| Murraya paniculata(L.)Jack belongs to the Murraya Genus of Rutaceae family which has drug effects of anti-inflammatory and analgesic,hypoglycemic,anti-fertility,anti-tumor and others.Now the M.paniculata is one of the main raw materials of Chinesse medicine"999 Weitai".In order to produce the Chinese medicine "999 Weitai",China Resources Sanjiu Pharmaceutical Co.,Ltd.needs to consume about a few thousand tons of M.paniculata shoots per year.These shoots should be collected from about 100 million thousands of plants,which covers 200 hectares of land according to the yield of per unit area.It is said that only a few tens hectares of M.paniculata were planted at present by the China Resources Sanjiu Pharmaceutical Co.,Ltd..So the wild plants of M.paniculata are the main source for the materials of "999 Weitai".For the wild plants of M.paniculata,the yield is unstable and the drug effect is not uniform,so to establish an artificial cultivation base for M.paniculata is necessary and urgency.To establish an artificial cultivation base for M.paniculata,should be produced first.At present the seedlings of M.paniculata is mainly from seeds.Unfortunately,M.paniculata flowers less in the wild that resulting in few seeds can be collected,which limits the scale production of M.paniculata seedlings.The technique of tissue culture is a plant biotechnology developed in the early nineteenth Century.Explants can regenerate to whole plants under controlled conditions by using tissue culture technique.The regenerated plant comes from ways:one is fast propagation way that through the direct organogenesis;another way is through callus induction and differentiation.Technique conducted in the first way is simple and the regenerated plants scale up quickly;conducted in the second way is relatively difficult,but the propagation coefficient is higher remarkably than that of first way.ObjectiveThe technology of tissue culture of M.paniculata can contribute to the industrial production of seedlings and lay the foundation for new germplasm study.Tissue culture of M.paniculata was studied in this paper through the two aspects of direct organogenesis and indirect organogenesis.MethodsThe disinfection method,the stem initiation culture,subculture and root regeneration of M.paniculata were studied in this paper.The explants include stem segments from field plants and sterile seedlings germinated from seeds.In addition,the callus induction was studied by using the leaves of field plants and sterile seedlings and sterile root segments as material.The most suitable explants and callus induction formula were selected by conducting orthogonal design experiment.Results1 The results of rapid propagation of M.paniculata.1.1 The disinfection effects of different methods on stems from field plant of M.paniculata were studied.1.2 The disinfection effects of different methods on seeds of M.paniculata were studied.1.3 The effects of different plant hormones and concentrations on the axillary bud germination of M.paniculata were studied.1.4 The effects of different plant hormones and concentrations on the induction of cluster buds of M.paniculata were studied.1.5 The effects of plant hormones and concentrations on the induction of cluter buds of stem segments from sterile seedlings were studied1.6 The effects of different plant hormones and concentrations on Rooting of M.paniculata were studied.2 The results of callus induction,subculture and proliferation culture of M.paniculata.2.1 The disinfection effects of different methods on leaves from field plant of M.paniculata were studied.2.2 The effects of different plant hormones and concentrations on the callus induction of sterile seedling leaves of M.paniculata were studied.2.3 The effects of different plant hormones and concentrations on the callus induction of sterile seedling roots of M.paniculata were studied.2.4 The effects of different induction media on the eallus proliferation culture were studied.Conclusion1 The results of rapid propagation of M.paniculata.1.1 The best sterilizing condition for explants of stem segments from field plants of M.paniculata was studied.That explants were soaked in 1%sodium hypochlorite solution(adding drops of Tween-20)for 15 min,and then immersed in 0.1%HgCl2 solution for 10 min was the best operation procedure for disinfection.The contamination rate of explants disinfected was 21.67%,the browning rate was 10.00%,and the survival rate was 68.33%.1.2 The best way for M.paniculata seeds sterilization was also studied.Mature seeds were disinfected with 75%ethanol for 1 min,and then 0.1%HgCl2(adding drops of Tween-20)8 min.The contamination rate of seeds disinfected was 12.3%,the survival rate was as high as to 93.43%.1.3 The best medium for setm buds germination of field M.paniculata is that contains two kinds of cytokinins and auxin.The best formula is the MS+6-BA 1.0 mg/L+KT 1.0 mg/L+ NAA 0.5 mg/L with the induction rate of 95.83%.Axillary buds germinated in the medium could grow to about 2 cm in average.1.4 The best medium for cluster buds induction of Murraya paniculata is MS+6-BA 0.5 mg/L+ KT 0.5 mg/L+ NAA 0.5 mg/L,with the regenerated bud number of 5.61 in average.The material used for cluster buds induction is axillary buds induced from stems of field M.paniculata.1.5 The optimum culture medium for induction of clustered shoots from seed sterile seedlings is the same as that for induction of axillary buds from field plnats.That is MS+6-BA 0.5 mg/L+KT 0.5mg/L+ NAA 0.5 mg/L,with an average bud number of 9.631.6 The optimum medium for rooting culture of aseptic seedlings is 1/2 MS+IBA1.5 mg/L+ NAA 0.5 mg/L.The rooting rate was 52%,the average root number was 4.58,and the average root length was 2.34 cm2 The results of callus induction,subculture and proliferation culture of M.paniculata.2.1 For stem from filed plants,the contamination rate can be reduced by sodium hypochlorite and HgCl2 solution.But for leaves from field plant,the best disinfection condition is 75%ethanol immersing for 30 s,and then 0.1%HgCl2 solution(adding drops of Tween-20)soaking for 12 min.The contamination rate of 22.22%was the lowest;the browning rate was low to 4.44%;the survival rate was up to 73.33%.2.2 Calli can be induced in all 18 media designed from the leaves of the sterile seedling.Calli appeared earlier when the media contained 2,4-D,but if the 2,4-D concentration was too high,the calli appeared serious browning.When the media contained NAA,the calli appeared later and the induction rate was relatively low,but the browning rate was also low.The experimental results show that the induction rate is relative higher in the medium with the ratio of auxin and cytokinin less than 2.The highest callus induction rate was formula 2 of MS+2,4-D 0.5 mg/L+6-BA 0.5 mg/L.2.3 Calli can be also induced succeed in the all 18 media above from roots of aseptic seedling.The callus induction rates have a larger difference among each media.The highest induction rate was 100%appeared in the medium 17 of MS+NAA 3.0 mg/L+ 6-BA 0.5 mg/L.The next highest induction rate was 97.22%appeared in the medium 14 of MS+NAA 2.0 mg/L+ 6-BA 0.5 mg/L.,It had a high induction rate in medium 2 for leaves,but for roots the induction rate was only 63.27%.In addition,there was no particularly large difference about callus appearing time between media containing 2,4-D and NAA.The browning rate of calli vas low in all 18 media,especial for media 10 and 17,there were no browning in root calli.Not like the results of leaves,callus induction rates were low in medium 2,3,6 and 12 when the the ratio of auxin and cytokinin is less than or equal to 1.Therefore,the optimum medium for callus induction from root segments is MS+NAA 3.0 mg/L+6-BA 0.5 mg/L.2.4 Callus growth conditions were difference between leaf callus and root callus after they were inoculated into the proliferation media.For leaf calli,they were browning and died subsequently in the media of 7,8 and 9;they were mostly white green and compact in the media of others.In addition,shoots was regenerated in the proliferation medium of 2 which was the same as callus induction medium.For root calli,the proliferation rate was great significantly,and they were yellowish white with good texture. |
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