油松胚性愈伤组织培养中褐化因素研究及增殖培养基的优化

Investigation of browning factors in embryogenic callus culture of Pinus tabulaeformis and optimization of proliferation medium

【目的】调查油松Pinus tabulaeformis胚性愈伤组织增殖培养阶段不同因素对于褐化产生的影响,并结合增殖率对培养基进行调整优化以降低褐化率,提高利用率。【方法】以3个年份的9个细胞系的油松胚性愈伤组织为材料,利用单因素试验设计对放置不同数量愈伤块、pH、植物凝胶含量、糖种类和含量以及培养时间的褐化率和增殖率进行测定计算。采用正交试验选出最佳培养条件。【结果】单因素试验结果表明,褐化率在不同基因型间差异显著,且与增殖继代年龄显著正相关;在培养皿中放置6~7块愈伤组织、pH 5.8~6.0、培养基中添加2.0~3.0 g·L^(-1)植物凝胶、添加10 g·L^(-1)蔗糖能保证较低的褐化率和较高的增殖率,且蔗糖优于葡萄糖和麦芽糖。【结论】结合正交设计,90 mm培养皿中放置7块愈伤组织,培养基中添加2.5 g·L^(-1)植物凝胶、10 g·L^(-1)蔗糖,调节pH为5.9时,褐化率、增殖率和利用率最理想。

【Objective】To study the effects of different factors on browning of embryogenic callus of Pinus tabulaeformis during proliferation, and optimize the culture medium based on proliferation rate for lower browning rate and higher efficiency.【Method】Embryogenic callus of P. tabulaeformis from nine cell lines and three different year were used as material.We performed a serious of single factor experiments and calculated the browning rates and proliferation rates of embryogenic callus under the treatments of different callus number, pH, phytagel content, type and content of sugars, and culturing time. Orthogonal experiment was then conducted to screen out the best cultureconditions.【Result】Results of the single factor experiments showed that browning rate was significantly different among different genotypes, and it was significantly positively correlated with the age of subculture. The browning rate was relatively low and the proliferation rate was relatively high when the callus number in each petri dish was 6-7,medium pH was 5.8-6.0, phytagel was 2.0-3.0 g·L^(-1), and sucrose was 10 g·L^(-1). Sucrose was better than glucose and maltose.【Conclusion】Based on the orthogonal design, placing 7 callus in each 90 mm petri dish, adding 2.5 g·L^(-1)phytagel and 10 g·L^(-1) sucrose to the medium, and adjusting the pH to 5.9 could achieve the preferred browning rate,proliferation rate and efficiency.