The effect of several factors on somatic embryogenesis and plant regeneration in protoplast cultures of Gentiana kurroo (Royle)

Abstract

Protoplasts were isolated from cell suspensions derived from cotyledon and hypocotyl Gentiana kurroo (Royle). Cell walls were digested with an enzyme cocktail containing cellulase, macerozyme, driselase, hemicellulase and pectolyase in CPW solution. Protoplast viability ranged from 88 to 96%. Three techniques of culture and six media were evaluated in terms of their efficiency in producing viable cultures and regenerating whole plants. With liquid culture, cell division occurred in only a low number of the protoplasts isolated, and no plant regeneration was successful. Cell division occurred within 2 or 3 days in case of agarose solidified media. After 10 days of culture, the number of dividing cells was the highest with modified MS medium in which NH4NO3 was replaced with 3.0 g l−1 glutamine. The best results were obtained with agarose bead cultures: plating efficiency was 68.7% and 58.1% for protoplasts isolated from cotyledon and hypocotyl derived suspensions, respectively. The results were achieved with using medium containing 0.5 mg l−1 2,4-D + 1.0 mg l−1 kinetin or 2.0 mg l−1 BAP + 1.0 mg l−1 dicamba + 0.1 mg l−1 NAA + 80 mg l−1 adenine sulfate. Protocalluses transferred on the following composition of plant growth regulators: 0.5 mg l−1 2,4-D + 1.0 mg l−1 kinetin or 1.0 mg l−1 kinetin + 0.5 mg l−1 GA3 + 80.0 mg l−1 adenine sulfate developed in embryogenic cultures. However, the best embryo production occurred with the first one. Later embryos were transferred to half-strength MS mineral salts to promote plants formation. Flow cytometry studies revealed increased amounts of DNA in about one third of the regenerants.