Studies On In Vitro Culture And Dynamic Change Of Amylose In Liriope Muscari (Desne.) Bailey

Liriope muscari (Desne.) Bailey are important garden plants, and their dry earthnuts are used to be Chinese traditional medical herbal. In this experiment, Liriope muscari (Desne.) Bailey was used as the materials for establishing the technical system of micropropagation, callus induction and subculture, cell suspension culture, and developing a meithod to distill amylose from callus and analyzing the dynamic changes of amylose in callus, and further comparing the difference of amylose contents among callus, in vitro seedlings and natural seedlings in the field. The main results were as follows:1. The establishment of micropropagation technique in Liriope muscari (Desne.) BaileyThe stem tips were cultured on the MS medium added with 2.0 mg/L BA and 0.2 mg/L NAA, and the highest differentiating rate of 90% was achieved in Liriope muscari (Desne.) Bailey. The MS medium added 4.0mg/L BA and 0.2 mg/L NAA was suitable for multiple-shoot proliferation, some certain high concentrations of BA promoted the shoot differentiation. The MS medium added the coconut milk had obviously better effect than that with the control group or the banana mud. Different sucrose concentrations did little difference in the multiple-shoot proliferation, the better sucrose concentration was 20g/L. The medium for seeding growth and rooting was 1/2MS added IBA0.5mg/L, and the root coefficient was 6.87. Peat soil was the best transplanting medium. April was the best transplanting time in Fuzhou region .2. Callus induction and plant regeneration in Liriope muscari (Desne.) BaileyAccording to the induction of callus from four different explants (stems, roots, leaves, fruits), it was found that the stems were better than others for inducting callus with the callus induction rate of 80%. The apical meristem induction rate on the medium MS+2,4-D 2.0mg/L +BA 0.1mg/L +NAA 0.5mg/L and MS +2,4-D1.5mg/L+ BA1.0mg/L+NAA1.0 mg/L was the highest, about 87.5%. The optimal medium for callus proliferation was MS+2,4-D0.5-1.0 mg/L, and the multiplication coefficient was 6.53. By comparing the multiplication coefficient, the optimal sucrose concentration was 20g/L, and the optimal water for the callus keeping was tap water by reducing cost. Because the difference between the light culture and the dark culture was small on the callus multiplication, choosing dark culture for reducing cost. The callus growth curve was S-shaped. In the process of callus culture, the moisture content in the overall was downtrend, proliferation factor uptrend.In the experimental process of callus differentiation and plant regeneration, the medium was MS without growth regulator. Different concentrations of sucrose could make the callus induction into somatic embryogenesis, the optimal sucrose concentration was 30g/L. Embryogenic callus differentiation and regeneration, the optimal medium was MS + BA2.0mg/L+ IBA1.0 mg/L + NAA0.5 mg/L, the differentiation rate was 92%.3 Establishment and maintenance of cell suspensionsThe callus from stem was incompact, the inoculation amount of callus was 0.45g. The difference among the four carbon sources (sucrose, glucose, mannitol and sorbitol) for suspension cell culture was not significant, sugar was the optimal carbon sources, and the highest growth rate was 252%,and sugar was the cheapest. The line of growth on suspension cells was approximately S-shaped. Suspension cultured cell needed 10d to achieve the optimization.4 Extraction process of polysaccharide and the dynamic change of amylose in callusCallus in drying box at 60℃to be constant weight, plused petroleum ether, detreated in 65℃water bath pot for 30min, dried solvents, plused 12 times the water and extracted for 2 times, one time for 30min, and percipitaion with 90% alcohol. Standard curve was y=215.78x+0.7977, R2 =0.9964.Taking 0.4mL sample solution of polysaccharide in callus into dry test tube, adding 5% phenol solution 0.8mL, shake 4.0mL concentrated sulfuric acid, after 30min colored, 490nm measured absorbance. The content of callus polysaccharide in the whole culture increased as the days went by. But the callus polysaccharide content would approach to be stability if cultured more than 60 days. The polysaccharide content of callus after 40d-60d would be better, at the same time the growth rate of callus reached the highest. Polysaccharide content in root from in vitro plants was higher than that grew in the field.