Effect of the rol genes from Agrobacterium rhizogenes on polyamine metabolism in tobacco roots

Plants of the mangrove species Pelliciera rhizophoreae and Avicennia germinans, exhibit pronounced oscillations in stomatal aperture under certain climatic conditions. During these oscillations, changes in transpirational water loss were closely followed by those in leaf water potential (ψ1) as indicated by continuous monitoring with an in situ dewpoint hygrometer. With this instrument, it was possible to measure dynamic changes in ψ1 for several days under constant conditions. Subsequently, the leaf was detached from the shoot and a pressure-volume (PV) curve was established by repeatedly weighing the leaf, still attached to the hygrometer during short interruptions of the water potential recordings. The pressure-volume relationship was then used to derive other water relations parameters from these water potential data. Thus, the procedure described herein allows a continuous analysis of the relevant components of bulk leaf water relations. Oscillations in water potential were also measured with single leaves using a pressure chamber. Water relations data obtained with these two different methods were in good agreement. In addition, osmotic potentials derived from the PV-analysis were well within the range of those determined cryoscopically using extracted cell sap.     

Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only

Atropa belladonna leaf disks were infected by a wild strain Agrobacterium rhizogenes 15834 harboring the Ri-TL-DNA and by a disarmed Agrobacterium tumefaciens strain harboring a construction with only rol ABC and npt II genes. Thirteen root lines were established and examined for their growth rate and alkaloid productivity to evaluate the possible role of rol genes in morphological differentiation and in tropane alkaloid formation. A great diversity has been observed in the growth rate of these 13 root lines. The root biomass increased up to 75 times. The total alkaloid contents were similar in the root lines obtained by infection with A. rhizogenes 15834 and A. tumefaciens rol ABC. The last ones accumulated between 4 (1.1 mg g(-1) DW) and 27 (8 mg g(-1) DW) times more alkaloids than the intact roots (0.3 mg g(-1) DW). This work has shown that the rol ABC genes were sufficient to increase tropane alkaloid production in A. belladonna hairy root cultures.     

Agrobacterium rhizogenes mediated transformation of grapevine (Vitis vinifera L.)

Genetically transformed grapevine (Vitis vinifera L.) roots were obtained after inocultation of in vitro grown whole plants (cv. Grenache) with Agrobacterium rhizogenes. The strain used contains two plasmids: the wild-type Ri plasmid pRi 15834 and a Ti-derived plasmid which carries a chimaeric neomycin phosphotrans-ferase gene (NPT II) and the nopaline synthase gene. Expression of the NPT II gene can confer kanamycin resistance to transformed plant cells. Slowly growing axenic root cultures derived from single root tips were obtained. Opine analysis indicated the presence of agropine and/or nopaline in established root cultures. For one culture, the presence of T-DNA was confirmed by dot-blot hybridization with pRi 15834 TL-DNA. Callogenesis was induced by subculturing root fragments on medium supplemented with benzylaminopurine and indoleacetic acid.Transformation of in vitro cultured grapevine cells has recently been reported (baribault T.J. et al., Plant Cell Rep (1989) 8: 137–140). In contrast with the results presented here, expession of the NPT II gene Conferred kanamycin resistance to Vitis vinifera calli that was sufficient for selection of trasformed cells.Abbreviations BAP benzylaminopurine - IAA indoleacetic acid - NAA naphtaleneacetic acid - NPT II neomycin phosphostransferase II - EDTA ethylenediaminetetraacetic acid     

Host-tissue differences in transformation of pumpkin (Cucurbita pepo L.) by Agrobacterium rhizogenes

Agrobacterium rhizogenes (wild-type strains 8196 and 15834) transformation of pumpkin (Cucurbita pepo L.) intact seedlings grown in vivo, and 6–8-day-old excised cotyledons cultured in axenic conditions was investigated. Transformed (hairy) roots were successfully induced only on the excised cotyledons with the strain 8196, while intact seedlings failed to form hairy roots with either of the two different bacterial strains. Axenic hairy-root cultures established on MS medium without hormones grew vigorously. Mannopine was detected in all transgenic root clones examined. The peroxidase activity in transformed roots was higher compared with normal roots. Electrophoretic analyses of soluble proteins and isoperoxidases showed substantial differences between transformed and normal pumpkin roots.     

Critical factors governing the efficient direct organogenesis in green-fleshed kiwifruit (Actinidia deliciosa) [A. Chev.] var. deliciosa

In Vitro Cellular & Developmental Biology - Plant - The present study was focused towards determining the factors governing direct organogenesis in kiwifruit cultivar ‘Allison’....     

Comparison between RAPD and SSR molecular markers in detecting genetic variation in kiwifruit (Actinidia deliciosa A. Chev)

Two different DNA-based techniques, random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers, were used for fingerprinting kiwifruit genotypes and for detecting undesirable genetic variation in micropropagated plants. The fragments were scored as present (1) or absent (0), and those readings were entered in a computer file as a binary matrix (one for each marker). Two cluster analyses were performed to express - in the form of dendrograms - the relationships among the genotypes and the genetic variability detected. Both DNA-based techniques were able to amplify all of the genotypes, but only SSR markers could detect genetic variation induced in micropropagated plants of cv. Tomuri. Two hypotheses were formulated to explain these results, both of them are in agreement with the results obtained using these two types of molecular markers. We conclude that when the tissue culture technique is used, the analysis of somaclonal variability could require more than one DNA-based technique; in fact, the genetic variation present in different sources could interfere or combine with the more or less polymorphic ability, as our results showed for SSR and RAPD markers.     

Getting to the root: The role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots

Agrobacterium tumefaciens and A. rhizogenes are the causative agents of the crown gall and hairy root diseases, respectively. The pathogenicity of both species is caused by an inter-kingdom transfer of DNA from the bacteria to wounded plant cells. This 'transfer-DNA' (T-DNA) contains oncogenes whose expression transforms the plant recipient cell into a rapidly dividing tumour cell. In the case of A. tumefaciens , three of these oncogenes have been shown to encode enzymes catalyzing the biosynthesis of the plant growth hormones auxin and cytokinin. Therefore, the unorganized cell division in the crown gall tumour can be largely explained by an unregulated overproduction of these plant growth regulators. In contrast, the hairy root disease is characterized by a massive growth of adventitious roots at the site of infection. Because of the similarities of the infection processes, and because A. rhizogenes and A. tumefaciens are very closely related, it has been suggested that the most important A. rhizogenes oncogenes, the so called rol genes, are also encoding proteins involved in the regulation of plant hormone metabolism. However, recent data indicate that this is not the case. Thus the rol genes have functions that most likely are different from producing mere alterations of plant hormone concentrations. This review summarizes recent results concerning the expression and function of the rol genes, and presents a model for the role of these genes, especially rolB and rolC , in the A. rhizogenes infection process.     

Efficient transformation of potato (Solanum tuberosum L.) using a binary vector in Agrobacterium rhizogenes

Summary We transformed three potato (Solanum tuberosum L.) genotypes by using A. rhizogenes or a mixture of A. rhizogenes and A. tumefaciens. Inoculations of potato stem segments were performed with Agrobacterium rhizogenes AM8703 containing two independent plasmids: the wild-type Ri-plasmid, pRI1855, and the binary vector plasmid, pBI121. In mixed inoculation experiments, Agrobacterium rhizogenes LBA1334 (pRI1855) and Agrobacterium tumefaciens AM8706 containing the disarmed Ti-plasmid (pAL4404) and the binary vector plasmid (pBI121) were mixed in a 11 ratio. The T-DNA of the binary vector plasmid pBI121 contained two marker genes encoding neomycin phosphotransferase, which confers resistance to kanamycin, and -glucuronidase. Both transformation procedures gave rise to hairy roots on potato stem segments within 2 weeks. With both procedures it was possible to obtain transformed hairy roots, able to grow on kanamycin and possessing -glucuronidase activity, without selection pressure. The efficiency of the A. rhizogenes AM8703 transformation, however, was much higher than that of the mixed transformation. Up to 60% of the hairy roots resulting from the former transformation method were kanamycin resistant and possessed -glucuronidase activity. There was no correlation between the height of the kanamycin resistance and that of the -glucuronidase activity in a root clone. Hairy roots obtained from a diploid potato genotype turned out to be diploid in 80% of the cases. Transformed potato plants were recovered from Agrobacterium rhizogenes AM8703-induced hairy roots.     

Transformation of apple (Malus domestica Borkh.) with the stilbene synthase gene from grapevine (Vitis vinifera L.) and a PGIP gene from kiwi (Actinidia deliciosa)

The objective of the present research was to introduce genes with antifungal potential into the commercially important apple cvs. Elstar and Holsteiner Cox in order to establish resistance against fungal diseases. The gene encoding the stilbene synthase (Vst1) from Vitis vinifera L., responsible for the synthesis of the phytoalexin resveratrol in grapevine, and the gene for a polygalacturonase-inhibiting protein (PGIP) from kiwi (Actinidia deliciosa) were transferred into Holsteiner Cox and Elstar via Agrobacterium tumefaciens-mediated transformation. A total of nine transgenic Holsteiner Cox clones and one transgenic E clone carrying the stilbene-synthase gene as well as three transgenic Holsteiner Cox lines harbouring the polygalacturonase-inhibiting protein from Kiwi were identified via polymerase chain reaction and Southern blot analysis. High performance liquid chromatography analysis revealed the accumulation of a resveratrol-derivate, a glycoside, in transgenic Vst1 plants.Abbreviations BAP 6-Benzylaminopurine - E Elstar - H Holsteiner Cox - HPLC High performance liquid chromatography - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - TDZ Thidiazuron - YEP Yeast extract broth Communicated by H. Lörz     

Genetic transformation of cauliflower (Brassica oleracea L. var. Botrytis) by Agrobacterium rhizogenes

Cauliflower plantlets were inoculated with different Agrobacterium rhizogenes strains. Numerous hairy roots were induced on cauliflower hypocotyls by agropine-type strains. Fewer roots were obtained with mannopine-type strains. In vitro cultures were established both from normal and hairy roots. Plant regeneration occured spontaneously from normal and transformed roots. Regenerated plants contained the same opines (if present) as root cultures. Some mannopine-positive regenerants displayed a modified phenotype. Relationships between phenotype, opine content, T-DNA content and/or expression are discussed.     

Pseudomonas syringae pv. syringae Causal Agent of Disease on Floral Buds of Actinidia deliciosa (A. Chev) Liang et Ferguson in Italy

Pseudomonas syringae pv. syringae as causal agent of floral buds necrosis, has been isolated from kiwifruit plants cv. Hayward in Italy. The pathogen has been identified on the basis of morphological, biochemical and physiological features and also on the basis of pathogenicity. Symptoms, similar to those caused by Pseudomonas viridiflava —browning and darkening on floral buds, have been observed on kiwifruit plants in a wide area in the North of Latium region (central Italy), in Viterbo province.     

Morphological anomalies in pollen tubes of Actinidia deliciosa (kiwi) exposed to 50 Hz magnetic field

The role of the pollen grain, with respect to the reproductive process of higher plants, is to deliver the spermatic cells to the embryo sac for egg fertilisation. Delivery occurs through the pollen tube, a self produced organ that is generated when the pollen grain reaches the stigma surface. The effect of magnetic fields on pollen tube growth was reported in a recent publication by Germanà et al. Pollen tube growth is an interesting candidate for the detailed study of the effects of electromagnetic fields on cytoplasmic structures and organelles. In this research Actinidia deliciosa (kiwifruit) pollen grains were germinated in the presence of an alternating magnetic field (50 Hz). Our results, although of preliminary nature, show that pollen tube growth is affected by magnetic fields. The analysis of the observed anomalies in the pollen tube appear to be the result of changes in the ionic charges within the pollen tube cytoplasm.     

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.     

Two-phase temporary immersion system for Agrobacterium rhizogenes genetic transformation of sage (Salvia tomentosa Mill.)

Hairy root cultures of Salvia tomentosa were initiated by transformation with Agrobacterium rhizogenes. To prevent necrosis in the explants and to protect young hairy roots, Amberlite XAD-4 resin, in combination with a temporary immersion cultivation system, was applied. HPLC analyzes showed that the resin adsorbed more than 93% of the released phenolic acids and 100% of the released flavonoids. The decreased content of the released phenolics significantly reduced their destructive effects on the plant tissues, prevented, and speeded up the appearance of hairy roots.     

发根农杆菌Ri质粒rolB基因研究进展（综述）

RiT-DNArolB基因是发根农杆菌转化植物的决定因子,rolB基因表达引起转基因植物形成大量毛状根（hairy root)。本文介绍近年来rolB基因的位点,表达与调控及RolB蛋白结构与功能方面的研究进展。     

Stable transformation of Mesembryanthemum crystallinum (L.) with Agrobacterium rhizogenes harboring the green fluorescent protein targeted to the endoplasmic reticulum

Stable transformation of Mesembryanthemum crystallinum L. (common ice plant) with a green fluorescent protein (GFP) construct targeted to the endoplasmic reticulum was obtained. Seven and fourteen days after germination seedlings were infected with Agrobacterium rhizogenes strain ARqua1 either by direct coating of the cut radicles with bacteria growing on solid medium or by immersion of the cut surface in bacterial suspension at different optical densities. Both methods of infection resulted in production of GFP-positive roots with a frequency ranging from 6 to 20% according to the age of the explants and the application procedure. The green fluorescing roots displayed the typical hairy root phenotype and were easily maintained in liquid medium without growth regulators for over 2 years. Stable expression of the transgene in the roots was confirmed by polymerase chain reaction (PCR), immunoblotting and the capacity of roots to grow and produce callus on kanamycin-enriched medium. Nineteen endogenous cytokinins were determined in transgenic and non-transformed roots. The results revealed significantly lower levels of the free bases of isopentenyladenine, dihydrozeatin, cis- and trans-zeatin, as well as a conspicuous decline in concentrations of the corresponding nucleosides and most nucleotides in transgenic roots compared to the wild type. Comparison of the cytokinin profiles in transgenic and non-transformed roots suggested that transformation by A. rhizogenes disturbed cytokinin metabolism during the early steps of biosynthesis. Calli obtained from transformed roots were GFP-positive and remained non-regenerative or displayed high rhizogenic potential depending on the auxin/cytokinin ratio in the medium. Calli and callus-derived roots showed a strong GFP signal for over 2 years.     

Morphogenetic sequences of the rooting process after in vitro culture or Agrobacterium rhizogenes inoculation

Abstract

After a brief review dealing with the factors inducing adventitious rhizogenesis, the morphogenetic patterns of the rooting process induced by in vitro culture or after Agrobacterium rhizogenes inoculations are described. In vitro cultures of Nicotiana tabacum, Datura innoxia and Crepis capillaris leaf explants on Murashige and Skoog's medium supplemented with sucrose and IAA or NAA have been used in order to identify the target cells for direct and indirect rooting. The structural and functional features of the prerhizogenetic cells are described and the ploidy levels of both direct and indirect root meristems are examined by means of DNA microspectrophotometry. Data on the synergistic effects between auxin, sucrose and amino acids (particularly L-ornithine) for the reactivation of the prerhizogenetic cells are also given.

Transformed roots from carrct root discs or pea epicotyls after Agrobacterium rhizogenes inoculation arise indirectly from cambium-like layers differentiated inside a previously formed callus. Numerous auxin-like symptoms at the cell level are detected after the inoculation, suggesting modifications in the endogenous contents of auxin prior to the rooting process. It is also shown that initially non-susceptible cells (fully differentiated tobacco pith) can be induced to become susceptible by in vitro treatments resulting in cell reactivation before inoculation with the bacteria. Further work is in progress to extend these observations to non-susceptible species in order to check, via the occurrence of transformed roots, their ability to react positively to foreign Ri T-DNA.     

Fast, efficient and reproducible genetic transformation of Phaseolus spp. by Agrobacterium rhizogenes

This transformation procedure generates, with high efficiency (70-90%), hairy roots in cultivars, landraces and accessions of Phaseolus vulgaris (common bean) and other Phaseolus spp. Hairy roots rapidly develop after wounding young plantlets with Agrobacterium rhizogenes, at the cotyledon node, and keeping the plants in high-humidity conditions. Callogenesis always precedes hairy-root formation, and after 15 days, when roots develop at wounded sites, the stem with the normal root is cleaved below the hairy root zone. Transgenic roots and nodules co-transformed with a binary vector can be easily identified using a reporter gene. This procedure, in addition to inducing robust transgenic hairy roots that are susceptible to being nodulated by rhizobia and to fixing nitrogen efficiently, sets the foundation for a high-throughput functional genomics approach on the study of root biology and root-microbe interactions. This protocol can be completed within 30 days.     

The response to auxin of rapeseed (Brassica napus L.) roots displaying reduced gravitropism due to transformation by Agrobacterium rhizogenes

It has recently been documented that, compared to untransformed controls, the roots of oilseed rape (Brassica napus L. CV CrGC5) seedlings transformed by Agrobacterium rhizogenes A4 show a reduced gravitropic reaction (Legué et al. 1994, Physiol Plant 91: 559–566). After stimulation at 90°C or 135°, the transformed root tips curve, but never reach a vertical orientation. In the present study, we investigated the causes of reduced gravitropic bending observed in stimulated transformed root tips. First, we localized the gravitropic curvature in normal and in transformed roots after 1.5 h of stimulation. The cells involved in root curvature (target cells) corresponded at the cellular level to the apical part of the zone of increasing cell length. In transformed roots grown in the vertical position, these cells showed a reduction in cell length compared to controls. Because auxin is considered to be the gravitropic mediator, the response of normal and transformed roots to exogenous auxin was studied. Indole-3-acetic acid (IAA) was applied along the first 3 mm using resin beads loaded with the hormone. In comparison to normal roots, transformed roots showed reduced bending toward the bead at all points of bead application. Moreover, the cells which responded to IAA corresponded to the target cells involved in the gravitropic reaction. The level of endogenous IAA was lower in transformed roots. Thus, it was concluded that the modified behavior of transformed roots during gravitropic stimulation could be due to differences either in IAA levels or in reactivity of the target cells to the message from the cap.Abbreviations DEZ distal elongation zone - ELISA enzymelinked immunosorbent assay - T-DNA DNA transferred from Agrobacterium rhizogenes to the plant genome This work was supported by the Centre National d'Etudes Spatiales.