Fruit Varieties Journal 44(3):141-148 1990 Multiplication of Rubus Germplasm In Vitro: A Screen of 256 Accessions BARBARA M. REED! ' Abstract weeks each were placed on media with addi- Rubus germplasm at the National Clonal tiona.l modi~ications. The majority (6~) will Germplasm Repository, Corvallis, Oregon, was multiply 3X In three weeks on MS medium with screened to determine tissue culture growth 1 ~g/l BA, 0.1 mg/l IBA and 0.1 mg/l GA3 media suitable to the diverse collection being ,,:hlle another 18S respond on Anderson's me- maintained. Explants were taken from mature ?Ium at the ~ame hormone levels. The remain- pot-grown plants. Murashige and Skoog (MS) mg 2OS require BA at 2 mg/l, O.I/mg/1 IBA and medium with 1 mg/l benzyladenine (BA) and GA3. 0.1 m.g/l to produce 3X growth in three 0.1 mg/l indole butyric acid (IBA) was used for weeks. This is the first report of the in vitro initiation (explant establishment) of 256 differ- cult?re of many. of these Rubus species and ent accessions of Rubus. Evaluation was based cultlvars. Chemical names used; N(-phenyl- on a three fold (3X) increase in the number of me;thyl)-IH-l!~rin-6-a~ine (BA); Indole-3-butyic plantle~s follow.in.g.three weeks on the medium. acid (IBA); Gibberellic Acid A3 (GA3). Followm,s two mltial transfers, those which did Introduction not multiply 3X in three weeks were divided I' into two ~roups based on the vigor of the clone. ~ vItro propagatIon m IndIvIdual Group 1 (low multiplication but ~owing well) cultIvars of raspberry and blackberry were placed on MS with 1 r:ng/\ BA, 0.1 mg/ and optimal basal media and hormone IBA .an.d 0;1 mg/l GA3 while Group 2 (low concentrations are well documented multiplication and poor growth) were grown (1 2 3 4 7) . on Anderson's medium with 1 mg/l BA and 0 1 "". Most studIes have de- mg/l IBA. Accessions which did not respo~d veloped media formulations for one with 3X growth after two transfers of three to five cultivars. The development of 'USDA/ ARS National Clonal ~ermplasm Repository, 33447 Peoria Road, Corvallis, OR 97333. The autho~ acknowled.ges the assistance of Trudy McFarlane in collecting and Margaret Norton in cultunng and stonng the explants. l41
Transcript
Fruit Varieties Journal 44(3):141-148 1990
Multiplication of Rubus Germplasm In Vitro:A Screen of 256 Accessions
BARBARA M. REED! '
Abstract weeks each were placed on media with addi-Rubus germplasm at the National Clonal tiona.l modi~ications. The majority (6~) will
Germplasm Repository, Corvallis, Oregon, was multiply 3X In three weeks on MS medium withscreened to determine tissue culture growth 1 ~g/l BA, 0.1 mg/l IBA and 0.1 mg/l GA3media suitable to the diverse collection being ,,:hlle another 18S respond on Anderson's me-maintained. Explants were taken from mature ?Ium at the ~ame hormone levels. The remain-pot-grown plants. Murashige and Skoog (MS) mg 2OS require BA at 2 mg/l, O.I/mg/1 IBA andmedium with 1 mg/l benzyladenine (BA) and GA3. 0.1 m.g/l to produce 3X growth in three0.1 mg/l indole butyric acid (IBA) was used for weeks. This is the first report of the in vitroinitiation (explant establishment) of 256 differ- cult?re of many. of these Rubus species andent accessions of Rubus. Evaluation was based cultlvars. Chemical names used; N(-phenyl-
on a three fold (3X) increase in the number of me;thyl)-IH-l!~rin-6-a~ine (BA); Indole-3-butyicplantle~s follow.in.g.three weeks on the medium. acid (IBA); Gibberellic Acid A3 (GA3).
Followm,s two mltial transfers, those which did Introductionnot multiply 3X in three weeks were divided I' into two ~roups based on the vigor of the clone. ~ vItro propagatIon m IndIvIdual
Group 1 (low multiplication but ~owing well) cultIvars of raspberry and blackberry
were placed on MS with 1 r:ng/\ BA, 0.1 mg/ and optimal basal media and hormoneIBA .an.d 0;1 mg/l GA3 while Group 2 (low concentrations are well documentedmultiplication and poor growth) were grown (1 2 3 4 7) .on Anderson's medium with 1 mg/l BA and 0 1 "". Most studIes have de-
mg/l IBA. Accessions which did not respo~d veloped media formulations for onewith 3X growth after two transfers of three to five cultivars. The development of
'USDA/ ARS National Clonal ~ermplasm Repository, 33447 Peoria Road, Corvallis, OR 97333. The
autho~ acknowled.ges the assistance of Trudy McFarlane in collecting and Margaret Norton incultunng and stonng the explants.
l41
142 FRUIT VARIETIES JOURNAL
a medium to support widely variedgermplasm collections would requirea long period of time if each accession(species or 9ultivar) were individuallyoptimized. The development of a me-dium for the in vitro propagation ofcultivars and species is important inthe maintenance of large germplasmcollections. This information wouldalso be useful to those working withvirus indexing, cold storage or ex-change of in vitro material. Ideally allaccessions would be grown on onemedium (6); however the great amountof genetic diversity found in a collec-tion of hundreds of species and culti-vars of a genus such as Rubus makethis unlikely. This report provides in-formation on the response of 256Rubus species and cultivars to modi-fications of two widely used mediawhich allow for growth and multipli-cation in vitro.
into two groups based on plant vigor.Group 1 (low multiplication but grow-ing well) remained on the MS mediumused for initiation and GA3 at 0.1 mg/lwas added. Group 2 (low multiplica-tion and poor growth) were transferredto Anderson's raspberry medium (1).Two more growth cycles (3 weekseach) were run and cultures multiply-ing at 3X were cold stored. Group 1clones which did not respond weretransferred to MS medium with 2 mg/lBA, 0.1 mg/l IBA and 0.1 mg/l GA3'This improved multiplication for someand they were stored. For group 2clones which did not respond to An-derson's medium, GA\i was added at0.1 mg/l. Those that did not respondafter two more three week transfershad an additional 1 mg/l BA added tothe medium.
Cultures of 45 rapi~ly growing ac-cessions were transferred to hormone-free MS medium for rooting. Not allaccessions were tested for rooting abil-ity due to time and space limitations.
ResultsNearly all accessions initiated could
be multiplied using the medium modi-fications used in this study. The ma-jority of accessions (69~) could besuccessfully cultured on one of the MSmedia formulations (Tables 1 A, B,C). Most of the accessions on thismedium (Group 1) were blackberries(68~). The addition of GA3 to themedium increased multiplication ofsome accessions (Table 1B). The ac-cessions listed in Tables 1A and 2Awere found to grow equally well withthe addition of GA3. The addition of 2mg/l BA was needed to stimulate mul-tiplication in others (Table 1C).
Anderson's medium with and with-out modifications (Group 2) improvedmultiplication for 34~ of the acces-sions, of which 94~ are raspberries(Tables 2 A, B, C). Some accessionsremained alive but multiplied veryslowly despite medium modifications(Table 2C).
Materials and MethodsNodal cuttings (3 cm) of 5 year old
pot-grown plants in the screenhousecollection of the National ClonalGermplasm Repository, Corvallis, Or-egon were surface sterilized by immer-sion for 15 min. in a 10% bleach solu-tion (5.25~ sodium hypochlorite) with0.1 mIll of Tween 20 followed by twosterile water rinses. Cuttings of allaccessions were initiated in 16 mmtubes on Murashige and Skoog (MS)medium (5) with 0.1 mg/l indole-3-butyric acid (IBA), 1 mg/l Benzyl-adenine (BA) and 7 g/l agar (Difco-Bacto) with a pH of 5.7 before auto-claving. Growth room conditions were16 hr days and 8 hr nights at 25C.Light intensity was 25 #l.Em2s-1 at thelevel of the plants.
Cuttings were grown in the initia-tion medium for two transfers of threeweeks each then evaluated for growthresponse (Fig. 1). Those which weremultiplying at a rate of 3X or betterper three week transfer period werecold stored at this point. Those whichdid not multiply at 3X were divided
MULTIPLICATION OF RUBUS GERMPLASM IN VITRO: 143
MEDIA FLOW CHARTFOR RUBUS SCREENING
INITIATION MEDIUMMS (1 mg/l BA, 0.1 mg/IIBA)
Grow for two three-week passagesThose with 3X multiplication are stored
(102 clones) Table 1A
Divide into two groups
GROUP 1 (Table 1 B) GROUP 2 (Table 2A)Not rapidly mltiplying but in good condition. Not rapidly multiplying and not in goodMS (1 mg/l BA,0.1 mg/IIBA 0.1 mg/l GA3) condition. Anderson's medium (1 mg/l BA,Grow for two three-week passages, store 0.1 mg/l IBA). Grow for two three-weekthose with 3X multiplication. (57 clones) passages then store. (8 clones)
GROUP 1a (Table 1C) GROUP 2a (Table 2B)Did not respond to Group 1 treatment. Did not respond to Group 2 treatment.Transfer to MS (2 mg/l BA, 0.1 mg/IIBA Transfer to Anderson's (1 mg/l BA,0.1 mg/land 0.1 mg/l GA3) for two three-week IBA, 0.1 mg/l GA3) for two three-weekpassages. Improved but not to 3X. (18 passages. Then store. (38 clones)
clones)GROUP 2b (Table 2C)Did not respond to Group 2a treatment.Transfer to Anderson's (2 mg/l BA, 0.1 mg/lIBA, 0.1 mg / I GA3)' Improved but not to 3X.
(33 clones)
Figure 1.
'September' (multiplication rates notgiven) on Boxus medium with MSmicronutrients. With these three culti-vars we found Anderson's mediumwith GA3 superior to MS, with higherBA levels (2 mg/l) required for 'Heri-tage' and 'Malling Exploit' (Table 2C).Three cultivars also studied by Ander-son (1) were included in this screen.Of those three, 'Willamette' (Table1A) required less BA (1 mg/l) and noGA3 compared to Anderson's study.'Nootka' and 'Heritage' (Table 2C)grew best at the same hormone levels(2 mg/l BA, 0.1 mg/l GA) noted byAnderson (1).
Removal of all hormones from MSmedium stimulated rooting in the 45accessions tested (Table 3). All of theaccessions listed in Table 3 rootedwithin the first 3 weeks following
Specific cultivars responded in amanner similar to previous reports.'Black Satin' and 'Smoothstem' (TablelA) had high multiplication rates onMS medium but did not require theaddition of GAa as used by Broomeand Zimmerman (2) and others (4).'Bedford Giant' and 'Tayberry' cul-tured in Lindsmeier and Skoog me-dium by Harper (3) responded in asimilar fashion respectively to 'Bed-ford Giant' (Table lA) and'Tayberry'seedling (Table 1B). The four culti-vars of thornless blackberry grown inthe Broome and Zimmerman study allgrew well while in this study threeother thornless blackberries, 'Thorn-less Logan,' 'Chester Thornless' and'Thornfree' were recalcitrant.
Snir (7) successfully grew meristemsof 'Heritage,' 'MaIling Exploit' and
144 FRUIT VARIETIES JOURNAL
Table lA. Rubus accessions from theNational Clonal Germplasm Reposi-tory (NCGR) with greater than 3Xmultiplication in three weeks onMurashige ahd Skoog medium with1 mg/l BA and 0.1 mg/l IBA (102clones).
Table IA. (Continued),NCGR
Accessionnumber
CroptypePlant name
~
81840
424248
43959
151
794
BlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryBlackberryR. speciesR. species
R. discolor Weihe &Nees R. species
R. errabundus Watson R. speciesR. flosculosus Focke R. speciesR. fruiticosus L. R. speciesR. fuscus Weihe &
Nees R. speciesR. hirsutus Thunb. R. species
R. speciesR. Hirtus Waldst. & Kit. R. speciesR. hispidus L. R. speciesR. idaeus v. strigosus
(Michaux) Maxim.R. illecebrosus FockeR. lambertianus Ser.R. lasiostylus FockeR. lasiostylus v.
hubeiensis R. speciesR. leucodermis Douglas
ex Torrey & A. Gray R. speciesR. species
R. microphyllus L. f. R. speciesR. odoratus L. R. species
R. speciesR. parviflorus Nutt. R. species
R. speciesR. parvifolius L. .R. species
R. speciesR. speciesR. species
R. pungens Cambess. R. speciesR. rigidus Smith R. speciesR. rosaefolius Smith R. species
R. speciesR. speciesR. speciesR. speciesR. speciesR. speciesR. speciesR. speciesR. speciesR. speciesR. species
1459676354191404922
255202961~
R.
spectabi)is Pursh
KotataMarionNCII0ORUSI063ORUSI280ORUS 1465ORUS 1467ORUSIBOOOR US 1620OR US 998RavenSmoothstemSnyderWaldoWatlabWhitford ThornlessWomackR. alumnus BaileyR. armeniacus FockeR. caesius open
CropPlant name type.R. sumatranus Miq. R. speciesR. thyrsoideus Wimmer R. species
R. speciesR. tomentosus Borkh. R. speciesR. trivialis Michaux R. speciesR. ulmifolius f.
bellidiflorus VossR. ulmifolius open
pollinated hybridR. ursinus Chamb. &
2271Q
CropPlant name- type
Tayberry seedling (notCultivar) HybridR. caesius L. R. speciesR. caesius open
pollinated hybrid R. speciesR. canadensis L. R. species
R. species
ISO25
100814
34
R.
species
R.
species 2:3R. cissoides Cunn. R. speciesR. distractiformis
Newton R. speciesR. idaeus L. R. speciesR. lasiococcus A. Gray R. speciesR. lasiostylus Focke R. speciesR. leucodermis Douglas
ex Torrey & A. Gray R. speciesR. speciesR. species
R. nessensis Hall R. speciesR. parviflorus Nutt. R. species
R. species
219238612427
804615155118444
Schldl. R. species
R. species
Baumforth (Standard A) Raspberry
Boyne RaspberryMaIling Promise Raspberry
NC 84-10-8 (R.
occidentalis L.) Raspberry
NC 86-14-02 Raspberry
OR US 1308 Raspberry
OR US 963 Raspberry
Veten Raspberry
Willamette Raspberry
64765359983280052
730988970361454100
R. plicatus Weihe &Nees R. species
R. sp. R. speciesR. thyrsoideus Wimmer R. speciesR. thyrsoideus Wimmer R. speciesR. trivialis Michaux R. species
.R. species
441324837
982724
--Table lB. Rubus accessions from the
National Clonal Germplasm Reposi-tory (NCGR) with greater than 3Xmultiplication in tfiree weeks onMurashige and Skoog medium with1 mg/l BA, 0.1 mg/l IBA and 0.1mg/l GA3 (57 clones). -
occidentalis L)ORUS 1028OR US 1314ORUS 1341Pocahontas
PuyallupTrentZzopska Alena
146 FRUIT VARIETIES JOURNAL
Table lC. Rubus accessions from theNational Clonal Germplasm Reposi-tory (NCGR) with multiplicationrates less than 3X in three weeks onMS media with 2 mg/l BA, 0.1 mgJIIBA and O~~l G~3 (18 clon~.
Table 2B. Rubus accessions from theNational Clonal Germplasm Reposi-tory (NCGR) with greater than 3Xmultiplication in three weeks onAnderson's medium with 1 mg/lBA, 0.1 mg/l IBA and 0.1 mg/l GA3(38 clones).NCCR
Accessionnumber
83969
721368360
347
105
81
131
835
Crop NCGRCrop A«ess;on
Plant name type number
367133459179268591239
607
17258762134~
61
Plant name type
Chester Thornless Blackberry
Comanche Blackberry
Flordagrand BlackberryOR US 1067 Blackberry
OR US 922 Blackberry
OR US 992 Blackberry
Thornfree Blackberry
Thornless Logan Blackberry
Young BlackberryR. hirsutus Thunb. R. speciesR. hirtus Waldst. & Kit. R. species
R. lambertianus var.giabra R. species
R. parviflorus Nutt. R. species
R. ursinus Cham. &Schldl. R. species
Chief RaspberryLowden Raspberry
Mailing Orion Raspberry
OR US 1029 Raspberry
163260632384492985321263
197
995433
1002
288
Table 2A. Rubus accessions from theNational Clonal Germplasm Reposi-tory (NCGR) with greater than 3Xmultiplication in three weeks onAnderson's medium with 1 mg/l BA
~~d 0.1 ~ I~A (8 ~~s).
267
Bodega Bay (R. ursinusCham. & Schldl.) Blackberry
Chehalem BlackberryOR US 1362 BlackberryR. calcynoides Hayata R. speciesR. crataegifolius Bunge R. speciesR. idaeus L. R. speciesR. idaeus L. R. speciesR. idaeus v. peramoenus
(E. Greene) Fern R. speciesR. idaeus v. strigosus
(Michaux) Maxim. R. speciesR. lasiococcus A. Gray R. speciesR. muelleri Lef. R. speciesR. parvif)orus Nutt. R. speciesR. phoenicolasius
Maxim. R. speciesR. trivialis Michaux R. speciesAmity RaspberryCanby RaspberryCarnival RaspberryChilliwack ~ RaspberryGlen Esk RaspberryHeija RaspberryJingu Juegal (R.
Table 2C. Rubus accessions from theNational Clonal Germplasm Reposi-tory (NCGR) with multiplicationrates less than JJX in three weeks onAnderson's medium with 2 mg/lBA, 0.1 mg/l IBA and 0.1 mg/l GA3t" I'lnnp~).
Table
3. Rubus accessions from theNational Clonal Gennplasm Reposi-tory (NCGR) which rooted withinthree weeks of transfer to Mura-shige and Skoog medium withouthnrmnnp~-
NCGRAccessionCrop,DI__o
65774
4252
184147178986447229
488
491440
95990
88493141319316773
96
318400
R.'l.'>,
14111.')
609199200ROO
Brison BlackberryThornless Logan BlackberryR. lasiostylus Focke R. species
Aurora BlackberryAustin Thornless BlackberryBrazos BlackberryBurbank Thornless BlackberryChehalem BlackberryNCII0 BlackberryORUS 1127 BlackberryOR US 1620 BlackberryOR US 998 BlackberryWaldo BlackberryR. armeniacus Focke R. speciesR. fruiticosus L. R. speciesR. hirsutus Thunb. R. speciesR. hirsutus Thunb. R. speciesR. hirtus Waldst. & Kit. R. speciesR. hispidus L. R. speciesR. lasiococcus A. Gray R. speciesR. lasiostylus Focke R. speciesR. leucodermis Douglas
ex Torrey & A. Gray R. speciesR. leucodermis Douglas
ex Torrey & A. Gray R. speciesR. odoratus L. R. speciesR. odoratus L. R. speciesR. palmatus Thunb. R. speciesR. parviflorus Nutt. R. speciesR. parviflorus Nutt. R. speciesR. parviflorus Nutt. R. speciesR. parviflorus Nutt. R. speciesR. parvifolius L. R. speciesR. rigidus Smith R. speciesR. rosaefolius Smith R. speciesR. scanicus Chapple &
Watson R. speciesR. shankii Standley &
L. O. Williams R. speciesR. shankii Standley &
L. O. Williams R. speciesR. sp. R. speciesR. sp. R. speciesR. spectabilis Pursh R. speciesR. spectabilis Pursh R. speciesR. sDectabilis Pursh R. species
148 FRUIT VARIETIES JOURNAL
Table 3. (Continued).NCCR
Crop Accessiontype number
should be reinitiated and optimizedindividually.
We have successfully used this pro-cedure to initiate and grow these 256Rubus accessions. This information onthe basal medium required for Rubusspecies and cultivars provides a start-ing point for those wishing to opti-mize the medium for a particularRubus cultivar or species.
Plant name
R. spectabilis v.menziesii Pursh
R. sumatranus Miq.R. ursinus Chamb. &
Schldl.HeijaMatsquiOR US 1308Zzopska Alena
47
R. speciesR. species
R. speciesRaspberryRaspberryRaspberryRaspberry
804263215970456
transfer onto MS medium without hor-mones. Other slower growing Rubuscultivars may require the addition ofIBA to stimulate rooting (4).
Literature Cited1. Anderson, W. C. 1979. Tissue culture propa-
gation of red and black raspberries. ActaHort. 112:13-20.
2. Broome, O. C. and R. H. Zimmerman. 1978.In vitro propagation of blackberry. Hort-Science 13(2):151-153.
3. Harper, P. C. 1978. Tissue culture propaga-tion of blackberry and tayberry. Hort. Res.18:141-143.
4. McPheeters, K. D., R. M. Skirvin, and H. K.Hall. 1988. Brambles (Rubus ~p.) in: Bio-technology in Agricul!u.re and Forestry. Vol6. Crops II. Y.P.S. BaJaJ ed. pp. 104-123.
5. Murashige, T., and F. Skoog. 1962. A revisedmedium for rapid growth and bioassays withtobacco tissue cultures. Physiol. Plant. IS:474-494.
6. Parfitt, D. E. and A. A. Almehdi. 1986. Invitro propagation of peach: II. A medium forin vitro multiplIcation of 56 peach cultivars.Fruit Varieties J. 40(2);46-47.
7. Snir, I. 1981. Micropagation of red rasp-berry. Scientia Hort. 14;139-143.
DiscussionThis screen has demonstrated that
selecting a medium for multiplyinglarge numbers of species and cultivarsin the genus Rubus can be accom-plished without optimizing media foreach accession. Among the 256 acces-sions of Rubus used in this study aresome that are very diverse geneticallyand have originated from various partsof the world. Most have not previouslybeen grown in vitro. Differences be-tween the results of this study andearlier work may be due to the age ofthe source plants or the season ofinitiation. Six week, rather than longer,culture periods were used in this studysince most modifications involvedadding one new component to stand-ard media rather than deleting com-ponents. Relatively fast changes werenecessary to keep struggling accessionsalive until the proper medium wasfound.
To optimize medium requirementsfor this many accessions would requireyears of study. By using a screen start;.ing with one or two basal media andmodifying the hormone concentrationwith large groups of accessions, thetime required from initiation to finalstorage for most of the accessions ofthis genus should be three to fivemonths. Those that remain recalcitrant
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