Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (6): 1218-1228.doi: 10.3864/j.issn.0578-1752.2021.06.012

• HORTICULTURE • Previous Articles     Next Articles

Rescue and Molecular Marker Assisted-Selection of the Cold-Resistant Seedless Grape Hybrid Embryo

PeiPei ZHU(),YiJia LUO,Wen XIANG,MingLei ZHANG,JianXia ZHANG()   

  1. College of Horticulture, Northwest Agriculture and Forestry University/State Key Laboratory of Crop Stress Biology in Arid Areas/Key Laboratory of Horticultural Plant Germplasm Resource Utilization in Northwest China, Ministry of Agriculture, Yangling 712100, Shaanxi
  • Received:2020-06-30 Accepted:2020-08-28 Online:2021-03-16 Published:2021-03-25
  • Contact: JianXia ZHANG E-mail:825965951@qq.com;zhangjx666@126.com

Abstract:

【Background】The traditional methods of breeding seedless grape can only use the seeded variety as the female parent and the seedless variety as the male parent to cross, and the rate of the seedless hybrid is extremely low. As a new breeding technology, the embryo rescue can use seedless grapes as female parents, which expands the selection range of female parents, and greatly improves the efficiency of seedless grape breeding. However, the embryo development rate, germination rate and seedling formation rate in seedless grape embryo rescue are still low, which restricts the application of this technique. 【Objective】The main objective of this paper was to screen suitable female parents and embryo development medium for embryo rescue, and to create new cold-resistant seedless grape germplasm by embryo rescue and molecular marker assisted-selection, so as to provide a basis for improving the effect of seedless grape embryo rescue and for further breeding new cold-resistant seedless grape varieties. 【Method】Using the cold-resistant seedless grape cultivar Jupiter (Euro-American hybrid) and the seeded hybrid 00-1-5 ( V. vinifera × V. amurensis) as the male parents and five seedless grape cvs. (Ruby Seedless, Qinhong No.1, Qinhong No.2, Crimson Seedless and Qinxiu) as the female parents, respectively, the hybridization was carried out between seedless grape and cold-resistant grape. The immature fruits of each female parent at the suitable sampling time were collected for ovule culture in vitro. The solid-liquid biphasic MM3 and ER medium were used as the embryo development medium to compare the effects of different medium and female parents on the rate of embryo development, germination, normal seedlings and abnormal seedlings. WPM + 0.2 mg?L-1 6-BA + 0.1 mg?L-1 IAA solid medium was used as embryo germination and seedling formation medium. The embryo-rescue seedlings obtained after domestication in the greenhouse were transplanted to the field in spring. In addition, 2MS + 0.2 mg?L-1 6-BA + 0.1 mg?L-1 IAA medium was used for transforming deformed seedlings into normal seedlings, which was added with 0, 1.0 and 1.6 mg?L-1 ZnSO4, respectively. Finally, the molecular marker SCF27-2000 linked to the seedless gene was employed to detect the seedless hybrids for embryo-rescue seedlings. 【Result】For five cross combinations, 175 embryos from 2 158 ovules cultured in MM3 were obtained, which were further germinated and formed 118 hybrid plants. 74 embryos from 894 ovules cultured in ER medium were obtained and further germinated and formed 58 hybrid plants. Using 00-1-5 as male parent, seedless cv. Qinhong No.2 was much more suitable to be the female parent than Qinxiu, its embryo development rate and seedling rate were the highest, reaching 17.04 % and 7.41%, respectively. Using Jupiter as the male parent, Ruby Seedless × Jupiter had the best effect, with embryo development and seedling rate of 13.71% and 10.67%; the next was Qinhong No.1 × Jupiter, the embryo development rate and seedling rate were 14.39% and 4.71%, respectively. However, Crimson Seedless × Jupiter had the lowest embryo development rate and seedling rate, reached to 9.55% and 1.76%, respectively. The rates of embryo development and seedling formation of ovules inoculated into the MM3 medium were higher than that of the ER medium. The highest rate of deformed seedlings was Qinhong No.1 × Jupiter, while Ruby Seedless × Jupiter had no deformed seedlings. The rate of deformed seedlings in the ER medium was higher than that in the MM3 medium. The deformed seedlings of combination Qinhong No.2 × 00-1-5 were transformed to 2MS+1.6 mg?L-1 ZnSO4+0.2 mg?L-1 6-BA+0.1 mg?L-1 IAA, producing the best results with a conversion rate of 40.00%. Using the molecular marker SCF27-2000 linked to seedless character, 176 hybrid strains from 5 cross combinations were detected and there were 159 strains with the seedless molecular markers. 【Conclusion】 Ruby Seedless, Qinhong No.1 and Qinhong No.2 were suitable as the seedless female parents for embryo rescue in vitro, but Crimson Seedless and Qinxiu were not relevant. MM3 medium was more appropriate for embryo rescue in vitro than the ER medium. The 159 plants from embryo rescue with the molecular marker linked to the seedless gene were potential seedless hybrids.

Key words: seedless grape, cold-resistance, embryo rescue, new germplasm, molecular marker- assisted selection

Fig. 1

Procedure charts of breeding new cold-resistant seedless grapes by embryo rescue A: Flowers after emasculation; B: Embryo; C-E: Young embryo germinates into a seedling; F: Transplanting hybrid seedlings to field"

Table 1

Effects of different female parents on embryo rescue"

杂交组合
Crossing
combination
胚珠数
No. of ovules
剥胚数
No. of embryos developed
萌发数
No. of embryos germinated
成苗数
No. of normal seedings
发育率
Proportion of embryos developed (%)
萌发率
Proportion of embryos germinated (%)
成苗率
Proportion of seedings
(%)
秦秀×00-1-5 Qinxiu×00-1-5 427 18 11 6 4.22e 2.58d 1.41e
秦红2号×00-1-5 Qinhong No.2×00-1-5 405 69 38 30 17.04a 9.38b 7.41b
Crimson Seedless × Jupiter 398 38 27 7 9.55d 6.78c 1.76d
Ruby Seedless × Jupiter 525 72 56 56 13.71c 10.67a 10.67a
秦红1号×Jupiter Qinhong No.1×Jupiter 403 58 43 19 14.39b 10.67a 4.71c
总计 Total 2158 255 175 118 11.82 8.11 5.47

Table 2

Effects of embryo development media MM3 and ER on embryo rescue"

杂交组合
Crossing
combination
培养基类型
Types of medium
胚珠数
No.of ovules cultured
剥胚数
No. of embryos developed
萌发数
No. of embryos germinated
成苗数
No. of Normal seedings
发育率
Proportion of
Embryos developed (%)
萌发率
Proportion of
Embryos germinated (%)
成苗率
Proportion
of seedings
(%)
Ruby Seedless × Jupiter MM3 525 72 56 56 13.71a 10.67a 10.67a
Ruby Seedless × Jupiter ER 508 58 35 35 11.42b 6.89b 6.89b
秦红2号×00-1-5
Qinhong No.2 × 00-1-5
MM3 405 69 38 30 17.04a 9.38b 7.41a
秦红2号×00-1-5
Qinhong No.2 × 00-1-5
ER 386 50 39 23 12.95b 10.10a 5.96b

Table 3

Effects of female parents on formation of abnormal seedlings"

杂交组合
Crossing combination
胚珠数
No. of ovules
正常苗 Normal plantlet 畸形苗 Abnormal plantlet
数量 No. 百分比Percentage (%) 数量 No. 百分比Percentage (%)
秦秀×00-1-5 Qinxiu × 00-1-5 427 6 1.41c 5 1.17bc
秦红2号×00-1-5 Qinhong No.2 × 00-1-5 405 30 7.41b 8 1.98cd
Crimson Seedless × Jupiter 398 7 1.76d 20 5.03a
Ruby Seedless × Jupiter 525 56 10.67a 0 0d
秦红1号×Jupiter Qinhong No.1 × Jupiter 403 19 4.71c 24 5.96ab
总计 Total 2158 118 5.47 57 2.64

Table 4

Effects of MM3 and ER media on rate of abnormal seedlings"

杂交组合
Crossing combination
培养基类型
Types of medium
胚珠数
No. of ovules
正常苗 Normal plantlets 畸形苗 Abnormal plantlet
数量 No. 百分比Percentage (%) 数量 No. 百分比Percentage (%)
Ruby Seedless × Jupiter MM3 525 56 10.67a 0 0.00b
Ruby Seedless × Jupiter ER 508 35 6.89b 0 0.00b
秦红2号×00-1-5 Qinhong No.2 × 00-1-5 MM3 405 30 7.41b 8 1.98b
秦红2号×00-1-5 Qinhong No.2 × 00-1-5 ER 386 23 5.96b 16 4.15a

Table 5

Effects of different concentration of ZnSO4 on rate of abnormal seedling transformation"

培养基类型
Types of medium
畸形苗数
No. of abnormal plantlet
畸形苗
Abnormal plantlet
转化的正常苗
Transformation normal plantlet
数量
No.
百分比Percentage (%) 数量
No.
百分比Percentage (%)
2MS+0.2mg?L-1 6-BA+0.1mg?L-1 IAA 15 15 100a 0 0b
2MS+1.0mg?L-1 ZnSO4+0.2mg?L-1 6-BA+0.1mg?L-1 IAA 15 10 66.67b 5 33.33a
2MS+1.6mg?L-1 ZnSO4+0.2mg?L-1 6-BA+0.1mg?L-1 IAA 15 9 60.00b 6 40.00a

Fig. 2

Detection of seedless marker SCF27-2000 to the hybrids of combination ‘Qinhong No.2’ × ‘00-1-5’ M: Marker (2k Plus DNA marker); 1: Qinhong No.2; 2: 00-1-5; 3-55: hybrid seedlings from crosses between ‘Qinhong No.2’ × ‘00-1-5’. “+” indicates the 2000 bp specific band was present; “-” indicates the 2000 bp specific band was absent. The same as below"

Fig. 3

Detection of seedless marker SCF27-2000 to the hybrids of combination ‘Qinhong No.1’ × ‘Jupiter’ M:Marker (2k Plus DNA marker); 1: Qinhong No.1; 2: Jupiter; 3-21: hybrid seedlings from crosses between ‘Qinhong No.1’ × ‘Jupiter’"

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