新疆雪莲毛状根的诱导及其植株再生体系的建立

利用发根农杆菌R1601、R1000、LBA9402感染新疆雪莲的叶片、叶柄和根段外植体,诱导产生毛状根。毛状根接种量为2.8 g/L(FW)时,20d生长量可达66.7 g/L,黄酮含量达到干重的10.23%。冠瘿碱的检测和rolB基因的PCR分析表明,Ri质粒中的T_DNA片段已经整合到毛状根细胞的基因组中。预培养时间、外植体类型以及发根农杆菌的菌株属性对毛状根诱导有着重要的影响。其中预培养2 d的新疆雪莲根段外植体,经过R1601感染后,毛状根的诱导率可达100%。诱导产生的毛状根在附加生长素的液体培养基中,有少量愈伤组织产生。由毛状根再生的植株与雪莲外植体再生的植株在形态上无明显区别,但前者的黄酮含量仅为后者的53%。     

Expression and inheritance of inserted markers in binary vector carrying Agrobacterium rhizogenes-transformed potato (Solanum tuberosum L.)

Summary Transgenic shoots were regenerated from eight diploid potato hairy root clones obtained by transformation with Agrobacterium rhizogenes harboring next to its wild-type Ri-plasmid a binary vector containing the neomycin phosphotransferase and the -glucuronidase genes. The plants exhibited the typical hairy root phenotype. Of the plants isolated, 58% were tetraploid and 38% were diploid. Flowering and tuberization was much better in the diploid than in the tetraploid plants. Transgenic plants formed a significantly larger root system when grown on kanamycin-containing medium as compared to growth on kanamycin-free medium. Direct evidence for genetic transformation was obtained by opine, neomycin phosphotransferase and -glucuronidase assays, and by molecular hybridization. Fourteen flowering diploid plants were reciprocally crossed with untransformed S. tuberosum plants, but only six were successful. Seedlings obtained from four crosses showed that all traits were transmitted to the offspring. Molecular analysis confirmed the presence of multiple integrations (copies) of both vector T-DNA and Ri-T-DNA. The genetic data, furthermore, suggest that the traits derived from Ri-T-DNA and binary vector T-DNA are linked, as no recombination between the different traits was observed.     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> mediated transformation of <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> and production of flavonoids in hairy roots

Using different explants of in vitro seed grown Scutellaria baicalensis Georgi plantlets, hairy roots were induced following inoculation of Agrobacterium rhizogenes strains A₄GUS, R1000 LBA 9402 and ATCC11325. The A₄GUS proved to be more competent than other strains and the highest transformation rates were observed in cotyledonary leaf explant (42.6 %). The transformed roots appeared after 15–20 d of incubation on hormone free Murashige and Skoog medium. Growth of hairy roots was assessed on the basis of total root elongation, lateral root density and biomass accumulation. Maximum growth rate was recorded in root:medium ratio 1:100 (m/v). Hairy root lines were further established in Gamborg B₅ medium and the biomass increase was maximum from 15 to 30 d. PCR, Southern hybridization and RT-PCR confirmed integration and expression of left and right termini-linked Ri T-DNA fragment of the Ri plasmid from A₄GUS into the genome of Scutellaria baicalensis hairy roots. GUS assay was also performed for further integration and expression. All the clones showed higher growth rate them non-transformed root and accumulated considerable amounts of the root-specific flavonoids. Baicalin content was 14.1–30.0 % of dry root mass which was significantly higher then that of control field grown roots (18 %). The wogonin content varies from 0.08 to 0.18 % among the hairy root clones which was also higher than in non-transformed roots (0.07 %).     

Characterization of root clones obtained after transformation of monohaploid and diploid potato genotypes with hairy root inducing strains of Agrobacterium

A procedure for transformation of monohaploid and diploid potato genotypes through infection of stem internodes with hairy root inducing strains of Agrobacterium is described. Hairy roots induced by A. rhizogenes strain LBA9402 and A. tumefaciens strain LBA1020, both containing the Ri1855 plasmid, were analysed for phenotype, growth and development, and opine expression. The ploidy level of the hairy roots was determined by measurements of the nuclear DNA content and the chromosome number. The genotypes of potato (8 monohaploids, 2 diploids) greatly differed in their response to transformation, i.e. the frequency of stem internodes with primary hairy roots, the number of roots per internode and their phenotype. Transformation efficiency was lower in most of the monohaploid genotypes as compared to that in diploid genotypes. Hairy root clones could be established in 4 of the 8 monohaploid genotypes and in both diploid genotypes after subculturing of primary hairy roots. Hormone autotrophy was observed in all the root clones. The root clones varied in their phenotype and opine expression; opine expression was found in only 50% of the clones. Twenty-five of the 26 hairy root clones of the diploid genotypes showed only parental (diploid) chromosome number, even after 6 months of culture, suggesting genetic stability during the transformation and in the resulting hairy roots. However, in monohaploid genotypes the hairy root clones were either diploid or tetraploid. The transformation of monohaploid and diploid potato genotypes can be an efficient system for the establishment of a series of genetic marker lines for gene mapping.     

发根土壤杆菌体外转化甘草子叶及下胚轴

利用发根土壤杆菌农杆碱型15834、A_4菌株和甘露碱型8196、K_(599)菌株体外转化甘草子叶及下胚轴。结果表明:除K_(599)菌株外,其余三种菌株都能诱导产生甘草发状根无性系。其中15834菌株的诱导率最高,下胚轴的诱导率高于子叶。组织学观察表明,在感染后4天左右,下胚轴维管束鞘外细胞、特别是形成层部分细胞大量启动形成分生细胞及分生细胞团,并进一步分裂形成根原基。6—8天后根原基具单向极性生长形成完整的发状根结构。发状根能在无外源激素的培养基上迅速生长。高压纸电泳检测到发状根中存在农杆碱及甘露碱,说明Ri质粒T-DNA编码的这两种冠瘿碱合成酶基因在甘草细胞中得到了表达。     

发根农杆菌介导的菠菜毛状根遗传转化体系的建立

发根农杆菌(Agrobacterium rhizogenes)侵染植物后可诱导植物产生毛状根。菠菜(Spinacia oleracea)是常见的食用蔬菜, 目前尚未见菠菜毛状根的研究报道。经筛选得到适合诱导菠菜毛状根的发根农杆菌菌株LBA9402, LBA9402侵染菠菜外植体茎后, 毛状根的诱导率最高可达16%。菠菜毛状根呈白色, 具有丰富的根毛, 能在无外源激素的固体培养基上快速增殖生长。通过诱导菠菜毛状根产生愈伤组织并进行分化, 获得了菠菜毛状根的再生植株, 再生率为8%。此外, LBA9402可将含有Ri质粒的T-DNA和携带外源GFP基因的Ti质粒T-DNA共同导入外植体中。PCR检测和荧光显微观察结果显示, rolB及GFP基因在菠菜毛状根基因组中稳定表达, 共转化频率为50%。     

发根农杆菌介导的菠菜毛状根遗传转化体系的建立

发根农杆菌(Agrobacterium rhizogenes)侵染植物后可诱导植物产生毛状根。菠菜(Spinacia oleracea)是常见的食用蔬菜, 目前尚未见菠菜毛状根的研究报道。经筛选得到适合诱导菠菜毛状根的发根农杆菌菌株LBA9402, LBA9402侵染菠菜外植体茎后, 毛状根的诱导率最高可达16%。菠菜毛状根呈白色, 具有丰富的根毛, 能在无外源激素的固体培养基上快速增殖生长。通过诱导菠菜毛状根产生愈伤组织并进行分化, 获得了菠菜毛状根的再生植株, 再生率为8%。此外, LBA9402可将含有Ri质粒的T-DNA和携带外源GFP基因的Ti质粒T-DNA共同导入外植体中。PCR检测和荧光显微观察结果显示, rolB及GFP基因在菠菜毛状根基因组中稳定表达, 共转化频率为50%。     

Genetic transformation of <Emphasis Type="Italic">Gentiana macrophylla</Emphasis> with <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>: growth and production of secoiridoid glucoside gentiopicroside in transformed hairy root cultures

Hairy root cultures of Gentiana macrophylla were established by infecting the different explants four Agrobacterium rhizogenes strains namely A₄GUS, R1000, LBA 9402 and ATCC11325, and hairy root lines were established with A. rhizogenes strain R1000 in 1/2 MS + B₅ medium. Initially, 42 independent hairy root clones were maintained and seven clones belongs to different category were evaluated for growth, morphology, integration and expression of Ri T-DNA genes, and alkaloid contents in dry root samples. On the basis of total root elongation, lateral root density and biomass accumulation on solid media, hairy root clones were separated into three categories. PCR and Southern hybridization analysis revealed both left and right T-DNA integration in the root clones and RT-PCR analysis confirmed the expression of hairy root inducible gene. GUS assay was also performed to confirm the integration of left T-DNA. The accumulation of considerable amounts of the root-specific secoiridoid glucosides gentiopicroside was observed in GM1 ( and ) and the GM2 ( and DNA) type clones in considerably higher amount whether as two but callus-type clones (GM3) accumulated much less or only very negligible amounts of gentiopicroside. Out of four media composition the 1/2 MS + B₅ vitamin media was found most suitable. We found that initial establishment of root cultures largely depends on root:media ratio. Maximum growth rate was recorded in 1:50 root:media ratio. The maximum biomass in terms of fresh weight (33-fold) was achieved in 1/2 MS + B₅ media composition after 35 days in comparison to sixfold increase in control. The biomass increase was most abundant maximum from 15 to 30 days. Influence of A. rhizogenes strains and Ri plasmid of hairy root induction, the possible role of the T_L-DNA and T_R-DNA genes on growth pattern of hairy root, initial root inoculum:media ratio and effect of media composition is discussed.     

Structure and function of root-inducing (Ri) plasmids and their relation to tumor-inducing (Ti) plasmids

The abilities of Agrobacterium tumefaciens and A. rhizogenes to transform dicotyle-dons and cause crown gall and hairy root disease are caused by the presence of tumor inducing (Ti) and root inducing (Ri) plasmids. During transformation plasmid T-DNA (transferred DNA) is inserted into the plant genome. The T-region is flanked by 25 bp direct repeats, which are essential for transfer. The T-regions contain oncogenes that are expressed in the plants. Some of these code for enzymes that synthesize auxin or cytokinin. Another type, present in Ri plasmids only, appears to impose a high hormone sensitivity on the infected tissue. The T-DNA also contains genes for enzymes synthesizing opines, which the bacteria catabolize. The T-DNA transfer is initiated by the induction of genes in the virulence (vir) region of the plasmid by phenolic compounds secreted by wounded tissue. The products of the vir -genes and of chromosomal genes mediate transfer of T-DNA to the plant cells. Crown gall disease is caused by production of auxin and cytokinin by the transferred T-DNA. The T-DNA of Ri plasmids codes for at least three genes that each can induce root formation, and that together cause hairy root formation from plant tissue. Current results indicate that the products of these genes induce a potential for increased auxin sensitivity that is expressed when the transformed cells are subjected to a certain level of auxin. After this stage the transformed roots can be grown in culture without exogenous supply of hormones.     

Ri T-DNA对盾叶薯蓣的遗传转化及薯蓣皂甙元产生的影响

利用农杆菌介导法成功地将Pd T-DNA转入药用植物盾叶薯蓣,产生了毛状根,经分子信标探针检测农杆菌Pd质粒上的T-DNA已整合进植物基因组中。研究建立了毛状根大量快速繁殖技术,基本技术要求为：1／2 MS液体培养基,28℃培养温度,350lux弱光条件下有利于毛状根的增殖培养,提高生物量。HPLC测定结果显示,转基因获得的毛状根其薯蓣皂甙元的含量分别是微块茎、愈伤组织和植物体合成量的5．68倍、6．12倍和2．68倍。     

A reliable and efficient protocol for inducing genetically transformed roots in medicinal plant Nepeta pogonosperma

Nepeta pogonosperma is an important medicinal plant with anti-inflammatory effects. An efficient and reliable transformation system for this plant was developed through optimization of several factors which affected the rate of Agrobacterium rhizogenes mediated transformation. Five bacterial strains, A4, ATCC15834, LBA9402, MSU440 and A13, two explant types, leaves and stems, and several co-cultivation media were examined. The maximum rate of hairy root induction was obtained from stem explants using MSU440 and ATCC15834 bacterial strains. A drastic increase in the frequency of transformation (91 %) was observed when MS medium lacking NH₄NO₃, KH₂PO₄, KNO₃ and CaCl₂. Hairy root lines were confirmed by polymerase chain reaction (PCR) using primers of the rolB gene. According to Southern blot analysis, one T-DNA copy was inserted into each of the hairy root lines. In the present study, transgenic hairy roots have been obtained trough genetic transformation by A. rhizogenes harbouring two plasmids, the Ri plasmid and pBI121 binary vector harbouring gus reporter gene. Expression of the gus gene in transgenic hairy root was confirmed by histochemical GUS assay.     

Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus

In order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the T-DNA of the Ri plasmid together with the tobacco pmt gene under the control of CaMV 35S promoter, into the genome of Datura metel and Hyoscyamus muticus. This gene codes for putrescine:SAM N-methyltransferase (PMT; EC. 2.1.1.53), which catalyses the first committed step in the tropane alkaloid pathway. Hairy root cultures overexpressing the pmt gene aged faster and accumulated higher amounts of tropane alkaloids than control hairy roots. Both hyoscyamine and scopolamine production were improved in hairy root cultures of D. metel, whereas in H. muticus only hyoscyamine contents were increased by pmt gene overexpression. These roots have a high capacity to synthesize hyoscyamine, but their ability to convert it into scopolamine is very limited. The results indicate that the same biosynthetic pathway in two related plant species can be differently regulated, and overexpression of a given gene does not necessarily lead to a similar accumulation pattern of secondary metabolites.     

Agrobacterium rhizogenes-transformed roots of coffee (Coffea arabica): conditions for long-term proliferation, and morphological and molecular characterization

Background and Aims: The aims of this study were to set up proliferation conditionsfor hairy roots of Coffea arabica regenerated after transformationby Agrobacterium rhizogenes strain A4-RS, and to carry out themorphological and molecular characterization of hairy root clonesmaintained over the long term. Methods: Auxin supply, light conditions and sucrose concentration weremodified with the aim of establishing efficient root proliferationconditions. The morphological variability among 62 establishedhairy root clones was phenotyped by scanning the roots and analysingthe images using ‘whinRHIZO’ software procedures.PCR analysis of integration in transformed root cells of roland aux oncogenes from the T-DNA of the Ri plasmid was usedto study the molecular variability among clones. Key Results: Auxin supply was necessary to obtain and stimulate growth andbranching, and IBA applied at 0·5 µM was the mostefficient auxin. Significant differences were shown among the62 clones for total root length and for the percentage of fineroots. These variables were stable across subcultures and couldhence be used for efficient characterization of hairy root clones.The majority of hairy root clones (86 %) exhibited non-significantphenotype differences with non-transformed roots. Eight cloneswere significantly different from the non-transformed controlsin that they possessed a low proportion of fine roots. Two otherhairy root clones grew significantly faster than the other clones.The PCR analysis revealed a low variability in the integrationof rol and aux oncogenes in transformed root cells. The T_R-DNAwas never integrated as aux1 and aux2 genes were not found,although rolB and rolC genes from the T_L-DNA were always present. Conclusions: The discovery of low morphological variability among coffeehairy roots together with the identification of morphologicalvariables allowing easy identification of phenotypically alteredclones represent two important results. They make hairy rootsa possible, and efficient, tool for functional-genomic studiesof coffee root genes.     

Stable transformation and direct regeneration in Coffea canephora P ex. Fr. by Agrobacterium rhizogenes mediated transformation without hairy-root phenotype

A system for genetic transformation of Coffea canephora by co-cultivation with Agrobacterium rhizogenes harbouring a binary vector has been developed. The objective of the present study was the genetic transformation and direct regeneration of transformants through secondary embryos bypassing an intervening hairy root stage. Transformants were obtained with a transformation efficiency up to 3% depending on the medium adjuvant used. A. rhizogenes strain A4 harbouring plasmid pCAMBIA 1301 with an intron uidA reporter and hygromycin phosphotransferase (hptII) marker gene was used for sonication-assisted transformation of Coffea canephora. The use of hygromycin in the secondary embryo induction medium allowed the selection of transgenic secondary embryos having Ri T-DNA along with the T-DNA from the pCAMBIA 1301 binary vector. In addition transgenic secondary embryos devoid of Ri-T-DNA but with stable integration of the T-DNA from the binary vector were obtained. The putative transformants were positive for the expression of the uidA gene. PCR and Southern blot analysis confirmed the independent, transgenic nature of the analysed plants and indicated single and multiple locus integrations. The study clearly demonstrates that A. rhizogenes can be used for delivering transgenes into tree species like Coffea using binary vectors with Agrobacterium tumefaciens T-DNA borders.     

Effects of light and growth regulators on adventitious bud formation in horseradish (Armoracia rusticana)

Summary To clarify that the presence of Ri T-DNA genes are not prerequisite for the light-induced bud formation in horseradish (Armoracia rusticana) hairy roots, leaf and root segments of nontransformed horseradish plants were used as explants. Bud formation from nontransformed tissues was observed in hormone-free medium under 16 h daylight conditions, but not under continuous darkness. To investigate the effects of growth regulators on bud formation, leaf and root explants were treated with auxin (1-naphthaleneacetic acid; NAA) and / or cytokinin (6-benzyl-aminopurine; BA). The most effective treatment in the dark to stimulate bud formation was BA at 1 mg·1^-1. These results show that adventitious bud formation in horseradish can be induced by light and growth regulators, and especially cytokinin, may be involved in bud formation, irrespective of whether the tissues were transformed with Ri T-DNA.Abbreviations BA 6-benzyl-aminopurine - NAA 1-Naphthaleneacetic acid - MS Murashige & Skoog (1962) medium     

The non-conserved region of cucumopine-type Agrobacterium rhizogenes T-DNA is responsible for hairy root induction

The T-DNA regions of three strains of Ri plasmids 1855, 8196, 2659 (agropine, mannopine and cucumopine type respectively) share two highly conserved regions flanking a non-homologous central part [1,2]. We have cloned segments of the cucumopine Ri plasmid 2659 T-DNA in the binary vector system Bin 19 and infected carrot discs with recombinant Agrobacterium strains. We show here that the central non-conserved region is crucial in hairy root induction as it is sufficient to induce rooting on the apical (auxin-rich) surface of carrot discs; in order to observe rooting on the basal (auxin-depleted) side of the discs, a longer T-DNA fragment, also encompassing part of the right conserved region, had to be utilized in conjunction with a Agrobacterium strain carrying aux genes. Differences of growth properties in culture are exhibited by roots transformed with different fragments of pRi 2659 T-DNA, although all transformed roots show the plagiotropic behaviour typical of hairy roots.     

发根农杆菌Ri质粒rolB基因研究进展（综述）

RiT-DNArolB基因是发根农杆菌转化植物的决定因子,rolB基因表达引起转基因植物形成大量毛状根（hairy root)。本文介绍近年来rolB基因的位点,表达与调控及RolB蛋白结构与功能方面的研究进展。     

T-DNA length variability in mannopine hairy root: more than 50 kilobasepairs of pRi T-DNA can integrate in plant cells

Independent carrot (Daucus carota) hairy root lines were established by inoculation of discs taken from the same carrot with Agrobacterium rhizogenes 8196 and A. tumefaciens C58C1(pRi8196) carrying pRi8196. Several lines were compared with respect to T-DNA length. One of them was found to have integrated sequences covering more than 50 kbp of the Ri plasmid