Optimization On Initiation And Proliferation Of Embryogenic Masses In Masson Pine (Pinus Massoniana Lamb.)

Masson pine(Pinus massoniana Lamb.),belongs to Pinus of Pinaceae,is widely distributed in the subtropical area of Southern China and is the main tree species for afforestation both in resin and timber production.It has the characteristics of drought resistance,barren,strong adaptability,fast growth and high yield and plays an important role in meeting up with the demand of national social and economic development for wood,pine resin supplies and national ecological security.Somatic embryogenesis is an effective way for rapid propagation of improved forest varieties.However,the problems with limited initiation rate and instability of embryogenic mass proliferation have restricted the practical application of somatic embryogenesis of Masson pine.To establish an efficient protocol on the initiation and proliferation of embryogenic mass of Masson pine,this study explores the effects of family genotype,types and concentrations of plant growth regulator,levels of sucrose and inositol dosage on embryogenic mass induction and proliferation.In view of the problem of instable proliferation and maintenance of embryogenic mass,RNA-Seq was applied to explore the gene differential expression patterns on the effects of different sucrose and inositol concentrations during the embryogenic mass proliferation,to provide technical support for the optimization of induction and proliferation of embryogenic mass in Masson pine somatic embryogenesis.The main results are as following:1.In the induction stage,the immature zygotic embryos in cones collected from induction-sensitive genotype of mother tree in late July were used as explants for embryonic mass induction,and the highest initiation rate was up to 26.09%in LP medium with 1.0 mg·L-1 2,4-D+1.0 mg·L-1 6-BA,and a stable protocol on embryonic mass induction in Masson pine was established.Based on the selection of induction-sensitive genotype of mother tree for the induction of embryogenic masses,there was no significant difference between the initiation results on different genotype of mother trees,while the concentration levels of plant growth regulator had a significant effect on embryonic masses induction(P<0.05);In the early stage of embryogenic mass induction,the cell structure of the cultures was observed by a squashing technique,and it was found that the clear polar structures could be identified in the proembryo mass which could be stably proliferated,which provided a technical basis for identification of stable embryogenic masses in early induction stage.2.There is a significant effect(P<0.05)on the quality of proliferation and differentiation of embryogenic masses with different combinations of concentration levels in sucrose and inositol treatments in Masson pine embryogenic masses proliferation.According to the stability and the status of proembryo mass polar structural development in proliferation,a medium containing 2,4-D 0.5 g·L-1+6-BA 0.5 g·L-1 for embryogenic masses proliferation was selected with suitable combination of 30.0 g·L-1 sucrose+0.1g·L-1 inositol,in which the proembryo mass was developed with good quality,and the multiplication rate was the highest with 9.28,the second highest multiplication rate was 7.82 at 20.0 g·L-1 sucrose+1.0 g·L-1 inositol,and the lowest multiplication rate was 2.32 at 10.0 g·L-1 sucrose+0.1 g·L-1 inositol,which was the used induction medium in initiation stage as a control for proliferation,and the proembryo mass structure development was delayed a lot.The above evaluation of different treatments on proembryo mass structure development with different levels of sucrose and inositol combination suggested that it was necessary to maintain the induced embryogenic masses to a stably proliferated masses with normally developed proembryo mass by appropriately increasing the concentration levels of sucrose and inositol in the proliferation medium,that is to say,the osmotic stress level in the proliferation medium was increased at the same time during the transition of initiation to proliferation process.3.More somatic embryos were obtained by adding a certain amount of ZT to the mature medium containing ABA than by using ABA alone in the differentiation and maturation stage of Masson pine somatic embryogenesis.It is to say that by adding ZT to the mature medium with ABA could improve the efficiency of the transition of proliferation mass from proembryo mass to somatic embryo during maturation,and the best hormone combination for somatic maturation was selected as 5.0mg·L-1ABA+0.2mg·L-1 ZT,in which about 120 matured somatic embryos were obtained per 2.0 g/dish of embryogenic mass.4.The transcriptome sequencing of RNA-Seq was carried out based on the instability of proliferation and differentiation of embryogenic mass treated with different levels of concentration combination of sucrose and inositol in the process of proliferation.The sequencing analysis results showed that a total of 99239 unigenes were obtained,including 1483,1756,689 and 154 differentially expressed genes within the four groups of treatments with different sucrose and inositol combinations(F3001vsF1001、F3001vsF3010、F3001vsF3020、F3010vsF3020).These differentially expressed genes were significantly enriched by KEGG(P<0.05),and those pathways were significantly enriched on phenylpropane biosynthesis,protein processing in endoplasmic reticulum,tyrosine metabolism,metabolic pathway,flavonoid biosynthesis,glutathione metabolism,plant hormone signal transduction and so on.A large number of genes related to flavonoid biosynthesis,glutathione metabolism and plant hormone signal transduction pathway were up-regulated in the optimal medium for embryogenic mass proliferation containing 30.0 g·L-1 sucrose and 0.1 g·L-1 inositol,while the relted genes of the above three pathways were down regulated or only a few genes up-regulated in other three treatments.Thus,by regulating the concentration of sucrose and inositol in the proliferation medium,the optimal osmotic stress environment on the proliferation and differentiation medium may be formed,in which these environment may stimulate the up-regulated expression of stress resistance system genes such as flavonoid biosynthesis and glutathione metabolism pathway in embryonic mass,and may coordinate with the active expression of auxin and cytokinin genes related to intracellular hormone signaling pathway.Those factors may have promoted the normal development of proembryo mass polar structures in the embryonic mass and ensured the establishment and optimization of embryogenic mass proliferation in Masson pine somatic embryogenesis.Those findings provide helpful reference for understanding the role of the sucrose and inositol concentration regulating in the proliferation medium for the embryogenic mass stable proliferation.