Micropropagation of carnivorous plants

A simple, efficient system for seed surface disinfestation and in vitro germination of the carnivorous pitcher plants Darlingtonia californica and Sarracenia leucophylla was developed. Of the disinfectants tested, hydrogen peroxide or 10% CloroxRTM were most effective for disinfesting seeds of D. californica, while concentrated sulfuric acid worked best for S. leucophylla. Differences in the effectiveness of sterilants were accompanied by differences in seed coat morphology observed with scanning electron microscopy. Seeds of D. californica imbibed at 4--7°C in sterile deionized water with surfactant and gibberellin germinated earlier than seeds without exposure to gibberellin. Unimbibed seeds of S. leucophylla germinated rapidly in sterile water after treatment in concentrated sulfuric acid. An effective system for in vitro growth, multiplication and rooting of axenically germinated seedlings of D. californica was developed. Seedlings grown on solid ½ strength Murashige and Skoog medium produced more biomass and more and longer pitcher leaves than seedlings grown on other solid media assayed. Root development on solid media was minimal. Seeds stimulated by gibberellin prior to germination and exposed to auxin and cytokinin during early seedling development produced multiple offshoots as well as fibrous root systems when transferred to ½ strength liquid medium containing charcoal. Seedlings cultured in medium without charcoal produced more but smaller pitchers than seedlings cultured in medium containing charcoal. Multiplication did not occur on solid media, and seedling growth was stunted. Seedling multiplication through offshoots occurred in all liquid media and was both prolific and rapid. Darlingtonia californica was regenerated from whole, in vitro germinated seedlings and excised segments from in vitro generated juvenile pitchers. When incubated on solid Phytomax Orchid Multiplication Medium, seedlings produced protocorm-like bodies and green, leafy callus. Transverse segments of excised pitcher leaves from axenically-grown seedlings produced shoots and protocorm-like bodies when subcultured in liquid medium containing hormones. Unlike D. californica, S. leucophylla did not readily produce offshoots when incubated on solid media. A protocol for extraction of embryos from selected Sarracenia species was developed.