摘要
采用继代培养4～5 d 后的胡萝卜悬浮细胞,经酶解获得大量原生质体,并在不同的培养基上经培养,细胞进行分裂,形成愈伤组织,获得了完整的再生植株。探讨了影响原生质体培养及植株再生的多种因素,实验表明:①原生质体的培养密度在5×10~4/mL～5×10~5/mL 范围内均可,以2.5×10~5/mL 为佳。②对于细胞的分裂、细胞团的生长以及愈伤组织的形成,以改良的 B_5培养基最好,N_5培养基其次,MS 较差。③适当降低原生质体培养基中的甘露醇浓度,有利于细胞的分裂和生长。④在本实验所涉及的培养方法中,以液体浅层培养为优。⑤诱导愈伤组织分化,以 1mg/L KT 为佳。⑥不同培养基对愈伤组织的生长速度和分化频率产生一定影响,以 MS 培养基提高分化频率效果较好。
Large number of protoplasts were isolated enzymatically from 4 to 5 days old carrot suspension cells and cultured in different media.Cultured protoplasts could divide,form calli and also whole plants could be obtained. In this paper,various factors affecting the protoplast culture and plant regeneration were disocussed.The results showed:①culture densities ranging from 5×10~4/mL to 5x10^5/mL were suitable for carrot protoplasts,with 2.5× 10~8/mL being the best;②for carrot protoplast division,cell cluster growth and calli formation,modified B_5 medium was the best,followed by N,medium,MS medium was not suitable;③properly lowering the concentration of mannitol in the protoplast culture medium,was beneficial to the division and growth of cells;④among the culture methods involved in the experiment,thin-layer liquid culture was the most suitable one;⑤ for the regeneration frequency of calli derived from protoplasts,Kinetin (ling/L)was found to give the best result;⑥ different media had certain effects on the growth rate and regenera- tion frequency of the calli,among the media tested,MS medium was better in terms of promoting regeneration freguency of the calli.
