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온주밀감(Citrus unshiu Marc. var. Okitsu)과 오렌지(Citrus sinensis (L.) Osbeck)의 원형질세 융합
홍경애,송성준,이옥영 제주대학교 방사능이용연구소 2000 연구보고 Vol.14 No.-
By the development of plant molecular biology these days which has been accomplishing much an attempt to get mutants. However, there have been haying many issues in lacking of recognition for transformants and the right of ownership of the gene. The breeding method of cell fusion without any problems can easily make new variety with an excellent character having plant itself. The purpose of this study was therefore to carry out to get trantsformants having sweeter taste and the original character of Citrus unshiu itself using PEG method of cell fusion. The yield of protoplasts per gram of leaf was 7 × 105 ~ 1 × 106/㎖ and callus cell was 1 × 106 / ㎖ . The first protoplast division and formation of micro-calli were observed 7 to 9 days and 30 to 40 days after incubation, respectively. To induce embryo from these calllus were inoculated solid medium containing 1.6% agarose and 5% sucrose. Green somatic embryo and plantlet were appeared from 1 to 2 months after plating and transfered to MT basal medium to stimulate rooting and shoot elongation. It was difficult to get normal plant directly through embryo of globular, heart and terpenoid state, and more effective method was directly to get plantlet from the globular embryo without heart and terpenoid state to control properly agar concentration.
Phytochrome이 보리잎 Protoplast의 팽윤에 미치는 영향
홍경애,송성준,송필순,유장걸,문두길 제주대학교 방사능이용연구소 1986 연구보고 Vol.2 No.-
암조건에서 생육시킨 보리(Hordeum valgare)잎의 생장점 부근에서 분리한 Protoplast가 적색광 처리에 의해서 팽윤되었고 적외선광에 의해서는 적색광 효과과 소멸되었다. 이 사실은 Protoplast의 팽윤이 Phytochrome에 의해서 조절됨을 시사하는 것이나 Phytochrome의 작용기작을 알기 위해서는 다각적인 연구가 필요하다. 실험결과로 부터 적색광 처리시간은 2분 적색광 처리후 배양시간은 2시간이 적당함을 알았다. Protoplast 배양액의 Sorbitol 농도에 따른 크기변화는 농도가 낮을수록 커졌으며 0.4M 이하에서는 Protoplast가 파괴되었다. Light effect on the size of protoplasts isolated from etiolated barley leaves was studied. Red light irradiation caused a swelling of protoplasts while little change in size found under the dark condition. Photoreversibility by red light and far-red light was found in protoplast swelling, indicating the involvement of phytochrome. Action mechanism of phytochrome on protoplast swelling should be clarified in aspect of growth regulators and intermediates involved. Two minute irradiation time and two hour incubation after red light treatment were found sufficient to induce protoplast swelling. The protoplasts were swollen according to the sorbitol concentration of incubation media but destroyed below 0.4M-sorbitol concentration.
강순선,유장걸,홍경애 濟州大學校 放射能利用硏究所 1990 연구보고 Vol.5 No.-
本 實驗은 寒蘭의 生育에 가장 중요한 要因 중의 하나로 생각되고 있는 相對濕度가 燐酸의 吸收 및 移行과 urea permeability에 끼치는 影響, 그리고 寒蘭의 無機成分 組成과 V.A.mycorrhizae 有無를 硏究 檢討하여 寒蘭의 營養 生理 特性에 관한 基礎 資料를 提供하고져 遂行되었다. 1. 寒蘭의 AFS 값은 0.44∼0.50㎖/g.fresh weight로 다른 식물 平均値(0.10∼0.15㎖/g.fresh weight)와 비교하여 큰 것이 特徵이었다. 2. 시간 經過에 따른 뿌리에서 잎으로의 養分移動은 매우 느려서 P-32 燐酸을 吸收시킨 뒤 14시간(840분) 이내에는 잎에서의 P-32 檢出量이 아주 적었다. 3. 뿌리 연령에 따른 寒蘭의 養分移行을 보면 뿌리에서 뿌리(촉 포함)로의 養分移行이 뿌리에서 잎으로의 養分移行보다 活發하였고 젊은 뿌리일수록 燐酸의 移行 能力은 컸다. 4. 寒蘭을 잎, 촉, 뿌리로 나누어 部位別 無機成分 分析을 해 본 결과 K는 잎중에, Mg, Cu, Al은 뿌리에, 그리고 P, Ca, Fe, Mn, Mo, Zn, B, Si, Na는 촉에 많이 含有되어 있었다. Fe, B, Mo 含量은 다른 植物의 平均値에 비해서 그 含量이 매우 높았고 K 含量은 낮은 것으로 나타났다. 5. 35%, 50%, 65%, 90%의 相對濕度下에서 자란 寒蘭이 urea permeability를 測定한 결과 相對濕度가 낮을수록 浸透速度(Ks)가 컸으며 相對濕度가 클수록 Ks 값은 낮았다. 6. 寒蘭의 燐酸 吸收能은 相對濕度가 낮은 곳에서 자란 寒蘭일수록 吸收能이 낮았으며 相對濕度가 높은 곳에서 자란 寒蘭일수록 吸收能이 컸다. 7. 野生 寒蘭에서는 V.A.mycorrhizae가 거의 觀察되었으나 組織培養된 寒蘭과 野生 春蘭에서는 V.A.mycorrhizae가 觀察되지 않았다. The study is concerned with the apparent free space of Cym. Kanran root, characteristics of P-32 absorption by roots, and translocation from roots to upper parts and urea permeability under different relative humudities(35%, 50%, 65%, 90%). The composition of inorganic elements in Cym. kanran was determined and the infection of V.A.mycorrhizae was examined. 1. AFS of Cym. kanran was 0.44-0.5㎖/g.fresh weight, being rather higher than those of most higher plant roots. 2. The phosphate translocation rate of Cym. kanran root to leaf was very slow comparing to another higher plants. 3. The phosphate transport from roots to roots was more active than that from roots to another parts, regardless of old and young roots. 4. Bulbs contained higher amounts of P, Ca, Mn, Fe, Mo, Zn, B, Si, and Na than another parts but the concentrations of Cu, Al, and Mg were higher in roots and more K was in leaf. Fe, B, and Mo contents seemed to be higher than another plants while K content was lower. 5. The lower relative humidity, the higher urea permeability. The quicker permeation of urea might be brought about by increase of cell sap concentration and membrane structure alteration induced by water loss. 6. The lower relative humidity, the lower P-32 uptake by roots. This fact indicated that active absorption of phosphate was inhibited by water deficit. 7. V.A.mycorrhizae was observed from wild Cym. kanrans, but not from cultured Cym. kanrans and wild Cym. virescenes.
PEG에 의한 삼보감(Citrus sulcata Takahashi)과 오렌지(Citrus sinensis(L.) Osbeck var. Cara Red Naval)와의 원형질체 융합
양경애,홍경애,이옥영 제주대학교 방사능이용연구소 1999 연구보고 Vol.13 No.-
PEG-mediated protoplast fusion between Citrus sulcata Takahashi and Citrus sinensis(L.) Osbeck var. Cara Cara Red Navel was demonstrated to establish the biotechnological breeding of Citrus. The optimum enzyme composition for protoplast isolation was 0.5 % cellulase, 0.5 % macerozyme and 0.1 % pectolyase for Citrus sulcata Takahashi(Sambogam) and 0.3 % cellulase, 0.3 % macerozyme and 0.1 % drieselase for Citrus sinensis(L.) Osbeck var. Cara Cara Red Navel(CCRN), giving the protoplast yield of ∼ 1 x 106 protoplast / ml. The best plant materials could be obtained from young matured leaves(1 ∼2 months old) of Sambogam and 4 weeks old callus of CCRN. The optimum protoplast density for fusion and culture was 6 × 104∼ 1.5 × 105 protoplast / ml. The same volume of protoplast sample and PEG brought about the best fusion. One of the important factors to get good plantlets was to cultivate the protoplast under the dark condition until embryo formation(6 weeks). If the protoplast were cultivated under the light condition then microcalli grew loosely and finally died not to form embryo. The formed embryo were grown under the light condition for greening. Among 21 arbitrary primers, 6 primers(OPH-04 GGAAGTCGCC, OPM-13 AGCGTTCACTC, OPN-07 CAGCCCAGAG, OPH-15 AATGGCGCAG, OPAT-04 TTGCCTCGCC, OPAT-13 CTGGTCCAAG) showing different PCR band pattern between Sambogam and CCRN were selected. Using the selected primers, it was confirmed by the RAPD technique that the obtained plantlets was fused with Sambogam and CCRN.