Establishment Of A Rapid Propagation System Of Dioscorea Nipponica Makino And Study On Its Content Of Medicinal Components

Dioscorea nipponica Makino is a perennial twining vine of Dioscoreaceae, Dioscorea L,which has high medicinal value.In recent years the wild resources is destroyed,and the traditional breeding methods are limited by resources,seed dormancy,low germination rate and other reasons,resulting in the difficulties in artificial propagation and cultivation.Therefore,the stem segment of Dioscorea nipponica Makino was used as the test material to induce redifferentiation and produce a large number of clumps of shoots,and then the proliferation and root culture of Dioscorea nipponica Makino were carried out,and exploring the domestication condition,so as to establish a complete and efficient rapid propagation system of tissue culture of Dioscorea nipponica Makino.The medicinal components in the roots were determined to provide theoretical basis for further research and utilization of Dioscorea nipponica Makino. 1.The survival rate was significantly affected by different explants and disinfection methods.Stem segments were selected,and the highest survival rate was 82.7%when disinfected with 75%of alcohol for 30 seconds and 0.2%of mercuric chloride for 2 minutes. 2.The effect of axillary bud induction was significantly different with different culture media.Using MS basic medium,the induction rate was the highest when 2.5 mg·L-¹ of 6-benzylamine adenine,0.5 mg·L-¹ of 2,4-dichlorophenoxyacetic acid were added,reaching 90.8%,the plant height was 3.8cm,the fresh weight was 1.5g,and the dry weight was 160.9mg. 3.In the process of proliferation,it was found that different kinds and concentrations of cytokinin affected obviously the multiplication culture of Dioscorea nippomica Makino,when 6-BA is 3.0 mg·L-¹ the plant fresh weight and dry weight is heavy,that is 1.5 gand 133.5 mg,proliferation coefficient is 7.8,the plant height is 3.4 cm,superior to other similar cytokinin concentration;At the concentration of 0.75 mg·L-¹,the auxin species was NAA,and the plant growth reached the optimal state. The plant height was 2.7cm,the fresh weight was 1.5 g,and the dry weight was 133.6 mg.When the sugar source was explored,it was found that the selection of sucrose and the addition of 40 g·L-¹ was most conducive to the proliferation and growth of plants. When the light intensity was investigated,it was found that the light intensity of 24001x was a relatively appropriate light intensity,and high or low light intensity would inhibit the proliferation of plants.The results showed that the medium MS that add 3.0 mg·L-¹ of 6-BA,0.75 mg·L-¹ of NAA,40 g·L-¹ of sucrose,5 g·L-¹ of agar had the best proliferation effect under 24001x light,the proliferation coefficient is 8.4,average plant height is 5.1 cm,fresh weight is 1.3 g,and dry weight is 143.2 mg. 4.Rooting rate of Dioscorea nipponica Makino in 1/2MS medium is 90.0%,fresh weight is 318.6 mg,dry weight is 27.4mg,number of fibrous roots is 14.0,number of rhizomes is 2.5,higher than MS medium of other concentrations.When IBA and NAA were added for rooting culture,the rooting effect of NAA is due to IB A.When 1.5 mg·L-¹ ofNAA was added,the fresh weight is 359.4 mg and the dry weight is 31.2 mg, the number of fibrous roots is 16.1,the number of rhizomes is 3.6.When 0.4 g·L-¹ of activated carbon was added,the fresh weight of the plant is 380.1 mg,the dry weight is 31.7 mg,the number of rhizomes is 4.3 and the rooting rate is 98.3%,reaching the maximum.When sucrose was added,it is beneficial to plant rooting.As the concentration increased,the fresh weight,dry weight,number of fibrous roots and number of rhizomes increased fist and then decreased.When sucrose is 50 g·L-¹,the maximum number of fresh weight,dry weight,number of fibrous roots and number of rhizomes reached 417.4 mg,35.7 mg,23.3 and 5.0,respectively. 5.Different concentrations of MS medium,auxins,sucrose had certain effects on the accumulation of medicinal components in tissue culture roots,including dioscin. protodioscin,total flavonoids and polysaccharides.When MS medium was used,the contents of dioscin,protodioscin,total flavonoids and polysaccharides are 749.0?g·g^-1. 1200.0 ?g· g^-1,5.5mg·g^-1 and 26.6 mg·g^-1.The contents of dioscin,protodioscin,total flavonoids and polysaccharides are 882.4 ?g· g^-1.1241.2 ?g· g^-1.7.3 mg·g^-1 and 24.4 mg·g^-1,respectively,when the NAA was 2.0 mg·L-¹.When sucrose was 50 g·L-¹ which is beneficial to the accumulation of medicinal components in the tissue culture of Dioscorea nipponica Makino,the content of each component is 751.3 ?g·g^-1,1340.3?g·g^-1,7.8 mg·g^-1,26.9 mg·g^-1. 6.In transplanting experiment,the mixed matrix contained peat and perlite.When the volume ratio was 1:1,the survival rate is 81.3%.Choosing the somaclone which have a combination of fibrous roots and rhizomes,the hidtest survival rate is 90.2%.