青海野生黑果枸杞再生体系的建立及遗传稳定性分析

Establishment of the Regeneration System of Wild Lycium ruthenicum from Qinghai and Analysis in Its Genetic Stability

以野生黑果枸杞(Lycium ruthenicum Murr.)的无菌苗叶片作为外植体,建立了两条再生体系:一条是经愈伤组织再分化的间接再生体系,一条是不经愈伤组织再分化的直接再生体系。并采用流式细胞术(FCM)及ISSR分子标记技术对两种途径再生苗进行了遗传稳定性分析。结果表明:(1)最佳愈伤组织诱导培养基为MS+1.5 mg·L-12,4-二氯苯氧乙酸(2,4-D),诱导率达100%;最佳分化培养基为MS+1.5 mg·L-16-苄氨基腺嘌呤(6-BA)+0.1 mg·L-1吲哚-3-丁酸(IBA),1 g愈伤组织上的平均不定芽数为39.4个。(2)叶片直接诱导不定芽的最佳培养基为MS+0.5 mg·L-16-BA+0.3 mg·L-1α-萘乙酸(NAA),不定芽诱导率为92.9%,每个外植体上平均不定芽数为18.1个。(3)两条途径再生的不定芽在不含植物生长调节剂的MS培养基上,2周内均可正常生根。(4)FCM结果显示亲本苗及2种再生苗均为二倍体。(5)ISSR分析表明,间接再生苗的平均遗传相似性系数为0.84,直接再生苗的平均遗传相似性系数为0.91,直接再生体系是一种更加快速高效的繁殖方法。

Two regeneration systems were established by using sterile leaves of wild Lycium ruthenicum.The direct organogenesis is differentiated by leaves,and the indirect organogenesis is differentiated by calli.The genetic stability of the regenerated plantlets was assessed by the ISSR markers and flow cytometry(FCM).The results showed that:(1)the best medium for callus induction was MS+1.5 mg·L-12,4-D,with induction rate of 100%;the optimum medium for callus differentiation was MS+1.5 mg·L-16-BA+0.1 mg·L-1 IBA,with 39.4 shoots per gram of calli.(2)The suitable medium for direct shoot induction was MS+0.5 mg·L-16-BA+0.3 mg·L-1 NAA,with induction rate of 92.9%,and 18.1 shoots per explant.(3)After adventitious buds were transferred to the MS medium without hormones,the roots could form within two weeks.(4)FCM results showed that parental plantlets and regenerated plantlets were diploid.(5)ISSR analysis showed that the average genetic similarity coefficients of indirect and direct regenerated plantlets were 0.84 and 0.91.The direct organogenesis was a more effective method for plant regeneration of L.ruthenicum.