|
Plant regeneration system from protoplasts isolated from callus derived from immature embryo was established in H. syriacus. Enzyme solution for protoplast isolation consisted of 3% (WN) cellulase R10, 0.1% (WN) macerozyme, 0.1% (WN) pectolyase, 1mM DTT, and 3mM MES. Protoplasts were cultured under continuous dark condition until 14 days after initial culture and 16 hours day length (2,000 lx) 1 month after culture. Plant growth regulator requirement for microcolony formation, callus development, and plant regeneration were 1 NAA + 0.01 TDZ, 0.1 BA + 1 NAA, and 0.1 BA + 0.1 NAA, respectively (unit: ㎎·L^(-1)). Cell division was greatly increased by an addition of 2% DMSO to colony formation medium and 100 ㎎·L^(-1) adenine to callus growth medium, respectively. Basal medium was composed of salts, vitamins, and organic acids of KMBp medium. Callus formation was not observed in semi-solid medium or alginate bead, but continuous cell division and plant regeneration were possible using stationary liquid culture at early culture period and shaking liquid culture (30 rpm) when the callus was larger than 0.5 ㎜ in diameter. Optimum osmoticum concentration was 0.6 M mannitol, and plating efficiency was promoted by lowering mannitol concentration by 0.2 M at every 5-7 day from 15 days after culture. Optimum culture density was 5-10×10⁴mL.
|