Construction Of Somatic Embryo Regeneration Technique Of Picea Abies

Picea abies(L.)Karst.,also known as Norwegian spruce,belonging to the genus Pinaceae,is an evergreen coniferous species,which is widely introduced in our country.The seed breeding cycle of Picea abies is long,its growth rate is slow,and it is prone to variation.The somatic embryogenesis experiment of Picea abies can solve the above production difficulties,and the construction of mature somatic embryogenesis technology system is an effective means to break the bottleneck of reproduction difficulties of Picea abies.In this study,immature zygotic embryos of Picea abies superior trees were used as explants for somatic embryogenesis,and the effects of different plant growth regulator combinations and different media on embryogenic callus induction were investigated.The effects of different culture methods,different combinations of plant growth regulators and different anti-browning agents on embryogenic callus proliferation were analyzed.Research on somatic embryo maturation and germination culture was carried out.Based on the above research content,the regeneration technology system of somatic embryogenesis of Picea abies was optimized,which laid the foundation for efficient and rapid propagation of superior clones of Picea abies.The main results were as follows:1.The optimal medium type and the optimal hormone concentration ratio were selected for induction of embryonic callus of Picea picea.When immature zygote embryos of Picea abies were inoculated on 1/2 LV medium,the embryogenic callus induction rate was 20.33%.The embryotic callus induction rates were 18% and 20.33% when the medium was supplemented with 4.4 mg/L2,4-D+1.0 mg/L 6-BA and 4.4 mg/L 2,4-D+2.0 mg/L 6-BA,respectively.2.Select the optimal parameters for the proliferation and culture of embryogenic callus of Picea abies.The surface of the embryogenic callus of Picea abies was waterstained and sticky in the medium with high concentration of 2,4-D for a long time,and even the embryogenic callus was lost.When 3.0 mg/L 2,4-D+2.5 mg/L 6-BA was added into the medium,the embryonic callus developed well and its translucent filamentous structure was intact.The supplementation of2.0 mg/L NAA+2.5 mg/L 6-BA was beneficial to maintain embryogenic ability,and the appropriate subgeneration period was 14 days.The addition of 1.0 g/L PVP effectively reduced the Browning problem during proliferation.3.Identify the advantages and disadvantages of suspension culture and solid culture.The growth rate of liquid suspension culture was faster than that of solid culture,and the embryogenic callus remained loose and dispersed,which was conducive to mature culture.Solid culture can also make embryogenic callus maintain better development state and mature culture,but the proliferation rate is slower than that of suspension culture.In this experiment,the proliferation rate of embryonic callus in suspension culture and solid culture was compared and analyzed in the proliferation stage.However,all the embryonic callus used in mature culture stage were from solid proliferation medium,mainly because suspension culture requires the use of filter,complicated process and more experimental materials.For these objective reasons,suspension culture was only carried out on some clones in this experiment,and the proliferation rate of embryogenic callus was compared.4.To screen the optimum culture parameters for somatic embryo maturation and germination of Picea abies.It was found that the combination of ABA and PEG was beneficial to the somatic embryo maturation of Picea abies,and 22.67/500mg·FW embryos could be obtained at 16.0 mg/L ABA+50.0 g/L PEG.The number of mature embryos differentiated by different concentrations of maltose was very small,but the addition of 30 g/L sucrose was beneficial to the maturation of somatic embryos of Picea abies.The optimum time of drying pretreatment before germination culture was 3 weeks.The cotyledon embryo was obtained by drying,and the cotyledon was fully developed,the cotyledon was extended,and the radicle showed reddish brown.On the germination medium,the cotyledon developed into a robust true leaf,the cotyledon continued to expand,and some clones developed fine roots.