Agrobacterium tumefaciens mediated transfer of Phaseolus vulgaris α-amylase inhibitor-1 gene into mungbean Vigna radiata (L.) Wilczek using bar as selectable marker
TL;DR: Morphologically normal and fertile transgenic plants of mungbean with two transgenes, bar and α-amylase inhibitor, have been developed for the first time and inheritance of both thetransgenes in most of the T1 lines was revealed.
Abstract: Morphologically normal and fertile transgenic plants of mungbean with two transgenes, bar and α-amylase inhibitor, have been developed for the first time. Cotyledonary node explants were transformed by cocultivation with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pKSB that carried bialaphos resistance (bar) gene and Phaseolus vulgaris α-amylase inhibitor-1 (αAI-1) gene. Green transformed shoots were regenerated and rooted on medium containing phosphinothricin (PPT). Preculture and wounding of the explants, presence of acetosyringone and PPT-based selection of transformants played significant role in enhancing transformation frequency. Presence and expression of the bar gene in primary transformants was evidenced by PCR-Southern analysis and PPT leaf paint assay, respectively. Integration of the Phaseolus vulgaris α-amylase inhibitor gene was confirmed by Southern blot analysis. PCR analysis revealed inheritance of both the transgenes in most of the T1 lines. Tolerance to herbicide was evidenced from seed germination test and chlorophenol red assay in T1 plants. Transgenic plants could be recovered after 8–10 weeks of cocultivation with Agrobacterium. An overall transformation frequency of 1.51% was achieved.
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TL;DR: The data presented herein suggests that this defensin has potential application in the development of transgenic plants for insect pest control, and molecular modeling analyses were performed, revealing that the N‐terminus of the molecule is responsible for binding with the active site of weevil enzymes.
Abstract: Plant defensins are small cysteine-rich proteins commonly synthesized in plants, encoded by large multigene families. Most plant defensins that have been characterized to date show potent antifungal and/or bactericidal activities. This report describes VuD1, an unusual defensin that is able to inhibit insect-pest alpha-amylases. VuD1 was cloned from cowpea (Vigna unguiculata) seeds and expressed in a heterologous system. Inhibitory enzyme assays showed that VuD1 efficiently inhibits alpha-amylases from the weevils Acanthoscelides obtectus and Zabrotes subfasciatus, caused low inhibition toward mammalian enzymes and was unable to inhibit the alpha-amylases from Callosobruchus maculatus and Aspergillus fumigatus. To shed some light over the mechanism of action of VuD1, molecular modeling analyses were performed, revealing that the N-terminus of the molecule is responsible for binding with the active site of weevil enzymes. Moreover, models of VuD1 and mammalian enzymes were also generated to elucidate the specificity mechanisms. The data presented herein suggests that this defensin has potential application in the development of transgenic plants for insect pest control.
91 citations
01 Jan 2017
TL;DR: The development of pest-resistant cultivars of crop plants and progress in integrated pest management both require an intricate understanding of insect-plant relationships, as outlined in this chapter.
Abstract: The green plants and insects represent the two dominant groups of living organisms on Earth. The green plants occupy the most capacious segment among all biological organisms, whereas the insects are the most specious group. These two ‘empires’ are interconnected as well as interdependent. Green plants are the primary producers of food, and all animals being heterotrophs depend directly or indirectly on plant-produced food. In turn, nearly three fourths of all angiosperms require the services of insect pollinators. The entomophilic flowering plants and their insect pollinators thus represent the most evident and widely applicable example of mutualism among living organisms. But a wide variety of phytophagous insects also flourishes, diversifies and sustains on these plants. Consequently, the plants have evolved a dizzying array of morphological and biochemical (constitutive as well as induced) barriers for protection against insects and other herbivores. Evolutionary interactions between plants and insects may have contributed to the increased biodiversity and success of both these groups. The study of these interrelationships, as outlined in this chapter, is of great practical significance for the future agricultural production. The development of pest-resistant cultivars of crop plants and progress in integrated pest management both require an intricate understanding of insect-plant relationships.
85 citations
TL;DR: A transformation efficiency of 29% was achieved for leaf explants using this protocol, and GUS histochemical analysis of the transgenic tissues further confirmed the transformation event.
74 citations
TL;DR: Transgenic peanut plants exhibited adequate tolerance under salt stress condition and thus could be explored for the cultivation in salt affected areas for the sustainable agriculture.
71 citations
Additional excerpts
...Saini et al., 2007...
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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
References
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Book•
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
215,169 citations
TL;DR: A method is presented for the rapid isolation of high molecular weight plant DNA which is free of contaminants which interfere with complete digestion by restriction endonucleases, and which yields total cellular DNA.
Abstract: A method is presented for the rapid isolation of high molecular weight plant DNA (50,000 base pairs or more in length) which is free of contaminants which interfere with complete digestion by restriction endonucleases. The procedure yields total cellular DNA (i.e. nuclear, chloroplast, and mitochondrial DNA). The technique is ideal for the rapid isolation of small amounts of DNA from many different species and is also useful for large scale isolations.
10,481 citations
"Agrobacterium tumefaciens mediated ..." refers methods in this paper
...Molecular analysis of putative transformants Total genomic DNA was extracted from fresh leaves of putative transformants (T0) and nontransformed (control) plants by the cetyl trimethyl ammonium bromide (CTAB) method (Murray and Thompson 1980)....
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...Total genomic DNA was extracted from fresh leaves of putative transformants (T0) and nontransformed (control) plants by the cetyl trimethyl ammonium bromide (CTAB) method (Murray and Thompson 1980)....
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TL;DR: GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage, and Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.
Abstract: We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.
9,765 citations
"Agrobacterium tumefaciens mediated ..." refers methods in this paper
...After cocultivation, the explants were washed with sterile distilled water for five to six times, blotted dry on sterile paper towel, and incubated in X-gluc solution at 37◦C for histochemical GUS assay (Jefferson et al. 1987)....
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TL;DR: The nutrient requirements of suspension cultures from soybean root have been investigated, and a simple medium consisting of mineral salts, sucrose, vitamins and 2,4-dichlorophenoxyacetic acid (2, 4- d) has been designed.
9,342 citations
"Agrobacterium tumefaciens mediated ..." refers methods in this paper
...These explants were cultured in vertically upright position with the hypocotyl cut end slightly embedded in the B5 medium (Gamborg et al. 1968) containing 3% (w/v) sucrose, supplemented with different concentrations of BAP (0.5–10 µM), pH 5.8 and solidified with 0.7% agar (Himedia, Mumbai, India)....
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...These explants were cultured in vertically upright position with the hypocotyl cut end slightly embedded in the B5 medium (Gamborg et al. 1968) containing 3% (w/v) sucrose, supplemented with different concentrations of BAP (0....
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TL;DR: It is shown here that Agrobacterium tumefaciens virulence (Vir) gene expression is activated specifically by the plant molecules acetosyringone (AS) and α-hydroxyacetosyringsone (OH-AS).
Abstract: We show here that Agrobacterium tumefaciens virulence (Vir) gene expression is activated specifically by the plant molecules acetosyringone (AS) and α-hydroxyacetosyringone (OH-AS). These molecules induce the entire vir regulon in Agrobacterium as well as the formation of T-DNA intermediate molecules. AS and OH-AS occur specifically in exudates of wounded and metabolically active plant cells and probably allow Agrobacterium to recognize susceptible cells in nature.
1,013 citations
"Agrobacterium tumefaciens mediated ..." refers background in this paper
...…penetration into the tissue facilitating the accessibility of plant cells for Agrobacterium or possibly stimulated the production of potent vir gene inducers like phenolic substances (Stachel et al. 1985) and enhanced the plant cell competence for transformation (Binns and Thomashow 1988)....
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...wounded tissue into the surrounding medium (Stachel et al. 1985)....
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...The increase could, probably, be due to cytokinininduced active divisions of the cells (Sangwan et al. 1992) or secretion of phenolics, vir inducing compounds, by the wounded tissue into the surrounding medium (Stachel et al. 1985)....
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...Wounding the plant material before cocultivation allows better bacterial penetration into the tissue facilitating the accessibility of plant cells for Agrobacterium or possibly stimulated the production of potent vir gene inducers like phenolic substances (Stachel et al. 1985) and enhanced the plant cell competence for transformation (Binns and Thomashow 1988)....
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