Somatic Embryogenesis And Physiological And Biochemical Characteristics Of Pinus Elliottii

Pinus elliottii was introduced to China in the 1930s,and it is characterized by strong adaptability,high lipid production and rapid growth.However,sexual reproduction is still the main problem in the breeding of fine species of P.elliottii,which is characterized by slow regeneration,high cost and high mutation rate.The establishment of efficient somatic cell generation technology is of great significance to realize efficient asexual propagation and accelerate the breeding progress of improved species of P.elliottii.Plant somatic embryogenesis has the advantages of high reproduction coefficient,high stability and short cycle,etc.Many scholars at home and abroad have carried out researches on somatic embryogenesis of P.elliottii and have successfully realized somatic embryogenesis and plant regeneration of P.elliottii.However,somatic embryogenesis and plant regeneration of P.elliottii are still characterized by low somatic embryo induction rate,low quality of embryogenic callus,low proliferation coefficient,difficulty in somatic embryo maturation,and low germination rate.This research in 2018 and 2019 for two consecutive years in P.elliottii immature zygotic embryos as material,using L₉(3⁴)orthogonal design and the completely random block method and single factor experiment,the embryonic callus induction,proliferation and mature embryo and germination stage embryo culture were studied,and the different body zygote embryo,The morphological characteristics and endogenous hormone contents of callus in four different apparent forms were analyzed,and the endogenous hormone levels the main results were as follows:1.Induction of embryogenic callusGenotype,development degree of zygotic embryo,sampling period,species and concentration ratio of plant growth regulators,carbon source,brassinolactone,silver nitrate,ascorbic acid,gellan adhesive and culture temperature all had effects on the induction of embryogenic callus in P.elliottii.The developmental stage of zygote embryo in the same cone was different with different genotypes at the same collection date,and the developmental stage of zygote embryo in the same cone was also different.The callus induction rate of the cone of no.16 single plant collected in 2018 was higher on July 4and July 11（stage 2-3）,reaching 33.33%.With the maturation of zygote embryo,the induction rate of embryogenic callus decreased gradually.When immature zygotic embryo was in stage 2-4（multi-embryo stage）,embryogenic callus induction rate was the highest.Among the 9 hormone combinations treated with DCR+5:KT 2.2 mg/L+2,4-D2.2 mg/L+6-BA 2.2 mg/L had the highest embryogenic callus induction rate of 30%.After collection and storage in the refrigerator at 4^oC for one week,it is beneficial to improve the induction rate of embryogenic callus,and unfavorable to the induction of embryogenic callus after storage for two months.Compared with sucrose,maltose and trisaccharide were more beneficial to the induction of embryogenic callus.When the concentration of trisaccharide was 1.5 g/L,the maximum induction rate was 40%,and the average induction rate of embryogenic callus was 20%.When the maltose concentration was 30 g/L,the somatic embryo induction rate reached the highest 30%,and the average embryogenic callus induction rate was 18.75%.Under the conditions of 23 ^oC and 27 ^oC culture,there was no significant difference in the average induction rate of embryogenic callus,but under the condition of 27 ^oC,embryogenic callus differentiation started earlier.The induction rate of embryogenic callus was higher when 3.0 g/L was added to gellan adhesive for solid support than when 6.0 g/L was added,and the highest was 42.5%.The addition of different concentrations of brassinolactone had no significant effect on the somatic embryo induction rate of soil-pine.Appropriate addition of ascorbic acid was beneficial to the induction of embryogenic callus.When VC concentration was 10g/L,the induction rate of embryogenic callus was the highest,reaching 37.5%.The addition of silver nitrate was not conducive to the induction of embryogenic callus.2.Maintenance and proliferation of embryogenic callusIn the early stage of embryonic callus proliferation culture,it takes 3-5 days to recover,and in the later stage,the cell proliferation rate is accelerated.Embryonic cell line 4 proliferated the fastest in the combination of DCR+1.0 mg/L 2,4-D and 1.0mg/L6-BA treated by 5,up to 7.65 times.The callus is white and transparent with typical filamentous structure and can proliferate steadily for a long time.The proliferation was the most obvious and the proliferation was the highest in the medium of sucrose 30 g/L,with the maximum of 9.44 times and the average of 6 times.It is suitable to take 12d as a cycle for subculture.The results of preliminary study on liquid suspension culture of P.elliottii were as follows:the suspension culture conditions were optimal with 1/4DCR as the basic medium,45 g/L sucrose added,and 2.0g embryonic callus inoculated.3.Maturation and germination of somatic embryosTo explore the different concentration of ABA,PEG 8000 combination of P.elliottii body mature embryo and the effect of the experimental results showed that in 10 mg/L+100 g/L PEG 8000 ABA success under the condition of the cultivation of the induction of somatic embryo（cotyledon embryo）,significant difference,the effect of different embryonic cell line body mature to mature cultivation of cotyledon embryo transfer to DCR medium composition on germination,blank with normal form of sprouting eventually get P.elliottii early somatic embryogenesis embryo seedlings.4.Study on physiological and biochemical characteristics of somatic embryogenesis in P.elliottiiThe zygotic embryo development stage of F-A and F-B families is in the early stage1-4,and the induction rate of embryogenic callus is high.The development stage of zygotic embryos of F-C and F-D families is in the late stage,and the development is not synchronous.For example,zygotic embryos at stage 2-3 and 5 were observed at the same time on July 4,and the induction rate of embryogenic callus is low.The phenomenon of multiple embryos was observed in the developmental stage of zygotic embryo.When the immature zygote embryo was in the early stage of development with high endogenous ABA content,it was beneficial to the induction of embryogenic callus,while when the immature zygote embryo was in the late stage of development with high endogenous IAA content,it was unfavorable to the induction of embryogenic callus.The proembryo mass of the embryogenic callus was composed of the embryo head and the embryo stalk cells.The proembryo mass went through three developmental stages of proembryo mass I,proembryo mass II and proembryo mass III.Nonembryogenic callus,cells subglobose,proliferating by didivision.High concentration of ABA can induce embryogenic callus to differentiate and mature into somatic embryos.