Construction of embryo rescue technology system for seedless grape cultivar Zhongputao No.18 and early selection of hybrid offspring

Abstract:【Objective】One of the major objectives of table grape breeding is to produce seedless grape- vines. Using stenospermocarpic grapevines as female parents could improve the seedless ratio of off- spring via embryo rescue techniques, so they are widely used for breeding seedless grapes. Here, we used the stenospermocarpic grapes Zhongputao No.18 as female parents to breed new seedless grapes and investigated the effects of various factors on the improvement of embryo formation, germination, and plantlet development to construct its embryo rescue technology system, and to provide a reference for the embryo rescue breeding of seedless grapevines.【Methods】The hybrid ovules were obtained from cross combination Zhongputao No.18 (seedless grape cultivar) × Muscat Hamburg (seeded mus- cat-scented cultivar) and the naturally pollinated Zhongputao No.18. In this research, the effects of the stage of ovule development (52, 53, 54, 57, 58, 61, and 65 days after anthesis), the type of basal medium and its phase, types of amino acids, coconut juice concentrations, and plant growth regulator were assessed on the success rate of the embryo rescue Zhongputao No.18. The gibberellic acid (GA3), kine- tin (KT), and Naphthylacetic acid (NAA) were combined by Uniform Design Experimentation U7 (73) to study the effects of the three types of hormones on embryo germination and seedling formation. Moreover, compare the effects of four cytokinins TDZ (0, 0.2 mg · L-1), 6-BA (0, 0.2 mg · L-1), KT (0, 0.2 and 0.4 mg·L-1) and ZT (0, 0.2 and 0.4 mg·L-1) on embryo germination and seedling formation. Charac- ters such as development embryo, embryo germination, and seedling formation were recorded. Finally, the molecular marker linked to the seedless gene was employed to detect the seedless hybrids for em- bryo-rescue seedlings.【Results】When the berries were harvested at 52, 53, 54, 57, 58, 61, and 65 days after anthesis (DAF), the highest development embryo percentage was observed at 61 days after anthe- sis, 34.33%. Moreover, the ovules were obtained at 16, 19, 22, 26, 29, 33, 36, 43, 45, 51, 54, 57, 61, 65 and 68 DAF, from the naturally pollinated Zhongputao No.18 to measure the longitudinal diameter, transverse diameter, and weight, the results indicated that all increased significantly before 36 DAF, they tended to be flat during 36-61 DAF and then decreased significantly after 61 DAF. When the hy- brid ovules were cultured on Nitsch and Nitsch (NN) medium or Emershad and Ramming (ER) medi- um, NN (29.33%) had a higher development embryo percentage than ER (29.33% compared to 25%). When hybrid ovules were cultured on a double-phase or solid medium with two different types, percent embryo formation was higher on double- phase. The results indicated that 2.5 mmol · L-1 Serine (Ser), Cysteine (Cys), or Glutamine (Glu) in the NN medium all increased the percentages of development em- bryo, which was higher than no addition (40.74%, 43.33%, 44.21% compared to 29.33%). The effects of coconut juice (CJ) concentrations (0, 100, 200 mL·L-1) were assessed on embryo development, the re- sults indicated that 200 mL·L-1 CJ in NN medium had a higher percentage of development embryo than no addition (37.10% compared to 29.33%). In addition, the results revealed the effect of GA3/KT/NAA combination or four different cytokinins (TDZ, 6-BA, KT, and ZT) in Woody Plant Medium (WPM) on embryo germination and seedlings formation. The results indicated that percentage seedlings formation was the highest when the embryo was germinated on WPM with 0.4 mg·L-1 GA3+1 mg·L-1 KT (60%), while the percentage was low ranged from 1.52% to 37.74% when NAA was added. The embryo in me- dium with NAA showed abnormal differentiation, for example, there were only a large number of differ- entiation of roots and very little differentiation of stems and leaves. Moreover, among the four cytoki- nins, the percentage of seedlings formation was the highest when embryos were inoculated at WPM with 0.2 mg · L- 1 ZT (76.36% ). Using the molecular markers P3_VvAGL11 and 5U_VviAGL11 linked to seedless character, 409 hybrid strains from cross combination Zhongputao No.18 × Muscat Hamburg were detected, then 229 strains with the seedless specific bands were preliminarily identified as seed- less, and the seedless rate was 56.4%.【Conclusion】When using the seedless grape cultivar V. viniferaL.‘Zhongputao No.18’as the female parents for embryo rescue breeding, 61 DAF was the best sam- pling time. The ovules could be cultured in vitro in the medium that solid and liquid phases were all NN medium adding 2.5 mmol·L-1 Cys/Gln, 60 g·L-1 sucrose, 0.5 g·L-1 casein hydrolysate, 2 g·L-1 activated carbon and 5.5 g·L-1 agar or not. After 9-10 weeks of ovules culture under dark condition at a room tem- peratureof (25±2)°C,allembryosdevelopedwereexcisedandculturedonsolidWPM+0.2 mg·L-1 ZT+ 20 g · L-1 sucrose + 6 g · L- 1 agar + 1 g · L- 1 activated carbon. The 229 embryo rescued seedlings carrying seedless specific bands provided the germplasm basis for the breeding of new seedless grapevines.