Hormone-independent embryogenic callus production from ginseng cotyledons using high concentrations of NH4NO3 and progress towards bioreactor production

Abstract

Cotyledon explants of Panax ginseng produced somatic embryos directly on solid hormone-free MS medium containing 3% (w/v) sucrose while high concentration of NH4NO3 (60 mM) induced embryogenic callus. Ten subcultures of the embryogenic callus on hormone-free MS medium with 40 mM NH4NO3 gave hormone-independent proliferation of callus, which exhibited proliferation potential even on MS medium with a standard level of NH4NO3 (20 mM). Pulse treatment of callus with exogenous auxin or cytokinin (1.0 mg 1−1 2,4-D, 1.0 mg 1−1 kinetin) resulted in the loss of the hormone-independent characteristic and caused the callus to brown. For the suspension culture, embryogenic callus was transferred to MS liquid medium containing 3% (w/v) sucrose in an 500 ml Erlenmyer flask. Embryogenic cell clumps in full-strength MS liquid medium discharged toxic substances, resulting in strong suppression of cell growth. In 1/3-strength MS medium, exudation of toxic material did not occur. Embryogenic cell clumps were mass-grown on a large-scale in a bioreactor (20-1), showing a 7.1 increase of fresh weight in 1/3-strength MS medium with 3% (w/ v) sucrose after 5 weeks of culture. Total ginsenoside content of cultured embryogenic cell clumps was low and 6 times below naturally-cultivated ginseng roots.