Identification of the genetic locus responsible for non-polar root induction by Agrobacterium rhizogenes 1855

Summary Root proliferation can be induced by Agrobacterium rhizogenes on carrot discs both on the apical and basal surface (facing the root apex and base, respectively) or on the apical surface only, depending on the bacterial strain. This differential response on the two surfaces is denominated polarity. We correlate the polarity of some strains with the absence of an Ri plasmid genetic locus, present in non polar strains such as A. rhizogenes 1855, which bears sequence homology with the auxin genes of Ti plasmid T-DNA. We demonstrate that this locus is responsible for root induction on the basal surface since insertion of a transposon in this region of pRi1855 induces polarity in this strain.     

Plant regeneration from hairy-root cultures transformed by infection with<Emphasis Type="Italic"> Agrobacterium rhizogenes</Emphasis> in<Emphasis Type="Italic"> Catharanthus roseus</Emphasis>

Hypocotyl explants of Catharanthus roseus produced hairy roots when cultured on Murashige and Skoog (MS) basal medium after infection by Agrobacterium rhizogenes. Explants gave rise to adventitious shoots at a frequency of up to 80% when cultured on MS medium supplemented with 31.1 M 6-benzyladenine and 5.4 M -naphthaleneacetic acid. There was a significant difference in the frequency of adventitious shoot formation for each hairy-root line derived from a different cultivar. Plants derived from hairy roots exhibited prolific rooting and had shortened internodes. Approximately half of the plants had wrinkled leaves and an abundant root mass with extensive lateral branching, but otherwise appeared morphologically normal. Plants with hairy roots that were derived from the cultivar Cooler Apricot developed flowers with petals that were white in the proximal region, whereas the wild-type flower petals are red. PCR and Southern blot analyses revealed that plants derived from hairy roots retained the Ri TL-DNA.Abbreviations BA 6-Benzyladenine - MS Murashige and Skoog medium - NAA -Naphthaleneacetic acid - SH Schenk and Hildebrandt mediumCommunicated by I.S. Chung     

A reporter gene under the control of tms or aux promoters is differentially expressed in tobacco and barley protoplasts

Summary Agrobacterium tumefaciens and some Agrobacterium rhizogenes strains possess auxin biosynthesis genes (tms and aux genes respectively), responsible for a de novo auxin biosynthetic pathway in transformed plant cells. A comparison is presented of the potential expression of these genes in a monocotyledonous (barley) and a dicotyledonous plant (tobacco). The promoters of the genes were translationally fused to the -glucuronidase reporter gene and analysed in transient expression experiments. The tms and aux fusions were highly expressed in tobacco, but not in barley. However, the aux enhancer active in tobacco, conferred low -glucuronidase expression in barley when fused to a truncated cauliflower mosaic virus 35S promoter. The results are discussed in relation to the differential responses to Agrobacterium infection in monocots and dicots.Abbreviations CaMV cauliflower mosaic virus - PCR polymerase chain reaction - PEG polyethylene glycol - Mu 4-methyl umbelliferone     

Pleiotropic effect of the insertion of the <Emphasis Type="Italic">Agrobacterium rhizogenes rolD</Emphasis> gene in tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.)

The Agrobacterium rhizogenes rolD gene, coding for an ornithine cyclodeaminase involved in the biosynthesis of proline from ornithine, has been inserted in Lycopersicon esculentum cv Tondino with the aim of studying its effects on plant morphological characters including pathogen defense response. The analysis of plants transgenic for rolD did not show major morphological modifications. First generation transgenic plants however were found to flower earlier, and showed an increased number of inflorescences and higher fruit yield. Transformed plants were also analysed for parameters linked to pathogen defense response, i.e. ion leakage in the presence of the toxin produced by the fungus Fusarium oxysporum f. sp. lycopersici, and expression of the pathogenesis-related PR-1 gene. All the plants harbouring the rolD gene were shown to be more tolerant to the toxin in ion leakage experiments, with respect to the untransformed regenerated controls and the cv Tondino. PR-1 gene expression was quantitated by means of real-time PCR both at the basal level and after treatment with salicylic acid, an inducer of Systemic Acquired Resistance. In both cases the amount of PR-1 mRNA was higher in the transgenic plants. It seems therefore that the transformation of tomato plants with rolD could lead to an increased competence for defense response, as shown by toxin tolerance and increased expression of the Systemic Acquired Resistance marker gene PR-1. The results are finally discussed in view of their possible economic relevance.Communicated by G. Wenzel     

Characterization of transgenic plants derived from hairy roots ofHyoscyamus muticus

Mature plants were regenerated via protoplasts fromAgrobacterium rhizogenes-transformed root cultures ofHyoscyamus muticus L., and chemical analyses were performed on 34 individual plants. The regenerated plants showed strong phenotypic differences from clone to clone as well as from the control plants. Polymerase chain reaction studies revealed that the plants exhibiting the strongest phenotypic alterations contained therol (A, B and C) genes, whereas the plants with fewer alterations had lost them. The plants produced hyoscyamine, scopolamine and a range of different calystegins, and considerable somaclonal variation was observed. Alkaloid production in the plants transgenic for therol genes was clearly reduced. The pattern of calystegins was similar within all the regenerated plants lackingrol genes. Among the plants withrol genes, the calystegin B₁ was not detectable. It seems clear that the presence ofrol genes is detrimental to the alkaloid accumulation in the transgenic plants in contrast to hairy root cultures.Abbreviation PCR Polymerase chain reaction     

Use of roots transformed by Agrobacterium rhizogenes in rhizosphere research: applications in studies of cadmium assimilation from sewage sludges

The use of roots transformed by Agrobacterium rhizogenes in models for the rhizosphere is discussed. A list of species for which transformed root cultures have been obtained is provided and the example of studies of cadmium assimilation from sewage sludges is given to illustrate how transformed root cultures can be used in physiological tests under non-sterile conditions.     

Transformation of <Emphasis Type="Italic">Saussurea medusa</Emphasis> for hairy roots and jaceosidin production

Axenically grown Saussurea medusa plantlets were inoculated with four Agrobacterium rhizogenes strains, and hairy root lines were established with A. rhizogenes strain R1601 in N6 medium. PCR and Southern hybridization confirmed integration of the T-DNA fragment of the Ri plasmid from A. rhizogenes into the genome of S. medusa hairy roots. In N6 medium, maximum biomass of the hairy root cultures was achieved [8 g (dry weight) per liter; growth ratio 35-fold] after 21 days of culture. The amount of jaceosidin extracted from the hairy root cultures was 46 mg/l (production ratio of 37-fold) after 27 days of culture. The maximum jaceosidin content obtained using N6 medium was higher than that obtained with Modified White, MS or B5 medium. In N6 medium, the tip segments were more efficient for hairy root growth and jaceosidin production than the middle and basal regions of the root.Abbreviations AS Acetosyringone - BA Benzyladenine - cef Cefotaxime sodium - DW Dry weight - FW Fresh weight - HPLC High-performance liquid chromatography - IAA Indole-3-acetic acid - km Kanamycin - NAA -Naphthaleneacetic acid - SDS Sodium dodecyl sulfate     

Molecular basis for novel root phenotypes induced by Agrobacterium rhizogenes A4 on cucumber

We have used the wild-type Agrobacterium rhizogenes strain A4 to induce roots on cucumber stem explants. Cultures of transformed roots obtained that were capable of hormone-autonomous growth could be grouped in three phenotypic classes. Of particular interest were extremely thick roots of a type not previously described. Characterization of the transferred DNA and of the expression of the corresponding genes allowed us to determine that the genes rolABC of the TL region of the Ri plasmid are sufficient to induce thin roots similar to those observed in other species, while the aux genes of the TR region are sufficient to induce thick roots. Among clones bearing the aux genes, there was a correlation between level of expression of aux2 and root phenotype.     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated DNA transfer to<Emphasis Type="Italic"> Aesculu</Emphasis>s<Emphasis Type="Italic"> hippocastanum</Emphasis> L. and the regeneration of transformed plants

Hairy roots were induced from androgenic embryos of horse chestnut (Aesculus hippocastanum L.) by infection with Agrobacterium rhizogenes strain A4GUS. Single roots were selected according to their morphology in the absence of antibiotic or herbicide resistance markers. Seventy-one putative transformed hairy root lines from independent transformation events were established. Regeneration was induced in MS liquid medium supplemented with 30 6-benzylaminopurine (BA), and the regenerants were multiplied on MS solid medium containing 10 M BA. Following elongation on MS medium supplemented with 1 M BA and 500 mg/l polyvinylpyrrolidone, the shoots were subjected to a root-inducing treatment. Stable integration of TL-DNA within the horse chestnut genome was confirmed by Southern hybridization. The copy number of transgenes was estimated to be from two to four.Communicated by E.D. Earle     

Isolation of the promoter of a root cap expressed pectinmethylesterase gene from Pisum sativum L. (rcpme1) and its use in the study of gene activity

A genomic clone of a pea pectinmethylesterase encoding gene, rcpme1, was isolated; the promoter region was found to include regions of homology to phenylalanine ammonia lyase (PAL) and nodulin gene promoters. Agrobacterium rhizogenes mediated hairy roots were used for rcpme1 expression and functional analysis in pea. Patterns of rcpme1 expression in cultured hairy roots, measured using uidA encoding -glucuronidase (GUS) as a reporter gene, were distinct from patterns which occur in normal pea roots. No reporter gene expression occurred in transgenic Arabidopsis thaliana, whose roots do not produce border cells. Border cell number from transgenic hairy roots expressing rcpme1 anti-sense mRNA under the control of its 2.75 kb 5 flanking sequence was reduced by > 50%. Nodulation genes of Rhizobium leguminosarum were used as a marker to document that roots with reduced production of border cells and other root cap exudates have a corresponding reduction in levels of biologically active signal molecules. Direct measurements were used to confirm that most of the exudate harvested from young, unwounded roots of normal pea plants is derived from the root tip region where rcpme1 is expressed. The potential application of the rcpme1 gene as a molecular marker for root exudate production is discussed.     

3种农杆菌对茶树发状根诱导的影响

以茶树'福云6号'和'铁观音'成熟种子下胚轴、未成熟种子下胚轴和愈伤组织为材料,以发状根诱导率为指标,探究菌液浓度、农杆菌菌株、外植体类型和预培养时间对发状根诱导的影响.结果表明:(1)菌液浓度OD600在0.4～1.2范围内,'福云6号'成熟种子下胚轴发状根诱导率先升高后降低,ATCC15834在OD 600为0.6...     

Common evolutionary origin of the central portions of the Ri TL-DNA of Agrobacterium rhizogenes and the Ti T-DNAs of Agrobacterium tumefaciens

Analysis of published sequences for Ri TL-DNA (root-inducing left-hand transferred DNA) of Agrobacterium rhizogenes revealed several unsuspected structural features. First, Ri TL-DNA genes are redundant. Using redundancy as a criterion, three regions (left, middle and right) were discerned. The left one, ORFs (open reading frames) 1–7, contains no detectable redundancy. In the middle region a highly diverged gene family was detected in ORFs 8, 11, 12, 13 and 14. The right region contains an apparently recent duplication (ORF 15 =18+17). We interpret the phenomenon of redundancy, particularly in the central region that encodes the transformed phenotype, to be an adaptation that ensures function in a variety of host species. Comparison of Ri TL-DNA and Ti T-DNAs from Agrobacterium tumefaciens revealed common structures, unpredicted by previous nucleic acid hybridization studies. Ri TL-DNA ORF 8 is a diverged Ti T-DNA tms1. Both Agrobacterium genes consist of a member of the diverged gene family detected in the central part of the Ri TL-DNA, but fused to a sequence similar to iaaM of Pseudomonas savastonoi. Other members of this gene family were found scattered throughout Ti T-DNA. We argue that the central region of Ri and the part of Ti T-DNA including ORFs 5–10 evolved from a common ancestor. We present the hypothesis that the gene family encodes functions that alter developmental plasticity in higher plants.     

Genetic transformation of Tylophora indica with Agrobacterium rhizogenes A4: growth and tylophorine productivity in different transformed root clones

We have developed an efficient transformation system for Tylophora indica, an important medicinal plant in India, using Agrobacterium rhizogenes strains LBA9402 and A4 to infect excised leaf and stem explants and intact shoots at different sites. The induction of callus and transformed roots was dependent on the bacterial strain, explant type and inoculation site used. Transformed roots were induced only in explants infected with A. rhizogenes strain A4, while an optimal transformation frequency of up to 60% was obtained with intact shoots inoculated at the nodes. The presence of the left-hand transferred DNA (T_L-DNA) in the genome of T. indica roots induced by A. rhizogenes was confirmed by PCR amplification of the rooting locus genes of A. rhizogenes. Root growth and the production of tylophorine, the major alkaloid of the plant, varied substantially among the nine root clones studied. Both parameters increased over time in liquid cultures, with maximum biomass and tylophorine accumulation occurring within 4–6 weeks of growth in fresh medium. Interestingly, in liquid culture, the culture medium also accumulated tylophorine up to concentrations of 9.78±0.21 mg l^–1.     

Further insight concerning the TL region of the Ri plasmid of Agrobacterium rhizogenes strain A4: Transfer of a 1.9 kb fragment is sufficient to induce transformed roots on tobacco leaf fragments

Summary Disarmed plant transformation vectors were used to assay the ability of subfragments of the T-regions of the Ri plasmid of agropine-type strain A4 of Agrobacterium rhizogenes to induce proliferation of transformed roots on tobacco leaf fragments. We have shown that a 6 kb region of TR-DNA, bearing the presumptive auxin synthesis genes, is capable of inducing transformed roots with an essentially normal phenotype as had been shown previously with the entire TR-region. A 1.9 kb fragment of the 20 kb TL-region is suffcient to induced transformed roots in the absence of exogenous hormones. These roots grow profusely on hormone-free medium, as is typical of roots transformed by the intact TL-DNA.     

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。