| Pine wilt disease is a destructive disease harmed pine species and has a great effect on forest production and the ecological environment in China. Pinus thunbergii is susceptible species, and is harmed by the diease. Seed orchard was established based on disease resistance breeding, but the number of seeds can't satisfy the needs of production. Carrying out in vitro tissue culture related disease-resistant breeding technology provides the premise for rapid propagation of excellent resistance genes and its industrialization. The paper based on immature zygotic embryos of nematode-resistance P. thunbergii as explants,studying on somatic embryogenesis and plantlet regeneration process(the initiation and proliferation of ESM and the induction and maturation of somatic embryo, germination and plant regeneration of somatic embryo, and related factors. The main results are as follows:1 Induction of embryogenic callus from nematode-resistant P. thunbergiiThe immature zygotic embryos from nematode-resistant P. thunbergii used as explants, adding a certain concentration of auxin and cytokinin(2,4-D, KT, 6-BA), the results showed that the induction rate was higher in Zygote embryo development of 2-4 stages. The acquisition date of cones from different family was different: the best one for families of 39# and 37# was in July 1st, the family of 36# was July 8st. ESM induction rate of different family was different, and induction rate from high to low in turn is 36#,37#,34#,39#,35#, and induction rate were 2.5%, 2.5%, 1.5%, 1.5%, 1.0% respectively.2 Proliferation and maintenance of embryogenic callus from nematode-resistant P. thunbergiiAs the material of embryonic callus early induced, the main influence factors of the proliferation stage were studied.The results showed that different embryogenic cell lines had different response to 2,4-D and NAA. the best auxin concentration of 34#-1 and 37#-1 all was 0.5 mg/LNAA, and the one of 36#-2 and 39#-1 were 2 mg/L 2, 4-d, 1 mg/L 2, 4-d respectively. Each stage of embryonic cell line proliferation growth curve was the "S" type, and the maximum growth rate was between 5-10 d, then after subcultured for 15 d, the growth tended to stagnate. the best subculture cycle was about for 2 weeks.3 Induction and maturation of somatic embryo from nematode-resistant P. thunbergiiWith DCR as the basic culture medium and selection of callus proliferation in good condition, somatic embryos of P. thunbergii were induced with the addition of different concentrations of maltose, inositol, ABA, PEG, GA,respectively.The experimental results show that the concentration of maltose is 45 g / L, the Inositol of 6 g / L, developmental status of Embryo was best; the effect of GA3 on induction of somatic embryos was superior to that of GA4 + 7, and the best concentration of was 0.5-1g/L; the treatment of 10-15 mg/LABA,100-140 g/L PEG is advantageous to the maturation and development of somatic embryogenesis.4 Germination and plant regeneration of somatic embryo from nematode-resistant P. thunbergiiWith embryo cell line 39#-1 of good somatic embryos in mature stage as experimental materials, the effects on germination and plantlet regeneration of two factors(maltose, activated carbon) were studied and analyzed. The results showed that Results show that the optimal concentration of maltose and activated carbon was 20 g/L and 1-2 g/L respectively, and the concentration can promote the formation of cotyledons, hypocotyl and radicle,and is beneficial to the germination of somatic embryo. |
PDF Full Text Request