| Lonicera japonica Thunb. is one of perennial woody vine and belonging to Lonicera genus, Caprifoliaceae family, its main medicinal ingredients are chlorogenic acid and isochlorogenic acid, which have effects on anti-bacterium, anti-inflammation, anti-virus, anti-tumor, immunoregulation and so on. Lonicera japonica Thunb. is a famous authentic Chinese herbal medicine, which is major in Fengqiu city of Henan province. Because of the single species, serious degradation and the low speed of propagation, there is no ways to satisfy the need of large scale planting and market. In order to solve the above problems, we studied in vitro culture of improved variety"Jin Feng No.1"for the first time systemicly. The results were as followed:1. The technological system of asepsis culture had been established. Firstly, to dip the apical buds with two leaves from farmland into the ethanol of 75 % for 30 s, secondly, to put them in the HgCl2 of 0.1 % for 15 minutes, thirdly, to wash them in the germfree water for five to six, finally, to inoculate them on MS+6-BA 1.0 mg·L-1+NAA 0.2 mg·L-1. According to these steps, we could get plantlets after they were cultured for 30 days, and the yield of vaccine is up to 78.90 %.2. The techology system of rapid propagation had been established. The best medium for rapid propagation of Lonicerajaponica Thunb.is MS+6-BA 0.5 mg·L-1+NAA 0.1 mg·L-1+biotin-D 2 mg·L-1 with agar in can bottle of 500 ml, and the propagation coefficient could reach 7.37 by 28 days. The best rooting medium was 1/2 MS+IBA 3 mg·L-1+ AC 0.2 g·L-1+ sugar 15 g·L-1, and with reasonable managements the survival rate of transplantation could reach to 100 %.3. The callus induction and the best conditions of their subculture had been probed into. The best explants were buds, secondly were leaves, and the best induction medium was MS+NAA 2 mg·L-1+6-BA 0.01 mg·L-1+2,4-D 0.5 mg·L-1, the inducing ratio could reach 100 %; The best subculture medium for callus is MS+6-BA 2 mg·L-1+KT 0.75 mg·L-1+NAA 0.25 mg·L-1, in which the callus could be classified into four types after subcultured for three to five generations.4. The techology system of cell suspension culture had been established. Typeâ… which is yellowy, loosen grain and rapid growth was used to establish the suspension culture system in the liquid medium MS+6-BA 1.5 mg·L-1+KT 0.75 mg·L-1+NAA 0.5 mg·L-1: The S - like growth curve of suspension culturing cell was divided into three phases: lag phase (0-12 d ), exponential phase (12-18 d ) and lentamente phase (18-24 d ), cell dry weight was upward trend during the incubation period and it reached a maximum of 8 g·L-1 at 18 days.5. The rules of physiology and biochemistry had been researched during cell suspension culture. (1) During the incubation period the content of soluble sugar declined, raised, then declined, raised, the period of reaching peak was 18 days, it was the same as that of growth cruve doing; (2) The content of soluble protein declined firstly, then raised, and at last declined. Soluble proteins contents reached its peak value at 12 days, 6 days earlier than that of the cell growth peak; (3) POD and SOD activities all increased firstly, and then decreased. 6 days earlier than that of the cell growth peak; (4) pH of the medium dropped firstly and then raise slightly, and at last gradually leveled off and maintained about 5.14 after being cultured for 12 days; (5) Conductivity of the medium presented a very slow upward trend, then rapidly declined and at last raised slightly, it presented certain relativity with cell growth; (6) The content of chlorogenic acid had change of two peaks, that is, raised firstly, then declined, raised followly and at last declined. The period of two peaks was 6 days, 21 days respectively.6. Lonicera japonica Thunb. chromosome preparation technology has been primary Studied: the root was used for the material, at the room temperature, they were treated with 0.1 % colchicine for 4 h in the dark, then, they were dissociated for 3 min under the conditions of 65℃in a water bath with 1 mol·L-1 HCl, at last, they were stained for 4 h in Carbol fuchsine, and the effect was better.The results of this paper were with a view to further carry out new varieties generalizing and rapid propagation in vitro through biotechnology and industrial production based of the effective officinal ingredients. |
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