973 American Journal of Botany 90(7): 973–9 79. 2003. COMPARATIVE ANATOMY AND MORPHOLOGY OF VITIS VINIFERA (VITACEAE) SOMATIC EMBRYOS FROM SOLID- AND LIQUID-CULTURE-DERIVED PROEMBRYOGENIC MASSES 1 S. JAYASANKAR, 2 BHASKAR R. BONDADA, 3 ZHIJIAN LI, AND D. J. GRAY 4 Mid-Florida Research and Education Center, Institute of Food Agricultural Sciences, University of Florida, 2725 Binion Road, Apopka, Florida 32703-8504 USA Ontogeny of somatic embryos of grapevine ( Vitis vinifera) produced from solid- and liquid-culture-derived proembryogenic masses (PEM) was compared using light and scanning electron microscopy. Somatic embryos produced from solid-medium-derived PEM (SPEM) had large cotyledons, little or no visible suspensor structure, and a relatively undeveloped concave shoot apical meristem, whereas those from liquid-medium-derived PEM (LPEM) had smaller cotyledons, a distinct suspensor, and a flat-to-convex shoot apical meristem. The convex shoot apical meristem in LPEM-derived somatic embryos formed as early as the heart stage of development; it was 4–6 cell layers deep and rich in protein. Suspensors persisted in fully developed and mature LPEM-derived somatic embryos. The SPEM-derived somatic embryos exhibited dormancy, as do mature zygotic embryos, which also have a rudimentary suspensor, where as LPEM- deri ved embryo s were not dorma nt. We hypo thesi ze that the presen ce of a persi stent suspensor in LPEM- deriv ed somatic embryos modulates development, ultimately resulting in rapid germination and a high plant-regeneration rate. Key words: cell culture; embryogenesis; grapevine; somatic embryogenesis; Vitaceae; Vitis vinifera. Although somatic and zygotic embryos are nearly identical in stru ctura l and funct ional characte risti cs (Gold berg et al., 1989, 1994; Gray and Purohi t, 199 1a) , the ontog eny of so- mat ic emb ryos ten ds to be mor e var iab le (Li tz and Gray, 1992). Somatic embryos of most species, including grapevine (Vitis spp.), tend to exhibit several typical morphological ab- nor mal iti es suc h as var iat ion in sha pe, siz e, and cot yle don number (Goebel-Tourand et al., 1993; Gray, 1995; Jayasankar et al., 2002). In most species, somatic embryos are larger than zygotic embryos, and their regeneration rates are lower. For instance, in Vitis rupestris Scheele, only 3% of somatic em- bryos wer e cap abl e of dev elo pin g int o complete pla nts , al- though 27% had shoot and root apices (Faure, 1990). Grapevine somatic embryogenesis, first reported by Mullins and Srinivasan (1976), has become commonplace for several genotypes (Gray, 1995). In most reports, initiation and main- tenance of embryogenic cultures were accomplished by growt h on soli dified medium, and plan t-reg ener ation rates were often low (20%) . Recen tly , we demo nstra ted an em- bryogenic liquid culture system in which somatic embryos dif- fer morphologically from their solid-medium-derived counter- par ts and exhibi t a higher pla nt- reg ene rat ion rat e ( 60%) (Jayasankar et al., 1999). In the present study, we show the 1 Manu scrip t receiv ed 3 Decemb er 2002 ; revi sion accepted 13 Febru ary 2003. The authors thank Ms. Diann Achor, CREC, IFAS, University of Florida, Lake Alfred for help in scanni ng electron microsc opy. This research was supported by the Florida Agricultural Experiment Station and a grant from The Florida Department of Agriculture and Consumer Services’ Viticulture Trust Fund, Tallahassee, Florida and approved for publication as Journal Se- ries No. R-09251. 2 Presen t address: Depar tment of Plant Agricult ure, Onta rio Agri cult ural College, University of Guelph, Vineland Campus, 4890 Victoria Ave. N., P.O. Box 7000, Vineland Station, Ontario, Canada L0R 2E0. 3 Present address: University of California, Department of Viticulture and Enology, One Shields Avenue, Davis, California 95616 USA. 4 Author for reprint requests (e-mail: [email protected]fl.edu). ontogenic pattern to be set very early (i.e., in proembryogenic masses [PEM]), because PEM initiated in solid or liquid cul- ture systems then plated onto solid medium produced somatic embryos with characteristic morphological and developmental differences. Anatomical and morphological studies have prov- en usef ul in under stand ing somatic and zygot ic embryo de- velopment (Altamura et al., 1992; Gray, 1995; Faure et al., 1996a, b). Hence, we employ the same approach to compare somat ic embryog enesi s from liqu id- and soli d-med ium- de- rived PEM (LPEM and SPEM, respectively) to investigate rea- sons for differential ontogeny and plant-regeneration efficien- cy. MATERIALS AND METHODS Culture maintenance—Emb ryogen ic cultu res ofVitis vinifera L. ‘Char - donnay’ (Clone 15) and ‘Thompson Seedless’ were induced from anthers and leaves, respectively, and maintained either on solid medium as described by Gray (1995) or in liqu id culture and maintain ed as PEM as descr ibed by Jayasankar et al. (1999). To produce somatic embryos for study, PEM from solid and liquid culture systems were plated onto solidified X6 medium as previ ously descri bed (Li et al., 2001). Zygoti c embryos also were excis ed from fully mature seeds of ‘Char donn ay’ for compa rativ e study. Embryo s at vario us devel opmen tal stage s were select ed using a stereomicr oscop e and fixed immediately for light and scanning electron microscopy. Scanning electr on micro scopy (SEM) —All fixation and rinse solutio ns were buffered to pH 6.8 with 0.05 mol/L Sorenson’s phosphate buffer and kept at 4C. Unle ss otherwi se specifi ed, all fixation and dehyd ratio n steps were done on ice. Embryos were fixed in cold 3% gluteraldehyde and incu- bated overnight at 4C. After rinsing with buffer (three times), embryos were fixed in 1% osmium tetroxide for 30 min and then rinsed three times. Embryos were then dehydrated in a graded ethanol series (15% increments every 30 min). After two changes (15 min each) of 100% ethanol, embryos were sub- jected to critical-point drying, mounted on stubs, sputtered with gold-palla- dium, and immediately observed with a Hitachi model S530 SEM.
Although somatic and zygotic embryos are nearly identicalin structural and functional characteristics (Goldberg et al.,1989, 1994; Gray and Purohit, 1991a), the ontogeny of so-matic embryos tends to be more variable (Litz and Gray,1992). Somatic embryos of most species, including grapevine(Vitis spp.), tend to exhibit several typical morphological ab-normalities such as variation in shape, size, and cotyledonnumber (Goebel-Tourand et al., 1993; Gray, 1995; Jayasankar
et al., 2002). In most species, somatic embryos are larger thanzygotic embryos, and their regeneration rates are lower. Forinstance, in Vitis rupestris Scheele, only 3% of somatic em-bryos were capable of developing into complete plants, al-though 27% had shoot and root apices (Faure, 1990).
Grapevine somatic embryogenesis, first reported by Mullinsand Srinivasan (1976), has become commonplace for severalgenotypes (Gray, 1995). In most reports, initiation and main-tenance of embryogenic cultures were accomplished bygrowth on solidified medium, and plant-regeneration rateswere often low (20%). Recently, we demonstrated an em-bryogenic liquid culture system in which somatic embryos dif-fer morphologically from their solid-medium-derived counter-parts and exhibit a higher plant-regeneration rate (60%)(Jayasankar et al., 1999). In the present study, we show the
1 Manuscript received 3 December 2002; revision accepted 13 February2003.
The authors thank Ms. Diann Achor, CREC, IFAS, University of Florida,Lake Alfred for help in scanning electron microscopy. This research wassupported by the Florida Agricultural Experiment Station and a grant fromThe Florida Department of Agriculture and Consumer Services’ ViticultureTrust Fund, Tallahassee, Florida and approved for publication as Journal Se-ries No. R-09251.
2 Present address: Department of Plant Agriculture, Ontario AgriculturalCollege, University of Guelph, Vineland Campus, 4890 Victoria Ave. N., P.O.Box 7000, Vineland Station, Ontario, Canada L0R 2E0.
3 Present address: University of California, Department of Viticulture andEnology, One Shields Avenue, Davis, California 95616 USA.
ontogenic pattern to be set very early (i.e., in proembryogenicmasses [PEM]), because PEM initiated in solid or liquid cul-ture systems then plated onto solid medium produced somaticembryos with characteristic morphological and developmentadifferences. Anatomical and morphological studies have prov-en useful in understanding somatic and zygotic embryo development (Altamura et al., 1992; Gray, 1995; Faure et al.1996a, b). Hence, we employ the same approach to compare
somatic embryogenesis from liquid- and solid-medium-derived PEM (LPEM and SPEM, respectively) to investigate rea-sons for differential ontogeny and plant-regeneration efficiency.
MATERIALS AND METHODS
Culture maintenance—Embryogenic cultures of Vitis vinifera L. ‘Char
donnay’ (Clone 15) and ‘Thompson Seedless’ were induced from anthers and
leaves, respectively, and maintained either on solid medium as described by
Gray (1995) or in liquid culture and maintained as PEM as described by
Jayasankar et al. (1999). To produce somatic embryos for study, PEM from
solid and liquid culture systems were plated onto solidified X6 medium as
previously described (Li et al., 2001). Zygotic embryos also were excised
from fully mature seeds of ‘Chardonnay’ for comparative study. Embryos a
various developmental stages were selected using a stereomicroscope and
fixed immediately for light and scanning electron microscopy.
Scanning electron microscopy (SEM)—All fixation and rinse solution
were buffered to pH 6.8 with 0.05 mol/L Sorenson’s phosphate buffer and
kept at 4C. Unless otherwise specified, all fixation and dehydration step
were done on ice. Embryos were fixed in cold 3% gluteraldehyde and incu
bated overnight at 4C. After rinsing with buffer (three times), embryos were
fixed in 1% osmium tetroxide for 30 min and then rinsed three times. Embryo
were then dehydrated in a graded ethanol series (15% increments every 30
min). After two changes (15 min each) of 100% ethanol, embryos were sub
jected to critical-point drying, mounted on stubs, sputtered with gold-palla
dium, and immediately observed with a Hitachi model S530 SEM.
Light microscopy—Embryos were fixed in glutaraldehyde and serially de-
hydrated in ethanol as described. A trace amount of safranin was added to
the first 100% ethanol rinse, and specimens were incubated at room temper-
ature for 15 min to introduce color into the normally translucent embryos for
visualization in the resin for micro-orientating and sectioning. After two quick
rinses with 100% ethanol to remove excess safranin, embryos were transferred
to cold 100% ethanol for another 15 min. Dehydrated embryos were embed-ded in flat molds in JB4-Plus plastic resin (Polysciences, Pennsylvania, USA),
as per manufacturer’s instructions. The embryos were carefully oriented in
the molds to facilitate subsequent median longitudinal sectioning. Small
blocks of resin, approximately 1–4 mm3, each containing an embryo or cluster
of embryos, were excised from the molds and attached to the end of solidified
resin blanks with Superglue in an orientation for cutting of median longitu-
dinal sections. Specimens were reoriented several times during facing and
sectioning to obtain median longitudinal sections. Sections approximately 5
m thick were cut with glass knives and secured to slides by heating at 70 C.
The sections were stained with periodic acid-Schiff’s reagent (PAS) to contrast
polysaccharides (e.g., cell walls and amyloplasts) and counter stained with
naphthol blue black to visualize proteins.
RESULTS
Embryogenic cultures—Regardless of solid or liquid me-dium origin, an embryogenic culture typical of grapevine de-veloped when PEM were transferred to and grown on solidi-fied X6 medium. Because embryogenic cultures grew nonsyn-chronously, somatic embryos in different stages of develop-ment were present at any given time (Figs. 1, 3, 10). Somaticembryos were white and opaque (Fig. 1). Despite the generalsimilarity among embryogenic cultures, SPEM- and LPEM-derived somatic embryos differed in their structural and func-tional characteristics.
SPEM-derived somatic embryos—The SPEM-derived so-matic embryos typically developed with enlarged (often su-pernumerary) cotyledons (Figs. 1, 5). When isolated from the
underlying cell mass, a narrowed point of attachment reminis-cent of a suspensor was apparent (Fig. 2). As a result of thislimited attachment, such embryos were sessile with respect tounderlying tissue (Fig. 3). Repetitive embryogenesis occurredin the immediate subtending tissue (Fig. 4, arrows), resultingin typical groupings of somatic embryos, which often appearedto be fused to each other at their bases, but were, in fact,attached only through this embryogenic tissue. The shoot api-cal meristem of solid-medium-derived somatic embryos typi-cally was reduced in size, concave in cross section, and com-posed of 2–3 cell layers (Fig. 6). The root apical meristemappeared to be well developed, with typical embryonic vas-culature spanning from proximal to distal regions of the em-bryo body (Figs. 5, 7).
LPEM-derived somatic embryos—The LPEM-derived so-matic embryos differed from their SPEM-derived counterpartsin that they had an enlarged suspensor apparatus, which wasapparent from the earliest stages of development (Fig. 8) andpersistent through maturity (Figs. 9–12). Such embryos de-veloped without attachment/fusion to each other and often be-came perched well above subtending tissue (Figs. 8, 9, 10).The suspensor was multiseriate, often exceeding seven celllayers in width (Fig. 11). In early stages, a clear demarcationbetween the suspensor region and embryo proper could not beestablished. Like SPEM-derived somatic embryos, LPEM-de-rived embryos also often had supernumerary cotyledons, butthey were distinctly smaller in size (Fig. 12). The shoot apical
meristem was better developed than that of SPEM-derived em-bryos, being flat-to-convex in cross section and composed of four or more cell layers (Figs. 12, 13). The root apical meri-stem and embryonic vasculature did not differ substantiallyfrom SPEM-derived somatic embryos (Figs. 12, 14).
Development of somatic embryos—Figures 15–24 recon-struct the developmental sequence of LPEM-derived somaticembryos, based upon individually selected specimens. Earlystages of embryogenesis occurred rapidly from LPEM. Smallglobular somatic embryos were observed within 2 wk afterplating LPEM onto solid medium. As embryos reached theglobular stage, they protruded above subtending tissue on anelongate suspensor (Fig. 15), and the distinction between thesuspensor and the embryo proper became clear. As the embryobody continued to enlarge, it initially maintained a radial sym-metry (Figs. 15–18). Embryos up to this stage were approxi-mately 500 m or less in length, of which more than 60%was the suspensor. Assumption of bilateral symmetry occurredas early as 3 wk after plating LPEM onto solid medium, as
evidenced by differential enlargement from two distinct sitesof accelerated growth; this signaled the start of cotyledon de-velopment and resulted in the appearance of a concave dimplein the distal end of the embryo (Figs. 19, 20, arrows). Furthercell divisions and enlargement led to elongation of the embryobody and early definition of the cotyledons (Fig. 20, arrow).The cotyledonary poles rapidly outgrew the central region,leaving a distinct notch between them (Fig. 21, arrow). Thiscorresponded to a typical ‘‘heart stage’’ of development. Also,at this stage, the dimple became convex in many LPEM-de-rived somatic embryos from cell divisions in the shoot apicalmeristem (Fig. 22, arrow). The cotyledons differentiated fur-ther, completely enveloping the shoot apical meristem, and thehypocotyl continued to enlarge as embryos reached the ‘‘tor-pedo stage’’ of development. Embryos at this stage were 1.0–
2.5 mm in length. Within 5 wk of plating, well-defined, mor-phologically correct dicotyledonous somatic embryos were ap-parent (Fig. 23).
Mature LPEM-derived somatic embryos (Fig. 24) typicallydiffered from SPEM-derived somatic embryos (Fig. 25) bytheir large, persistent suspensors and smaller cotyledons. TheSPEM-derived somatic embryos were nearly identical in mor-phology to zygotic embryos in that they both had heart-shapedcotyledons typical of grapevine; however, somatic embryoswere noticeably larger than zygotic embryos, which tended tobe flattened (compare Figs. 25, 26). As with some SPEM-derived somatic embryos, zygotic embryos had a rudimentarysuspensor (Fig. 26). The relative length of hypocotyl-to-coty-ledon varied among the three embryo types. In SPEM-derivedsomatic embryos, the cotyledons were longer than the hypo-cotyls. In LPEM-derived somatic embryos, the hypocotylswere longer than the cotyledons, whereas in zygotic embryosthe lengths were approximately equal.
DISCUSSION
In SPEM-derived culture material, typical development of grapevine somatic embryos was nearly identical to that of zy-gotic embryos (Gray, 1995). Somatic embryos have more mor-phological variation, such as pluricotyly, than zygotic embry-os, which are smaller and flattened, probably from differencesin the in vitro and in vivo (seed) environments. In the seed,the developing zygotic embryo is physically compressed (Gray
July 2003] 975JAYASANKAR ET AL.—SOMATIC EMBRYO ONTOGENY IN GRAPEVINE
Figs. 1–7. The solid-medium-derived somatic embryogenesis in grapevine. 1. Embryogenic culture maintained on solid medium with white, opaque somaticembryos. Bar 520 m. 2. Isolated embryo with well-defined cotyledons and rudimentary suspensor. Bar 125 m. 3. Asynchronous development of somaticembryos. Note sessile nature of embryos relative to underlying tissue and pluricotyly of large embryo in upper center. Bar 340 m. 4. Attachment of somaticembryo to embryogenic tissue. Note secondary embryogenesis (arrows) and lack of defined suspensor. Bar 135 m. 5. Mature somatic embryo with largecotyledons and well-developed embryonic vasculature. Bar 275 m. 6. Concave shoot apical meristem typical of solid-medium-derived somatic embryosBar 65 m. 7. Base of mature somatic embryo detailing vasculature and root apical meristem. Bar 90 m.
Figs. 8–14. The liquid-medium-derived somatic embryogenesis in grapevine. 8. Somatic embryos with prominent suspensors originating from liquid-medium-derived proembryogenic masses (LPEM), transferred to solid medium for embryogenesis. Bar 200 m. 9. Pluricotyledonous somatic embryo connected toPEM by a suspensor. Bar 260 m. 10. Asynchronous development of somatic embryos. Note that embryos of all stages present exhibit elongated suspensors.Bar 400 m. 11. Attachment of heart-stage embryo to suspensor. Note that the demarcation between suspensor and embryo proper is not evident at thisstage. Bar 185 m. 12. Mature somatic embryo with small cotyledons, detailing embryonic vasculature. Bar 160 m. 13. Convex shoot apical meristemtypical of liquid-medium-derived somatic embryos. Bar 58 m. 14. Base of mature somatic embryo showing presence of suspensor. Bar 50 m.
July 2003] 977JAYASANKAR ET AL.—SOMATIC EMBRYO ONTOGENY IN GRAPEVINE
Figs. 15–26. Developmental morphology of liquid-medium-derived somatic embryos (15–24) compared with solid-medium-derived somatic embryo (25and zygotic embryo (26). 15. Initially, the suspensor (Su) is the most prominent part of the developing embryo apparatus. 16. As the embryo body (Em) beginto differentiate, it becomes more obvious. 17–18. Continued development results in a late-stage, globular embryo. 19. A concave dimple (arrow) in the distaend of the embryo is the first sign of cotyledons and development of shoot apical meristem. 20. The embryo body begins to elongate and the dimpled areabecomes more prominent (arrow). 21. Cotyledon enlargement (arrow) results in a distinct cleft. 22. The dimpled area (arrow) becomes convex as the shooapical meristem differentiates. 23. Continued growth results in an elongated embryo with cotyledons that enclose the shoot apical meristem. 24. A matureliquid-medium-derived somatic embryo exhibits smaller cotyledons and a persistent suspensor (Su), when compared to solid-medium-derived somatic embryo(25) or a zygotic embryo (26). The cotyledons of solid-medium-derived somatic embryo and zygotic embryo are typically heart-shaped; however, the zygoticembryo is smaller and laterally flattened in comparison. Bars 500 m.
and Purohit, 1991a). Developmentally, both SPEM-derived so-matic embryos and their zygotic counterparts exhibit physio-logical dormancy; they do not germinate to undergo plant de-velopment without a dormancy-breaking pretreatment.
It was surprising to discover that two morphologically dis-tinct types of grapevine somatic embryos could be producedon solid medium; the only apparent difference was the initialculture environment in which the PEM were grown. Whencompared to somatic embryos from SPEM, somatic embryosfrom LPEM possessed a large, persistent suspensor, smallercotyledons, and a more anatomically defined shoot apical mer-istem. Developmentally, LPEM-derived somatic embryos also
differed in that they did not exhibit dormancy (i.e., they ger-minated readily without a pretreatment to break dormancyand produced a higher percentage of plants compared toSPEM-derived somatic embryos (60% vs. 20%). It itempting to link structural differences to the observed devel-opmental and physiological differences between solid- and liq-uid-medium-derived somatic embryos.
In contrast to the persistent and robust suspensor of LPEMderived somatic embryos, the typical suspensor in zygotic em-bryos is programmed to die when the embryo proper reachesthe torpedo stage (Yeung and Meinke, 1993). The suspensorsin LPEM-derived somatic embryos are often long, multiser
iate, and relatively massive. Their cytoplasm-rich cells suggestthat they are metabolically active, perhaps in supplying nutri-ents or modulating the hormonal balance in the developingsomatic embryos (Souter and Lindsey, 2000). In contrast, thesuspensors in mature zygotic embryos, when detected, areonly a few cells long. The cells are often highly vacuolated,
suggesting that they are not cytologically active. The suspen-sor originally was thought to be an embryonic accessory thatwas necessary for positioning the developing embryo in theembryo sac. However, studies with common bean (Phaseolusvulgaris L.) have shown that it is necessary for active proteinsynthesis in developing zygotic embryos (Brady and Walthall,1985). Later, Nagl et al. (1991) demonstrated the synthesis of storage proteins in the suspensor cells of beans.
We earlier observed that LPEM-derived somatic embryosgerminate precociously without undergoing the dormancy typ-ical of grapevine zygotic embryos (Jayasankar et al., 1999),whereas somatic embryos derived from SPEM exhibit dor-mancy (Gray, 1989; Gray and Purohit, 1991a, b), and requirea pretreatment to germinate (Gray and Mortensen, 1987; Grayand Purohit, 1991b). Such dormancy in grapevine somatic em-bryos is attributed to ABA accumulation, which typicallyreaches a peak during maturation (Rajasekaran et al., 1982).However, exogenous GA3-induced grapevine somatic embryogermination, and the concentration of GA-like compounds alsoincreased during cold stratification (Takeno et al., 1983; Pearceet al., 1987). The persistent suspensor in LPEM-derived so-matic embryos may be a reason for the lack of dormancy andconcomitant precocious germination. Suspensors also are a siteof gibberellin synthesis. Very high levels of GA-like substanc-es have been found in suspensors of Trapeolum (Picciarelli etal., 1984), Cytisus (Picciarelli et al., 1991), and Phaseolus (Pi-aggesi et al., 1989). Studies in P. coccineus L. have shownthat GA moves from suspensor to the embryo proper duringembryo maturation (Alpi et al., 1975, 1979). We hypothesize
that gibberellins are produced abundantly and continuously inthe suspensors of LPEM-derived somatic embryos and thenare transported to the embryo proper, leading to precociousgermination.
Because certain nutrients and growth regulators are specif-ically produced only in the suspensor, lack of an adequatesuspensor may deprive somatic embryos of growth factorsneeded for proper shoot apical meristem development. Thislack is suggested by the poor apical meristem developmentexhibited by SPEM-derived somatic embryos, which have pre-mature vacuolation in the meristematic region. Embryos withsuch a defective meristem are not likely to develop into acomplete plant. Premature vacuolation also has been implicat-ed in the failure to develop a functional meristem in carrot(Nickle and Yeung, 1993) and canola (Yeung et al., 1996).Such poor development of the meristematic region in manySPEM-derived somatic embryos may be a factor leading tolow efficiency of plant regeneration.
Plant regeneration using somatic embryogenesis is neces-sary to produce novel genotypes after genetic manipulation.This necessity is particularly evident considering that manyplant transformation systems, including grapevine (e.g., Li etal., 2001), employ embryogenic culture systems as a sourceof target cells. However, little attention has been paid to thedevelopmental processes leading to regeneration of a wholeplant from a single somatic cell. Yeung and Stasolla (2000)proposed that the capacity of a somatic embryo to regenerateinto a plant largely depends on the quality of its shoot apical
meristem. Our research extends that viewpoint to suggest thatproper development of the shoot apical meristem depends, inturn, on the presence of certain embryonic organs, particularlythe suspensor.
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