桑树愈伤组织诱导及悬浮细胞系的建立

Callus Induction and Establishment of Suspension Cell Line of Mulberry

以桑品种育-711的叶片、芽尖、叶柄、茎段为材料,探讨不同外植体、不同培养基和激素配比等因素对桑树愈伤组织的诱导、继代培养以及悬浮细胞培养的影响,并对桑树悬浮细胞的生长曲线进行监测,初步建立桑树细胞悬浮培养体系。试验结果表明:叶片是诱导桑树愈伤组织的理想外植体,愈伤组织最佳诱导条件为MS培养基中添加1.5 mg/L 6-BA+0.5 mg/L2,4-D+0.5 mg/L NAA的激素组合,在此培养条件下的诱导率达90%以上;继代培养最优条件为MS培养基中添加1.0 mg/L6-BA+0.5 mg/L 2,4-D+0.5 mg/L NAA,在此培养条件下继代,愈伤组织生长旺盛,存活率达到100%,培养12 d进入对数生长期,愈伤组织的鲜质量在此期间增加6倍;悬浮细胞在MS液体培养基中添加1.5 mg/L 6-BA+0.5 mg/L 2,4-D+0.1 mg/L NAA的培养条件下,细胞增殖较快,生长曲线呈"S"型,活细胞数量在悬浮培养第16天时达到峰值。以桑树叶片作为外植体诱导愈伤组织及初步建立的悬浮细胞系,有助于进一步研发桑树组织培养物次生代谢产物的生产技术。

By using the leaf,shoot tip,petiole and stem from mulberry variety Yu-711 as material,the effects of different explants,culture media and hormone ratios on callus induction,subculture and suspension culture were discussed. In addition,the growth curve of mulberry suspension cells was surveyed and the suspension culture system of mulberry cells was established preliminarily. The results showed that mulberry leaves are ideal explant used for callus induction.The optimum condition of callus induction was MS culture medium supplemented with the hormone combination of 1. 5mg / L 6-BA ＋ 0. 5 mg / L 2,4-D ＋ 0. 5 mg / L NAA. Under this condition, the induction rate was over 90%. The optimum condition for subculture was MS culture medium supplemented with 1. 0 mg / L 6-BA ＋ 0. 5 mg / L 2,4-D ＋0. 5 mg / L NAA. Under this condition,the callus grewvigorously and the survival rate reached 100%. After cultured for 12 d,callus growth came to the logarithmic phase. The fresh mass of callus was increased by 6 times during this period. When mulberry suspension cells werecultured in MS culture medium added with hormone concentration of 1. 5 mg / L 6-BA ＋ 0. 5 mg / L 2,4-D ＋ 0. 1 mg / L NAA,the proliferation rate of cell was high. The cell growth process presented in S-shaped curve and the number of living cells reached the peak at day 17 after culturing. Using mulberry leaves as explants for callus induction and the preliminarily established suspension culture system of mulberry cells will help to further study and develop the production technologies of mulberry secondary metabolites through tissue culture.