Hairy Root Syndrome Research Articles

Induced expression of rolC for study of its effect on the expression of genes associated with nicotine synthesis in tobacco

Background. Agrobacterium rhizogenes rol genes cause not only hairy root syndrome in plants, but also affect their secondary metabolism. There are cases of increasing of nicotine content in transgenic tobacco roots expressing rolC alone or in combination with other rol genes. In this work, we evaluated the change in the expression of nicotine synthesis genes and their regulators in response to the induction of expression of rolC.
 Materials and methods. Plant material was represented by three Nicotiana tabacum genotypes: cv. Samsun and two transgenic lines, derived from this cultivar and containing rolC under dexamethasone inducible promoter: A. rhizogenes rolC (Pdex-A4rolC) and N. tabacum rolC (Pdex-trolC) correspondingly. Fluidigm Biomark RT-PCR was used for evaluation of expression of QPT1, QPT2, A622, ODC, ADC, PMT1, PMT2, PMT3, PMT4, MPO1, MPO2, BBL, MATE1, MATE2, ARF6, ERF168, ERF189, A4rolC, NtrolC, and reference gene gapdh. HPLC-MS / MS analysis was used to determine content of nicotine and its derivatives in plant tissues.
 Results. Expression of PMT genes for the synthesis of the pyrrolidine ring, as well as the genes, controlling enzyme for final stages of nicotine synthesis, was higher in transgenic lines without induction of rolC expression. Regulatory genes were activated by dexamethasone in both transgenic and control lines, indicating the inapplicability of rolC dexamethasone induction for their study. The level of expression of PMT and MPO genes increased over time in transgenic dexamethasone-induced lines. Nicotine content decreased in transgenic dexamethasone-induced plants.
 Conclusions. The rolC gene does not play a primary role in the regulation of nicotine synthesis genes. The mechanism of regulation of different nicotine biosynthesis genes and TFs varies.

The <i>rolC</i> gene of agrobacteria: towards the understanding of its functions

Agrobacterium rhizogenesConn is a soil bacterium, which can transform plants by inserting a plasmid fragment into the plant genome. This fragment contains a “root locus”: four genes that cause root overgrowth of the transformed plant, the so-called “hairy root syndrome”. The most studied gene of the root locus isrolC. For more than 30 years of research on this gene, data have been obtained on its expression, protein localization and putative functions of the protein as well as on its effect on plant morphology and biochemistry. TherolCtransformation leads to multiple morphological effects, most common among which are dwarfism, bushiness, and a change in the shape of the leaf blade. Such specific plant reactions are associated with changes in hormone balance under the influence ofrolC. The levels of auxins, cytokinins, and abscisic acid do change in transformed plants, but no regularities have been revealed. Also, the signaling pathways ofrolCaffecting the hormonal system of plants are not established. Morphogenetic effects can occur in varying degrees depending on the promoter under which therolCworks. A constitutive promoter usually leads to a more pronounced effect when compared to a gene that operates under a native promoter. Secondary plant metabolism is also affected byrolC. The synthesis of various metabolites is amplified in transformants, and, in contrast to morphological effects, this biochemical effect does not depend on the promoter. Some secondary metabolites are associated with the plant defense system; thus,rolCis able to indirectly influence this aspect of plant physiology. This review summarizes the results of therolCgene studies in plants. The authors formulate the main hypotheses regarding the mechanisms of the gene in order to promote our understanding of its function in plants.

Genetic transformation of the medicinal plant Salvia runcinata L. f. using Agrobacterium rhizogenes

Two different agropine strains (A4T and LBA 9402) of Agrobacterium rhizogenes were tested for their capacity to induce hairy roots on Salvia runcinata L. f. explants. Both nodal and leaf explants were examined and hairy roots formed 3 to 4weeks after inoculation. Although the LBA 9402 strain produced significantly more roots on each explant compared with the A4T strains, at the time of transformation, stable cultures could only be established using the A4T strain at a frequency of 4.17%. Putative transgenic roots exhibited a faster growth pattern when compared with wildtype roots. Out of the roots established, we continued the study with two clones that showed typical features of the hairy root syndrome. Amplification of the rol A, rol B, rol C and ags gene (showing 300, 400, 600 and 1600bp bands after electrophoresis, respectively) was positive using the polymerase chain reaction (PCR) and reverse-transcriptase-polymerase chain reaction (RT-PCR). Moderate antifungal activity against two Fusarium strains was exhibited by extracts generated from transgenic clones. We were also able to show the presence of some caffeic acid metabolites. These biochemicals are known to exhibit various biological activities, and therefore, the transgenic hairy roots of S. runcinata may be an alternative source of these industrially useful chemicals.

Phenotype of Transgenic Tobacco Plants (Nicotiana tabacum cv. Petit Havana SR-1) Expressing 1724orf13 Gene of Agrobacterium rhizogenes strain MAFF301724

Nicotiana tabacum cv. Petit Havana SR-1 transgenic plants expressing ORF13 of Agrobacterium rhizogenes strain MAFF301724 under different promoters displayed plant morphology abnormalities. They were small, with short and variable internodes lengths; leaves were small, asymmetric, rounded, wrinkled and dark green; flowers were short, and irregularly shaped. This phenotype was also exhibited, similar, but not completely the same, to those of hairy root syndrome, indicating that expression of ORF13 influences plant development. Keywords: ORF13, Agrobacterium rhizogenes strain MAFF301724, transgenic plants, morphology abnormalities

Effects associated with insertion of cryptogein gene utilizing Ri and Ti plasmids on morphology and secondary metabolites are stable in Bacopa monnieri-transformed plants grown in vitro and ex vitro

Bacopa monnieri (Linn.) Wettst. (family Scrophulariaceae), a therapeutically important perennial herb, was transformed to study the stability of the effects associated with the insertion of a gene encoding β-cryptogein, a proteinaceous elicitor, either alone or in addition to the T-DNA genes in long-term in vitro culture as well as after transfer to the greenhouse. Plant lines BmAr-IXcrypt obtained via transformation by LBA 9402-crypt (pRi1855+pBin19++crypt) showed integration and expression of rolA, rolB, rolC, and rolD genes after 4 years of maintenance in vitro. The plant line BmAt-ncrypt obtained via transformation with A. tumefaciens strain LBA4404-crypt (harboring pBin19++crypt) as well as plant line BmAr-IXcrypt showed stable integration and expression of nptII gene and crypt gene even after transfer to greenhouse. The clones of Ri-crypt-transformed plants differed morphologically from Ri-transformed plants in that the typical “hairy root syndrome” was not observed in plants expressing crypt gene in the presence of rol genes. Except plant line BmAr-IXcrypt, none of the other transgenic plant lines showed any alteration in floral morphology and onset of flowering. The crypt-transformed plant lines (BmAr-IXcrypt and BmAt-ncrypt), maintained under long-term culture, showed significantly higher (p ≤ 0.05) amount of bacoside production in vitro (1.66- to 2.05-fold, with respect to non-transformed plants). In addition, accumulation of all the four bacosides was significantly higher (p ≤ 0.05) in crypt-transformed plants as compared to non-transformed plants grown for 1 year in greenhouse. Thus, the crypt-transformed plant lines of B. monnieri may be considered as the potential source for sustainable bioproduction of bacosides.

Changes in morphological phenotypes and essential oil components in lavandin ( Lavandula × intermedia Emeric ex Loisel.) transformed with wild-type strains of Agrobacterium rhizogenes

Hairy roots were induced from leaf-derived calli of lavandin ( Lavandula × intermedia Emeric ex Loisel.) by infection with wild-type strains of Agrobacterium rhizogenes, A-5 (MAFF 02-10265) and A-13 (MAFF 02-10266). A-5-inoculated calli formed hairy roots more efficiently than A-13 ones. The transgenic shoots could be obtained from hairy root segments mediated by each Agrobacterium strain. However, different plant growth regulators were required for efficient adventitious shoot formation in each strain. In A-5, the most efficient adventitious shoot formation rate of 23.8% was observed in a medium with 4.4 × 10 −6 M of 6-benzylaminopurine. On the other hand, a significantly higher rate of 13.2% was detected in a medium with 4.0 × 10 −7 M of N-(2-chloro-4-pyridyl)- N′-phenylurea in A-13. Most of the regenerated plants showed dwarfism with closed internodes and extensive lateral branching, which were typical characteristics of ‘hairy root syndrome’. On the other hand, only nine of the 45 regenerated plants formed flower buds in early June, a delay of about one month compared with nontransgenic regenerated plants. The floral stalks and spikes of these plants were very short, resulting in a compacted form. Many regenerants showed a significantly lower productivity of essential oil than nontransgenic regenerants. Moreover, the relative percentage of the linalyl-cation-derived compounds, linalool and linalyl acetate, decreased in most of the regenerated plants. Compact plants with the ability of flower bud formation are assumed to be valuable not only for lavandin breeding, but also for clarifying the interaction between rol genes expression and essential oil production.

Effect of the Number of rol Genes Integrations on Phenotypic Variation in Hairy Root-Derived Hypericum perforatum L. Plants

The extent of phenotypic variation of St. John's wort (Hypericum perforatum L.) plants transformed with wild agropine ATCC 15834 Agrobacterium rhizogenes plasmid was evaluated with respect to the number of rol genes integrations. The transfer of T(L)-DNA to plant explants during each transformation event was incomplete with different rolA, rolB, and rolC copy numbers. Along with typical features representing the hairy root syndrome, an altered size, number and density of dark and translucent glands, changes in ability to synthesize secondary metabolites, and reduced fertility were observed. The highest copy number of transferred rol genes resulted in weak expression of transgenic character and comparable quantitative parameters with the controls. Only 1 out of 11 transgenic clones was able to produce seed progeny and not more than 4 out of its 35 offsprings were positive for rolC gene integration. Sterility of the clones was due to retarded development of both gametophytes.

Rol (root loci) gene as a positive selection marker to produce marker-free Petunia hybrida

The presence of marker genes conferring antibiotic or herbicide resistance in transgenic plants has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the strategies to excise the selection marker gene from transgenic plants. Agrobacteriumtumefaciens strain EHA105 harboring a rol-type MAT vector, pMAT101, was used to produce morphologically normal transgenic Petunia hybrida ‘Dainty Lady’ employing rol gene as the selection marker gene. LacZ gene was used as a model gene of interest. Infected explants were cultured on plant growth regulator (PGR)- and antibiotic-free half-strength MS medium. Sixty-five percent of the infected explants produced hairy roots. The hairy roots were separated and proliferated on 1/2 MS hormone-free medium. Shoots produced from the hairy roots on 1/2 MS medium supplemented with benzylaminopurine (BA) and naphthalene acetic acid (NAA) exhibited hairy root syndrome (Ri syndrome) such as dwarfed, reduced apical dominance, short internodes and increased rooting, but subsequently produced normal-looking marker-free shoots. Molecular analysis of DNA from the hairy roots, shoots with Ri syndrome and morphologically normal shoots revealed that the normal shoots had only LacZ gene, and the removable cassette consisting of rol, R (recombinase) and GUS genes was excised. From this study it can be concluded that the chimeric rol genes can be used as a selection marker for Agrobacterinum-mediated transformation of Petunia hybrida and that the production of marker-free normal transgenic plants is possible without using selective chemical agents employing rol-type MAT vector.

Agrobacterium tumefaciens-mediated hairy root production from seedlings of Chinese cabbage

Cruciferous hairy roots are often used for improving drought adaptability, peroxidase production, andin vitro subculturing ofPlasmodiophora brassicae. For metabolic engineering,Agrobacterium tumefaciens-mediated systems have previously been developed for hairy root production in other plant species. Here, we used therolABC gene binary construct inA. tumefaciens strain GV3101 to establish cultures of Chinese cabbage hairy roots. On both solid and liquid media, therolABC hairy root lines exhibited a wild-type hairy root syndrome in terms of their growth and morphology. This demonstrates that those three genes are sufficient to induce high-quality hairy roots in Chinese cabbage. Such a system could be useful for the stable production of secondary metabolites in that species.

Terpenoid indole alkaloid production by Catharanthus roseus hairy roots induced by Agrobacterium tumefaciens harboring rol ABC genes

We have established Catharanthus roseus hairy root cultures transgenic for the rol ABC genes from T(L)-DNA of the agropine-type Agrobacterium rhizogenes strain A4. The rol ABC hairy root lines exhibit a wild-type hairy root syndrome in terms of growth and morphology on solid medium. However, they differ from wild-type hairy root lines in that they more frequently have excellent adaptability to liquid medium and do not appear to form calli during cultivation. Moreover, they do not produce detectable levels of mannopine and agropine which, in contrast, are often synthesized abundantly in wild-type hairy root lines. The absence of these opines does not appear to cause the rol ABC lines to have higher levels of terpenoid indole alkaloids than wild-type hairy root lines. Unlike wild-type lines, rol ABC lines produce very similar levels of total alkaloids despite wide variations in individual alkaloid contents. This work demonstrates that the three genes rol ABC are sufficient to induce high-quality hairy roots in Catharanthus roseus.

Shoot regeneration capacity from roots and transgenic hairy roots of tomato cultivars and wild related species

The organogenetic competence of roots and Agrobacterium rhizogenes-induced hairy roots of twelve Lycopersicon genotypes was investigated. Both roots and hairy roots of L. peruvianum, L. chilense, L. hirsutum and two L. peruvianum-derived genotypes regenerated shoots after 2–4 weeks of incubation on zeatin-contained medium. Anatomical analysis showed that shoot regeneration in roots could be direct or indirect, depending on the genotype considered. Hairy roots showed considerable differences in their morphogenetic responses, when compared to the corresponding non-transgenic roots. The differences observed may reflect the influence of the introduced rol genes on hormonal metabolism/sensitivity. Hairy root-derived T0 plants had shortened internodes, wrinkled leaves and abundant root initiation, and most produced flowers and fruits with viable seeds. The hairy root syndrome was detected early in germinating T1 seedlings as a strong reduction in the hypocotyl length. Our data point to the possibility of the use of A. rhizogenes, combined with regenerating Lycopersicon genotypes, in a very simple protocol, based on genetic capacity instead of special procedures for regeneration, to produce transgenic tomato plants expressing rol genes, as well as, genes present in binary vectors. Furthermore, the regeneration differences observed in each Lycopersicon genotype and in transgenic materials expressing rol genes open the possibility for their use in the analysis of both the biochemical and the genetic background of organogenetic competence.

Analysis of Unique Variable Region of a Plant Root Inducing Plasmid, pRi1724, by the Construction of its Physical Map and Library

Ri plasmids in Agrobacterium rhizogenes specifically induce the hairy root syndrome on various dicotyledonous plants. Its T-DNA transfer system as well as those of Ti plasmids have successfully provided the fundamental technique to introduce exogenous genes into plants. To study the Ri genome structure, we constructed a complete BamHI physical map and a lambda library of pRi1724 of A. rhizogenes strain 1724. By using these, we carried out the complete sequence of the 74-kb region between the right border of T-DNA and tra operon, which is the highly variable region (VAR) among Ri and Ti plasmids. As a result, we found three kinds of putative ABC-type transport operons, histidine utilization operon, glycerol utilization operon and two chemoreceptor genes. In addition, a virulence-related gene, tzs was located independently of the vir region.

Phenotype and hormonal status of transgenic tobacco plants overexpressing the rolA gene of Agrobacterium rhizogenes T-DNA.

The rolA gene of the TL-DNA of Agrobacterium rhizogenes Ri-plasmid plays a major role in establishing the hairy root syndrome in transgenic plants. Transgenic tobacco plants (Nicotiana tabacum L.) expressing constitutively the rolA gene under the transcriptional control of the 35S RNA promoter show pronounced phenotypical alterations. P35S-rolA transgenic tobacco plants are characterized by stunted growth, dark green wrinkled leaves with an altered length-to-width ratio, condensed influorescences, retarded onset of flowering, a reduced number of flowers and shortened styles. To investigate whether the pleiotropic alterations of growth and development are linked to an altered hormonal status we have compared the immunoreactive content of indole-3-acetic acid, cytokinins, abscisic acid, gibberellin and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) of seedlings and different tissues of P35S-rolA transgenic plants, transgenic plants expressing the rolA gene under control of its own phloem-specific promoter and wild-type plants. Multiple tissue-specific alterations of phytohormone concentrations are the consequence of rolA gene activity. Changes of phytohormonal content can explain part of the rolA-induced phenotypic alterations. Most strikingly, in young and fully developed leaves of rolA and P35S-rolA transgenic clones a 40-60% reduction of immunoreactive gibberellin A1 was found, as compared to wild-type leaves. Treatment of wild-type tobacco plants with inhibitors of gibberellin biosynthesis phenotypic alterations similar to those of rolA transgenic plants. This suggests that the reduction of gibberellic acid content is indirectly but causally involved in rolA-induced alterations of stem elongation and planar leaf blade growth.

Protein, Free Amino Acid and Polyamine Contents during Development of Hairy Root Nicotiana tabacum Plants

Summary Clones of transgenic tobacco plants, regenerated from transformed roots arising from Agrobacterium rhizogenes inoculation, were analyzed both in vivo under greenhouse conditions and in in vitro culture. Transformed plants displayed phenotypic alterations, referred to as hairy root syndrome. These traits were particularly accentuated in some plants, designated supertransformed, and seemed to be modulated by in vivo versus in vitro culture conditions. The comparison of protein, amino acid and polyamine levels between normal and transformed plants revealed alterations in nitrogen metabolism. In fact, under in vivo conditions apices and leaves of transformed plants displayed lower levels of glutamine and asparagine. The evolution in total polyamine content was generally an increase from 6-13 weeks, followed by a decrease at 20 weeks, the transformed tissues always containing lower polyamine levels than the normal ones. The major difference in vivo consisted in a strong reduction in the TCA-soluble and - insoluble bound polyamines, particularly evident in the leaves of transformed and supertransformed plants. On the whole, significant differences were found between in vivo and i n vitro conditions: TCA-soluble bound polyamines were undetectable in both transformed and untransformed in vitro plantlets; in addition, supertransformed in vitro plantlets contained unexpectedly high levels of chlorophyll, proteins, amino acids and free polyamines compared with their counterparts grown in soil.

Independent and synergistic activity of rol A, B and C loci in stimulating abnormal growth in plants

The Ri plasmid A4 of Agrobacterium rhizogenes contains within its T-DNA genetic information able to trigger root formation in infected plants. Tobacco plants regenerated from transformed roots display the hairy root (hr) syndrome. We show that DNA fragments containing the rol B locus alone are able to induce root formation both in tobacco and kalanchoe tissues. The rol A and the rol C loci by themselves are also able to induce root formation in tobacco but not in kalanchoe. This capacity to induce root formation in either host is greatly increased when the rol A and/or C loci are combined with the rol B locus. Root induction is shown to be correlated with the expression of the rol loci. Transgenic plants exhibit all the characteristics of the hairy root syndrome only when all three loci are present and expressed. Although the activity of the rol encoded functions is synergistic, each of them appears to independently influence host functions involved in the determination of root differentiation.