Plant regeneration from ginger [Zingiber officinale] protoplast by asymmetric cell fusion
1998
Ohyama, T. (Nagasaki-ken. Agricultural and Forestry Experiment Station, Isahaya (Japan)) | Komura, K. | Nakao, T. | Takeda, T. | Mori, N.
1) The cytoplasm of protoplast isolated from calli of ginger varieties was inactivated by an acetoamido iodine treatment with either 10 mM concentration for 10 min or 5 mM for 15 min. The nuclei were inactivated by an exposure to 10 KR Xray. 2) For an asymmetric ell fusion of ginger using PEG, the highest fusion rate between single cells were at conditions of 5 x 10(5) protoplasts per ml and a 30% concentration of PEG with a molecular weight of 1540. For an electric fusion, optimized conditions were 4-6 x 10(5) protoplasts per ml, a 2 MHz high frequency wave, an AC 40 Vpp and one cycle of 100 mu sec pulse volts. PEG was superior infusion rate to an electric fusion, but was difficult to culture. 3) Protoplasts after a fusion treatment were cultured best on MS medium or a half strength of MS medium supplemented with 14.6% sucrose and 2 ppm 2,4-D. The cultured density of 4-6 x 10(5) protoplasts per ml gave the highest cell division rate. The largest number of colonies was obtained by replacing one third of the medium with that of reduced sucrose content to 3% three weeks after beginning of the culture. Regeneration from the calli after a fusion treatment was optimized with the MS medium supplemented with 1 mg/l BAP and 3% sucrose. By removing ammonium nitrate from the medium, green spots were formed promptly. Higher shoot formation frequencies were obtained by culturing the calli first in liquid medium with shaking for two or three weeks, then on solid regeneration medium
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