Somatic Embryogenesis And Plantlet Regeneration Of Akebia Trifoliate(Thunb.)Koidz.

Akebia trifoliate（Thunb.）Koidz belongs to the Akebia Decne in Lardizabalaceae family.They are semi-deciduous lianas and wild fruit trees.The whole tree can be used as medicine,and it has medicinal,edible,economic and ornamental values and has broad application prospects.With thick pericarp,many seeds in the fruit,low edible rate,low seed setting rate and low germination rate in the natural state,the supply of wild A.trifolia resources in China falls short of the demand with the increase of the demand.Somatic embryogenesis is one of the important ways of transgenic breeding and variety improvement.In this study,the young embryos of immature seed of A.trifolia were used as explants to induce somatic embryogenesis of through indirect genesis pathway.To investigate the effects of different types and concentrations of plant growth regulators on callus induction and somatic embryogenesis,and to screen the suitable medium for different growth stages,so as to establish somatic embryogenesis and plant regeneration system.The materials in each stage of somatic embryogenesis were observed by histology and cytology and determined by physiological and biochemical methods.This study provided a theoretical basis for somatic embryogenesis and plant regeneration system of A.trilobata,and also laid a foundation for the establishment of genetic improvement system of A.trilobata.The main results are as follows:（1）To establish the system of somatic embryogenesis and plant regeneration of A.trifoliaThe optimal sterilization treatment was 0.1%Hg Cl₂ for 18 min,washed with sterile water and inoculated on callus induction medium supplemented with 0.05%PPM.Then,the explants were rinsed with sterile water and inoculated.At this time,the contamination rate was low and the browning situation was light.Young embryos of immature seeds of A.trifolia were used as explants and inoculated into MS medium supplemented with different concentrations of 2,4-D,NAA,6-BA and KT.The results show that:The best medium for callus induction was MS+2,4-D 2.0 mg/L+NAA 0.2 mg/L,and the highest callus induction rate was 95.67%.The primary callus was yellowish brown,with hard texture and mostly embryogenic callus.The optimal medium for somatic embryogenesis induced from embryogenic callus was MS+NAA 0.1mg/L+6-BA 0.2 mg/L,and the somatic embryo induction rate was 55%.When the concentration of NAA and 6-BA was adjusted,secondary embryos could proliferate.The medium suitable for the proliferation of secondary embryos was MS+NAA 0.05 mg/L+6-BA0.1 mg/L,and the proliferation ratio reached 19.84.Somatic embryos differentiated gradually with the increase of subculture times and the proliferation activity of secondary embryos became weak.Mature somatic embryos can regenerate into seedlings after differentiation,and the regenerated plants can grow strong in MS without adding plant growth regulators.When the somatic embryos to be roted grew to 3～5 cm,they could survive stably in the 2:1:1 matrix of peat:vermiculite:perlite.（2）Histological and cytological observation on somatic embryogenesis of A.trifoliaThe non-embryonic callus,the embryonic callus and the somatic embryo of A.trifolia were used as the research materials for paraffin section.Through cytological observation,it was found that the morphological,arrangement and starch grain distribution of the embryonic cells and the non-embryonic cells were significantly different.Non-embryonic cells have large cell volume,irregular shape and arrangement,uniform cytoplasm,and almost no starch granules in cells.However,embryonic cells have small cell space,compact arrangement,dense cytoplasm,and abundant starch granules in cells.Somatic embryogenesis of A.trilobata underwent four stages:globular embryogenesis,heart-shaped embryogenesis,torpedo embryogenesis and cotyledon embryogenesis,and it was observed that somatic embryogenesis was exogenous.（3）Physiological and biochemical changes in somatic embryogenesis of A.trilobataThe physiological and biochemical changes of somatic embryos were analyzed by using non-embryonic callus,embryogenic callus,early somatic embryo and mature somatic embryo of A.trifolia.The soluble protein content was determined by Coomassie bright blue G-250 method,the soluble sugar content was determined by anthrone colorimetric method,the SOD enzyme activity was determined by nitrogen blue tetraazole photochemical reduction method,the POD enzyme activity was determined by guaiacol method,and the CAT enzyme activity was determined by ultraviolet spectrophotometry.The results show that:The content of soluble protein,soluble sugar and the activities of antioxidant enzymes SOD,POD and CAT in embryogenic callus were higher than those in non-embryogenic callus.The cell activity and metabolism of embryogenic callus were higher than those in non-embryogenic callus.During embryogenic callus development to early somatic embryo and then to somatic embryo maturation,the content of soluble protein,soluble sugar and CAT activity increased,and the accumulation of matter and energy was greater than the consumption,which provided the basis for the germination of somatic embryo seedlings.The activities of antioxidant enzymes SOD and POD decreased,and its high activity was beneficial to somatic embryogenesis of A.trifolia.