A refined method for ovule culture in sugar beet (Beta vulgaris L.)

Induction of gynogenesis through ovule culture is a valuable tool to produce haploid and doubled haploid plants in sugar beet (Beta vulgaris L.). However, there is still large room for refining the method. In this study we investigated the gynogenic response of cultured ovules of three sugar beet genotypes, the effect of the application to inflorescences of different pretreatments with mannitol at 4ºC and with 5-azacytidine and 2,4-D, and the effect of the use of different basal culture media and sucrose concentrations. The response was evaluated in terms of percentages of induction of gynogenesis, embryogenesis and callogenesis, as well as of regenerated plants. We showed that a pretreatment with 0.5 M mannitol at 4 °C for 4 days, and with 50 µM 5-AzaC for 1 h, notably improved the percentage of embryogenesis and plant regeneration. Besides, the use of MS basal medium and 60 g/L sucrose was also found beneficial. This study provides new ways to improve the efficiency of haploid induction and plant regeneration through ovule culture in sugar beet, and is potentially applicable to ovule culture in other crops.

     

Antimitotic and hormone effects on green double haploid plant production through anther culture of Mediterranean japonica rice

Rice double haploid (DH) plants are produced mainly through anther culture. In order to improve the anther culture protocol, microspores of two japonica rice genotypes (NRVC980385 and H28) were subjected to three growth regulator combinations and four colchicine treatments on induction medium. In addition, a post anther culture procedure using colchicine or oryzalin was tested to induce double haploid plantlets from haploid plantlets. A cold pre-treatment of microspores for 9 days at 10 °C increased callus induction 50-fold in the NRCV980385 genotype. For both genotypes, 2 mg L^?1 2,4-D and 1 mg L^?1 kinetin on colchicine-free induction medium gave the best culture responses. The culturability of both genotypes changed on colchicine-supplemented induction media. A high genotype dependency was recorded for callus induction, callus regenerating green plantlets and regeneration of green double haploid plantlets. Colchicine at 300 mg L^?1 for 48 h enhanced callus induction 100-fold in H28. Colchicine-supplemented media clearly improved green double haploid plantlet regeneration. We showed that the post-anther culture treatment of haploid plantlets at 500 mg L^?1 of colchicine permitted fertile double haploid plantlets to be generated. Finally, an enhanced medium-throughput flow cytometry protocol for rice was tested to analyse all the plantlets from anther and post anther culture.     

Factors affecting in-vitro gynogenic haploid production in niger (<Emphasis Type="Italic">Guizotia abyssinica</Emphasis> (L. f.) Cass.)

The effects of growth regulators, cold-pretreatment of flower buds, ovule (embryo sac) developmental stage and genotype on induction of gynogenesis in unpollinated ovule cultures were assessed in niger (Guizotia abyssinica (L. f.) Cass.). Indirect callus-mediated gynogenesis occurred in cvs JNC-6 and Ootacamund when the ovules were cultured on MS medium supplemented with 30 g l⁻¹ sucrose and 2,4-D either alone (0.5–2.0 μM) or in combination (2.0 μM) with different cytokinins, such as adenine, BA, 2iP and kinetin (0.5–2.0 μM). An optimum induction of gynogenesis was fostered on medium supplemented with 2.0 μM 2,4-D and 1.0 μM kinetin. Cold-pretreatment of flower buds had no stimulatory effect, but ovules collected one day before anthesis were most responsive to gynogenesis. The results showed significant variations in genotypic competence for gynogenesis with cv. Ootacamund being the most responsive (12.5%) and cv. IGP-76 the least (2.5%). Gynogenic embryos differentiated and matured on media (30 g l⁻¹ sucrose) supplemented with 0.5 μM NAA plus 1.0 μM kinetin, and 0.5 μM ABA, respectively. The haploidy (2n = 1x = 15) of gynogenic plants was confirmed by cytological analysis.     

Embryological study on gynogenesis in onion (Allium cepa L.)

Haploid induction in onion can, to date, be induced only via gynogenesis by culturing unfertilized flowers, ovaries or ovules. The process of haploid embryo induction has been macroscopically well studied, but only limited data exist from microscopic examination of ovule development status at the inoculation stage and of the origin of gynogenic embryos. Microscopic studies were carried out using individual donor plants with relatively high embryo induction frequencies (45.9 embryos formed per 100 flowers, on average, for 2 years). Ovaries from flower bud culture were fixed at 1 week intervals up to the 7th week of culture. These were compared with pollinated ovaries at 1 or 2 weeks after pollination. In total, 1428 unfertilized embryo sacs were examined. The results indicate that, at the time of inoculation, ovules within ovaries 2.0–3.0 mm in diameter contained two- or four-nucleate embryo sacs in the smallest ovaries to mature embryo sacs in the largest ovaries. It seems likely that the embryos are actually induced from ovaries cultured at the immature stage. After 1 or 2 weeks in culture, the egg apparatus primarily consisted of distinctly enlarged synergids and the egg cell, which was often detached from the micropylar pole. But free nuclear endosperm was also formed. From the 2nd to 7th week in culture, formation of haploid embryos (from globular to the almost mature cylindrical stage) was detected in 5.7% of the ovules. Their origin, for several reasons, was most likely the egg cell. In addition, ovules containing endosperm only (3.6%) and ovules containing the egg apparatus (0.5%) or both endosperm and embryo (0.4%) were detected. This observation is probably unique and has not yet been reported in other species studied. Received: February 2001 / Revision accepted: 20 April 2001     

Induction of apogamy inEquisetum arvense

InEquisetum arvense, apogamous sporophytes were produced on medium containing 5×10^?6–5×10^?8 g/ml kinetin. NAA, IAA, GA₃, glucose and saccharose were ineffective for the induction of apogamy. On medium containing 5×10^?7–5×10^?8 g/ml kinetin, the gametophytes passed into sporophytic structures directly. On medium containing 5×10^?6 g/ml kinetin, some gametophytes passed into sporophytic structures directly, and others became a callus-like cell mass from which an apogamoun shoot arose. The results of the morphological observations on them were reported and compared with the sexually produced sporophyes. The apogamous sporophytes induced by 5×10^?7 g/ml kinetin were haploid in their nuclear phase and some of those induced by 5×10^?6 g/ml kinetin had a tendency to become diploid.     

An improved protocol for carrot haploid and doubled haploid plant production using induced parthenogenesis and ovule excision in vitro

In this work, we describe an improved protocol for induced parthenogenesis and ovule culture of carrot (Daucus carota L.). The effects of pollination with parsley pollen and/or 2,4-dichlorophenoxyacetic acid (2,4-D) treatment on the stimulation of parthenogenesis were studied using heterozygous donor plants of 30 varieties and breeding populations of carrots. Isolated ovules, cultured in vitro, enlarged and developed embryos or calli. The application of 2,4-D on pollinated flowers stimulated callus development but did not increase the frequency of embryo development from ovules and, thus, was not useful for increasing the frequency of haploid plant recovery. The efficiency of embryo development was accession-dependent and varied from 0 to 24.29%. In optimized conditions, most accessions responded by embryo development exclusively. The highest frequency of embryo development was observed from ovules excised from ovaries 20–22 d after pollination with parsley pollen. Among several media used for ovule culture, 1/2-strength Murashige and Skoog medium with 0.06 μM indole-3-acetic acid (IAA) was the best. It allowed the production of embryos at a similar frequency as on the media supplemented with kinetin, gibberellic acid, putrescine, or thidiazuron, but restricted callus development. Most plants obtained were haploids and diploids derived from parthenogenesis, as evidenced by homozygosity at three independent loci based on isozyme and PCR analyses. In total, considering haploids and embryo-derived homozygous diploids together, 72.6% of regenerated plants were of gametic origin.     

Effect of genotype,induction medium,carbohydrate source,and polyethylene glycol on embryogenesis in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) anther culture

This study conducted two experiments involving in vitro anther culture of Zea mays L. The first experiment tested 46 maize genotypes, including inbred lines, single and three-way cross hybrids, and line A188 as control, in three different induction basal media (IMSS, N6 and YPm) for their androgenic responses. The results showed that the embryos were established 2–3 weeks after the anthers of the few responsive genotypes were cultured. Most responsive genotypes produced embryos in at least one of the three basal media; therefore, genotype is more important than the type of medium for androgenesis in maize. The mean number of anthers that developed to embryo ranged from 19 embryos per Petri dish in YPm medium for the cross (DH5 × DH7) genotype to 0 for some maize genotypes. In the second experiment, this research reports for the first time the effect of carbohydrates and polyethylene glycol (PEG) as a non-metabolized osmoticum on the embryogenesis anther culture of maize. The genotype DH5 × DH7 was used for this experiment, and the media were varied by altering sucrose, maltose, and PEG concentrations. Results showed that the maximum embryogenesis (32 embryos per Petri dish) was obtained by YPm basal medium supplemented with 60 gl^?1 sucrose + 0.0125 M PEG and 30 gl^?1 sucrose + 30 gl^?1 maltose + 0.0125 M PEG. The lowest rate of embryogenesis was observed in YPm basal medium with 60 gl^?1 maltose and 0.0125 or 0.025 M PEG. Sucrose or a high concentration of maltose was found to be necessary for embryogenesis in anther culture of maize. Therefore, the addition of low levels of PEG and/or different sugars in the experimental design appeared to improve the protocol currently available in the world, especially for anther embryo yield and haploid plant regeneration in maize.     

百合未授粉子房离体培养胚胎形成及植株再生

未受精子房离体培养是诱导雌核产生单倍体的技术之一。以1个野生种和3个杂种系共7个百合(Lilium)基因型为实验材料, 探讨了基础培养基、花蕾取样时期和外源激素等因素对百合未授粉子房离体培养胚胎形成的影响。结果表明, CBM、MS和BDS三种基础培养基均可诱导百合未授粉子房胚胎形成, 但以BDS培养基诱导效果最佳; 开花前1天的花蕾较适于百合未授粉子房离体培养; 2 mg·L^-1 2,4-D + 2 mg·L^-1 6-BA和2 mg·L^-1 2,4-D + 2 mg·L^-1 KT两种外源激素配方均适用于百合未授粉子房离体培养诱导胚胎形成。在培养过程中, 大多数胚性胚珠中只含有1个胚胎, 位于珠孔端、合子端或极核处, 少数胚性胚珠中含有双胚胎。通过百合未授粉子房离体培养, 从5个基因型材料中共获得146株再生植株。采用根尖染色体计数法对其中的62株进行了倍性测定, 其中43株与母体植株染色体倍性不同。     

Cytological Observation on Megasporogenesis and Embryo Sac Development of Regenerated Ovule in Hyacinth

The morphogenesis of regenerated ovule and cytological changes of its megasporogenesis and embryo sac development were studied. Results showed as follows: 1. the differentiation of the regenerated ovule had followed a normal process in the order of inner integument , outer integument and then funiculus. But the form of the regenerated ovules in vitro was quite different from that of ovule in vivo. Most of the regenerated ovules were orthotropous and hemianatropous , only a few were anatropous which are the same with that in vivo. 2. the megasporogenesis and the embryo sac development also had normal cytological process ,and the Polygonum type-embryo sac consisted of one egg, two synergids , one central cell and three antipodals could be seen in mature regenerated ovule. These ex-perimental results make clear that the regenerated ovule differentiated directly from explant could accomplish the complex processes of megasporogenesis and embryo sac development. By this fact ,authors infer that once the differentiation of ovule primordium, the complex biochemical programs for the megasorogenesis and embryo sac development can be controlled by the ovule itself and need no more information from flower bud and /or plant.     

Foliar application of plant nutrients and kinetin modifies growth and essential oil profile in <Emphasis Type="Italic">Rosa damascena</Emphasis> under acidic conditions

Rosa damascena Mill. is cultivated for its high-value essential oil in different parts of the world. The flower yield and the composition of essential oil of R. damascena are strongly affected by a number of factors. Nevertheless, the interactive effects of foliar application of plant nutrients and kinetin and its time of application on yield and secondary metabolites profile of R. damascena under acidic conditions are still unclear. Thus, a field experiment comprising two different times of spray and five foliar spray treatments was conducted to test the hypothesis that flowering behavior and secondary metabolites profile can be modified through proper nutrient supply at right time. The foliar spray at flower bud appearance stage (S₂) significantly (P ≤ 0.05) increased flower yield by about 10.0 % compared with the foliar application at axillary bud development stage (S₁) during both years, regardless of plant nutrients. Among the foliar spray treatments, kinetin at 0.20 g L^?1 registered about 23–39 % higher flower yield compared with the water spray control; however, remained statistically at par (P ≤ 0.05) with Ca(NO₃)₂ at 4.06 g L^?1. Moreover, the percentage of major fragrance-bearing compounds of essential oil (β-citronellol + nerol, linalool, E-geraniol, and Z-citral) was marginally increased with Ca(NO₃)₂ compared with kinetin treatment. However, the percentages of major hydrocarbons, nonadecane and heneicosane, were noticeably increased when kinetin was applied at S₁. Foliar application of kinetin and Ca(NO₃)₂ might be done to improve flower yield and essential oil content in R. damascena flowers.     

Autonomous endosperm induction by in vitro culture of unfertilized ovules of Viola odorata L.

Autonomous endosperm was found in unfertilized ovules of V. odorata L. cultured on MS medium supplemented with 2,4-D as a sole growth regulator or on media with 2,4-D and BAP or kinetin. Frequency of endosperm induction was approximately 9% in ovules analyzed. The induction rate depended mainly on genotype of the donor plant, and to lesser degrees, on floral stage, flower series and medium type. Multinuclear endosperms consisting of 10–37 nuclei were found in ovules after as few as 4 days of culture. In some ovules at this stage, the egg cell and two polar nuclei were present. The process of endosperm degeneration began after 3 weeks of culture. In some ovules, degenerating autonomous endosperm was observed up to the 7th week. Parthenogenetic development of egg cells or apogamy did not accompany autonomous endosperm, supporting the hypothesis of independent pathways for embryo and endosperm development. Received: 1 December 1998 / Revision accepted: 6 April 1999     

Conversion of oat (<Emphasis Type="Italic">Avena sativa</Emphasis> L.) haploid embryos into plants in relation to embryo developmental stage and regeneration media

Obtaining oat DH lines is only effective via wide crossing with maize. Seven hundred haploid embryos from 21 single F₁ progeny obtained from wide crosses with maize were isolated, divided into four groups according to their size (<0.5 mm, 0.5–0.9 mm, 1.0–1.4 mm, and ≥1.5 mm), and transferred into 190–2 regeneration medium with different growth regulators: 0.5 mg L^?1 kinetin (KIN) and 0.5 mg L^?1 1-naphthaleneacetic acid (NAA); 1 mg L^?1 zeatin (ZEA) and 0.5 mg L^?1 NAA; or 1 mg L^?1 dicamba (DIC), 1 mg L^?1 picloram (PIC), and 0.5 mg L^?1 kinetin (KIN). Among all isolated embryos, approximately 46.1% were between 1.0–1.4 mm, while the smallest group of embryos (7.1%) were those <0.5 mm. The ability of haploid embryos to germinate varied depending on oat genotypes and the size of embryos. Haploid embryos <0.5 mm were globular and did not germinate, whereas embryos ≥1.5 mm had clearly visible coleoptiles, radicles, and scutella, and were able to germinate. Germination of oat haploid embryos varied depending on growth regulators in the regeneration medium. Most haploid embryos germinated on medium with 0.5 mg L^?1 NAA and 0.5 mg L^?1 KIN, while the fewest germinated on medium with 1 mg L^?1 DIC, 1 mg L^?1 PIC, and 0.5 mg L^?1 KIN. One hundred thirty germinated haploid embryos converted into haploid plants. Fifty oat DH lines were obtained after colchicine treatment.     

Effects of low temperature,genotype and culture media on in vitro androgenic answer of pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Anther culture is one of the most important and useful tool to create pure lines for plant breeding programs rapidly. Some pepper genotypes are recalcitrant and embryogenic frequency in anther culture is still low or reaction is not observed at all. Temperature stress (low or high) can facilitate switching the microspore to sporophyte developmental pathway. In this study, some differences were found in embryogenic reaction among the pepper genotypes and culture media variants, depending on duration of cold treatment of flower buds. Experimental results indicated that embryogenic efficiency decreased under low-temperature stress. Nevertheless, positive effect of cold pretreatment on direct embryo induction was obtained in four genotypes—cultivar Hebar, hybrid 50/01 and lines 668/02 and 1312/02. Increasing of embryogenic reaction after cold pretreatment was observed in media variants C (24 h) and MS-3 + (24 and 48 h), while on medium variant C-0 direct embryo formation was registered only after 48 h cold pretreatment. These results show that the donor genotypes have specific requirements for type and duration of temperature pretreatment and also culture media for induction of androgenesis with higher frequency.     

In vitro gynogenesis in cotton (<Emphasis Type="Italic">Gossypium</Emphasis> sp.)

The effects of genotype, pollen or growth regulator-pretreatment of pistils, developmental stage of the ovule (embryo sac) and culture media on induction of gynogenesis, and subsequent plantlet regeneration in vitro were assessed in interspecific Gossypium barbadense × G. hirsutum cotton hybrids. Gynogenesis occurred in all genotypes used when the pistils had been pre-treated with pollen from Hibiscus cannabinus and ovaries were harvested 5 or 10 days after anthesis. The use of culture media, SH and MS, showed no significant differences in responding ovules, embryogenic ovules or embryo germination frequency. Recovered progeny were characterized cytogenetically and microscopically to help documenting their reproductive basis. Root tip chromosome counts of 17 plants established from ovule culture revealed that chromosome numbers ranged from 27 to 44. Although the reproductive mechanisms need to be characterized more extensively by cytological and molecular means, the observations suggest that gynogenesis in cotton involves some unusual reproductive events. Aneuploids could be useful for functional genomic characterization of genome shock, deletion mapping, and germplasm introgression.     

Gynogenesis in gentians (Gentiana triflora, G. scabra): production of haploids and doubled haploids

Gynogenesis was investigated on gentian (Gentiana triflora, G. scabra and their hybrids), which is an important ornamental flower. When unfertilized ovules were cultured in 1/2 NLN medium containing a high concentration of sucrose (100 g/l), embryo-like structures (ELS) were induced. Although genotypic variation was observed in ELS induction, all four genotypes produced ELSs ranging from 0.93 to 0.04 ELSs per flower bud. The ovules collected from flower buds of later stages (just before anthesis or flower anthesis) tended to exhibit higher response. The dark culture condition produced more than four times as many ELSs than in 16-h light condition. A significant number of plantlets were directly regenerated from ELSs on MS regeneration medium. The ploidy levels of 179 regenerated plants were determined by flow cytometry, revealing that the majority of them were diploid (55.9%) and haploid (31.3%). When a total of 54 diploid plants were examined by molecular genetic markers, 52 (96.3%) were considered as doubled haploids (DHs). This is the first report showing successful gynogenesis in gentian. The production of haploids and DHs by unfertilized ovule culture opens a novel prospect in gentian F1 hybrid breeding.     

Doubled haploid plant production from unpollinated ovules of sugar beet (Beta vulgaris L.)

 The effects of cold pretreatment, AgNO₃ and activated charcoal on haploid plant production from unpollinated sugar beet ovules were investigated. Both cold pretreatment and the addition of charcoal increased the frequency of embryo formation, whereas AgNO₃ decreased or completely inhibited it. Colchicine (50, 100, 150 or 500 mg l^–1) and trifluralin (1.7, 3.4 or 5.0 mg l^–1) for 12, 24, 36 or 48 h were compared in agar-solidified, agarose-solidified or liquid media. Although colchicine gave a higher doubling rate (25.3%) than trifluralin (18.2%), the difference was not significant. Both agents were more effective when used in liquid (29.1%) than agarose-solidified medium (20.7%) and agar-solidified medium (15.4%). A treatment duration of 48 h was significantly more effective (27.5%) than 12 h (13.6%) but it was not different from 24 h (16.3%) or 36 h (18.6%). Received: 25 October 1999 / Revision received: 18 May 2000 / Accepted: 29 May 2000     

Production of haploid plants from in vitro culture of unpollinated ovules of Cucurbita pepo

Ovaries from squash plants (cv. Eskandarani) were picked one day before anthesis, and exposed to cold temperature (4 °C) for 0, 2, 4 and 8 days. The ovules were cultured on MS medium with 30 g l⁻¹sucrose, 8 g l⁻¹agar and supplemented with four concentrations of 2,4-D, i.e., 0.1, 1.0, 5 and 10 mg l⁻¹. Then the dishes were incubated at 25 ± 1 °C under 16-h photoperiod for 4 weeks. After that ovules were transferred to growth regulator free MS medium for 4 weeks. Data indicated that the most plantlets per 100 cultured ovules resulted from the ovule of ovaries without cold treatment, when cultured in MS medium supplemented with 1 or 5 mg l⁻¹2,4-D. The cytological study revealed that one third of examined plants were haploid (2n = x = 20) and the others were double haploid (2n = 2x = 40). This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of LAB 173711 and ethephon on time of flowering and cold hardiness of peach flower buds

Peach flowers are often killed during bloom by spring frosts. LAB 173711, a compound with abscisic (ABA)-like activity, and ethephon delayed flowering in peach trees. In greenhouse experiments, LAB 173711, at concentrations of 10^?3–10^?2 M, was most effective in delaying bloom when applied after a 5°C cold storage period, rather than before the dormancy breaking treatment. In contrast, ethephon delayed bloom most effectively when applied before 5°C cold storage; ethephon caused flower bud abscission when treatments were made after the chilling requirement had been satisfied. In field experiments, ethephon delayed flowering by 6–7 days, which reduced bud injury after a spring frost during bloom. No flower bud injury was found on ethephon-treated trees after temperatures of ?4.3°C; whereas without ethephon 25% of the flower buds were frost damaged. LAB 173711 delayed the time to 50% bloom by 2–3 days. However, this was not long enough to avoid low-temperature injury to the flower buds.     

Structural aspects of ovule and seed development and nonrandom abortion inMelilotus officinalis (Fabaceae)

Summary Only one ovule matures into a seed inMelilotus officinalis. Although eight ovules form within an ovary, only the basal ovule develops into a mature seed, whereas the other ovules degenerate. The investigation of ovule and seed structure at different developmental stages and a comparison of quantitative characters of differently fated ovules within an ovary were undertaken by light, phase contrast, and fluorescence microscopy. In this species, campylotropous ovules develop simultaneously on marginal placentae in an apocarpous unilocular gynoecium. Megasporo- and megagametogenesis proceed normally and are completed in bud. The maturation of the Polygonum type embryo sac takes place after the flower opens. Shortly before fertilization, synergids show signs of degeneration in all ovules. At this stage, neither the structure nor the sizes of ovules within one ovary differ significantly. In spite of this, only the basal ovule develops into a seed. Rarely, one of the upper-situated ovules or the basal and another ovule mature into seeds. Seed enlargement is insignificant until the stage when globular embryo and nuclear endosperm are formed. At the seed-filling stage, other ovules have collapsed and the seed gradually comes to occupy the total volume of the pod. The fruit-to-seed length ratio decreases considerably during seed ripening. At fertilization, ovary length is four times greater than ovule length. In the mature state, the fruit and seed lengths are approximately equal. Seed size and weight diminish with an increase in seed number within a pod, although pod size remains constant. It is assumed that nonrandom abortion of young seeds inM. officinalis is under maternal control and is not related to structural abnormalities in ovule development or with limitation in pollen. We suppose that evolution of this species may have proceeded in the direction of a decrease in seed number and an increase in its sizes, which may play an important role in seed dispersal and seedling establishment.     

An improved embryo-rescue protocol for hybrid progeny from seedless Vitis vinifera grapes × wild Chinese Vitis species

A highly efficient technique of embryo rescue is critical when using stenospermocarpic Vitis vinifera cultivars (female parents) to breed novel, disease-resistant, seedless grape cultivars by hybridizing with wild Chinese Vitis species (male parents) having many disease-resistance alleles. The effects of various factors on the improvement of embryo formation, germination, and plantlet development for seven hybrid combinations were studied. The results indicated that Beichun and Shuangyou were the best male parents. The best sampling time for ovule inoculation differed among the female parents. When hybrid ovules were cultured on a double-phase medium with five different solid medium types, percent embryo formation was highest (11.3–28.3%) on a modified MM3 medium. Percentages of embryo germination (15.4–55.4%) and plantlet development (11.15–44.6%) were all highest when embryos were cultured on Woody Plant Medium?+?5.7 μM indole-3-acetic acid?+?4.4 μM 6-benzylaminopurine?+?1.4 μM gibberellic acid?+?2% sucrose?+?0.05% casein hydrolysate?+?0.3% activated charcoal?+?0.7% agar. In the absence of other amino acids, the addition of proline significantly increased embryo formation (36.1%), embryo germination (64.6%), and plantlet development (90.5%). A highly efficient protocol has been developed for hybrid embryo rescue from seedless V. vinifera grapes?×?wild Chinese Vitis species that results in a significant improvement in breeding efficiency for new disease-resistant seedless grapes.