Detection of a 17 kb unique sequence (T-DNA) in plant chromosomes by in situ hybridization

An approach is described for the detection of a unique sequence, the T-DNA region of the Agrobacterium rhizogenes root-inducing (Ri) plasmid, in plant chromosomes by in situ hybridization. This sequence was introduced into the Crepis capillaris genome (2n=6) by infecting Crepis stem segments with A. rhizogenes. Roots growing from the infection site contain T-DNA and synthesize mannopine, which can be used as a convenient biochemical marker for T-DNA transformation. Southern analysis of DNA isolated from one transformed Crepis root line verified the presence of a single copy of T-DNA (approximate size 17 kb) per diploid Crepis genome. To localize T-DNA, both DNA and RNA probes, labelled with either tritium or biotin, were hybridized to Crepis chromosomes prepared from transformed root tips by a novel spreading method. Biotinylated probes were visualized using reflection-contrast microscopy. In the hybridization experiments described, T-DNA was detected in one homologue of chromosome 3, where it could be assigned to a paracentromeric position in the neighbourhood of the nucleolar organizing region. These results demonstrate that it is possible to localize unique sequences in plant chromosomes by in situ hybridization.     

Chromosomal mapping of T-DNA inserts in transgenicPetunia byin situ hybridization

The chromosomal location of T-DNa inserts in ten independently derived and confirmed transgenic plants ofP. hybrida was detected byin situ hybridization. Nine transgenic plants had the T-DNA inerts at single sites distributed among each of the seven chromosomes; in one plant the T-DNA inserts were detected on two different chromosomes. Although the T-DNA inserts were integrated randomly among the chromosomes, seven of the 11 total inserts were located at or near the telomere. Thus, T-DNA inserts appear to have potential for tagging chromosomes and chromosome fragments.     

Regeneration of flax plants transformed by Agrobacterium rhizogenes

Regeneration of flax (Linum usitatissimum) following transformation by either Agrobacterium tumefaciens carrying a disarmed Ti-plasmid vector, or Agrobacterium rhizogenes carrying an unmodified Ri plasmid, was examined. Hypocotyl and cotyledon explants inoculated with A. tumefaciens formed transformed callus, but did not regenerate transformed shoots either directly or via callus. However, cotyledon explants inoculated with A. rhizogenes formed transformed roots which did regenerate transformed shoots. Ri T-DNA encoded opines were detected in the transformed plantlets and Southern hybridization analysis confirmed the presence of T-DNA from the Ri plasmid in their DNA. Transformed plantlets had curled leaves, short internodes and some had a more developed root system characterized by plagiotropic behaviour.     

Segregation of genes transferred to one plant cell from two separate Agrobacterium strains

Agrobacterium tumefaciens and Agrobacterium rhizogenes are soil bacteria which transfer DNA (T-DNA) to plant cells. Two Agrobacterium strains, each with a different T-DNA, can infect plants and give rise to transformed tissue which has markers from both T-DNAs. Although marker genes from both T-DNAs are in the tissue, definitive proof that the tissue is a cellular clone and that both T-DNAs are in a single cell is necessary to demonstrate cotransformation. We have transferred two distinguishable T-DNAs, carried on binary vectors in separate Agrobacterium rhizogenes strains, into tomato cells and have recovered hairy roots which received both T-DNAs. Continued expression of marker genes from each T-DNA in hairy roots propagated from individual root tips indicated that both T-DNAs were present in a single meristem. Also, we have transferred the two different T-DNAs, carried on identical binary vector plasmids in separate Agrobacterium tumefaciens strains, into tobacco cells and recovered plants which received both T-DNAs. Transformed plants with marker genes from each T-DNA were outcrossed to wild-type tobacco plants. Distribution of the markers in the F1 generation from three cotransformed plants of independent origin showed that both T-DNAs in the plants must have been present in the same cell and that the T-DNAs were genetically unlinked. Cotransformation of plant cells with T-DNAs from two bacterial strains and subsequent segregation of the transferred genes should be useful for altering the genetic content of higher plants.     

Multiple transformation of plant cells by Agrobacterium may be responsible for the complex organization of T-DNA in crown gall and hairy root

Summary Inoculation of carrot discs and Lotus corniculatus plantlets with mixtures of different Agrobacterium rhizogenes or of A. rhizogenes and A. tumefaciens or with Agrobacterium strains harboring both an Ri and a modified Ti plasmid resulted in frequent multiple (pluribacterial) transformation of cells, as revealed by the mixed opine-type of hairy roots arising from them. Multiple transformation may account for the presence of dispersed T-DNA inserts in crown gall and hairy root lines. A plant genetic engineering strategy based on segregation of T-DNA inserts in the progeny of multiple transformants is proposed.     

Chromosomal localization of foreign genes in Petunia hybrida

Summary A F1 hybrid of Petunia hybrida, heterozygous for at least one marker on each of the seven chromosomes, was transformed with a modified strain of Agrobacterium tumefaciens in which the phytohormone biosynthetic genes in the transferred DNA (T-DNA) were replaced with a NOS/NPTII/NOS chimeric gene and a wildtype nopaline synthase (NOS) gene. The chimeric gene, which confers kanamycin resistance, was used as selectable marker during the transformation process and the NOS gene was used as a scorable marker in the genetic studies. After plants had been regenerated from the transformed tissues, the transgenic plants that expressed both of these markers were backcrossed to the parental lines. The offspring were examined for the segregation of the NOS gene and the Petunia markers. Genetic mapping was thus accomplished in a single generation.By Southern hybridization analysis we confirmed the presence of the expected T-DNA fragments in the transformed plants. Four out of the six plants presented here, had just one monomeric T-DNA insertion. The sizes of the plant/T-DNA junction fragments suggest that the integration occurred in different sites of the Petunia genome. One transformant gave a more complicated hybridization pattern and possibly has two T-DNA inserts. Another transgenic plant was earlier reported (Fraley et al. 1985) to have two, possibly tandemly repeated T-DNAs.Data is presented on the genetic localization of the T-DNA inserts in six independently obtained transgenic plants. The T-DNA inserts in three plants were mapped to chromosome I. However, the distances between the NOS gene and the marker gene on this chromosome were significantly different. In another transgenic plant the NOS gene was coinherited with the marker on chromosome IV. Two other transgenic plants have the T-DNA insert on chromosome III. A three point cross enabled us to determine that both plants have the NOS gene distally located from the peroxidaseA (prxA) marker and both plants showed about 18% recombination. However, Southern hybridization analysis shows that the sizes of the plant/T-DNA junction fragments in these transgenic plants are different, thus suggesting that the integrations occurred in different sites.     

Conserved regions in the T-DNA of different Agrobacterium rhizogenes root-inducing plasmids

The T-regions of the three so far identified types of Ri plasmids-corresponding to the synthesis of three different hairy root opines, agropine, mannopine and cucumopine-have been compared in detail by Southern blot cross hybridizations. Two distinct zones of very strong sequence homology, approximately 4 and 3 kilobases in length respectively, have been identified in all three T-regions. The highly conserved sequences, not present in Ti plasmid T-DNA, may encode essential rhizogenic functions common to all Agrobacterium rhizogenes T-DNAs.     

T-DNA tagging in the model legume Medicago truncatula allows efficient gene discovery

The annual legume Medicago truncatula has been proposed as a model plant to study various aspects of legume biology including rhizobial and mycorrhizal symbiosis because it is well suited for the genetic analysis of these processes . To facilitate the characterization of M. truncatula genes participating in various developmental processes we have initiated an insertion mutagenesis program in this plant using three different T-DNAs as tags. To investigate which type of vector is the most suitable for mutagenesis we compared the behavior of these T-DNAs. One T-DNA vector was a derivative of pBin19 and plant selection was based on kanamycin resistance. The two other vectors carried T-DNA conferring Basta resistance in the transgenic plants. For each T-DNA type, we determined the copy number in the transgenic lines, the structure of the T-DNA loci and the sequences of the integration sites. The T-DNA derived from pBin19 generated complex T-DNA insertion patterns. The two others generally gave single copy T-DNA inserts that could result in gene fusions for the pGKB5 T-DNA. Analysis of the T-DNA borders revealed that several M. truncatula genes were tagged in these transgenic lines and in vivo gus fusions were also obtained. These results demonstrate that T-DNA tagging can efficiently be used in M. truncatula for gene discovery.     

Identification of hairy root loci in the T-regions of Agrobacterium rhizogenes Ri plasmids

Summary Agrobacterium rhizogenes induces root formation at the wound site of inoculation in plants and inserts a fragment of its plasmid (Ri) into the plant nuclear DNA. Parts of the transferred region (T-region) of the Ri plasmid of A. rhizogenes strain A4 or 8196 are cloned in Escherichia coli. Insertions of the E. coli lacZ coding region into the hybrid plasmids were made in vivo using transduction by miniMu. Twenty insertions localized in the T_L-DNA of pRiA4 (or pRi1855) and 2 inserts in the T-DNA of pRi8196 were obtained in E. coli. One of the T_L-DNA insertions is saved up because it is linked to an internal T-DNA deletion; the others because they confer a lactose plus phenotype on E. coli; this indicates that the T-DNA harbours sequences that are expressed in E. coli. Fifteen of these T-DNA insertions were transfered to Agrobacterium where they substitute the corresponding wild-type T-DNA of the Ri plasmid by homologous recombination. These strains corresponding to insertion-directed mutagenesis were used to inoculate Daucus carota slices and stems and leaves of Kalanchoe daigremontiana. The two insertions strains obtained in the T-DNA of pRi8196 are avirulent on K. daigremontiana; but their phenotypes differ on D. carota slices, suggesting that insertions affect distinct loci on the T-DNA involved in hairy root formation. Only one insertion out of the twenty obtained in the T_L-DNA of pRiA4 (or 1855) induces a loss of virulence on leaves of K. daigremontiana. However the T_L-DNA deletion harbouring strain induces a loss of virulence on D. carota and K. daigremontiana (stems and leaves), confirming the importance of the T_L-DNA for hairy root induction. re]19850711 rv]19851230 ac]19860114     

A comparison of strategies for multiple-gene co-transformation via hairy root induction

Hairy root is a transformed root tissue in which transfer DNA (T-DNA) is inserted in the genome by Agrobacterium rhizogenes. To establish a system for multiple-gene co-transformation in hairy roots, we evaluated four different strategies using A. rhizogenes. The genes gusA and mgfp5 were located in separate plasmids, which were transformed into two different batches of A. rhizogenes (strategy 2AR) or a single batch (strategy 2BV). The two reporter genes were also inserted in one T-DNA (strategy 1TD) or two different T-DNAs (strategy 2TD) in a binary vector. Over 90 % of infected Nicotiana tabacum leaf discs formed hairy roots in all four groups, which was not significantly different from the infection efficiency of wild-type A. rhizogenes. Proportions of co-transformed hairy roots with strategies 2AR, 2BV, 1TD, and 2TD were 65.4, 40.0, 78.6, and 82.1 %, respectively, which indicated that all of the strategies were suitable for co-transformation of multiple genes. High variation in growth rate and heterologous protein expression indicated that further screening is required to identify the clone with the highest productivity. Our results indicated that strategies 1TD and 2TD achieved the highest co-transformation efficiency. Combination with strategy 2AR or 2BV provides additional options for co-transformation of multiple transgenes.     

Analysis of TR-DNA/plant junctions in the genome of a Convolvulus arvensis clone transformed by Agrobacterium rhizogenes strain A4

A Charon 4A phage library, containing insert DNA isolated from a morning glory (Convolvulus arvensis) plant genetically transformed by Ri T-DNA from Agrobacterium rhizogenes strain A4, was used to isolate a lambda clone that contains part of the Ri TL-DNA and the complete TR-DNA. The two Ri T-DNAs were recovered adjacent to each other in a tail-to-tail configuration (i.e. with the TR-DNA inverted with respect to the TL-DNA). Comparison of nucleotide sequences from this lambda clone with the corresponding sequences from the Ri plasmid allowed us to determine the location of the T-DNA/plant junction for the right end of the TL-DNA and the left and right ends of the TR-DNA. We located, near each of these borders, a 24 bp sequence that is similar to the 24 bp consensus sequence found near the pTi T-DNA extremities. In addition, sequences similar to the core overdrive sequence from pTi are located near each right border. Hybridization and nucleotide sequence analysis of the DNA adjacent to the TL/TR junction shows that no plant DNA is located between the TL and TR-DNAs and suggests that the plant DNA adjacent to the end of the TR-DNA may have been rearranged during the integration into the plant genome.     

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.     

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.     

Characterization and culture of Agrobacterium rhizogenes transformed roots of forage legumes

Agrobacterium rhizogenes wild type strain 8196 induced root growth at the site of stab-wounding on 5-day-old seedlings of red clover (Trifolium pratense), siratro (Macroptilium atropurpureum, a tropical forage legume), and alfalfa (Medicago sativa). Excised roots grew rapidly on hormone-free medium, were highly branched, and lacked geotropism. Paper electrophoresis of the root extracts confirmed the presence of opines. Confirmed transformed roots still proliferating from the wound site, were inoculated with Rhizobium and compared with inoculated non-transformed roots on seedlings raised under identical conditions. Nodulation was inhibited in the transformed roots. Control experiments using mixed inoculation of Rhizobium and Agrobacterium even at a ratio of 1:1000 on control seedlings showed no inhibition of nodulation, suggesting that the observed inhibition of nodulation on transformed roots was a result of the Ri T-DNA rather than the Agrobacterium rhizogenes in the tissue.     

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.     

Changes in morphological phenotypes and withanolide composition of Ri-transformed roots of <Emphasis Type="Italic">Withania somnifera</Emphasis>

Developmental variability was introduced into Withania somnifera using genetic transformation by Agrobacterium rhizogenes, with the aim of changing withasteroid production. Inoculation of W. somnifera with A. rhizogenes strains LBA 9402 and A4 produced typical transformed root lines, transformed callus lines, and rooty callus lines with simultaneous root dedifferentiation and redifferentiation. These morphologically distinct transformed lines varied in T-DNA content, growth rates, and withasteroid accumulation. All of the lines with the typical transformed root morphology contained the T_L T-DNA, and 90% of them carried the T_R T-DNA, irrespective of the strain used for infection. Accumulation of withaferin A was maximum (0.44% dry weight) in the transformed root line WSKHRL-1. This is the first detection of withaferin A in the roots of W. somnifera. All of the rooty callus lines induced by strain A4 contained both the T_L and the T_R-DNAs. In contrast, 50% of the rooty-callus lines obtained with strain LBA 9402 contained only the T_R T-DNA. All the rooty callus lines accumulated both withaferin A and withanolide D. The callusing lines induced by LBA 9402 lacked the T_L T-DNA genes, while all the callusing lines induced by strain A4 contained the T_L DNA. Four of these callus lines produced both withaferin A (0.15–0.21% dry weight) and withanolide D (0.08–0.11% dry weight), and they grew faster than the transformed root lines. This is the first report of the presence of withasteroids in undifferentiated callus cultures of W. somnifera.     

Function of the aux and rol genes of the Ri plasmid in plant cell division in vitro

Auxin-autonomous growth in vitro may be related to the integration and expression of the aux and rol genes from the root-inducing (Ri) plasmid in plant cells infected by agropine-type Agrobacterium rhizogenes. To elucidate the functions of the aux and rol genes in plant cell division, plant cell lines transformed with the aux1 and aux2 genes or with the rolABCD genes were established using tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells. The introduction of the aux1 and aux2 genes enabled the auxin-autonomous growth of BY-2 cells, but the introduction of the rolABCD genes did not affect the auxin requirement of the BY-2 cells. The results clearly show that the aux genes are necessary for auxinautotrophic cell division, and that the rolABCD genes are irrelevant in auxin autotrophy.Key words: Agrobacterium rhizogenes, auxin-autotrophic cell, auxin biosynthesis, hairy root, plant cell division, Ri plasmid, T-DNA, aux, rol, tobacco BY-2 cells     

Genetic transformation of oilseed rape (Brassica napus) by the Ri T-DNA of Agrobacterium rhizogenes and analysis of inheritance of the transformed phenotype

Summary Genetically transformed repeseed (Brassica napus) roots were obtained by in vitro inoculation of excised stem segments with Agrobacterium rhizogenes. Axenic root organ clones were established and they exhibited a phenotype characteristic of transformed roots: rapid growth, reduced apical dominance and root plagiotropism. Stem regeneration was induced by exposing root fragments to 2,4-dichloroacetic acid (2,4-D) in liquid medium, followed by transfer to solid regeneration medium. The resulting plants exhibited the transformed phenotype observed in other species where similar experiments have been performed. Direct evidence for genetic transformation was obtained from opine assays and molecular hybridization. Sexual transmission of the transformed phenotype was Mendelian, and a probable case of T-DNA insertion into two independent loci within the same plant was detected. The estimated optimal time necessary to obtain transformed oilseed rape plants using this approach is 2 months.     

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.