摘要
【目的】探究褪黑素在酿酒葡萄种子体细胞胚发生及循环诱导中的作用,建立以种子为外植体的体细胞胚诱导体系,为缩短葡萄属植物体胚诱导周期提供依据。【方法】以花后60、70、80 d的欧洲酿酒葡萄品种霞多丽、马瑟兰与美乐种子为外植体,研究不同切种和放置方法下,5种不同质量浓度(0.2、0.4、0.6、0.8、1.0 mg·L^(-1))褪黑素对其初生子叶胚萌发及二次胚循环诱的作用。【结果】花后80 d种子诱导率为18.33%,明显高于70 d和60 d,采用纵切法正放接种外植体30 d时,初生子叶胚萌发率达到最高,为33.07%,纵切倒放、横切正放和横切倒放的萌发率依次降低,为10.98%、5.71%和3.24%。褪黑素诱导3个酿酒葡萄品种萌发初生子叶胚的作用均强于2,4-D,其中,霞多丽种子在MS+0.6mg·L^(-1)褪黑素+2.0 mg·L^(-1)6-BA+30 g·L^(-1)蔗糖+3 g·L^(-1)植物凝胶+0.5 g·L^(-1)活性炭的培养基诱导30 d时,其初生子叶胚诱导率最高,为18.33%。利用霞多丽初生子叶胚切段循环诱导二次体细胞胚60 d时,开始出现二次体细胞胚,进一步诱导形成球形胚、心形胚、鱼雷胚和子叶胚。诱导至90 d时,在添加0.6 mg·L^(-1)褪黑素的培养基上诱导率最高,为14.40%,0.4 mg·L^(-1)次之,为3.13%,0.2 mg·L^(-1)诱导率为1.89%,而0.8、1.0 mg·L^(-1)褪黑素培养基和2,4-D诱导下无次生胚的形成。【结论】种子成熟度与初生子叶胚的萌发率呈正相关;种子纵切正放接种有利于初生子叶胚萌发;低质量浓度褪黑素对霞多丽种子萌发初生子叶胚及二次胚发生具有缩短诱导时间的作用。
【Objective】The role of melatonin in the induction of somatic embryogenesis and induced circulation establishment from wine grape seeds was investigated with the aim to establish a somatic embryo induction system based on seed induction,and to provide a theoretical basis for shortening the cycle of grapevine somatic embryo induction.【Methods】Seeds from European wine grape varieties Chardonnay,Marselan and Merlot were used as explants,which were collected at 60 d,70 d and 80 d after flowering,respectively.The seeds were sterilized with 70%ethanol and 1.5%NaClO,and then cut and placed in four ways,namely,transversely cut and flatwise,transversely cut and anti-put,longitudinally cut and flatwise,and longitudinally cut and anti-put,to investigate the effect of different cutting and placing patterns on the germination of primary cotyledon embryos.In addition,five different concentrations(0.2,0.4,0.6,0.8,1.0 mg·L^(-1))of melatonin were explored on their primary cotyledon embryos germination,and the germinated primary cotyledon embryos were further cut to 0.5 cm sections to investigate the cyclic induction of their secondary somatic embryos by five different concentrations(0.2,0.4,0.6,0.8,1.0 mg·L^(-1))of melatonin.【Results】Seeds of the three wine grape varieties and different seed maturity levels differed significantly in the induction rate of primary cotyledon embryos on the medium supplemented with different concentrations of melatonin.Among them,the cotyledon embryo germination rates were all proportional to seed maturity on MC,MEL0,MEL1,MEL2,MEL3,MEL4 and MEL5 medium,with induction rates ranging from 0.9 to 16.56%at 60 d after flowering,1.34%to17.3%at 70 d,and up to 18.33%at 80 d.Among the three test varieties,Chardonnay had the highest germination rate of 18.33%at 80 d after flowering induced by 0.6 mg·L^(-1)melatonin,while the control2,4-D induced germination with the worst effect of 2.98%.The difference in germination rate between varieties may be related to genotype.The seeds of Chardonnay were inoculated into the medium in different ways,and the seed coat started to discolor at 3 d.The germination of cotyledon embryos started at 10 d.The germination rate of primary cotyledon embryos reached the highest rate of 33.07%at 30 d when the explants were inoculated by the longitudinally cut and flatwise,and the germination rates decreased in longitudinally cut and anti-put,transversely cut and flatwise,transversely cut and anti-put,with 10.98%,5.71%,and 3.24%.The results showed that longitudinal cut and flatwise was the most suitable inoculation method,which helped to improve the germination rate of primary cotyledon embryos.Melatonin-induced germination of primary cotyledon embryos was stronger than 2,4-D in all three wine grape varieties,among which seeds from Chardonnay could occurred the highest primary cotyledon embryos induction rate of 18.33%in MS+0.6 mg·L^(-1)melatonin+2.0 mg·L^(-1)6-BA+30 g·L^(-1)sucrose+3 g·L^(-1)phytogel+0.5 g·L^(-1)activated charcoal for 30 d.The secondary somatic embryos were induced by using Chardonnay primary cotyledon embryo cuttings in a cyclic manner,and callus tissues were formed in different media at 14 d.At 60 d,secondary somatic embryos began to appear,and further induction resulted in the formation of spherical embryos,heart-shaped embryos,torpedo embryos and cotyledon embryos.The highest induction rate of 14.4%was induced at 90 d with MS+0.6 mg·L^(-1)melatonin+2.0 mg·L^(-1)6-BA+30 g·L^(-1)sucrose+3 g·L^(-1)phytogel+0.5 g·L^(-1)activated carbon,followed by 0.4 mg·L^(-1)melatonin with the induction rate of 3.13%,0.2 mg·L^(-1)occurred the induction rate of 1.89%,while no secondary embryos were formed either on medium supplemented with 0.8,and 1.0 mg·L^(-1)melatonin or on medium supplemented with 2,4-D.The plant materials induced by 2,4-D gradually browned and died during the succession process.The induction of Chardonnay seeds with melatonin resulted in the formation of primary cotyledon embryos,and after cyclic induction,primary embryo clusters were produced and secondary embryos were formed,and seedlings could be established after transplanting.【Conclusion】The role of melatonin in the induction of grapevine somatic embryos was investigated with wine grape seeds as explant material.The results showed seed maturity was positively correlated with the germination rate of primary cotyledon embryos.The longitudinally cut and flatwise was beneficial to the germination of primary cotyledon embryos.Low concentration of melatonin had the effect of shortening the induction time for germination of primary cotyledon embryos and secondary embryogenesis in Chardonnay seeds.The primary cotyledon embryos from Chardonnay could induce the production of primary embryo clusters and somatic embryos,which could help to increase the numbers of embryo materials and provide experimental materials for grapevine genetic transformation.
作者
张莹
雅蓉
徐伟荣
王佳慧
崔莹
李俊铎
ZHANG Ying;YA Rong;XU Weirong;WANG Jiahui;CUI Ying;LI Junduo(Collge of Agronomy,Ningxia Universit,Yinchuan 750021,Ningxia,China;School of Food&Wine,Ningxia Universit,Yinchuan 750021,Ningxia,China;Engineering Research Center of Grape and Wine,Ministry of Education,Yinchuan 750021,Ningxia,Chi na;Key Laboratory of Modern Molecular Breeding of Dominant and Special Crops in Ningxia,Yinchuan 750021,Ningxia,China;Chi na Wine Industry Technology Institute,Yinchuan 750021,Ningxia,China)
出处
《果树学报》
CAS
CSCD
北大核心
2021年第6期922-933,共12页
Journal of Fruit Science
基金
国家自然科学基金项目(32060672、31860542)
国家重点研发计划项目(2019YFD1002500)
宁夏回族自治区重点研发计划(重大)重点项目(2019BBF02022)。
