苹果体细胞无性系变异的RAPD评估

以苹果栽培品种Gala试管苗叶片为材料 ,将叶片刺伤后接种于附加了BA1mg L、2 ,4 D 0 5mg L和NAA 5mg L的MS培养基 ,黑暗培养 7d后转移至附加BA1mg L的MS培养基 ,继续暗培养 40d ,97%叶片发生直接类型体细胞胚胎 ,单位叶片平均再生体细胞胚胎 2 5个。对来自同一植株上的前 3片展开叶中的 1片叶进行组织培养 ,再生得到 1 5株直接体细胞胚胎发生、植株再生后代。应用随机扩增多态性DNA(RAPD)分析技术 ,以供体 (原初供体 )为对照进行了供体和再生体间及再生体彼此之间体细胞无性系变异研究 ,没有观察到供体和再生体间及再生体彼此之间有DNA多态性 ;接着用同样的方法对上述 1 5株体细胞胚胎植株随机抽取 7个单株单叶进行培养 ,得到二代直接体细胞胚胎植株 ,分别从 7个单株单叶再生的二代植株中各随机抽取 1株 ,再次用RAPD方法进行原初供体和二代再生体间及二代再生体之间体细胞无性系变异分析 ,结果二代直接体细胞胚胎植株 5和引物OPF 0 6组合中出现一条多态带 ,其分子量约为 90 0bp,命名为OPF 0 690 0 ,重复 3次多态性稳定。结果表明 ,苹果离体叶片直接体细胞胚胎发生途径再生植株的体细胞无性系变异率接近自然变异率

RAPD AS AN AID TO EVALUATE SOMACLONAL VARIATION OF APPLE

Plantlets were regenerated by direct somatic embryogenesis in leaf culture of apple ( Malus X domestica Borkh.cv.Gala).The leaf was pricked before cultured on MS+BA 1mg/L+2\^4 D 0\^5mg/L+NAA 5mg/L medium for embryonic cell induction,and was transfered 7 days later to MS+BA 1mg/L medium for somatic embryo regeneration.After 40 days,97% of leaves regenerated direct somatic embryos and mean number of somatic embryos regenerated per leaf were 25.15 plantlets were selected from regenerants of the first three open leaves of an in vitro plant.Somaclonal variation in the 15 regenerants and CK were studied using RAPD.No DNA polymorphism was found among them.7 secondary regenerants were obtained respectively from randomly selected 7 leaves of first three open leaves of above 15 regenerants.RAPD analysis again was applied to study somaclonal variations in secondary regenerants and CK,one DNA polymorphism was found in No.5 secondary regenerants with primer OPF-06,the MW of the band is about 900bp and named OPF-06 900 ,and the DNA polymorphism was stable after three times of repetition.The results suggested that somaclonal variation in apple was lower than natural variation.