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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.
유장걸,소인섭,홍경애 제주대학교 1993 논문집 Vol.37 No.-
. The enzymatically isolated protoplasts generally give difficulties in cell differentiation and regeneration because the enzymes used could alter the physiological properties of protoplasts. In this study, the possibility that DNA could move out out of the cell isolated from radish hypocotyl callus was investigated after treatments with low pH, high concentration K??,GA₃combined with centrifugal force, all of which are known to affecct the physico-chemical properties of cells. 1. Treatment with 1.0% NaOCL for 20 min. by infiltration was found to be effective for the surface sterilization of radish seeds and the improvement of germination rate. 2. MS medium containing BA 0.5ppm and 2,4-D 1.0ppm gave the best callus formation from the radish hypocotyls. 3. The working concentration of SDS as an anion detergent which minimize the cell deterioration and function was 1ppm. 4. High K?? concentration(0.1% to 2.0%) and low pH(pH 4 tk 5) treatments resulted in the movement of DNA out of the radish hypocotyl callus cells. 5. The GA treatment was less effective than that of K??, but the DNA band appeared in the gel electrophoresis.
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.