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소나무 자엽을 (子葉) 이용한 원형질체 나출 (裸出) 및 배양
이경준,이재순,윤양,이석구 ( Kyung Joon Lee,Jae Soon Lee,Yang Youn,Suk Koo Lee ) 한국산림과학회 1985 한국산림과학회지 Vol.71 No.1
Protoplasts were isolated from cotyledons of germinating Pinus densiflora S. et Z. seeds. The seeds were germinated for nine days, and excised embryonic cotyledons about 3-4㎜ long were incubated with Cellulase Onozuka R-10 and Macerozyme. After 8 hours of incubation, large number of viable protoplasts were isolated. Isolated protoplasts were cultured in a medium containing basal salts of BS medium, vitamines, amino acids, organic acid, sugars, and growth hormones. The first; evidence of protoplast budding was observed after twelve hours in culture, and it suggested that high potential of the embryonic cotyledons for rapid cell division affected the early budding, rather than effect of culture medium was shown in twelve hours. The three- to four-cell stage was reached after three to four days of culture. Most cell divisions were achieved by additional buddings rather than equal binary cell division. No further cell division was observed beyond the four-cell stage. Protoplasts isolated from fully expanded cotyledons (germinated for 17 to 24 days) seldom initiated or failed to initiate cell division.