Construction Of The Technical System Of In Vitro Rapid Propagation Of Pueraria Thomsonii Benth

In this paper,the Pueraria thomsonii Benth plants were used as experimental materials to study the establishment of regeneration system of axillary bud,organogenesis and somatic embryo regeneration system.By comparing the efficiency and difficulty of regeneration of tissue culture seedlings of Pueraria thomsonii Benth in these three regeneration routes.It is clear that the regeneration route suitable for the construction of the rapid propagation system of the Pueraria thomsonii Benth.At the same time,on this basis,we also systematically studied the main influencing factors of the three regeneration pathways to induce the tissue culture seedlings of Pueraria thomsonii Benth and the main findings are as follows:1.Axillary bud regeneration.Young shoots of Pueraria thomsonii Benth as explants,Study the effect of sterilizer and surfactant on the sterilization effect of Pueraria thomsonii Benth;The young stem segments of Pueraria thomsonii Benth were used as test materials to study the effects of different explant parts,basic medium,6-BA concentration,NAA concentration and ratio on the induction of axillary buds of Pueraria thomsonii Benth;using aseptic tissue culture seedlings of Pueraria thomsonii Benth as materials,the effects of four factors,basic medium,cytokinin,auxin and organic additives,on the induction of cluster buds of axillary buds of Pueraria thomsonii Benth were studied;the axillary bud or clump bud of Pueraria thomsonii Benth was used as the material to study the effect of two factors,basic medium and hormone ratio,on the subculture.The results showed that the suitable for sterilization of Pueraria lobata explants:0.1%mercury+Tween-80;the explants suitable for inducing axillary buds of Pueraria thomsonii Benth were the second segmented stem segments and the medium suitable for inducing axillary buds of Pueraria thomsonii Benth was MS+6-BA0.5 mg·L^-1+NAA0.2 mg·L^-1+sucrose30 g·L^-1+agar 7 g·L^-1;the effect on the induction rate of bud buds of Pueraria thomsonii Benth is cytokinin>auxin>basic medium>organic additives,where TDZ has a significant effect on the induction of axillary buds;The medium suitable for inducing cluster buds in the axillary buds of Pueraria thomsonii Benth is MS+TDZ1.5 mg·L^-1+NAA0.1 mg·L^-1+CH200 mg·L^-1+sucrose30 g·L^-1+agar 7 g·L^-1;The medium suitable for the subsequent generation of tissue culture seedlings of Pueraria thomsonii Benth is MS+IBA0.2 mg·L^-1+6-BA1.0mg·L^-1+TDZ0.5 mg·L^-1+KT1.0 mg·L^-1+sucrose30 g·L^-1+agar 7 g·L^-1.2.Organogenesis research.Pueraria thomsonii Benth plants were used as experimental materials to study the effects of explants,basic medium,6-BA concentration,NAA concentration,light and temperature conditions on the induction of Pueraria thomsonii Benth callus;Pueraria thomsonii Benth callus was used as the material to study the effects of TDZ concentration,NAA concentration and CH concentration on the induction of adventitious buds of organogenesis.The results showed that the explants suitable for the callus induction of Pueraria thomsonii Benth were young stems（including nodal and nodal stem segments）;the medium suitable for callus induction by Pueraria thomsonii Benth explants is 1/2MS+6-BA0.7 mg·L^-1+NAA0.1 mg·L^-1+sucrose15 g·L^-1+agar 7 g·L^-1;the light and temperature conditions are 1000 Lx light and 25℃temperature in the early stage（first 14 days）,and 2500 Lx light and 25℃temperature in the late stage（after 14 days）;the medium for inducing organogenesis adventitious buds is 1/2MS+TDZ2.0 mg·L^-1+NAA1.0 mg·L^-1+CH50mg·L^-1+sucrose15 g·L^-1+agar 7 g·L^-1.3.Somatic embryogenesis research.Pueraria thomsonii Benth callus was used as a test material to study the effects of basic medium,inoculum amount,sucrose concentration,and hormone ratio on the proliferation factor of suspension cultured cells;using the Pueraria thomsonii Benth cell mass obtained by suspension culture as a test material,the cell mass was inoculated into adventitious bud medium for cultivation.The results show that the medium suitable for suspension culture of Pueraria thomsonii Benth cells is MS+2,4-D1.5 mg·L^-1+NAA1.0 mg·L^-1+KT0.2 mg·L^-1+sucrose 50 g·L^-1,The initial inoculation volume suitable for suspension culture of Pueraria thomsonii Benth cells is 0.2 g;through cultivation studies,this experiment did not succeed in obtaining regenerated plants for somatic embryogenesis.4.Research on rooting and domesticated tissue culture seedlings.Using tissue culture seedlings of Pueraria lobata as test materials,the effects of basic culture medium,organic additives,hormone ratio and support on rooting of tissue culture seedlings of Pueraria lobata were studied;the rooting seedlings of Tissue Culture of Pueraria thomsonii Benth were used as experimental materials to study the effects of different substrates on the transplanting of Tissue Culture of Pueraria thomsonii Benth.The results showed that the medium suitable for rooting of Pueraria thomsonii Benth tissue culture seedlings was 1/2MS+NAA1.0 mg·L^-1+IBA0.5 mg·L^-1+6-BA0.5 mg·L^-1+banana40 g·L^-1+sucrose15 g·L^-1+agar 7 g·L^-1,NAA has a very significant influence on the rooting of the tissue culture seedlings of Pueraria thomsonii Benth.The support suitable for the rooting of the Pueraria thomsonii Benth tissue culture seedlings is vermiculite;the substrate suitable for the transplanting of the Pueraria thomsonii Benth tissue culture seedlings is:humus:vermiculite:organic fertilizer=2:1:1.