Plant Physiol. (1983) 73, 803-8080032-0889/83/73/0803/06/$00.50/0

Endogenous Gibberellin-Like Substances in Somatic Embryos ofGrape ( Vitis vinifera x Vitis rupestris) in Relation toEmbryogenesis and the Chilling Requirement for SubsequentDevelopment of Mature Embryos'

Received for publication February 8, 1983 and in revised form May 12, 1983

KIYOTOSHI TAKENO, MASAJI KOSHIOKA, RICHARD P. PHARIS2, K. RAJASEKARAN, ANDMICHAEL G. MULLINSDepartment ofBiology, University ofCalgary, Calgary, Alberta T2N IN4 Canada (K. T., M. K., R. P. P.);and Department ofAgronomy and Horticultural Science, University ofSydney,Sydney, New South Wales 2006, Australia (K. R., M. G. M.)

ABSTRACT

Endogenous gibberellin (GA)-like substances were examined in sus-pension cultures of somatic embryos of a hybrid grape ( Vitis vinifera xVitis rupestris) during embryogenesis, and in mature embryos chilled at4°C, and subsequently incubated at 26°C with and without abscisic acid(ABA). The extract was separated into a nonpolar fraction (would containGA-precursors) a fraction that would contain free GAs; and a highlyH20-soluble fraction (would contain GA glucosyl conjugates and verypolar free GAs). Quantitation after SiO2 partition chromatography wasaccomplished by microdrop and immersion dwarf rice bioassays. Asembryogenesis developed, the free and highly H20-soluble GA-like sub-stances, expressed on a dry weight basis, decreased (however, theyincreased on a per embryo basis). Chilling at 4°C for 1 week greatlyincreased activity of free GA-like substances (per g dry weight and perembryo), it then declined over the next three weeks of chilling. Activity(per g dry weight and per embryo) in the H20-soluble fraction declinedthroughout chilling. Activity in the GA-precursor fraction, however,increased steadily with chilling (per g dry weight and per embryo).Incubation at 26°C after chilling enhanced activity in the free GA andH20-soluble fractions (per g dry weight and per embryo), but activity inthe GA-precursor fraction dropped dramatically. Incubation at 26°C with(±) ABA after chilling prevented germination and maintained high activ-ity for GA precursors and less polar free GAs and low activity in thepolar free GA and H20-soluble fractions.

Kaurene and kaurenoic acid were characterized in the GA-precursorfraction of chilled embryos by gas-liquid chromatography-mass spec-trometry (GLC-MS). The existence of GA4 and GA, in ABA-treated,chilled embryos was also confirmed by GLC-MS.

Development of somatic embryos is regulated by phytohor-mones in several plant species. Thus, auxin and cytokinin arerequired for development of embryos in the somatic culture ofcelery and grape (1, 22), although these same hormones can

' Supported by Natural Sciences and Engineering Research Council ofCanada Grant A-2585 to R. P. P. and a grant from Rural CreditsDevelopment Fund, Reserve Bank of Australia, to M. G. M.

2 To whom requests for reprints should be sent.

inhibit the formation ofembryos in carrot (5-7). However, GAs3appear to inhibit somatic embryogenesis in mandarin orange,carrot, and anise (5, 19, 24) and inhibition of somatic embryodevelopment in anise and carrot is correlated with high levels ofendogenous GA, (a native GA [19]) and a reduced rate of [3H]GA, metabolism (18).Germination of mature somatic embryos (e.g. plantlet for-

mation) can also be regulated by GA3. In grape, GA3 is requiredfor germination of the mature somatic embryo if chilling is notgiven (13, 22). These data suggest that endogenous GAs mayhave important regulatory roles in development and germinationof somatic embryos. The present work examines qualitative andquantitative changes of endogenous GA-like substances duringembryogenesis, and subsequent development ofmature embryosin somatic suspension cultures of grape.

MATERIALS AND METHODS

Plant Materials. Somatic embryos were derived from the callusformed by anthers of a hybrid grape Vitis vinifera L. x Vitisrupestris Scheele cultured in a liquid medium (17) supplementedwith 1 gM BA and 5 ,AM 2,4-D (22). Harvests of developingembryos were made at the globular, heart-shaped, and torpedo-shaped plus mature stages. Mature embryos were chilled at 4°Cfor 1, 2, or 4 weeks, then harvested. Mature embryos chilled for4 weeks were then incubated at 26°C for 10 d with and without10 Mm (±) ABA, then harvested. The use ofABA in this mannerwill inhibit germination of the embryos and the subsequentproduction of plantlets (23). Harvested embryos were rinsed incold H20, frozen in liquid N2, freeze-dried, and stored at -40°Cuntil extracted.

Extraction, Separation, and Bioassay of GA-Like Substances.The freeze-dried embryos were extracted with 80% aqueousMeOH (below 0°C, at least 10 ml/g dry weight) twice in a mortarand pestle, and the extract was purified by a procedure ( 11) usingsmall preparative columns packed with 55 to 105 Mm particlesize C1,8 monochlorooctadecyclysilane material on Porasil B (Wa-ters Scientific Ltd., Mississauga, Ontario, Canada). The 80%MeOH extract was passed through a column (4 g C,8 material/gdry weight of tissue) and this column was washed with 80%MeOH of the same volume as the extract (80% MeOH eluate)

3Abbreviations: GA, gibberellin; MeOH, methanol; EtOAc, ethylacetate; FID, flame ionization detector; Rt, retention time.

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Plant Physiol. Vol. 73, 1983

and subsequently with the same volume of 100% MeOH. The80% MeOH eluate was diluted with H20 to yield a 50% MeOHsolhrtion, which was then passed through a second column (asabove), followed by a wash of an equal volume of 50% MeOH(these were called the 50% MeOH eluate). The second columnwas subsequently washed with 100% MeOH in the same manneras above. The 100% MeOH eluates of both columns were com-bined to yield a nonpolar fraction. This fraction would containthe GA precursors kaurene and kaurenoic acid (11). The 50%MeOH eluate was evaporated in vacuo at 35°C and the residuedissolved in EtOAc:MeOH (1:1) to give a fraction that willcontain free GAs plus a portion of the GA glucosyl conjugate(s)(1 1). This fraction was then chromatographed on a SiO2 partitioncolumn (4). Fifteen 33-ml fractions were collected from the SiO2partition column, plus a final wash of the column with 100%MeOH (to elute the EtOAc:MeOH-soluble GA-glucosyl conju-gates [11]). The EtOAc:MeOH (1:1)-insoluble residue was dis-solved in H20 (11) for bioassay by the immersion method (16).This fraction would contain the rest of the GA-glucosyl conju-gates, and very polar free GAs such as GA32, if present.The nonpolar fraction and the 15 fractions from the SiO2

partition column were bioassayed in serial dilution (1/30 and I/100 [0 plants] for nonpolar fraction; 1/10 [5 plants], 1/30 and1/I00 [10 plants] for free GA fractions) using the microdropassay on dwarf rice cv Tan-ginbozu (14) (Table II). The MeOHwash of the Sio2 partition column, and the H20-soluble fractionof 50% MeOH eluate were bioassayed in serial dilution (1/3 and1/10) by immersion assay on dwarf rice Tan-ginbozu (16). Re-sults of both immersion assays were combined and called 'highlyH20-soluble fraction' (Table II). There were four zones of bio-logical activity from the SiO2 partition column, zone I (Rt ofGA9), zone II (Rt of GA4/7/20), zone III (Rt of GAI/3/19), and zoneIV (Rt of GA8 or longer) (Table II). Gibberellin A3 was alwaysassayed simultaneously over a wide range (10-2 to 100 ng/riceplant) as a standard, and biologically active fractions which weresignificantly (P c 0.05) different from the control had theiractivity expressed in GA3 equivalents per g dry weight and perembryo.

Identification of GA-Like Substances. The free GA- and highlyH20-soluble GA-like substances were insufficient in quantity toafford characterization other than that provided by co-chroma-tography of known standards ([4, 1 1] noted in Table II). How-ever, the residual GA-like substances in the nonpolar fraction(Table I) were further subjected to HPLC on a C18 reverse phaseg-Bondapak column (3.9 x 300 mm) (Waters Scientific Ltd.)eluted with a gradient of MeOH:H20 (1% acetic acid) (10) at aflow rate of 2 ml/min. The 2-ml HPLC fractions from samples7 and 8 (Tables I and II) were bioassayed by the microdrop assay(as above). Active fractions were methylated using diazomethane,then derivatized with TMSi [N, O-bis(trimethylsilyl)trifluoro-acetamide], and subjected to GLC (Packard model 430, FID at250C) on three packed columns; 3% OV-10l (2.5 mm x 2 m,220°C), 1% XE-60 (2.5 mm x 2 m, 205C), and 2% QF-l (2.0mm x 2 m, 195°C) with He carrier gas flowing at 50 ml/min(Table III). Subsequently, GLC-MS was accomplished on a Hew-lett-Packard model 5990A for GLC-FID peaks that could haverepresented GAs or GA precursors.

RESULTSGrowth of Embryos and Their Germination in Response to

Chilling. The changes in dry weight (per embryo) and percentagegermination at various stages of development and/or after var-ious times of chilling, are given in Table I. There was a 19-foldweight increase between the globular and mature stages, nochange in weight for the first 2 weeks of chilling, and then amodest (1.1-fold) increase between the 2nd and 4th weeks ofchilling. Immature embryos (without chilling) show virtually no

germination, and complete germination was obtained only with4 weeks of chilling (Table I). The addition of 10 ,M (±) ABA tothe medium prevents germination (at 26°C) of mature embryoschilled for 4 weeks (Table I).Changes of GA-Like Substances during Embryogenesis. The

levels ofGA-like substances in embryos at globular, heart-shaped,and torpedo-shaped plus mature stages are given in Table II(samples 1-3) on both a dry weight and per embryo basis. Itshould be noted here, and remembered in later sections, that theresults in Table II are expressed in GA3 equivalents. Kaureneand kaurenoic acid are, for example, <1/100 as active as GA3(15), hence expression as GA3 equivalents will underestimateactual amounts of GA precursors and many free GAs and GAglucosyl conjugates.

Per g dry weight activity in the nonpolar fraction that willcontain GA precursors increased up to the heart-shaped stage,then dropped appreciably at the torpedo-shaped plus maturestage. A similar trend was noted for activity in the highly H20-soluble fraction (which will contain GA glucosyl conjugates).However, the level of free GA-like substances decreased as em-bryogenesis proceeded, almost disappearing by the torpedo-shaped plus mature stage.On a per embryo basis, however, the trends tended to be just

the opposite. Ten- and 12-fold increases in activity occurredduring embryo development for the GA-precursor and highlyH20-soluble fractions. Similarly, both less-polar (except GA9-like) and polar free GA-like substances increased between theglobular and heart-shaped stages, but decreased to almost unde-tectable values at the torpedo-shaped plus mature stage. Thus,by the mature stage, but prior to chilling, each embryo hasincreased amounts of GA precursor-like substances, and alsoincreased levels of highly H20-soluble GA-like substances, butvery low levels of free GA-like substances (Table II).Changes of GA-Like Substances in Mature Embryos during

Chilling. The mature embryos were chilled at 4°C to allow furtherdevelopment; at least 2 weeks is required to induce normalgermination ([22] and Table I). Levels of activity of GA-likesubstances in embryos chilled for 1, 2, and 4 weeks are given inTable II (samples 4-6).The activity of the GA-precursor fraction increased dramati-

cally as chilling proceeded from weeks 1 to 4, both per g dryweight and per embryo. Conversely, activity in the highly H20-soluble fraction decreased as chilling progressed. The total activ-ity of free GA-like substinces did not change during chilling(although it increased from virtually a nil level just prior tochilling, to a modest level after 1 week of chilling). However,within the fraction that would contain free GAs there was atendency toward a decrease in less-polar GA-like substances (tonil values by week 4 of chilling) and an increase in more polarGA-like substances with chilling.

Thus, during chilling the biological activity in the fraction thatwould contain GA-precursors increased dramatically, activity inthe fraction that would contain GA-glucosyl conjugates de-creased appreciably, and total activity in the free GA fraction didnot change appreciably.GA-Like Substances in Mature Embryos after Chilling, with

and without ABA. Mature embryos chilled at 4°C for 4 weekswere incubated at 26°C for 10 d to allow germination. However,if 10 ,M (±) ABA is present in the medium germination will notproceed even though the chilling requirement has been met ([23]and Table I).Embryos whose chilliing requirement had been met showed an

appreciable drop in activity in the GA-precursor fraction, bothper g dry weight and per embryo (Table II, sample 7). Thisnonpolar fraction was chromatographed subsequently on C18HPLC prior to characterization of kaurene and kaurenoic acidby GCC-MS (Fig. 1). No free GA activity was apparent in the

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GIBBERELLINS IN SOMATIC GRAPE EMBRYOS

Table I. Growth and Germination of Somatic Embryos ofGrape (Vitis vinifera x Vitis rupestris)

Sample Developmental Sample Size UsedaNmope Stage and/or for GA Analysis Dry Wt GerminationaNo.

Treatment (Dry Wt)

mg/no. ofembryos mg/ %embryo

I Globular 275/5970 0.046 02 Heart-shaped 160/1000 0.16 03 Torpedo-shaped plus mature 510/586 0.87 2

4 Mature, 1 week at 4°C 150/172 0.87 625 Mature, 2 weeks at 4°C 130/149 0.87 836 Mature, 4 weeks at 4C 205/215 0.95 96

7 Mature, 4 weeks at 4°C, then 10d at 26°C 405/425 0.95 96

8 Mature, 4 weeks at 4°C, then 10d at 260C in presence of (±)ABA 475/100 4.8b 0

a Based on comparable experiments with different embryo samples, germination examined at 26°C, recordedat day 60 after planting the embryos in assay medium.

b Increase in dry weight, relative to sample 7, is due to expansion of cotyledons in presence of (±) ABA.

Table II. Endogenous Levels ofGA-Like Substances Detected by DwarfRice Bioassays in Somatic Embryos ofGrape (Vitis vinifera x vitisrupestris)

GA Level

Nonpolar Fraction containing free GA-like substances Highly H20-Developmental fraction soluble

Sample Stage and/or fractionNo. Treatment Kaurene/ (GA-glucosyl

kaurenoic GA9 GA4-Rt GAI/3/19 GA8-Rt Total conjugate-likekaurenoice -Rt -Rt -Rt or very polarfree acids)

ng GA3 eq/g tissue dry wt (pg GA3 eq/embryo)1 Globular 13 (0.6) 6.7 (0.31) 15 (0.69) 16 (0.74) 6.2 (0.29) 44 (2.0) 93 (4.3)2 Heart-shaped 23 (3.7) -a ( ) 5.6 (0.9) 15 (2.4) 7.4 (1.2) 28 (4.5) 170 (27)3 Torpedo-shaped 6.5 (5.7) (-) (-) (-) 0.3 (0.26) 0.3 (0.26) 60 (52)

4 Mature, 1 week at 4C 4.2 (3.7) 3.3 (2.9) 6.0 (5.2) 4.3 (3.7) 3.3 (2.9) 17 (15) 40 (35)5 Mature, 2 weeks at 4°C 6.7 (5.8) (-) 4.2 (3.7) 6.4 (5.6) 4.9 (4.3) 16 (14) 37 (32)6 Mature, 4 weeks at 4°C 20 (19) (-) (-) 11 (10) 7.3 (7.0) 18 (17) 22 (21)

7 Mature, 4 weeks at 4°C, then 10d at 26°C 3.0 (2.9) (-) 13 (12) 15 (14) 12 (11) 40 (37) 49 (47)

8 Mature, 4 weeks at 4C, then 10d at 26°C in presence ofABA 22 (100)b 4.2 (20) 7.5 (37) 3.3 (16) (-) 15 (73) (-)

a Undetectable.b Includes activity from free GA4 and GA9, which were anomalously characterized in this fraction by GLC-MS (See "Results" and Table III).

assay ofthe HPLC fractions (see Fig. 4 in [11 ]). However, activityin the highly H20-soluble fraction increased, as did activity inboth the less-polar and polar regions ofthe SiO2 partition column(Table II).However, embryos whose germination was arrested by ABA-

maintained high levels of activity in the GA precursor fraction,although an unknown proportion of this activity may be ac-counted for by less polar free GAs such as GA4 and GA9 (TableIII). THese free GAs were detected anomalously in this fraction,probably due to tailing caused by a pH below 6.0 in the 50%MeOPH eluting solvent (see Table II in [ 1 1]). Unfortunately, pHof the 50% MeOH eluting solvent was not monitored, and thehigh dry weight in sample 8 (see Table I) may have contributedan excess of acidic compounds to this extract.

Characterization of Endogenous GAs. The GA-precursor frac-

tion from embryos incubated at 26°C after chilling (Tables I andII, samples 7 and 8) yielded sufficient biological activity forfurther characterization. This fraction (Table II, sample 7) waschromatographed on analytical C18 HPLC (10), yielding a singlebiologically active peak whose Rt (60-64 min) was coincidentalwith kaurene and kaurenoic acid. Methyl derivatives of thisfraction were subjected to GLC, yielding two FID peaks whoseRt were coincidental with kaurene and kaurenoic acid (7.2 and14.3 min, respectively). Their identity was subsequently con-firmed by GLC-MS (Fig. 1).The GA-precursor fraction from ABA-treated embryos (Table

II, sample 8) was subjected to HPLC, which yielded biologicalactivity not only in the kaurene/kaurenoic acid region (as notedabove), but also significant activity in fractions where free GAswill elute (10). Several of these biologically active peaks were

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mleFIG. 1. GLC-MS profiles of two peaks in the GLC-FID trace (peaks I and II; Rt, 7.2 and 14.3 min, respectively) for the biologically active

fractions in the HPLC (Rt 60-64 min) of the 100% MeOH eluate from the preparative columns of Cl8 material (11) (e.g. nonpolar fraction) inextracts from embryos incubated at 26°C for 10 d after being chilled at 4C for 4 weeks (Table II, sample 7), and GLC-MS profiles of authenticstandards of kaurene and kaurenoic acid.

subjected to GLC-FID analysis. A GA4-like substance was de-tected (Rt 5.8, 6.8, and 9.4 min on 3% OV-101, 1% XE-60, and2% QF-1, respectively; Rt of standard GA4 is 5.9, 7.0, and 9.4min on the corresponding columns), as was a GA9-like substance(Rt 4.6, 5.4, and 6.3 min on the respective columns as above;4.5, 5.6, and 6.4 min on the corresponding column for standardGA9). This identity was further confirmed by GLC-MS (TableIII). Their anomalous presence in this nonpolar fraction is dis-cussed above.

DISCUSSIONThe level of endogenous GA-like substances, especially free

GA-like substances, was markedly reduced (on a dry weight

basis) in torpedo-shaped plus mature stage embryos of grape(Table II). A similar tendency was noted for GAI and other polarGAs in somatic embryos of anise and carrot (18). These findingsare consistent with the fact that somatic embryogenesis can beinhibited by exogenously applied GAs (5, 18, 24) and imply aregulatory role, albeit inhibitory, for high levels ofGAs in somaticembryogenesis. In somatic cultures of carrot, development ofembryos was not influenced by exogenous GA3 at the globularand early heart-shaped stages, whereas it was inhibited at heart-shaped and torpedo-shaped stages (5). This change in responseto GA3, and the relatively high levels of endogenous GA-likesubstances at early stages of embryogenesis (Table II), suggeststhat normal somatic embryogenesis may involve high levels of

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GIBBERELLINS IN SOMATIC GRAPE EMBRYOS

Table III. GLC-MS ofMe, TMSi-Derivatives ofGibberellin-Like SubstancesDerivatives were purified by HPLC (Rt 34-40 min) (10) in the 100% methanol eluate of a preparative

column of C18 material (I 1) (e.g. nonpolar fraction) of a methanolic extract of mature somatic grape embryosincubated at 26°C in the presence of(±) ABA after being chilled at 4°C for 4 weeks (Table II, sample 8).

Constituent Ions (% Relative Intensity)

Substance Ia 418 (5) 386 (3) 358 (2) 328 (7) 296 (6)289 (8) 284 (20) 277 (100) 225 (21)224 (21) 73 (44)

Standard GA4 418 (9) 386 (6) 358.(2) 328 (16) 296 (13)289 (17) 284 (47) 277 (11) 225 (50)224 (60) 73 (100)

Substance Ib 330 (7) 298 (34) 286 (6) 270 (29) 243 (25)227 (25) 226 (30) 91 (38) 28 (100)

Standard GA9 330 (12) 298 (64) 286 (9) 270 (57) 243 (49)227 (44) 226 (61) 91 (100) 28 (11)

a Whose Rt on GLC is coincidental to that of GA4 on 3% OV-101, 1% XE-60, and 2% QF-1 columns.bWhose Rt on GLC is coincidental to that ofGA9 on 3% OV-101, 1% XE-60, and 2% QF-1 columns.

free GAs initially, followed by a rapid decrease of free GAs. Infact, the 2,4-D suppression of continued embryogenesis of so-matic anise and carrot embryos is accompanied by high levels ofGA, and a reduced ability to metabolize [3H]GA, (18).Mature grape embryos also have somewhat reduced levels (per

g dry weight) of activity in fractions that would contain GA-precursors and GA glucosyl conjugates (Table II), relative toheart-shaped embryos, implying a rapid rate of metabolism inthe latter stages of embryo development. However, when ex-pressed on a per embryo basis, the trend is reversed. If theamount of GA precursor-like substance present in each embryo(rather than per unit dry weight) is important in future events,and if the amount of biologically active, highly H20-solublesubstance per embryo neither reflects past rapid metabolism offree GAs into very polar GAs orGA conjugates, or was indicativeof a pool of biologically active GA-conjugates that could behydrolyzed for use in subsequent growth and germination, thenexpression of endogenous growth substances per embryo mightbe more informative than expression per unit dry weight.Kaurene and kaurenoic acid were identified by GLC-MS in

somatic grape embryos (Fig. 1), along with GA4 and GA9 (TableIII). Although a wide variety of free GAs have been characterizedin several tissues of many plant species (2), relatively few char-acterizations (except by metabolism in cell-free systems [8]) ofthese two GA-precursors in higher plants have been made (2, 9,12, 20, 21). The present work used small preparative columns ofC18 material -- Sio2 partition to separate the component partsofthe extract (1 1). This procedure allowed us to save the fractioncontaining kaurene and kaurenoic acid in a relatively purifiedform (11) so that bioassay, HPLC -. bioassay, and GLC-MScould be readily accomplished.

Chilling the mature embryos results in a rapid (1 week) increasein free GA-like substances, although activity in the GA-precursorfraction and the highly H20-soluble fraction remains low. How-ever, by weeks 2 and 4 of chilling, activity in the GA precursorfraction has increased substantially (Table II). Finally, the dropin activity ofthe GA-precursor fraction, coupled with the increasein activity in the free GA fraction following chilling (Table II),indicates that the increased free GA-like substances may originatefrom a build-up in GA-precursors during the latter stages ofchilling, rather than from possible hydrolysis of GA-glucosylconjugates.The finding that the levels of free GA-like substances may be

lowered by ABA (Table II) is consistent with the findings (22,

23) that: (a) germination of chilled somatic grape embryos isinhibited by exogenous ABA; (b) levels of endogenous ABAincrease during embryogenesis; (c) chilling leads to a markedreduction in ABA level in somatic grape embryos; and (d)endogenous ABA levels are high in embryos which do not receivechilling.The present findings, dealing with GA-like substances, along

with previous reports (20, 21) dealing with ABA, imply that bothGAs and ABA have important roles in regulation ofdevelopmentand germination in somatic grape embryos.

LITERATURE CITED

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2. APPLETON RA, R MCCRINDLE, KH OVERTON 1968 The alternative occurrenceof (-) kaurene or (+)-phyllocladene in the leaves Cryptomeria japonica D.Don. Phytochemistry 7: 135-137

3. BEARDER JR 1980 Plant hormones and other growth substances-their back-ground structures and occurence. In J MacMillan, ed, Hormonal Regulationof Development. I. Molecular Aspects of Plant Hormones. Springer-Verlag,Berlin, pp 9-112

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