影响农杆菌介导的木薯基因转化因素的研究

本文研究了影响农杆菌介导的木薯基因转化的因素。结果表明,供试的4个菌种中,LBA4404(pBin9GusInt)及LBA4404(pTOK)瞬时表达效果较好。对农杆菌的诱导处理能增强瞬时表达效果,外植体的预处理对瞬时表达无影响,而外植体的预培养显著降低瞬时表达。所有供试的木薯品种都能被农杆菌侵染,但外植体的类型及生理状况对农杆菌的侵染力影响很大,成熟胚状体的子叶(萌发15d)及试管苗完全展开的叶片对农杆菌亲和性最高。四种筛选剂(kanamycin、hygromycin、phosphinothricin及geneticin)均表现出剂量效应且能同步抑制芽器官发生、愈伤生长及芽切段生根。     

影响农杆菌介导的木薯基因转化因素的研究

Factors influencing agrobacterium-mediated cassava transformation were investigated. Among the four Agrobacterium strains tested, LBA 4404 (pTOK 233) and LBA 4404 (pBin9GusInt) gave higher transient expression than C 58 C1 (pIG121Hm) and EHA 105 (pBin9Husint). Pretreatment of explants by bombardment or vaccum had no significant effect on transient expression while preinduction of Agrobacterium with acetosyringone showed better effects, and preculture of explants showed worse effects. All the cultivars tested were susceptible to Agrobacterium infection, while the types of explants and the physiological state of the explants had a strong influence on the transient expression efficiency. The 15-day-old somatic cotyledons and the fully expanded leaves from in vitro plantlets were the most susceptible to Agrobacterium infection. The results also showed that all the four selective reagents (hygromycin, geneticin, PPT, and kanamycin) synchronously suppressed the growth of callus, shoot organogenesis and shoot rooting in a dose dependent manner.     

胡萝卜组织培养和高效遗传转化体系的建立

为了建立高效的胡萝卜遗传转化体系,本实验选用3个胡萝卜(Daucuscarotavar.sativa)栽培品种:‘金笋五寸’、‘Carol’和‘改良黑田七寸’,以它们的下胚轴和子叶为外植体,首先建立了高频愈伤诱导体系。在此基础上,以Carol的下胚轴和愈伤组织为受体材料,利用根癌农杆菌LBA4404介导转化质粒pBI121。经X-Gluc染色,证明GUS基因瞬间表达成功,经PCR方法鉴定,证明GUS基因已整合到胡萝卜的染色体中,从而建立了高效的胡萝卜遗传转化体系。     

百合组织培养和遗传转化的研究进展

百合是单子叶球茎类观赏花卉,具有食用和药用的作用。本文综述了国内外百合组织培养和遗传转化研究进展。包括营养器官、生殖器官和原生质体再生系统的建立。详细介绍了基因转化的方法,例如农杆菌、基因枪、电激穿孔等。浅析百合外源基因的表达,例如GUS、半夏凝集素基因、几丁质酶基因等。同时,讨论了百合遗传转化中存在的问题,以期为百合基因工程方面的研究提供依据。     

木薯的再生体系和基因转化方法

木薯是一种很有发展潜力的非常古老的作物,但是木薯的育种工作却处于非常年轻的阶段.随着生命科学技术的发展,木薯育种工作也得到了进一步提高,由传统育种向现代育种转变,从各个方面对木薯品种进行分子改良.本文对近几十年来木薯生物技术研究取得的进展进行了系统的回顾和分析.木薯分子改良包括两个方面:木薯遗传转化体系和基因转化方法的发生、发展.目前研究所用的木薯再生系统途径,主要包括器官发生途径、体细胞胚胎发生途径:常用且有成功先例的木薯基因转化方法,包括农杆菌介导法和基因枪法.最后对前人研究的成果和存在的问题进行了讨论,并展望了以后的发展方向.     

猕猴桃的组织培养和遗传转化研究进展

综述了国内外猕猴桃组织培养和遗传转化研究进展,内容包括花药培养、胚培养、胚乳培养、子叶、叶、茎段等器官培养、原生质体培养以及遗传转化等,并对生物技术在猕猴桃研究中存在的问题以及今后在猕猴桃中的应用前景进行了讨论。     

月季组织培养和遗传转化体系的研究进展

月季通过器官和体细胞胚发生途径都可以获得再生植株,在遗传转化中主要是利用体细胞胚作为转化受体。目前,利用农杆菌介导法和基因枪法已成功将外源基因如报告基因、抗病基因和改变花色的基因等导入月季基因组中。本文对近年来月季组织培养和转基因研究进展进行了综述,为建立月季高效遗传转化体系奠定了理论基础。     

麻类作物组织培养及遗传转化研究进展

概述了国内外红麻等麻类作物快速繁殖、花药培养、原生质体培养、体细胞胚发生、器官发生等几个方面的组织培养以及遗传转化的研究进展,并对存在的问题进行了讨论,提出了进一步研究的一些见解,以期为促进麻类组织培养及遗传转化研究提供科学依据。     

青菜组织培养和转化系统的初步建立

青菜(Brassica campestris ssp. chinensisL.),属十字花科芸苔属,是我国长江流域及南方各省普遍栽培的重要绿叶蔬菜。目前迫切需要培育抗病,抗虫,抗逆和具有优良农艺性状的新品种。用常规育种和细胞工程方法在青菜品种选育方面取得了一定的进展,但是受到种质资源,杂交不亲和性以及耗时长的局限,而遗传工程可提供一个有较大潜力的方法。     

木薯SYP基因的序列特征及表达分析

植物突触融合蛋白(SYP)是一类与植物细胞内囊泡介导转运有关的蛋白。部分SYP基因与植物对生物和非生物胁迫的响应有关。该文利用生物信息学工具分析了木薯(Manihot esculenta)SYP基因及其蛋白结构、核苷酸多态性和系统进化关系, 并利用RT-PCR技术检测了木薯不同组织中SYP基因的表达。结果表明, 木薯SYP基因及其蛋白结构均具有明显的规律性和家族成员间的保守性; SYP基因的cDNA在基因间以及不同品种间具高度一致性, 核苷酸变异以同义替换为主。进化分析表明, 植物SYP基因可分为2个亚家族, 木薯SYP基因倾向于与蓖麻(Ricinus communis)SYP基因聚在进化树同一分支的末端。半定量RT-PCR分析表明, 5个木薯SYP家族成员具有组织特异性。上述研究结果为木薯SYP基因功能研究和功能单核苷酸标记的开发奠定了重要基础。     

PIG-mediated cassava transformation using positive and negative selection

 In order to develop new selection systems for production of transgenic cassava (Manihot esculenta Crantz), two different selection regimes were assessed for their efficiency on regeneration of transgenic cassava plants: positive selection using mannose and negative selection using hygromycin. Explants from somatic cotyledons and embryogenic suspensions were used as target tissues in the transformation experiments and bombarded using the particle inflow gun. Different culture and selection strategies were assessed to optimise the selection protocols. For the first time transgenic plants could be obtained using positive, and in the case of embryogenic suspensions, hygromycin-based negative selection. The stably transformed nature of the regenerated cassava plant lines and the expression of the transgenes were verified with PCR, RT-PCR, Southern and northern analyses. A rooting test for transgenic plants on a medium supplemented with mannose was developed to further improve the efficacy of the positive selection system. Our results demonstrate that it is possible to obtain transgenic cassava plants using non-antibiotic positive selection. Received: 21 February 2000 / Revision received: 2 May 2000 / Accepted: 5 May 2000     

Regeneration of cassava plants via shoot organogenesis

A novel regeneration system based on direct shoot organogenesis is described for cassava. Plants could be regenerated at high frequency by inducing shoot primordia on explants derived from cotyledons of cassava somatic embryos. After a passage on elongation medium, the regenerated shoots were easily rooted in hormone-free medium and could be successfully transplanted to soil. Using the shoot-organogenesis-based regeneration method, up to eight transplantable plantlets per explant could be regenerated. The system was optimised first for one cassava cultivar, and then its transferability to three other cultivars was demonstrated. This method widens the scope of in vitro regeneration modes of cassava, and is also compatible with Agrobacterium-mediated transformation. To develop an efficient system for production of somatic embryos for regeneration experiments, conditions for inducing primary and cycling somatic embryos were also studied, and highly efficient plant regeneration via germination of somatic embryos was achieved using maltose instead of sucrose in the culture medium, and combining paclobutrazol with 2,4-dichlorophenoxyacetic acid in the embryo induction medium. Received: 25 January 1997 / Revision received: 10 February 1997 / Accepted: 20 February 1997     

木薯体细胞胚胎发生及植株再生研究

对木薯体细胞胚胎发生的影响因素进行了优化研究。结果表明,基因型对木薯体细胞胚胎发生影响很大,在供试的六个品种中,“华南 8 号”的体细胞胚胎发生率和产胚量最高,分别为 65% 和19个;侧芽茎尖为最佳外植体,体细胞胚胎发生的最佳培养基为MS +0.5mg/L CuSO4 + 4 mg/L 2,4-D。同时,对木薯体细胞胚再生成完整植株的主要影响因素作了分析,建立了一个高效的植株再生体系。     

影响根癌农杆菌介导的木薯遗传转化因素分析

采用根癌农杆菌介导的叶盘转化法,以我国南方地区主栽木薯品种—华南8号的胚状体子叶为受体,对影响木薯遗传转化效率的主要因素进行了分析。研究结果表明,在木薯的遗传转化中,选用GV3101作为浸染外植体的农杆菌菌株,将感染时间和共培养时间分别控制在30~45 min和3~4 d、菌液浓度（OD₆₀₀）采用0.45、并添加200 μmol·L^-1的乙酰丁香酮（AS）均可明显提高其转化效率,但若对外植体进行预培养反而会降低其转化效率。利用该体系从453块外植体中共转化获得10株抗性再生植株,经PCR和Southern杂交检测,有8株木薯的基因组中已整合进了外源基因glgC336,转化率为1.77%。     

Production of stably transformed cassava plants via particle bombardment

 A novel protocol, based on biolistics and regeneration via organogenesis, was developed for genetic transformation of cassava (Manihot esculenta Crantz). The in vitro performance of cassava cultivars CMC40, MPer183 and MCol22 was evaluated, and the regeneration protocol was modified to improve shoot production from explants for transformation experiments. Somatic cotyledons were used as a target tissue in the transformation experiments using the Particle Inflow Gun and a plasmid containing the uidA gene in transient assays. The effect of different parameters for particle bombardment efficiency, including the amount of DNA used, the flying distance of the projectiles and the pre- and post-plasmolysis time of the target tissue, was evaluated and the conditions were partially optimised. Stably transformed cassava plants of cvs. MCol22 and TMS60444 were produced using the partially optimised conditions and two different vector constructs carrying the hpt gene as the selectable marker. The selection protocol was optimised further, and a rooting test was developed for screening the regenerants for antibiotic resistance to reduce the number of escapes obtained after primary selection. The production of stably transformed cassava lines and the expression of the transgenes was verified by Southern blot analysis and RT-PCR. Received: 10 December 1999 / Revision received: 12 January 2000 / Accepted: 7 February 2000     

Cassava genetic transformation and its application in breeding

As a major source of food, cassava (Manihot esculenta Crantz) is an important root crop in the tropics and subtropics of Africa and Latin America, and serves as raw material for the production of starches and bioethanol in tropical Asia. Cassava improvement through genetic engineering not only overcomes the high heterozygosity and serious trait separation that occurs in its traditional breeding, but also quickly achieves improved target traits. Since the first report on genetic transformation in cassava in 1996, the technology has gradually matured over almost 15 years of development and has overcome cassava genotype constraints, changing from mode cultivars to farmer-preferred ones. Significant progress has been made in terms of an increased resistance to pests and diseases, biofortification, and improved starch quality, building on the fundamental knowledge and technologies related to planting, nutrition, and the processing of this important food crop that has often been neglected. Therefore, cassava has great potential in food security and bioenergy development worldwide.     

How in vitro light affects growth and survival of ex vitro cassava

Cassava is one of the most important food crops in Africa. Meristem culture is an effective method of eliminating viruses and other systemic diseases spread through the vegetative propagation of stems. However, in semi‐arid conditions, survival of ex vitro plants in the field is often disappointing. When an increasing range of light regimes in vitro was provided, the fresh and dry masses more than doubled their values between 29 and 369 mmol s^?1 m^?2 PPFDs. Increases in numbers of senescent leaves and stem thickness were also recorded with increasing PPFD. However, PPFD above 101 mmol s^?1 m^?2 resulted in 30–70% reduction in plant survival, with the thin plants with the smallest fresh and dry masses being the ones with highest survival rates. High light and temperature levels in the greenhouse were also found to be critical for plant survival. It was also shown that transpirational loss from detached leaves and epicuticular wax deposits were not good indicators for predicting survival of ex vitro cassava plantlets during acclimatisation.     

A methodology for recovering cassava plants from shoot tips maintained in liquid nitrogen

Shoot tips of in vitro-grown plantlets of cassava (Manihot esculenta Crantz), representing a wide range of germplasm, were cryopreserved as follows: pre-cultured for 3 days, cryoprotected and dehydrated for 1 h, then frozen in liquid nitrogen using a six-step protocol. After 3 h in liquid nitrogen, the shoot tips were removed, rapidly warmed, and recultured sequentially in three recovery media. After 2 weeks, the regeneration of frozen shoot tips was completed. Genotypes with a low response were identified. Their response was attributed to the effects of pre and post-freezing steps. Refining the methodology led to a consistent 50–70% plant recovery.Abbreviations DMSO Dimethylsulfoxide - MS Murashige and Skoog medium (1962) - LN liquid nitrogen     

植物生长调节物质IP－1号对木薯产量及其生物性状的影响

１９９０和１９９１年在木薯（ＭａｎｉｈｏｔｅｓｃｕｌｅｎｔａＣｒａｎｔｚ）生长期以植物生长调节物质ＩＰ－１号０,２０,３０和４０ｐｐｍ进行叶面喷洒,结果表明：３０ｐｐｍ处理可使木薯块根产量平均增加５４．４４％,块根淀粉含量平均提高２０．８１％。单株最大薯重提高３１．５５％,块根数增加２１．１７％,块根长度增长１７．６２％,地上部鲜重增加３４．３６％,植株高度增加４．３６％,植株收获期保留青叶数增加１９．４２％,主茎直径增加６．２６％,块根直径增加２．５８％,叶片的叶绿素和蛋白质含量分别提高５．５７％和２５．９６％,叶片光合作用强度提高１５．８６％,而对主茎高度、主茎节数没有明显影响。