| Anoectochilus roxburghii (Anoectochilus roxburghii (Wall.)Lindl.)is one of the traditional Chinese herbals. It has medicinal, tonic and ornamental value. Its origin in our country, locates in 9 provinces along southeast and southwest coastal places, including Fujian, Guangxi and Taiwan. Because of the stringent requirements on the growth environment, low seed germination rates and slow growth of A. roxburghii, tissue culture techniques are more needed for its seedling production. In the study, the wild A. roxburghii in Mountain Baima of Fujian Province was selected as the object material to optimize the techniques of its industrialized tissue culture and artificial cultivation, and the genetic stability and diversity of tissue cultured seedling among different generations and wild seedlings among different places in Fujian Province was analyzed through ISSR method. At last, the study compared the differences of polysaccharides and total flavonoids among those A. roxburghii which came from different origins. The main results were as the follows:1 The difference about the wild seedlings of A. roxburghii between Mountain Baima in Fujian and other OriginsIn this experiment, the distinctiveness between wild A. roxburghii in Mountain Baima of Fujian and other places in the provinces was analyzed according to the external shape, genetic diversity by ISSR and the contents of polysaccharides and total flavonoids, we can determine the unique distinctive of those.(1)Through comparison of the A. roxburghii from Mountain Baima in Ningde with that in Nanping, Sanming, Longyan, Quanzhou of Fujian Province, and Guangxi, Guangdong, Guizhou of other provinces, we can find that the A. roxburghil’s leaves which originated from Mountain Baima in Ningde were larger and more heart-shaped, with red and sparse veins. Its leaf was of medium size, with smooth margin.(2)The polysaccharide and total flavonoids of the wild seedlings in A. roxburghii were extracted, including Mountain Baima in Ningde, Nanping, Sanming, Longyan, Quanzhou of Fujian Province, and Guangxi, Guangdong, Guizhou of other provinces. The results showed that the polysaccharide of Fujian Province’s was higher than that in some other provinces. Among them, the highest content of polysaccharides was found in A. roxburghii from Mountain Baima in Ningde, and then Quanzhou, the lowest in those from Guangxi province. In the total flavonoids, it was found that the wild A. roxburghii from Longyan and Guangdong tied for first, and then those from Mountain Baima and Sanming tied for second, with no significant differences.(3)DNA of the wild A.roxburghii seedlings from 5 main areas were extracted, including Mountain Baima in Ningde, Nanping, Sanming, Longyan and Quanzhou. On the basis of screened 11 universal primers, ISSR-PCR amplification program was made and we totally got 100 clear bands, including 72 polymorphic bands, accounting for 72% of the total. The A. roxburghii from Mountain Baima in Ningde was used as a standard for comparison to calculate the mutation rate of A. roxburghii in other origins. Then we could find the mutation rate from the wild A.roxburghii in Quanzhou was 50.00%, in Longyan was 62.50%, in Sanming was 58.33%, and in Nanping was 60.42%. According to the clustering analysis, the genetic similarity coefficients about the wild seedlings in different areas of Fujian Province were between 0.58-0.92, which meant the great genetic distance and the rich diversity were among them.2 The study on main techniques of micropropagation about tissue culture in A. roxburghii from Mountain Baima of FujianThe wild seedlings of A. roxburghii from Mountain Baima in Fujian were used as the materials. At first, the sterile-plantlets system was established, and the effects of different treatments on proliferation of stem segments was studied. Then the effects were compared among different media on rooting and among different additives on best growth of A. roxburghii. Finally, effects of six LED lights on growth in A. roxburghii seedlings were compared.(1)Different soaking time of 0.1% mercuric chloride solution and the use of different parts of A. roxburghii explants during the sterilization were compared to found the best combination in sterilization. The results showed that the top of the stem segments was most suitable part in the establishment of a sterile system and the best time of sterilization was 9 min. The survival rate was 76.67% and contamination rate was 21.67%.(2)In the proliferative phase, a four-factor, three-level orthogonal test was conducted, and the factors included MS medium,6-BA and NAA with different concentrations, together with the different positions stem segments. The results showed that the central stem segments was better than the terminal bud and the stem base, and the best combination is to inoculate the central stems to the medium 1/2 MS+6-BA 2.4 mg/L+NAA 0.1 mg/L+sugar 25 g/L+ agar 6.1 g/L. The proliferation coefficient could reach 4.02.(3)Based on the 1/2MS medium, a four-factor, three-level orthogonal test was conducted to explore the effects of AC, IBA and NAA on rooting of A. roxburghii. The results showed that AC and IB A had significant impact on rooting, and the the best selected combination of rooting was 1/2 MS+AC 0.5 g/L+NAA 1.5 mg/L+IBA 1.5 mg/L+sugar 30 g/L+agar 6.1 g/L. The average root number reached 2.1.(4)The seedlings of A. roxburghii from Mountain Baima in Fujian used as the materials, based on the above screened rooting medium, the effects on the best growth of seedlings were compared among the combinations of additives sugar, mashed banana, mashed potatoes, onions and Hyponexl#. The statistical analysis was conducted by the method of assignment score, and the results showed that mashed banana played a key role in these combinations. The optimal medium was 1/2 MS+agar 6.1 g/L+NAA 1.5 mg/L+IBA 1.5 mg/L+AC 0.5 g/L+sugar 20 g/L +banana 60 g/L+mashed potatoes 60 g/L+onion 60 g/L+Hyponexl# 2g/L.(5)Based on the above screened seedlings medium, single-factor comparison was made to analyze the effects of different light sources (red, yellow, blue, green, white, warm white light of LED) on seedlings growth, with the fluorescent as control. The results showed that the best effect was warm white light of LED. The red, yellow and green light could significantly increase the seedling’s height so that the seedlings appeared too leggy and thin phenomenon. The blue light benefit on the growth of seedlings and could inhibit the excessive growth. Warm white and white light were able to thrive the plants and increase the leaf area.3 The study of artificial transplanting of seedlings of A. roxburghii from Mountain Baima of Fujian ProvinceWe selected the robust plants, which were cultured for 3 months, and its height reached to 5-6 cm, with 2-3 taproots, four or more blades, for transplanting. Then comparison on the effects of different growing media, as well as a combination of broad-spectrum fungicide and different concentrations of two slow-release fertilizer were studied.(1)In the comparative study on different ratio of different substrates, it was found that the best transplanting substrate was a combination of peat:perlite:vermiculite:pine bark= 2:1:1:1, with the best seedling growth, and the survival rate could reach 100%.(2)In the optimized transplanting substrate, the transplanted seedlings were sprayed on rotation with different pairwise coupling combinations of carbendazim, chlorothalonil, mancozeb, thiophanate-methyl and procymidone, with different multiples, to compare prevention effect on disease. The best effect was found in those sprayed 1000 times of 50% carbendazim with 1000 times of 70% thiophanate-methyl on rotation, which also promote plant growth and increase yield.(3)The different applying amount of the two slow-release fertilizers, Hi·Control 1# and Osmocote 601,on the transplanted seedlings of A. roxburghii from Mountain Baima, was analyzed compared with the water as a control, to test its increment of growth index. The statistical analysis was conducted by the method of assignment score, and it showed that planted in 15-inch standard pots and sprayed with 13 g of Hi·Control 1# was the best combination for increasing yield of A. roxburghii. Its increments of the fresh weight, diameter, and height were 145.16%,400%, and 193%. Besides, those leaves were larger and more stretched, with red veins and two new leaves.4 The ISSR analyses of the genetic stability of the tissue cultured seedlings of A. roxburghii from Mountain Baima of FujianWe analysed the genetic stability by ISSR of the tissue cultured seedlings of A. roxburghii from Mountain Baima in Fujian, with 0,3r,6th and 9th generations. We used 11 universal primers to amplify the DNA of the different generations of A.s roxburghii seedlings, and totally got 96 bands, including 30 polymorphic bands, accounting for 31.25% of the total. Compared with the first generation, the mutation rate of each generation was 9.38%,10.42% and 12.50%, respectively. The genetic similar coefficient of 0-9th generations was between 0.813-0.990, which meant it had the great genetic similarity. Starting from the 3rd generation, the mutation rate gradually increased with later generations and the seedlings suffered from certain variations. However, the rise of mutation rate was not great, and its contents of main medicinal ingredients had little difference with wild seedlings’. Its genetic traits were still relatively stable so that it can be used in the production.5 Measurement and analysis on the medicinal ingredients in the tissue cultured seedlings of A. roxburghii from Mountain Baima in Fujian with other originsIn the study, the tissue cultured and transplanted seedlings of A. roxburghii in Mountain Baima of Fujian Province were used as materials, those from other origins as a control, to explore the changes in the content of main effective components (polysaccharides and total flavonoid).(l)The content of polysaccharides and total flavonoid was compared among the seedlings from Mountain Baima in Ningde, Guangxi, Jiangxi and Taiwan, tissue cultured for 3 months, coupled with the transplanted seedlings in greenhouse, cultivated for 2 and 3 months. It was found that compared to other species, the seedlings from Mountain Baima had the highest content of the polysaccharide and total flavonoids. On the whole, in the early transplanting period, the polysaccharide and total flavonoids content of all varieties are low, but their content increased with the cultivation time.(2)The A. roxburghii seedlings from Mountain Baima in Ningde, cultured 90 d under red, yellow, blue, green, white, warm white light of LED, were used for the comparative analysis of polysaccharide and total flavonoids content, with the fluorescent as control. It showed that the LED red light was best for the accumulation of polysaccharide, and the LED blue and green light were best for the increase in total flavonoids content. |
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