| Epimedium koreanum Nakai,named also San zhi jiu ye cao,or Tang ben cao,is a perennial herb of the genus Epimedium of Berberidacea.It is a traditional Chinese medicine that unique from Changbai Mountain,and also the best category of five traditional Chinese herbal Epimedium medicines in the Pharmacopoeia.Because of its tolerance to yin and drought,the leaf shape is unique and beautiful.In recent years,it has been widely used as a high-quality cover flower species by the foreign countries and exported to China.However,the wild resources of Epimedium koreanum Nakai in Changbai Mountain area have been exhausted due to predatory development.In recent years,farmers have a high degree of enthusiasm for spontaneous planting,and the source has stolen wild products,resulting in scarcity of seedlings.Therefore,the breeding technology of Epimedium is mainly carried out.Research on tissue culture technology is of great significance.It not only protects wild resources and meets market demand,but its medicinal value and ornamental characteristics will also be the new darling of forest tourism,agricultural pastoral complex and characteristic industrial projects in the future.In this study,different explants of Epimedium koreanum were used as experimental materials to systematically study explant sterilization methods,callus induction?proliferation and differentiation,cluster bud induction and proliferation,rooting culture,etc,so as to screen out the appropriate culture factors and conditions at each stage.The main research results are as follows:1?Screening and sterilization of explants:Young leaves,young petiole branching points,seed embryos and latent buds are the best explants,and the tip and petiole branching points are easier to disinfect.2%NaClO disinfection for 6min,the survival rate was 71.67%and 73.33%;Seed embryo disinfection is relatively difficult.the surface of the seeds was sterilized by0.1%HgCl2 for 8 min,and then the embryos were sterilized for 5 s.Under this condition,the survival rate of the embryos was 71.11%.Latent bud disinfection is the most difficult.It is treated with 0.2%HgCl2 and added with two drops of Tween for 4 min,followed by 0.1%HgCl2 for 3 min.The survival rate is 52.5%.2?Callus induction:Suitable medium for leaf tip callus induction 1/2MS+2,4-D2.0mg·L-1+6-BA0.4mg·L-1+NAA0.5mg·L-1,the induction rate of was 48.33%,and the optimal medium for petiole branching point was MS+2,4-D3.0mg·L-1+6-BA0.2mg·L-1+NAA 0.5 mg·L-1,the induction rate was 31.67%.In the study of leaf and petiole branching callus in different light and low temperature treatment time,it was found that the tip and petiole branching points were well induced under low light and 4°C low temperature treatment for 4 and 6 days,respectively effect.The suitable medium for embryogenic callus was 1/2MS+6-BA1.0mg·L-1+NAA0.5mg·L-1+IBA0.5mg·L-1+sucrose 30g/L,and the induction rate was up to 47.78%.3?Callus proliferation and differentiation:The suitable proliferation medium of leaf tip callus was 1/2MS+NAA1.0mg·L-1,the growth rate was 122.03%;the suitable proliferation medium for embryogenic callus was1/2MS+KT 0.5 mg·L-1+NAA 0.3 mg·L-1,the growth rate was 172.67%.The callus of leaf tips can differentiate after treatment at?4±0.5?°C for 180 days,and the differentiation rate is 54.4%.The callus of embryos is easy to differentiate,and the room temperature condition can be1/2MS+6-BA1.0mg·L-1.+NAA 0.4 mg/L,the differentiation rate was 65.83%.4?Cluster bud induction and proliferation culture:Tissue culture was carried out by using latent buds and embryos of Epimedium koreanum as explants,all of which induced cluster buds.The optimal single factor condition for inducing latent bud-induced shoot buds is:the inoculation method for removing the top buds,using 1/2WPM basal medium,6-BA2.0mg·L-1+NAA0.5mg·L-1+IBA1.0mg·L-1+GA30.5mg·L-1phytohormoneratio,4°C low temperature treat-ment of explants for 6d.The optimal medium for embryogenic induction of shoots was 1/2MS+IBA1.0mg·L-1+6BA1.0mg·L-1,and the induction rate was 43.33%.Proper medium for shoot bud proliferation:1/2MS+6-BA2.0mg·L-1+NAA0.4mg·L-1,proliferation coefficient 2.35;1/2MS+6-BA2.0mg·L-1+NAA0.4mg·L-1,proliferation The coefficient is 2.78.The shoot buds grew well on both media.5?Rooting culture:The rooting medium suitable for seedlings is 1/2MS+NAA0.5mg·L-1+0.05%activated carbon,the rooting rate is 48.89%,24 roots per plant,and the seedlings grow vigorously. |
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