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.     

Variation in structure and plant regeneration of Agrobacterium rhizogenes transformed and control roots of the potato cv. Bintje

Agrobacterium rhizogenes transformed and control roots of the tetraploid potato cv. Bintje were compared. Transformed roots were obtained after infection by A. rhizogenes 15834 or 1855. Both in leaf and stem segments, more roots were formed at the basal side of the segments, indicative for a polarity in root formation. As compared to control roots the transformed roots are characterized by smaller and more densely stained cells, a zone of cell division, and smaller statoliths. These characteristics are correlated with vigorous growth, high branching incidence and diminished geotropism. The plant regeneration procedure according to Ooms et al. [1] was modified. The transformed roots required less 2,4-D than control roots for the induction of shoot-competent calli. The callus and shoot induction phases were reduced from 8 and 6 weeks to 3 and 3 weeks, respectively. Upon induction, 25%, 58% and 61% of the root clones originating from tuber, stem and leaf, respectively, produced shoots, whereas all of the control roots produced shoots. Shoot outgrowth occurred on liquid MS medium in the absence of hormones.Abbreviations Ri-root Agrobacterium rhizogenes transformed root - BAP benzylaminopurine - IAA indoleacetic acid - GA₃ gibberellic acid - NAA naphthaleneacetic acid - 2,4-D 2,4 dichlorophenoxyacetic acid     

Traits of transgenic Atropa belladonna doubly transformed with different Agrobacterium rhizogenes strains

Hairy root cultures of Atropa belladonna L. were established by infection either with Agrobacterium rhizogenes ATCC 15834 or MAFF 03-01724, and transgenic plants were obtained from both hairy root cultures. Doubly transformed roots were induced by re-infection of the leaf segments of transgenic Atropa belladonna plants (A. rhizogenes 15834) with MAFF 03-01724. Shoots and viviparous leaves were regenerated from the doubly transformed roots. The genetic transformation was determined by the opine assay (agropine, mannopine and/or mikimopine) and polymerase chain reaction. Physiological changes and tropane alkaloid biosynthesis in the hairy roots (singly and doubly transformed) were investigated. The alkaloid content in the doubly transformed root strain was intermediate as compared to the root strains which were singly transformed. On the other hand endogenous IAA levels in doubly transformed roots were significantly decreased compared to both singly transformed roots.Abbreviations BA benzyladenine - IAA indoleacetic acid - NAA naphthaleneacetic acid - PCR polymerase chain reaction - t-ZR trans-zeatin     

Role of the Surface and Extracellular Substances of the Phytopathogenic Bacterium Xanthomonas campestris in Its Interactions with Cabbage Plants

Changes in some physiological and biochemical characteristics of cabbage (cv. Slava) seedling roots in response to inoculation with the phytopathogen Xanthomonas campestrisand its surface and extracellular substances were evaluated. Seven days after the inoculation, the growth of the roots was slightly suppressed and they contained increased amounts of peroxidase. The effect of the lipopolysaccharides stripped from the cell surface or isolated from the culture liquid of X. campestriswas similar to that of the whole cells of the phytopathogen. The bacterial lectin isolated from the cell surface material did not induce any defense response in cabbage plants but, presumably, could play a role in the contact interactions between bacteria and plants.     

A novel life cycle arising from leaf segments in plants regenerated from horseradish hairy roots

Summary Horseradish (Armoracia rusticana) hairy root clones were established from hairy roots which were transformed with the Ri plasmid in Agrobacterium rhizogenes 15834. The transformed plants, which were regenerated from hairy root clones, had thicker roots with extensive lateral branches and thicker stems, and grew faster compared with non-transformed horseradish plants. Small sections of leaves of the transformed plants generated adventitious roots in phytohormone-free G (modified Gamborg's) medium. Root proliferation was followed by adventitious shoot formation and plant regeneration. Approximately twenty plants were regenerated per square centimeter of leaf. The transformed plants were easily transferable from sterile conditions to soil. When leaf segments of the transformed plants were cultured in a liquid fertilizer under non-sterile conditions, adventitious roots were generated at the cut ends of the leaves. Adventitious shoots were generated at the boundary between the leaf and the adventitious roots and developed into complete plants. This novel life cycle arising from leaf segments is a unique property of the transformed plants derived from hairy root clones.     

Production of transgenic plants via Agrobacterium rhizogenes-mediated transformation of Panax ginseng

 Hairy roots of Panax ginseng were obtained after root disks were infected with wild-type strain Agrobacterium rhizogenes 15834. Three lines of hairy roots with different pigmentation were selected. Embryogenic callus was induced on Murashige and Skoog medium containing 1.0 mg/l 2,4-D. The frequency of embryogenic callus formation was 37.4% in a line with red pigmentation. Somatic embryo development from embryogenic callus was efficiently achieved by lowering the concentration of 2,4-D (0.5 mg/l). After the germination of somatic embryos on medium with 10 mg/l GA₃, the embryos were transferred to 1/2-MS medium without GA₃. The transformed ginseng plantlets had an actively growing root system with abundant lateral roots. The phenotypical alteration of transformed ginseng plants might be valuable character for increasing root yield. Received: 27 March 1999 / Revision received: 18 May 1999 / Accepted 8 July 1999     

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.     

Hairy root induction of<Emphasis Type="Italic"> Papaver somniferum</Emphasis> var.<Emphasis Type="Italic"> album</Emphasis>, a difficult-to-transform plant,by<Emphasis Type="Italic"> A. rhizogenes</Emphasis> LBA 9402

Two strains of Agrobacterium rhizogenes (15834, LBA 9402) and one Agrobacterium tumefaciens strain [GV 3101 (PMP90RK, p35SGUS-2)] and four culture media were tested and compared for their ability to induce hairy root formation on wounded Papaver somniferum L. hypocotyls. Five weeks after the infection with A. rhizogenes LBA 9402, hairy roots appeared on 80% of the hypocotyls maintained in the hormone-free liquid medium. Six hairy-root cultures were established. Transformation was confirmed by polymerase chain reaction analysis. One clone was analysed for its alkaloid production. The total alkaloid content was higher in the transformed roots (0.46±0.06% DW) than in the untransformed roots (0.32±0.05% DW). The transformed roots accumulated three times more codeine (0.18±0.02% DW) than intact roots (0.05±0% DW). Moreover, morphine (0.255±0.03% DW) and sanguinarine (0.014±0% DW) were found in the liquid culture medium.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - LS Linsmaier and Skoog     

Agrobacterium rhizogens mediated transformation of Rauvolfia serpentina: Regeneration and alkaloid synthesis

Shoot cultures of Rauvolfia serpentina infected with Agrobacterium rhizogenes 15834 produced tumourous tissue at the site of injection, which eventually developed callus with hairy roots. Sporadic shoot formation occurred from the hairy roots. The shoots were grown to maturity in the green house. The mature transformed plants (RST) showed distinct variations in their physiological characteristics. The flowers of the transformed plants were more delicate and less pigmented when compared to the flowers of the mature normal plants. The roots of the transformed plants were hairy with a number of lateral branches, whereas the roots of the normal plant had very few lateral branches. The biomass of the transformed plant was 86.39 g/plant (fresh wt), significantly higher than the normal plant which was 77.3 g/plant (fresh wt). The total alkaloid content in the mature transformed plant (0.073 g per plant) was similar to the normal plant (0.078 g per plant), although the hairy roots contained little alkaloid.Abbreviations MS Murashige & Skoog's basal medium - MLS modified Linsmaier & Skoog's basal medium - BA benzyladenine - NAA naphthalene acetic acid - TLC thin layer chromatography - HPLC high performance liquid chromatography     

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.     

Preference for plants damaged by conspecific larvae in ovipositing cabbage root flies: influence of stimuli from leaf surface and roots

In laboratory dual-choice assays females of the cabbage root fly, Delia radicum, prefer for oviposition plants with roots damaged by conspecific larvae to undamaged controls. Cauliflower and kale plants were inoculated with root fly eggs (25 per plant) and the hatching larvae were allowed to feed on the roots for various periods of time (1–17 days). After 4 (cauliflower) or 5 (kale) days of larval feeding the oviposition preference was most pronounced and flies laid between 64% and 68% of their eggs near plants with damaged roots. Later, with increasing damage but fewer surviving, and thus actively feeding, larvae, the magnitude of the preference declined. The preference for plants already damaged by conspecific larvae may contribute to the previously observed aggregated distribution of D. radicum eggs in Brassica crop fields.Further experiments revealed that the sensory cues inducing this oviposition preference originate from the complex consisting of the damaged roots, the surrounding substrate (soil) and associated microbes, rather than from the aerial plant parts. In choice assays using the root-substrate complex of damaged and control plants (aerial parts removed), the observed preference for damaged roots was similar to that found for the entire plant but was more pronounced. The damaged roots alone, compared to control roots, received up to 72% (cauliflower) and 75% (kale) of the eggs. By contrast, surrogate leaves sprayed with methanolic leaf surface extracts from the most preferred plants which had been damaged were not discriminated from surrogate leaved sprayed with extracts of the respective control plants. Analysis of glucosinolate levels in methanolic leaf surface extracts revealed that root damage resulted in enhanced concentrations of indole-glucosinolates on the leaf surface in kale but not in cauliflower. Although indole-glucosinolates are oviposition stimulants for the cabbage root fly, the induced changes were apparently too small to influence oviposition behaviour.     

Agrobacterium rhizogenes-mediated transformation of Taraxacum platycarpum and changes of morphological characters

Transformed hairy roots were efficiently induced from seedlings of Taraxacum platycarpum by infection with Agrobacterium rhizogenes 15834. Root explants produced transformed roots at a higher frequency (76.5±3.5%) as compared to stem (32.7±4.8%) or cotyledon (16.2±5.7%). Hairy roots exhibited active elongation with high branching of roots on growth regulator-free medium. The competence of plant regeneration from non-transformed adventitious roots and transformed hairy roots was compared. The frequency of adventitious shoot formation from transformed roots was much higher (88.5±9.8%) than that of non-transformed roots (31.7 ±9.5%) on hormone-free medium. Rooting of hairy root-derived adventitious shoots occurred easily on growth regulator-free medium but no rooting was observed on non-transformed shoots. The stable introduction of rol genes into Taraxacum plants was confirmed by PCR and Southern hybridization. Transgenic plantlets showed considerable differences in their morphology when compared to the corresponding wild-type (non-transgenic) plants. Plantlets formed from transformed roots had numerous fibrous roots with abundant lateral branches instead of the thickened taproots in non-transformed plants. The differences observed may reflect the modification of morphological root characters by introduction of rol genes.Communicated by M.R. Davey     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.     

Transformation of potato using mannopine and cucumopine strains of Agrobacterium rhizogenes

Mannopine and cucumopine strains of Agrobacterium rhizogenes were used for genetic transformation in two cultivars of potato (Solanum tuberosum L.). An overnight pretreatment of stem fragments with NAA prior to bacterial infection was necessary to induce root formation, otherwise very few roots were produced. Whatever the potato cultivar used, rhizogenesis induced by NAA pretreament depended on the bacterial strain. In fact, when explants from both potato cultivars were pretreated with 26.5 M NAA, on average 84.4% and 71.9% produced roots after inoculation with the strains 2659 and 2659 GUS respectively. On the contrary, few rhizogenic responses (2.0–17.0%) or no response at all (0.0%) were obtained with the strains 15834 and 8196 GUS whatever NAA concentration used. Tests for confirming stable transformation of plant explants by examining both -glucuronidase activity and the presence of opines showed that 85% of the selected roots were cotransformed. Most of the transformed roots were highly branched and grew rapidly, compared to non-transformed roots with no branching and poor growth. Transgenic plants were readily regenerated with a frequency reaching 80% of total explants tested for both potato cultivars.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - df degree of freedom - F F distribution - GA₃ gibberellic acid - MS Murashige and Skoog basal medium - NAA -naphthaleneacetic acid - P probability - T-DNA transferred DNA     

High frequency plant regeneration and accumulation of tropane alkaloids in regenerated plants of Scopolia parviflora

An efficient in vitro plant regeneration system was established from callus culture of Scopolia parviflora. Callus was induced from adventitious roots on B5 medium with 0.45–9.04 μM 2,4-dichlorophenoxyacetic acid (2,4-D). In vitro plantlet regeneration was achieved on B5 medium supplemented with 44.38 μM benzyladenine (BA), 3% sucrose, and 0.38% gelrite. Plantlets were transplanted to artificial soil and grown to maturity successfully in a greenhouse. The tropane alkaloid contents in regenerated plants were analyzed using high-performance liquid chromatography (HPLC), and were found to be higher than those of adventitious roots, native growing plants, and acclimated plants. Regenerated plants from organogenic callus cultures produced a greater amount of tropane alkaloids.     

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.     

The use of Agrobacterium rhizogenes transformed roots to obtain transgenic shoots of the apple rootstock Jork 9

The apple rootstock Jork 9 was transformed using four different Agrobacterium rhizogenes virulent strains. The mannopine strain 8196 gave the best results in the production of chimeric plants compared to two agropine strains (A4 and 15834) and one cucumopine strain. Shoot regeneration was performed on both untransformed and transformed roots. Optimum combination and concentration of thidiazuron (TDZ) and -naphtaleneacetic acid (NAA) was different between untransformed and transformed roots. From the transformed roots seven shoots were obtained and propagated as individual clones. All shoots from these clones rooted on a hormone-free medium contrary to untransformed shoots that did not root under similar culture conditions. Differences in the morphology of the leaves and stems were observed between the clones. The transformed status of the different clones was verified with mannopine tests, PCR and Southern blot analyses. Five clones contained the mas1', the ORF 13 and the rolB genes, whereas two clones contained only the rolB gene.     

Anaerobic induction and tissue-specific expression of maize Adh1 promoter in transgenic rice plants and their progeny.

Summary In order to analyze expression of the maize alcohol dehydrogenase 1 gene (Adh1), its promoter was fused with the gusA reporter gene and introduced into rice by protoplast transformation. Histochemical analysis of transgenic plants and their progeny showed that the maize Adh1 promoter is constitutively expressed in root caps, anthers, anther filaments, pollen, scutellum, endosperm and shoot and root meristem of the embryo. Induction of expression by the Adh1 promoter was examined using seedlings derived from selfed progeny of the transgenic plants. The results showed that expression of the Adh1 promoter was strongly induced (up to 81-fold) in roots of seedlings after 24 h of anaerobic treatment, concomitant with an increase in the level of gusA mRNA. 2,4-D also induced Adh1 promoter-directed expression of gusA to a similar extent. In contrast, little induction by anaerobic treatment was detected in transformed calli, leaves or roots of primary transformants or shoots of seedlings. A detailed examination of seedling roots during anaerobic treatment revealed that the induction started first at the meristem and after 3 h there was strong induction in the elongation zone which is located 1–2 mm above the meristem; the induction then progressed upward from this region. Our results suggest that transgenic rice plants carring the gusA reporter gene fused with promoters are useful for the study of anaerobic regulation of genes derived from graminaceous species.