穿龙薯蓣种子离体再生体系构建及其组培苗解剖结构特征

Construction of Regeneration System from Seed and Anatomical Structure of Tissue Culture Seedling in Dioscorea nipponica

以药源植物穿龙薯蓣种子为外植体,构建优化种子消毒时间以及愈伤组织、不定芽和生根诱导等激素浓度、组成和配比体系,并比较组培苗的叶片、缠绕茎和根状茎等营养器官与实生苗在解剖结构上的差异,初步建立以种子为外植体的再生体系。结果表明:(1)外植体采用2%NaClO消毒15 min和18 min为宜;愈伤组织诱导培养基为MS+1.0 mg·L^(-1)6-BA+2.0 mg·L^(-1)2,4-D时效果最佳,出愈率为83.33%;不定芽诱导的最佳培养基为MS+2.0 mg·L^(-1)6-BA+0.2 mg·L^(-1)NAA,诱导率达到90.00%;以1/2MS+0.5 mg·L^(-1)NAA进行生根培养为宜,生根率达86.70%,组培苗可产生13.2条根,且组培苗生长旺盛,叶片浓绿,茎粗长。(2)组培苗与实生苗在叶片、缠绕茎的解剖结构上存在显著差异:组培苗叶片薄,主脉维管束小,栅栏组织厚度/海绵组织厚度P/S低;组培苗缠绕茎的内径、导管面积和维管束数量均小于实生苗,导管/维管束为0.06,为实生苗的1/2;组培苗缠绕茎皮层比例大,但厚角组织与厚壁组织分化不完全,仅存在排列极为分散的双韧维管束。研究认为,组培苗在外形和结构上的变化主要是对其高湿、低光、恒温、密闭培养瓶培养环境的高度适应的结果。

In this paper,the establishment of seed regeneration system of Dioscorea nipponica from medicinal plant was studied,and the differences in anatomical structure between leaf,entangled stems and rhizome of tissue culture seedlings and solid seedling were investigated.The results showed that:(1)15 min and 18 min 2%NaClO were suitable for disinfection.The optimal medium for callus induction was MS+1.0 mg·L^(-1)6-BA+2.0 mg·L^(-1)2,4-D,and its induction rate was up to 83.33%.MS+2.0 mg·L^(-1)6-BA+0.2 mg·L^(-1)NAA,induction rate was 90.00%in the best medium for bud induction.The best culture medium hormones ratio was 1/2MS+0.5 mg·L^(-1)NAA,generating 13.2 roots,its rooting rate was up to 86.70%.The growth of tissue culture seedlings is vigorous,the leaves are dark green,and the stems are thick and long.(2)There were significant differences in the structure of leaf and entangled stems between tissue culture seedlings and solid seedlings.The leaves of tissue culture seedlings were thin and the main vascular bundles were small and P/S low.The diameter,area and number of vascular bundles of the entangled stems were smaller than those of the solid seedlings;the catheter/vascular bundle was 0.06,which was 1/2 of the seedlings.Culture seedling proportion of cortex is large,but the differentiation between thick-angle tissue and thick-walled tissue is not complete,and there are only two ductile vascular bundles with extremely dispersed arrangement.The changes in the shape and structure of tissue culture seedlings were mainly adapted to the culture environment of high humidity,low light,constant temperature and closed culture bottle.