Construction Of The Primary Tissue Cultivation System And Physiological Analysis Of Explant Development In Chinese Chestnut

Tissue culture techniques are widely used in rapid propagation,seedling virus-free,mutation breeding,and genetic engineering in plants.However,there has been no obvious breakthrough in the research of tissue culture technology in Chinese chestnut till now.The main problems of primary culture include incomplete sterilization of explants,inappropriate hormone types and concentrations,and browning of explants during the culture process,etc.In this experiment,we used annual shoots with axillary buds of Canstanea mollissima cv.‘Yanbao’and embryos of mature seeds of Castanea mollissima cv.‘Yanlong’as materials to detect the effects of different sampling times,sampling parts,sterilization times,hormone types and concentrations on the preferable contamination rate and survival rate in Chinese chestnut,and to construct a developmental tissue culture system using annual shoots with axillary buds and embryos as explants,respectively.Then we used the embryos of mature seeds of‘Yanlong’as materials and used our preferable sterilization methods and hormone concentrations to study the changes processes of hormones,sugars and other related morphogenetic substances during the primary culture of embryos to analyze the physiological mechanism of explants development in Chinese chestnut.The results were as follows:1.We used a complete test design of poly factors and nested analysis to construct the tissue culture system of annual shoots with axillary buds,‘Yanbao’as explants.The results indicated that the best sampling time was May 9,with 0%contamination and 98.15%survival rate.The best sampling part was the middle of shoots,with 0%contamination and 94.56%survival rate.The best sterilization method was 2%Na Cl O 20 min+0.1%Hg Cl₂ 15 min,with 0%contamination and 91.24%survival rate.The best hormone types and concentrations was ZT 1.00 mg/L+IAA 0.10 mg/L,with 77.50%germination rate.2.We used the L9（3³）orthogonal test design to construct the tissue culture system of embryos,‘Yanlong’as explants.The results indicated that the best sampling part was the 0.8 cm3including embryo in the core.The best sterilization method was2%Na Cl O 10 min+0.1%Hg Cl₂ 8 min,with 0%contamination and 100%survival rate.The best sterilization method for explants with only embryo was 2%Na Cl O 5min+0.1%Hg Cl₂ 5 min,with 0%contamination and 82.96%survival rate.All the results indicated that the survival rate of embryos that did not directly meet with the sterilizing agent was higher.3.During the development of the embryos of the seeds of‘Yanlong’,the addition of 0.10 mg/L IAA and 1.00 mg/L ZT to the primary medium could increase the contents of GA,GA₃,IAA,and CTK in the germ,which was beneficial to the growth and development of the embryos.as the embryos developed longer and longer,the contents of fructose and sucrose gradually decreased,the contents of glucose increased,and the contents of maltose remained stable,indicating that the addition of fructose and sucrose in the primary medium was more favorable to the growth and development of the embryos.In summary,we initially established a primary fast tissue culture system of annual shoots with axillary buds and embryos with low contamination rate and high survival rate and established the foundation for tissue culture and efficient genetic transformation system in Chinese chestnut.