| In this study,complete seed kernel of Pinus koraiensis was used as explant,studying somatic embryogenesis,Inducing embryogenic callus of mature seeds and immature seeds,and comparing the effects of explants collection time,clones,sucrose,growth regulator concentration and acid hydrolysis casein on embryogenic callus induction.Embryogenic callus which were Induced subculture to proliferation medium and then somatic embryos were obtained through somatic embryogenesis.Selecting the normal development of somatic embryos for germination,transplanting after the embryo is rooted.(1)The immature seed of P.koraiensis can induce embryogenic callus,and embryogenic callus can be maintained and proliferated for a long time under suitable conditions.Through preliminary screening of the initial:the suitable physiological period of the callus-induced explants of the callus of P.koraiensis was the primary period,the best medium for the callus maintenance and proliferation stage of the P.koraiensis was DCR + 6-BA 0.25 mg/L + NAA 1 mg/L + sucrose 30 g/L + L-glutamine 500 mg/L + hydrolyzed casein 500 mg/L + agar 6.5 g/L.The best subculture cycle of embryogenic callus was about 15 days,at this time both to maintain the embryo of embryogenic callus,but also to obtain a larger amount of proliferation.There was a significant difference in the maintenance and proliferation of embryogenic callus between different clones.The effect of different growth regulators on the callus proliferation rate of P.koraiensis was significantly different,the combination of 2,4-D + 6-BA was the best,but its proliferation rate was still lower than that of NAA + 6-BA.(2)The immature seed of P.koraiensis can achieve high frequency embryogenic callus induction system,and it can achieve somatic embryogenesis and plant regeneration.In this study,the somatic embryos of P.koraiensis were successfully induced,the root of plant regeneration was successfully obtained and regeneration plant transplanting domestication has survived.Callus induction medium for immature seed of P.koraiensis was DCR + agar 6.5 g/L+ L-glutamine 500 mg/L + sucrose 30 g/L + NAA 2 mg/L + 6-BA 1.5 mg/L + hydrolyzed casein 800 mg/L.The preservation and proliferation of embryogenic callus of P.koraiensis were studied with DCR as the basic medium,appending sucrose 30 g/L + 2,4-D 0.5 mg/L + 6-BA 0.1 mg/L + hydrolyzed casein 500 mg/L + L-glutamine 500 mg/L + agar 6.5 mg/L,and the subculture period was 15 days.The suitable medium for development and maturation of somatic embryos was DCR + maltose 40 g/L + ABA 20 mg/L + Gelrite 10 g/L + activated carbon 2 g/L + hydrolyzed casein 500 mg/L + L-Glutamine 500 mg/L.The germination medium of somatic embryos was DCR + sucrose 25 g/L + activated carbon 2 g/L + agar 6.5 g/L,dark culture was carried out for 7 days,and then dark culture was carried out 8 hours and light culture was carried out 16 hours each day.When the plants of P.koraiensis grows to about 3cm,turn them into rooting medium(WPM + sucrose 20 g/L + IB A 0.1 mg/L + agar 6.5 g/L).Finally,the regenerated plants were transplanted into the soil containing nutrient soil,vermiculite and perlite(mass ratio of 3:1:1),survival rate of plant was 46%.(3)The mature seed of P.koraiensis can induce callus,and induced callus are all embryogenic callus both from morphological and cytological aspects.But so far,the embryogenic callus proliferation of mature seed of P.koraiensis was not successful,and it has not yet successfully achieved somatic embryogenesis.The suitable medium for callus induction of mature seeds of P.koraiensis was DCR + sucrose 35 g/L + NAA 2.0 mg/L + 6-BA 1.5 mg/L+ acid hydrolyzed casein 500 mg/L + L-glutamine 500 mg/L.The callus induction rate was the highest,the browning rate was the lowest and the growth condition was the best at this medium.Under the optimized culture conditions,the callus induction rate of the hypocotyls cut off from the naked embryo was high,the browning rate was low,and the proliferative ability was strong,and it had morphological anatomical characteristics of embryogenic callus. |
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