Improvement of Somatic Embryogenesis Induction Frequency via Subculture in Hevea

Low induction frequency of somatic embryos and normal embryos was the main limiting factor for development of rubber anther culture.Results of histological sections showed that the anther callus growth period(50 days)was divided into two stages: the callus induction phase(0~30 days)and embryogenesis expression and callus proliferation stage(30~50 days).So 30 days old anther callus of rubber tree were used as the starting material for subculture in this study.An orthogonal design was arranged to optimize the subculture medium and explore the way of improving somatic embryogenesis frequency via subculture.The results showed that after a subculture in the optimal subculture medium,the number of embryos of subculture callus was 4.29 times that of the not subcultured,of which the number of normal embryos was 2.4 times that of the not subcultured.Four kinds of hormones were contained in the subculture medium,of which 6-BA showed a dominant effect,followed by KT.The optimized callus subculture medium was the modified MS medium with 6-BA 0.1 mg/L+ KT 0.3 mg/L+3,4-D 0.3 mg/L+ ABA 0.013 mg/L.Compared to the traditional anther regeneration system of the rubber tree,a procedure of callus subculture was added to the regeneration system and appropriate subculture medium formulations were screened.The addition of subculture procedure made anther regeneration system more in line with the actual development process of anther culture,improving embryo differentiation frequency,promoting the industrial development of somatic embryo seedling in rubber tree.