New disease-resistant, seedless grapes are developed using embryo rescue and molecular markers

Abstract

Improving the operation of embryo rescue technology is a highly efficient way to breed new high-quality cultivars of seedless, disease-resistant grapes when using a stenospermocarpic Vitis vinifera L. (female parents) hybrid with Chinese wild Vitis (male parents). In this study, sampling time had a significant effect on embryo recovery. Four crosses were investigated to improve the embryo rescue efficiency by determining the best sampling time, which was found to be 40, 50, 55, and 57 days after pollination (DAP) for 'Flame seedless' × 'Ruby seedless', 'Kunxiang seedless' × 'Flame seedless', 'Ruby seedless' self-pollination and 'Ruby seedless' × 'Flame seedless', respectively. The highest percentage embryo germination ranged from 60.83 to 89.10% for four cross-combinations when the embryo was germinated on woody plant medium (WPM), with 1.0-μM thidiazuron (TDZ, a potent cytokinin). For 11 cross-combinations, the embryo recovery rate varied from 4.8 to 29.6% and the plant development rate varied from 17.1 to 78.9%. 'Ruby seedless' was the best female parent followed by 'Flame seedless'. The Grape Seedless gene Probe 1 (GSLP1) and SCF27 molecular markers were used to assist the identification of the seedless traits of the 388 progeny. A total of 309 plantlets were amplified, with the specific bands at 569-bp and 2k-bp being preliminarily considered as seedless. The S382-615 marker was used to identify the downy mildew resistance of the 'Ruby seedless' × 'Beichun' cross. Two F1 progeny were amplified, with the specific band at 615-bp being identified as downy mildew resistance.

Keywords: Disease resistant; Embryo rescue; Grapevine; Molecular marker-assisted selection; Seedlessness.

Fig. 1

Hybrid progeny from Vitis vinifera grapes × wild Chinese vitis species in the field. a The inflorescence of Shuangyou; b emasculated inflorescence; c hand pollination; d bagging the inflorescence

Fig. 2

Embryo rescue protocol for hybrid progeny. a Ovules inoculated onto MM3 medium; b–d embryo inoculated onto germination medium; e–f plant in a pot covered with a plastic cup in the greenhouse; g surviving plants growing in soil

Fig. 3

Effect of different sampling times on embryo recovery rate in different crosses. a(1), the ovules on cultures of ‘Flame seedless’ × ‘Ruby seedless’ from DAP 37 to 43; a(2), the ovules on cultures of ‘Ruby seedless’ × ‘Flame seedless’ from DAP 52 to 58. The red arrow indicates the aborted ovule. b(1)–(4), the embryo recovery rate of four crosses; b(5)–(8), the embryo germination rate of four crosses; b(9)–(12), the plant development rate of four crosses. Values are mean ± SE of three replicates. Means with the same letter within a column are not significantly different (Duncan’s multiple range test, P ≤ 0.05). Embryo recovery rate (%) = the number of embryos developed/the number of ovules cultured × 100%

Fig. 4

The embryos germinated from four different cross-combinations in different media