| In order to realize rapid propagation and nutritional components of Anoectochilus roxburghii,the study on pollination,seed germination,tissue culture,cultivation and nutrient composition experiment were performed in the academic thesis.The seed originated in Tonggu county,Jiangxi province,was served as experimental materials,and orthogonal experimental was designed using enzyme concentration,treatment time and temperature as 3 factors to study the impact of snail enzyme treatment on seed germination.Seed symbiotic germination was studied with fungi separating from the root of A.roxburghii.Protocorms proliferation and differentiation research were tested respectively using NAA,6-BA,S-3307 and TDZ,6-BA,NAA as 3 factors.Stem segments and terminal bud was served as explants,and multiple shoots induction was performed,with different concentrations and ratios of TDZ,NAA and 6-BA,NAA respectively.Root promoting experiment was completed by different concentrations of NAA and activated carbon.Growing media selection,and nutritional components comparative analysis in seedlings of transplanting 2,4,6 months were performed.The results as follows:1.The pollination rate of the first day after flowering(DAF)was higher than that of 2,3 DAF,and the pollination rate of cross-pollination was higher than self-pollination,so cross-pollination at the 1 DAF was the best way and pollination rate reached 83.61 %,while seed viability reached 98.72%.In addition,seed viability was reduced with prolonging storage time,so sowing the fresh-cut seeds could improve germination rate.The seed treated by 2 mg/mL snailase at 30 ?,40 min,and the germination experiment indicated the concentration of snailase influenced germination rate more significantly than treatment time.2.The best medium for multiplication of protocorms was MS + 0.5 mg/L S-3307 + 1.0 mg/L 6-BA + 1.0 mg/L NAA,and the multiplication coefficient was 6.17.In addition,S-3307 and 6-BA were very significant for the effect on multiplication of protocorms while NAA was significant.The best differentiation medium was MS + 0.05 mg/L TDZ + 2.00 mg/L 6-BA + 0.60 mg/L NAA,and the differentiation rate was 83.3%.Besides,TDZ,NAA was significant for the effect on differentiation.The best medium for rooting was 1/2 MS + 0.5 mg/L NAA + 0.05% Ac,and its rooting rate reached 96.5%.Sawdust combined with cow dung was the suitable cultivation substrate,for seedlings of transplanting.When sawdust and cow dung were added in a 2:1 ratio,the transplant survival rate of and gain weight were up to 91% and 0.26 g respectively.Mixed substrate combined pine bark with dung was not suit for seedlings growing.The transplant survival rate of tube seedlings was 82% under the pine forest,but growth rate was too slow.3.The best induced medium of multiple shoots was MS + 0.1 mg/L TDZ + 0.2 mg/L NAA,and the induction rate by stem segments and terminal bud was 7.74 or 3.77.The effect of TDZ for inducting was higher than 6-BA.4.35 kinds of fungi were obtained from stem segment,and one fungus could promote seed germination in the sawdust substrate.Some protocorms were dead as the extension of time,indicated that endophytic fungi promoting seeds germination and seedlings growth may not be the same kind of fungus.Across the protocorms differentiation and seedlings growth,it required the interaction of different fungus or adding nutrients for its whole process of seeds growth,which needs further study.5.The polysaccharide content of tissue culture seedling was the highest reached 126.30 mg/g,while wild was lower with 85.88 mg/g,and seedlings transplanting 2 months was the lowest only 66.38 mg/g,then gradually increased and 6 months was up to 123.09 mg/g.The flavonoids content of wild was higher with 17.56 mg/g,while tissue culture seedling was the lowest with 13.25 mg/g.The flavonoid content of transplanting seedlings changed like a parabolic,increased at first and then decreased,and seedlings transplanting 2 months was 14.89 mg/g,4 months was the highest,up to 20.32 mg/g,while 6 months was 17.03 mg/g.The main component of free amino acids was aspartic acid in all samples,at least 70% of total content.The total amino acids and essential amino acid content of tissue culture seedling were considerable high,respectively 24.872 mg/kg and 2.387 mg/kg,while that of wild seedlings were lower,respectively 6.480 mg/kg and 1.445 mg/kg.The total amino acids of seedlings transplanted for 2 months,reached the maximum with 26.605 mg/kg,but the essential amino acid content was lower,just 1.501 mg/kg.The total amino acid of 4 months was higher and the essential amino acids were the highest,respectively 25.283 mg/kg and 2.882 mg/kg.However,both of them in six months were the minimum,respectively 0.742 mg/kg and 5.034 mg/kg.The Zn content of tissue culture seedling was the highest,reached 110 mg/kg,while Cr and Pb was not detected.Fe content of the wild reached 584 mg/kg and Zn was the minimum with 61 mg/kg.Besides,Pb has reached 4.8 mg/kg.Compared with tissue culture seedlings,Fe and Cr content of seedlings transplanted for 6 months,it showed a slow increasing,respectively 162 mg/kg and 9.1 mg/kg,and Zn content was reduced to 66 mg/kg,besides,Pb content was increased to 1.0 mg/kg,but less than the wild. |
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