Strain and cultivar specificity in the Agrobacterium-chickpea interaction

The susceptibility of four genotypes of chickpea to four wild strains of Agrobacterium tumefaciens was evaluated. Successful transformation was dependent on specific bacterial strain-plant cultivar interactions. Agropine strain A281 was the most effective for tumor induction. Tumors displayed hormone autonomous growth, were opine positive and contained DNA that was homologous to the T-DNA of the inciting strain.     

Specificity of strain and genotype in the susceptibility of pea to Agrobacterium tumefaciens

Summary To determine the best combination for potential use in transformation of Pisum sativum L., 13 genotypes were inoculated with wild-type Agrobacterium tumefaciens strains A281, C58 and Ach5. A281 appeared to be the most virulent strain, as determined by size and number of tumours, followed by C58 and Ach5. Genotypes differed considerably in their response to inoculation and genotype x strain interaction was evident. Genotypes also responded differently to in vivo or in vitro inoculation. Axenic calli from tumours could be grown on hormone-free medium and the presence of the specific opines for each strain in the callus indicated successful transfer and expression of T-DNA. Southern blot analysis of DNA from callus of A281-inoculated material showed that both T_R and T_L T-DNA had been incorporated into the pea genome.NRCC No. 30265     

Parameters affecting the frequency of kanamycin resistant alfalfa obtained by Agrobacterium tumefaciens mediated transformation

Kanamycin resistant plants of Medicago varia A2 were obtained by an optimized procedure for high frequency transformation using Agrobacterium tumefaciens infection of leaf and petiole tissue. Parameters which affected the frequency were explant type, the Agrobacterium strain used and the time allowed for cocultivation. Under optimum conditions, i.e., using the Agrobacterium strain A281 and a 4 day cocultivation period, the frequency of transformed leaflets obtained was greater than 70%.     

Transformation of white spruce and other conifer species byAgrobacterium tumefaciens

Studies of the ability ofAgrobacterium to transform white spruce (Picea glauca), Engelmann spruce (P. engelmanni), Sitka spruce (P. sitchensis) and Douglas-fir (Pseudotsuga menziesii) showed frequencies of gall formation from 0–80% depending upon the strain ofAgrobacterium, and the conifer species. Thirty sixA. tumefaciens strains and oneA. rhizogenes strain were tested on 6 month old white spruce seedlings. NineA. tumefaciens strains induced gall formation on more than 50% of the inoculated trees and at greater than 10% of the inoculated sites. One strain, B2/74 gave rise to galls at 28% of the inoculated sites on white spruce and induced the highest overall frequency of gall formation on all the conifer species tested. Relative frequency of gall formation was consistent among species, although the overall frequency was much higher on Douglas-fir. Of the well characterized strains for which disarmed derivatives are available only A281 (carrying the supervirulent tumor inducing plasmid, pTiBo542) gave efficient transformation. Stable integration of T-DNA encoded genes has been confirmed by the expression of opine synthesis and hormone autonomous growth. The transfer and long-term stable expression of kanamycin resistance and firefly luciferase activity using binary vector systems was also achieved.     

Crown gall transformation of lentil (Lens culinaris Medik.) with virulent strains of Agrobacterium tumefaciens

Summary Four diverse strains of Agrobacterium tumefaciens (C58, Ach5, GV3111, and A281) were capable of inducing tumors at a high frequency on inoculated stems of lentil (Lens culinaris Medik. cultivar Laird) in vivo, and on excised shoot apices in vitro. GV3111 and Ach5 produced the largest and heaviest tumors in vivo, while A281 produced the heaviest tumors in vitro. Tumor formation and opine production are indicative of plant cell transformation and tumors produced appropriate opines: nopaline (C58), octopine (Ach5 and GV3111), and agropine and mannopine (A281). Southern analysis of DNA from a tumor line produced by strain C58 showed that a T-DNA fragment had been transferred into the lentil genome.     

T-DNA presence and opine production in tumors of Picea abies (L.) Karst induced by Agrobacterium tumefaciens A281

The hypervirulent Agrobacterium tumefaciens strain A281 formed frequent tumors (31%) on Picea abies (Norway spruce), an economically important tree species in Swedish forests. Three-month-old seedlings were inoculated and tumors were established that grew hormone-independently in culture. Tumors contained agropine and mannopine/mannopinic acid as determined by acid pH paper electrophoresis. In addition, DNA hybridization studies showed that the DNA from these tumor lines contained sequences homologous to Ti plasmid T-DNA, whereas wild-type spruce seedling DNA did not. These results suggest that Agrobacterium vectors can be used for gene transfer into this important forest species.     

Transformation of cultured cells of Chenopodium quinoa by binary vectors that carry a fragment of DNA from the virulence region of pTiBo542

Summary A 15.2-kb KpnI fragment from the virulence region of pTiBo542, the Ti plasmid harbored by Agrobacterium tumefaciens strain A281, was introduced into binary vectors. The fragment contained the virB, virC and virG genes, and it is known to have the ability to increase the virulence of strains of A. tumefaciens. The strains of A. tumefaciens that carried the resulting plasmids were able to transform cells in a suspension culture of Chenopodium quinoa Willd cells which were not transformable by common vectors. Although the sizes of the plasmids was very large, a foreign segment of DNA was introduced into one of the plasmids by homologous recombination in A. tumefaciens cells, and the segment was subsequently transferred to plant cells.Abbreviations NPT neomycin phosphotransferase - SPT streptomycin/spectinomycin phosphotransferase     

Genetic transformation of 9 in vitro clones of Alnus and Betula by Agrobacterium tumefaciens

Crown gall tumorigenesis, integration and expression of T-DNA encoded genes from Agrobacterium tumefaciens were investigated in 9 clones of Alnus glutinosa, A. incana and Betula papyrifera. Tumor formation on in vitro shoots was frequent in all clones with strain Ach5 and present in 8 clones with strain C58. Tumors excised from shoots were selected for autotrophic growth in vitro and axenic cultures were established. Octopine or nopaline, respective of the strain type used for inoculation, was detected in tumorous cultures. Southern blot analyses demonstrated T-DNA integration by hybridization of DNA from tumors with tmr and nos gene probes. One clone of B. papyrifera produced tumors with a morphogenic character, unusual in calli of this species, generating viable shoots which did not synthesize opine.Abbreviations Cb Carbenicillin - Cf Cefotaxime - 2,4-D 2,4-Dichloro-phenoxyacetic acid     

Agrobacterium-mediated transformation of African violet

A method for genetic transformation of Saintpaulia ionantha by co-cultivation of in vitro-grown leaves and petioles with Agrobacterium tumefaciens is described. Two bacterial strains, EHA105 and A281 both harbouring the binary plasmid pKIWI105 carrying the genes uidA and nptII, were used in the experiments. Regenerants were not obtained using the disarmed strain EHA105. The oncogenic strain A281 resulted in efficient transient and stable expression of the transferred traits for petiole explants only. After transformation and regeneration, the integration of the transgenes in the plant genome was confirmed by PCR analysis and Southern hybridization.     

In vitro Crown Galls Induced by Agrobacterium tumefaciens Strain A281 (pTiBo542) in Trigonella foenum-graecum

Transformation of fenugreek (Trigonella foenumgraecum) was carried out with A281 oncogenic strain of Agrobacterium tumefaciens using root, cotyledon and hypocotyl explants excised from 1-week-old seedlings, which showed that the plant was highly susceptible to transformation. Tumors (calli) were selected on 50 mg dm^–3 kanamycin. They were analyzed for -glucuronidase (GUS) expression. Presence of uidA (gus) gene, was confirmed by polymerase chain reaction (PCR) amplification.     

Agrobacterium-mediated transformation of commercial mints

Commercial peppermint (P) (Mentha × piperita L. ev. Black Mitcham), native spearmint (NS) (M. spicata L.) and Scotch spearmint (SS) (M. × gracillis Sole cv Baker) petioles and orange mint (OM) (M. citrata Ehrh.) leaf disks were cocultivated with a number of Agrobacterium tumefaciens strains. P, SS and OM initiated tumor-like callus tissue on growth regulator-free MS medium after cocultivation with strain A281, a hypervirulent agropine strain containing Ti plasmid pTiBo542. Callus did not initiate from explants cocultivated with strain C58, a virulent nopaline strain; with A 136, a plasmidless strain, or from uninoculated controls. A281-derived callus was maintained on growth regulator-free medium in the absence of antibiotics for up to two years with no bacterial outgrowth. No shoots regenerated from any of the tumors on regeneration medium. Five of seven OM callus lines assayed gave a positive signal for agropine. DNA extracted from OM tumor tissue hybridized to a DNA probe specific to the T-DNA region of pTi plasmid. Genomic Southern analysis of DNA from tumors of P and SS indicated that one to a few copies of the T-DNA integrated into the mint chromosomes. PCR amplification of genomic DNA with primers specific for one of the T-DNA encoded genes yielded fragments that, when analyzed by restriction enzyme mapping and on Southern blots, corresponded to the cytokinin biosynthesis gene ipt. These results demonstrate transformation of three species of mint and the potential for using A. tumefaciens to transfer economically important genes into commercial mint cultivars.Abbreviations BA benzyladenine - CW coconut water - Cef cefotaxime - P peppermint - SS scotch spearmint - NS native spearmint - OM orange mint - BM basal medium - MS Murashige and Skoog (1962) - PAR photosynthetically active radiation - CTAB hexadecylatrimethylammonium bromide - ipt isopentenyl transferase Received for publication 1994. Published as Miscellaneous Paper No. 1482 of the Delaware Agricultural Experiment Station. Contribution No. 317 of the Department of Plant and Soil Sciences. Mention of trade names in this publication does not imply endorsement by the Delaware Agricultural Experiment Station of products named, nor criticism of similar ones not named.     

Agrobacterium-mediated transformation of peanut (Arachis hypogaea L.) embryo axes and the development of transgenic plants

Transgenic peanut (Arachis hypogaea L.) plants have been produced using an Agrobacterium-mediated transformation system. Zygotic embryo axes from mature seed were cocultured with Agrobacterium tumefaciens strain EHA101 harboring a binary vector that contained the genes for the scorable marker B-glucuronidase (GUS) and the selectable marker neomycin phosphotransferase II. Nine percent of the germinated seedlings were GUS+. Polymerase chain reaction analysis confirmed that GUS+ shoots and T₁ progeny contained T-DNA. Molecular characterization of one primary transformant and its T₁ and T₂ progeny plants established that T-DNA was integrated into the host genome.     

Stable transformation of eggplant (Solanum melongena L.) by cocultivation of tissues withAgrobacterium tumefaciens carrying a binary plasmid vector

Stable transformation of eggplant to kanamycin resistance was obtained by cocultivation of cotyledonary and young leaves with the hypervirulent, fully oncogenicAgrobacterium tumefaciens strain A281 carrying plasmid pGA472. No transformation was observed when using the disarmedA. tumefaciens LBA4404 strain carrying pGA472 or when using either strain for cocultivation with eggplant suspension cells.The NPTII enzyme and DNA dot blot assays performed on callus cells growing in the presence of kanamycin indicated both the presence and expression of the foreign gene. The highest proportion of transformed explants was obtained from intact cotyledonary leaf pieces while the highest NPTII enzyme specific activity was detected in callus cells originating from superficially wounded cotyledonary leaf pieces. Kanamycin-resistant plantlets were regenerated after six months in culture.Abbreviations CTAB Cetyltrimethylammonium Bromide - DTT Dithiothreitol - EDTA Ethylenediaminetetraacetic Acid - NAA Naphtaleneacetic Acid - 2,4-D 2,4-Dichlorophenoxyacetic Acid - NPT II Neomycin Phosphotransferase     

Agrobacterium-mediated transformation and plant regeneration of buckwheat (Fagopyrum esculentum Moench.)

Genetic transformation of buckwheat (Fagopyrum esculentum Moench.) and regeneration of transgenic plants were obtained by using Agrobacterium tumefaciens strains as vectors. Buckwheat cotyledons were excised from imbibed seeds, co-cultivated with A. tumefaciens and subjected to previously reported protocols for callus and shoot regeneration. The transformation with oncogenic strains was confirmed by opine and DNA analyses of tumour tissue extracts. Plants were regenerated on cotyledon fragments incubated with strain A281, harboring pGA472, which carries the neomycin phosphotransferase II gene for kanamycin resistance. The transformation of resistant shoot clones was confirmed by NPTII enzyme assay and DNA hybridization. A large number of transformed shoots were rooted and fertile plantlets were raised in the greenhouse. Transgenic plants comprised pin and thrum clones, which were allowed to cross-pollinate. In about 180 R₂ seeds tested for kanamycin resistance, the ratio of resistant to sensitive seedlings was roughly 3:1.Abbreviations BAP 6-benzylaminopurine - 2,4-D dichloro-phenoxyacetic acid - 2iP 6-(, ,-dimethylallyl-amino)-purine - IBA indole-3-butyric acid - IAA indole-3-acetic acid - Km kanamycin - NPTII neomycin phosphotransferase II     

Transformation of eggplant (Solanum melongena L.) using a binary Agrobacterium tumefaciens vector

Summary Kanamycin resistant plants of Solarium melongena L. (eggplant) cv. Picentia were obtained following the cocultivation of leaf explants with Agrobacterium tumefaciens. A disarmed binary vector system containing the neomycin phosphotransferase (NPTII) gene as the selectable marker and chloramphenicol acetyltransferase (CAT) as a reporter gene was utilized. In vitro grown plants were used as sources of explants to produce transgenic plants on selective medium containing 100 mg/l kanamycin. The transformation and expression of the foreign genes was confirmed by DNA hybridizations, leaf disc assays, and by measuring NPTII and CAT enzyme activities. This technique is simple, rapid, efficient, and transgenic eggplants of this commercial cultivar have been transferred to soil where they have flowered and set seed.Abbreviations CAT chloramphenicol acetyltransferase - MS Murashige and Skoog - NPTII neomycin phosphotransferase - NOS nopaline synthase - ZEA zeatin     

Optimizing the production of transformed pea (Pisum sativum L.) callus using disarmed Agrobacterium tumefaciens strains

For optimization of the transformation procedure with Pisum sativum L. stern explant callus was used to test the effect of disarmed Agrobacterium tumefaciens strains, cocultivation procedures (preconditioning of explants; use of Nicotiana tabacum L. nurse cultures), duration of cocultivation (2, 3 or 4 days), and agents for selection (kanamycin or hygromycin). The succinamopine strain EHA101(pBI1042) produced the highest percentage of transformed calli (77%) when used in conjunction with tobacco nurse culture during four days of cocultivation. Using this strain, kanamycin (76%) and hygromycin (77%) were equally effective selective agents, but for strain LBA4404(pBI1042) percentage of transformed calli was higher for hygromycin (63%) than for kanamycin (17%). The procedures and strains shown to be optimal for transformation of pea callus will now be complemented by a pea regeneration system.     

Agrobacterium-mediated transformation and regeneration of kiwi fruit

Summary Genetically transformed kiwi fruit (Actinidia deliciosa) plants were obtained from hypocotyl and stem segments co-cultured with Agrobacterium tumefaciens strain EHA101 harboring a binary vector, pLAN411 or pLAN421, which contained the neomycin phosphotransferase II (nptII) gene and the -glucuronidase (GUS) gene. After co-culturing with the A. tumefaciens, the hypocotyl or stem segments were cultured on a selection medium containing 25g/ml kanamycin and 500g/ml Claforan. After one month in culture, shoots had regenerated from the cuttings. Green shoots were analyzed for NPTII activity and GUS activity. Eighty-five percent of the green shoots examined expressed the nptII and GUS genes. GUS histochemical assays revealed strong GUS expression in guard cells, mesophyll cells, and trichomes.     

Improved <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of GNA transgenic sugarcane

Six plasmids carrying a snowdrop lectin (Galanthus nivalis agglutinin, GNA) and one of three selection markers were successfully transferred into two sugarcane cultivars (FN81–745 and Badila) via Agrobacterium-mediated transformation. Agrobacterium strains LBA4404, EHA105 and A281 that harboured a super-binary vector were used for sugarcane transformation. The use of the hygromycin (Hyg) resistance gene (hpt II), phosphinothrincin (PPT) resistance gene (bar) or G418 resistance gene (npt II) as a screenable marker facilitated the initial selection of GNA transgenic sugarcane callus with different efficiencies and helped the rapid segregation of individual transformation events. All the three selective marker genes were controlled by CaMV 35S promoter, while GNA gene was controlled by promoter of RSs-1 (rice sucrose synthase-1) or Ubi (maize ubiquitin). Factors important to successful transformation mediated by Agrobacterium tumefaciens were optimized, which included concentration of A. tumefaciens, medium composition, co-cultivated methods with plant tissue, strain virulence and different selective marker genes. An efficient protocol for sugarcane transformation mediated by A. tumefaciens was established. The GNA gene has been integrated into sugarcane genome as demonstrated by PCR and Southern dot blotting detections. The preliminary results from bioassay demonstrated a significant resistance of the transgenic sugarcane plants to woolly aphid (Ceratovacuna lanigera Zehnther) indicating thus the possibility for obtaining a transgenic sugarcane cultivar with resistance to woolly aphid.     

Reverse transcription-PCR assay to verify gene integrity within plasmid constructs for plant transformation

We have developed a rapid and simple RT-PCR based method to check the integrity of chimeric genes within plasmid constructs for plant transformation. It exploits the Agrobacterium tumefaciens-mediated transient expression of plasmid constructs in plant tissue. Total RNA was isolated from tobacco leaves co-cultivated for 3 days with Agrobacterium tumefaciens harbouring the plant transformation vectors constructed in our laboratory. First strand cDNA synthesis with oligo(dT) primers generate a pool of cDNA that was used for PCR amplification with primers specific for each of the genes present within the constructs. PCR amplification reactions were successful for all chimeric genes tested, thus confirming their intactness and suitability to be used for stable plant transformation.     

Production of terpenes by differentiated shoot cultures of Mentha citrata transformed with Agrobacterium tumefaciens T37

Crown gall initiation on Mentha × piperita var. citrata (Ehrh.) Briq. (mint) was investigated using a range of wild type and mutant strains of Agrobacterium tumefaciens. Axenic transformed shoot cultures of Mentha citrata were established on plant stems inoculated with the nopaline strain T37 of Agrobacterium tumefaciens. The presence of T-DNA in the transformed tissues and the absence of bacterial contamination was established by Southern Blot hybridisation, using ³²P labelled fragments of the T-DNA and virulence region of the Ti plasmid as probes. The shoot cultures synthesised a mint oil fraction which contained the major terpenes characteristic of the parent plant in quantities similar to those found in intact tissue. Oil glands were observed to be present on the leaves of the transformed culture using scanning electron microscopy.Abbreviations aux 1 Tryptophan monoxygenase - aux 2 Indoleacetamide hydrolase - ipt Isopentenyl transferase - tzs Trans-Zeatin secretion locus - 2,4-D 2,4-Dichloro phenoxyacetic acid - oct Octopine - nop Nopaline - suc L,L-succinamopine - T-DNA transferred DNA