The Research On Hosta Tissue Culture And The Contamination Control Of Entophytes In Test-tube

We chose terminal buds of French hosta as our experiments materials. We carried out the experiments of exogenous hormone and culture medium effects on hosta buds primary induction, subculture multiplication, rooting and acclimatization transplanting under different species and concentration. Then the most appropriate formula at different stages was picked out. Meanwhile, analyzing the contamination phenomenon of hosta endophyte together with separation identification and drug sensitive test, we picked out the formula and the antibiotic species which can reduce the endophyte contamination. Major results were described below:1. Establishment of hosta tissue culture systemIn the inducing buds process, the most appropriate position of exophyte is terminal buds. The inductivity can reach 62% and the contamination ratio can be very low. The most appropriate time is 16 min which treating the exophyte by mercury dichloride to disinfect. The best culture medium formula for primary induce culture is MS+6-BA2.0mg/L+NAA0.1mg/L.In the subcultured bud multiplication step, the most appropriate culture medium formla for adventitious buds is MS+KT0.5mg/L+6-BA1.0mg/L+NAA0.2mg/L, and the growth coefficient can reach 4.8. Comparing F values of three hormones, we can confirm that the 6-BA is the major factor that influence the adventitious buds subculture multiplication. The impact on adventitious buds is 6-BA> KT>NAA. We can get best proliferation results by transplanting 2-3 calluses, adjusting the medium pH to 6.0, experiencing 12 h lighting with the light intensity of 20001x and making subculture periods 40d within 15 times. When optimizing the experimental scheme, we use sugar instead of sucrose, tap water instead of distilled water, in order to simplify the experimental procedure and reduce the experiment cost.In the rooting culture process, we picked out the optimal medium formula for hosta rooting culturing that is MS+6-BA0.5mg/L+NAA0.2mg/L+AC200mg/L.In transplanting test-tube buds, the most appropriate training time is two days after open the bottle cap in the intelligent room, then transplanting into the turf and perlite medium with the ratio of 3:1. The survival rate can reach as high as 98.47% and the plant grow well.2. Research on contamination control of hosta test-tube bud endophyteIn the separation identification and drug sensitive experiments for endophyte, we use the BITEK-2-Compact 15 automatic microorganism authenticate system to confirm that the contamination sources in our experiments is the Micrococcus Kristinae the reliability is above 99%. This bacteria has different degree of tolerance to streptomycin and lincomycin, however, gentamicin, amoxicillin and tetracycline and so on 13 kinds antibiotic drug have good inhibition to Micrococcus Kristinae.Adding antibiotic can reduce the contamination of hosta test-tube buds. We immersed the contaminated test-tube buds into 100mg/L penicillin and cefazolin for 30 min, the results showed the antibiotic ratio are 46.2% and 49.62% respectively. When adding 400 mg/1 penicillin into the culture medium, the antibiotic rate can reach 73.33%, and the rooting rate can reach 98.6% after 30d and the plants grow well. Adding 800 mg/L cefazolin, the antibiotic ratio can reach 76.67%, and the rooting rate can reach 97.8% after culture 30d, and the plant grow well. The antibiotic rate of control group without any further treatment is 0, the leaf on the bud is yellow and wilting, and the plant grow slow.