【作者】 黄馨慧；

【导师】 王喆之；

【作者基本信息】 陕西师范大学 ， 植物学， 2004， 硕士

【摘要】 秦艽是我国传统常用中药，性苦、辛、平，微寒。现代药理研究表明秦艽具有抗风湿、解热、镇痛、降压、抗菌等多种功效。本实验以秦艽（Gentianamacrophylla Pall．）叶片、胚轴为材料，较系统的研究秦艽组织培养与快速繁殖技术，通过胚胎发生途径建立其植株再生体系，并对胚状体发生过程从形态学、组织学等方面进行了研究，对应用生物技术繁育秦艽优良种苗具有一定的意义。 （一）愈伤组织培养：在附加一定浓度的BA和NAA的MS培养基上经过2周以上的培养，95％以上的秦艽叶片和胚轴外植体可产生绿色愈伤组织。这些愈伤组织在含NAA配合一定浓度BA或2，4-D的培养基上培养3周可增殖15倍以上。暗培养对秦艽愈伤组织的诱导和增殖没有明显影响，但可以减缓愈伤组织老化；与其他植物生长调节剂相比，NAA对秦艽愈伤组织的形成有显著影响。 （二）无菌苗的增殖培养：无菌苗在含1 mg·L^-1NAA和1 mg·L^-1 BA的生长培养基上培养4周后，幼苗的高度可增长4.30倍，20％的幼苗可形成丛生芽，4周内芽增殖倍数为2～3倍。 （三）不定根的诱导：下胚轴、幼叶和不定根在附加3 mg·L^-1 NAA+0.1mg·L^-1 BA的培养基上培养4周后，形成白色、粗短、表面有细小绒毛的不定根，这些不定根可用于愈伤组织和胚状体的诱导。 （四）胚胎发生：由胚轴诱导产生的淡绿色、生长迅速的愈伤组织在含NAA和2，4-D的培养基上培养4周后可形成黄白色疏松易碎的胚性愈伤组织。这些愈伤组织在附加NAA和2，4-D为0.5 mg·L^-1或2 mg·L^-1的MS培养基上培养4周后的增殖倍数可达4.5倍以上，并且能较好的保持胚状体的发生能力。胚性愈伤组织在含0.5 mg·L^-12，4-D和0.5 mg·L^-1NAA的培养基上培养4周后发生胚状体。大约66.7％的球形胚在附加0.5 mg·L^-1 BA和0.5 mg·L^-1 NAA的MS培养基上可发育成具子叶的胚状体，在不含植物生长调节剂的MS培养基上90％的具子叶的胚状体可萌发形成完整再生植株。 （五）影响胚胎发生的因素：（1）外植体的影响。秦艽叶片、胚轴、不定根和愈伤组织都能通过间接途径诱导胚状体的发生，以叶片为外植体诱导胚状体，在一次培养中通过间接途径形成的胚状体，形态变化大，畸形胚多，愈伤组织化严重;以胚轴作为外植体，先诱导愈伤组织，转接后诱导形成的正常胚状体多，并且形态较均匀;不定根可以通过直接途径诱导胚状体的发生，但是产生的胚状体多为畸形。（2）植物生长调节剂的影响。NAA对胚性愈伤组织、体细胞胚的诱导等都有促进作用，高浓度的BA明显抑制体细胞胚的形成;附加0.5 mgr’NAA和0.5 mgl一’2，4一D的Ms培养基可使27.8%的愈伤组织发生正常的胚状体;含ZmgL一’NAA和2，4一D的Ms培养基使30%的不定根产生胚状体;含3 mgL一’NAA配合0.1 mgL一’BA的培养基可使13%的不定根形成胚性愈伤组织，进一步培养可形成胚状体和完整再生植株。（3）球形胚状体的来源影响胚状体成熟。在含3 ms.l一，NAA+3 mg.l一’IBA+1mg’l一’KT的Ms培养基上诱导产生的胚状体比在附加0.5 mg.l一’BA十4 ms.l一’NAA+0.3 mg.l一’KT的Ms培养基上诱导产生的胚状体能更好的发育成正常的具子叶的胚状体。 （六）组织学观察:通过连续石蜡切片观察胚状体的形成过程，结果发现秦芜胚状体的发育过程与合子胚的发育过程相似，经原胚、球形胚、心形胚、鱼雷胚发育成具子叶的胚状体。根据观察结果推测秦芜胚状体的起源为单细胞起源。 （七）超微结构观察:通过透射电镜观察秦芜胚状体与其他组织（叶片、不定根和愈伤组织）在超微结构上的差别主要在于细胞内含物的变化。胚状体中的细胞质浓厚，细胞器多，有丰富的线粒体、粗面内质网等，表明细胞生长代谢较为活跃，而不定根和叶片细胞只有少量的叶绿体和线粒体被中央大液泡挤到细胞边缘。更多还原

【Abstract】 Gentiana macrophylla Pall, is an important traditional Chinese herb for the use of its dried roots as large-leaved gentian, a kind of traditional Chinese medicine. Large-leaved gentian was one of the most common herbal medicines to cure chronic liver disease by herbalist doctors in China thousands years ago. Modern pharmacology research has revealed that the large-leaved gentian contains kinds of effective compounds. The State Council of the People’s Republic of China has listed large-leaved gentian into one of the protective wild herbs to prohibit the illegal collection due to the shortage of wild resources. According to the recent market survey, the supply sources are reduced seriously and the price of gentian is rising up continuously. The study of somatic embryogenesis of Gentiana macrophylla Pall, can not only alleviate the pressure of resources shortage but is also beneficial to breeding and genetic engineering.Using Gentiana macrophylla Pall, as an experiment material, we have studied the morghogenesis of its somatic embryo and established its plant regeneration system. Histological studies has confirmed the somatic embryogenic nature of the process as well. The research results show:1. Callus induction: 95% leaf and hypocotyls can produce callus on the MS medium with some concentration BA and NAA, which affects observably the induction of callus compared with BA, KT and IB A. Callus proliferates 15 times on the medium including NAA and BA or 2,4-D. Callus culture in dark condition lowers the process of aging.2. Sterile seedling proliferating culture: Sterile seedling increases 4.30 times on the medium with 1 mg.L-1 NAA and 1 mg.L-1 BA. 20% seedling can form bud-cluster and the multiplication multiple is 2-3 times.3. Adventitious root induction: After 4 weeks, hypocotyls, young leaves can produce white adventitious roots with fine hair. These roots can induce somatic embryo.4. Somatic embryogenesis: NAA and 2,4-D help callus form light yellow, frail embryogenic callus. Embryogenic callus proliferate 4.5 times on the medium with 0.5 mg.L-1 or 2 mg.L-1 NAA and 2,4-D and it can maintain the function ofsomatic embryogenesis. After 4 weeks of culture on MS medium supplemented with 0.5 mg.L-1 NAA and 0.5 mg.L-1 BAP, 66.7% of the globular embryos develop into mature ones. Up to 90% of normal mature embryos root on MS medium without any plant growth regulators.5. Effective factors of somatic embryogenesis: (1) the effect of explants: leaf, hypocotyl, adventitious root and callus all can induce somatic embryogenesis. (2) the effect of plant growth regulators: NAA promotes induction of embryogenic callus and somatic embryo; BA restrains the formation of somatic embryo; callus induces somatic embryogenesis on the MS medium with 0.5 mg.l-1 NAA and 0.5 mg.l-1 2,4-D; 30% adventitious roots produces somatic embryo on the MS medium with 2 mg.l-1 NAA and 2 mg.l-1 2,4-D. There are, however, many abnormal embryos. On the medium with 3 mg.L-1 NAA and 0.1 mg.L-1 BA 13% adventitious roots induce embryogenic callus, and then complete regeneration plantlet formed through somatic embryo. And (3) the effect of somatic embryo origin: somatic embryo, which is produced on the medium with 3 mg.l-1 NAA+3 mg.l-1 IBA+1 mg.l-1 KT. This could develop better mature somatic embryo than somatic embryo produced on the medium with 0.5 mg.l-1 BA+4 mg.l-1 NAA+ 0.3 mg.l-1 KT.6. Histological study: It is concluded that the development of somatic embryo of Gentiana macrophylla Pall, has the similar process with zygotic embryo through continuous histological study. They all experience the stages of proembryoid, globular embryo, heart-shaped embryo, torpedo embryo and cotyledon embryo. Based on the observation of the experimental result, we assume that the origin of somatic embryo of Gentiana macrophylla Pall is from the single-cell.7. Ultrastructure study: The main ultrastructure difference of somatic embryo and other tissue (leaf, adventitious root and callus) is the cell inclusion. The cell of somatic embryo has dense cy更多还原