In vitro kavapyrone production and genomic stability of in vitro kava cultures

A possibility of establishing a reliable tissue culture system for a medicinal plant, kava (Piper methysticum) was investigated by minimizing the incidence of necrosis and contamination. In addition, in vitro phytochemical production and genomic stability of in vitro kava cultures were explored with the established tissue culture system.; Utilization of foliar explants as well as medium supplementation with plant preservative mixture resulted in the reduction of contamination incidence that was not attained with high osmoticum medium, alternative sterilants or post sterilization with AgNO₃. A medium treatment of N 6-benzyladenine (0.5 mg L−1) and (2,4-dichlorophenoxy)acetic acid (0.5 mg L−1) resulted in the highest callus generation and the low necrosis incidence. Optimization of shoot primordia and microshoots formation was achieved with a medium containing N6-benzyladenine at 1.0 mg L−1.; A gradual increase in kavapyrone content was observed with in vitro cultures over a period of time although the total amount was very low (0.446–1.208 g/100 g DW). In in vitro plantlets, the total kavapyrone content was the highest in leaves (0.942 g/100 g DW), while the content was the lowest in roots (0.117 g/100 g DW). A significant increase in the total kavapyrone content (2.080 g/100 g DW) was observed one month after in vitro plantlets were acclimatized. This suggests a positive effect of plant development, ontogeny and growth environment on kavapyrone production.; In addition to ontogenic effects on kavapyrone production, a particular pattern of kavapyrone accumulation was observed in different organs of the greenhouse grown plant, in vitro plantlet and acclimatized plants, confirming an earlier report on tissue specificity of kavapyrone accumulation.; Of the four signaling compounds used, salicylic acid and γ-aminobutyric acid elicited time- and concentration-dependent overproduction of kavapyrones. Treatments of callus cultures with sodium acetate resulted in a significant increase in kavapyrone production (∼400%) although responses did not appear to be time- or concentration-dependent.; Inter simple sequence repeat (ISSR) markers were used for detection of polymorphism among in vitro kava cultures. An absence of normally observed bands was observed among organogenic callus cultures, while a small degree of ISSR polymorphism was detected among regenerated plantlets.