Breeding of disease-resistant seedless grapes using Chinese wild Vitis spp. I. In vitro embryo rescue and plant development

The purpose of this study was to understand factors affecting in vitro embryo rescue culture from hybrids between diploid and tetraploid varieties of grape in creation new triploid germplasm resources. The effects of different media, removal ages of immature seeds and reciprocal crosses of parents on the germination and seedling survival of immature seeds from crosses between diploid and tetraploid grape varieties by in vitro embryo rescue culture were investigated. The results indicated that the medium consisting of NN-1969 + IAA 1.75 mg l−1 + GA3 0.35 mg l−1 + CH 400 mg l−1 + AC 2.0 g l−1 was better than other media. The optimal removal age of immature seeds for the best development of embryos was 35–45 days after pollination (DAP). The percentage of germination (PG) for immature seeds and the percentage of seedling survival (PSS) for immature seeds for diploid varieties used as female parents were 10.72% and 4.35% higher than when tetraploid varieties were used as female parents respectively. A total of 41 hybrid progenies from eight combinations were obtained, made up of 17 diploid, 9 tetraploid, 14 aneuploid, and 1 triploid progeny as determined by root-tip chromosome identification. The triploid progeny was from Fujiminori (2n = 4x = 76) × Jingxiu (2n = 2x = 38). These results implied that it was feasible to extend the hybridization range of grape and to create new germplasm resources by in vitro embryo rescue based on the conventional hybridization. The NN-1969 medium supplemented with GA3 and IAA was more propitious to the development of immature seeds sampled at about 45 DAP. It was easier to obtain plants using diploid as female parent, but triploid progeny was only obtained using tetraploid as female parent.

In vitro embryo rescue culture of F1 progenies from crosses between diploid and tetraploid grape varieties

Grape (Vitis vinifera L.) is one of the most important fruit crops in the world and is subject to intense breeding efforts to develop new seedless cultivars. To overcome inherent obstacles associated with crossing seedless selections, in ovulo embryo rescue was developed and successfully utilized by plant breeders to rescue inherently weak, immature and/or abortive embryos, in order to obtain progeny from seedless × seedless crosses. To date, embryo rescue has been utilized in grape breeding for more than three decades. Genotype, sampling/inoculation time and medium are the most crucial and well-studied factors affecting the success of grape embryo rescue. Besides, other factors, such as the culture methods and utilization of plant growth regulators are also important for grape embryo rescue. Thus far, embryo rescue was extensively applied in rescuing inherently weak grape embryos, breeding seedless grapes and triploid grapes, and distant hybridization between different Vitis species. Although grape embryo rescue has been widely investigated, the development of improved cultivars is few. Breeding novel grape cultivars through embryo rescue is still a challenging and long-term task, which requires persistent effort of grape breeders. This review provides updated and comprehensive information concerning factors and applications of embryo rescue in grape.

Embryo rescue technique and its applications for seedless breeding in grape

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.

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

This project aimed at breeding new seedless grape cultivars by embryo rescue through three hybridization methods: 1) using cross-breeding between seedless Vitis vinifera cultivars and wild Chinese Vitis spp; 2) crossing with two seedless cultivars, and 3) hybridization between grapes of different ploidy. Genotype, sampling times, and media were confirmed to play important roles in this system. Among the different genotypes, the productions of hybrid plants were significantly different, ranging from 23.0% (Ruby Seedless x Black Olympia) to only 1.1% (Pink Seedless x Beichun), except for the combinations from which no surviving seedlings were obtained. We got the best sampling times, in days after flowering (DAF), from the following different combinations: 'Flame Seedless x Beichun' (39 DAF); 'Blush Seedless x Shuangyou' (54 DAF); 'Pink Seedless x Beichun' (54 DAF); 'DA7 x Shuangyou' (44 DAF); 'Blush Seedless x Thompson Seedless (54 DAF)'; 'Pink Seedless x Flame Seedless' (54 DAF); 'DA7 x Blush Seedless' (44 DAF); 'Ruby Seedless x Black Olympia' (63 DAF); 'DA7 x Jingyou' (44 DAF); 'Flame Seedless x Fujiminori' (39 DAF), and 'Big Black x Kyoho' (72 DAF). The highest rates of embryo formation (13.2%) and plant development (90.1%) were found when ovules were cultured in MM4 with 500 mg/L mashed banana. Conversely, they were reduced by addition of plant growth regulators. Seven new hybrids were successfully obtained. As a result of early nuclear-free character and ploidy level identification, 11 seedless grape lines, and 3 triploid and 2 haploid grape lines were obtained.

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Breeding new seedless grape by means of in vitro embryo rescue.

In ovulo embryo rescue techniques have been used to recover new hybrids from seedless × seedless grape crosses. This study was conducted to increase efficiency by investigating effects of genotype, medium, and ovule 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 also investigated. Ovules exhibited continuous growth in vivo and in vitro. The most vigorous growth was observed for ovules cultured at 30 and 43 DAF, but more embryos were recovered from ovules cultured at 60 and 70 DAF. Ovule growth and 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%, and 12.2% for Sunmuscat, Merbein Seedless, and Marroo Seedless, respectively, when ovules were excised and cultured at 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) medium containing 0.3% AC. Embryo recovery was improved by excising and culturing ovules at 60 DAF in BD or NN media and then by transferring embryos to WP medium supplemented with BA and AC.

P. R. Clingeleffer

Papers

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