矮牵牛细胞的长期离体培养及再生植株的ISSR分析

 【目的】实现矮牵牛细胞的长期离体培养和再生，不但可以获得矮牵牛体细胞突变体，从中选出优良株系，而且可以为研究外源基因在矮牵牛细胞增殖与分化过程中的稳定性提供技术体系。【方法】以重瓣矮牵牛叶片为外植体，使用不同浓度BA与NAA的组合在黑暗条件下诱导愈伤组织。在不同光条件下，将愈伤组织在MS+ BA 2.0 mg•L-1+0.5 mg•L-1 NAA+200 mg•L-1 CH 与 MS+ BA 0.5 mg•L-1+0.5 mg•L-1 NAA+200 mg•L-1 CH两种培养基进行愈伤组织3年多的继代培养，并利用筛选的8个引物对20个再生植株的总DNA进行ISSR分析。【结果】不同的生长素与分裂素配比诱导的愈伤组织状态明显不同，继代后的愈伤组织在低激素培养基上分化出不定芽，再生芽复壮后可得到正常植株。对再生植株的总DNA进行ISSR分析发现，再生植株之间存在很大的遗传变异。【结论】矮牵牛愈伤组织在高BA浓度培养基上可长期继代保存；在低激素培养基上可实现植株再生。长期离体培养矮牵牛是获得其突变体的有效方法。

Long-Term in vitro Culture of Petunia hybrida Callus and ISSR Analysis of the Regenerated Plants

【Objective】A Long-term in vitro culture plant regeneration system of Petunia hybrida is useful not only in obtaining somatic variants and thereafter to select superior lines, but also in analyzing the stability of foreign gene in the cells during long-term culture and plant regeneration. 【Method】Different callus were obtained by culturing the leave explants of one double flower Petunia plant on the media supplemented with different combinations of auxin and cytokinnins. Calli were sub-cultured on the media of MS+ BA 2.0 mg•L-1+0.5 mg•L-1 NAA+200 mg•L-1 CH and MS+ BA 0.5 mg•L-1+0.5 mg•L-1 NAA+200 mg•L-1 CH for more than 3 years under different light condition. The total DNA of 20 regenerated plants were analyzed by ISSR utilizing 8 primers. 【Result】 Plants were successfully regenerated from Long-term cultured callus when transferred onto shoot induction media supplemented with low concentration of PGR. After continuous culture on the media for at least three times, all adventitious shoots developed into complete plants of normal morphological characteristics. ISSR analysis showed that there was dramatic genetic variation among somatic variants. 【Conclusion】Plant regeneration could be successfully implemented after long-term subculture of the callus of Petunia hybrida in higher concentration BA media when they were transferred onto shoot induction media supplemented with low concentration of PGR. The culture system presented here is effective in obtaining somatic variants for Petunia hybrida.