Date of Award
Doctor of Philosophy in Biological Sciences
A simple, efficient system for seed surface disinfestation and in vitro germination was developed for the carnivorous pitcher plants Darlingtonia californica and Sarracenia leucophylla. Of the disinfectants tested, hydrogen peroxide or 10% Clorox® 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 associated by differences in seed coat morphology. 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.
Scanning electron microscopy of D. californica seed coats revealed waxy trichomes covering the seed surface. In contrast, the seed coats of S. leucophylla were pitted with surface and sub-surface cells possessing heavily thickened cell walls. These cells were devoid of contents. Fungal hyphae were observed on the seed surface and within empty cells of the integuments. Scanning electron microscopy observations and comparison of seed coat morphology of the carnivorous plant genera Drosera, Dionaea, Sarracenia and Darlingtonia revealed a wide range of differences in structure and ornamentation, which may suggest a species specific approach to surface disinfestation.
A simple effective system for the in vitro growth, multiplication and rooting of axenically germinated seedlings of D. californica was developed. Seedlings grown on solid Yi 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 and usually limited to the seminal root regardless of the medium. Seeds stimulated by gibberellic acid 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 Y2 strength liquid medium containing charcoal. Similarly treated seedlings transferred to Yi strength liquid media without charcoal produced multiple offshoots but fewer root systems. 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 When divided and subcultured in liquid Phytomax Orchid Multiplication Medium, explants of both protocorm-like bodies and green, leafy callus gave rise to multiple shoots as well as more protocorm-like bodies and green, leafy callus. These could be further divided and subcultured. Transverse segments of excised pitcher leaves from axenically-grown seedlings produced shoots and protocorm-like bodies when subcultured in liquid Phytomax Orchid Multiplication Medium. Unlike D. californica, seedlings of S. leucophylla did not readily produce offshoots when incubated on solid media. A protocol for extraction of embryos from selected Sarracenia species was developed.
Uhnak, Kenneth Stephen, "Micropropagation of Carnivorous Plants" (2003). Open Access Dissertations. Paper 471.