조직배양을 이용한 멸종위기식물 흰땃딸기(Fragaria nipponica Makino)의 대량증식 방법연구

Abstract

The study was implemented in the absence of a study on the endangered plant species of Fragaria nipponica Makino with the understanding of the urgent need for the protection of the F. nipponica species vulnerable to many visitors to Mount Halla, suppression of Sasa palmata (Bean) E.G.Camus, climate change, etc. The purpose of this study is to provide the basic data for inheritance preservation, natural habitat restoration and rendering to resources. From January, 2013 to September, 2017, the temperature and humidity of each elevation of 1,500m, 1,600m, 1,700m, and 1,800m of Mt. Halla were investigated. As a result, from December to February, all of the altitudes were found to keep below-zero temperatures; and from April, every area kept above-zero temperatures. The aboveground part of F. nipponica started to appear, and flower and bear fruit from June to fruit in July until the average temperature was the highest at 17.5℃. Soil chemical property was implemented for the soil of the natural F. nipponica habitat and that of the adjacent non-habitat area. As a result, the soil pH was the highest at pH 5.24 in the Gwaneumsa F. nipponica habitat and electrical conductivity was the highest at 1.08 ㎳/㎝ in the Eorimok F. nipponica habitat region. Organic content in the Eorimok F. nipponica habitat was 24.5%, the highest. The Seongpanak F. nipponica habitat had the available phosphate content of 28.1㎎/㎏, approximately 2 times or more higher than that in other regions. The total nitrogen content in the Eorimok F. nipponica non-habitat was 0.45%. The replaceable K in the Eorimok F. nipponica habitat was 0.24 cmol^+/㎏. The Seongpanak non-habitat was found to have 1.32 of Ca and 0.09 cmol^+/㎏ of Mg, the highest. Na of the Seongpanak habitat was 0.025 cmol+/㎏, the highest among them. As a result of Pearson correlation analysis according to SPAD value and altitude, it was found that the altitude and SPAD-502 values had a negative correlation (r=-0.272, p<0.01) at 1% significance level, indicating that the chlorophyl of F. nipponica was closely related with date and altitude. F. nipponica's runners were collected and their total polyphenol and flavonoid contents were measured. As a result, the total polyphenol content was 70.75 ㎎·GAE/g, and total flavonoid was 5.93 ㎎·QE/g. F. nipponica's DPPH radical scavenging activity was measured. It was found that at the concentration level of 500 ㎕/㎖ or higher, the DPPH radical scavenging activity was similar to that of the synthetic anti-oxidizer, After the preconditioning of F. nipponica seeds, a total of 96.1% germinated until the 43 day from sowing. In 10 days after sowing, 45.3%; 20 days, 87.3%; and 30 days, 93.4%. The average number of days until germination was 12.81 days. To find out the optimal culture medium for in vitro culture, MS culture medium, Gamborg B5 culture medium and White culture medium were experimented. The MS culture medium showed a lower D/F rate but its plant length, root length, etc. were relatively higher and its amount of growth such as leaf length and leaf width were the largest. The MS culture medium experiment results showed that, the higher the MS culture medium concentration, the fewer the number of roots; the root length was the longest at the base level concentration and grew shorter; and at the MS culture medium base level concentration of 1 time, the D/F ratio was 21.43%, higher than those at other levels of concentration. The sucrose concentration experiment found that the plant length, theca diameter, number of stems and number of leaves showed no big difference according to concentration levels. But the D/F ratio analysis showed that it was 21.43%, the highest at the sucrose 3% concentration level and the theca diameter was the thickest. This seems to be the most appropriate concentration level for vernalization. The growth and development characteristics of seedlings germinated under in vitro condition were compared according to culture method. It was found that the bioreactor culture had the D/F ratio of 23.63%, higher than other culture methods. So the soil purification of plantlets is expected to be efficient compared with that in other culture methods. At the MS medium culture 1-fold concentration level, the D/F ratio was also found the highest at 1-fold level. The sucrose concentration experiment found that the 3% sucrose had the D/F ratio of 23.63%, indicating that most of the growth amount was larger than that at other concentration levels. As a result of BA concentration level-specific processing, shoot generation was 290% at 0.2 ppm, the largest and the best for shoot generation. This level seems effective for the mass proliferation of F. nipponica. In regard to bioreactor optimal aeration injection amount, the D/F ratio at 0.2 vvm was 23.63%, the highest. The longer the culture period in bioreactor, the larger the growth amount and the D/F ratio also jumped from 8.22% on the 2 week to 23.63% on the 6 week. Fragaria nipponica Makino also showed germination ratio as high as 96.1% through seed germination. But, for the tissue culture planting production for future mass production, it seems appropriate for short-term mass production to utilize 1-fold MS culture medium at sucrose 3% and BA 0.2ppm; inject 0.2 vvm aeration; and culture for 6 weeks.