| Larix olgensis has many advantages,such as strong stress resistance and rapid growth in early stage.As the fast-growing timber tree species in temperate zone and frigid zone of northern hemisphere the Larix olgensis has high ecological value and economic value.But the larch has some characteristics,shch as long growth cycle,high heterozygosity,rooting difficut by cuttage and the the offspring produced by reproduction vary greatly,the demand for larch rapid genetic improvement cannot be met by traditional breeding methods.Therefore,it has important and far-reaching significance for the rapid propagation and genetic improvement of Larix olgensis that establishment of a stable and efficient regeneration system and genetic transformation system.In this study,the immature zygotic embryos of Larix olgensis as explants to induce embryogenic callus,establishment and optimization the somatic embryo-genesis system,embryogenic callus suspension culture system and Agrobacterium-mediated genetic transformation system.On this basis,study the physicochemical characteristics in different developmental stages of somatic embryos.The main results are as follows:（1）Zygotic embryos of Larix olgensis collected after 70 days of pollination are suitable for induction of embryogenic callus,the medium was BM,adding 2,4-D 1.5mg·L-1、BA 0.5 mg·L-1 and KT 0.5 mg·L-1,and the induction rate between different families have certain differences.（2）Embryogenic calli can rapidly proliferate and maintain their embryogenic properties in BM medium containing 2,4-D 0.3 mg·L-1、BA 0.1 mg·L-1 and KT 0.1mg·L-1.Suspension culture of embryogenic tissue rapid proliferation,in shock strength is 120 rpm·L-1,the initial inoculation amount was 4 g·L-1（25 mL liquid medium inoculated 0.1g）under the condition of culture for 14 days the proliferation rate is about 1726.67±126.64%,is about 8 times the solid cultivated under the similar conditions.（3）The pre culture of embryogenic callus has a positive effect on the matura-tion of somatic embryos.The effects of pre incubation period,ion and inositol concentration on embryogenic callus were extremely significant.While sucrose,glutamine and casein had no significant effect.Using Box-Behnken Design response surface test and established a mathematical model to predict the best conditions.In pre culture stage,the ion concentration 26.77%（BM）,inositol concentration 10.46 g·L-1and cultured 12.66 days,amount of somatic embryos was 337.04 Per gram.（4）In somatic embryos maturation stage,the final amount of somatic embryos was affected by many factors.Among them,the effects of abscisic acid,silver nitrate,sucrose,casein hydrolysate and hydrogen peroxide were more significant,while the effects of maltose,inositol and glutamine were not significant.Using Box-Behnken Design test based response surface model fitting of Larix somatic embryos maturation and the optimum culture conditions were:BM medium with ABA 18.28 mg·L-1,AgNO3 5.46 mg·L-1 and sucrose 82.63 g·L-1,the average amount of somatic embryogenesis was 203.72 Per gram.（5）The somatic embryogenesis of Larix olgensis is accompanied by cell division and differentiation,morphogenesis of tissues and organs,as well as a series of physiological and biochemical changes.According to the morphological identification of embryogenic cells and somatic embryos:In the stage of tissue proliferation,the somatic embryos were in the stage of primary embryo.During the14 days of pre-culture,the embryo developed to the early stage of per-cotyledon embryo.After 7 days of maturation,the development of somatic embryos into the late stage.At maturation 14 days,the embryo developed to the initial stage of cotyledon embryo,and its morphology was close to maturity.In the process of somatic embryo maturation,the soluble sugar content showed an upward trend,while the soluble protein content and SOD activity showed a trend of decreasing,rising and decreasing,while POD activity showed a"wave type"decline.（6）Cef,Kan and Hyg three kinds of antibiotics had different degrees of toxicity to the embryogenic callus of Larix olgensis.The 100-400 mg·L-1 of Cef can cause the decrease of the rate of embryogenic callus of Larix olgensis,but it has no effect on the morphology and structure of embryogenic cells and tissues.10 mg·L-1 Kan or 2mg·L-1 Hyg can significantly reduce the rate of embryogenic callus proliferation,and completely inhibit the maturation of somatic embryos.The 20 mg·L-1 Kan or 4 mg·L-1Hyg can inhibit the proliferation of embryogenic tissue.（7）In the BM medium of presence0.15mg·L-1 2,4-D,0.05mg·L-1 BA and KT on proliferation of Larch embryogenic callus suspension at OD600≈0.6 of the bacteria（strain GV3101）infection in first 20 min,then co cultured for 3 days,and were added to the culture fluid of 100μM·L-1AS medium suspension.The infected tissue after 200mg·L-1 Cef wash 10 min,the removal of bacteria after rinsing.Then 3-7 days of recovery training,and transferred to the medium containing 4 mg·L-1 Hyg for continuous screening,screening will be organized to adjust the size of 1.4 cm2,the resistance organization average 0.56±0.04 Per square centimeter.Positive the rate of PCR is 88.34%.There was a slight difference in the frequency of somatic embryo maturation in different embryogenic lines. |
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