【作者】 曹晓燕；

【导师】 王（吉吉）之；

【作者基本信息】 陕西师范大学 ， 植物学， 2002， 硕士

【摘要】 以龙爪槐和毛刺槐的子叶、花药、叶片等外植体为材料，开展其组织培养和植株再生系统的研究。 (一)龙爪槐组织培养的研究 以龙爪槐子叶、叶片、花药等外植体进行组织培养，结果显示：(1)子叶是进行龙爪槐快速繁殖的最好材料。以龙爪槐子叶为外植体，通过体细胞胚胎发生和器官发生两条途径获得了大量的再生植株。子叶在MS附加BA(0.5-5mg/L)的培养基上先形成愈伤组织，进而分化出不定芽。当不定芽长至2-3cm时，将其切下，转入生根培养基上即可获得完整的再生植株。将子叶接种在MS附加2，4-D(0.1-40mg/L)或NAA(20mg/L)的培养基上，2周内可产生大量肉眼可见的胚状体。胚状体的诱导受子叶接种方向及子叶成熟程度等因素的影响。胚状体经进一步培养，逐渐发育成熟，在MS附加BA 1mg/L的培养基上形成完整的小植株。在培养过程中形成了许多畸形胚。(2)本实验对龙爪槐的花药培养进行了初步探索，主要研究了激素、低温预处理、蔗糖浓度、pH值等因素对愈伤组织形成的影响。结果显示：2，4-D与BA配合使用，可显著提高花药愈伤组织诱导率；4℃低温预处理对花药愈伤组织的形成没有促进作用；蔗糖浓度为9％时，可提高愈伤组织诱导率：pH值以6.5为宜。虽然愈伤组织形成率最高可达90％以上，但本实验未能分化出芽。(3)以龙爪槐叶片为外植体进行培养时，叶片切口处在几种不同激素浓度的培养基上均可100％产生少量白绿色致密的愈伤组织，这种愈伤组织在MS附加BA 3.0mg/L培养基上进行2-3次继代培养后，约有10％的愈伤组织可分化出不定芽。 (二)毛刺槐组织培养的研究 以毛刺槐花药、叶片等外植体进行组织培养，结果显示：(1)将毛刺槐的花药接种在MS附加2，4-D 0.1mg/L和BA3.0mg/L的培养基上，20d时花药愈伤组织诱导率可达41.5％。然后将花药愈伤组织转入含BA 5.0mg/L的MS培养基上，继代培养2个月后，可见愈伤组织上分化出许多绿色的芽点，待不定芽长至2-3cm时将其切下，转入MS附加IBA 1mg/L的生根培养基上，3周后即可得到完整的再生植株。毛刺槐花药培养过程中，低温预处理不能提高愈伤组织诱导率，将蔗糖浓度提高到6％和9％时，愈伤组织诱导率有所提高，当蔗糖浓度增加到12％时，愈伤组织诱导率反而显著降低。(2)以毛刺槐叶片为外植体进行培养时，叶片切口处先形成少量白绿色的愈伤组织，继代培养3个月后，可见绿色芽点的出现，芽 点进一步长大，发育为小植株。以叶片为外植体时，愈伤组织的分化率可达12％。 （三）龙爪槐遗传转化的初步研究 选用龙爪槐花药愈伤组织为转化受体系统，通过农杆菌介导法将菜豆几丁质 酶基因导入龙爪槐，获得了部分抗性愈伤组织。对转化过程中的预培养时间、浸 染时间、共培养时间、抗菌素浓度等进行了初步筛选，结果显示：预培养2－3d， 浸染 5习0叫 n，共培养 2 d，选择培养基中头抱霉素浓度 5 0 omg／L，卡那霉素浓度 5 omg／L为龙爪槐基因转化及抗性愈伤组织获得的较佳条件。 龙爪槐、毛刺槐组织培养和植株再生系统的建立不仅可为进一步研究龙爪槐 和毛刺槐的发育生理、细胞分化以及形态建成等许多基础理论问题提供新的实验 系统，更能为进一步利用现代生物技术改良品种、加速育种进程、促进优良种苗 的快速繁殖与利用等奠定基础。更多还原

【Abstract】 Using different explants of cotyledon, anther and leaf as- materials, studied the tissue culture and plant regeneration of Sophora japonica f. pensula hort and Robinia hispida. 1. Tissue culture of Sophora japonica f. pensulaUsing the cotyledon, anther and leaf as materials, studied the tissue culture of Sophora japonica f. pensula and the results showed that:(1)Cotyledon was the best explant for the rapid propagation of Sophora japonica f. pensula. Using the cotyledon as explant, a large number of regenerated plantlets were obtained from somatic embryogenesis and organogenesis. Calli formed from cotyledons cultured on MS basal medium supplemented with BA 0.5-5mg/L for 20 days and then a lot of adventitious buds formed from the callus. The plantlets could be obtained when the buds were cut and continually cultured on the root-induced medium. Embryoids could be induced when the cotyledons were placed on the MS basal medium supplanted with 2,4-D 0.1-40mg/L or NAA 20mg/L for two weeks. The induction of somatic embryogenesis was influenced by the explants size and the orientation on the medium. The embryoids gradually matured and could develop into plantlets when they were transferred to MS medium supplemented with BA 1 mg/L, but many embryoids were morphologically abnormal.( 2 ) In the course of anther culture, the effects of hormone, pretreatment of low temperature, sucrose concentration and pH on the anther culture were studied and the results showed that: The combination of BA and 2,4-D could obviously improved the induction rate of callus; pretreatment of low temperature at 4癈 could not improve the callus induction efficiency; 9% sucrose and pH6.5 was beneficial for the anther culture. Though the induction rate of anther callus could reach as high as 90%, the buds couldn’t be obtained from the callus.(3)When the leaves were cultured on MS medium with different hormones, a little white and green callus formed from the incision and the induction rate were all 100%. The differentiation rate of callus was about 10% and the callus formed adventitious buds when they were cultured on the MS medium supplemented with BA 3mg/L forin2-3 months subculture.2. Tissue culture of Robinia hispidaUsing the anther and leaf as explants, studied the tissue culture of Robinia hispida and the results showed that:(l) The highest induction rate of callus was 41.5% when the anthers were cultured on MS medium supplemented with 2,4-D 0. Img/L and BA 3mg/L for 20 days. When the callus were subcultured for twice or three times on MS medium supplemented with BA 5.0mg/L, green buds formed from the callus. When the shoots grew to 2-3 cm long, they were detached and transferred to MS medium supplemented with IBA Img/L for rooting. The shoots produced roots within 3 weeks of culture and formed plantlets. In the course of anther culture, Pretreatment of low temperature couldn’t improve the induction rate of callus; The callus induction efficiency improved when the concentration of sucrose improved from 3% to 6% and 9%, but it obviously reduced when the concentration come up to 12%.(2)A little white and green callus formed from the incision when the leaves of Robinia hispida were cultured. After subculture for three months, some green buds formed from the callus and further developed into plantlets. Using the leaves as explants, the differentiation rate of callus was about 12%. 3.The primary study of gene transformationSome work about gene transformation were did. Using the anther callus as transgenic receptor, the CHI8 gene were tried to transfer to Sophora japonica f. pensula by Agrobacterium-medialed method. The antibiotic resistant callus were obtained. In the course of gene transformation, the time of pre-culture, immersion, co-culture and the concentration of antibiotic were studied and the results showed that: 2-3 days pre-culture, 5-10 minutes immersion, 2 days co-culture, 50mg/L kanamycin and 500mg/L cefotaxime in the selection medium were suitable.The study of tissue culture and plant regeneration not on更多还原