利用胚挽救技术创制无核抗寒葡萄新种质

Breeding for Grape Germplasm Involved in Seedlessness with Cold-Resistant Using Embryo Rescue

【目的】获得无核抗寒葡萄新种质并提高抗寒无核葡萄胚挽救育种效率。【方法】以7个杂交组合的胚珠作为试验材料,研究不同亲本基因型、基本培养基类型(MM3和ER)、不同氨基酸(MM3+2.5 mmol·L^(-1)半胱氨酸、天冬酰胺、谷氨酰胺、丝氨酸)对无核葡萄胚发育率和成苗率的影响。从杂交亲本中分别利用无核探针GSLP1-569、SCC8-1018和抗寒的分子标记S241-717,确定能扩增出特异带的亲本,再用相应的分子标记对其杂种后代进行无核和抗寒性状的鉴定。【结果】7个杂交组合接种至MM3培养基的胚珠为1 168个,获得发育胚331个和杂种后代97株,杂交组合‘红宝石无核’ב北醇’的胚发育率和成苗率最高,分别为46.00%和17.33%。通过比较母本基因型,以‘双优’为父本,‘红脸无核’‘无核白’‘昆香无核’为母本,杂交组合‘昆香无核’ב双优’获得胚发育率和成苗率最高,分别为45.39%和16.31%;以‘北冰红’为父本,‘红脸无核’‘美丽无核’‘无核白’为母本,杂交组合‘红脸无核’ב北冰红’获得胚发育率和成苗率最高,分别为33.52%和6.59%。父本基因型对胚挽救成苗有一定影响,‘红脸无核’ב北冰红’和‘红脸无核’ב双优’,胚发育率分别为33.52%、39.00%,成苗率为6.59%、9.50%;‘无核白’ב北冰红’和‘无核白’ב双优’的胚发育率分别为7.64%、19.26%,成苗率为1.91%、8.15%。接种至MM3培养基的胚珠,获得的胚发育率和成苗率均高于ER培养基。杂交组合‘昆香无核’ב双优’的胚珠接种至添加2.5 mmol·L^(-1)谷氨酰胺的MM3培养基,获得胚发育率最高,为59.69%;添加天冬酰胺、谷氨酰胺培养的杂种胚珠获得的成苗率分别为21.43%、20.93%,对成苗促进作用显著;对于杂交组合‘红宝石无核’ב北醇’,添加2.5 mmol·L^(-1)天冬酰胺的胚发育率最高,为55.71%;添加天冬酰胺、谷氨酰胺培养的杂种胚珠的成苗率分别为21.43%、22.22%,对成苗促进作用显著。利用无核标记GSLP-569、SCC8-1018和抗寒分子标记S241-717,共检测了6个杂交组合的83个杂种株系,其中携带无核分子标记的杂种株系49个,抗寒分子标记的杂种株系55个,同时携带无核和抗寒标记的杂种株系36个。【结论】‘昆香无核’和‘红脸无核’适宜作为胚挽救育种的母本材料。‘双优’作为父本比‘北冰红’的胚挽救效率更高。MM3适宜作为胚发育培养基。MM3培养基中添加酰胺类氨基酸可以促进胚挽救成苗。分子标记检测的83个杂种株系中,36个杂种株系携带无核抗寒分子标记。

【Objective】The objective of this study is to obtain new cold-resistant seedless grape germplasm and improve the breeding efficiency through embryo rescue in vitro.【Method】The different genotypes,basal culture media（MM3 and ER）,amino acids（2.5 mmol·L^（-1 ）cysteine,asparagine,glutamine and serine were added in MM3 medium）were used to investigate the effect on the embryo development rates and seedling rates from ovules of 7 cross combinations.From the parents,the molecular markers GSLP1-569 and SCC8-1018 for seedlessness and the marker S241-717 for cold-resistance were used to identify the strains with specific bands,and then the three markers were used to screen the hybrids by recognizing the specific bands.【Result】Of the 7combinations,1 168 ovules cultured in MM3 were obtained 331 embryos and 97 hybrid strains.Among these crosses,‘Ruby Seedless’בBeichun’got the highest embryo development rate and seedling rate,which were 46.00%and 17.33%,respectively.Compared with female parents,for‘Shuangyou’as male parent and‘Blush Seedless’‘Thompson Seedless’‘Kunxiang Seedless’as female parents,‘Kunxiang Seedless’בShuangyou’had the highest embryo development rate and seedling rate,which were 45.39%and 16.31%,respectively.For‘Beibinghong’as male parent and‘Blush Seedless’‘Beauty Seedless’‘Thompson Seedless’as female parents,‘Blush Seedless’בBeibinghong’had the highest embryo development rate and seedling rate,which were 33.52%and6.59%,respectively.Different male parents also influenced seedlings formation.The embryo development rates of‘Blush Seedless’בBeibinghong’and‘Blush Seedless’בShuangyou’were 33.52%and 39.00%,and the seedling rates of them were 6.59%and 9.50%,respectively.The embryo development rates of‘Thompson Seedless’בBeibinghong’and‘Thompson Seedless’בShuangyou’were7.64%and 19.26%,and the seedling rates of them were 1.91%and 8.15%,respectively.The rate of embryo development and seedling of ovule cultured in MM3 medium was higher than that of ER medium.For‘Kunxiang Seedless’בShuangyou’,the ovules cultured in MM3 medium with 2.5 mmol·L^-1 glutamine showed the highest embryo development rate,which was 59.69%.The seedling rate of ovules cultured in basal media with asparagine and glutamine was 21.43%and 20.93%,respectively.The effect of promoting seedlings development was significant.For‘Ruby Seedless’בBeichun’,the ovules in media with 2.5 mmol·L^-1asparagine showed the highest embryo development rate,which was 55.71%.The seedling rate of ovules cultured in basal media with asparagine and glutamine was 21.43%and 22.22%,respectively.The effect of promoting seedlings development was significant.By using the molecular markers GSLP1-569 and SCC8-1018 for seedlessness and the marker S241-717 for cold-resistance,83 hybrid strains from 6 cross combinations were detected,and there were 49 strains with specific bands of the molecular markers linked to the seedlessness,55 strains with specific bands of the markers S241-717 for cold-resistance.Importantly,36 strains with both seedlessness and cold-resistance were identified in this study.【Conclusion】‘Kunxiang Seedless’and‘Blush Seedless’are suitable as the female parents for embryo rescue in vitro of seedless grapes.The embryo rescue efficiency of‘Shuangyou’as the male parent is higher than that of‘Beibinghong’.MM3 is suitable for embryo rescue in vitro,and the addition of amide acids to MM3 culture medium is helpful to the seedling formation.Among the 83 hybrid strains detected by molecular markers,36 strains may have the seedless and cold-resistant characteristics.