| Premna puberula Pamp.is an upright or climbing shrub to small tree belonging to the Premna Linn.,Verbenaceae.It plays an important role in medicine and food.Due to the scarce resources of this species and the difficulty of seed germination under natural conditions,it is urgent to solve the seedling breeding technology（especially the tissue culture rapid propagation technology）to meet the needs of industrialization.In this paper,the seeds,twigs and stems of P.puberula were used as the materials to break down the barriers to seed germination,optimize the cutting propagation technology,and construct the tissue culture and rapid propagation system,in order to provide the basis for the establishment of the seed and seedling production technology for the industrialized development of this species.The main findings are as follows:（1）Seeds germination:Based on the preliminary study of seed germination characteristics,it was found that the seed was covered with hard seed coat,which made it extremely difficult for the radicle and germ to break through the physical barrier and inhibit the germination.The seeds were sterilized with alcohol for 45 s,then with 0.1%HgCl2 for 8min;After sterilization,the seeds were washed by sterilized water three to five times.The seeds were placed onto MS medium(6-BA 2.0 mg·L-1 and GA3 0.5 mg·L-1),then cultured 3days at darkness,and then cultured at light condition（2000 lx）.This method can effectively solve the problem of difficult natural germination of seeds with the germination rate was up to82.83%.（2）Optimization of cuttage propagation:Preliminary research in the laboratory found that when plant growth regulator were soaked cuttings,the growth regulator concentration and soaking time needed to be optimized.In this experiment,single factor and orthogonal experiments were conducted to improve the survival rate of cutting propagation.The results showed that the biennial three-node branch was used as the material,soaked in NAA 100mg·L-1 and IBA 250 mg·L-1 solution for 70 min,the survival rate reached 95.6%,the longest root system was 14.65 cm,and the maximum number of roots is 36.2,with strong growth potential.（3）Tissue culture rapid propagation technology:In order to construct tissue culture system of rapid propagation,P.puberula young branches were chosen as experimental material,the effects of different basic media,plant growth regulators,carbon source,light on different growth stages were studied.The specific results are as follows:（1）Callus induction and proliferation:The formula of callus induction and proliferation was optimized by single factor and orthogonal test screening of different combinations and concentrations of plant growth regulators.The suitable medium for callus induction and proliferation was B5+NAA 0.5 mg·L-1+6-BA 2.0 mg·L-1.Under this condition,the callus showed light yellow and good growth,the induction rate was 85.33%.The proliferation medium of callus was B5+NAA 0.5 mg·L-1+2,4-D 1.0 mg·L-1,and the proliferation factor was 7.46.（2）Embryoid induction:The appropriate medium for embryoid induction was selected by orthogonal test with different combinations and concentrations of plant growth regulators:6-BA 1.0 mg·L-1+NAA 0.5 mg·L-1+KT 0.5 mg·L-1.An individual callus tissues may induct embryoid bodies to 17.2 with the average number.（3）Embryoid differentiation into seedlings:Taking the formed embrae-shaped body as the experimental material,through single factor and orthogonal test of different combinations and concentrations of plant growth regulators,the highest induction rate was 82%under optimized medium which was B5+6-BA 1.0 mg·L-1+NAA 0.2 mg·L-1+KT 0.5 mg·L-1.（4）Strong seedlings:Using the above test-tube seedlings as materials,a suitable medium for screening seedlings was selected as B5+NAA 1.0 mg·L-1+6-BA 0.2 mg·L-1.（5）Rooting:Through the different basic culture medium and the different growth regulator ratio test screening,the optimum rooting medium was 3/4 MS+6-BA 0.2mg·L-1+IAA 0.6 mg·L-1,and the induction rate was 97.67%.（6）Refining seedlings and transplanting:In order to study the effect of time and different mixing ratio of transplanting medium on the survival rate of tissue culture seedlings,the asymbiotic seedlings were used as materials.The results showed that more than 80%of the transplanted plantlets can survive under the condition that the ratio of peat,perlite and vermiculite was 2:1:1.Comparing the three different breeding methods,the tissue culture fast propagation biotechnology is the most potential and effective technology for the industrialization of the P.puberula seedlings industrialization. |
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