珍稀濒危药用植物独叶草组织培养与内生菌分离研究

【作者】 姬文帅；

【导师】 任毅；

【作者基本信息】 陕西师范大学 ， 中药学， 2010， 硕士

【摘要】 独叶草(Kingdonia uniflora Balf. f. et W. W. Smith)为星叶草科(Circaeasteraceae)多年生小草本,为中国特有单种属植物,被列为国家Ⅰ级重点保护植物,具有重要的药用功效。独叶草种子在野外环境条件下大多不能成熟,主要依靠根状茎顶端伸长繁殖,在其苛刻的生存环境里,自然更新能力差,加之人为破坏森林植被和采挖,自然环境的突变等因素,其植株数量逐渐减少,自然分布区日益缩小。而其单一的繁殖方式又不能使其及时得到恢复。本实验通过对濒危药用植物独叶草组织培养的初步研究,以期为其野外种群数量扩大提供理论参考,从而为独叶草的保护开辟一条行之有效的途径。同时,为了促进独叶草在组织培养过程中快速生长,进行了独叶草内生真菌分离鉴定的初步研究。结果如下：1.独叶草离体再生体系的建立在实验过程中,探讨了不同外植体消毒、不同基本培养基(MS、1/2MS、1/4MS、B5、N6、White)、不同激素种类、浓度及组合(不同生长素(NAA、2,4-D、IAA、IBA)、不同细胞分裂素(6-BA、KT、ZT、TDZ))、不同温度光照效应和不同外植体对独叶草生长和分化的具体作用。(1)外植体消毒：通过用0.1%的升汞溶液和2%的次氯酸钠溶液对不同外植体在不同时间下消毒,发现根状茎用0.1%的升汞溶液消毒3min较合适,成熟叶片用2%次氯酸钠溶液消毒1min较合适,成熟叶柄用0.1%升汞溶液消毒1min或2min较合适,芽苞用0.1%升汞溶液消毒4min较合适。(2)外植体诱导：通过不同基本培养基、不同激素种类、浓度及组合对不同外植体的培养,发现1/4MS是较合适的基本培养基；幼叶柄是最合适的外植体,可以诱导出愈伤组织、不定芽、不定根,而根状茎、成熟叶柄、成熟叶片、芽鳞片和幼叶片的诱导率均为零；幼叶柄仅在含有激素2,4-D的培养基中可以诱导出愈伤组织,但该愈伤组织容易褐化死亡。2.独叶草内生真菌的分离鉴定(1)独叶草根表面和内部结构的观察：采用扫描电子显微镜对独叶草根表面进行了观察,发现其根表面具有2个以上丝状物簇生区；通过徒手切片,乳酸石炭酸棉蓝染色,对独叶草根内部结构进行了显微观察,独叶草根表皮细胞内具有菌丝。(2)通过两次分离,共得到5个菌株,根据菌落和菌丝特征、产孢结构、孢子形态等一系列鉴定,初步鉴定所得到的菌株主要属于子囊菌纲和半知菌类,其中半知菌类青霉属占优势。更多还原

【Abstract】 Kingdonia uniflora Balf. f. et W. W. Smith belongs to the Circaeasteraceae and is the only species of Kingdonia. It is one of the most endangered species in China and was ranked as the first grade of National Protected Plants and the key species of Chinese biodiversity. Kingdonia uniflora seeds have largely failed to mature in the field environmental conditions, rely mainly on the top of rhizome elongation reproduction, survive in the harsh environment, poor natural regeneration. Coupled with the destruction of forest vegetation and excavation, result in mutation of the natural environment, its plants declined and shrinking natural distribution While the single mode propagation can not be restored. This experiment was maiuly to probe an effieient way through tissue culture, which offer the theory foundation for enlarging source of Kingdonia uniflora. Meanwhile, endophytic fungi Kingdonia uniflora was isolated and identified in growth of it. As follows:1.The establishment of regeneration system of Kingdonia unifloraIn the experiment, The effects of the different sterilization of explants, different basic medium (MS,1/2MS,1/4MS, B5, N6, White), different hormones, different concentrations and combinations (auxins (NAA,2,4-D, IAA, IBA) and cytokinins (6-BA, KT, ZT, TDZ)), different culture conditions and different explants on the specific role of growth and differentiation of Kingdonia uniflora were debated.(1) The sterilization of explants:Different explants are sterilized at different time by using 0.1% mercuric chloride solution and 2% sodium hypochlorite solution and found that the rhizome with 0.1% mercuric chloride solution,3 min more suitable disinfection, mature laminae with 2% sodium hypochlorite disinfection 1 min more suitable, mature petiole sterilized with 0.1% mercuric chloride solution,1 min or 2 min more suitable, Buds sterilized with 0.1% mercuric chloride solution,4 min more suitable(2) The experiment on induction of Kingdonia uniflora:The different explants are cultured in culture conditions with different basic medium, different hormones, different concentrations and combinations. The results indicated that the 1/4MS media was the most suitable basic medium for the most suitable explants of young petiole of Kingdonia uniflora, and Callus, adventitious buds, adventitious roots are induced. While the induction rates of rhizomes, mature petioles, mature laminae and bud scales were zero. Only 2,4-D can induce callus, but the callus become brown and dead.2. Isolation and identification of endophytic fungi of Kingdonia uniflora (1) Roots surface and inner of Kingdonia uniflora:The surface of Kingdonia uniflora roots were obsered by scanning electron microscopy, and we found that there are two or more regions clustered filaments on the root sruface. Through hand slicing and lactic acid phenolic cotton blue staining,the internal structure of Kingdonia uniflora root was observed and found that hyphae exist in epidermal cells.(2) 5 fungal strains have been isolated. Isolates were identified on the basis of colonial morphology and microscopic characteristics of hpyhae, spores, conceptacle, the results showed that the resulting strains are mainly Ascomycetes and Imperfect fungi, The superior genus is Pencillium更多还原