Ammi visnaga: Somatic Embryo Induction and Furanochromone Production in Embryos, Seedlings, and Plants

AMMI VISNAGA (L.) Lam. globular, heart, and torpedo shaped embryos were induced from the calli of sterile seedling hypocotyls in Murashige and Skoog's medium with or without either 0.5 ppm benzyladenine or 1.0 ppm 2,4-D. After transfer of the calli into liquid media supplemented with 10 mM L-glutamine, all three embryo stages were observed after eight weeks. Liquid media lacking growth regulators appeared to enhance the development into torpedo stage embryos, especially at four weeks. Eight weeks after the transfer of the callus into liquid media, flasks containing predominately one microscopic stage were pooled, transferred into fresh liquid media, and the production of the furanochromones, khellin and visnagin, in the embryos were determined by a normal phase HPLC method. All three embryo stages produced similar amounts of khellin and visagin. Maximum production was reached at six weeks. The six-week-old torpedo shaped embryos produced 0.57% khellin and 0.72% visnagin on a dry weight basis. The embryo cultures produced significantly higher concentrations of the furanochromones than A. VISNAGA cell suspensions (reported previously) that produced approximately 0.1-0.3% visnagin and no detectable khellin. In addition, the production of khellin and visnagin in the fruits, seedlings, young plants, and flowering or fruiting AMMI VISNAGA (L.) Lam plants was determined. Upon germination of the fruits, a rapid decline in the content of the furanochromones was observed. Subsequent experiments showed that the majority of the furanochromone content in fruits was released into the media within one to two days. Total visnagin content (media + seedlings) was unchanged, however, a loss of 40% of the khellin content was found after 2-4 weeks. When media containing seedlings was spiked with exogenous khellin and visnagin, the concentrations of both furanochromones declined, apparently due to uptake by the roots and subsequent catabolism in the plant. No evidence for degradative enzymes in media exposed to seedlings was observed. During vegetative growth of the plants, relatively low levels of furanochromones were found. High concentrations were observed at early flowering, followed by a decline, and a gradual rise at fruiting as was previously reported by other investigators. In the vegetative stages, visnagin concentrations were higher than those for khellin, whereas in fruiting plants and fruits, the ratio was reversed.