Indole-3-acetic acid and indole-3-butyric acid in tissues of carrot inoculated withAgrobacterium rhizogenes

The role of auxins in induction of roots byAgrobacterium rhizogenes was studied in carrot root disks. Transformed roots were produced on root disks by inoculation withA. rhizogenes, A4. Measurement of indole-3-acetic acid (IAA) by gas chromatography-mass spectrometry (GC-MS) indicated that there was a significant increase in the concentration of IAA in transformed callus and induced roots compared with initial IAA concentrations in carrot disks. Indole-3-butyric acid (IBA) was found to occur naturally in carrot roots. The presence of IBA, a potent root inducer, must be taken into account when assessing the role of auxin during transformation and induction of roots byA. rhizogenes.     

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.     

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.     

In vitro culture of crown gall and hairy root tumors ofAtropa Belladonna: Differentiation and alkaloid production

Crown galls and hairy roots were induced inin vitro cultivated seedlings ofAtropa belladonna by differentAgrobacterium tumefaciens andA. rhizogenes strains. During furtherin vitro cultivation, tumors and hairy roots showed high differentiation ability which persisted for at least one year ofin vitro culture. Only root cultures, induced by Ri plasmid A4, synthesized detectable amounts of alkaloids.     

Regeneration of tobacco plants from crown gall tumors

Summary Tissue culture methods have been developed for regeneration of normal appearing tobacco plants from bacteria-free crown gall strains incited byAgrobacterium tumefaciens C58, IIBV7, B6, CGIC, A6NC, 27, and AT4. Regenerants fall into two categories depending on the properties of tissues from these plants. The first type of regenerant was obtained from tumors incited byA. tumefaciens C58 and it retained the potential for expression of tumor characteristics such as a nonrequirement for phytohormones (auxin and cytokinin) by explants in vitro and the presence of detectable concentrations of nopaline. Normal appearing plants obtained from C58 tumors had much lower concentrations of nopaline than the corresponding tumor tissue (130 versus 1700 μg per g dry wt) indicating a parallel repression of abnormal growth and nopaline concentrations in regenerants. The second type of regenerant was obtained from tumors incited by the otherA. tumefaciens strains and was characterized by requirements for phytohormones by explants in vitro and the apparent lack of octopine or nopaline in regenerant tissues.     

Transformed Roots of Lupinus mutabilis: Induction, Culture and Isoflavone Biosynthesis

Transformed roots of Lupinus mutabilis cv. Potosi induced by Agrobacterium rhizogenes strain R1601 were cultured on Murashige and Skoog-based medium lacking kanamycin sulphate, or with this antibiotic at 40 mg l⁻¹. The neomycin phosphotransferase gene in the genome of transformed roots was confirmed by non-radioactive Southern hybridisation. Neomycin phosphotransferase protein was detected by ELISA. Transformed roots synthesised isoflavones, but not quinolizidine alkaloids; the latter are typical secondary metabolites of lupin normally produced in aerial parts of the plant. Genistein and 2′-hydroxygenistein, were the main secondary metabolites in cultured, transformed roots, whereas the glycoside genistin was more abundant in roots of non-transformed plants. Wighteone concentrations in transgenic roots were higher than those of non-transformed roots. Transformed roots produced twice the concentration of isoflavones compared with roots from non-transformed plants, indicating that Ri plasmid T-DNA genes modified isoflavone concentration and pattern of biosynthesis.     

Spontaneous plant regeneration in transformed roots and calli from Tylophora indica: changes in morphological phenotype and tylophorine accumulation associated with transformation by Agrobacterium rhizogenes

We examined the effects of genetic transformation by Agrobacterium rhizogenes on the production of tylophorine, a phenanthroindolizidine alkaloid, in the Indian medicinal plant, Tylophora indica. Transformed roots induced by the bacterium grew in axenic culture and produced shoots or embryogenic calli in the absence of hormone treatments. However, hormonal treatment was required to regenerate shoots in root explants of wild type control plants. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes, which include, short internodes, small and wrinkled leaves, more branches and numerous plagiotropic roots. Plants regenerated from transformed roots showed increased biomass accumulation (350–510% in the roots and 200–320% in the whole plants) and augmented tylophorine content (20–60%) in the shoots, resulting in a 160–280% increase in tylophorine production in different clones grown in vitro.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> transformed roots of the parasitic plant <Emphasis Type="Italic">Triphysaria versicolor</Emphasis> retain parasitic competence

Parasitic plants in the Orobanchaceae invade roots of neighboring plants to rob them of water and nutrients. Triphysaria is facultative parasite that parasitizes a broad range of plant species including maize and Arabidopsis. In this paper we describe transient and stable transformation systems for Triphysaria versicolor Fischer and C. Meyer. Agrobacterium tumefaciens and Agrobacterium rhizogenes were both able to transiently express a GUS reporter in Triphysaria seedlings following vacuum infiltration. There was a correlation between the length of time seedlings were conditioned in the dark prior to infiltration and the tissue type transformed. In optimized experiments, nearly all of the vacuum infiltrated seedlings transiently expressed GUS activity in some tissue. Calluses that developed from transformed tissues were selected using non-destructive GUS staining and after several rounds of in vivo GUS selection, we recovered uniformly staining GUS calluses from which roots were subsequently induced. The presence and expression of the transgene in Triphysaria was verified using genomic PCR, RT PCR and Southern hybridizations. Transgenic roots were also obtained by inoculating A. rhizogenes into wounded Triphysaria seedlings. Stable transformed roots were identified using GUS staining or fluorescent microscopy following transformation with vectors containing GFP, dsRED or EYFP. Transgenic roots derived from both A. tumefaciens and A. rhizogenes transformations were morphologically normal and developed haustoria that attached to and invaded lettuce roots. Transgenic roots also remained competent to form haustoria in response to purified inducing factors. These transformation systems will allow an in planta assessment of genes predicted to function in plant parasitism. Alexey Tomilov and Natalya Tomilova made an equal contribution in the paper.     

Genetic manipulation in cultivars of oilseed rape (Brassica napus) using Agrobacterium

Summary The response of oilseed rape cultivars to infection with Agrobacterium tumefaciens and A. rhizogenes and the possibility of regenerating genetically transformed oilseed rape plants were examined. The frequency at which Agrobacterium induced galls or hairy-roots on in vitro cultured plants ranged from 10% to 70%, depending on the cultivar. From galls induced by the tumorigenic strain T37, known to be strongly shoot inducing on tobacco, roots developed frequently. Occasionally, shoots formed and some of these produced tumour cell specific nopaline. Attempts to grow the transformed shoots into plants have so far been unsuccessful. Whole plants transformed with Ri-T-DNA, however, were regenerated. These had crinkled leaves and abundant, frequently branching roots that showed reduced geotropism, similar to previously isolated Ri T-DNA transformed tobacco and potato plants. The transformed oilseed rape plants flowered, but failed to form seeds.     

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.     

Regeneration of transgenic papaya plants via somatic embryogenesis induced byAgrobacterium rhizogenes

AnAgrobacterium rhizogenes-mediated procedure for transformation of papaya (Carica papaya) was developed. Transgenic plants were obtained from somatic embryos that spontaneously formed at the base of transformed roots, induced from leaf discs infected withA. rhizogenes. Transformation was monitored by autonomous growth of roots and somatic embryos, resistance to kanamycin, β-glucuronidase activity (GUS), and Southern hybridization analysis. Over one-third of the infected leaf explants produced transformed roots with GUS activity, from which 10% spontaneously produced somatic embryos. Histological analysis ofA. rhizogenes-transformed embryos showed that they have an altered symmetry between the shoot apex and the root meristem when compared to somatic embryos induced with hormone treatment from control explants. Transgenic papaya plants containingA. rhizogenes rol genes were more sensitive to auxins, developed wrinkled leaves, and grew slower than nontransformed plants.     

Biotransformation of nicotine alkaloids by tobacco shooty teratomas induced by a Ti plasmid mutant

Tobacco shooty or rooty teratomas and hairy roots were induced by Agrobacterium tumefaciens (pGV 3845), A. tumefaciens (pGV 3304) and A. rhizogenes (pRi 8196), respectively. The tobacco alkaloids, nicotine, nornicotine and anatabine, were produced in hairy roots and in rooty teratomas but not in shooty teratomas. However, the shooty teratomas have the ability to accumulate the alkaloids and to biotransform nicotine to nornicotine. These were established by co-culture experiments incubating hairy roots and shooty teratomas in a same dish and by biotransformation experiments with shooty teratomas.     

Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens -transformed roots and Agrobacterium rhizogenes-transformed hairy roots

Medicago truncatula, barrel medic, is a forage crop that has been developed into a model legume. The development of new transformation methods is important for functional genomic studies in this species. Based on Agrobacterium tumefaciens-mediated transformation of root explants, we developed an effective system for producing M. truncatula (genotype R108) transgenic plants. Among the four A. tumefaciens strains (AGL1, C58C1, EHA105 and LBA4404) tested, EHA105 and AGL1 were most effective in regenerating transgenics. Callus induction frequency from root explants was 69.8%, and plantlet/shoot regeneration frequency was 41.3% when EHA105 was used. Transgenic nature of the regenerated plants was confirmed by PCR and Southern hybridization analyses. Progeny analysis revealed stable Mendelian meiotic transmission of transgenes. Because M. truncatula is particularly useful for the study of root endosymbiotic associations, we further developed a plant regeneration system from A. rhizogenes-transformed hairy roots of M. truncatula. Fertile true transgenic plants were regenerated from the hairy roots, thus allowing the assessment of gene functions at the whole plant level. Segregation analysis revealed that the hairy root genes could be segregated out in the progenies. By coupling A. rhizogenes-mediated hairy root transformation and the regeneration system reported here, once potential genes of interest are identified, the transformed hairy roots carrying such genes could be directly regenerated into plants for more detailed characterization of the genes.     

Specificity of Agrobacterium-mediated delivery of maize streak virus DNA to members of the Gramineae

Parameters affecting the efficiency of agroinfection of maize streak virus (MSV) in maize have been determined. Monomeric units, cloned at a number of sites in the MSV genome were not infectious but multimeric units containing partial duplications were equally as infectious as complete tandem dimeric clones. Inoculation of tandem dimeric units conjugated into different strains of Agrobacterium showed that both A. tumefaciens and A. rhizogenes were able to transfer DNA to maize and this ability was Ti (or Ri) plasmid-specific. Nopaline strains of A. tumefaciens and both agropine and mannopine A. rhizogenes strains efficiently transferred MSV DNA to maize. A number of strains were capable of MSV DNA transfer to other members of the Gramineae, providing information which may be essential for Agrobacterium-mediated transformation of monocotyledonous plants.     

Establishment of hairy root cultures of Psoralea species

Eight Psoralea species (Leguminosae) were inoculated with Agrobacterium rhizogenes, strains 8196 and 9402. Hairy roots were only induced by strain 9402. Attention was focussed on Psoralea lachnostachys. Transformed roots grew very rapidly in Gamborg B5 liquid medium with a doubling time of the culture of 38 hours. Whatever the culture conditions, the two furanocoumarins usually found in roots of Psoralea plants, psoralen and angelicin, were not detected in cultured transformed and non transformed roots even when some chitosan was added to the medium. However, 669 g.g^–1 dry matter of psoralen and 215 g.g^–1 dry matter of angelicin were found in roots from soil grown plants. A possible translocation of these compounds from the aerial parts to the roots is suggested.Abbreviations B5 medium Gamborg's medium (Flow laboratories's formulation) - NAA Naphthaleneacetic acid     

Indole-3-acetic acid and indole-3-butyric acid in tissues of carrot inoculated withAgrobacterium rhizogenes

The role of auxins in induction of roots byAgrobacterium rhizogenes was studied in carrot root disks. Transformed roots were produced on root disks by inoculation withA. rhizogenes, A4. Measurement of indole-3-acetic acid (IAA) by gas chromatography-mass spectrometry (GC-MS) indicated that there was a significant increase in the concentration of IAA in transformed callus and induced roots compared with initial IAA concentrations in carrot disks. Indole-3-butyric acid (IBA) was found to occur naturally in carrot roots. The presence of IBA, a potent root inducer, must be taken into account when assessing the role of auxin during transformation and induction of roots byA. rhizogenes.     

Solasodine production in transformed organ cultures (roots and shoots) of Solanum eleagnifolium Cav.

Summary Transformed organ cultures of Solanum eleagnifolium Cav. (roots and shoots) were obtained by infection with different Agrobacterium species (A. rhizogenes and A. tumefaciens). The growth index and the solasodine productivity of both kinds of cultures were determinated for several clones. Transformed roots (clone 5) shows a Growth Index of 39.07 and a solasodine yield of 1.90 ± 0.08 mg g^-1DW . Solasodine content in clone 5 of transformed roots (1.9 ± 0.08 mg g^-1 DW) is similar to those obtained by hormonal manipulation (1.00 – 1.68 mg g^-1DW) but higher than those found in shoots and leaves of the wild-grown plant (0.3–0.4 mg g^-1DW).     

Transformation of meristematic cells in the shoot apex of cultured pea shoots byAgrobacterium tumefaciens andA. rhizogenes

Summary Different tissues in cultured pea shoots were inoculated withAgrobacterium tumefaciens wild types C 58 and ACH 5 andA. rhizogenes wild type 9402. The C 58 and 9402 bacteria induced the formation of tumours and hairy roots respectively while the ACH 5 was inactive. The younger the tissue the more rapidly it responded to the active bacteria. The shoot apex was the most reactive organ and developed into a tumour, theA. rhizogenes tumours subsequently giving rise to transformed hairy roots. Histological examination showed that transformed cells (including those within the apical dome) initially became highly vacuolate before dividing rapidly to form a tumour. These changes were accompanied by cell division in surrounding tissues.     

Efficient production of Agrobacterium rhizogenes-transformed roots and composite plants for studying gene expression in coffee roots

The possibility of rapid validation and functional analysis of nematode resistance genes is a common objective for numerous species and particularly for woody species. In this aim, we developed an Agrobacterium rhizogenes-mediated transformation protocol for Coffea arabica enabling efficient and rapid regeneration of transformed roots from the hypocotyls of germinated zygotic embryos, and the subsequent production of composite plants. The A. rhizogenes strain A4RS proved to be the most virulent. High transformation efficiencies (70%) were obtained using a 2-week co-cultivation period at a temperature of 15–18°C. Using a p35S-gusA-int construct inserted in the pBIN19 binary plasmid, we could estimate that 35% of transformed roots were GUS positive (co-transformed). Using the GUS assay as visual marker, 40% composite plants bearing a branched co-transformed rootstock could be obtained after only 12 weeks without selection with herbicides or antibiotics. Transgenic coffee roots obtained with A. rhizogenes did not exhibit the ‘hairy’ disturbed phenotype and were morphologically similar to normal roots. PCR analyses demonstrated that all co-transformed roots were positive for the expected rolB and gusA genes. Transformed and non-transformed root systems from both susceptible and resistant varieties were inoculated with Meloidogyne exigua nematode individuals. Inoculation of composite plants from the Caturra susceptible variety resulted in the normal development of nematode larvae. Numbers of extracted nematodes demonstrated that transformed roots retain the resistance/sensibility phenotype of varieties from which they are derived. These results suggest that composite plants constitute a powerful tool for studying nematode resistance genes.