马尾松胚性细胞悬浮增殖培养体系的建立

Establishment of embryogenic suspension cultures in somatic embryogenesis of Pinus massoniana Lamb.

本研究利用马尾松未成熟胚诱导的胚性愈伤组织,首次建立马尾松胚性愈伤组织悬浮增殖培养体系。实验中定期对细胞生长参数沉淀细胞体积（SCV）、细胞活力进行了测定,研究了初始接种量,继代细胞密度对悬浮增殖培养体系建立的影响。研究结果表明：不同初始接种量、继代转接细胞密度显著地影响马尾松胚性细胞悬浮培养的效果;在悬浮培育过程中,胚性细胞的生长周期曲线呈＂S＂型,细胞活力呈现先急剧上升,后缓慢下降的趋势;本研究建立的马尾松胚性细胞悬浮增殖培养体系为：在30 mL的培养基中,初始接种1.0 g胚性细胞（平均增殖系数达12.097）,继代培育以1/6作为继代细胞密度,且继代周期为8～12 d为宜。本研究为优化调控马尾松体细胞胚胎发生胚性愈伤组织稳定增殖提供了技术支撑,同时为采用遗传转化及细胞融合等技术进行马尾松新品种选育奠定了研究基础。

The objective of the study was to establish the embryogenic tissues suspension proliferation system using the embryogenic tissues initiated from immature zygotic embryos in Pinus massoniana Lamb. （masson pine）. The effects of initial tissues weight and cell density in subculture were investigated through measuring the sediment cell volume （SCV） and cell viability during the establishment of suspension proliferation system. The results showed that the growth of embryogenic tissues was significantly influenced by initial tissues weight and cells density in suspension cultures; the embryogenic cells growth basically accorded with the ＂S＂ growth curve, while the cells viability sharply increased at first and gradually decreased in later. A better suspension proliferation system in masson pine embryogenic cultures was established： 1.0 g tissues per 30 mL fresh liquid media were the appropriate initial tissues weight （the mean growth rate was up to 12.097）; then the better cell density in subculture was 1/6 and the appropriate subculture period was identified as 8 ~ 12 days. This study provide basic information for optimization of mass propagation in masson pine somatic embryogenesis. The establishment of suspension system is also a basic technique for new variety breeding of masson pine through genetic transformation and cell fusion in masson pine breeding.