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.     

Agrobacterium-mediated transformation of white mustard (Sinapis alba L.) and regeneration of transgenic plants

Summary A procedure for the regeneration of fertile transgenic white mustard (Sinapis alba L.) is presented. The protocol is based on infection of stem explants of 7–9 day old plants with an Agrobacterium tumefaciens strain harboring a disarmed binary vector with chimeric genes encoding neomycin phosphotransferase and -glucuronidase. Shoots are regenerated from callus-forming explants within 3–4 weeks. Under selection, 10% of the explants with transgenic embryonic callus develop into fertile transgenic plants. Rooting shoots transferred to soil yield seeds within 14–16 weeks following transformation. Integration and expression of the T-DNA encoded marker genes was confirmed by histochemical glucuronidase assays and Southern-DNA hybridization using primary transformants and S1-progeny. The analysis showed stable integration and Mendelian inheritance of trans-genes in transformed Sinapis lines.Abbreviations BAP 6-benzylaminopurine - CaMV cauliflower mosaic virus - GUS -glucuronidase - IBA indole-3-butyric acid - IM infection medium - NAA 1-naphthalene acetic acid - neo gene encoding NPTII - NPTII neomycin phosphotransferase - RIM root-inducing medium - SEM shoot-elongation medium - SIM shoot-inducing medium - t-nos polyadenylation site of the nopaline synthase gene - uidA gene encoding GUS - WM wash medium - X-Gluc 5-bromo-4-chloro-3-indolyl -D-glucuronide     

Selection of transgenic flax plants is facilitated by spectinomycin

Regeneration of transformed flax shoots after inoculation withAgrobacterium tumefaciens carrying a binary vector with either a neomycin phosphotransferase (nptII) gene and a -glucuronidase (GUS) reporter gene or a spectinomycin resistance gene was examined. Hypocotyls from 4-day-old seedlings were inoculated with either of the twoA. tumefaciens strains. Selection and regeneration were achieved on a medium containing 0.1 M thidiazuron, 0.01 M napthalene acetic acid, 100 mgl^–1 kanamycin sulphate or spectinomycin sulphate and 300 mgl^–1 cefotaxime. Use of different neomycins for the selection of transformed tissues did select transformed calli but not transformed shoots either directly or via a callus phase. Selection based on spectinomycin resistance allowed the growth of transformed shoots. Transgenic shoots were rooted on a medium containing 100 mgl^–1 spectinomycin sulphate. Integration of the spectinomycin resistance gene into the flax genome was confirmed by Southern blot hybridizations and spectinomycin resistance was shown to be inherited as a dominant Mendeliant trait. Therefore, spectinomycin resistance is more suitable for genetic engineering of flax than aminoglycoside resistance.     

Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants

Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies of transformed kanamycin-resistant calli were produced, more than 80% of which were induced to form somatic embryos. Somatic embryos were germinated, and plants were regenerated and transferred to soil. Transformation was confirmed by opine production, kanamycin resistance, immunoassay, and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton.     

Improved flax regeneration from hypocotyls using thidiazuron as a cytokinin source

Summary The effects of thidiazuron, benzyladenine and zeatin were tested with respect to bud regeneration of different flax explants from hypocotyls, cotyledons and apices of two fibre varieties (Ariane, Viking) and one linseed variety (Antarès). These three cytokinins were tested either alone or in combination with naphthalene acetic acid, indole acetic acid or 2,4-dichlorophenoxyacetic acid.Hypocotyls were the most responsive explants. Thidiazuron was significantly the most effective followed by benzyladenine, and then zeatin, in inducing organogenesis from hypocotyl segments. The optimal thidiazuron concentration for bud regeneration from hypocotyls was 0.1–0.3 M in combination with 0.01 M of naphthalene acetic acid. Six days after plating, shoot initials began to appear on hypocotyl sections compared with ten to fifteen days when using benzyladenine or zeatin.     

High efficiency Agrobacterium-mediated gene transfer to flax

Summary Using an Agrobacterium tumefaciens binary vector (pAL4404, pBI131), we have demonstrated the transfer of the -glucuronidase gene into the flax (Linum usitatissimum L.) cultivar Glenelg after selection for kanamycin resistance. The transformed lines were obtained by inoculation and subsequent regeneration of hypocotyl segments. The callus that formed on the cut surfaces of the hypocotyl segments was isolated three weeks after infection and was subsequently subcultured to yield shoots. This procedure generated a large number of transgenic shoots over a relatively short period of time. The transformation efficiencies obtained were the highest reported so far for this plant species.Abbreviations 2,4-D, 2,4 dichlorophenoxyacetic acid - GUS glucuronidase - MS Murasbige and Skoog (1962) medium - MU 4-methyl-umbelliferone - MUG 4-methylumbelliferyl-glucuronide - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction     

The use of the phosphomannose isomerase gene as alternative selectable marker for Agrobacterium-mediated transformation of flax (Linum usitatissimum)

In order to meet the future requirement of using non-antibiotic resistance genes for the production of transgenic plants, we have adapted the selectable marker system PMI/mannose to be used in Agrobacterium-mediated transformation of flax (Linum usitatissimum L.) cv. Barbara. The Escherichia coli pmi gene encodes a phosphomannose isomerase (E.C. 5.1.3.8) that converts mannose-6-phosphate, an inhibitor of glycolysis, into fructose-6-phosphate (glycolysis intermediate). Its expression in transformed cells allows them to grow on mannose-selective medium. The Agrobacterium tumefaciens strain GV3101 (pGV2260) harbouring the binary vector pNOV2819 that carries the pmi gene under the control of the Cestrum yellow leaf curling virus constitutive promoter was used for transformation experiments. Transgenic flax plants able to root on mannose-containing medium were obtained from hypocotyl-derived calli that had been selected on a combination of 20 g L⁻¹ sucrose and 10 g L⁻¹ mannose. Their transgenic state was confirmed by PCR and Southern blotting. Transgene expression was detected by RT-PCR in leaves, stems and roots of in vitro grown primary transformants. The mean transformation efficiency of 3.6%, that reached 6.4% in one experiment was comparable to that obtained when using the nptII selectable marker on the same cultivar. The ability of T1 seeds to germinate on mannose-containing medium confirmed the Mendelian inheritance of the pmi gene in the progeny of primary transformants. These results indicate that the PMI/mannose selection system can be successfully used for the recovery of flax transgenic plants under safe conditions for human health and the environment.     

Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants

Summary Transgenic sweet orange (Citrus sinensis L. Osbeck) plants have been obtained by Agrobacterium tumefaciens-mediated gene transfer. An hypervirulent A. tumefaciens strain harboring a binary vector that contains the chimeric neomycin phosphotransferase II (NPT II) and ß-glucuronidase (GUS) genes was cocultivated with stem segments from in vivo grown seedlings. Shoots regenerated under kanamycin selection were harvested from the stem segments within 12 weeks. Shoot basal portions were assayed for GUS activity and the remaining portions were shoot tip grafted in vitro for production of plants. Integration of the GUS gene was confirmed by Southern analysis. This transformation procedure showed the highest transgenic plant production efficiency reported for Citrus.Abbreviations BA benzyladenine - CaMV cauliflowermosaic virus - GUS ß-glucuronidase - LB Luria Broth - MS Murashige and Skoog - NAA naphthalenacetic acid - NOS nopaline synthase - NPT II neomycin phosphotransferase II - PEG polyethylene glycol - RM rooting medium - SRM shoot regeneration medium     

Agrobacterium-mediated transformation of Asparagus officinalis L. long-term embryogenic callus and regeneration of transgenic plants

Summary Twenty-three independent kanamycin resistant lines were obtained after cocultivation of longterm embryogenic cultures of three Asparagus officinalis L. genotypes with an Agrobacterium tumefaciens strain harboring ß-glucuronidase and neomycin phosphotransferase II genes. All the lines showed ß-glucuronidase activity by histological staining. DNA analysis by Southern blots of the kanamycin resistant embryogenic lines and of a plant regenerated from one of them confirmed the integration of the T-DNA.Abbreviations GUS ß-glucuronidase - X-Gluc 5-bromo-4-chloro-3indolyl ß-D-glucuronic acid - NPT II neomycin phosphotransferase II     

Genetic transformation and regeneration of transgenic plants in grapevine (Vitis rupestris S.)

Isolated somatic embryos from petiole-derived callus cultures ofVitis rupestris Scheele have been employed in experiments on genetic transformation. Co-cultivation of somatic embryos during embryogenesis induction withAgrobacterium tumefaciens strain LBA4404, which contains the plasmid pBI121 carrying the neomycin phosphotranspherase and the-glucuronidase genes, produced transformed cellular lines capable of recurrent somatic embryogenesis. Precocious selection for high levels of kanamycin (100 mgl^-1) was an important part of our transformation protocol. Transformed lines still have strong-glucuronidase expression as well as stable insertion of the marker genes after 3 years of in-vitro culture, during which they have maintained their capacity to organize secondary embryos and to regenerate transgenic plants with an agreeable efficiency (13%).     

Effect of medium osmotic potential on callus induction and shoot regeneration in flax anther culture

Development of an efficient and cost-effective doubled haploid production system in flax (Linum usitatissimum L.) is the prerequisite for the application of doubled haploid technology in a practical breeding program. Pre-culture of anthers on a medium containing 15% sucrose for 2–7 days before transfer to the same medium containing 6% sucrose for a total of 28 days culture period significantly increased shoot regeneration for all four genotypes evaluated. Moreover, pre-culture of anthers on medium containing 15% sucrose for 2–7 days was sufficient to dramatically reduce the frequency of shoot regeneration from somatic tissues and thereby to increase the frequency of microspore-derived plants in flax anther culture. Furthermore, replacing 15% sucrose with 6% sucrose and 9% polyethylene glycol (PEG), or 3% sucrose and 12% PEG, in pre-culture medium did not significantly affect callus induction and shoot regeneration. The results indicate that sucrose may act as carbon/energy source as well as an osmotic regulator in flax anther culture. Sucrose as an osmotic regulator may be replaced by a non-metabolizable osmoticum: PEG. The implication of this study in flax anther culture and breeding is discussed.     

Distribution of selected elements within flax plants as affected by FeEDDHA

Summary Flax growing on a calcareous soil in the greenhouse developed Mn toxicity symptoms. The toxicity was eliminated by application of 2 ppm FeEDDHA-Fe. FeEDDHA had major effects on distribution of Mn, Zn, Fe and P among selected plant parts. Application of the chelate reduced Mn concentration in older leaves, the tissue most susceptible to Mn toxicity, associated stem tissue, plant tops, and roots from 2295 to 133 ppm, 62 to 7 ppm, 550 to 34 ppm, and 42 to 34 ppm, respectively. Analysis of older leaves is recommended for diagnosing Mn toxicity in flax.FeEDDHA reduced Zn concentration in plant tops and this was chiefly reflected in greatly reduced leaf concentrations, especially in older leaves. FeEDDHA increased plant Fe concentration and the effect was greatest in root and older leaf tissues. The overall effect of FeEDDHA on P concentration was small but large increases occurred in younger leaf tissue due to application of the chelate. Relative distributions of K, Na, Ca, and Mg among plant parts were only slightly affected by FeEDDHA.     

The use of the phosphomannose-isomerase/mannose selection system to recover transgenic apple plants

A selection system based on the phosphomannose-isomerase gene (pmi) as a selectable marker and mannose as the selective agent was evaluated for the transformation of apple (Malus domestica Borkh.). Mannose is an unusable carbon source for many plant species. After uptake, mannose is phosphorylated by endogenous hexokinases to mannose-6-phosphate. The accumulation of mannose-6-phosphate leads to a block in glycolysis by inhibition of phosphoglucose-isomerase, resulting in severe growth inhibition. The phosphomannose-isomerase is encoded by the manA gene from Escherichia coli and catalyzes the conversion of mannose-6-phosphate to fructose-6-phosphate, an intermediate of glycolysis. Transformed cells expressing the manA gene can therefore utilize mannose as a carbon and survive on media containing mannose. The manA gene along with a β-glucuronidase (GUS) gene was transferred into apple cv. ‘Holsteiner Cox’ via Agrobacterium tumefaciens-mediated transformation. Leaf explants were selected on medium supplemented with different concentrations and combinations of mannose and sorbitol to establish an optimized mannose selection protocol. Transgenic lines were regenerated after an initial selection pressure of 1–2 g l⁻¹ mannose in combination with 30 g l⁻¹ sorbitol followed by a stepwise increase in the mannose concentration up to 10 g l⁻¹ and simultaneous decrease in the sorbitol concentration. Integration of transgenes in the apple genome of selected plants was confirmed by PCR and southern blot analysis. GUS histochemical and chlorophenol red (CPR) assays confirmed activity of both transgenes in regenerated plants. The pmi/mannose selection system is shown to be highly efficient for producing transgenic apple plants without using antibiotics or herbicides.     

Patterns of transformation intensity on flax hypocotyls inoculated with Agrobacterium tumefaciens

Summary Cellular transformation intensities on flax (Linum usitatissimum) hypocotyl explants using disarmed Agrobacterium tumefaciens were investigated through various preculture durations, cocultivation durations and removal of epidermis. The expression of an intron-containing -glucuronidase (GUS) gene driven by CaMV 35S promoter served as a reporter for determination of transformed tissues on hypocotyls. The binary plasmid p35SGUSINT in octopine-type Agrobacterium strain GV2260 was used as the vector system. A prolonged cocultivation duration (5–7 days) resulted in a much higher transformation staining intensity (frequency * tissue area) than 2- or 3-day-cocultivation duration on hypocotyls variously precultured prior to inoculation. A high staining intensity on the two cut ends was obtained from nonprecultured hypocotyls. A reduction in intensity on the upper cut end of hypocotyls was observed with preculture times greater than 6 days. Peeled hypocotyls with a post-peeling preculture of 2 or 3 days had a high proportion of superficial area covered by transformed tissues after a 7 day-cocultivation duration. These results will help to improve the efficiency of recovery of transgenic plants by increasing the proportion of transformation in the regenerable tissues.     

Gelling agent influences the detrimental effect of kanamycin on adventitious budding in flax

Flax (Linum usitatissimum L.) hypocotyls were cultivated on regeneration media containing various concentrations of kanamycin (an aminoglycoside antibiotic commonly used to select transgenic plant material) solidified with three different gelling agents: gellan gum, agar and a mixture of both. The inhibitory effect of kanamycin on bud regeneration was analyzed. A significant interaction was observed between the nature of the gelling agent and the kanamycin concentration. The antibiotic concentration needed to strongly inhibit bud production varied greatly with the nature of the gelling agent. Gellan gum lowered the inhibitory effect of kanamycin. This revised version was published online in June 2006 with corrections to the Cover Date.     

The sequence of a pea vicilin gene and its expression in transgenic tobacco plants

A 5.5 kb Eco RI fragment containing a vicilin gene was selected from a Pisum sativum genomic library, and the protein-coding region and adjacent 5 and 3 regions were sequenced. A DNA construction comprising this 5.5 kb fragment together with a gene for neomycin phosphotransferase II was stably introduced into tobacco using an Agrobacterium tumefaciens binary vector, and the fidelity of expression of the pea vicilin gene in its new host was studied. The seeds of eight transgenic tobacco plants showed a sixteen-fold range in the level of accumulated pea vicilin. The level of accumulation of vicilin protein and mRNA correlated with the number of integrated copies of the vicilin gene. Pea vicilin was confined to the seeds of transgenic tobacco. Using immunogold labelling, vicilin was detected in protein bodies of eight out of ten embryos (axes plus cotyledons) and, at a much lower level, in two out of eleven endosperms. Pea vicilin was synthesized early in tobacco seed development; some molecules were cleaved as is the case in pea seeds, yielding a major parental component of M _r50000 together with a range of smaller polypeptides.     

Regeneration of flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) plants from anther culture and somatic tissue with increased resistance to<Emphasis Type="Italic"> Fusarium oxysporum</Emphasis>

The aim of this study was to establish a protocol for the efficient production of flax plants of microspore origin. The results were compared to those obtained for plants regenerated from somatic explants from hypocotyls, cotyledons, leaves, stems and roots. All the plants obtained during the experiments were regenerated from callus that was grown for periods from a few weeks to a few months before the regeneration was achieved. Anther cultures were less effective in plant regeneration than somatic cell cultures. However, regenerants derived from anther cells showed valuable breeding features, including increased resistance to fungal wilt. The age of the donor plants and the season they grew in had a noticeable effect on their anther callusing and subsequent plant regeneration. Low temperature had a negative effect and dark pre-treatment a positive effect on callusing and plant regeneration. Different media were most effective for callus induction, shoot induction and rooting. For callus induction two carbon sources (2.5% sucrose and 2.5% glucose) were most effective; for shoots, only sucrose at lower concentration (2%) was effective. Rooting was most efficient in 1% sucrose and reduced (50%) mineral concentration in the medium. It was found that the length of in vitro cultivation significantly increases the ploidy and affects such features as regenerant morphological characteristics, petal colour, and resistance to Fusarium oxysporum-induced fungal wilt. The established plant regeneration system provides a basis for the creation of transgenic flax.Abbreviations BAP 6-Benzyl-aminopurine - IAA Indole-3-acetic acid - MS Murashige and Skoog medium - NAA -Naphthalene-acetic acidCommunicated by H. Lörz     

Production of transgenic pineapple (Ananas cosmos (L.) Merr.) plants via adventitious bud regeneration

A new protocol for the production of transgenic pineapple plants was developed. Adventitious buds were induced directly from Agrobacterium-infected leaf bases and stem discs of in vitro plants, bypassing the establishment of callus cultures. Non-chimeric transgenic plants were obtained by multiple subculturing of primary transformants under increasing levels of selection. A total of 42 independent transgenic lines were produced from two cultivars with two different constructs: one containing a modified rice cystatin gene (Oc-IΔD86) and the other with the anti-sense gene to pineapple aminocyclopropane synthase (ACS). GUS histochemical staining provided the first evidence of the non-chimeric nature of the transformed plants. Their non-chimeric nature was further demonstrated by PCR analyses of the DNA extracted from individual leaves of a primary transformed plant and also from multiple plants propagated from a single transformation event. Southern hybridization confirmed random integration patterns of transgenes in the independent lines. For the Oc-IΔD86 gene, the expression at the mRNA level was detected via RT-PCR and its translation was detected by protein blot. Agronomic evaluation and bioassays of the transgenic plants will further validate the utility of this new tool for pineapple improvement.