低频再生黄瓜离体培养体系的优化

Optimization of in vitro culture system for cucumber(Cucumis sativus L.)with low regeneration frequency

【目的】优化低频再生黄瓜离体培养体系,为黄瓜遗传转化体系的进一步完善奠定基础。【方法】以低频再生黄瓜种质Q24和L66为材料,分别以黄瓜的子叶、子叶节和下胚轴为外植体,研究外植体类型对黄瓜再生频率的影响;在MS培养基中添加不同质量浓度6-苄基腺嘌呤(6-BA)和脱落酸(ABA),研究激素配比对黄瓜再生频率的影响;将获得的黄瓜子叶节外植体置于1/2 MS培养基上预培养0,1,2,3和4 d,研究预培养处理对黄瓜再生频率的影响。【结果】不同基因型黄瓜再生频率差异显著,其中Q24整体上具有相对较高的再生频率,其子叶节再生频率为54.8%,下胚轴再生频率为50.0%;L66的再生频率相对较低,其子叶节再生频率为36.9%,下胚轴再生频率为16.7%。激素配比显著影响黄瓜子叶节外植体的再生频率,其中1.00 mg/L ABA+1.00 mg/L 6-BA对Q24子叶节不定芽的诱导效果最好,再生频率达到60.7%;2.00 mg/L ABA+1.00 mg/L 6-BA对L66子叶节不定芽的诱导效果最好,再生频率达到42.9%。预培养处理可显著提高L66子叶节的再生频率,经过1~3 d预培养,其再生频率逐渐增加并达到最高(32.1%),4 d后再生频率开始降低;Q24子叶节经3 d预培养再生频率达到最高,为27.4%,但与对照相比差异不显著。【结论】Q24子叶节外植体最适激素配比为1.00 mg/L ABA+1.00 mg/L 6-BA,经3 d预培养后其再生频率可达60.7%;L66子叶节外植体最适激素配比为2.00 mg/L ABA+1.00 mg/L 6-BA,经3 d预培养后再生频率可达42.9%。

【Objective】This study optimized the low-frequency regeneration system of cucumber to provide basis for further improvement of genetic transformation system of cucumber.【Method】The explants including cotyledons,cotyledon nodes,and hypocotyls of low frequency regenerated cucumber germplasms Q24 and L66 were used to investigate the effect of explant types on regeneration frequency of cucumber.Different concentrations of 6-benzyladenine(6-BA)and abscisic acid(ABA)were added to MS medium to investigate the effect of hormone ratio on regeneration frequency of cucumber.Cucumber cotyledon nodes explants were also pre-cultured on 1/2 MS medium to investigate the effects of pre-culture treatment for 0,1,2,3 and 4 days on regeneration frequency.【Result】The regeneration frequency of different genotypes of cucumber was significantly different.In the two low-frequency regenerated cucumber germplasms,Q24 had relatively higher regeneration frequencies.The regeneration frequencies of cotyledon nodes and hypocotyl were 54.8%and 50.0%,respectively.The regeneration frequencies of L66 were relatively low,with 36.9%for cotyledon node and 16.7%for hypocotyl.The regeneration frequency of cotyledon node explants in cucumber was significantly affected by hormone ratio.Results showed that 1.00 mg/L ABA+1.00 mg/L 6-BA had the best induction effect on adventitious buds of Q24 cotyledon node with the regeneration frequency of 60.7%,and 2.00 mg/L ABA+1.00 mg/L 6-BA had the best induction effect on adventitious buds of L66 cotyledon node with the regeneration frequency of 42.9%.The regeneration frequency of L66 cotyledon node was increased significantly after 1-3 days pre-culture and gradually increased to 32.1%.After 4 days,the regeneration frequency began to decrease.The regeneration frequency of Q24 cotyledon node reached the highest level of 27.4%after 3 days pre-culture without significant difference compared with the control.【Conclusion】The optimum hormone ratio for Q24 cotyledon node explants was 1.00 mg/L ABA+1.00 mg/L 6-BA and the regeneration frequency after 3 days pre-culture was 60.7%.The optimum hormone ratio of L66 cotyledon node explants was 2.00 mg/L ABA+1.00 mg/L 6-BA with 42.9%regeneration frequency after 3 days pre-culture.