Kanamycin-resistant Plants Research Articles

Transgenic tomato plants expressing a Lycopersicon chilense chitinase gene demonstrate improved resistance to Verticillium dahliae race 2.

An acidic endochitinase gene (pcht28) isolated from Lycopersicon chilense was introduced into tomato (L. esculentum) through Agrobacterium-mediated transformation, using the CAMV 35S promoter. Transgenic plants demonstrated a high level of constitutive expression of pcht28 and chitinase enzyme activity. Kanamycin-resistant R1 plants (resulting from self-pollination of transgenic plants) as well as R2 plants were evaluated for their tolerance to Verticillium dahliae (race 1 and 2 for R1 plants and race 2 for R2 plants) in the greenhouse. They demonstrated a significantly (P<0.05) higher level of tolerance to the fungi compared to the nontransgenic plants, as measured by foliar disease symptoms, vascular discoloration, and vascular discoloration index. The transgenic plants produced in this study represent a source of genetic resistance to Verticillium dahliae.

The Possibility of Micropropagation and Agrobacterium-mediated Transformation of Kalmia Latifolia

Micropropagation and Agrobacterium-mediated transformation were developed in Kalmia latifolia cv. Ostbo Red. The transformation of Kalmia latifolia plants was carried out by an Agrobacterium tumefaciens strain containing the nptII and gusA genes in its T-DNA. Shoots were regenerated on kanamycin selection medium and the expression of the gusA reporter gene was verified by fluorogenic β-glucuronidase (GUS) assay in a few vegetative generations after regeneration. The presence of the gusA gene in regenerated kanamycin resistant plants was detected by polymerase chain reaction.

The indole alkaloid tryptamine produced in transgenic Petunia hybrida

Tryptophan decarboxylase (TDC) from Catharanthus roseus (periwinkle) converts tryptophan to the indole-alkaloid tryptamine, an anti-insect compound. This TDC cDNA was transformed and expressed in transgenic Petunia hybrida under the control of the strong and constitutive 35S promoter from cauliflower mosaic virus. Kanamycin screening and Southern hybridization with the TDC cDNA confirmed plant transformation. Northern analysis indicated greater TDC mRNA accumulation in transgenic plants compared to non-transformed plants. Additionally, eight-fold more tryptamine accumulated in leaves of kanamycin resistant transgenic plants compared to non-transformed plants. Flower petals from the transgenic plants contained lower tryptamine levels than their leaves. Because tryptamine titers were higher in transformed plants compared to controls, over-expression of the TDC enzyme may partially overcome endogenous tryptamine catabolism and/or other negative biosynthetic regulation. Future alteration of tryptamine breakdown in Petunia may further increase total endogenous tryptamine concentrations, potentially discouraging insect reproduction on these transgenic plants.

Increased stable inheritance of herbicide resistance in transgenic lettuce carrying a petE promoter-bar gene compared with a CaMV 35S-bar gene

Inheritance of resistance to herbicide (300 mg/l glufosinate ammonium) up to the third (T3) seed generation was compared in two populations of transgenic lettuce (Lactuca sativa L. cv 'Evola') harbouring a T-DNA containing the bar gene, linked to either the Cauliflower Mosaic Virus (CaMV) 35S promoter, or a -784-bp plastocyanin promoter from pea (petE). Only 2.5% (4/163) of CaMV 35S-bar plants, selected by their kanamycin resistance(T0 generation), transmitted herbicide resistance at high frequency to their T3 seed generation compared with 97% (29/30) for kanamycin resistant petE-bar plants. In the case of 35S-bar transformants, only 16% (341/2,150) of the first seed generation (T1) plants, 22% (426/1,935) T2 plants and 11% (1,235/10,949) T3 plants were herbicide-resistant. In contrast, 63% (190/300) T1 plants, 83% (2,370/2,845) T2 plants and 99% (122/123) T3 petE-bar transformed plants were resistant to glufosinate ammonium. The T-DNAs carrying the petE-bar and CaMV 35S-bar genes also contained a CaMV 35S-neomycin phosphotransferase (nptII) gene. ELISA showed that NPTII protein was absent in 29% (45/156) of the herbicide-resistant T2 plants from 8/19 herbicide-resistant petE-bar lines. This indicated specific inactivation of the CaMV 35S promoter on the same T-DNA locus as an active petE promoter. The choice of promoter and T-DNA construct are crucial for long-term expression of transgenes in lettuce.

Promoter Deletions are Essential for Transformation of Lettuce by the T-cytGene: The Phenotypes of Transgenic Plants

TheAgrobacteriumT-cytgene was transferred into lettuce,Latuca sativa‘Saladin’ using a genotype-independent transformation procedure employing a supervirulentAgrobacterium tumefaciensstrain carrying the binary vector pMOG23. Kanamycin-resistant shoots were initiated from inoculated explants only when sites were deleted within the T-cytpromoter. In culture, kanamycin-resistant shoots exhibited several phenotypes, including gall production, reduced internode length (dwarfism) and vitrification together with differences in rooting. Rooted, neomycin phosphotransferase (NPTII)-positive plants recovered from their abnormal phenotype and self pollinated to produce viable seed, following transfer to the glasshouse. Kanamycin-resistant lettuce plants exhibited increased cytokinin and chlorophyll contents compared to non-transformed plants, physiological features which could benefit long-term storage of this leafy vegetable.

In solium Selection for Arabidopsis Transformants Resistant to Kanamycin

The kanamycin resistance encoded by the neomycin phosphotransferase II gene (nptII) of transposon Tn5 is widely used in higher plant genetic transformation. The general process of plant transformation using nptII as a selectable marker gene, however, requires selecting kanamycin-resistant plants or tissues in culture. Even with the recently developed vacuum infiltration method for Arabidopsis transformation, the plant culture steps are not completely eliminated in selection for kanamycin-resistant transformants. The herbicide resistance genes, such as bar, which provides resistance to bialaphos, allow Arabidopsis transformation to become a true non-culture procedure. In this report, we assessed the feasibility of applying kanamycin as a spray in selecting for kanamycin-resistant Arabidopsis transformants grown in soil. We find that kanamycin-resistant transformants were effectively selected by spraying soil-grown Arabidopsis seedlings.

CaMV 35S promoter directs β-glucuronidase expression in the laticiferous system of transgenic Hevea brasiliensis (rubber tree).

Hevea brasiliensis anther calli were genetically transformed using Agrobacterium GV2260 (p35SGUSINT) that harboured the β-glucuronidase (gus) and neomycin phosphotransferase (nptII) genes. β-Glucuronidase protein (GUS) was expressed in the leaves of kanamycin-resistant plants that were regnerated, and the presence of the gene was confirmed by Southern analysis. GUS was also observed to be expressed in the latex and more importantly in the serum fraction. Transverse sections of the leaf petiole from a transformed plant revealed GUS expression to be especially enhanced in the phloem and laticifers. GUS expression was subsequently detected in every one of 194 plants representing three successive vegetative cycles propagated from the original transformant. Transgenic Hevea could thus facilitate the continual production of foreign proteins expressed in the latex.

Production of Transgenic Grapevine (Vitis vinifera L. cv. Koshusanjaku) Plants by Co-cultivation of Embryogenic Calli with Agrobacterium tumefaciens and Selecting Secondary Embryos

Embryogenic calli induced from leaf segments of grapevine (Vitis vinifera L. cv. Koshusanjaku) were co-cultivated for 5 days with Agrobacterium tumefaciens strains EHA101 (pIG121Hm) or LBA4404 (pTOK233), both of which contained the plasmid carrying the neomycin phosphotransferase II (NPT II), hygromycin phosphotransferase (HPT) and the β-glucuronidase (GUS) genes. Putative transgenic calli were selected on 2g/l gellan gum-solidified Nitsch's medium (1969) containing 50mg/l kanamycin and 20g/l sucrose after co-cultivation with A. tumefaciens. Transformation frequency of the embryogenic calli evaluated by GUS histochemical assay was increased by the addition of acetosyringone to co-culture medium. Complete transgenic plants were selected among secondary embryos formed on the surface of embryos in the presence of kanamycin. Finally, kanamycin-resistant plants expressing GUS gene were obtained. PCR analysis confirmed their transgenic nature by detecting GUS and NPT II genes.

High-frequency embryogenesis from cotyledons of bird's-foot trefoil ( Lotus corniculatus) and its effective utilization in Agrobacterium tumefaciens-mediated transformation

Summary We have developed a rapid and simple procedure to regenerate bird's-foot trefoil (Lotus corniculatus L.) plants from hypocotyl and cotyledon expiants of 10 to 14-days-old seedlings. Both expiant types regenerated well on the same medium with only minimal interfacing callus formation but with different modes of morphogenesis. While hypocotyls responded primarily by forming shoot apices, cotyledons mostly underwent embryogenes is. Remaining on the original explantation medium, both structures developed into rootless shoots. The highest shoot regeneration frequency was obtained on B5-based medium containing 0.5 mg/L 6-benzylaminopurine with approximately 80% of all expiants forming on average 19 shoots in four weeks. Only two media were used in the entire regeneration process, the second being the rooting medium of half-strength B5 supplemented with 0.01 mg/L α-naphthaleneacetic acid. Rooting frequency and survival rate after potting were about 100%. This regeneration protocol has been successfully applied to Agrobacterium tumefaciens-mediated transformation. Cotyledon segments were used as acceptor tissue. The expiants were co-cultivated with A. tumefaciens strain LB4404 carrying the plasmid vector pBI121. This vector contains the neomycin phosphotransferase II gene (NPTII) and β-glucuronidase reporter gene (GUS), both under the control of the CaMV 35S promoter. Kanamycin-resistant plants regenerated within 45 days after transfer to selective media. On a selection medium containing 100 mg/L kanamycin, shoots were formed by 19.0% of the expiants. The histological GUS assay showed that 7.0% of the resistant shoots also expressed the GUS gene in a variety of tissues. The stable integration of this gene was confirmed by polymerase chain reaction (PCR) analysis. Using the embryogenic cotyledon regeneration system transgenic birds-foot trefoil plants were obtained within 2–3 months.

Biolistic Transformation of Rose (Rosa hybridaL.)

A reproducible method has been developed for the Biolistic transformation and regeneration of transgenic plants from embryogenic callus of rose (Rosa hybridaL.) cv. Glad Tidings. DNA delivery was optimized using the β-glucuronidase (gus) gene. The distance between the stopping screen and target explants and supplementation of pre-and post-bombardment culture media with 0.25mmyo-inositol influenced the transformation efficiency. Prior to culture on selection medium containing 250 mg l−1kanamycin sulphate, embryogenic calli were bombarded, using optimized gene delivery parameters, with a plasmid carrying the neomycin phosphotransferase (nptII) gene. Somatic embryo-derived kanamycin-resistant plants were regenerated and subsequently transferred to glasshouse conditions. Transformation was confirmed by kanamycin resistance of calli and plants, NPT II ELISA assay and Southern analysis. All transgenic plants were morphologically normal (true-to-type).

Seed-specific immunomodulation of abscisic acid activity induces a developmental switch.

A single-chain Fv antibody (scFv) gene, which has previously been used to immunomodulate abscisic acid (ABA) activity in transgenic tobacco to create a 'wilty' phenotype, was put under control of the seed-specific USP promoter from Vicia faba and used to transform tobacco. Transformants were phenotypically similar to wild-type plants apart from their seeds. Anti-ABA scFv embryo development differed markedly from wild-type embryo development. Seeds which accumulated similar levels of a scFv that binds to oxazolone, a hapten absent from plants, developed like wild-type embryos. Anti-ABA scFv embryos developed green cotyledons containing chloroplasts and accumulated photosynthetic pigments but produced less seed storage protein and oil bodies. Anti-ABA scFv seeds germinated precociously if removed from seed capsules during development but were incapable of germination after drying. Total ABA levels were higher than in wild-type seeds but calculated free ABA levels were near-zero until 21 days after pollination. We show for the first time seed-specific immunomodulation and the resulting switch from the seed maturation programme to a germination programme. We conclude that the immunomodulation of hormones can alter the development programme of target organs, allowing the study of the directly blocked endogenous molecules and manipulation of the system concerned.

Integration of T-DNA binary vector 'backbone' sequences into the tobacco genome: evidence for multiple complex patterns of integration.

During the process of crown gall tumorigenesis, Agrobacterium tumefaciens transfers part of the tumor-inducing (Ti) plasmid, the T-DNA, to a plant cell where it eventually becomes stably integrated into the plant genome. Directly repeated DNA sequences, called T-DNA borders, define the left and the right ends of the T-DNA. The T-DNA can be physically separated from the remainder of the Ti-plasmid, creating a 'binary vector' system; this system is frequently used to generate transgenic plants. Scientists initially thought that only those sequences located between T-DNA left and right borders transferred to the plant. More recently, however, several reports have appeared describing the integration of the non-T-DNA binary vector 'backbone' sequences into the genome of transgenic plants. In order to investigate this phenomenon, we constructed T-DNA binary vectors containing a nos-nptll gene within the T-DNA and a mas2'-gusA (beta-glucuronidase) gene outside the T-DNA borders. We regenerated kanamycin-resistant transgenic tobacco plants and analyzed these plants for the expression of the vector-localized gusA gene and for the presence of binary vector backbone sequences. Approximately one-fifth of the plants expressed detectable GUS activity. PCR analysis indicated that approximately 75% of the plants contained the gusA gene. Southern blot analysis indicated that the vector backbone sequences could integrate into the tobacco genome linked either to the left or to the right T-DNA border. The vector backbone sequences could also integrate into the plant genome independently of (unlinked to) the T-DNA. Although we could readily detect T-strands containing the T-DNA within the bacterium, we could not detect T-strands containing only the vector backbone sequences or these vector sequences linked to the T-DNA.

Expression of cholera toxin B subunit oligomers in transgenic potato plants.

A gene encoding the cholera toxin B subunit protein (CTB), fused to an endoplasmic reticulum (ER) retention signal (SEKDEL) was inserted adjacent to the bi-directional mannopine synthase P2 promoter in a plant expression vector containing a bacterial luciferase AB fusion gene (luxF) linked to the P1 promoter. Potato leaf explants were transformed by Agrobacterium tumefaciens carrying the vector and kanamycin-resistant plants were regenerated. The CTB-SEKDEL fusion gene was identified in the genomic DNA of bioluminescent plants by polymerase chain reaction amplification. Immunoblot analysis indicated that plant-derived CTB protein was antigenically indistinguishable from bacterial CTB protein, and that oligomeric CTB molecules (M(r) approximately 50 kDa) were the dominant molecular species isolated from transgenic potato leaf and tuber tissues. Similar to bacterial CTB, plant-synthesized CTB dissociated into monomers (M(r) approximately 15 kDa) during heat or acid treatment. The maximum amount of CTB protein detected in auxin-induced transgenic potato leaf and tuber tissues was approximately 0.3% of total soluble plant protein. Enzyme-linked immunosorbent assay methods indicated that plant-synthesized CTB protein bound specifically to GM1-ganglioside, the natural membrane receptor of cholera toxin. In the presence of the SEKDEL signal, CTB protein accumulates in potato tissues and is assembled into an oligomeric form that retains native biochemical and immunological properties. The expression of oligomeric CTB protein with immunological and biochemical properties identical to native CTB protein in edible plants opens the way for preparation of inexpensive food plant-based oral vaccines for protection against cholera and other pathogens in endemic areas throughout the world.

Expression of cryIA(c) gene of Bacillus thuringiensis in transgenic chickpea plants inhibits development of pod-borer (Heliothis armigera) larvae

Two strains of chickpea (Cicer arietinum L.) ICCV-1 and ICCV-6, were used for transgenic plant generation. Embryo axis of mature seed devoid of the root meristem and the shoot apex was used as experimental material. The explants were cultured in medium containing MS macro salts, 4 × MS micro salts, B5 vitamins, 3.0 mg l−1 BAP, 0.004 mg l−1 NAA, 30 mg l−1 sucrose and cultured at 26 °C in dark, 24 h prior to bombardment. Gene delivery to the explants was carried out using a Bio-Rad Biolistic 1000/He particle gun. A chimaeric, truncated bacterial cryIA(c) gene construct was developed for plant expression with the CaMV35S promoter, nos terminator, an initiatory kozak sequence and a translational enhancer (STAR-P) sequence of tobacco mosaic virus. This cryIA(c) gene was cotransferred with a plasmid containing nptII gene as the selection marker. Transgenic kanamycin resistant chickpea plants were obtained through multiple shoot formation and repeated selection of the bombarded explants. Molecular analyses of the transformants revealed the presence of the transferred functional cryIA(c) gene in plant. Insect feeding assay indicated that the expression level of the cryIA(c) gene was inhibitory to the development of the feeding larvae of Heliothis armigera Hubner, the chickpea pod-borer

Efficient transgenic plant regeneration throughAgrobacterium-mediated transformation of Chickpea (Cicer arietinum L.)

Three genotypes of chickpea ICCV-1, ICCV-6 and a Desi (local) variety were tested for plant regeneration through multiple shoot production. The embryo axis was removed from mature seeds, the root meristem and the shoot apex were discarded. These explants were cultured on medium containing MS macro salts, 4X MS micro salts, I35 vitamins, 3.0 mg/1 BAP, 0.004 mg/1 NAA, 3% (w/v) sucrose and incubated at 26(0)C. The explants were transformed withAgrobacterium tumefaciens strain LBA4404 with binary vector pBI121 containing theuidA andnptIl genes. Multiple shoots were repeatedly selected with kanamycin. The selected kanamycin resistant shoots were rooted on MS medium supplemented with 0.05 mg/1 113A. The presumptive transformants histochemically stained positive for GUS. Additionally, nptll assay confirmed the expression ofnptII in kanamycin resistant plants. Transgenic plants were transferred to soil and grown in the green house.

The effects of Agrobacterium rhizogenes rolAB genes in lettuce

Agrobacterium rhizogenes rolAB genes were transferred into lettuce ( Lactuca sativa L.) cultivar ‘Lake Nyah’ using a supervirulent strain of A. tumefaciens. Southern hybridisation confirmed the presence of the rol genes in kanamycinresistant plants. In culture, transgenic plants exhibited extensive root development and an increased response to auxin. Phenotypic characterisation of transgenic populations indicated significant alterations in plant development, especially in the seed progeny of transgenic plants. The potential commercial value of rolAB transformed lettuce plants is discussed.

Tobacco (Nicotiana tobaccum) nuclear transgenics with high copy number can express NPTII driven by the chloroplast psbA promoter

A chloroplast expression vector containing the NPTII gene under the control of apsbA promoter (psbA-NPTII) was constructed, and was biolistically delivered into both suspension cells and leaf strips of tobacco (Nicotiana tabaccum). Analyses of subsequently recovered kanamycin-resistant transgenic plants indicate that the psbA-NPTII gene was not located in the chloroplast, but was in the nucleus in very high copy number. This conclusion was based upon results from: (1) Southern hybridization analyses of chloroplast and nuclear DNAs using NPTII, chloroplast-marker, and nuclear-marker probes; (2) pulse-field gel electrophoresis; and (3) kanamycin screening of sexual progenies. This study suggests that the nuclear expression of the NPTII gene may have been associated with many copies of the psbA-NPTII construction. Very high copy number in the nucleus might either allow NPTII expression from the otherwise inadequate psbA promoter, or might increase the chance of recombining with upstream tobacco regulatory sequences.

Transgenic broccoli expressing aBacillus thuringiensis insecticidal crystal protein: Implications for pest resistance management strategies

We usedAgrobacterium tumefaciens to transform flowering stalk explants of five genotypes of broccoli with a construct containing the neomycin phosphotransferase gene and aBacillus thuringiensis (Bt) gene [CryIA(c) type] optimized for plant expression. Overall transformation efficiency was 6.4%; 181 kanamycin-resistant plants were recovered. Of the 162 kanamycin-resistant plants tested, 112 (69%) caused 100% morality of 1st-instar larvae of aBt-susceptible diamondback moth strain. Southern blots of some resistant transformants confirmed presence of theBt gene. Selected plants that gave 100% mortality of susceptible larvae allowed survival of a strain of diamondback moth that had evolved resistance toBt in the field. F1 hybrids between resistant and susceptible insects did not survive. Analysis of progeny from 26 resistant transgenic lines showed 16 that gave segregation ratios consistent with a single T-DNA integration. Southern analysis was used to verify those plants possessing a single T-DNA integration. Because these transgenic plants kill susceptible larvae and F1 larvae, but serve as a suitable host for resistant ones, they provide an excellent model for tests ofBt resistance management strategies.

Biolistic transformation of cucumber using embryogenic suspension cultures: long-term expression of reporter genes

The generation of transgenic cucumber ( Cucumis sativus L.) plants was achieved by biolistic transformation of a highly embryogenic cell suspension culture using the nptII and uidA gene. Functional expression of the genes in transgenic plants was determined by neomycin phosphotransferase and β-glucuronidase enzyme assays. Southern analysis of DNA isolated from kanamycin-resistant plants confirmed stable integration of the genes as well as multicopy integration and rearrangements. A study of gene expression showed activity of the uidA gene in plants regenerated from kanamycin-resistant calli about one year after bombardment, indicating a high stability of the nonselectable gene.

Gene silencing in transgenic tobacco hybrids: frequency of the event and visualization of somatic inactivation pattern.

We have investigated the stability of the expression of different T-DNA-borne genes in hybrid tobacco lines. These lines were constructed to rescue rolC-induced male sterility in kanamycin-resistant P35s-rolC transgenic tobacco plants by expression of rolC antisense genes. Using five different tester lines, a total of 158 hybrids was obtained. We observed inactivation of transgene expression in 20% of the F1 progeny and in 35% of the backcrossed F2 progeny, as indicated by the loss of kanamycin resistance. In 3% of all crosses complete loss of antibiotic resistance was noted, while in most affected hybrid progeny only part of the population became kanamycin sensitive. Single genes could be selectively inactivated on T-DNAs harboring several genes. Gene inactivation was not restricted to one of the two T-DNAs examined. Somatic silencing, visualized by a cell-specific 35SGUSINT marker gene, occurred in a random fashion or exhibited an inherited specific pattern. The type of somatic silencing pattern observed indicated developmental control of the process. Two phenotypic classes could be distinguished with respect to frequency and timing of the inactivation process. Rapid gene inactivation, occurring within a few weeks after germination of hybrid seedlings, was characterized by complete methylation of restriction sites in the promoter of the silenced gene, resetting of gene expression during meiosis, heredity of the developmentally controlled program of gene silencing in subsequent generations, and rapid reactivation of gene expression after genetic separation of the different T-DNAs. In contrast, a slow type of gene inactivation was of a more stochastic nature and was recognized only in hybrids of the backcrossed F2 generation. In this case the degree of promoter methylation, which could extend beyond the T-DNA borders, was not correlated with the reduction in steady-state poly(A)+ mRNA levels, the silenced state was transmitted through meiosis and reactivation lasted several generations. The implications of the observations for our understanding of the gene inactivation process are discussed.