High-efficiency Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) and regeneration of insect-resistant transgenic plants

To develop an efficient genetic transformation system of chickpea (Cicer arietinum L.), callus derived from mature embryonic axes of variety P-362 was transformed with Agrobacterium tumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding β-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD₆₀₀ 0.3 with 48 h of co-cultivation period at 20°C was found optimal for transforming 10-day-old MEA-derived callus. Inclusion of 200 μM acetosyringone, sonication for 4 s with vacuum infiltration for 6 min improved the number of GUS foci per responding explant from 1.0 to 38.6, as determined by histochemical GUS assay. For introducing the insect-resistant trait into chickpea, binary vector pRD400-cry1Ac was also transformed under optimized conditions and 18 T₀ transgenic plants were generated, representing 3.6% transformation frequency. T₀ transgenic plants reflected Mendelian inheritance pattern of transgene segregation in T₁ progeny. PCR, RT-PCR, and Southern hybridization analysis of T₀ and T₁ transgenic plants confirmed stable integration of transgenes into the chickpea genome. The expression level of Bt-Cry protein in T₀ and T₁ transgenic chickpea plants was achieved maximum up to 116 ng mg⁻¹ of soluble protein, which efficiently causes 100% mortality to second instar larvae of Helicoverpa armigera as analyzed by an insect mortality bioassay. Our results demonstrate an efficient and rapid transformation system of chickpea for producing non-chimeric transgenic plants with high frequency. These findings will certainly accelerate the development of chickpea plants with novel traits.     

Agrobacterium-mediated transformation and regeneration of cotton plants

Cotton (Gossypium hirsutum L.) was transformed by the EHA101 strain of Agrobacterium tumefaciens harboring a binary vector pGA482GG plasmid carrying the marker genes for neomycin phosphotransferase II (nptII) determining resistance to kanamycin and β-glucuronidase (GUS). The cotyledons, hypocotyls, shoot meristem tissue, and its segments taken from in vitro growing seedlings were used as explants. Explants were cultured in a Murashige and Skoog (MS) medium containing various hormone combinations to induce shoot regeneration. The highest frequency of shoot formation was obtained from the shoot meristem. After selection in the MS medium containing kanamycin (50 mg/l), these tissues were tested by histochemical GUS assay. Shoots regenerated from excised shoot meristems or their halves were cultured for 4–6 weeks to obtain rooted plants, which then produced fully-developed plants and seeds in pots. Genomic integration of the kanamycin-resistance gene was detected by the PCR analysis. Seed germination percentage was 95% after the F1 seeds of transgenic cotton plants were cultured on half-strength MS medium supplemented with 50 mg/l kanamycin. Thus, a protocol for effective Agrobacterium-mediated genetic transformation of cotton was optimized. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 462–467. The text was submitted by the authors in English.     

Obtaining of transgenic French bean plants (Phaseolus vulgaris L.) resistant to the herbicide pursuit by Agrobacterium-mediated transformation

The transgenic plants of French bean (Phaseolus vulgaris) resistant herbicide Pursuit and kanamycin have been obtained. The genetic transformation was carried out with Agrobacterium tumefaciens strain LBA4404 containing binary vector carrying mutant ahas/als and selective nptII genes. Integration of the transgenes into plant genome was confirmed by polymerase chain reaction.     

Generation of transgenic Lolium temulentum plants by Agrobacterium tumefaciens-mediated transformation

Lolium temulentum L. (Darnel ryegrass) has been proposed to be used as a model species for functional genomics studies in forage and turf grasses, because it is a self-fertile, diploid species with a short life cycle and is closely related to other grasses. Embryogenic calluses were induced from mature embryos of a double haploid line developed through anther culture. The calluses were broken up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harboring pCAMBIA1301 and pCAMBIA1305.2 vectors were used to infect embryogenic callus pieces. Hygromycin was used as a selection agent in stable transformation experiments. Hygromycin resistant calluses were obtained after 4–6 weeks of selection and transgenic plants were produced in 10–13 weeks after Agrobacterium-mediated transformation. Fertile plants were readily obtained after transferring the transgenics to the greenhouse. Transgenic nature of the regenerated plants was demonstrated by Polymerase chain reaction (PCR), Southern hybridization analysis, and GUS staining. Progeny analysis showed Mendelian inheritance of the transgenes. The transformation system provides a valuable tool for functionality tests of candidate genes in forage and turf grasses.     

Improvement of protein quality in transgenic soybean plants

Glycinin is one of the abundant storage proteins in soybean seeds. A modified Gy1 (A1aB1b) proglycinin gene with a synthetic DNA encoding four continuous methionines (V3-1) was connected between the hpt gene and the modified green fluorescent protein sGFP(S65T) gene, and a resultant plasmid was introduced into soybean by particle bombardment in order to improve nutritional value of its seeds. After the selection with hygromycin, the efficiency of gene introduction was evaluated. More than 60 % of the regenerated plants tolerant to hygromycin yielded the hpt and V3-1 fragment by polymerase chain reaction (PCR) analysis, and the expression of sGFP was detected in about 50 % of putative transgenic soybeans. Southern hybridization confirmed the presence of transgenes in T0 plants and the transgenic soybeans hybridized with the hpt and V3-1 genes were analyzed showed different banding patterns. Most of the transgenic plants were growing, flowering normally and produced seeds. Analysis of seed obtained from transgenic soybean plants expressing hpt and V3-1 genes showed higher accumulation of glycinin compared with non-transgenic plants. In addition, protein expression in transgenic soybean plants was observed by using 2D-electrophoresis.     

Accumulation and within-seed distribution of iron in common bean and soybean

The influence of times of applying FeEDDHA on seed yield and Fe accumulation by four common bean (Phaseolus vulgaris L.) and two soybean (Glycine max L.) genotypes grown on a calcareous soil was studied under greenhouse conditions. The soybean genotypes, unlike the common bean genotypes, developed Fe-deficiency chlorosis and responded to application of the chelate. A preplant application of FeEDDHA was more efficacious than a flowering application in increasing seed yield of soybean. In contrast, the flowering application was much more effective than the preplant application for increasing seed Fe concentration [Fe] of both species. Percentage of seed Fe located in the seed coat of the common bean genotypes ranged from approximately 5 to 40% and was little affected by FeEDDHA. This within-seed distribution of Fe was correlated with methanol-extractable seed-coat pigments absorbing at 500 nm, presumably anthocyanins, but not with condensed tannins (proanthocyanidins). The soybean genotypes did not accumulate anthocyanins or tannins in the seed coat. Seed of Fe-deficient soybean plants without FeEDDHA had appreciably lower [Fe] and had a lower percentage of seed Fe in the seed coat than treated plants. Within-seed distribution of Fe should be considered in plant breeding because of concerns about both human nutrition and early seedling growth. Abbreviations: DTPA – diethylenetrinitrilopentaacetic acid; EDDHA – ethylenediamine di(o-hydroxyphenylacetic acid) acid; SPAD – single photon avalanche diode     

Agrobacterium-mediated transformation of pepino and regeneration of transgenic plants

Regeneration of pepino (Solanum muricatum Ait.) shoots was achieved both by organogenesis and by embryogenesis. Shoots derived via organogenesis were easily rooted and most regenerated plants appeared phenotypically normal. Transgenic plants were obtained using the binary vector pKIWI110 in the avirulent Agrobacterium tumefaciens strain LBA4404. Optimization of transformation protocols was rapidly achieved by monitoring early expression of the GUS (-D-glucuronidase) reporter gene carried on pKIWI110. Transgenic plants expressed GUS and selectable marker genes for kanamycin resistance and chlorsulfuron resistance. PCR (polymerase chain reaction) and Southern analysis provided molecular evidence for transformation.     

Production of fertile transgenic maize plants by silicon carbide whisker-mediated transformation

A simple and inexpensive system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed using silicon carbide whiskers to deliver plasmid DNA carrying the bacterial bar and uidA (gus) genes. Transformed cells were selected on medium containing the herbicide bialaphos. Integration of the bar gene and activity of the enzyme phosphinothricin acetyl transferase (PAT) were confirmed in all bialaphos-resistant callus lines analysed. Fertile transgenic maize plants were regenerated. Herbicide spraying of progeny plants revealed that the bar gene was transmitted in a Mendelian fashion.     

Improved soybean transformation for efficient and high throughput transgenic production

Transgenic Research - Although genetic transformation of soybean dates back to over two decades, the process remains inefficient. Here, we report the development of an organogenesis-based...     

Ribosomal RNA genes in soybean and common bean: chromosomal organization, expression, and evolution

Ribosomal RNA (5S and 45S) genes were investigated by FISH in two related legumes: soybean [Glycine max (L.) Merr.] and common bean (Phaseolis vulgaris L.). These species are both members of the same tribe (Phaseoleae), but common bean is diploid while soybean is a tetraploid which has undergone diploidization. In contrast to ploidy expectations, soybean had only one 5S and one 45S rDNA locus whereas common bean had more than two 5S rDNA loci and two 45S rDNA loci. Double hybridization experiments with differentially labelled probes indicated that the soybean 45S and 5S rDNA loci are located on different chromosomes and in their distal regions. Likewise, the common bean 45S and 5S rDNA loci were on unique chromosomes, though two of the 5S rDNA loci were on the same chromosome. FISH analysis of interphase nuclei revealed the spatial arrangement of rDNA loci and suggested expression patterns. In both species, we observed one or more 5S rDNA hybridization sites and two 45S rDNA hybridization sites associated with the nucleolar periphery. The 45S rDNA hybridization patterns frequently exhibited gene puffs as de-condensed chromatin strings within the nucleoli. The other condensed rDNA sites (both 5S and 45S) were spatially distant from the nucleolus in nucleoplasmic regions containing heterochromatin. The distribution of rDNA between the nucleoplasm and the nucleoli is consistent with differential gene expression between homologous alleles and among homoeologous loci.     

Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment

We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T₀, T₁ and T₂ transgenic plants. Oryza sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hyg^r), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T₁ and T₂ generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T₁ lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression.     

Celery transformation by Agrobacterium tumefaciens: cytological and genetic analysis of transgenic plants

Transgenic celery plants were obtained following co-cultivation of petiole explants with Agrobacterlum tumefaciens containing pMON200, a cointegrate vector carrying genes for kanamycin resistance and nopaline synthase. Transformants were selected by ability of callus to grow in the presence of 50mg/l kanamycin. Transformation was confirmed either by the presence of nopaline or by Southern blots. Cytological analysis of 14 transformed plants revealed chromosomal aberrations, both in structure and number. Only 20% of the regenerated plants had the normal karyotype. Kanamycin resistance behaved as a monogenic, dominant trait, segregating in a 3:1 ratio in three families derived from plants with normal karyotypes.Abbreviations KB Kilobases - 2-4D 2,4-diphenoxyacetic acid     

Colonization preference of Euschistus servus and Nezara viridula in transgenic cotton varieties,peanut, and soybean

Producers of Bt cotton, Gossypium hirsutum L. (Malvaceae), in the southeastern USA face significant losses from highly polyphagous stink bug species. These problems may be exacerbated by crop rotation practices that often result in cotton, peanut, Arachis hypogaea L., and soybean, Glycine max (L.) Merrill (both Fabaceae), growing in close proximity to one another. Because all of these crops are hosts for the major pest stink bug species in the region, we experimentally examined colonization preference of these species among the crops to clarify this aspect of their population dynamics. We planted peanut, soybean, Bt cotton, and glyphosate‐tolerant (RR) non‐Bt cotton at three sites over 3 years in replicated plots ranging from 192 to 1 323 m² and calculated odds ratios for colonization of each crop for Nezara viridula (L.) and Euschistus servus (Say) (both Hemiptera: Pentatomidae). In four of five experiments, both E. servus and N. viridula preferred soybean significantly more often than Bt cotton, non‐Bt cotton, and peanut. Neither N. viridula nor E. servus showed any preference between non‐Bt and Bt cotton in any experiment. Both species had higher numbers in Bt and non‐Bt cotton relative to peanut; this was not significant for any single experiment, but analyses across all experiments indicated that N. viridula preferred Bt and non‐Bt cotton significantly more often than peanut. Our results suggest that soybean in the landscape may function as a sink for stink bug populations relative to nearby peanut and cotton when the soybean is in the reproductive stage of development. Stink bug preference for soybean may reduce pest pressure in near‐by crops, but population increases in soybean could lead to this crop functioning as a source for later‐season pest pressure in cotton.     

Agrobacterium-mediated transformation of strawberry calli and recovery of transgenic plants

Transformed calli and shoots of strawberry (Fragaria × ananassa Duch.) cv. Redcoat were obtained using Agrobacterium tumefaciens carrying plasmid pB1121. Inoculated leaf explants produced transgenic calli at a frequency of 3% on selection medium containing 50 g/ml kanamycin. Twenty per cent of selected caili regenerated, giving rise to transgenic shoots. All transgenic calli and shoots expressed substantial amounts of GUS and NPT-II activity. The Southern blot analysis confirmed the insertion of both marker genes into the strawberry genome as single and multiple copy inserts. The transgenic shoots elongated on rooting medium in the presence of 25 g/ml kanamycin, but exhibited reduced rooting ability.Abbreviations BA benzyladenine - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NPT-II neomycin phosphotransferase(EC 2.7.1.95) - GUS -glucuronidase(EC 3.2.1.31) - X-GLUC 5-bromo-4-chloro-3-indolyl-glucuronide - 4-MU 4-methylumbelliferone NRCC No. 31227     

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     

Field performance of transgenic sugarcane produced using Agrobacterium and biolistics methods

Future genetic improvement of sugarcane depends, in part, on the ability to produce high‐yielding transgenic cultivars with improved traits such as herbicide and insect resistance. Here, transgenic sugarcane plants generated by different transformation methods were assessed for field performance over 3 years. Agrobacterium‐mediated (Agro) transgenic events (35) were produced using four different Agrobacterium tumefaciens strains, while biolistic (Biol) transgenic events (48) were produced using either minimal linearized DNA (LDNA) transgene cassettes with 5′, 3′ or blunt ends or whole circular plasmid (PDNA) vectors containing the same transgenes. A combined analysis showed a reduction in growth and cane yield in Biol, Agro as well as untransformed tissue culture (TC) events, compared with the parent clone (PC) Q117 (no transformation or tissue culture) in the plant, first ratoon and second ratoon crops. However, when individual events were analysed separately, yields of some transgenic events from both Agro and Biol were comparable to PC, suggesting that either transformation method can produce commercially suitable clones. Interestingly, a greater percentage of Biol transformants were similar to PC for growth and yield than Agro clones. Crop ratoonability and sugar yield components (Brix%, Pol%, and commercial cane sugar (CCS)) were unaffected by transformation or tissue culture. Transgene expression remained stable over different crop cycles and increased with plant maturity. Transgene copy number did not influence transgene expression, and both transformation methods produced low transgene copy number events. No consistent pattern of genetic changes was detected in the test population using three DNA fingerprinting techniques.     

Impact of transgenic cotton plants on a non-target pest, Aphis gossypii Glover

Abstract.  1. Transgenic crops have shown great promise for the control of target pest insects, but in some cases they can also influence non-target species. This study investigated the impact of Bt and Bt+CpTI transgenic cottons on the non-target cotton aphid, Aphis gossypii Glover, by comparing life-table parameters, feeding behaviour, and the fluctuating asymmetry of morphological traits of aphids reared on transgenic cotton and those on untransformed control plants.
2. Aphids on the Bt+CpTI cotton showed a shorter reproductive duration and maximum lifespan, lower survival rates and potential maximum fecundity, and an earlier occurrence of peak daily mortality in the first or second generation. However, the aphid population soon developed fitness and overcame the negative effect in the second or third generation. The aphids on the Bt cotton had significantly longer reproductive durations in the first generation, higher survival rates in the third generation, and apparently larger potential maximum fecundity in all three generations.
3. The percentages of accumulated duration of feeding waveforms E1 and E2 were significantly lower in aphids on the Bt+CpTI cotton than in those on the Bt or control cotton, whereas the frequencies of moving and finding feeding sites, and probe behaviour were significantly higher.
4. Fluctuating asymmetry in three morphological characters of aphids reared on transgenic and control cotton was detected. The fluctuating asymmetry value of the third segment of antenna in aphids on Bt+CpTI cotton was significantly higher than that of aphids on Bt or control cotton. Based on the fluctuating asymmetry value, the stress of cotton on the aphids could be ranked as Bt+CpTI cotton > Bt cotton > control cotton.     

Stable genetic transformation of cotton plants using polybrene-spermidine treatment

Polybrene and/or spermidine treatments were used to deliver plasmid DNA into cotton suspension culture obtained from cotyledon-induced callus. The transforming plasmid (pBI221.23) contained the selectablehpt gene for hygromycin resistance and the screenablegus gene. Primary transformant cotton plants were regenerated and analyzed by DNA hybridization and β-glucuronidase assay. The combination polybrene-spermidine treatment greatly enhanced the uptake and expression of DNA and the recovery of nonchimeric germ-line transgenic cotton plants.