Studies On Rapid Propagation And Plant Regeneration Of Aconitum Carmichaelii Debx

Radix Aconiti Lateralis Preparata is the sub-root of Aconitum carmichaelii Debx., which is a kind of famous traditional Chinese medicine, and Sichuan genuine medicinal material. According to the Third National Census of traditional Chinese medicine resources, the annual requirement of aconite is about 1000t-1200 t. Aconite products on the markets are in short supply, which has a direct relationship with the roots breeding of aconite. Aconite breeding is needed to bulb reservation and bulb substitution in the mountains every year. This mode of reproduction restricts the industrial development of aconite seriously, which can accumulate pests, result in continuous cropping obstacle and seriously result in yield reduction and quality decreased. Using tissue culture, which can improve seedlings reproduction rate, is an effective way to solve these problems. The study mainly included rapid propagation by stem segment, seed propagation and different explants regeneration in vitro, which purpose is to construction of a rapid propagation system of large-scale propagation for Aconite. The research results can be listed as follows:1. propagation system:(1) Stem propagation: The second stem with axillary bud was explant for sterile culture. We explored the best conditions of stem segment propagation system by the following tests: the disinfection test 、 adventitious bud induction 、 subculture multiplication and rooting test. In the disinfection test, the best disinfection method was that infused alcohol for 50 s and followed infusion with 0.1% Hg Cl2 for 10 min, and under this condition, the contamination rate was 27.78%. In the test of adventitious bud induction, the MS culture medium contained 2 mg·L-1 6-BA and 0.3 mg·L-1 NAA showed the best inductivity of 86.67%. In the tests of proliferation conditions: the best hormone combination was 2 mg·L-1 TDZ and 0.3 mg·L-1 NAA and the proliferated rate was 100%、 proliferated coefficient was 4.029; In the sucrose concentration test, proliferated effect was the best when the concentration of sucrose was 30g·L-1, and the proliferated coefficient was reached to 4.364; In the tests of subculture cycle and subculture time, adventitious buds cultured in the propagation medium for 40 days and after the second subculture proliferated the best and the proliferated coefficient was reached to 8.1. The plantlets, that cultured in the 1/2 MS contained 0.5 mg·L-1 IBA, rooted best, and the rooting rate was 100%.(2) Sterile culture system of seeds: The seeds were explant for sterile culture. We explored the best conditions of seed propagation system by the following tests: the disinfection test、adventitious bud induction、subculture multiplication、 rooting test and transplanting test. In the disinfection test, the disinfection effect was the best, when we used sodium hypochlorite of 10% for 30 min, the seed germination rate was reached to 92.3%, and the contamination rate was 5.60%. So sodium hypochlorite of 10% was suitable for aconite seed disinfection. But 0.1%Hg Cl2 was not suitable for aconite seed disinfection. In the bud induction test of seeds: the best hormone combination was MS+ 2 mg·L-1 6-BA+0.1 mg·L-1 NAA, which showed the best inductivity of 75.1%; In the tests of reagents soaking and soaking time, the germination rate of 97.50% was the highest, when treated seeds with 400 mg·L-1 gibberellin and soaking 12 h. In the proliferation test, the buds’ proliferated effect was the best, when seedlings were cultured in MS medium contain 0.5 mg·L-1 TDZ and 0.1 mg·L-1 NAA and the proliferated coefficient was 2.23. Seedlings were cultured in the 1/2 MS contained 0.1 mg·L-1 NAA. The rooting rate was 88.3% and the number of roots was up to 20. In the transplanting medium test, the suitable proportion of transplanting medium with nutrient soil and sand was 1:1. The seedlings survival rate was up to 100% and plantlets grew well.(3)It is needed 120 days from a stem to seedlings(7.02) in the stem propagation. It is needed 101-120 days from a seed to seedlings(1.92) in the seed propagation. So in the same time, stem propagation can get more seedlings.2. Establishment of regeneration system(1)The stem and adventitious root were explant for sterile culture. We explored the best conditions of dedifferentiation regeneration system by the following tests: the callus induction、proliferation and differentiation. In the callus induction test: the better culture media combination for the inductive stem callus tissues was MS+4 mg·L-1 2,4-D+0.1mg·L-1 NAA+2 mg·L-1 6-BA +3 mg·L-1 KT; And the MS+2.5 mg·L-1 6-BA+0.1 mg·L-1 NAA had the best induction effect with induction rate of 100% for adventitious root. MS+2.5 mg·L-1 6-BA+0.1 mg·L-1 NAA had the best proliferated effect with the largest growth content of 2.5g. In the differentiation test, the MS+2 mg·L-1 6-BA+0.2 mg·L-1 NAA had the best differentiation effect with differentiation rate of 61.54% and average number of buds of 2.23.(2)We explored the best conditions of dedifferentiation regeneration system by adventitious bud induction test and aseptic leaves were explant. In the double factor experiment of TDZ and NAA, the 4 mg·L-1 TDZ+0.3 mg·L-1 NAA had the best inducted effect with the induction rate of 92.30%. While the explants browned seriously and most of them were to be callus. The regenerated adventitious buds were deformity and grew slowly. So the medium combination of TDZ and NAA were not suitable for regenerating adventitious bud. In the double factor experiment of 2,4-D and 6-BA, the MS+2 mg·L-1 6-BA+1 mg·L-1 2,4-D had the best induction effect, that the average number of buds was 3.167 and the capillary root induction rate was only 12.50%.(3)It is needed 90 days from a stem to adventitious buds(3.87) in the way of bud by stem callus. It is needed 90 days from a root to adventitious buds(9.14) in the way of bud by root callus. It is needed 20 days from a leaf to adventitious buds(14.55) in the way of bud by leaves. So in the shortest time, the way of bud by leaves can get most buds.3. The technology roadmap of seedlings propagationBuds induced by leaves will cultured to rooting seedlings, and we will use its roots to induce callus. Then callus will be proliferated and differentiated to adventitious buds. Finally, buds will be proliferated and rooted to seedlings. In the ideal state, after 300 days, a leaf can produce 91615.4 seedlings in the tissue culture.In a word, this study researched main influence factors in the culture stage of stem propagation, seed propagation and regeneration system. We selected optimum conditions in the culture stage and significantly improved the bud proliferated coefficient and the seedling survival rate. Therefore, this study solves the seedling breeding problems on the market and provides technical support to industrial seedling.