© CSIRO 2003 10.1071/AR03053 0004-9409/03/090869

www.publish.csiro.au/journals/ajar Australian Journal of Agricultural Research, 2003, 54, 869–876

CSIRO PUBLISHING

Improved in ovulo embryo culture for stenospermocarpic grapes(Vitis vinifera L.)

S. M. LiuA,B, S. R. SykesA,C, and P. R. ClingelefferA

ACSIRO Plant Industry, Horticulture Unit, PMB, Merbein, Vic. 3505, Australia.BCurrent address: DPI, Institute of Sustainable Irrigated Agriculture, Private Bag 1,

Ferguson Road, Tatura, Vic. 3616, Australia.CCorresponding author; email: steve.sykes@csiro.au

Abstract. In ovulo embryo rescue techniques have been used to recover new hybrids from seedless × seedlessgrape crosses. This study was conducted to increase efficiency by investigating effects of genotype, medium, andovule removal age on ovule elongation, embryo recovery, growth, and plantlet formation. Ovules from self-pollinated berries of seedless varieties Sunmuscat, Merbein Seedless, and Marroo Seedless were cultured at 30, 43,60, and 70 days after flowering (DAF) in a range of media, some of which were supplemented with gibberellic acid(GA3) and indole-3-acetic acid (IAA). The effect of activated charcoal (AC) in media on rescued embryos was alsoinvestigated.

Ovules exhibited continuous growth in vivo and in vitro. The most vigorous growth was observed for ovulescultured at 30 and 43 DAF, but more embryos were recovered from ovules cultured at 60 and 70 DAF. Ovule growthand embryo production in vitro were improved in Bouquet and Davis (BD) and Nitsch and Nitsch (NN) media.Supplementation with GA3 increased embryo recovery rates. Highest embryo recovery rates were 18.1%, 9.6%, and12.2% for Sunmuscat, Merbein Seedless, and Marroo Seedless, respectively, when ovules were excised and culturedat 60 or 70 DAF in either BD or NN media. In vitro embryo survival and plantlet formation were higher for torpedo-shaped embryos, and improved greatly in 6-benzyladenine (BA)-supplemented woody plant (WP) mediumcontaining 0.3% AC. Embryo recovery was improved by excising and culturing ovules at 60 DAF in BD or NNmedia and then by transferring embryos to WP medium supplemented with BA and AC.AR03053Im pr oved em br yo cult ur e f or gr apesS. M. Liu et al .

Additional keywords: table grapes, ovule growth, embryo recovery rate, plantlet formation, media, plant growthregulators.

IntroductionSeedless grape berries develop either through parthenocarpyor stenospermocarpy (Stout 1936; Pratt 1971). Whereas ovulefertilisation is not needed for fruit development inparthenocarpic types, it is required for stenospermocarpicgrapes in which seeds fail to develop because of ovuleabnormality and embryo abortion. Even though seeds abort,the stimulus of fertilisation leads to larger berries in steno-spermocarpic than in parthenocarpic types (Stout 1936). Asseedlessness and large berries are important consumer-preferred characteristics, the development of steno-spermocarpic seedless varieties has been emphasised in table-grape breeding programs worldwide (Clingeleffer 1998).

Conventional breeding strategies for seedless grapes haveused seeded females crossed with stenospermocarpic malesto yield seedless hybrids (Barlass et al. 1988; Ramming1990). The need for repeated intermating or backcrossing, a

long breeding cycle, and a low proportion of seedless hybridsin progenies are major disadvantages for this strategy(Ramming 1990). In order to improve efficiency in breedingseedless grapes, in ovulo embryo rescue techniques havebeen developed (Cain et al. 1983; Spiegel-Roy et al. 1985).Under in vitro conditions, arrested embryos continuedevelopment and can be established as normal plants. Thus,it is possible to intermate stenospermocarpic parents directlyto produce progenies with higher proportions of seedlesshybrids (Ramming 1990). The strategy makes it easier torecombine complementary traits in seedless germplasm(Gray et al. 1987; Ramming 1990) and may shorten thebreeding cycle (Ramming 1990).

The technique of in ovulo embryo rescue consists ofaseptic ovule removal and culture followed by embryoexcision from developing ovules and continued culture invitro until plantlet formation (Emershad and Ramming 1984;

870 Australian Journal of Agricultural Research S. M. Liu et al.

Spiegel-Roy et al. 1985; Gray et al. 1987; Burger andGoussard 1996). Success is influenced by genotype(Emershad et al. 1989; Burger and Goussard 1996; Ponce etal. 2000), ovule removal age (Bouquet and Davis 1989;Emershad et al. 1989; Bouquet and Davis 1989; Burger andGoussard 1996; Ponce et al. 2000), and medium (Spiegel-Roy et al. 1985; Gray et al. 1990; Emershad and Ramming1994; Valdez and Ulanovsky 1997). In vitro embryo growthand germination can be promoted by exogenous plant growthregulators, such as gibberellic acid (GA3) combined withindole-3-acetic acid (IAA) (Spiegel-Roy et al. 1985;Gribaudo et al. 1993) and 6-benzyladenine (BA) (Gray et al.1987; Emershad and Ramming 1994), and by activatedcharcoal (AC) in media (Gray et al. 1990; Emershad andRamming 1994; Burger and Goussard 1996).

The development of improved seedless varieties adaptedto Australian growing conditions has been pursued in tableand drying grape breeding programs at CSIRO PlantIndustry, Horticulture Unit, Merbein, since the mid 1960s(Clingeleffer 1998). To assist in achieving breedingobjectives, in ovulo embryo rescue techniques have beenincorporated in the program (Barlass et al. 1988). The aimsof the investigation reported here were to examine furtherspecific genotype, medium, and ovule removal age effects onembryo recovery and plantlet formation and thus improve inovulo embryo rescue protocols for the program.

Materials and methods

Experiments were conducted with ovules excised from berries of3 seedless varieties: Sunmuscat, Merbein Seedless, and MarrooSeedless. Sunmuscat is a muscat-flavoured selection released jointly byCSIRO and USDA (Clingeleffer 1998). Marroo Seedless and MerbeinSeedless were bred and released by CSIRO in the 1980s (Antcliff 1981;Clingeleffer and Possingham 1988). All vines were 30 years old, trainedto T-trellis, in a Coomealla sandy loam soil (Penman et al. 1939) withinter- and intra-row spacings of 3.0 m and 1.5 m, respectively, atCSIRO’s vineyard in north-western Victoria. Vines were irrigated vialow-level sprinklers, which delivered approximately 1000 mm perannum, and mildew diseases were controlled by fungicide sprays at21-day intervals.

Inflorescences were bagged before flowering at the end of October2000. Flowering date was recorded as 4 November when 50% calyptrafall occurred for the 3 varieties. Self-pollinated inflorescences werecollected at 30, 43, 60, and 70 days after flowering (DAF). Berries withpedicels attached were removed and 100-berry weight measured.Berries were surface-disinfested by immersion in 70% ethanol for2 min, followed by 2.5% sodium hypochlorite containing 0.1% Tween20 for 8 min, and then rinsed 4 times with sterile distilled water. Ovuleswere removed aseptically and their length recorded before beingtransferred to appropriate media.

Three basal media, namely NN (Nitsch and Nitsch 1969), BD(Bouquet and Davis 1989), and ER (Emershad and Ramming 1994),were used to culture excised ovules. NN is a popular in vitro culturemedium used for Vitis species (Dunstan et al. 1995), and BD is amodified Murashige and Skoog’s (1962) medium. NN and BD aresimilar in composition, except the latter is supplemented with differentamino acids and casein hydrolysate. In contrast, ER medium has lowerconcentrations for most nutrients and vitamins, but higher concen-trations of MgSO4.7H2O, Ca(NO3)2.4H2O, glycine, and sucrose. In

addition, cysteine and casein hydrolysate are added in ER medium andiron is supplied by iron citrate rather than iron/EDTA chelate. All mediacontained 0.3% (w/v) AC and were solidified by 0.8% Difco Bacto agar(w/v) and 1.8% gelrite (w/v). Media were adjusted to pH 5.8 with 0.1 MNaOH or 0.1 M HCl before autoclaving. GA3 and IAA supplementswere filter-sterilised and added to media after autoclaving.

Experiments were arranged in a factorial design with 10 Petri dishes60 by 15 mm per treatment, with 8 ovules cultured in each dishcontaining 8 mL of appropriate medium as one replication. Ovulesexcised at 43 DAF from Merbein Seedless and Marroo Seedless werealso cultured on BD and ER basal media supplemented with either GA3(1 µM) or GA3 (1 µM) + IAA (1 µM).

Petri dishes were accommodated on 2 shelves 60 by 155 cmilluminated by 2 OSRAM fluorescent tubes (L58W/77). Light intensityrecorded on Petri dish surfaces ranged from 250 to 360 µmol/m.s. A16-h photoperiod was applied during ovule culture, with temperaturemaintained at 25 ± 2°C.

Ovules were dissected under a stereomicroscope after 60 days inculture and their length recorded. Where embryos occurred, theirdevelopmental stages were recorded based on morphologicalappearance, i.e. globular-, heart-, or torpedo-shaped. Ovules containinggerminated or multiple embryos were also recorded. Embryos wereremoved and placed into individual culture tubes 16 by 80 mmcontaining 10 mL of woody plant (WP) medium (Lloyd and McCown1980) at pH 5.8, solidified with 0.8% Difco Bacto agar (w/v) and 1.8%gelrite (w/v), with or without 0.3% AC (w/v) and supplemented with1.5% sucrose (w/v) and 1.0 µM BA.

Embryo cultures were maintained under conditions used for ovulecultures. Embryo growth and plantlet formation were observed after 2months of culture, with survival scored on the basis of expansion,callogenesis, or plantlet formation.

When shoot apices reached the top of culture tubes, plantlets weretransferred to 425-mL clear plastic containers with bottom drainageholes containing a potting mix consisting 2 parts of a commercialseedling potting mixture (Debco, Debco Pty Ltd) and 1 part of perlite(v/v). Containers and plantlets were sealed within plastic bags tomaintain humidity and assist acclimatisation under culture roomconditions. When new shoot growth occurred, plastic bags werepunctured to reduce humidity. Holes in the bags were increasedprogressively as plantlets grew until they were transferred to aglasshouse. After a month in the glasshouse, plants were re-potted in2.8-L pots filled with a sand : red loam : perlite : peat moss (2 : 1 : 1 : 1;v/v/v/v) mix.

Ovule elongation rate was calculated as a percentage by [(ovulelength at embryo excision – ovule length at removal)/ovule length atremoval] × 100%. Embryo recovery was the percentage of ovules withembryos in a Petri dish. Contaminated Petri dishes were treated asmissing data for analysis. Data were subjected to log transformation andanalysis of variance with fixed main effects using GENSTAT statisticalsoftware. Least significant differences (l.s.d.) (Fisher 1951; Carmer andWalker 1982) were calculated after adjustments were made for missingvalues, and used for comparison of treatment means.

Results

In vivo and in vitro ovule growth

Mean 100-berry weight was 26.3, 74.9, 131.2, and 189.7 gfor Sunmuscat; 36.2, 48.7, 96.1, and 141.2 g for MerbeinSeedless; and 51.2, 71.7, 138.6, and 176.6 g for MarrooSeedless at 30, 43, 60, and 70 DAF, respectively. Berryweight increased linearly with DAF for each genotype, withR2 ranging from 0.94 to 0.99 (P < 0.05). Berries were soft at60 DAF, indicating that they had reached veraison.

Improved embryo culture for grapes Australian Journal of Agricultural Research 871

The time taken for ovules to reach maximum lengthin vivo varied between genotypes (Table 1). It was at 60 DAFfor Sunmuscat and 43 DAF for both Merbein Seedless andMarroo Seedless. At 70 DAF, ovules were significantlysmaller for Sunmuscat and Merbein Seedless.

Ovule length varied significantly between varieties foreach berry age when ovules were excised and cultured(Table 1). Merbein Seedless consistently had the longestovules. Marroo Seedless ovules were longer than those ofSunmuscat at 30, 40, and 70 DAF and shorter at 60 DAF.

Most ovules (73%) became green when cultured in vitroat 30, 43, and 60 DAF (Fig. 1a). In contrast, 74% of ovulesbecame brown when cultured at 70 DAF. Genotype, ovuleremoval age, medium, and their interactions affected in vitroovule growth significantly (Table 2).

The magnitude of ovule growth differed with removalages in NN and BD media. Ovules elongated by 0.5 and 1.5

Table 1. Mean ovule length (mm) at excision (n = 240) for 3 seedless varieties at 4 berry removal ages

DAF, days after flowering, which was used to quantify the age at which ovules were excised and removed. The letters a, b, c indicate the comparison for the values within a row across different ovule removal ages, and x, y, z for the values within a column across different lines; means followed by the same letters are not significantly different at

P = 0.05. l.s.d. (P = 0.05) = 0.11

Variety 30 DAF 43 DAF 60 DAF 70 DAF

Sunmuscat 1.4bz 1.3bz 2.0ay 1.0czMerbein Seedless 1.9bx 2.2ax 2.2ax 1.9bxMarroo Seedless 1.5by 1.7ay 1.7az 1.7ay

(b)(a) (c)

(d) (e) (f)

Fig. 1. Ovule, embryo appearance and germination at embryo excision, and plantletsformed from cultured embryos: (a) Merbein seedless ovules in BD medium after 60days culture in a 60 mm by 15 mm Petri dish; (b) globular embryo as seen in a dissectedovule showing a collapsed, papery inner integument without endosperm; (c) earlytorpedo-shaped embryo without endosperm inside a dissected ovule; (d) germinatedembryo from a cultured ovule; (e) a plantlet from a rescued embryo developing withina culture tube; (f) a self-pollinated Marroo Seedless plantlet after acclimatisationfollowing in ovulo embryo rescue.

Table 2. Summary of analysis of variance table demonstrating significant factors affecting in vitro ovule elongation and embryo

recovery rates (%)The error degrees of freedom were 299

Source d.f. F valuesOvule elongation

rateEmbryo

recovery rate

Replication 9 0.9 1.4Genotype (G) 2 4.9** 2.6Medium (M) 2 114.6** 3.4*Ovule removal age (A) 3 38.6** 13.9**G × M 4 4.4** 0.6G × A 6 28.5** 9.7**M × A 6 23.3** 1.3G × M × A 12 4.7** 1.7*

*P < 0.05; **P < 0.01.

872 Australian Journal of Agricultural Research S. M. Liu et al.

times their original size when cultured at 30 and 43 DAF andby less than a twentieth to almost a half at 60 and 70 DAF(Table 3). In ER medium, ovule growth was not significantlyaffected by removal ages, with the exception of MerbeinSeedless at 30 DAF.

Ovules excised at 30 and 43 DAF had significantlygreater growth rates in both NN and BD than in ER mediumacross all genotypes. Ovule growth was unaffected byculture medium for ovules excised and cultured beyond 60DAF in 15 out of 18 cases across genotypes (Table 3).

Ovule growth varied according to media and mediasupplements (Table 4). BD as a basal medium producedbetter growth than ER and this was improved with GA3 andIAA supplements for Merbein Seedless. GA3-supplementedER medium produced better growth than ER basal mediumand ER + GA3 + IAA.

Effect of genotype, ovule removal age, and medium on embryo recovery

With the exception of embryo and endosperm coexistence insclerified ovules of Marroo Seedless, which is similar tonormal grape seeds, endosperm development was notevident in ovules where embryos were found (Fig. 1b, c).Embryos at various developmental stages, from globular totorpedo-shaped, were observed (Fig. 1b, c). Three embryosgerminated directly from in vitro ovules of MerbeinSeedless, but none from other genotypes in the entireexperiment (Fig. 1d). One of these was found in ovulescultured in NN medium and the other two in BD mediumsupplemented with GA3. Two instances of multiple embryoformation were found in cultured ovules of Sunmuscat andMarroo Seedless during ovule dissection.

Embryo recovery was affected significantly by mediumand ovule removal age and there were interactions betweengenotype and ovule removal age and among genotype,medium, and ovule removal age (Table 2). Embryo recoveryrate was more than 3.6% in both NN and BD media, whichwas significantly higher than 1.77% in ER medium.

Embryo recovery rate was zero at 30 DAF, and increasedwith delaying ovule culture across genotypes (Fig. 2).Highest embryo recovery rates were from Sunmuscat ovulesexcised and cultured at 60 DAF across media and forMerbein Seedless ovules at 70 DAF in NN and BD media.For Marroo Seedless, optimum recovery varied from 43 to70 DAF, depending on culture medium.

The best embryo recovery rate was 18.1% for Sunmuscatovules cultured in BD medium at 60 DAF (Fig. 2). This wassignificantly higher than for all other media and ovule

Table 3. Effects of medium and ovule removal age on ovule elongation rate (%) of 3 seedless varieties after 60 days in vitro culture

DAF, days after flowering, which was used to quantify the age at which ovules were excised and removed. The letters a, b, c indicate the comparison for the values within a row across different ovule removal ages, and x, y, z for the values within a column across different lines;

means followed by the same letters are not significantly different at P = 0.05

Genotype Medium Ovule elongation30 DAF 43 DAF 60 DAF 70 DAF

Sunmuscat NN 56.4ax 69.5ax 17.9bx 34.7bxBD 51.9ax 64.1ax 25.6bx 12.0byER 22.1ay 19.0ay 21.7ax 27.7axy

Merbein Seedless NN 91.2ay 59.3bx 11.4cx 3.6cxBD 152.6ax 63.3bx 11.8cx 4.6cxER 50.7az 15.9cy 28.1bx 11.9cx

Marroo Seedless NN 52.8bxy 77.4ax 23.4cy 39.0bcxBD 62.4ax 57.5axy 44.7ax 18.1byER 32.5aby 43.6ay 30.3abxy 23.6bxy

Table 4. Effects of plant growth regulators on mean elongation rate (%) of ovules excised and cultured at 43 DAF from 2 seedless

varieties after 60 days in vitroThe letters a, b, c, d indicate the comparison for the values within a col-umn; means followed by the same letters are not significantly different

at P = 0.05

Medium Merbein Seedless Marroo Seedless

BD 64.1b 57.5 abBD + 1 µM GA3 65.4b 41.8bBD + 1 µM GA3 + 1 µM IAA 100.5a 60.8abER 15.2d 43.6bER + 1 µM GA3 39.0c 72.7 aER + 1 µM GA3 + 1 µM IAA 17.3d 41.5b

Improved embryo culture for grapes Australian Journal of Agricultural Research 873

removal age combinations. For Merbein Seedless, optimalembryo rescue rate (9.6%) was achieved from ovulescultured at 70 DAF in NN medium. However, this was notsignificantly greater than 6.3% in BD medium at 70 DAF.For Marroo Seedless, the highest embryo recovery rate was12.2% in BD medium at 70 DAF, although this did not differsignificantly from means for NN medium at 43 DAF and ERmedium at 60 DAF (Fig. 2).

Incorporation of GA3 alone or GA3 combined with IAAin BD and ER media significantly influenced embryorecovery rates in ovules cultured at 43 DAF (Table 5). Over5% of viable embryos were found in ovules cultured in bothGA3-supplemented media across genotypes. These rateswere significantly higher than the zero embryo recovery

observed in media with and without both GA3 and IAA, withthe exception of lower rates for Merbein Seedless in BD +GA3 + IAA and for Marroo Seedless in BD medium.

Ovules cultured in either NN or BD medium produced anequal number of embryos, which was twice the numberobtained in ER medium (Table 6). GA3-supplemented BDand ER media yielded similar numbers of embryos, whichwere more than those produced in basal media or the mediasupplemented with GA3 plus IAA (Table 7).

Of the embryos rescued from ovules cultured in NN or ERmedium, over 45% were at either the globular- or torpedo-shaped stage, with <7% being heart-shaped (Table 6). In BDmedium, percentages of globular and torpedo-shapedembryos were less, but more (26%) were heart-shaped. Inmedia with or without plant growth regulators, theproportion of torpedo-shaped embryos was more than 70%in BD media compared with 50% or less in ER media,irrespective of plant growth regulator supplements (Table 7).Therefore, basal medium may have been critical for theproduction of embryos at advanced developmental stageseven though the numbers observed were low.

Development of excised embryos

The fate of excised embryos in vitro was strongly associatedwith developmental stage. Torpedo-shaped embryosexhibited higher rates of survival and plantlet formation(Table 8).

Incorporation of AC in WP medium almost doubledembryo survival and resulted in a mean 3.5-fold increase inplantlet formation (Table 8), which was associated withembryo developmental stage. Embryo survival ratesincreased by 45.8, 37.5, and 25.7% for globular-, heart-, andtorpedo-shaped embryos, respectively, in medium supple-mented with AC. Plantlet formation increased from 0% tomore than 12% of globular- and heart-shaped embryos whenthe medium was supplemented with AC. Almost a 3-foldincrease was observed in plantlet formation of torpedo-shaped embryos in the medium supplemented with AC.

As AC had a positive effect on embryo survival andplantlet formation, all embryos that had been cultured in WP

ddd dd

cd

a

b

bc

d d d0

4

8

12

16

20

ccc cc

bcbcbc

bc

c

ab

a

0

4

8

12

16

20

Mea

n em

bryo

rec

over

y (%

)

c c c

ab

bc

c

bc bc

abbc

a

bc

0

4

8

12

16

20

NN BD ER

43 DAF

30 DAF

60 DAF

70 DAF

Culture media

Sunmuscat

Merbein Seedless

Maroo Seedless

Fig. 2. The effects of ovule removal age and medium on meanembryo recovery rate for 3 seedless varieties; l.s.d. (P = 0.05) was6.1–6.8% after adjustment for missing data. Bars labelled withdifferent letters indicate a difference at P = 0.05. Note that some barsare not visible above the base line. DAF, days after flowering, whichwas used to quantify the age at which ovules were excised andremoved. NN, BD, and ER refer to basal culture media as described byNitsch and Nitsch (1969), Bouquet and Davis (1989), and Emershadand Ramming (1994), respectively.

Table 5. Effects of plant growth regulators on mean embryo recovery rate (%) from ovules of 2 seedless grape varieties excised

at 43 DAF and cultured in vitro The letters a, b indicate the comparison for the values within a column;

means followed by the same letters are not significantly different at P = 0.05

Medium Merbein Seedless Marroo Seedless

BD 0.0b 4.3abBD + 1 µM GA3 5.7a 5.1aBD + 1 µM GA3 + 1 µM IAA 2.8ab 0.0bER 0.0b 0.0bER + 1 µM GA3 5.3a 7.5aER + 1 µM GA3 + 1 µM IAA 0.0b 0.0b

874 Australian Journal of Agricultural Research S. M. Liu et al.

medium were transferred to AC-supplemented WP mediumafter 2 months. As a result, 15 and 12 plantlets wererecovered for Merbein Seedless and Marroo Seedless,respectively (Fig. 1e, f). These represented plantlet formationrates of 60% (15/25) and 28.6% (12/42) for MerbeinSeedless and Marroo Seedless, respectively, when based onthe numbers of embryos obtained at ovule dissection.Plantlets were not recovered for Sunmuscat, which may havebeen because most embryos rescued from ovules were at theglobular stage (22 out of 27), and only a few survived inembryo culture and were lost due to contamination.

Discussion

In vivo ovule growth during early berry development forstenospermocarpic seedless grapes was reported either asgains in weight (Spiegel-Roy et al. 1985) or length increases(Gray et al. 1990; Notsuka et al. 2001). Notsuka et al. (2001)also demonstrated that there was no association between

growth of ovules and berries in stenospermocarpic seedlessgenotypes. In this investigation, changes of ovule length withremoval age reflected continuous growth of ovules in vivo,but the period of ovule growth varied between varieties andwas shorter than the duration of berry growth.

Ovule growth in vitro was continuous and greater forovules excised from berries collected at 30 and 43 DAF thanfor those collected at 60 and 70 DAF. Poor growth wasprobably linked to browning of excised ovules collected at 70DAF. Gray et al. (1990) reported high frequency of browningin vitro for ovules removed from softening berries. Thepossible causes behind ovule browning might be similar tothose reported in plant tissue culture, such as the presenceand activation of polyphenol oxidase and peroxidase inwounded tissues and/or the accumulation of phenols (Georgeand Sherrington 1984; Pan and van Staden 1998). Whetherthe inclusion of polyvinylpyrolidone (PVP) in media wouldreduce ovule browning could be addressed in the future.

Table 6. The number and developmental stage of embryos obtained in 3 basal media across all ovule removal age and variety combinations

Medium No. of Globular Heart-shaped Torpedo-shapedembryos No. % No. % No. %

NN 31 15 48.4 2 6.5 14 45.1BD 31 13 41.9 8 25.8 10 32.3ER 15 7 46.7 1 6.7 7 46.7

Table 7. The number and developmental stage composition of embryos from ovules excised at 43 DAF and cultured in media with or without plant growth regulators

Medium No. of Globular Heart-shaped Torpedo-shapedembryos No. % No. % No. %

BD 3 0 0.0 0 0.0 3 100.0BD + 1 µM GA3 7 2 29.0 0 0.0 5 71.0BD + 1 µM GA3 + 1 µM IAA 2 0 0.0 0 0.0 2 100.0

ER 0 0 – 0 – 0 –ER + 1 µM GA3 8 4 50.0 0 0.0 4 50.0ER + 1 µM GA3 + 1 µM IAA 0 0 – 0 – 0 –

Table 8. Influence of embryo developmental stages at excision and of activated charcoal (AC) in BA-supplemented WP medium on embryo survival and plantlet formationValues in parentheses are the percentage

WP + BA WP + BA + ACNo. of embryos No. surviving No. of plantlets formed No. of embryos No. surviving No. of plantlets formed

Globular 25 2 (8.0)0 0 (0.0)0 13 7 (53.8) 2 (15.4)Heart-shaped 4 1 (25.0) 0 (0.0)0 8 5 (62.5) 1 (12.5)Torpedo-shaped 19 13 (68.4) 3 (15.8) 17 16 (94.1) 7 (41.2)Average (%) 33.3 5.9 57.9 21.6

Improved embryo culture for grapes Australian Journal of Agricultural Research 875

Nevertheless, more embryos were recovered from ovulescultured at 60 or 70 DAF. In vitro ovule growth may not havebeen indicative of the presence of viable embryos in ovules,which was consistent with other findings (Emershad andRamming 1984; Gray et al. 1990; Burger and Goussard1996), but contrary to the results of Bouquet and Davis(1989), who found an association between in vitro ovule sizeand the existence of viable embryos.

Ovule removal age appeared to be one of the primaryfactors affecting percentage embryo recovery. Optimal agesfor ovule removal and culture varied with genotype andranged from 43 to 70 DAF. This matched the range ofbetween 41 and 70 DAF suggested by other studies (Spiegel-Roy et al. 1985; Emershad et al. 1989; Gray et al. 1990;Gribaudo et al. 1993; Burger and Goussard 1996; Notsuka etal. 2001), and provided further evidence that embryos can berecovered with greater success at later culture dates (Spiegel-Roy et al. 1985; Emershad et al. 1989; Gray et al. 1990;Tsolova and Atanassov 1994; Burger and Goussard 1996;Burger and Trautmann 2000; Notsuka et al. 2001). Delayingovule removal and culture from berries, however, increasedthe inconvenience of excising ovules from softening berries.This was especially problematic for the white-berriedgenotypes Sunmuscat and Merbein Seedless, where ovulesbecome less distinct from surrounding pulp tissue with age,which was similar to what was reported by Gray et al. (1990).Thus, the optimal stage for berry collection for ovule culturewas around 60 DAF when berries were still firm and therewas a greater chance of rescuing embryos.

To increase breeding efficiency, it is important to knowpotential embryo recovery rates under optimal ovule cultureconditions for various genotypes. Among the genotypestested, the highest embryo recovery rate was 18.1% forSunmuscat, which was 50% higher and double the rate fromMarroo Seedless and Merbein Seedless ovules, respectively.Similar genotypic differences were reported in previousstudies (Spiegel-Roy et al. 1985; Gribaudo et al. 1993; Ponceet al. 2000; Notsuka et al. 2001), which suggests that there isgenetic variation for ovule fertilisation rates andsubsequently for embryo recovery in vitro forstenospermocarpic seedless grape germplasm.Consequently, hybridisation among seedless grapes shouldemphasise genotypes with high embryo formation rates asfemale parents, which would maximise resource inputs toestablish larger breeding populations using embryo rescuetechniques. Although not investigated here, the effect ofpollen parent cannot be discounted and should beconsidered, especially since berries from self-pollinationwere used and there may have been inbreeding effects.

The effects of media on in vitro embryo recoverydemonstrated that NN and BD media were significantlymore beneficial to embryo formation than ER medium. Theproportion of torpedo-shaped embryos was higher fromovules cultured in NN medium than in BD medium despite

the comparable numbers of embryos obtained from ovulescultured in these media. Torpedo-shaped embryos survivedbetter and led to increased plantlet formation in subsequentembryo culture, indicating that NN medium was better thanBD medium.

Addition of GA3 in either BD or ER medium improvedembryo recovery for Merbein Seedless and Marroo Seedless.However, the incorporation of both GA3 and IAA in thesemedia exhibited no benefit, which was in accordance with aprevious report (Burger and Goussard 1996) butcontradictory to the results of Spiegel-Roy et al. (1985) andGribaudo et al. (1993). Zlenko et al. (2002) reported thatinclusion of exogenous GA3 in medium improved plantformation of grape somatic embryos and that regeneratedplants had long, thick hypocotyls and large cotyledons.Therefore, GA3 probably promoted embryo growth, whichimproved embryo rescue with a flow-on effect for survivaland plant formation.

In vitro embryo survival and plant formation was higherfor torpedo-shaped than for both globular- and heart-shapedembryos. In examining in vitro embryo germination ofgrapes, Horiuchi et al. (1991) demonstrated that globularembryos not only germinated poorly but also produced weakplants. Balthazard (1969) reported that successfulgermination of Vitis vinifera was usually reached only whenembryos were longer than 1 mm even under in vivoconditions, and Monnier (1995) demonstrated that theoptimal stage to rescue zygotic embryos was the heart-shaped stage for most plant species. Our resultsdemonstrated that embryo stage attained was affected byovule culture medium. This suggests that further studies areneeded to optimise in vitro conditions to maximise in ovuloembryo growth rates to achieve a higher proportion of thetorpedo-shaped stage.

Improved embryo survival and plant formation wereachieved when embryos were cultured in medium containingAC. The role of AC was not clear, but it may have created adarker environment similar to the conditions under whichembryos grow inside ovules before dissection. AC may alsoreduce inhibitory substances such as phenolics by absorption(Pan and van Staden 1998). These conditions may aidembryo recovery from excision injury and thus stimulatein vitro growth. In addition, when medium containing ACwas used to culture zygotic embryos, somatic embryogenesiswas observed in some cultures (data not shown) and plantletswere successfully regenerated from the somatic embryos.WP medium + BA + AC improved embryo survival and gavegreater plantlet formation, which agreed with the results ofEmershad and Ramming (1994).

In summary, this investigation demonstrated that mediaand berry age can be optimised to ensure maximum embryorecovery rate. In addition, embryo formation and growthwere stimulated by GA3. Plantlet formation was improved bythe use of WP medium supplemented with BA and AC.

876 Australian Journal of Agricultural Research S. M. Liu et al.

http://www.publish.csiro.au/journals/ajar

These results are now being used to progress the table anddrying grape breeding program at CSIRO Plant Industry bymeans of seedless × seedless crosses.

References

Antcliff AJ (1981) Merbein seedless: a new white seedless grape fordrying. Journal of the Australian Institute of Agricultural Sciences47, 167–168.

Balthazard J (1969) Temperatures alterneés, longueur des embryons etpouvoir germinatif des graines de Vigne. Comptes Rendus desSeances de l’Academie d’Agriculture de France 269, 2355–2358.

Barlass M, Ramming DW, Davis HP (1988) In-ovulo embryo culture: abreeding technique to rescue seedless × seedless table grape crosses.The Australian Grapegrower and Winemaker 292, 123–125.

Bouquet A, Davis HP (1989) In vitro ovule and embryo culture forbreeding seedless table grapes (Vitis vinifera L.). Agronomie 9,565–574.

Burger P, Goussard PG (1996) In vitro culture of ovules and embryosfrom seedless grape (Vitis vinifera L.). South African Journal forEnology and Viticulture 17, 31–37.

Burger P, Trautmann IA (2000) Manipulations of ovules to improve invitro development of Vitis vinifera L. embryos. Acta Horticulturae528, 613–619.

Cain DW, Emershad RL, Tarailo RE (1983) In-ovulo embryo cultureand seedling development of seeded grapes (Vitis vinifera L.). Vitis22, 9–14.

Carmer SG, Walker MA (1982) Baby bear’s dilemma: a statistical tale.Agronomy Journal 74, 122–124.

Clingeleffer PR (1998) Breeding table grape and raisin varieties. In‘Breeding and Biotechnology for Fruit Trees’. Proceedings of the2nd Japan-Australia International Workshop. (Eds M Omura, THayashi, NS Scott) pp. 46–49. (National Institute of Fruit TreeScience, MAFF: Tsukuba, Japan)

Clingeleffer PR, Possingham JV (1988) Marroo Seedless: a new tablegrape variety. Agriculture Science 1, 18–19.

Dunstan DI, Tautorus TE, Thorpe TA (1995) Somatic embryogenesis inwoody plants. In ‘In vitro embryogenesis in plants’. (Ed. TAThorpe) pp. 471–538. (Kluwer Academic Publishers: Dordrecht)

Emershad RL, Ramming DW (1984) In ovulo embryo culture of Vitisvinifera L. cv. ‘Thompson Seedless’. American Journal of Botany71, 873–877.

Emershad RL, Ramming DW (1994) Somatic embryogenesis and plantdevelopment from immature zygotic embryos of seedless grapes(Vitis vinifera L.). Plant Cell Reports 14, 6–12.

Emershad RL, Ramming DW, Serpe MD (1989) In ovulo embryodevelopment and plant formation from stenospermic genotypes ofVitis vinifera. American Journal of Botany 76, 397–402.

Fisher RA (1951) ‘The design of experiments.’ 6th edn (Longman:London)

George EF, Sherrington PD (1984) ‘Plant propagation by tissueculture.’ (Exegetics Ltd: Basingstoke, England)

Gray DJ, Fisher LC, Mortensen JA (1987) Comparison ofmethodologies for in ovulo embryo rescue of seedless grapes.HortScience 22, 1334–1335.

Gray DJ, Mortensen JA, Benton CM, Durham RE, Moore GA (1990)Ovule culture to obtain progeny from hybrid seedless bunch grapes.Journal of the American Society for Horticultural Sciences 115,1019–1024.

Gribaudo I, Zanetti R, Botta R, Vallania R, Eynard I (1993) In ovuloembryo culture of stenospermocarpic grapes. Vitis 32, 9–14.

Horiuchi S, Kurooka H, Furuta T (1991) Studies on embryo dormancyin grape. Journal of the Japanese Society for Horticultural Science60, 1–7.

Lloyd G, McCown B (1980) Commercially-feasible micropropagationof mountain laurel, Kalmia latifolia, by use of shoot-tip culture.Proceedings of International Plant Propagator’s Society 30,421–427.

Monnier M (1995) Culture of zygotic embryos. In ‘In vitroembryogenesis in plants’. (Ed. TA Thorpe) pp. 117–153. (KluwerAcademic Publisher: Dordrecht)

Murashige T, Skoog F (1962) A revised medium for rapid growth andbioassays with tobacco tissue cultures. Physiologia Plantarum 15,473–497.

Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science163, 85–87.

Notsuka K, Tsuru T, Shiraishi M (2001) Seedless–seedless grapehybridization via in-ovulo embryo culture. Journal of the JapaneseSociety for Horticultural Science 70, 7–15.

Pan MJ, van Staden J (1998) The use of charcoal in in vitro culture—areview. Plant Growth Regulation 26, 155–163.

Penman F, Taylor JK, Hooper PD, Marshall JJ (1939) A soil survey ofthe Merbein irrigation district, Victoria. Council of Scientific andIndustrial Research, Bulletin No. 123.

Ponce MT, Aguero CB, Gregori MT, Tizio R (2000) Factors affectingthe development of stenospermic grape (Vitis vinifera) embryoscultured in vitro. Acta Horticulturae 528, 667–671.

Pratt C (1971) Reproductive anatomy in cultivated grapes—a review.American Journal of Enology and Viticulture 22, 92–109.

Ramming DW (1990) The use of embryo culture in fruit breeding.HortScience 25, 393–398.

Spiegel-Roy P, Sahar N, Baron J, Lavi U (1985) In vitro culture andplant formation from grape cultivars with abortive ovules and seeds.Journal of the American Society for Horticultural Sciences 110,109–112.

Stout AB (1936) Seedlessness in grapes. New York State AgriculturalExperiment Station, Technical Bulletin No. 238.

Tsolova V, Atanassov A (1994) Induction of polyembryony andsecondary embryogenesis in culture for embryo rescue ofstenospermocarpic genotypes of Vitis vinifera L. Vitis 33, 55–56.

Valdez JG, Ulanovsky SM (1997) In vitro germination of stenospermicseeds from reciprocal crosses (Vitis vinifera L.) applying differenttechniques. Vitis 36, 105–107.

Zlenko VA, Kotikov IV, Troshin LP (2002) Efficient GA3-assisted plantregeneration from cell suspension of three grape genotypes viasomatic embryogenesis. Plant Cell, Tissue and Organ Culture 70,295–299.

Manuscript received 4 March 2003, accepted 29 July 2003