【目的】为了研究抗松材线虫病赤松体细胞胚胎发生和植株再生,进行了抗病赤松愈伤组织培养条件的优化。【方法】以抗松材线虫病赤松胚性愈伤组织22#-1和13#-1为材料,首先在体视镜下观察胚性与非胚性愈伤组织的细胞学形态差异,然后研究激素配比、基因型、继代方式和次数对愈伤组织增殖中形态上和生物量上的影响。【结果】抗病赤松愈伤组织可分为两类:一类为胚性愈伤组织,含有胚性胚柄细胞团(embryonic suspensor mass, ESM)结构,一类为非胚性愈伤组织; 胚性愈伤组织维持与增殖最佳的激素组合是2.0 mg/L 6-BA和4.0 mg/L NAA; 不同基因型的胚性愈伤组织的增殖状况存在差异,其中抗病赤松无性系22#-1胚性愈伤组织增殖较快,无性系13#-1增殖较慢,但差异不显著; 液-固与固体交替培养方式为抗病赤松胚性愈伤组织最适宜的继代增殖方式。【结论】不同的基因型增殖状况差异不显著,增殖过程应挑选胚性愈伤组织进行增殖,使用2.0 mg/L 6-BA和4.0 mg/L NAA激素配比的培养基,并采用液-固与固体交替的方式培养。
Abstract
【Objective】Somatic embryogenesis and plant regeneration of nematode-resistant Pinus densiflora were studied for obtaining a large number of embryogenic callus. 【Method】We used embryogenic calluses 22#-1 and 13#-1 of nematode-resistant P. densiflora as material. First, cell structure of callus was observed under a stereomicroscope. Then we evaluated how combination of hormone, genotype, subculture modes and times affected callus proliferation and biomass. 【Result】The results showed that successful maintenance and proliferation of embryogenic callus depended heavily on plant growth regulating substances, genotype, subculture modes and times. Embryogenic callus of nematode-resistant P. densiflora was classified into two categories: embryogenic callus containing embryonic suspensor mass and non-embryogenic callus. The optimal combination of hormone was 2.0 mg/L 6-BA and 4.0 mg/L NAA. The proliferations of embryogenic calluses in different genotypes were different. The rate of proliferation of clonal embryo callus 22#-1 was slightly faster and 13#-1 was slower, but the difference was not significant. Liquid -solid and solid alternate culture mode was the most suitable proliferation mode. 【Conclusion】we should select embryogenic callus to subculture and the optimal combination of hormone was 2.0 mg/L 6-BA and 4.0 mg/L NAA. The calluses should be cultured in a liquid -solid and solid alternate mode.
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基金
基金项目:江苏省科技支撑计划(BE2014405); 国家林业公益性行业科研专项项目(201204501); 江苏高校优势学科建设工程资助项目(PAPD)
第一作者:许建秀(723442545@qq.com)。*通信作者:吴小芹(xqwu@njfu.edu.cn),教授。
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