| Epimedium sagittatum(Sieb.et Zucc.)Maxim.,also known as " Sanzhi Jiuyecao," is a perennial herb of the genus Epimedium of Berberidacea.It is one of the most famous traditional Chinese medicines in China since ancient times.It has been recorded in one of the four classical works of traditional Chinese medicine,which is known as《Shennong ’ s Herbal Classics》 and has a history of more than 2000 years.As is recorded in the calendar edition 《Chinese Pharmacopoeia》,its main medicinal parts are dry leaves which have effects of enhancing kidney,strengthening muscles and bones and treatiing rheumatism.Since the whole genome sequencing,significant progress has been made in the functional studies of many important flavonol glycosides and related enzymes in Epimedium.Biosynthetic genes that synthsis the main active components of Epimedium have been gradually excavated.However,due to the lack of tissue culture regeneration and genetic transformation system,it is difficult to carry out gene function analysis and transgenic research of Epimedium,and it is urgent to establish an efficient and stable genetic transformation system.Therefore,this paper first established tissue culture regeneration system of Epimedium sagittatum : including two-step regeneration system and different explants induction regeneration system;At the same time,the callus induced in the tissue culture system was used as the receptor material to carry out the research on Agrobacterium-mediated callus transformation.The main results are as follows :1.In the traditional method of stratification with 15 % water content of sand in low temperature(about 4 °C or 15 °C)to relieve the physiological dormancy of Epimedium seeds,the embryo as explants need secondary disinfection treatment,regularly flip and water supplement,which may not only greatly reduce the survival rate,but also increase labor force.So the appropriate disinfectant and treatment time were chosen to promote seed germination in the refrigerator with solid culture dish.The results show that the best disinfection effect with 2 % Na Cl O for 7 min,the germination rate is 84.44 %.2.Explant disinfection : The effects of five different disinfectants(0.1 % Hg Cl2,2 %Na Cl O,10 % H2O2,0.1 % benzalkonium bromide and 0.2 % povidone iodine)with different disinfection time(2 min,4 min,6 min,8 min,10 min)on three different explant materials(immature embryo,petiole branch point and inflorescence)were studied.The results showed that immature embryos were best treated with 0.1 % benzalkonium bromide solution for 8 min,and the survival rate was 83.33 %.When petiole branch point was treated with 2 %Na Cl O solution for 8 min,the effect was the best,and the survival rate was 86.67 %;The inflorescence was best treated with 0.1 % Hg Cl2 solution for 6 min,and the survival rate was65.56 %;3.Callus induction : immature embryo,petiole branch point and inflorescence were selected as explants,and the suitable medium for immature embryo induction was MS + 2.12 mg / L 2,4-D + 0.53 mg / L 6-BA + 0.53 mg / L NAA,with the induction rate of 93.52 %.The suitable medium for petiole branch point induction of seedlings was MS + 2.22 mg / L2,4-D + 0.57 mg / L IBA + 0.93 mg / L 6-BA,and the induction rate was 89.24 %.The suitable medium for inflorescence induction was MS + 2.74 mg / L 2,4-D + 0.76 mg / L 6-BA+ 0.84 mg / L NAA,and the induction rate was 68.89 %.4.Callus proliferation : The optimal medium for immature embryos was MS + 2.1 mg /L 2,4-D + 0.5 mg / L 6-BA + 0.5 mg / L NAA,and the proliferation coefficient was 3.47.The optimal medium formula for petiole branch point was MS + 2.2 mg / L 2,4-D + 0.6 mg /L IBA + 0.9 mg / L 6-BA,and the proliferation coefficient was 2.12.The optimal medium for inflorescence was MS + 2.7 mg / L 2,4-D + 0.8 mg / L 6-BA + 0.8 mg / L NAA,and the proliferation coefficient was 1.37.5.Cluster bud induction and proliferation culture : MS + 1.0 mg / L 6-BA + 1.0 mg / L NAA The proliferation medium formula of cluster buds is MS + 1.26 mg / L 6-BA + 1.14 mg / L NAA,and the proliferation rate was 3.81.6.The rooting culture condition was 1 / 2MS + 0.5g / L activated carbon,the rooting rate was 86.67 %.7.The lethal concentration of Epimedium callus in Kan(kanamycin)was 125 mg / L,and in Hyg(hygromycin)was 25 mg / L.8.GUS marker gene was transformed by Agrobacterium tumefaciens GV3101 to explore the important parameters of callus transformation of Epimedium.Transgenic callus was detected under the condition of bacterial concentration OD600 = 0.6 and infection time 10 min.The Es GT1-7GT-GFP gene with green fluorescent protein tag in Epimedium was also transformed by this method,and green fluorescence was observed under the electric fluorescence stereomicroscope after 2 months of culture. |
PDF Full Text Request