巴西橡胶树体胚胚性愈伤组织悬浮系的建立和植株再生

Establishment of Somatic Embryogenic Callus Suspension Lines in Hevea brasiliensis and Plant Regeneration

以巴西橡胶树‘热研7-33-97’品种花药体胚胚性愈伤组织的新鲜增殖组织为培养材料,建立和优化适合橡胶树胚性愈伤组织增殖的悬浮培养体系,并对悬浮组织进行体细胞胚的成熟诱导和植株再生。结果表明:橡胶树体细胞胚诱导的易碎胚性愈伤组织是建立稳定、均一的悬浮细胞系非常适合的材料;改良MS+1.0 mg/L 2,4-D+1.5 mg/L KT+0.5 mg/L NAA有利于细胞分裂,最适初始接种量为1.2 g/50 mL,旧培养液比例为1/3;悬浮培养物易分散、细胞大小均一、形态一致,细胞团由4～15个细胞聚合而成,细胞颜色淡黄色,培养基清澈透亮;胚性愈伤悬浮细胞的生长曲线呈S形,第3～第7天是指数增长期,最适继代周期为7～8 d;在一个生长周期内,培养液的pH先降后升,最后逐渐趋于稳定,而电导率则持续下降;三次继代后,将悬浮培养物接种到固体分化培养基上分化可得到成熟体细胞胚状体,将其接种到植株再生培养基上,45 d后可成功获得再生植株。橡胶树花药体胚胚性愈伤组织悬浮系的建立,为橡胶树胚性愈伤组织的快速增殖及体细胞胚的规模化繁育提供帮助。

The fresh proliferative tissue of the anther embryogenic calli from H. brasiliensis ‘Reyan 7-33-97’ was used as the culture material. A suspension culture system suitable for embryogenic callus development was established and optimized to explore the main factors affecting the tissue proliferation in Hevea brasiliensis. On the basis of these, somatic embryo maturation induction and plant regeneration of suspension tissue were carried out.The results showed the fragile embryogenic callus induced by anther somatic embryos of Hevea brasiliensis was a suitable material for establishing stable and homogeneous suspension cell lines. The optimum culture medium for suspension was modified MS medium supplemented with 1.0 mg/L 2,4-D, 1.5 mg/L KT and 0.5 mg/L NAA, with the initial inoculation amount of 1.2 g/50 mL and the ratio of the old culture medium was 1/3. The medium was suitable for cells proliferation and could produce uniform cells and small cell aggregates composed of4~15 cells, which were uniform in sizes and consistent in shapes. The cell aggregates looked yellowish and were easy to disperse in the liquid medium, which was clear and transparent. The growth curve of suspension cells was typical of "S". From 3 to 7 days, the cells proliferated exponentially. The optimum subculture period was within 7 to 8 days to sustain the strong cell division competence and cell viability. During a growth cycle, the pH of the culture medium first decreased, then increased and gradually stabilized, while the electrical conductivity showed a downward trend. After three times of subcultures, the small cell aggregates were moved into the solid differentiation medium to obtain the mature embryos. The mature embryos were transferred to the plant regeneration medium, and the regenerated plants were successfully obtained after 45 days. The establishment of suspension system of embryogenic callus from somatic embryos in Hevea brasiliensis set up a basis for rapid proliferation of embryogenic callus and large-scale propagation of somatic embryos.