Agrobacterium rhizogenes T-DNA genes capable of inducing hairy root phenotype

Summary Segments of the TL-DNA of the agropine type Ri plasmid pRi 1855 encompassing single and groups of open-reading frames were cloned in the Ti plasmid-derived binary vector system Bin 19. Leaf disc infections on Nicotiana tabacum led to transformed plants, some of which showed typical hairy root phenotypes, such as the wrinkled leaf morphology, excessive and partially non geotropic root systems and the ability of leaf explants to differentiate roots in a hormone-free culture medium. Particularly interestingly, most of these traits were shown by plants transformed with a TL-DNA segment encompassing the single ORF 11, corresponding to the rolB locus. Hairy root can be induced by this latter T-DNA segment on wounded stems of tobacco plants; hairy root induction on carrot discs requires, on the contrary, a more complex complement of TL-DNA genes.Abbreviations YMB yeast mannitol broth - MS Murashige and Skoog medium - 6-BAP 6-benzylaminopurine - NAA naphthalene acetic acid - Km kanamycin - Cb carbenicillin     

Independent induction of transformed roots by the TL and TR regions of the Ri plasmid of agropine type Agrobacterium rhizogenes

Summary To analyse the respective role of TL- and TR-DNA in root induction by agropine-type Agrobacterium rhizogenes Ri plasmids, deletions covering the TL- or the TR-regions were constructed in vitro and introduced into pRiA4 by marker exchange. Each T-region of pRiHRI was also cloned separately on an independent replicon and used in a binary system with the virulence functions of either an Ri or a Ti plasmid provided in trans. Transformed roots were induced on tobacco and tomato explants by TL-DNA as well as by TR-DNA, suggesting that agropine type Ri plasmids from strains A4 and HRI can induce root proliferation by two independent transformation mechanisms. The root induction by the TR-DNA is probably due to auxin biosynthesis by gene products of aux loci homologous to the tms genes of Ti plasmid T-DNA. The molecular mechanism of root proliferation induced by the TL-DNA is probably equivalent to that of mannopine type Ri plasmid T-DNA.     

Frequent spontaneous deletions of Ri T-DNA in Agrobacterium rhizogenes transformed potato roots and regenerated plants

The presence of T-DNA was examined by Southern blot analysis in 16 regenerated shoot lines derived from 6 Agrobacterium rhizogenes-transformed root clones of Solanum tuberosum L. cv. Bintje.TR-DNA, present in regenerated shoot lines from 3 out of 6 root clones was correlated with the presence of opines. One root clone produced opines up to 2.5 years of subculture. However, plant regeneration from and prolonged subculturing of this root clone resulted in loss of opine synthesis, caused by deletion of TR-DNA.TL-DNA inserted at 1 to 5 independent loci was found in 14 of the 16 shoot lines. Surprisingly, 1 to 2 additional insertions next to similar insertions of TL-DNA were found in shoot lines from the same root clone (named sister shoot lines) in 2 out of 4 root clones. Nevertheless, this did not result in gross phenotypic variation between sister shoot lines. Another root clone regenerated 1 shoot line with an Ri phenotype, containing 1 insertion of TL-DNA, and 2 shoot lines with a normal Bintje phenotype without TL-DNA. The 5th root clone showed no difference between sister shoot lines and the 6th root clone produced only 1 shoot line.We conclude that during prolonged root culture and during shoot regeneration from root clones deletion of TL- and TR-DNA insertions can occur. The significance of the frequency of deletion of T-DNA of the Ri plasmid is discussed.     

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.     

Cotransformation and differential expression of introduced genes into potato (Solanum tuberosum L.) cv Bintje

Summary The Dutch potato cultivar Bintje has been transformed by Agrobacterium strain LBA1060KG, which contains two plasmids carrying three different DNAs (TL- and TR-DNA on the Agrobacterium rhizogenes plasmid and TKG-DNA on the pBI121 plasmid). Several transformed root clones were obtained after transformation of leaf, stem, and tuber segments, and plants were then regenerated from these root clones. The expression of the various marker genes [rol, opine, -glucuronidase (GUS), and neomycin phosphotransferase (NPTII)] was determined in several root clones and in regenerated plants. The selection of vigorously growing root clones was as efficient as selection for kanamycin resistance. In spite of the location of NPTII and GUS genes on the same T-DNA, 17% of the root clones did not show GUS activity. Nevertheless, Southern blot analysis showed that these root clones contained at least three copies of the GUS gene. Sixty-four per cent of the root clones contained opines. The expression of these genes, however, was negatively correlated with plant regeneration capacity and normal plant development. The differential expression of the marker genes in the transgenic potato tissues is discussed.     

Roots induced on cucumber cotyledons by the agropine Ri plasmid TR-DNA exhibit the transformed phenotype

Cucumber explants were transformed by Agrabacterium strains carrying Ri plasmids with functional TL and TR-DNAs, and by strains whose pRi had an intact TR-DNA but a disarmed TL-DNA lacking open reading frames (ORFs) 3 to 9, 10 (rol A), 11 (rol B), 12 (rol C), 13, 14, 15 (rol D), 16 and 17. Roots induced by all strains exhibited extensive root hair formation under axenic conditions, synthesised opines, and contained TR-specific DNA. These results confirm that the TR-DNA of an agropine Ri plasmid is able to elicit the transformed root phenotype in this plant.     

T-DNA analysis of plants regenerated from hairy root tumors

Summary Plants regenerated from hairy root tumors induced on Nicotiana glauca and Nicotiana tabacum by Agrobacterium rhizogenes strain A4 were examined for the presence of T-DNA. Regenerated N. tabacum plants contained intact copies of both TL-DNA and TR-DNA. However, plants regenerated from N. glauca tumors did not contain the TR-DNA region corresponding to the tms (auxin synthesis) genes. Some of the regenerants exhibited an abnormal phenotype which is characterized by severe leaf wrinkling. This phenotype is correlated with the presence of TL-DNA, but not TR-DNA.     

Use of a Disarmed Ri Plasmid Vector in Analysis of Transformed Root Induction

Nicotiana glauca, N. tabacum, Solanian dulcamara and S. nigrumwere transformed by Agrobacteriun rhizogenes strain BN1010 (TL^–TR⁺).The TR-DNA stimulated agropine-positive root induction and wastransformation competent in the absence of the TL-DNA. An unusualpattern of root induction was seen when stem explants were inoculatedwith this strain; occasionally, agropine-positive roots wereinduced at the inoculation sites, but prolific agropine-negativeroots were formed in profusion down the stems. The utility ofBN1010 as an efficient co-integrating vector was demonstratedby the separate transfer of a fragment containing rol ABC (BN1010::pEM15) and of a chimeric nopaline synthase-kanamycin resistancegene (BN1010:: Neo) into plants. Root cultures of S. dulcamaratransformed with BN1010:: Neo had an unusual, positively geotropicphenotype. Strain BN1010:: pEM15 (rol ABC⁺D^–TR⁺) incitedmore roots down stem explants than strain A4T. This indicatesthat rol D may act to suppress agropine-negative root productionin N. glauca and N. tabacum. Key words: Agrobacterium rhizogenes, TL-DNA, TR-DNA, disarmed Ri vector, transformed roots, Nicotiana glauca, N. tabacun, Solatium dulcamara, S. nigrum     

The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants

Summary A plant gene transfer system was developed from the Agrobacterium rhizogenes pRi15834 TL-DNA region. Intermediate integration vectors constructed from ColE1-derived plasmids served as cloning vectors in Escherichia coli and formed cointegrates into the TL-DNA after transfer to A. rhizogenes. An A. rhizogenes strain with pBR322 plasmid sequences replacing part of the TL-DNA was also constructed. Plasmids unable to replicate in Agrobacterium can integrate into this TL-DNA by homologous recombination through pBR322 sequences. No loss of pathogenicity was observed with the strains formed after integration of intermediate vectors or strains carrying pBR322 in the TL-DNA segment. Up to 15 kb of DNA have been transferred to plant cells with these systems. The T-DNA from a binary vector was cotransformed into hairy roots which developed after transfer of the wild-type pRi T-DNA. Tested on Lotus corniculatus the TL-derived vector system transformed 90% of the developed roots and the T-DNA from the binary vector was cotransformed into 60% of the roots. Minimum copy numbers of one to five were found. Both constitutive and organ-specific plant genes were faithfully expressed after transfer to the legume L. corniculatus.     

Stimulation of scion bud release by rol gene transformed rootstocks of Rosa hybrida L

For improvement of the root characteristics of roses, the rolA, B and C genes from Agrobacterium rhizogenes were introduced in the cultivar Moneyway, which is used as a rootstock. Four independent rolA+B+C transformants were produced. One rolC expressing transformant, showing good growth and clearly decreased apical dominance, was selected for a grafting experiment with the cut rose cultivar Madelon as a scion. Grafting on this rolA+B+C transformed rootstock resulted in a stimulation of both root development of the rootstock and axillary bud release of the untransformed scion. Axillary bud release increased from 0.1 to 0.6 and from 0.3 to 1.3 basal shoots per plant at 15C and 20C, respectively. Since basal shoots from flowers, this altered plant architecture will presumably lead to a more efficient flower production.Keywords: Agrobacterium transformation, apical dominance, rol genes, rootstock, rose.      

The use of Agrobacterium rhizogenes transformed roots to obtain transgenic shoots of the apple rootstock Jork 9

The apple rootstock Jork 9 was transformed using four different Agrobacterium rhizogenes virulent strains. The mannopine strain 8196 gave the best results in the production of chimeric plants compared to two agropine strains (A4 and 15834) and one cucumopine strain. Shoot regeneration was performed on both untransformed and transformed roots. Optimum combination and concentration of thidiazuron (TDZ) and -naphtaleneacetic acid (NAA) was different between untransformed and transformed roots. From the transformed roots seven shoots were obtained and propagated as individual clones. All shoots from these clones rooted on a hormone-free medium contrary to untransformed shoots that did not root under similar culture conditions. Differences in the morphology of the leaves and stems were observed between the clones. The transformed status of the different clones was verified with mannopine tests, PCR and Southern blot analyses. Five clones contained the mas1', the ORF 13 and the rolB genes, whereas two clones contained only the rolB gene.     

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.     

Recovery of phenotypically normal transgenic plants of Brassica oleracea upon Agrobacterium rhizogenes-mediated co-transformation and selection of transformed hairy roots by GUS assay

In this paper we describe the production of transgenic broccoli and cauliflower with normal phenotype using an Agrobacterium rhizogenes-mediated transformation system with efficient selection for transgenic hairy-roots. Hypocotyls were inoculated with Agrobacterium strain A4T harbouring the bacterial plasmid pRiA4 and a binary vector pMaspro::GUS whose T-DNA region carried the gus reporter gene. pRiA4 transfers T_L sequences carrying the rol genes that induce hairy root formation. Transgenic hairy-root production was increased in a difficult-to-transform cultivar by inclusion of 2,4-D in the medium used to resuspend the Agrobacterium prior to inoculation. Transgenic hairy roots could be selected from inoculated explants by screening root sections for GUS activity; this method eliminated the use of antibiotic resistance marker genes for selection. Transgenic hairy roots were produced from two cauliflower and four broccoli culivars. Shoots were regenerated from transgenic hairy root cultures of all four cultivars tested and successfully acclimatized to glasshouse conditions, although some plants had higher than diploid ploidy levels. Southern analysis confirmed the transgenic nature of these plants. T₀ plants from seven transgenic lines were crossed or selfed to produce viable seed. Genetic analysis of T₁ progeny confirmed the transmission of traits and revealed both independent and co-segregation of Ri T_L-DNA and vector T-DNA. GUS-positive phenotypically normal progeny free of T_L-DNA were identified in three transgenic lines out of the six tested representing all the cultivars regenerated including both cauliflower and broccoli.     

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.     

Phenotypic Effects of Isolated pRiA4 TL-DNA rol Genes in the Presence of Intact TR-DNA in Transgenic Plants of Solanum dulcamara L.

The effect of the rol genes, together with the TR-DNA of pRiA4on the phenotype of Solanum dulcamara plants, was analysed.Plants transformed by Agrobacterium strain BN1010: :rolA (rolA^7plus;TR⁺)exhibited severe leaf wrinkling, whereas plants transformedby strain BN1010: :rolC (ro/C⁺TR⁺) had a typical ‘hairyroot’ phenotype. Leaf discs excised from these latterplants produced roots on hormone-free medium. BN1010: :rolABC(rolABC⁺TR⁺) transformed plants had an exaggerated transformedphenotype. Some of the BN1010: :rolABC transformants had positivelygeotropic root growth which correlated with the presence ofmultiple copies of the TR-DNA. S. dulcamara plants, transformedby the TR-DNA region only, exhibited epinasty. Scanning electronmicroscopy of plants containing various regions of agropineRi T-DNA revealed that transformation causes changes in basicplant siructure.     

Upstream non-coding region which confers polar expression to Ri plasmid root inducing gene rolB

Summary Root differentiation could be elicited on carrot discs by transformation with the agropine Ri plasmid rolB gene cloned in the binary vector Bin19, provided two conditions were met. Firstly, an adequate auxin supply had to be provided. This was achieved by co-inoculation with a strain carrying only the auxin synthetic genes of the T_R-DNA. Most of the resulting roots were then shown to harbour only rolB and no aux genes. Secondly, an extended non-coding region (1200 bp) at the 5 end of rolB had to be included in the construction. A shorter (300 bp) 5 region, including TATA and CCAAT boxes, was not sufficient to trigger root differentiation. Both the extended (B1185) and reduced (B310) 5 regions of rolB were then cloned upstream of the -glucuronidase (GUS) reporter gene and infections carried out both on the apical and on the basal side of carrot discs. Strong expression of GUS, visualized histochemically as an intense blue colouring of transformed cells was observed with B1185-GUS constructions on the apical side of the discs. Only occasionally could coloured cells be observed on the basal side of the discs with B1185-GUS and on both apical and basal sides with B310-GUS constructions. Strong GUS expression was, on the contrary, achieved on cells of both auxin-rich (apical) and auxin-depleted (basal) sides of the discs with the strong constitutive viral promoter, CaMV35S. These results indicate the presence of an upstream regulatory region which confers polar expression to the rolB gene and suggest a role for auxin in its activation.     

Residual phosphorus and long-term management strategies for an Ultisol

Due to their extensive growth potential, transgenic root systems arising from inoculation withAgrobacterium rhizogenes became popular in the last decade as model systems in domains as diverse as production of secondary metabolites, interactions with pathogens and symbionts, examination of gene importance in control of root development or in regulation of gene expression in roots. Wild-type bacterial strains have also been considered as useful tools to stimulate rooting on recalcitrant cuttings or microcuttings as they cause abundant root initiation at the site of inoculation.Root initiation and the in vitro growth characteristics of transformed roots result from the transfer of genes located on the root-inducing plasmid (Ri) to plant cells and their expression therein. Two sets of pRi genes are involved in the root induction process: therol (rootloci) genes located in the TL region and theaux genes of the TR region. Some of these genes being able to interact, the system appears also as a new tool to study the role of auxin in the process of root initiation. The distinctive phenotype of the transformed roots which are capable of hormone autonomous growth seems to be controlled mainly by therol genes. Theserol genes, i.e. the geneticloci rol A, rol B, rol C androl D correspond to open reading frames ORFs 10, 11, 12 and 15. In vitro experiments determined the functions of the Rol B and Rol C proteins but the functions of Rol A and Rol D are still unknown. Altered metabolism of developmental regulators or modified sensitivity to auxin have been suspected to mediate root induction and morphological abnormalities of transformed roots and plants.The target cells for transformation and the cells which are competent for root initiation will be characterized as well as the subsequent development of transgenic roots provided with various constructs from the whole T-DNA to singlerol genes. Results dealing with auxin contents in relation with root growth kinetics, phenotype and structure, will also be presented and discussed with the potential use of therol genes to control root biomass. F J de Bruijn Section editor