Journal Article•
Agrobacterium mediated transformation of Vigna sesquipedalis Koern (asparagus bean).
TL;DR: Histological analysis of GUS gene expression directly after cocultivation revealed a high competence of subepidermal cell layers of cotyledonary node and associated cotYledons for transformation with Agrobacterium.
Abstract: Agrobacterium mediated transformation of Vigna sesquipedalis was achieved using cotyledonary node explants prepared from 5 days old seedlings germinated on B5 basal medium, and transformed using Agrobacterium tumefaciens strain EHA101, carrying the phosphinothricin-N-acetyltransferase gene and neomycin-3-phosphotransferase-II gene as selectable markers and GUS gene as a screenable marker. Gene transfer was achieved by inoculation of cotyledonary node explants with a bacterial suspension and a further cocultivation with Agrobacterium suspension for 3 days on B5 basal medium. Only 10% of the explants were transformed with EHA101 and exhibited transient expression of GUS genes, while 2% of shoots exhibited stable integration of genes and developed into plants. Transgenic character of tissues was confirmed by GUS assay and Southern analysis. Histological analysis of GUS gene expression directly after cocultivation revealed a high competence of subepidermal cell layers of cotyledonary node and associated cotyledons for transformation with Agrobacterium.
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TL;DR: Legumes are a large, diverse family ranging from herbaceous annuals to woody perennials that, because of their capacity to fix nitrogen, are essential components in natural and managed terrestrial ecosystems.
Abstract: Legumes are a large, diverse family ranging from herbaceous annuals to woody perennials that, because of their capacity to fix nitrogen, are essential components in natural and managed terrestrial ecosystems. Legumes have been domesticated for the production of food, feed, forage, fiber, industrial
283 citations
TL;DR: With optimization of various factors which influence genetic transformation of pulse crops, it will be possible to develop transgenic plants in this important group of crop species with more precision and reproducibility, according to the reason for lack of commercialization of transgenic pulse crops.
68 citations
TL;DR: Southern hybridization analysis confirmed the transgenic nature of the herbicide-resistant plants, and all the transformed plants were fertile, and the transgene was inherited by Mendelian genetics.
Abstract: Herbicide (Basta®)-tolerant Vigna mungo L. Hepper plants were produced using cotyledonary-node and shoot-tip explants from seedlings germinated in vitro from immature seeds. In vitro selection was performed with phosphinothricin as the selection agent. Explants were inoculated with Agrobacterium tumefaciens strain LBA4404 (harboring the binary vector pME 524 carrying the nptII, bar, and uidA genes) in the presence of acetosyringone. Shoot regeneration occurred for 6 wk on regeneration medium (MS medium with 4.44 μM benzyl adenine, 0.91 μM thidiazuron, and 81.43 μM adenine sulfate) with 2.4 mg/l PPT, explants being transferred to fresh medium every 14 d. After a period on elongation medium (MS medium with 2.89 μM gibberellic acid and 2.4 mg/l PPT), β-glucuronidase-expressing putative transformants were rooted in MS medium with 7.36 μM indolyl butyric acid and 2.4 mg/l PPT. β-Glucuronidase expression was observed in the primary transformants (T0) and in the seedlings of the T1 generation. Screening 128 GUS-expressing, cotyledonary-node-derived, acclimatized plants by spraying the herbicide Basta® at 0.1 mg/l eliminated nonherbicide-resistant plants. Southern hybridization analysis confirmed the transgenic nature of the herbicide-resistant plants. All the transformed plants were fertile, and the transgene was inherited by Mendelian genetics. Immature cotyledonary-node explants produced a higher frequency of transformed plants (7.6%) than shoot-tip explants (2.6%).
27 citations
Cites background from "Agrobacterium mediated transformati..."
...Use of kanamycin for selection of transformants in grain legumes has been reported in Vigna unguiculata (Muthukumar et al. 1996), V. radiata (Jaiwal et al. 2001), and V. sesquipedalis (Ignacimuthu 2000)....
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TL;DR: In vitro-regenerated plantlets with well-developed roots were successfully hardened in a greenhouse and established in soil and more than 80% of somatic embryos were converted into true-to-type fertile plants.
Abstract: An efficient in vitro regeneration protocol for moth bean [Vigna aconitifolia (Jacq.) Marechal] via somatic embryogenesis has been developed. Embryogenic callus cultures were established from the cotyledonary node as explant on semi-solid Murashige and Skoog (MS) medium supplemented with 0.75 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l−1 6-benzylaminopurine (BA) and with various additives (50 mg l−1 ascorbic acid and 25 mg l−1 each of adenine sulphate, citric acid and l-arginine). Numerous somatic embryos differentiated on MS basal nutrient medium supplemented with 0.25 mg l−1 2,4-D and 0.5 mg l−1 of kinetin (Kin). Sustained cell division resulted in the formation of cell aggregates, which progressed to the globular- and heart-shaped somatic embryos and then, if they differentiated properly, to the torpedo shape and cotyledonary stages. The transfer of embryos onto fresh MS basal medium containing 0.2 mg l−1 BA and 2.0 mg l−1 gibberellic acid enabled the embryos to achieve complete maturation and germination. More than 80% of somatic embryos were converted into true-to-type fertile plants. In vitro-regenerated plantlets with well-developed roots were successfully hardened in a greenhouse and established in soil.
22 citations
TL;DR: The cotyledonary node-based direct regeneration system described here is time-efficient and amenable to Agrobacterium-mediated transformation, and therefore should be further explored for peanut transgenic improvement.
Abstract: Efficient and genotype-independent in vitro regeneration is an essential prerequisite for incremental trait improvement in peanut (Arachis hypogaea L.) via genetic transformation. We have optimized a facile and rapid method to obtain direct shoot organogenesis from cotyledonary node (CN) explants excised from peanut seedlings germinated on cytokinin-supplemented Murashige and Skoog (MS) basal salt medium. Starting with mature embryos, shoot induction occurred in approximately 7 weeks, followed by 4 weeks for rooting of excised shoots and 3 weeks of acclimatization of regenerated plantlets in soil. The regeneration and transformation system described here is time-efficient, yielding greenhouse-acclimatized plantlets within 14 weeks, in contrast to 12–14 months required for initiating and regenerating somatic embryogenic cultures, currently the most tractable method available for peanut transformation. The highest shoot induction frequency and shoot quality was obtained with 6.66 μM 6-benzylaminopurine, followed by adequate root induction at 5.37 μM α-Naphthaleneacetic acid. New Mexican Valencia A was chosen for Agrobacterium-mediated transformation. Stable GUS expression from pWBvec10a was obtained at a transformation rate of 1.25 %. Furthermore, results from genomic PCR and Southern blot analyses showed that 14 out of 576 putative transgenic regenerants contained transgene pSag12::IPT, therefore yielding a total transformation rate of 2.43 %. The cotyledonary node-based direct regeneration system described here is time-efficient and amenable to Agrobacterium-mediated transformation, and therefore should be further explored for peanut transgenic improvement.
16 citations
Cites background from "Agrobacterium mediated transformati..."
...However, the transformation rate could be as low as 0.9 % in mung bean (Jaiwal et al. 2001), 2 % in asparagus bean (Ignacimuthu 2000), and 4.31 % in blackgram (Vigna mungo L. Hepper) (Saini and Jaiwal 2007)....
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References
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TL;DR: A vector molecule for the efficient transformation of higher plants has been constructed with several features that make it efficient to use.
Abstract: A vector molecule for the efficient transformation of higher plants has been constructed with several features that make it efficient to use. It utilizes the trans acting functions of the vir region of a co-resident Ti plasmid in Agrobacterium tumefaciens to transfer sequences bordered by left and right T-DNA border sequences into the nuclear genome of plants. The T-region contains a dominant selectable marker gene that confers high levels of resistance to kanamycin, and a lac alpha-complementing region from M13mp19 that contains several unique restriction sites for the positive selection of inserted DNA.
2,534 citations
TL;DR: There are several biological systems in which uidA-encoded beta-glucuronidase may be an attractive alternative or complement to previously described gene-fusion markers such as beta-galactosidase or chloramphenicol acetyltransferase.
Abstract: We have developed a gene-fusion system based on the Escherichia coli beta-glucuronidase gene (uidA). The uidA gene has been cloned from E. coli K-12 and its entire nucleotide sequence has been determined. beta-Glucuronidase has been purified to homogeneity and characterized. The enzyme has a subunit molecular weight of 68,200, is very stable, and is easily and sensitively assayed using commercially available substrates. We have constructed gene fusions of the E. coli lacZ promoter and coding region with the coding region of the uidA gene that show beta-glucuronidase activity under lac control. Plasmid vectors have been constructed to facilitate the transfer of the beta-glucuronidase coding region to heterologous control regions, using many different restriction endonuclease cleavage sites. There are several biological systems in which uidA-encoded beta-glucuronidase may be an attractive alternative or complement to previously described gene-fusion markers such as beta-galactosidase or chloramphenicol acetyltransferase.
1,051 citations
TL;DR: It is suggested that the hypervirulence of Agrobacterium tumefaciens A281 is due to non-T-DNA sequences on the Ti plasmid.
Abstract: We used a binary-vector strategy to study the hypervirulence of Agrobacterium tumefaciens A281, an L,L-succinamopine strain. Strain A281 is hypervirulent on several solanaceous plants. We constructed plasmids (pCS65 and pCS277) carrying either the transferred DNA (T-DNA) or the remainder of the tumor-inducing (Ti) plasmid (pEHA101) from this strain and tested each of these constructs in trans with complementary regions from heterologous Ti plasmids. Hypervirulence on tobacco could be reconstructed in a bipartite strain with the L,L-succinamopine T-DNA and the vir region on separate plasmids. pEHA101 was able to complement octopine T-DNA to hypervirulence on tobacco and tomato plants. Nopaline T-DNA was complemented better on tomato plants by pEHA101 than it was by its own nopaline vir region, but not to hypervirulence. L,L-Succinamopine T-DNA could not be complemented to hypervirulence on tobacco and tomato plants with either heterologous vir region. From these results we suggest that the hypervirulence of strain A281 is due to non-T-DNA sequences on the Ti plasmid.
928 citations
TL;DR: Early phases after transformation of Arabidopsis cotyledon explants were analysed using a modified prokaryotic indicator gene, showing that Agrobacterium-mediated T-DNA transfer to plants is much more efficient than has been judged from experiments where selection is applied immediately.
Abstract: Agrobacterium tumefaciens is a commonly used tool for transforming dicotyledonous plants. The underlying mechanism of transformation however is not very well understood. One problem complicating the analysis of this mechanism is the fact that most indicator genes are already active in Agrobacterium, thereby preventing the precise determination of timing and localisation of T-DNA transfer to plant cells. In order to overcome this obstacle a modified prokaryotic indicator gene was constructed. The expression of this indicator gene and its use in analysing early events in Agrobacterium-mediated plant transformation are described. A portable intron, derived from a plant intron, was introduced into the beta-glucuronidase (GUS) gene. In transgenic plants containing this chimaeric gene the intron is spliced efficiently, giving rise to GUS enzymatic activity. Mapping of the splice junction indicates the exact removal of the intron. No GUS activity is detected in agrobacteria containing this construct due to the lack of a eukaryotic splicing apparatus in prokaryotes. Early phases after transformation of Arabidopsis cotyledon explants were analysed using this GUS-intron chimaeric gene showing that as early as 36 h after Agrobacterium infection significant GUS activity is detected. In vivo GUS staining of transformed cells clearly shows that quickly proliferating calli expressing GUS activity are formed, mainly at the cut surface. Minor transformation events occur however throughout the whole cotyledon. These data indicate that Agrobacterium-mediated T-DNA transfer to plants is much more efficient than has been judged from experiments where selection is applied immediately. The intron-containing GUS gene can be used as an optimised marker gene in transient and stable transformation experiments.
882 citations
TL;DR: Sexually-mature dry bean plants (Phaseolus vulgaris L.) were regenerated from cotyledonary node tissue of seedlings germinated on Murashige and Skoog (MS)-based media supplemented with 5 μM benzyladenine (BA).
98 citations