Preliminary Studies On Induction And Proliferation Of Embryogenic Callus Of Pinus Elliottii

Since its introduction in the 1930s,Pinus elliottii has become the main tree species in the south of China,but its lagging breed breeding technology still restricts its development.With the rapid development of modern biotechnology,plant tissue culture and somatic embryogenesis techniques have the advantages of rapid propagation,short cycle,high efficiency and stability,and provide powerful technical support for rapid propagation of S.elliottii.At present,many domestic and foreign scholars have done research in this area and succeeded in establishing the system of embryogenesis and plant regeneration.However,in Pinus elliottiiembryo technology,there are still problems such as low embryogenic induction rate and unstable proliferation.In this study,immature zygotic embryos of Pinus elliottii were used as explants.L9(3~4)orthogonal design experiments and single factor experiments were used to study the minimal medium(LP,DCR and LM),and the different concentrations of plant growth regulation hormones The combination of 2,4-D and 6-BA,the effects of different inoculation methods on the induction and proliferation of somatic embryos of Pinus elliottii at different collection stages.Experiments initially established the somatic embryo induction and proliferation system and screened the conditions for optimal embryogenic callus induction and proliferation.At the same time,microscopic observations of the callus induced by Pinus elliottii and determination of endogenous hormones were performed.The differentiation of embryogenic and non-embryogenic callus was made from morphological and cytological and physiological and biochemical studies.The iodine-potassium iodide staining was used to observe the early embryos in embryogenic calli of Pinus elliottii,which revealed the development of early embryos.The results showed that the most suitable time for seed collection was from late June to early July,when the zygotic embryos were in the first to fourth stages of development.The induction rates of embryogenic callus of the four genotypes tested were different.The highest embryogenic induction rates of genotypes F-B,F-C,and F-D were 13.33%,6.67%,and 13.33%,The average of embryogenic induction rate were 3.25%,4.33%,and 3.25%,but F-A only induced two embryogenic callus on June 24th.The average induction rate was only 0.667%,and the highest embryogenic induction rate was 6.67%.Somatic embryo-inducing material.The basic medium had a significant difference in the induction rate of embryogenic callus.All genotypes in the test period induced embryogenic callus on DCR medium only,and non-embryonic callus on DCR medium.The induction rate was higher than that of LP and LM mediums,indicating that DCR medium is the most suitable basic medium for both embryogenic and non-embryonic callus induction of Pinus elliottii.Among the 9 combinations of media,the highest callus induction rate was:DCR+2.2mg/L2,4-D+2.2 mg/L6-BA.The inoculation method has a great influence on the induction of embryogenic callus.In this experiment,the young embryos of the five fruit collections from July 14 to August 11 were selected as explants.The results of the study showed that in these 5 periods,With the female gametophyte approach than direct embryo access,embryogenic callus induction rate is higher.Embryogenic callus showed a white,translucent viscous,with a small part of texture loose,granular,embryogenic callus,can be observed under the microscope,there are many protruding filaments on the surface of the callus,in Embryonic cells were observed under a microscope to consist of small,dense embryonic head cells and long,vacuolated suspensor cells.The overall cells were small and tightly packed.After embryonic cytoplasm staining with iodine-potassium iodide,dense embryonic cells were observed.The nucleus is large and easy to be stained,in which the embryonic head cells appear yellow.In the highly active meristem,the cells are mitotically active,the callus is rapidly proliferated,the prickle stalk cells show a large vacuolated state,and the isolated nucleus is stained.Independent of cells.Non-embryogenic callus is light white,milky yellow or tan granules,with loose surface and irregular shape.Observable under the microscope,the callus is composed of many spherical cells of similar size,and the difference between cells is relatively small.Large,large cells,loosely arranged,irregular,after iodine-potassium iodide staining,can be observed small nuclei,large vacuoles,less cytoplasm,difficult to be stained.In physiological and biochemical studies,the content of endogenous hormones also differs.In this study,it was found that in addition to ABA content,the content of embryogenic calli was higher than that of non-embryogenic callus,and the other three hormones IAA and ZR,The content of GA3 was higher in non-embryogenic calli than in embryogenic calli.It was concluded that high concentrations of ABA and low concentrations of IAA,ZR,and GA3 can favor the formation of embryogenic callus.Iodine-potassium iodide staining was used to observe early embryogenic tissue in embryogenic calli of Pinus elliottii.It was found that embryogenic calli of Pinus elliottii originated from the zygotic embryonic axis.Embryonic cells consist of two cells,one of which is the embryonic head cell and the other of which is a long,vacuolated cell.Embryogenic cells contain pro-embryo I,pro-embryo II and pro-embryonic III and some free cell masses.