins. Proceedings of the National Academy of Science, 94: 8907-8911.
Mitsunaga, S., T. Tashiro, and J. Yamaguchi. 1994. Identification and characterization of gibberellin-insensitive mutants selected from among dwarf mutants of rice. Theoretical Applied Genetics 87:705-712
Pharis, RP., King, RW. 1985. Gibberellins and Reproductive Development in Seed Plants. Annual Reviews of Plant Physiology, 36: 517-568.
Poole, AT, Ross, JJ, Lawrence, NL, Reid, JB. 1995. Identification of gibberellin ~ inPisum sativwn L. and the effects of applied gibberellins A9,~, A5 and A3 on the Ie mutant. Journal of Plant Growth Regulation, 16257-262.
Santes, C.M., Hedden, P., Gaskin, P., Garcia-Martinez, J.L. 1995. Gibberellins and related compounds in young fruits of pea and their relationship to fruit-set. Phytochemistry 40(5): 1347-1355.
Singer, S.R., Sollinger, J., Maki, S., Fishbach, l, Short, B., Reinke, C., Fick, J., Cox, L., McCall, A., Mullen, H. 1999a. Inflorescence architecture: a developmental genetics approach. The Botanical Review, Vol. 65, No.4: 385-410.
Takagi, M., Pearce, D.W., Janzen, L.M., Pharis, RP. 1994. Effect of exo- 16,17 dihydro gibberellin A5 on gibberellin ~o metabolism in seedlings of dwarf rice (Olyza sativa L. cv. Tan-
ginbozu). Journal of Plant Growth Regulation 15:207-213.
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Tomoaki, S., Kobayashi, M., Itoh, H., Tagiri, A., Kayano, T., Tanaka, H., Iwahori, S. and Matsuoka, M. 2001. Expression of a Gibberellin 2-0xidase Gene around the Shoot Apex Is Related to Phase Transition in Rice. Plant Physiology 125: 1508-1516.
Wang, H., Caruso, L.V., Downie, A. B., and Perry, S.E. 2004. The embryo MADS domain protein AGAMOUS-Like 15 directly regulates expression of a gene encoding an enzyme involved in gibberellin metabolism. Plant Cell 16: 1206-1219.
Yu, H., Ito, T., Zhao, Y., Peng, J., Kumar, P., Meyerowitz, E.M. 2004. Floral homeotic genes are targets of gibberellin signaling in flower development. PNAS 101(20):7827-7832.
Zhou, R., Yu, M., Pharis, P. 2004. 16,17-dihydro GAs competitively inhibits a recombinant Arabidopsis GA 3~-hydroxylase encoded by the GA4 gene. Plant Physiology 135:1000-1007.
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ETHYLENE SENSITIVITY OF CUT RACEMES OF ADVANCED BREEDING LINES OF PINK FLOWERED BLUEBONNET
W.A. Mackay, * N. Sankhla and T.D. Davis Texas A&M University, TAES-Dallas, 17360 Coit Rd., Dallas, TX 75252-6599.
ABSTRACT
Cut racemes of Big Bend bluebonnet (Lupinus havardii Wats.) hold considerable promise as a new specialty cut flower crop. Over the years, as a result of our breeding and selection efforts, we have developed several lines of improved germplasm with blue, white and pink flower colors. We now have genotypes which show considerably reduced or no flower shattering. This study was conducted to evaluate the relative ethylene sensitivity of four newly developed lines (Pink Bulk, PB; Pink Light, PL; Pink Dark, PD; Pink Coral, PC) which produce different shades of pink flowers. Freshly harvested racemes were put into vases containing either water or desired concentration of 2-chloroethylphosphonic acid (CEPA) and the abscission of flowers was recorded regularly. The results indicate that the breeding lines differ widely with respect to their ethylene sensitivity. Based on the intensity of flower abscission in the presence of CEP A, the breeding line PC was found to be the most sensitive to the presence of ethylene in the vase solution, whereas the line PL appeared to be the least sensitive. A pretreatment of racemes with silverthiosulphate (STS), a known ethylene action inhibitor, prevented flower abscission even in the presence of CEP A. Earlier we reported that the sensitivity of cut racemes of Lupinus spp. vary widely among species. The results of this study point out that even the selections within a species may have varied sensitivity to ethylene. Recurrent selection and breeding has been quite successful in obtaining low shattering genotypes with improved vase life and longevity in Big Bend bluebonnet.
INTRODUCTION
In Texas, native lupine species (bluebonnets) have official state flower status. Big Bend Bluebonnet (Lupinus havardii Wats.) is native to a narrow geographical range along the Rio Grande River in southwestern Texas, and produces tall blue, fragrant racemes (2,5). Flowers are generally violet-blue, but rarely plants with white or pink flowers are also found. A research project to evaluate the cut flower potential of L. havardii was started in 1991. The initial focus was to improve crop uniformity and enrich our seed diversity for breeding and selection of superior genotypes. Our breeding efforts are aimed at 1) developing genotypes with novel and uniform flower colors, 2) improved vaselife, 3) ethylene insensitivity or reduced ethylene production and 4) improved response to shipping. Other traits that were used for selection included low shattering and long display of flowers on the intact raceme to improve vase life (5).
The key determinants of longevity and performance of cut racemes in bluebonnet are flower abscission and senescence (4,7). Over the years, as a result of our breeding and selection efforts, we have developed several cultivars and breeding lines of improved germplasm with blue, white and pink flowers, and low ethylene sensitivity (5). This study
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was conducted to evaluate the relative ethylene sensitivity of four newly developed pink flowered genotypes of Big Bend bluebonnet.
MATERIALS AND METHODS
The genetic history of the 'pink flowered' line is somewhat complicated (5). In contrast to the blue and white flowered lines, we only had one plant to begin our breeding efforts. There were four seed collected and from this we developed the pink flowered lines. Although, progress was made on postharvest longevity, color saturation, and general plant vigor, all the breeding lines originating from this single plant flowered 4-6 weeks after the blue and white flowered breeding lines. In addition, there appeared to be an increased susceptibility to disease and insect pressure in the pink flowered lines that did not occur in the blue or white flowered lines.
An exceptional year for Big Bend Bluebonnets occurred in the winter of 2000-2001. A collection trip was undertaken to determine if there were pink flowered plants in the wild population that could be used to bring new genetics into the current breeding program. Plants were found widely scattered across the Big Bend region and were pollinated and tagged for later seed collection. Seed from these rare five pink flowered plants was collected in April of200l. In the fall of2001 these were planted and grown in the greenhouse for evaluation. In early 2002, three plants were selected that exhibited early flowering, good flower color, and general vigor and were crossed with selected plants from an advanced pink flowered breeding line (Dark Pink 2001). Seed was collected from the crosses and planted in the fall of 2002 and designated Pink Select 2002. Plants were re-selected in early 2003 based on color, vigor, and ability ofthe flowers to be retained on the racemes. There were three color lines selected and pollinated separately (Dark Pink 2003, Pink Select 2003 and Coral 2003). In the fall of 2003 breeding continued on these separate lines in the same manner. From "Pink Select 2003", we obtained another distinct "Pink Light" genotype. For comparison, the original "Pink Bulk" line was also used in this study.
Plants were grown in non-shaded greenhouses at Texas A&M University Agriculture Research Center, Dallas. Cut racemes were placed in glass vases containing either 400 ml water or the test solution at 22± 2°C under cool white florescent lamps (30 /-lmol·m-z·sec-1
). For evaluating the response of the cut racemes to ethylene 2-chloroethylphosphonic acid ( CEP A: 100 /-lM, 200 /-lM ) was added to the vase solution. Pretreatment with STS (5 mg/l) or 1-MCP (generated from 60 mg Ethylbloc™) was accomplished as described earlier (7). Observations on parameters related to postharvest display were recorded regularly.
RESULTS AND DISCUSSION
Postharvest perfonnance of lupine racemes depends on abscission and senescence of flowers as well as on the extent of ethylene sensitivity, which varies widely both within and among species (4,7). Among the species tested, inflorescences of L. succulentus exhibited the highest sensitivity to CEP A in the vase solution, while L. densijlorus and L. luteus were least responsive. In L. havardii, among the white, blue and
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pink flowered lines (Fig. 1), the "white flower" line was found to be relatively much more tolerant than the "blue" and "pink" flower lines (7). The results of the current study indicate that the newly developed "pink flower" genotypes of Big Bend bluebonnet also differ in their response to ethylene supplied as CEP A via the vase solution (Fig. 2). As compared to the original "Pink Bulk" line, the newly developed "Pink Light" line exhibited very little flower abscission and much reduced ethylene sensitivity. "Pink Dark" as well as "Pink Coral" lines also showed less flower abscission than the "Pink Bulk" line, although the flower abscission in the former two lines was much still much higher than that recorded for "Pink Light". Thus, the order of decreasing ethylene sensitivity in pink flowered genotypes was: Pink Bulk> Pink Coral> Pink Dark> Pink Light. Among the white and blue flower lines, our current improved selection "White Select" and "Blue Select" have also been shown to exhibit very little or no flower abscission and considerably reduced sensitivity (5). It has been reported that in carnation reduced ethylene sensitivity is heritable (10). Our results with bluebonnet are in conformity with those reported for carnation.
Earlier, we reported that in a pretreatment of cut racemes of bluebonnet genotypes with either STS or 1-MCP almost completely inhibited the ethylene-induced flower abscission (7). An antagonism between CEP A and STS or 1-MCP in preventing the inhibition of ethylene-induced flower abscission was also observed in all the pink flowered genotypes tested in this investigation (data not presented). Recently, it has been reported that endogenous ethylene evokes the co-expression and accumulation of an ethylene receptor gene, ERS1, and an ethylene signaling regulator gene, CTR1, thereby speeding up flower abscission (3). STS antagonized ethylene-induced floret abscission in Delphinium by controlling the expression and decline of these transcripts, and thus possibly shutting off the ethylene signal transduction.
In recent years, there has been a significant outburst of new knowledge in the processes regulating abscission (6,9). The identification and characterization of delayed abscission mutants, ethylene response mutants, MADS-box genes (e.g. shatterproof 1 and 2, Jointless, AGL-15), as well as novel genes (e.g. inflorescence deficit abscission, ida genes, and delayed abscission, dab 1-5 genes) is likely to lead to improved control of abscission and dehiscence in many plants. Additionally, several unique features of ethylene signalling and response pathways, having a multitude of inputs and outputs, have been discovered and evaluated (1,8). New infonnation has shed light on the components of the pathway, on the cross-talk between ethylene signaling and other hormone signaling pathways, and on the roles of transcriptional and post-transcriptional regulation of ethylene signaling (1,8). The global analysis of ethylene-mediated changes in gene expression has uncovered hundreds of ethylene-regulated genes, providing the basis for dissecting the myriad of ethylene-mediated responses in plants (8). Genetic engineering technology could also be a very powerful tool in breeding flower crops for enhanced postharvest performance. However, in plants such as carnation and bluebonnet which have a relatively short generation time, and where several crops can be evaluated in one season, improvement by conventional breeding can also be of much practical value. Our results with bluebonnet using recurrent phenotypic selection for traits such as low flower shattering, long display life and different flower colors (Fig. 1) have clearly
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demonstrated the reliability and effectiveness of selection in the development of bluebonnet cultivars with reduced ethylene sensitivity and extended vase life (5).
REFERENCES
1. Chen, Y. F, N. Etheridge and Schaller, G. E. 2005. Ethylene signal transduction. Annals of Botany 95: 901-915.
2. Davis, T.D., Mackay, W.A. and Sankhla, N. 2000. Distribution, biology, and potential horticultural uses of Big Bend bluebonnet (Lupinus havardii Wats.) - a showy winter arumal from the Chihuahuan Desert. Desert Plants 16:3-9.
3. Kuroda, S., Hirose, Y., Shiraishi, M., Davies, E. and Abe, S. 2004. Co-expression of an ethylene receptor gene, ERS 1, and ethylene signaling regulator gene, CTRl, in Delphinium during abscission of florets. Plant Physio!. Biochem. 42: 745-751.
4. Mackay, W.A., Davis, T.D., and Sankhla, N. 2001. Effect of ethephon and silver thiosulphate on postharvest characteristics of inflorescences of several lupine species. Acta Hort. 543:69-73.
5. Mackay, W. A., Sankhla, N., and Davis, T.D. 2005. Improvement of display life of Big Bend blueboilllet racemes by recurrent phenotypic selection. Acta Hort. 669: 207-211.
6. Patterson, S. E. and Bleecker, A. B. 2004. Ethylene-dependent and -independent processes associated with floral organ abscission in Arabidopsis. Plant Physio!. 134: 194-203.
7. Sankhla, N., Mackay, W.A. and Davis, T.D. 2001. Extension of vase life and prevention of ethylene-induced flower shattering in Lupinus havardii by 1-methylcyc1opropene. Acta Hort. 543:75-78.
8. Stepanova, A. N. and Alonso, J. M. 2005. Ethylene signaling and response pathway: a unique signaling cascade with multitude of inputs and outputs. Physio!. Plant. 123: 195-206.
9. Taylor, J.E. and Whitelaw, C.A. 2001. Signals in abscission. New Phto!. 151:323-339.
10. Woltering, E.J. Somhorst, D. and de Beer, C.A. 1993. Roles of ethylene production and sensitivity in senescence of carnation flower (Dianthus ca7yophyl/us) cultivars white sim, chinera and epomeo. J. Plant Physio!. 141:329-335.
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Fig. 1. Pink, blue, white, and coral pink flowered breeding lines of Big Bend bluebonnet. Fig. 2. Effect of CEP A on flower abscission in pink flowered lines after 96 hours.
EFFECT OF NITRIC OXIDE GENERATING COMPOUNDS ON FLOWER SENESCENCE IN CUT RACEMES OF PINK FLOWERED LUPINUS HAV ARDII WATS.
N. Sankhla, * W.A. Mackay and T.D. Davis Texas A&M University, TAES-Dallas, 17360 Coit Rd., Dallas, TX 75252-6599.
ABSTRACT
Nitric oxide (NO*) is viewed as a diffusible multifunctional plant signal molecule. It has been shown to extend the postharvest life of a range of flowers possibly by downregulating ethylene production. In this study, we have evaluated the effect of two nitric oxide (NO*) generating compounds (sodium nitroprusside, SNP ; N-tert-butyl-aphenylnitrone, PBN), alone and in combination with sucrose, on postharvest flower senescence of cut racemes of four advanced breeding lines(pink: Bulk, PB; Pink: Light, PL; Pink: Dark, PD; Pink: Coral, PC) of pink: flowered Big Bend bluebonnet (Lupinus havardii Wats.). The promotion/retardation of flower senescence depended on the concentration used and the genotype. Incorporation of sucrose in the vase solution considerably reduced the senescence of flowers, promoted growth of inflorescence axis and opening of additional flowers in PB, PL and PD. However, in PC sucrose (>30 mM) induced the wilting of the tip of the banner spot petal which eventually hastened flower senescence and flower fall. NO* donors and sucrose, when used in combination, generally exhibited a lesser degree of flower senescence as compared to those growing in solutions containing NO* donors alone. These results indicate that the beneficial or detrimental effects ofNO* may depend on concentration, sensitivity of genotypes and presence or absence of sucrose in the vase solution.
INTRODUCTION
Recently, there has been an impressive upsurge in elucidating the physiological and biochemical functions of nitric oxide (NO*) in plants (2,4,6,8, 13). This enigmatic, but unique diffusible multifunctional plant signal molecule, plays pivotal role in diverse plant processes including hormone modulation, programmed cell death, and wounding and defense responses. The cytotoxic or the cytoprotective roles ofNO* are thought to be due to its reactivity with ROS (4, 6, 8, 13). A major breakthrough in understanding the role ofNO* in plants relates to identification of multiple, enzymatic as well as nonenzymatic, NO* generating systems, and widespread production, either constitutive or induced by biotic/abiotic stresses, ofNO* in plants (2, 4).
Several studies point out that there is a cross talk between NO*, ethylene, IAA, abscisic acid, GA, calcium, calmodulin, cGMP and cADPR (6, 13). NO* has also been shown to inhibit ethylene action and synthesis in plants (7), and it has been suggested that NO* acts as a natural senescence-delaying plant growth regulator primarily by downregulating ethylene production. NO* donors have also been shown to protect a variety of cut flowers from ethylene and dramatically increase the vaselife (1).
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Fig. 1. Pink:, blue, white, and coral pink: flowered breeding lines of Big Bend bluebonnet.
c: o
'en en '1:5 en
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Fig. 2. Effect of CEP A on flower abscission in pink flowered lines after 96 hours.
60 ................... . 00lm~'1i Control _ CEPA100 ~ CEPA200
~ 40 ................... . o
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125
EFFECT OF NITRIC OXIDE GENERATING COMPOUNDS ON FLOWER SENESCENCE IN CUT RACEMES OF PINK FLOWERED LUPINUS HAVARDII WATS.
N. Sankhla,* W.A. Mackay and T.D. Davis Texas A&M University, TAES-Dallas, 17360 Coit Rd., Dallas, TX 75252-6599.
ABSTRACT
Nitric oxide (NO*) is viewed as a diffusible multifunctional plant signal molecule. It has been shown to extend the postharvest life of a range of flowers possibly by downregulating ethylene production. In this study, we have evaluated the effect of two nitric oxide (NO*) generating compounds (sodium nitroprusside, SNP ; N-tert-butyl-aphenylnitrone, PBN), alone and in combination with sucrose, on postharvest flower senescence of cut racemes of four advanced breeding lines(pink Bulk, PB; Pink Light, PL; Pink Dark, PD; Pink Coral, PC) of pink flowered Big Bend bluebonnet (Lupinus havardii Wats.). The promotion/retardation of flower senescence depended on the concentration used and the genotype. Incorporation of sucrose in the vase solution considerably reduced the senescence of flowers, promoted growth of inflorescence axis and opening of additional flowers in PB, PL and PD. However, in PC sucrose (>30 mM) induced the wilting of the tip of the balIDer spot petal which eventually hastened flower senescence and flower fall. NO* donors and sucrose, when used in combination, generally exhibited a lesser degree of flower senescence as compared to those growing in solutions containing NO* donors alone. These results indicate that the beneficial or detrimental effects ofNO* may depend on concentration, sensitivity of genotypes and presence or absence of sucrose in the vase solution.
INTRODUCTION
Recently, there has been an impressive upsurge in elucidating the physiological and biochemical functions of nitric oxide (NO*) in plants (2, 4, 6, 8, 13). This enigmatic, but unique diffusible multifunctional plant signal molecule, plays pivotal role in diverse plant processes including honnone modulation, programmed cell death, and wounding and defense responses. The cytotoxic or the cytoprotective roles ofNO* are thought to be due to its reactivity with ROS (4, 6, 8, 13). A major breakthrough in understanding the role ofNO* in plants relates to identification of multiple, enzymatic as well as nonenzymatic, NO* generating systems, and widespread production, either constitutive or induced by biotic/abiotic stresses, ofNO* in plants (2, 4).
Several studies point out that there is a cross talk between NO*, ethylene, IAA, abscisic acid, GA, calcium, calmodulin, cGMP and cADPR (6, 13). NO* has also been shown to inhibit ethylene action and synthesis in plants (7), and it has been suggested that NO* acts as a natural senescence-delaying plant growth regulator primarily by downregulating ethylene production. NO* donors have also been shown to protect a variety of cut flowers from ethylene and dramatically increase the vaselife (1).
126
Over the years, as a result of our breeding and selection efforts, we have developed several lines of improved germplasm of L. havardii with blue, white and pink flower colors. We now have genotypes which show considerably reduced or no flower shattering. This study was conducted to evaluate the effect ofNO* donors on senescence of cut racemes of four newly developed lines of Big Bend bluebonnet (Pink Bulle, PB; Pink Light, PL; Pink Dark, PD; Pink Coral, PC) which produce different shades of pink flowers.
MATERIALS AND METHODS
Cut racemes of four advanced breeding lines of pink flowered (Pink Bulk, PB; Pink Light, PL; Pink Dark, PD; Pink Coral, PC) L. havardii Wats. were obtained from plants grown in a non-shaded greenhouse of trial garden at Texas A&M University, Agricultural Experiment Station, Dallas. Inflorescences were harvested in the morning and brought to the postharvest laboratory for experimentation. Sodium nitroprusside (SNP) and N-tert-a-phenylnitrone (PBN) were used as the source ofNO* donors. Cut inflorescences, with their freshly trimmed bases in water, were placed in glass vases containing freshly prepared solutions of the desired concentrations of the NO* donors (20 /-lM, 100 /-lM). Based on the results of our earlier studies, in some experiments sucrose (30 mM) was also added to the vase solution. In the vases containing sucrose, in order to reduce microbial contamination, 8-hydroxyquinoline sulphate (8-HQS) was also added regularly. The vases containing cut inflorescences in various test solutions were placed on benches in the laboratory at 22-25° C under cool white fluorescent lamps (30 /-lmol'm-2sec-1
) for 12 hours per day. The number of senescent flowers was scored daily up to 5 days, and the vase life characteristics evaluated regularly.
RESULTS AND DISCUSSION
The promotion/retardation of flower senescence by NO* donors depended on the concentration used and the genotype. The various pink flowered lines tested exhibited differential response to NO* donors (Fig.l, 2) which ranged from almost no effect or a slight inhibition to a distinct promotion of flower senescence. In genotype PD and PC a clear promotion was noticed in the senescence of flowers during postharvest vase life in SNP solution (Fig. I). In genotypes PB and PL, depending on the concentration, the racemes either did not show any effect on flower senescence or indicated a slight inhibition. More or less, a similar response was observed in the presence ofPBN (Fig. 2), although the intensity ofthe effect was much milder than that observed with SNP. Visible signs of flower senescence included onset of wilting and burning at the tip of the standard petal and a change in the color of banner spot. At high concentration of SNP the banner spot in PB flowers turned black, desiccated and ultimately senesced. In DP and PC dark brown patches were initiated on the balmer spot during postharvest vase life. The genotype PC was found to be the most tolerant to the presence of high concentration of NO* donors. In this genotype, even at the highest concentration ofNO* donors only a few flowers exhibited small brown dots on the bamler spot. Earlier we observed that high concentration of SNP also brought about a change in color of banner spot from light yellow to muddy-brown/intense black in the genotype "Blue Select" (10). NO*-mediated toxicity is mainly due to its reaction with superoxide anion (02), leading to the formation
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of strong oxidant peroxinitrite, which can oxidize thiol residues to sulfenic and sulfonic acids (5). However, in soybean the HR cell death appears to be activated following interaction ofNO* with H202, rather than 02- (3). Furthermore, the release ofNO* into solution depends on the characteristics and concentrations of the NO* donor, the pH, temperature and concentrations ofNO* target molecules (6). Thus, it becomes difficult to discriminate between the pharmacological effects and physiological relevance of the role ofNO* donors and modifications induced by endogenous NO*.
Incorporation of sucrose (30 mM) in the vase solution considerably reduced the senescence of flowers (Fig. 1, 2), promoted growth of inflorescence axis and opening of additional flowers in PB, PL and PD. However, in PC sucrose (>30 mM) induced the wilting of the tip of the banner spot petal which eventually hastened flower senescence and flower fall. NO* donors and sucrose, when used in combination, generally exhibited a lesser degree of flower senescence as compared to those growing in solutions containing NO* donors alone (Fig. 1,2).
NO * has been shown to extend the postharvest life of a range of flowers, fruits and vegetables possibly by down-regulating ethylene production (1, 7). In phlox, although SNP in the vase solution promoted the abscission of open flowers, the younger buds continued to open even in the presence of high SNP concentrations (11). On the other hand, at high SNP concentrations (> 50 11M), the leaves became either yellow, or more frequently turned progressively black and senesced (11). Inclusion of sucrose in the vase solution, or pretreatment of flower heads with either I-MCP or STS, significantly delayed the abscission of flowers and blackening of leaves, and improved postharvest display life. Similarly, low concentrations « 50 11M) of SNP and PBN delayed, but high concentrations (> 50 11M) promoted senescence of flowers in cut inflorescences of L. densiflorus (12).
Thus, it would appear that the beneficial or detrimental effects ofNO* donors may depend on concentration, sensitivity of genotypes and presence or absence of sucrose and/or ethylene inhibitors in the vase solution. Also, it should be borne in mind that the multiple modes of action ofNO* are suggestive of its wider modality than just on ethylene action (1). In fact, a recent analysis ofNO* responsive genes based on whole genome micro array in Arabidopsis indicates that a wide variety of genes, including those encoding transcription factors, ABC transporters, kinases and biosynthetic genes of ethylene and jasmonic acid, are up-regulated in response to NO* treatment(9), and provide an insight into the molecular basis for the diverse functions of NO* in plants. Recent results have identified a new mechanism to modulate NO* bioactivity via non-symbiotic hemoglobin, a gene involved in arginine-dependent NO* synthesis and a novel function for NO*signaling in flowering (2). It is clear that further studies are required to dissect the exact mode of action of this multifunctional plant signal molecule.
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REFERENCES
1. Badiyan, D., R B. H. Wills and Bowyer, M. C. 2004. Use of nitric oxide donor compound to extend the vase life of cut flowers. HortScience 39:1371-1372.
2. Crawford, N. M. and Guo, F. Q. 2005. New insights into nitric oxide metabolism and regulatory functions. Trends Plant Science 10: 195-200.
3. Delledone, M., 1., Murgia, D., Ederle, P.F., Sbicego, A., Biondani, A., Polverari, A., and Lamb, C. 2002. Reactive oxygen intennediates modulate nitric oxide signaling in the plant hypersensitive disease resistance response. Plant PhysioI. Biochem. 40:605-610.
4. del Rio, L. A., Corpas, F. J., and Barroso, J. B. 2004. Nitric oxide and nitric oxide synthase activity in plants. Phytochemistry 65 :783-792.
5. Huie, RE. and Padmaja, S. 1993. The reaction of NO with O2-. Free Radical Res. Comm. 18:195-199.
6. Lamattina, L., Garcia-Mata, C., Graziano, M. and Pagnussat, G. 2003. Nitric oxide: the versatility of an extensive signal molecule. Ann. Rev. Plant BioI. 54:109-136.
7. Leshem, Y. Y. and Wills, R B. H. 1998. Harnessing senescence delaying gases nitric oxide and nitrous oxide: a novel approach to post harvest control of fresh horticultural produce. BioI. Plant, 41: 1-10.
8. Neill, S. J., Desikan R and Hancock, J. T. 2003. Nitric oxide signalling in plants. New Phytologist. 159: 11-35.
9. Parani, M., S. Rudrabhatla, R Myers, H. Weirich, B. Smith, D. W. Leaman and Goldman, S. L. 2004. Microarray analysis of nitric oxide responsive transcripts in Arabidopsis. Plant Biotechnology Journal 2: 359-366.
10. Sankhla, N., Mackay, W. A. and Davis, T. D. 2003. Nitric oxide enhances flower abscission and senescence in cut racemes of Lupinus havardii Wats. Proc.Plant Growth ReguI. Soc. Amer. 30: 133-134.
11. Sankhla, N., Mackay, W. A. and Davis, T. D. 2003a. Effect of nitric oxide on postharvest perfonnance of perennial phlox cut inflorescences. Acta Hort. 628:843-847.
12. Sankhla, N., Mackay, W. A. and Davis, T. D. 2004a. Nitric oxide donors delay methyljasmonate-induced senescence of flowers in cut inflorescences of Lupin us densijlorus Benth. Proc. APEC Sym. Qual. Managemt. Postharvest Systems, Bangkok (in press).
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13. Wendehanne, D., Dumer, J. and Klessig, D. F. 2004. Nitric oxide: a new player in plant signaling and defense responses. Curro Opin. Plant BioI. 7: 449-455.
130
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132
COMPATIBLILITY OF SALAD CROPS GROWN IN MIXED CROP HYDROPONIC SYSTEMS
Sharon L. Edney\Jeffrey T. Richards\ Matthew D. Sisko\ Neil C. Yorio\ Gary W. Stutte\ and Raymond M. Wheeler2
ABSTRACT NASA's Advanced Life Support (ALS) Program is developing crop systems for use on
spacecraft, or surface habitats on the Moon and Mars. Growing multiple crops on a shared nutrient solution potentially simplifies production of a variety of crops, but may result in nutrient competition and production of allelochemicals that reduce yield. Three salad crops, radish (Raphanus sativus L. cv. Cherry Bomb II), lettuce (Lactuca sativa L. cv. Flandria) and bunching onion (Allium fistulosum L. cv. Kinka) were grown for 28 days hydroponically in either mono culture (control) or in a mixed-crop arrangement. Plants were grown in a walk-in growth chamber with baseline environments maintained at an air temperature of 28°C, 50% RH, 17.2 mol m-2 d-l daily PPF integral and a 16-h light/8-h dark photoperiod under cool-white fluorescent lamps. Experimental CO2 treatments consisted of 400, 1200 and 4000 /lmol mOrl.
INTRODUCTION NASA's Advanced Life Support program is currently studying plants as components of
bioregenerative life support systems for long-term missions or as dietary supplementation for short-term missions. In addition to the physiological benefits, providing fresh food for a crew on long duration space missions can have a considerable psychological impact as well (1). A salad machine on ISS and early Mars missions would require optimal use of limited area (volume) and electrical power. A mixed cropping arrangement could provide a more efficient use of resources, a greater satisfaction in dietary variety, a greater crop biomass production per unit of growth area, and a greater overall yield stability through crop diversity. However, mixed crop growth could also have a negative impact through competition for nutrients, the production of allelopathic compounds (2), or a more complicated mineral nutrition and irrigation management routine. In developing a mixed cropping system with a common root and aerial enviromnent, it is critical to understand the crop interactions under a range of environmental conditions. Lettuce, onions, and radishes are being grown under baseline environmental conditions that would be encountered aboard the ISS to determine if mixed cropping impacts their growth and yield (1,3).
MATERIALS AND METHODS Plant Cultural Conditions: All plants were grown in a 48 ft2 walk-in controlled environment
chamber at Kennedy Space Center's Space Life Sciences Lab Phytotron. Seeds were planted in six plastic trays (0.25 m2 growing area) using recirculating nutrient film technique culture with a modified 1'2 strength Hoagland's solution (4). Three of the trays were planted as mono culture controls of radish, lettuce or onion. The other three trays were planted with all three species combined in different arrangements. Solution pH was automatically controlled to near 5.8 with additions of dilute nitric acid (004 M RN03). Water depletion from the nutrient reservoirs due to
IDynamac Corporation, Kemledy Space Center, FL 32899 and 2NASA Biological Sciences, Kennedy Space Center, FL 32899.
133
evapotranspiration was monitored and manually replaced on a daily basis to maintain constant liquid level. Solution electrical conductivity was monitored and maintained near 120 mS m- I
by daily addition of a complete stock solution.
RESULTS Table 1: Growth measurements from 28 DAP for lettuce grown in mixed (MC) versus mono culture (Mono) trays with three CO2 levels. Data represents means, n=24 for MC and n=16 for Mono.
""' .. ~ s s s ~ ....... '" s '"'s .. s .. s s s s s s .. s S'O
Cropping Method CO2 EdibleDM TotalDM Harvest EdibleDM (ppm) (g planrl) (g planrl) Index (%) (g m-2)
Contrasts Mono 400 vs. MC 400 ns ns *** ns Mono 1200 vs. MC 1200 * ** * ns Mono 4000 vs. MC 4000 ns ns ** ns
Interaction CO2 vs Cropping Method ns ns *** ns
Mono 400 vs. 1200 *** *** ns *** 400 vs. 4000 *** *** ns ** 1200 vs. 4000 ns ns ** ns
MC 400 vs. 1200 *** *** ns *** 400 vs. 4000 *** *** ** ** 1200 vs. 4000 ** ** *** ns
zns, *, *;, *** = non-significant or significant at p:::,.0.05; 0.01 ~r 0.001 respectively using ANOV A. Numb;~~ () are S.E.
134
Table 2: Growth measurements from 28 DAP for radish grown in mixed (MC) versus mono culture (Mono) trays with three CO2 levels. Data represents means, n=24 for MC and n=16 for Mono.
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Cropping Method CO2 EdibleDM TotalDM (g Harvest EdibleDM (ppm) (g planrl) planrl) Index (%) (g m-2)
Contrasts Mono 400 vs. MC 400 ns * ns ** Mono 1200 vs. MC 1200 ns ns ns *** Mono 4000 vs. MC 4000 * * ns *
Interaction C02 vs Cropping Method ns ns ns **
Mono 400 vs. 1200 *** *** *** ** 400 vs. 4000 ns ns ns ns 1200 vs. 4000 *** ** *** ns
MC 400 vs. 1200 *** ** *** *** 400 vs. 4000 ns ns ns ns 1200 vs. 4000 ** ns *** *** . -
zns, *, **, *** = non-significant or significant at p> 0.05; 0.01 or 0.001 respectively nsing ANOV A. Numbers in () are S.E.
135
Table 3: Growth measurements from 28 DAP for onion grown in mixed (MC) versus mono culture (Mono) trays with three CO2 levels. Data represents means, n=24 for MC and n=16 for Mono.
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Cropping Method CO2 EdibleDM TotalDM Harvest EdibleDM (ppm) (gplanrl) (g planrl) Index (%) (g m-2)
Contrasts Mono 400 vs. MC 400 ns ns ns ns Mono 1200 vs. MC 1200 ns ns * ns Mono 4000 vs. MC 4000 ns ns ns ns
Interaction CO2 vs Cropping ns ns ns ns Method
Mono 400 vs. 1200 *** *** * * 400 vs. 4000 *** *** ns * 1200 vs. 4000 ns ns ns ns
MC 400 vs. 1200 *** *** ns ** 400 vs. 4000 *** *** * *** 1200 vs. 4000 ** *** ns ns """" ~ ~,.. .. ~~ .... ~,,~ .... ., .. "'s~ " ........ ................ " .... 01"".... ~ .. s .. ~ .. t ................ .,"'I" .... ~ ........ "'" .. :Osl>""'n' .. i!'
zns, *, **, *** = non-significant or significant at p> 0.05; 0.01 or 0.001 respectively using ANOV A. Numbers in () are S.E.
136
Figure 1. Monoculture trays at 28 days (L to R -lettuce, onion, radish)
Figure 2. Mixed crop trays at 28 days (L to R - onion, lettuce, radish; radish, onion, lettuce; lettuce, radish, onion)
137
Figure 4. Mixed crop root system (lettuce, onion, radish) at 28 DAP.
138
DISCUSSION Mixed vs. Monoculture cropping method: In lettuce (Table 1), both edible and total DM
planrl were higher in mixed plants at 1200 /-lmol morl CO2. Harvest index was lower in mixed crop plants at all three C02 levels, due to an increase in root mass. Mixed cropping in onion (Table 2) only had a significant impact on plant growth at 1200 /-lmol morl CO2 for total plant DM, with mono culture plants having a greater total DM at this level. Radish grown under mixed cropping showed a significant increase in edible DM planrl compared to mono culture plants at 4000 /-lmol mol-1 CO2, and a significant increase in total plant DM at 400 and 4000 /-lmol morl
CO2. Edible DM m-2 was higher in mixed crop radish at 400 /-lmol morl CO2, but lower at higher CO2 levels.
C02 effects: Increasing CO2 level to 1200 /-lmol morl resulted in further increases in the growth of onions in both mixed crop and mono culture plants. Mixed crop plants increased in growth even further at higher CO2. In both lettuce and radish, plant growth increased significantly for mono and mixed plants between 400 and 1200 /-lmol morl CO2. There was no affect in radish grown either mixed or mono culture arrangements between 400 and 4000 CO2, and a significant decrease in growth between 1200 and 4000 /-lmol morl CO2. For lettuce mixed crops, there was a significant increase in growth between 400 and 1200 and also between 400 and 4000 /-lmol mOrl. However, changing the CO2 level from 1200 to 4000 /-lmol morl resulted in a significant decrease in growth for the mixed crop lettuce. Monoculture lettuce plants experienced the same increases in growth between 400 and 1200 and 400 and 4000, but had no significant decrease between 1200 and 4000 CO2 levels.
CONCLUSION Intercropping of salad crops is one way to provide a diversity of vegetables within a small
spaceflight growth chamber. Determining if there are any negative effects of this arrangement on species being grown is important in developing the design and horticultural management of such chambers. These series of experiments have shown that for onion, there is no significant difference in edible dry mass/m-2 bewteen plants grown in either a mixed or mono culture arrangement at all three CO2 levels. For radish, the edible dry mass/m-2 was higher in mixed crops at 400 /-lmol morl C02, but lower at higher CO2 concentrations. Lettuce showed no impact in edible dry mass/m-2 in a mixed vs. mono culture arrangement at any of the three CO2 concentrations.
LITERATURE CITED
1. Goins, G. D., Yorio, N. C., Stutte, G. W., Wheeler, R. M., and Sager, J. C. 2003. "Crop Production Technology: Advanced Life Support 'Salad' Crop Testing for Flight Applications." ALS Program Element Technology Development Proposal. National Aeronautics and Space Administration.
2. Salisbury, F.B., and C.W. Ross. 1985. Plant Physiology: Third Edition. Belmont: Wadsworth Publishing Company, Inc.
139
3. Richards, J. T., Edney, S. L., Yorio, N. c., Stutte, G. W., Cranston, N., Wheeler, R. M., and Goins, G. D. 2004. "Effects of Lighting Intensity and Supplemental CO2 on Yield of Potential Salad Crops for ISS." SAE International 2004-01-2296.
4. Hoagland, D.R. and D.l. Arnon. 1950. The water culture method for growing plants without soil. California Agr. Expt. Stat. Circ. 347.
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SENSITIVITY SCREENING OF RADISH SEEDLINGS TO SPACECRAFT VOCs
1. Eraso*l, G.W. Stutte1, O. Monje\ S. Anderson1 and R. D. Hickey*2
Atmospheric contaminants in spacecraft can affect the performance of plant-based life support systems for long duration space missions. NASA has established Spacecraft Maximum Allowable Concentration (SMAC) guidelines for crew exposure to air pollutants commonly found in spacecrafts and the Intemational Space Station. However, NASA has not established similar guidelines for plant systems. An automated volatile organic compound (VOC) system was developed to allow bioactivity screening of compounds on plant growth.
A series of experiments were conducted to determine the radish seedling threshold values to four alcohols currently present in the Intemational Space Station (ethanol, isopropyl, tert-butyl alcohol, and methanol). Growth after five days of chronic exposure to the alcohol on the radish seedling (Raphanus sativus L., cv. Cherry Bomb Hybrid II) at a concentration of 0, 50, 100, 175,250 and 500ppm for each alcohol was used to establish preliminary threshold values. The SMAC values for ethanol, methanol, tert-butyl alcohol, isopropyl alcohol are 1000ppm, 6.7ppm, 39.6ppm and 61ppm, respectively.
Results show a general response-pattem for the four alcohol exposures at threshold responses of 10%,50% and 90% of the total biomass of the plant (Table 1). At concentrations of 50-115ppm, a slight reduction (~IO%) in seedling growth was observed. When the alcohols exposure was increased at 120-250ppm, there was a reduction of seedling growth by ~50% and finally, seeds did not germinate in the presence of alcohol concentrations above 250ppm. The results indicated a very different sensitivity of alcohols on humans than on plants. Ethanol is the only alcohol to be bioactive (SMAC= 1000 ppm), while isopropyl, tert-butyl alcohol, and methanol bioactivity begin at levels quite high above their human SMAC levels (60, 40, 6.7ppm respectively).
Table 1: Threshold values of isopropanol, tert-butanol, ethanol and methanol on length of radish seedlings.
TlO=lO% reduction in seedling length; Tso = 50% reduction in seedling length; T90 = 90% reduction in seedling length; SMAC=Spacecraft Maximum Allowable Concentration.
These studies indicate that existing guidelines for VOC contamination, such as SMAC established for humans are not suitable for use in establishing bioactivity thresholds for plants. The seedling VOC bioassay provides a rapid screening tool to establish the biological threshold of several VOCs and to identify compounds must be monitored and/or filtered during long duration space missions.
IDynamac Corporation, Kennedy Space Center, FL, 2 University of Cork, Cork, Ireland
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ROOTSTOCK EFFECT ON GROWTH OF APPLE SCIONS WITH EXCURRENT AND DECURRENT GROWTH HABITS
Thomas Tworkoski1* and Stephen Miller1
ABSTRACT
Rootstocks are used to propagate scions of preferred cultivars, improve fruit tree tolerance to environmental stress, and to control tree size. Improved fruiting from sizecontrolling rootstocks has been accompanied by altered tree morphology. In the current experiment the goal was to improve understanding of rootstock effects on growth and development of apple scions with different growth habits. Apple scions with excurrent and decurrent growth habits were grafted on various size controlling rootstocks and morphological characteristics were measured after six years of growth in the field. Scion had more influence than rootstock on monthly growth rate. Across all rootstocks, scions with excurrent growth habits grew rapidly in April and May and achieved most seasonal growth earlier than scions with decurrent growth habits that grew slowly early in the season. In all growth habits and rootstocks, growth rate slowed appreciably but did not cease by August and growth did not terminate earlier for anyone scion-rootstock combination. Across all scions, the dwarfing rootstock, M.9, consistently had the lowest and seedling rootstock had the greatest tree height and diameter. However, no one size controlling rootstock consistently influenced dates of bud break and full bloom, shoot elongation rate, or duration of growth. Significant interactions indicated that effects of size-controlling rootstock on components of shoot growth will vary with apple tree growth habit. These effects on phenology and development can significantly affect orchard management systems.
1 USDA, ARS, Appalachian Fruit Research Station, 2217 Wiltshire Rd., Kearneysville, WV 25430 USA
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EMBRYOGENESIS INDUCTION WITH IAA AND IAA CONmGATES IN CARROTS
K. Tworkoski, A. Newton, and E.M. Shea Loyola College Chemistry Department, 4501 N. Charles St., Baltimore MD 21210, USA
Abstract: In order for cells to form embryos they must initially be exposed to auxins before
being transferred into an auxin-free medium. Synthetic auxins, such as 2,4-dichlorophenoxyacetic acid (2,4-D), generate embryos with genetic variations and other negative side effects. Natural auxins, like indole-3-acetic acid (IAA), can provide an alternative way to induce somatic embryogenesis. Our research has shown that it is possible to use amide-conjugated IAA in place of2,4-D to induce embryo formation. It may be possible to use free IAA to accomplish the same feat. It has also been determined that ethanol-based stock solutions ofIAA alter a culture's ability to fonn emblYos, but water-based solutions ofIAA do not. Water-based stock solutions ofIAA are generated using the potassium salt of IAA, which does not substantially change the pH of culture medium. Cellular metabolism of IAA has been determined by examining the concentrations of IAA and its conjugates in cells and culture media that have been treated with IAA over a period of 24 hours. Based on preliminary data using ethanol-based IAA solutions, it would be necessalY to replenish IAA daily to ensure that it acts as an effective auxin.
Introduction: The ability to form embryos is conferred upon carrot cells by exposure to high
concentrations of auxins (1,3). After this incubation, cells must be placed in an auxinfree environment before embryos can begin to develop (Figure 1). Researchers frequently use synthetic auxins, lilce 2,4-dichlorophenoxyacetic acid (2,4-D), to induce embryogenesis. Such synthetic auxins, however, do not yield unifonn results. Specifically, 2,4-D may introduce genetic variation into embryos, which is not always desirable. Additionally, some plant species do not respond to 2,4-D at all.
Indole-3-acetic acid (IAA) is a naturally OCCUlTing auxin which is less likely to share the faults of its synthetic compatriots. Using IAA or conjugates ofIAA instead of 2,4-D would allow somatic embryogenesis, without genetic variation, ofrare or endangered plant life. Such somatic embryogenesis might also be used as a comparative model against zygotic embryos (5).
Our research is designed to test the feasibility of using IAA and IAA conjugates as auxins that may be used in conjunction with, or in place of, synthetic auxins such as 2,4-D.
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Figure 1. Stages in embryo development: A) undifferentiated carrot cells, B) globular, C) heartstage, and D) torpedo-stage embryos.
Methods: Culture Conditions:
Cultures were grown in medium containing MS salts and vitamins (Murashige and Skoog, 1962; commercial preparation) supplemented by 30g/L sucrose (PH 5.7). Cells were subcultured weekly.
Analyzing Cellular Metabolism of fAA: Cells were grown in medium with lAA. Periodically, cells were harvested and examined for their free lAA, ester-linked IAA, and amide-linked IAA concentrations. Free IAA fractions were examined without treatment. Free + ester-linked lAA fractions were examined after a IN hydrolysis with NaOH, and free + ester-linked + amide linked IAA fractions were examined after a 7N hydrolysis with NaOH (2).
Testing Conjugate Embryogenic Potential: Cultures of embryogenic cell lines were filtered through mesh screens of varying sizes. Cell clusters between 43 and 109 um in diameter were grown in medium without auxins for 1 week to remove residual 2,4-D. Cells were then grown in medium with conjugates for 4 weeks. Afterwards, cells were transferred into media without auxins and observed for embryo fonnation.
Testing fAA Embryogenic Potential: For 4 weeks, lAA was added to cells in growth medium at regular intervals. At the end of the 4 week period, cells were transferred into media without auxins and observed for embryo fonnation.
10000 ~~~---~------------'-'--~-~~---,-----. I -. . ....... .. ~ 5000 ----.~ I ~
O fii!':""\\>-W.i"-'''-''··'')!~, .. -·-"'·--"-"-'-"'''.,@,'''.------"--'!:i=~,,_,,::'':_.'''"''''''''''''''',"_~._. "I ' Ilft...... ..........;.. .. _._. ___ ~_ r ._ _ ..... ~ .. ;:..;;-~::;;:=a.
o 4 8 12 16 20 24
Hours after IAAtreatment
Figure 2: The metabolism ofIAA and IAA conjugates in carrot cells over a period of 24 hours. At time 0, the medium contained 17 ~ IAA. Peak at time 1 represents 4% of initial IAA concentration, or 2.9/lg.
It is evident that IAA does not remain in its free state for an extended period of time. Instead, it is converted primarily to amide linked IAA conjugates for the purpose of storage. Under natural conditions, IAA is degraded by light and enzymes, thus necessitating a plant's use of conjugates to maintain a sufficient concentration of auxins (2). Based on the above rate of degradation, it would be necessary to replenish the IAA concentration approximately every 24 hours if IAA is to act as an effective auxin.
We decided to test IAA as a natural auxin by adding it to medium either every day or every third day (Table 1). By adding IAA every third day we hoped to determine if IAA was an effective auxin in extremely low concentrations.
Table 1. Embryogenesis during and after four weeks treatment with free lAA or lAAconjugates. Stock solutions (20 mg/ml 95% ethanol) ofIAA-conjugates were filter sterilized and added to culture medium to reach a final concentration of 57 JlM. Sterile IAA solution (20 mg/ml 95% ethanol) was added to reach a final concentration of 17 JlM.
The data in Table 1 demonstrates that amide conjugates lAA-leucine and IAAhistidine show promise as natural substitutes for 2,4-D. Our data also suggests that that adding IAA to culture medium every third day is not sufficient to prevent embryogenesis, although it may slow embryo growth. It is interesting to note that after cells were transferred to an lAA free environment, the embryos that had already formed did not develop any further.
Up through this point in the experiment, lAA and IAA conjugates were applied to medium dissolved in ethanol. It is possible that ethanol, even at the concentrations in which we use it (S.7JlM) may retard embryo fonnation. Previous research has shown that concentrations of 10 mM ethanol can be toxic to carrot cells. This research has also determined that the noxious effects of ethanol are derived from the acetaldehyde that is oxidized from ethanol, rather than the ethanol itself (4). Given that our experiment used ethanol in concentrations that were over 1000 times more dilute than that which was shown to be toxic, we did not expect undesirable results. This
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expectation was not entirely bourn out-we did observe retarded embryo growth. We did not, however, encounter the levels of toxicity outlined in the aforementioned research.
To verify the hypothesis that ethanol was responsible for the observed retarded emblYo formation, we grew various cell lines in2,4-D medium, then transferred them to auxin-free medium and treated half the cells with ethanol (Table 2).
Cell Line Age of Cell EtOH? (YIN) Development after Development after Line 1 week 2 weeks
y No embryos Globular to heart DH 2 yrs
N > 50% globular Globular to torpedo
ED 1 yr Y Pre-globular Pre-globular
N Pre-globular >50% Globular
y Pre-globular Globular and FA 2 months Torpedo (3mm
long)
N Heart to Torpedo Plantlets (up to 1 cm tall)
Table 2: The inhibitory effects of ethanol on emblYo formation. For the ethanol treatments, we added ethanol to 25ml of medium to reach a concentration of 8.7 llM.
By the second week of observation, those cells that did not have ethanol added to them displayed more embryogenic potential than those that did. This furthers the idea that dilute ethanol slows embryogenesis and may therefore have influenced our earlier results.
It should be noted, however, that although our previous data was obtained using IAA in ethanol, the cell lines used were youthful enough to allow them some measure of protection from the effects of ethanol. Our most recent data suggests that although ethanol may retard embryo fornlation, it does not prevent embryos from growing beyond a certain stage (Table 2). Thus, the lack of further development of cells that had IAA added every third day may be attributed to the presence ofIAA-to a certain extent, lAA acts as an effective auxin at extremely low concentrations. It is the frequency with which lAA is added to medium which controls its effectiveness (Table 1).
We are currently re-testing the cell metabolism ofIAA using the potassium salt ofIAA dissolved in distilled water. Adding this water-based stock solution to growth medium alters the pH by only .0308 ± .01 pH units: an acceptable difference given that autoclaving growth medium can modify the pH by .507 ± .1 pH units.
We believe that the overall rate of metabolism of lAA is not ethanol-dependant.
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It is probable, however, that the initial spike in the free IAA concentration seen in Figure 2 was due to the presence of ethanol, given that ethanol generates a wounding response in plants.
Due to the relative youth of the cell lines used and the low concentration of ethanol, it is not likely that our results are overly skewed by the presence of ethanol. To verify this assumption, we are currently using a water-based IAA stock solution to confirm that IAA-Ieucine, IAA-histidine, and free IAA can be used as natural substitutes for 2,4-D.
References: 1. Ammirato, V (1985). Patterns of development in culture. In: Henke RR, Hughes
KW, Constantin MP, and Hollaender A, eds., Tissue Culture in Forestry and Agriculture, pp. 9-29. Plenum Press, New York.
2. Cohen JD and Bandurski RS (1978). The bound auxins: protection ofindole-3-acetic acid/rom peroxidase-catalyzed oxidation. Planta 139:203-208.
3. Kamada H and Harada H (1979). Studies on the organogenesis in carrot tissue cultures. 1. Effects of growth regulators on somatic emblyogenesis and root formation. Z. Pflanzenphysiol. 91:255-266.
4. Perata Pierdomenico and Alpi Amedeo (1990). Ethanol-Induced Injuries to Carrot Cells. Plant Physiology: The Role of Acetaldehyde. Plant Physiology 95:748-752.
5. Zimmerman JL (1993). Somatic emblyogenesis: a modelfor early development in higher plants. Plant Cell 5: 1411-1423.
Acknowledgements: This work was funded by grants from Research Corporation and the National Science Foundation (IBN97-53001).
The authors wish to thank Dr. Jerry Cohen and Dr. Lana Barkawi ofthe University of Minnesota, Twin Cities for analyzing our samples by GC-MS.
