The pleiotropic effects induced by therolC gene in transgenic plants are caused by expression restricted to protophloem and companion cells

Expression of therolC gene fromAgrobacterium rhizogenes causes morphological and developmental alterations in transgenic plants. The histological alterations underlying the macroscopic changes and the cellular localization of the site of expression of therolC gene have shown that: (i) the expression of therolC gene is developmentally regulated, (ii) in vegetative transgenic plants, the expression of therolC gene under the control of its own promoter is restricted to companion and protophloem cells, (iii) the site of action of the product(s) of the activity of the rolC enzyme is distinct from its site of expression, (iv) precise localization of the rolC peptide has been achieved by immunocytochemistry but not by the histochemical GUS assay. These results imply that the sites of action and expression of therolC gene in trangenic plants are physically separated. Thus the product(s) of the activity of the rolC enzyme must be a factor capable of being transported. Current models forrolC gene action are discussed taking into account the reported results.     

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.     

Potential use of theaux2 gene fromAgrobacterium rhizogenes as a conditional negative marker in transgenic cabbage

An originalAgrobacterium tumefaciens-mediated transformation procedure, based on the actions of both wild type and disarmed bacterial strains, was developed. Theaux2 gene ofA. rhizogenes was introduced into a rapid-cycling genotype of cabbage (Brassica oleracea L.). Theaux2 gene product converts naphthalene acetamide into the auxin naphthalene acetic acid. Expression of this gene in the transgenic progeny grownin vitro led to an altered root phenotype. On a medium supplemented with napthalene acetamide (NAM), two of the three analysed progenies were characterized by the formation of callus instead of roots, whereas on a NAM-free medium all the plantlets from these progenies presented a normal phenotype. Expression of theaux2 gene was also assessed under horticultural conditions by sowing seeds in sand and watering them with a nutritive solution supplemented with NAM. Under these conditions, NAM inhibited the formation of a root system in transgenic plantlets and induced the death of the transgenic plantlets three to four weeks after germination. Thus,aux2 acts as a lethal conditional marker which could be used in negative selection of cabbage. Potential utilization of theaux2 gene to screen spontaneous androgenetic plants in order to transfer cytoplasmic male sterility in a single generation is discussed.     

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.     

Transformation of cucumber (Cucumis sativus L.) plants with Agrobacterium rhizogenes

Summary Transgenic cucumber plants (Cucumis sativus L., cv. Straight Eight) were regenerated from roots induced by inoculation of inverted hypocotyl sections with Agrobacterium rhizogenes containing the vector pARC8 in addition to the resident Ri-plasmid. The DNA transferred to the plant from the vector (T-DNA) included a gene which encoded the enzyme neomycin phosphotransferase II, and thus conferred on the plant cells resistance to kanamycin. The transgenic plants looked normal and were positive for the neomycin phosphotransferase II. Southern blot analysis of the transgenic plants revealed that all plants contained vector DNA, but only some of them contained DNA from the Ri plasmid.     

Transgenic tobacco plants regenerated from leaf disks can be periclinal chimeras

Amongst rolC transgenic tobacco plants regenerated from leaf disks 6.5% are periclinal chimeras, i.e. plants with genetically different cell populations in different cell layers. The expression of the rolC gene of Agrobacterium rhizogenes causes a reduction in pigment content in leaves. The chimeric composition of the regenerated plants becomes thus apparent as light green leaf tissue in the transgenic region, tissue flanked by dark green wild-type sectors. Southern and northern blot analysis confirmed the chimeric nature of such plants. Investigation of selfed progeny of chimeric plants on selective media indicates that layer invasion in reproductive tissues can occur in tobacco early during the formation of the flower buds. The results show (1) that tobacco plants regenerated from leaf disks and grown on selective media have not necessarily the same clonal origin and (2) that they can give rise to non-transgenic offspring. The chimeric plants provide insight on the effect of rolC gene expression on microsporogenesis.     

Deviating T-DNA transfer fromAgrobacterium tumefaciens to plants

We analyzed 29 T-DNA inserts in transgenicArabidopsis thaliana plants for the junction of the right border sequences and the flanking plant DNA. DNA sequencing showed that in most lines the right border sequences transferred had been preserved during integration, corroborating literature data. Surprisingly, in four independent transgenic lines a complete right border repeat was present followed by binary vector sequences. Cloning of two of these T-DNA inserts by plasmid rescue showed that in these lines the transferred DNA consisted of the complete binary vector sequences in addition to the T-region. On the basis of the structure of the transferred DNA we propose that in these lines T-DNA transfer started at the left-border repeat, continued through the vector part, passed the right border repeat, and ended only after reaching again this left-border repeat.     

Use of Agrobacterium rhizogenes to create transgenic apple trees having an altered organogenic response to hormones

Summary The apple rootstock, M26, was genetically and phenotypically transformed using the Agrobacterium wild-type strain, A4. First, chimeric plants were obtained having transformed roots and normal aerial parts. Transformed plants were then produced through regeneration from transformed roots. Transformation was demonstrated by molecular hybridization and opine analysis. The effects of hormones on organogenesis was altered in transformants: cytokinins were required to form roots, whereas auxin was toxic at the concentration used to induce rooting in the control.     

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.     

Different promoter regions control level and tissue specificity of expression of Agrobacterium rhizogenes rolB gene in plants

Expression of the rolB gene of A. rhizogenes T-DNA triggers root differentiation in transformed plant cells. In order to study the regulation of this morphogenetic gene, the GUS reporter gene was placed under the control of several deleted fragments of the rolB 5 non-coding region: carrot disc transformations and the analysis of transgenic tobacco plants containing these constructions identified the presence of distinct regulatory domains in the rolB promoter. Two regions (located from positions –623 to –471 and from –471 to –341, from the translation start codon) control the level but not the tissue specificity of rolB expression: progressive deletions of the rolB promoter starting from position –1185 to –341, although at different levels, maintained the same pattern of GUS expression — maximal in root meristems and less pronounced in the vascular tissue of aerial organs. Further deletion of 35 bp, from –341 to –306, drastically affected tissue specificity: GUS activity was still clearly detectable in the vascular tissue of the aerial organs while expression in the root meristem was totally suppressed. Analysis of transgenic embryos and seedlings confirmed that distinct promoter domains are responsible for meristematic (root) and differentiated (vascular) expression of rolB. Finally, we present data concerning the effects of plant hormones on the expression of rolB-GUS constructions.     

Regeneration and characterization of plants from potato root lines transformed by Agrobacterium rhizogenes

Summary Transformed root lines were obtained after infection of leaf segments and tuber discs of tetraploid potato cvs Bintje and Desirée with Agrobacterium rhizogenes. In response to shoot induction, about 10% of the root lines produced shoots through callus formation. The tests for opine suggest that all 26 shoot lines of cv Bintje (Ri-Bintje) and 13 of Desirée (Ri-Desirée) were transformed. All shoot lines were tetraploid except for one octoploid subshoot line of cv Desirée; no aneuploids were observed. With the exception of two shoot lines derived from the same root line, all other Ri-Bintje plants showed a pattern of phenotypic variation, generally observed among transformed plants. In contrast, the phenotype of Ri-Desirée plants was uniform and normal; variation was observed in tuber form and size. Phenotypic variation observed among Ri-plants appeared to be mainly root line-dependent, particularly for height of plants and tuber size and form. Variation was also observed within root and shoot lines and was more pronounced among the Ri-Bintje plants. Segregation of phenotypic characteristics was observed among transformed plants, resulting in the occurrence of phenotypes resembling the control. Chromosomal stability and the frequent reversion to normal phenotype of Ri-plants make A. rhizogenes particularly suitable as a virulence vector in the binary transformation system for the transfer of desirable genes.     

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 ri-mediated transformation for production of transgenic plants

Summary Agrobacterium rhizogenes-mediated transformation has been used to obtain transgenic plants in 89 different taxa, representing 79 species from 55 genera and 27 families. A diverse range of dicotyledonous plant families is represented, including one Gymnosperm family. In addition to the Ri plasmid, over half these plants have been transformed with foreign genes, including agronomically useful traits. Plants regenerated from hairy roots often show altered plant morphology such as dwarfing, increased rooting, altered flowering, wrinkled leaves and/or increased branching due to rol gene expression. These altered phenotypic features can have potential applications for plant improvement especially in the horticultural industry where such morphological alterations may be desirable. Use of A. rhizogenes and rol gene transformation has tremendous potential for genetic manipulation of plants and has been of particular benefit for improvement of ornamental and woody plants.     

Variation of alkaloid productivity among several clones of hairy roots and regenerated plants ofAtropa belladonna transformed withAgrobacterium rhizogenes 15834

Hairy root cultures ofAtropabelladonna were established by transformation withAgrobacterium rhizogenes 15834. Five clones of them were employed to study the production of hyoscyamine, the main constituent of the plant, together with other tropane alkaloids. The growth and alkaloid production of each clone were differently affected by basal liquid culture media tested. The transgenic plants regenerated from each clone of the hairy roots had different phenotypes and diverse alkaloid productivity both in the cultured condition and in productivitiy both in the cultured condition and in hydroponics.Abbreviations ANOVA analysis of variance - B5 medium Gamborg B5 medium - BA N⁶-benzyladenine - B.S. Balanced Solution - dw dry weight - EC electric conductivity - fw fresh weight - GC/MS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - MS medium Murashige and Skoog medium - NAA naphthalene-l-acetic acid - PCR polymerase chain reaction - SDS sodium dodecyl sulfate - TMS trimethylsilyl - WP medium Woody Plant medium     

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 the monocot Alstroemeria by Agrobacterium rhizogenes

An efficient procedure is described for transformation of calli of the monocotyledonous plant Alstroemeria by Agrobacterium rhizogenes. Calli were co-cultivated with A. rhizogenes strain A13 that harbored both a wild-type Ri-plasmid and the binary vector plasmid pIG121Hm, which included a gene for neomycin phosphotransferase II (NPTII) under the control of the nopaline synthase (NOS) promoter, a gene for hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a gene for -glucuronidase (GUS) with an intron fused to the CaMV 35S promoter. Inoculated calli were plated on medium that contained cefotaxime to eliminate bacteria. Four weeks later, transformed cells were selected on medium that contained 20 mg L^–1 hygromycin. A histochemical assay for GUS activity revealed that selection by hygromycin was complete after eight weeks. The integration of the T-DNA of the Ri-plasmid and pIG121Hm into the plant genome was confirmed by PCR. Plants derived from transformed calli were produced on half-strength MS medium supplemented with 0.1 mg L^–1 GA₃ after about 5 months of culture. The presence of the gusA, nptII, and rol genes in the genomic DNA of regenerated plants was detected by PCR and Southern hybridization, and the expression of these transgenes was verified by RT-PCR.     

Analysis of octopine left border-directed DNA transfer fromAgrobacterium to plants

We constructed a binary plasmid, pVR30, with a neomycin phosphotransferase II (nptII) plant expression cassette flanked by a pTiA6 left border on its right and a pTiA6 right border on its left. This plasmid was used to study transfer of DNA to plants from a left border in the presence of a right border. Infection of tobacco leaf discs with a wild type octopine strain ofAgrobacterium tumefaciens harbouring the binary plasmid resulted in the generation of kanamycin resistant calli at 18 to 26% frequency. Southern hybridization analysis of DNA isolated from eight transformed lines to different probes indicated that left border could mediate DNA transfer to plants in the presence of a right border in cis. Our results also suggest that transfer events corresponding to transfer of T- centre DNA of octopine Ti plasmid pTiA6 do occur. We have shown the relevance of left border- initiated T- DNA transfer by specifically selecting for such events and have confirmed it by Southern hybridization analysis. We also found that a border could be skipped in a few T- DNA transsfer events. This work was presented at “Plant Molecular Biology and Plant Biotechnology” symposium held in ICGEB, New Delhi during December 14–17, 1994.     

Production of hairy roots in Aconitum heterophyllum wall. using Agrobacterium rhizogenes

Summary A method for the production of hairy roots of Aconitum heterophyllum wall. is reported for the first time. Embryogenic callus cultures were successfully transformed using Agrobacterium rhizogenes strains viz. LBA 9402, LBA 9360, and A4 for the induction of hairy roots. The transgenic nature of hairy roots was confirmed by mannopine assay using paper electrophoresis. Best growth of transformed roots was obtained on 1/4 MS (Murashige and Skoog, 1962) medium with 3% sucrose. Total alkaloid (aconites) content of transformed roots was 2.96%, which was 3.75 times higher compared to 0.79% in the nontransformed (control) roots. Thin layer chromatography (TLC) analysis of the components of aconites in the transformed roots revealed the presence of heteratisine, atisine, and hetidine.