Studies On Rapid Propagation And Regeneration In Vitro Of Penstemon Barbatus

Penstemon barbatus (Can.) Roth, a kind of perennial herb plant in Penstemon, is a member of the family Scrophulariaceae. It can be found in North America to Mexico area; it is the local Aboriginal important drug resources. Almost the whole plant can be medicinal, but its medical value has not yet been fully exploited. The study found that Penstemon contains echinacoside, and the contents are very close to the Cistanche tubulosa in levels of Echinacoside. Therefore, Penstemon barbatus may become an alternative plant of Cistanche tubulosa for large-scale production of Echinacoside.Currently, echinacoside is largely extracted from Cistanche tubulosa. Echinacoside content not less than1.0%is the only quality control standards of Cistanche tubulosa in Chinese pharmacopoeia2005. Due to the special growth circumstances, the difficulty to plant in recent years, coupled with the recent overexploitation which cause wildlife resources reduced drastically, Cistanche tubulosa has become an endangered species. Echinacoside was known not only as a phenylethanoid glycoside, but also as a caffeic acid derivative. It possesses many pharmacologically activities, such as antioxidant, anti-inflammatory, anti-tumor, improve memory etc, which can be used for neurasthenia, insomnia and cardiovascular function degeneration isorders treatment and prevention. Therefore, in today’s world into an aging, it has important practical significance of propagation and genetic transformation study on plants that can produce echinacoside instead of Cistanche tubulosa.Currently, researches on Penstemon barbatus mainly in terms of secondary metabolites, which can not been reported in tissue culture and regeneration in vitro. In this study, aseptic buds, leaves and stems were used as explants to establish regeneration system in vitro mainly through direct regeneration stems. This lays a good foundation for cultivating Penstemon barbatus which has rich echinacoside by transgenic technology.In this study, Penstemon barbatus vitro regeneration system was established by rapid propagation, explants dedifferentiation and direct regeneration.The main results are as follows:1. The establishment of in vitro rapid propagation system of Penstemon barbatusIn this study, aseptic buds and lateral buds of plantlets were applied as explants to establish in vitro rapid propagation system. Complete plants can be obtained by primary culture, subculture, rooting culture. The results showed that buds were trained on1.0mg·L-16-BA+0.05mg·L-1NAA of MS medium for30days, the induction rate of the adventitious buds achieved100%and the buds proliferated9.8. The adventitious buds were subcultured every other30day. The average breeding coefficient achieved6.0times from1to4generations, and the cluster buds grew well. The adventitious buds were trained on0.2mg·L-1IBA+0.2mg·L-1NAA of1/2MS medium for30days, the rooting rate of adventitious buds achieved100%, the root system developed well and the test-tube plantlet grew robustly.105plantlets were transplanted to matrix soil, the survival rate was91.42%.2. The establishment of regeneration system via explant dedifferentiationIn this study, leaves and stems of plantlets were applied as explants to establish dedifferentiation and redifferentiation in vitro regeneration system. The results showed that the optimal medium for callus induction of leaves is SH+3.0mg·L-16-BA+0.3mg·L-1NAA, callus induction rate was57.14%for30days, callus grows well. Stems explants callus induction in SH+0.5mg·L-16-BA+0.1mg·L-1IBA is the best, up to83.33%, callus is yellow and smooth. Callus can induce adventitious buds in SH+1.0mg·L-16-BA+0.1mg·L-1IBA after30days. The budding rate is23.81%. The buds proliferation rate is100%in SH+1.5mg·L-16-BA+0.1mg·L-1IBA, the highest propagation coefficient is13.8, the average propagation coefficient of four generations is12.55. In rooting medium1/2SH, the buds rooting rate are100%, transplanting survival rate is89.53%.3. The establishment of directly regeneration system from stems and leaves explantsLeaves and stems of plantlets were applied as explants to establish directly regeneration system in vitro. During the test, leaf explants failed to direct budding. It was found that in SH+2.0mg·L-16-BA+0.5mg·L-1IBA, stems can directly induce adventitious buds by29.17%, the buds grow well. Buds have a good effect in SH+1.5mg·L-16-BA+0.1mg·L-1IBA proliferation medium, the buds proliferation rate is100%, the average propagation coefficient is10.62. In1/2SH medium the buds rooting rate is100%; the transplanting survival rate is90.68%.4. The studies of explants browning and buds vitrificationIn training process, explants such as stems and leaves browning, buds have occurred vitrification in the process subculture. By shortening the training period and join the anti-brown agents can reduce and control explants browning. Reduce sucrose and agar content in medium can control buds vitrification.The results showed that explants can effectively reduce the browning of buds in medium which PVP concentration is0.04g·L-1, subcultured once every15days. In subculture process, growth medium containing20g·L-1sucrose and7g·L-1agar can prevent the vitrified buds. Hormones and basic medium without any hormones used alternately also can reduce or prevent buds vitrification.