Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO₃ and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All R_o transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R₁ seed progeny and showed co-segregation.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - NPTII neomycin phosphotransferase type II - GUS -glucuronidase - CaMV cauliflower mosaic virus - MS Murashige and Skoog - X-Gluc 5-bromo-4-chloro-3-indolyl-D--glucuronic acid - IBA indolebutyric acid - SDS sodium dodecyl sulfate     

Transformation of the wild tomatoLycopersicon chilense Dun. byAgrobacterium tumefaciens

Summary Leaf disc transformation-regeneration technique was applied to the drought tolerant wild relative of cultivated tomato,Lycopersicon chilense, using a plasmid construct which contained the coding sequences of neomycin phosphotransferase (NPTII) and chloramphenicol acetyltransferase (CAT) genes. The two genotypes used, LA2747 and LA1930, showed a distinct difference in their aptitude to transformation; a higher success rate was obtained for the first genotype in every stage of the process. Shoots were formed on the regeneration medium containing 100 g/ml kanamycin through direct or indirect organogenesis. Root formation became only possible when the concentration of kanamycin was reduced to 50 g/ml. Expression of chloramphenicol acetyltransferase gene was observed in all of the kanamycin-screened plants after they matured; the activity of the gene was absent or low in some of the young plants. The presence of the CAT gene in transgenic plants was further confirmed by Southern blot analysis. Although transgenic plants grew to maturity, they did not produce fruit, owing to the self incompatibility ofL. chilense. Abbreviations BAP 6-benzylaminopurine - CAT chloramphenicol acetyltransferase - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - LB Luria Broth - EDTA ethylenediamine-tetraacetic acid     

Transformation of Solanum integrifolium poir via Agrobacterium tumefaciens: Plant regeneration and progeny analysis

Summary The wild species Solanum integrifolium represents a source of pest and disease resistance genes for breeding strategies of the cultivated species Solanum melongena. Somatic hybridization via protoplast fusion between the two species may provide a valuable tool for transferring polygenic traits into the cultivated species. The availability of S.integrifolium cells carrying dominant selectable markers would facilitate the heterokaryon rescue. An appropriate methodology for in vitro culture and plant regeneration from leaf explants of S.integrifolium is reported. Efficient leaf-disk transformation via co-cultivation with Agrobacterium tumefaciens led to the regeneration of transformed plants carrying the reporter genes GUS and NPT-II. Transformed individuals were obtained through selection on kanamycin-containing medium. Stable genetic transformation was assessed by histochemical and enzymatic assays for GUS and NPT-II activity, by the ability of leaf disks to initiate callus on Km-containing medium, Southern blot analyses of the regenerated plants, and genetic analysis of their progenies. Selfed-seed progeny of individual transformed plants segregated seedlings capable to root and grow in selective condition, while untransformed progeny did not. Genetic analyses of progeny behaviour showed that the reporter gene NPT-II segregated as single as well as two independent Mendelian factors. In two cases an excess of kanamycin-sensitive seedlings was obtained, not fitting into any genetic hypothesis.Abbreviations MS Murashige and Skoog (1962) medium - NOS nopaline synthase - NPT-II neomycin phosphotransferase - GUS beta-glucuronidase - LB Luria and Bertani medium - KIN 6-furfurylaminopurine - BAP 6-benzylaminopurine - 2iP N⁶-(2-isopentyl)adenine - ZEA zeatin - TDZ Thidiazuron     

Genetic transformation of a pasture legume,Lotus corniculatus,L. (birdsfoot trefoil)

Summary A rapid procedure for introducing foreign genes inLotus corniculatus based on the induction of hairy roots byAgrobacterium rhizogenes was developed. Expression of chloramphenicol acetyltransferase and neomycin phosphotransferase II was revealed in transgenic plants. Southern blot hybridization was used to confirm the genetic transformation. The transgenic plants looked normal and did not show any morphological modification compared to the seed grown plants.     

Genetic transformation in the grain legume Cicer arietinum L. (chickpea)

In the grain legume Cicer arietinum L. (chickpea), the seed-derived embryo axes deprived of the apical meristem were able to regenerate adventitious shoots on Murashige and Skoog (1962) medium supplemented with kinetin. This protocol was suitable for Agrobacterium-mediated gene transfer by the co-cultivation technique. Chickpea transgenic plants showed neomycin phosphotransferase II and ß-glucuronidase activities and the presence in their genome of integrated bacterial DNA.Abbreviations 6-BAP 6-benzyl-aminopurine - CaMV cauliflower mosaic virus - GUS ß-glucuronidase - IAA indole-3-acetic acid - Kn kanamycin - MU methyl umbelliferone - NAA naphthaleneacetic acid - NPTII neomycin phosphotransferase II     

Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker

Summary We have established an efficient Agrobacterium-mediated transformation procedure for Arabidopsis thaliana genotype C24 using the chimeric bialaphos resistance gene (bar) coding for phosphinothricin acetyltransferase (PAT). Hypocotyl explants from young seedlings cocultivated with agrobacteria carrying a bar gene were selected on shoot-inducing media containing different concentrations of phosphinothricin (PPT) which is an active component of bialaphos. We found that 20 mg/l of PPT completely inhibited the control explants from growing whereas the explants transformed with the bar gene gave rise to multiple shoots resistant to PPT after 3 weeks under the same selection conditions. The transformation system could also be applied to root explants. Resulting plantlets could produce viable seeds in vitro within 3 months after preparation of the explants. The stable inheritance of the resistance trait, the integration and expression of the bar gene in the progeny were confirmed by genetic tests, Southern analysis and PAT enzyme assay, respectively. In addition, the mature plants in soil showed tolerance to the herbicide Basta.Abbreviations bar bialaphos resistance gene - CIM callus-inducing medium - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - GM germination medium - HPT hygromycin phosphotransferase - MS Murashige and Skoog salts - NPTII neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin - SIM shoot-inducing medium     

Agrobacterium-mediated transformation of commercial melon (Cucumis melo L., cv. Amarillo Oro)

Cotyledon explants of muskmelon (Cucumis melo L., cv. Amarillo Oro) seedlings were co-cultivated with disarmed Agrobacterium tumefaciens strain LBA4404 that contained the binary vector plasmid pBI121.1. The T-DNA region of this binary vector contains the Nopaline synthase/neomycin phosphotransferase II (NPTII) chimeric gene for kanamycin resistance and the Cauliflower Mosaic Virus 35S/-glucuronidase (GUS) chimeric gene. After infection, the cotyledon pieces were placed in induction medium containing 100 mg/l kanamycin. Putative transformed shoots were obtained, followed by the development of morphologically normal plantlets. The transgenic nature of regenerants was demonstrated by polymerase chain reaction, Southern blot analysis, plant growth on medium selective for the transgene (NPTII) and expression of the co-transformed GUS gene. Factors affecting the transformation procedure are discussed.Abbreviations CaMV Cauliflower Mosaic Virus - Cf Cefotaxime - GUS -glucuronidase - Km Kanamycin - MS Murashige and Skoog - NOS nopaline synthase - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction     

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     

Introduction and expression of an insect proteinase inhibitor in alfalfa Medicago sativa L.

As one approach to alleviating the need for insecticide spraying, our objective is to express protein insecticides in transgenic alfalfa. To initiate these studies, a cDNA encoding the protease inhibitor (PI) anti-elastase from Manduca sexta was placed under the control of the CaMV 35S promoter, inserted into pAN 70, and transferred into leaf and petiole sections of alfalfa (Medicago sativa L.) using Agrobacterium tumefaciens mediated gene transfer. Transformation rates were 10% of all explants exposed to Agrobacterium. More than 1000 transgenic plants containing the PI have been recovered. Transgenic plants were initially identified when leaf explants from the regenerated plants formed callus in the presence of 50 g/ml kanamycin, and subsequently the presence of the PI gene was confirmed by southern analysis. The 35S promoter-PI fusion produced up to 0.125% of total protein as PI protein in leaves, roots, and flowers. Progeny analysis demonstrated Mendelian segregation of the NPTII gene (observed as kanamycin resistance) and the PI (confirmed by southern analysis). Accumulation of the anti-elastase PI insecticide in transgenic alfalfa reduced the onset of thrip predation, suggesting that this methodology can establish insect resistance within this agronomically important legume.Abbreviations Km kanamycin - PI protease inhibitor - SDS Sodium dodecyl sulfate - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction - SH Shenk and Hildebrandt (1972) medium     

Evaluation of a cotyledonary node regeneration system forAgrobacterium-mediated transformation of pea (Pisum sativum L.)

Summary A rapid regeneration system was used for studies ofAgrobacterium-mediated transformation inPisum sativum L. Cotyledonary node explants were inoculated withAgrobacterium tumefaciens strains containing binary vectors carrying genes for nopaline synthase (NOS),β-glucuronidase (GUS), and neomycin phosphotransferase (NPTII) and placed on selection medium containing either 75 or 150 mg/liter kanamycin. A GUS encoding gene (uidA) containing an intron was used to monitor gene expression from 6 to 21 days postinoculation. GUS activity could be observed 6 days after inoculation in the area of the explant in which regeneration-occurred. Regenerating tissue containing transformed cells was observed in explants on selection medium 21 days postinoculation. Using this system, a single transgenic plant was obtained. Progeny of this plant, which contained two T-DNA inserts, demonstrated segregation for the inserts and for expression of the NOS gene in the selfed R₁ progeny. NPTII activity was observed in the R₂ generation, indicating inheritance and expression of the foreign DNA over at least two generations. Attempts to repeat this procedure were unsuccessful.     

Factors affecting the use of chloramphenicol acetyltransferase as a marker for Brassica genetic transformation

The CAT gene which codes for the enzyme chloramphenicol acetyltransferase was found to be ineffective as a reporter gene in cells and tissues of Brassica species. High levels of endogenous CAT activity were found to be widespread among this genus and did not appear to be distributed in a tissue- or cell-specific manner. Moreover, the presence of an inhibitor of CAT activity was discovered in Brassica napus and Brassica juncea. This inhibitor appeared to act selectively on bacterial CAT in transgenic plants. These findings provided an explanation for difficulties experienced in the detection of transgenic CAT activity in B. napus.     

Agrobacterium-mediated transformation of five wild Solanum species using in vitro microtubers

Summary A genetic transformation method, using in vitro microtubers and Agrobacterium-mediated transformation has been developed for five wild Solanum species: S. verrucosum, S. hjertingii, S. papita, S. stoloniferum, S. demissum, which range in ploidy from diploid to hexaploid. A disarmed A. tumefaciens strain, C58 harbouring the co-integrate vector pGV3580::pKU2 with the genes of neomycin phosphotransferase (NPTII) and hygromycin phosphotransferase (HPTII) as selectable markers, was used. Selection of putative transformants was based on their ability to grow and produce roots on a medium containing 150 mg/l kanamycin. The transgenic nature of the putative transformants was confirmed by Polymerase Chain Reaction analysis and by NPTII dotblot assay to show the expression of the NPTII gene. Additionally, the transmission of transgenes, NPTII and HPTII in selfed-sexual progeny of some transgenic plants was also determined.Abbreviations MS Murashige and Skoog - NPTII neomycin phosphotransferase - HPTII hygromycin phosphotransferase - PCR polymerase chain reaction - GA₃ gibberellic acid - CCC chlorocholine chloride - BAP benzylaminopurine - NAA naphthalene acetic acid - ZR zeatin riboside - IAA indole acetic acid - LB Luria Broth     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.) using root explants

An efficient variety-independent method for producing transgenic eggplant (Solanum melongena L.) via Agrobacterium tumefaciens-mediated genetic transformation was developed. Root explants were transformed by co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBAL2 carrying the reporter gene beta-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Transgenic calli were induced in media containing 0.1 mg l(-1) thidiazuron (TDZ), 3.0 mg l(-1) N(6)-benzylaminopurine, 100 mg l(-1) kanamycin and 500 mg l(-1) cefotaxime. The putative transgenic shoot buds elongated on basal selection medium and rooted efficiently on Soilrite irrigated with water containing 100 mg l(-1) kanamycin sulphate. Transgenic plants were raised in pots and seeds subsequently collected from mature fruits. Histochemical GUS assay and polymerase chain reaction analysis of field-established transgenic plants and their offsprings confirmed the presence of the GUS and NPTII genes, respectively. Integration of T-DNA into the genome of putative transgenics was further confirmed by Southern blot analysis. Progeny analysis of these plants showed a pattern of classical Mendelian inheritance for both the NPTII and GUS genes.     

Transgenic antibiotic resistance may be differentially silenced in germinating pollen grains

Transgenic tobacco (Nicotiana tabacum L.) plants, carrying the neomycin phosphotransferase (NPT II) gene from E. coli, are resistant to kanamycin when grown from seeds on kanamycin containing medium. Tissue and cell cultures derived from those transformants also express resistance and regenerate complete plantlets in the presence of the antibiotic. This unspecific response to the selective condition has led to the belief that the foreign gene is continuously active or uniformly inducible in all cells of the transgenic plant. However, our experiments show that this view is not true for pollen grains during in vitro germination. Pollen grains isolated from kanamycin resistant tobacco plants carry and transmit the foreign gene but do not express resistance when germinating in vitro. This data presents evidence for differential silencing of a foreign gene in a mature gamete. On the other hand, immature pollen grains (microspores) appear to express resistance. The point of the downregulation of the neomycin transferase gene during pollen maturation is discussed.Abbreviations kan kanamycin sulfate - NPT II neomycin phosphotransferase II - sr streptomycin sulfate     

Insect-resistant transgenic Pinus radiata

Transgenic radiata pine (Pinus radiata D. Don) plants containing a Bacillus thuringiensis (Bt) toxin gene, crylAc, were produced by means of biolistic transformation of embryogenic tissue. Using the selectable marker gene nptII and corresponding geneticin selection, 20 independent transgenic lines from five genotypes were established. Over 200 plants regenerated from ten transgenic lines were successfully transferred to soil. The integration and expression of the introduced genes in transgenic tissue and/or plants were confirmed by PCR, Southern hybridisation and neomycin phosphotransferase II (NPTII) and Bt ELISA assays. Bioassays with larvae of the painted apple moth, Teia anartoides, demonstrated that transgenic plants displayed variable levels of resistance to insect damage, with one transgenic line being highly resistant to feeding damage.     

Genetic transformation ofPelargonium X hortorum

Summary TransgenicPelargonium X hortorum have been producedvia Agrobacterium tumefaciens-mediated transformation. The regeneration protocol used provided a regeneration frequency approximately to 95 percent. Clumps of regenerants, from cotyledons and hypocotyls ofPelargonium X hortorum seedlings, were inoculated with the disarmed strain EHA101 ofAgrobacterium tumefaciens. This strain contains a binary vector carrying neomycin phosphotransferase II, hygromycin B phosphotransferase and ß-glucuronidase genes. Selection on the regeneration medium supplemented with hygromycin allowed production of transgenic plants in up to 20% of the inoculated explants. The insertion of foreign DNA was demonstrated by Southern and polymerase chain reaction analysis: these experiments indicated that the inserted T-DNA is not full length for most of the plants. All RO transgenic plants exhibited a normal phenotype and are fertile.Abbreviations GUS ß-glucuronidase coding sequence - PCR polymerase chain reaction - CaMV cauliflower mosaic virus - NPTII neomycin phosphotransferase coding sequence - NOS nopaline synthase gene promoter and terminator - HPH hygromycin B phosphotransferase coding sequence - SDS sodium dodecyl sulphate - EDTA (ethylenedinitro trilo)tetra-acetic acid disodium salt     

Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens

High frequency transformation of Arabidopsis thaliana leaf explants has been obtained using a disarmed Ti plasmid containing the coding region of a neomycin phosphotransferase gene (NPT II) as a selectable marker. The rate of transformation ranged from 55 to 63 percent when acetosyringone (AS), a natural wound response molecule, was added to an Agrobacterium tumefaciens culture prior to incubation with leaf segments. Without acetosyringone, the transformation rate was approximately 2 to 3 percent. Calli resistant to G418 were regenerated into mature flowering plants in the presence of 10 g/ml G418. Southern analysis and neomycin phosphotransferase assays confirmed the insertion and expression of the NPT II gene in regenerated Arabidopsis plants.     

Hairy roots — a short cut to transgenic root nodules

To facilitate molecular studies of symbiotic nitrogen fixation a procedure for rapid production of transgenic root nodules was established on the legumeLotus corniculatus (Bird'sfoot trefoil). Regeneration of transgenic plants is not required as transgenic nodules are formed onAgrobacterium rhizogenes incited roots inoculated withRhizobium. Easy identification of transformed roots is possible using a set ofA. rhizogenes acceptor strains carrying assayable marker genes such as chloramphenicol acetyltransferase (CAT), -glucuronidase (GUS), or luciferase (LUC) under control of the cauliflower mosaic virus (CaMV) 35S promoter. Counterselection ofA. rhizogenes after infection of plants was improved using an auxotrophy marker.Abbreviations CAT chloramphenicol acetyltransferase - GUS -glucuronidase - LUC luciferase - Ri root inducing - TL left T-DNA - CaMV Cauliflower Mosaic Virus     

Electroporation of immature maize zygotic embryos and regeneration of transgenic plants

Using the pulse-discharging electroporation system HPES-3, we have transferred the neomycin phosphotransferase II (nptII) gene and -glucuronidase (gus) gene into mechanically-woulded immature zygotic embryo cells of an elite local maize cultivar Huanong Supersweet No. 42 and have produced transgenic maize plants. DNA hybridization and NPTII dot assay showed that the foreign genes were integrated into the genomes and expressed stably in the cells of the transgenic calluses and plants.