发根农杆菌Ri质粒及其在植物科学中的应用

阐述了发根农杆菌的性质及Ri质粒的结构和功能,介绍了毛状根的诱导及其鉴定方法,并对Ri质粒在植物基因工程、植物次生代谢产物生产、植物品种改良和植物栽培等方面的应用进行了综述。     

影响毛状根生长及其次生代谢产物合成因素的研究进展

利用发根农杆菌转化药用植物,并对毛状根进行离体培养,大量提取重要成分,是药用资源植物可持续发展的有效途径之一。讨论了毛状根的诱导、影响毛状根生长及次生代谢产物形成的因素、利用毛状根培养技术生产植物次生代谢物的最新研究进展。     

何首乌毛状根的诱导和培养(简报)

发根农杆菌（Agrobacteriumrhizogenes）侵染后,通过其Ri质粒的T－DNA片段在植物细胞基因组中整合,诱导形成毛状根（hairyroot）l’l。利用生长迅速、生产效能高而稳定的毛状根培养物生产药用植物的次生物质,已有不少报道「一习。但未见发根农杆菌对何首乌遗传转化的报道。何首乌（Polygonu。。ltij7orumThunb．）为常用中药,其块根为乌须发、悦颜色、益精养血和抗衰老的要药。本文报道发根农杆菌对何首乌遗传转化的实验结果,为今后用毛状根培养物生产何首乌的次生物质打下基础。l材料和方法细菌菌株及培养发根农杆菌（Agrobacteri…     

Ri质粒诱导的植物发根培养系及其应用

阐述了具Ri质粒的发根农杆菌的生物学特性,介绍了毛状根的诱导、筛选及增殖培养的具体方法,提出了农杆菌转化植物细胞的影响因素,对国内外利用植物发根培养系进行次生代谢物质生产方面的研究进行了综述,并对Ri质粒诱导的发根培养系的应用作了展望。     

何首乌毛状根培养及其活性成分的产生

利用发根农杆菌LBA940 2诱导药用植物何首乌产生毛状根。PCR扩增和Southern印迹杂交实验证实发根农杆菌中Ri质粒的T-DNA片段已整合进入植物核基因组中。经过基本培养基的筛选和毛状根生长动力学的考察 ,确立了何首乌毛状根在MS培养基中的最佳继代时间为 30d左右。HPLC实验测定结果显示 ,毛状根培养物中大黄酸的含量是原植物的 2 85倍     

转化毛状根获得萝芙木生物碱的研究

药用植物萝芙木(Rauvolfia verticillata)的无菌苗被含有Ri质粒的发根农杆菌(Agro—bacterium rhizogenes)感染后,诱导出毛状根(Hairy root)。将毛状根分离,除菌后,在不合激素的Ms固体或液体培养基上培养,从296个株系中筛选出Rv 19、RV 26,RV 37和RV53Eq个生长速度快、分枝数量多等优良特性的无性系。对毛状根中次生代谢产物的提取和测定表明,四个无性系均含有原植物体所具有的吲哚生物碱。为应用转化毛状根技术获得植物次生代谢产物开辟了一条新的有效途径。     

Ri质粒诱导的植物发根培养系及其应用

本文阐述了具Ri质粒的发根在杆菌的生物学特性,介绍了毛状根的诱导、筛选及增殖培养的具体方法,提出了农杆菌转化植物细胞的影响因素,对国内外利用植物发根培养系进行次生代谢物质生产方面的研究进行了综述,并对Ri质粒诱导的发根培养系的应用做了展望。     

五加科植物发状根诱导研究进展

五加科植物多为重要的中药材,利用发根农杆菌诱导五加科药用植物产生发状根,并从中获取有用的次生代谢产物,是保护五加科珍稀药用植物资源和实现有效次生代谢物质工业化生产的有效途径。该文在概述发根农杆菌转化药用植物研究历程和转化机理研究的基础上,对近年来在发根农杆菌诱导五加科植物的种类及诱导率、影响发根农杆菌诱导五加科植物的各种因素和利用发根农杆菌诱导五加科植物获得再生植株等方面研究进行了重点分析,并对今后亟需研究的几个重点方向进行了展望,以期为五加科药用植物的良性开发和合理利用提供参考。     

发根农杆菌转化龙胆再生植株的研究

本文利用具Ri质粒的发根农杆菌(Agrobacterium rhizogenes)通过叶盘法对药用植物龙胆(Gentiana manshurica Kitagawa)进行了转化实验,发根农杆菌15834感染龙胆,诱发产生毛状根,并得到再生的龙胆植株,冠瘿碱检测实验表明,再生植株显示甘露碱带(mannopine),说明发根农杆菌Ri质粒的T-DNA部分已转移到龙胆植物细胞中。再生的龙胆丛生苗可以在不含激素的简化培养基上快速无限繁殖,转化的龙胆植株明显具有发达的根系,且根部龙胆苦甙含量比对照高,从而为东北龙胆栽培事业的发展以及龙胆有效成份的工业化生产提供了新的途径。     

Ri质粒基因转化植物细胞的机理

发根农杆菌(Agrobacteriumrhizogenes)含有一个约250Kb的大质粒,与Ti质粒转化植物细胞产生冠瘿瘤(crowngall)相似,它也可以侵染双子叶植物受伤部位的细胞并产生大量不定根(所谓发根或毛状根,(hairyroot),因此,这个大质粒被称为Ri质粒(根诱导质粒,Root-inducingplasmid)。     

Transgenic hairy roots. recent trends and applications

Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic roots produced by A. rhizogenes infection is characterized by high growth rate and genetic stability. These genetically transformed root cultures can produce higher levels of secondary metabolites or amounts comparable to that of intact plants. Hairy root cultures offer promise for production of valuable secondary metabolites in many plants. The main constraint for commercial exploitation of hairy root cultures is their scaling up, as there is a need for developing a specially designed bioreactor that permits the growth of interconnected tissues unevenly distributed throughout the vessel. Rheological characteristics of heterogeneous system should also be taken into consideration during mass scale culturing of hairy roots. Development of bioreactor models for hairy root cultures is still a recent phenomenon. It is also necessary to develop computer-aided models for different parameters such as oxygen consumption and excretion of product to the medium. Further, transformed roots are able to regenerate genetically stable plants as transgenics or clones. This property of rapid growth and high plantlet regeneration frequency allows clonal propagation of elite plants. In addition, the altered phenotype of hairy root regenerants (hairy root syndrome) is useful in plant breeding programs with plants of ornamental interest. In vitro transformation and regeneration from hairy roots facilitates application of biotechnology to tree species. The ability to manipulate trees at a cellular and molecular level shows great potential for clonal propagation and genetic improvement. Transgenic root system offers tremendous potential for introducing additional genes along with the Ri T-DNA genes for alteration of metabolic pathways and production of useful metabolites or compounds of interest. This article discusses various applications and perspectives of hairy root cultures and the recent progress achieved with respect to transformation of plants using A. rhizogenes.     

Hairy Root and Its Application in Plant Genetic Engineering

Agrobacterium rhizogenes Conn. causes hairy root disease In plants. Hairy root-Infected A. rhizogenes Is characterlzed by a high growth rate and genetic stability. Hairy root cultures have been proven to be an efficient means of producing secondary metabolites that are normally biosyntheslzed In roots of differentiated plants. Furthermore, a transgenlc root system offers tremendous potential for introducing additional genes along with the RI plasmld, especially with modified genes, into medicinal plant cells with A. rhizogenes vector systems. The cultures have turned out to be a valuable tool with which to study the biochemical properties and the gene expression profile of metabolic pathways. Moreover, the cultures can be used to elucidate the Intermediates and key enzymes Involved In the biosynthesis of secondary metabolites. The present article discusses various appllcations of hairy root cultures in plant genetic engineering and potential problems aseoclsted with them.     

Hairy root culture for mass-production of high-value secondary metabolites

Plant cell cultivations are being considered as an alternative to agricultural processes for producing valuable phytochemicals. Since many of these products (secondary metabolites) are obtained by direct extraction from plants grown in natural habitat, several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of these secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution in the production of secondary metabolites. Most of the research efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Hairy root cultures offer promise for high production and productivity of valuable secondary metabolites (used as pharmaceuticals, pigments and flavors) in many plants. The main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel. Emphasis has focused on designing appropriate bioreactors suitable to culture the delicate and sensitive plant hairy roots. Recent reactors used for mass production of hairy roots can roughly be divided as liquid-phase, gas-phase, or hybrid reactors. The present review highlights the nature, applications, perspectives and scale up of hairy root cultures for the production of valuable secondary metabolites.     

发根农杆菌介导的长春花高效转基因体系的建立

以长春花幼叶为外植体建立了发根农杆菌介导的长春花高效遗传转化体系,主要技术环节为：用携带有基因表达载体的发根农杆菌R1000侵染幼嫩叶片,侵染的叶片外植体与发根农杆菌共培养2d,外植体移至除菌培养基除菌培养2～3周,切取外植体上诱导长出的毛状根置于筛选培养基上培养1-2周,最后对筛选出的阳性毛状根无性系进行扩繁。筛选出的阳性毛状根经GUS染色和PCR分子鉴定表明,该方法的发根诱导率和阳性转化率分别为82％±2．49％和100％。该转化方法所获得的毛状根系数量大、质量高、遗传稳定且所需时间短,明显优于现有的长春花遗传转化技术,是长春花遗传转化的高效便捷体系。     

发根农杆菌介导的药用植物遗传转化研究

利用发根农杆菌(Agrobacterium rhizogenes)诱导药用植物产生的毛状根具有生长迅速,合成能力强和遗传性稳定等优点,已成为一种新的培养系统。就影响发根农杆菌介导的药用植物遗传转化的因素作一概述。     

褐脉少花龙葵毛状根的诱导、培养及其澳洲茄胺的产生

利用发根农杆茵的遗传转化和液体培养技术,研究了褐脉少花龙葵(solanum nigrum L.Var.Dauciforum)毛状根的诱导和离体培养及其澳洲茄胺的产生以及液体培养过程中培养基中N源和钙的消耗变化.结果表明.发根农杆菌ATCC15834感染褐脉少花龙葵叶片外植体5 d后产生毛状根.感染25d后,约90%的叶片外植体产生毛状根.毛状根能在无外源生长调节剂的MS固体和体培养基上自主生长.PCR扩增结果显示发根农杆菌Ri质粒的rilB和rolC基因已在少花龙葵毛状根基因组中整合并得到表达.所产生的毛状根能产生药用次生物质澳洲茄胺,其含量约为非转化植株根的1.3倍,达到582.05μg/g干重.少花龙葵毛状根液体培养0-5 d内处于生长迟滞期、5-15 d为快速生长期、15d后进入生长平台期.培养基的硝态氮和铵态氮在毛状根液体培养过程中被逐渐吸收和消耗,至培养15 d时铵态氮被消耗殆尽.而硝态氮仍剩余44.7%;培养基中钙的浓度在培养过程中虽逐渐降低,但在培养25d时仍未被完全消耗,其浓度约为起始浓度的43.5%.该结果为今后设计合适的培养基来规模培养褐脉少花龙葵毛状根生产药用次生物质澳洲茄胺提供了可能性.     

发根农杆菌介导药用甘薯西蒙1号的遗传转化

用发根农杆菌A4分别感染药用甘薯西蒙1号的叶片、茎切段、叶柄等外植体,诱导出毛状根,并对毛状根进行了离体培养.采用L9(34)正交设计法优化甘薯西蒙1号的毛状根诱导条件;PCR扩增检测转化毛状根;用高效液相色谱仪检测了毛状根中咖啡酸的含量.结果表明:转化中茎切段是最合适的外植体,最佳感染时间20 min,共培养最佳时间为2天;PCR扩增检测表明发根农杆菌Ri质粒的T-DNA片段已整合进植物的基因组中;经高效液相色谱仪证实毛状根中含有咖啡酸,含量为0.03792 mg/g.     

Hairy root research: recent scenario and exciting prospects

High stability of the production of secondary metabolites is an interesting characteristic of hairy root cultures. For 25 years, hairy roots have been investigated as a biological system for the production of valuable compounds from medicinal plants. A better understanding of the molecular mechanism of hairy root development, which is based on the transfer of Agrobacterium rhizogenes T-DNA into the plant genome, has facilitated its increasing use in metabolic engineering. Hairy roots can also produce recombinant proteins from transgenic roots, and thereby hold immense potential for the pharmaceutical industry. In addition, hairy roots offer promise for phytoremediation because of their abundant neoplastic root proliferation. Recent progress in the scaling-up of hairy root cultures is making this system an attractive tool for industrial processes.