抗寒无核葡萄杂种胚挽救及分子标记辅助选择

doi:10.3864/j.issn.0578-1752.2021.06.012

Abstract

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

PeiPei ZHU,YiJia LUO,Wen XIANG,MingLei ZHANG,JianXia ZHANG. Rescue and Molecular Marker Assisted-Selection of the Cold-Resistant Seedless Grape Hybrid Embryo[J].Scientia Agricultura Sinica, 2021, 54(6): 1218-1228.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2021.06.012

https://www.chinaagrisci.com/EN/Y2021/V54/I6/1218

Figures/Tables 8

Fig. 1

Table 1

Table 2

Table 3

Table 4

Table 5

Fig. 2

Fig. 3

References 48

[1]	LI G R, CHENG S S, JI W, HOU X J, HU H L. In vitro embryo rescue of F-1 progenies from crosses between seedless grapes and Chinese wild types. Bangladesh Journal of Botany, 2019,48(3):755-761.
[2]	ZHANG J, WU X, NIU R K, LIU Y C, LIU N, XU W H, WANG Y C. Cold-resistance evaluation in 25 wild grape species. Vitis, 2012,51(4):153-160.
[3]	RAMMING D W, EMERSHAD R L. In ovulo embryo culture of seeded and seedless Vitis vinifera. HortScince, 1982,17:487.
[4]	LI S S, LI Z Y, ZHAO Y N, ZHAO J, LUO Q W, WANG Y J. New disease-resistant, seedless grapes are developed using embryo rescue and molecular markers. 3 Biotech, 2020,10(1):1-12. doi: 10.1007/s13205-019-1978-z pmid: 31815083
[5]	NOTSUKA K, TSURU T, SHIRAISHI M. Seedless-seedless grape hybridization via in-ovule embryo culture. Journal of the Japanese Society for Horticultural Science, 2001,70(1):7-15.
[6]	徐海英, 阎爱玲, 张国军. 葡萄二倍体与四倍体品种间杂交胚珠的离体培养. 果树学报, 2001,18(6):317-320.
	XU H Y, YAN A L, ZHANG G J. Study on the in ovulo embryo culture of the crossed progeny between diploid and tetraploid grape cultivars. Journal of Fruit Science, 2001,18(6):317-320. (in Chinese)
[7]	SAHIJRAM L, MURTHY B N S, SHIKHAMANY S D. Introgression of downy mildew resistance into grape cv. Thompson Seedless through hybrid embryo rescue in vitro. Indian Journal of Horticulture, 2005,62(2):107-111.
[8]	李莎莎, 王跃进. 葡萄无核基因及无核育种研究进展. 园艺学报, 2019,46(9):1711-1726.
	LI S S, WANG Y J. Advances in seedless gene researches and seedless breeding in grapevine. Acta Horticulturae Sinica, 2019,46(9):1711-1726. (in Chinese)
[9]	ZHU P P, GU B, LI P Y, SHU X, ZHANG X, ZHANG J X. New cold-resistant, seedless grapes developed using embryo rescue and marker-assisted selection. Plant Cell Tissue and Organ Culture (PCTOC), 2019,140(3):551-562.
[10]	LI J, WANG X H, WANG X P, WANG Y J. Embryo rescue technique and its applications for seedless breeding in grape. Plant Cell Tissue Organ Cult (PCTOC), 2015,120(3):861-880.
[11]	SPIEGEL-ROY P, SAHAR N, BARON J, LAVI U. In vitro culture and plant formation from grape cultivars with abortive ovules and seeds. Journal of the American Society for Horticultural Science, 1985,110:109-112.
[12]	POMMER C V, RAMMING D W, EMERSHAD R L. Influence of grape genotype, ripening season, seed trace size, and culture date on in ovule embryo development and plant formation. Bragantia, 1995,54(2):237-249.
[13]	RAZI M, MARANDI R J, BANEH H D, DOULATI BANEH H, HOSSEINI B, DARVISHZADEH R. Effect of paternal genotypes sprays with BA and IAA concentration on embryo rescue of F₁ progenies from ‘Askari’ (Vitis vinifera L.) cultivar. Journal of Agricultural Science & Technology, 2013,15(5):1023-1032.
[14]	LIU Q, ZHANG J, WANG Y, YU D, XIA H. Breeding for cold-resistant, seedless grapes from Chinese wild Vitis amurensis using embryo rescue. New Zealand Journal of Experimental Agriculture, 2015,44(2):136-151.
[15]	RAMMING D W, EMERSHAD R L, SPIEGEL ROY P, SAHAR N, BARON I. Embryo culture of early ripening seeded grape (Vitis vinifera) genotypes. HortScience, 1990,25(3):339-342.
[16]	TIAN L L, WANG Y J. Seedless grape breeding for disease resistance by using embryo rescue. Vitis Geilweilerhof, 2015,47(1):15-19.
[17]	方宣钧, 吴为人, 唐纪良. 作物DNA标记辅助育种. 北京: 科学出版社, 2001: 2-6.
	FANG X J, WU W R, TANG J L. DNA Marker-Assisted Crop Breeding. Beijing: Science Press, 2001: 2-6. (in Chinese)
[18]	LAHOGUE F, THIS P, BOUQUET A. Identification of a codominant scar marker linked to the seedlessness character in grapevine. Theoretical and Applied Genetics, 1998,97(5/6):950-959.
[19]	MEJÍAN, HINRICHSEN P. A new, highly assertive SCAR marker potentially useful to assist selection for seedlessness in table grape breeding. Acta Horticulturae, 2003,603:559-564.
[20]	王跃进, LAMIKANRA O. 检测葡萄无核基因DNA探针的合成与应用. 西北农林科技大学学报(自然科学版), 2002,30(3):42-46.
	WANG Y J, LAMIKANRA O. Application and synthesis on the DNA probe for detecting seedless genes in grapevine. Journal of Northwest A & F University (Natural Science Edition), 2002,30(3):42-46. (in Chinese)
[21]	KARAAGAC E, VARGAS A M, NDRÉS M T D, CARREÑO I, IBÁÑEZ J, CARREÑO J, MARTÍNEZ-ZAPATER J, CABEZAS J. AMarker assisted selection for seedlessness in table grape breeding. Tree Genetics & Genomes, 2012,8(5):1003-1015.
[22]	CABEZAS J A, CERVERA M T, RUIZ-GARCIA L, CARRENO J, MARTINEZ-ZAPATER J M. A genetic analysis of seed and berry weight in grapevine. Genome, 2006,49(12):1572-1585. pmid: 17426772
[23]	马亚茹, 冯建灿, 刘崇怀, 樊秀彩, 孙海生, 姜建福, 张颖. 葡萄无核性状的SSR新分子标记开发及应用. 中国农业科学, 2018,51(13):2622-2630.
	MA Y R, FENG J C, LIU C H, FAN X C, SUN H S, JIANG J F, ZHANG Y. Development and application of SSR new molecular marker for seedless traits in grape. Scientia Agricultura Sinica, 2018,51(13):2622-2630. (in Chinese)
[24]	LI Z Q, LI T M, WANG Y J, XU Y. Breeding new seedless grapes using in ovulo embryo rescue and marker-assisted selection. In Vitro Cell Developmental Biology-Plant, 2015,51(3):241-248.
[25]	VALDEZ J G. Immature embryo rescue of grapevine (Vitis vinifera L.) after an extended period of seed trace culture. Vitis, 2005,44(1):17-23.
[26]	SIMIN U, KESGIN M, DILLI Y. The success of in vitro embryo rescue technique in hybridization of seedless grape varieties. World Congress of Vine and Wine, 2015,38(5):01008.
[27]	KIM S H, KWON J H, PARK Y S, HEO J Y. In vitro embryo rescue for the production of hypotetraploids after cross between hypotetraploid and tetraploid grape cultivars. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 2020,48(1):503-508.
[28]	EMERSHAD R L, RAMMING D W. In-ovulo embryo culture of Vitis vinifera L. cv. ‘Thompson Seedless’. American Journal of Botany, 1984,71(6):873-877.
[29]	BURGER P, GOUSSARD P G. In vitro culture of ovules and embryos from seedless grapes (Vitis vinifera L.). South African Journal for Enology & Viticulture, 1996,17(2):31-37.
[30]	KUMAR R, KHURANA A, SHARMA AK. Role of plant hormones and their interplay in development and ripening of fleshy fruits. Journal of Experimental Botany, 2014,65(16):4561-4575. doi: 10.1093/jxb/eru277
[31]	BHARATHY P V, KARIBASAPPA G S, PATIL S G, AGRAWAL D C. In ovulo rescue of hybrid embryos in Flame seedless grapes-influence of pre-bloom sprays of benzyladenine. Scientia Horticulturae, 2005,106(3):353-359.
[32]	KHOSHANDAM L, BANEH H D, MARANDI R J, DARWISHZADEH R. Effect of BA and ovule developmental stages on embryo rescue in Perlette grape (Vitis vinifera L.) cultivar. Journal of Natural and Social Sciences, 2017,6(1):1-9.
[33]	EBADI A, AALIFAR M, FARAJPOUR M, MOGHADDAM M R F. Investigating the most effective factors in the embryo rescue technique for use with ‘Flame Seedless’ grapevine (Vitis vinifera). Journal of Horticultural Science & Biotechnology, 2016,91(5):441-447.
[34]	PONCE M T, GUIÑAZÚ M E, TIZIO R. Brief note improved in vitro embryo development of stenospermic grape by putrescine. Biocell, 2002,26(2):263-266. pmid: 12240561
[35]	赵雅楠, 骆强伟, 王跃进. 利用胚挽救技术创制无核抗寒葡萄新种质. 中国农业科学, 2018,51(21):4119-4130.
	ZHAO Y N, LUO Q W, WANG Y J. Breeding for grape germplasm involved in seedlessness with cold-resistant using embryo rescue. Scientia Agricultura Sinica, 2018,51(21):4119-4130. (in Chinese)
[36]	GRAY D J, MORTENSEN J A, BENTON C M, DURHAM R E, MOORE G A. Ovule culture to obtain progeny from hybrid seedless bunch grapes. Journal of the American Society for Horticultural Science, 1990,115(6):1019-1024.
[37]	LESHEM B and SACHS T. ‘Vitrified’ Dianthus-teratomata in vitro due to growth factor imbalance. Annals of Botany, 1985,56(5):613-617. doi: 10.1093/oxfordjournals.aob.a087051
[38]	LIU S M, SYKES S R, CLINGELEFFER P R. Improved in ovulo embryo culture for stenospermocarpic grapes (Vitis vinifera L.). Australian Journal of Agriculture Research, 2003,54(9):869-876. doi: 10.1071/AR03053
[39]	JI W, LI Z Q, YAO W K, GONG P J, WANG Y J. Abnormal seedlings emerged during embryo rescue and its remedy for seedless grape breeding. Korean Journal of Horticultural Science & Technology, 2013,31(4):483-489.
[40]	STOUT A B. Progress in breeding for seedless grapes. Proceeding American Society for Horticultural Science, 1939,37:627-629.
[41]	WEINBERGER J H, HARMON F N. Seedlessness in vinifera grapes. Proceedings of the American Society for Horticultural Science, 1964,85:270-274.
[42]	SANDHU A S, JAWANDA J S, UPPAL D K. Inheritance of seed characters in hybrid populations of intercultivar crosses of grapes (Vitis vinifera L.). Journal of Research-Punjab Agricultural University (India), 1984,21:39-44.
[43]	SPIEGEL-ROY P, BARON Y, SAHAR N. Inheritance of seedlessness in seeded × seedless progeny of Vitis vinifera L. Vitis, 1990,29:79-83.
[44]	LEDBETTER C A, BURGOS L. Inheritance of stenospermocarpic seedlessness in Vitis vinifera L. Journal of Heredity, 1994,85(2):157-160. doi: 10.1093/oxfordjournals.jhered.a111419
[45]	BOUQUET A, DANGLOT Y. Inheritance of seedlessness in grapevine (Vitis vinifera L.). Vitis, 1996,35(1):35-42.
[46]	DOLIGEZ A, BOUQUET A, DANGLOT Y, LAHOGUE F, RIAZ S, MEREDITH C, EDWARDS K, THIS P. Genetic mapping of grapevine (Vitis vinifera L.) applied to the detection of QTLs for seedlessness and berry weight. Theoretical & Applied Genetics, 2002,105(5):780-795. doi: 10.1007/s00122-002-0951-z pmid: 12582493
[47]	ROYTCHEV V. Inheritance of grape seedlessness in seeded and seedless hybrid combinations of grape cultivars with complex genealogy. American Journal of Enology and Viticulture, 1998,49(3):302-305.
[48]	COSMIDES L M, TOOBY J. Cytoplasmic inheritance and intragenomic conflict. Journal of Theoretical Biology, 1981,89(1):83-129. pmid: 7278311

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

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

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

杂交组合 Crossing combination	胚珠数 No. of ovules	正常苗 Normal plantlet		畸形苗 Abnormal plantlet
杂交组合 Crossing combination	胚珠数 No. of ovules	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

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
杂交组合 Crossing combination	培养基类型 Types of medium	胚珠数 No. of ovules	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

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
培养基类型 Types of medium	畸形苗数 No. of abnormal 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

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

RichHTML

PDF

Abstract

Cite this article

share this article

Figures/Tables 8

References 48

Related Articles 11

Metrics

Comments

Recommended 0

[1]	XIE KaiDong,PENG Jun,YUAN DongYa,QIANG RuiRui,XIE ShanPeng,ZHOU Rui,XIA QiangMing,WU XiaoMeng,KE FuZhi,LIU GaoPing,GROSSER Jude W,GUO WenWu. Production of Citrus Triploids Based on Interploidy Crossing with Bendizao and Man Tangerines as Female Parents [J]. Scientia Agricultura Sinica, 2020, 53(23): 4961-4968.
[2]	LI GuiRong,QUAN Ran,CHENG ShanShan,HOU XiaoJin,FAN XiuCai,HU HuiLing. The Influencing Factors of in-vitro Ovule Development in Seedless Grape and Its Physiological Changes [J]. Scientia Agricultura Sinica, 2020, 53(22): 4646-4657.
[3]	JIA ShanShan,LUO QiangWei,LI ShaSha,WANG YueJin. Optimization of Embryo Rescue Technique and Production of Potential Seedless Grape Germplasm with Rosy Aroma [J]. Scientia Agricultura Sinica, 2020, 53(16): 3344-3355.
[4]	YaNan ZHAO,QiangWei LUO,YueJin WANG. Breeding for Grape Germplasm Involved in Seedlessness with Cold-Resistant Using Embryo Rescue [J]. Scientia Agricultura Sinica, 2018, 51(21): 4119-4130.
[5]	WANG HuiJuan, ZHAO Jing, SHI ZuoKun, QIU LingYu, WANG Su, ZHANG Fan, WANG ShiGui, TANG Bin. Sequence Analysis and Induced Expression of Three Novel Small Heat Shock Proteins Mediating Cold-Hardiness in Harmonia axyridis [J]. Scientia Agricultura Sinica, 2017, 50(16): 3145-3154.
[6]	LIU Chang-you, FAN Bao-jie, CAO Zhi-min, SU Qiu-zhu, WANG Yan, ZHANG Zhi-xiao, CHENG Xu-zhen, TIAN Jing. Interspecific Hybridization Among Vigna Species [J]. Scientia Agricultura Sinica, 2015, 48(3): 426-435.
[7]	LIU Zi-Gang, SUN Wan-Cang, YANG Ning-Ning, WANG Yue, HE Li, ZHAO Cai-Xia, SHI Peng-Fei, YANG Gang, LI Xue-Cai, WU Jun-Yan, FANG Yan, ZENG Xiu-Cun. Morphology and Physiological Characteristics of Cultivars with Different Levels of Cold-Resistance in Winter Rapeseed (Brassica campestris L.) During Cold Acclimation [J]. Scientia Agricultura Sinica, 2013, 46(22): 4679-4687.
[8]	WANG Ai-ling,WANG Yue-jin,TANG Dong-mei,ZHANG Jian-xia,ZHANG Chao-hong . Research on Improvement of Seedling Rate in Embryo Rescue of Seedless Grapes [J]. Scientia Agricultura Sinica, 2010, 43(20): 4238-4245 .
[9]	TANG Dong-mei,WANG Yue-jin,ZHAO Rong-hua,PAN Xue-jun,CAI Jun-she,ZHANG Jian-xia,ZHANG Chao-hong,LUO Qiang-wei . Factors Influencing Embryo Development in Embryo Rescue of Seedless Grapes#br# [J]. Scientia Agricultura Sinica, 2009, 42(7): 2449-2457 .
[10]	,,,,. Studies on Induction of Haploid Cucumbers by Irradiated Pollen Pollination and Their Characterization [J]. Scientia Agricultura Sinica, 2006, 39(7): 1428-1436 .
[11]	. Determination of the Proper Sampling Period for Embryo Rescue from Crosses Between Diploid and Tetraploid Grape Cultivars [J]. Scientia Agricultura Sinica, 2005, 38(03): 629-633 .