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Journal ArticleDOI

Recovery of herbicide-resistant Azuki bean [ Vigna angularis (Wild.), Ohwi & Ohashi] plants via Agrobacterium -mediated transformation

31 Jan 2005-African Journal of Biotechnology (Academic Journals (Kenya))-Vol. 4, Iss: 1, pp 61-67

TL;DR: The presence of transgenes in transformed azuki bean plants was confirmed by polymerase chain reaction (PCR) and southern blot analysis and demonstrates the feasibility of introducing potentially useful agronomic traits into azukibean through genetic engineering.

AbstractTransgenic azuki bean [ Vigna angularis (Willd.) Ohwi & Ohashi] plants expressing the hygromycin phosphotransferase ( hpt ), green fluorescent protein ( sgfp ) and phosphinothricin acetyltransferase ( bar ) genes were obtained by Agrobacterium- tumefacients - mediated transformation. A total of 210 epicotyl explants were inoculated with A. tumefaciens strain EHA105, harboring the binary plasmid pZHBG on MS co-cultivation medium supplemented with 100 mM acetosyringone and 10 mg/l of BA. Following selection on MS medium with 15 mg/l of hygromycin, the regenerated adventitious shoots that formed on the induced calli were further screened for sgfp expression before transferred to rooting medium. 31 transgenic plants were obtained with transformation frequency of 14%. The presence of transgenes in transformed azuki bean plants was confirmed by polymerase chain reaction (PCR) and southern blot analysis. Transcription of the bar and hpt genes was assessed by reverse transcription polymerase chain reaction (RT-PCR) analysis. sgfp- positive transgenic plants exhibited functional expression of the bar gene as determined by assaying for resistance to bialaphos applied directly to leaves. This result demonstrates the feasibility of introducing potentially useful agronomic traits into azuki bean through genetic engineering. Key Words: Agrobacterium tumefaciens, bar gene, bialaphos, transgenic, Vigna angulazris . African Journal of Biotechnology Vol.4(1) 2005: 61-67

Topics: Agrobacterium (58%), Agrobacterium tumefaciens (55%), Bialaphos (54%), Vigna (54%), Phosphinothricin acetyltransferase (54%)

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Citations
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Journal ArticleDOI
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.

60 citations


Cites background from "Recovery of herbicide-resistant Azu..."

  • ...Pisum sativum (Schroeder et al. 1993; Bean et al. 1997), Glycine max (Zhang et al. 1999), Lupinus species (Pigeaire et al. 1997), and Trifolium subterranean (Khan et al. 1994), and Vigna angularis (Khalafalla et al. 2005)....

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Journal ArticleDOI
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.
Abstract: It is three decades since the first transgenic pulse crop has been developed. Todate, genetic transformation has been reported in all the major pulse crops like Vigna species, Cicer arietinum, Cajanus cajan, Phaseolus spp, Lupinus spp, Vicia spp and Pisum sativum, but transgenic pulse crops have not yet been commercially released. Despite the crucial role played by pulse crops in tropical agriculture, transgenic pulse crops have not moved out from laboratories to large farm lands compared to their counterparts - 'cereals' and the closely related leguminous oil crop - 'soybean'. The reason for lack of commercialization of transgenic pulse crops can be attributed to the difficulty in developing transgenics with reproducibility, which in turn is due to lack of competent totipotent cells for transformation, long periods required for developing transgenics and lack of coordinated research efforts by the scientific community and long term funding. 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. A translation of knowledge from information available in genomics and functional genomics in model legumes like Medicago truncatula and Lotus japonicus relating to factors which contribute to enhancing crop yield and ameliorate the negative consequences of biotic and abiotic stress factors may provide novel insights for genetic manipulation to improve the productivity of pulse crops.

60 citations


Journal ArticleDOI
TL;DR: A step-wise procedure for the regeneration of fertile plants by organogenesis from cultures of the economically important Phaseolus angularis L., cultivars: KS-6, KS-7 and KS-8 using etiolated seedlings was established, finding the efficient shoot bud induction capability to be cultivar dependent.
Abstract: A step-wise procedure for the regeneration of fertile plants by organogenesis from cultures of the economically important Phaseolus angularis L., cultivars: KS-6, KS-7 and KS-8 using etiolated seedlings was established. Pre-culture of 5-day old seedling explants with MS (Murashige and Skoog (1962) Physiol Plant 15:473–493) + B5-vitamins (Gamborg et al. (1968) Exp Cell Res 50:151–158) liquid medium containing either 5.0 µM TDZ or 5.0 µM BAP under dark condition was essential for organogenesis. Bud growth and shoot multiplication were stimulated by reducing the BAP concentrations from 5.0 to 2.5 µM after 3 weeks. The maximum frequency of shoot induction was 65.2% (33.8 ± 2.54 shoots/explant) in cultivar KS-8 followed by KS-7 34.6% (23.4 ± 1.91 shoots/explant) and KS-6 30.6% (21.2 ± 2.28 shoots/explant). The multiplied buds elongated after transferring to solid MSB5 medium supplemented with 4.0 µM GA3, 12.5 µM AgNO3 and 0.4 µM IBA. Up to 98% rooting efficiency of was obtained when the shoots were pulse-treated with liquid medium containing 4.5 µM IBA for 10 min. The rooted plantlets were transferred to pots in the greenhouse, where they grew, mature, flowered and bared pod normally. The efficient shoot bud induction capability was found to be cultivar dependent. All the three cultivars tested formed multiple shoots. This efficient and rapid regeneration system may also be helpful for Agrobacterium- or particle gun-mediated transformation for this important legume crop.

45 citations


Cites methods from "Recovery of herbicide-resistant Azu..."

  • ...formation using epicotyl explants (Yamada et al. 2001, 2005; Khalafalla et al. 2005), our present...

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  • ...Although there were reports in V. angularis via Agrobacterium mediated transformation using epicotyl explants (Yamada et al. 2001, 2005; Khalafalla et al. 2005), our present studies deviates from earlier report where, we used pre-culture treatments using liquid medium followed by solid culture, and…...

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Journal ArticleDOI
TL;DR: This article examines the prospects of genomics assisted integrated breeding to enhance and stabilize crop yields and outlines the recent progress made in genomics of these lesser explored pulse crops.
Abstract: Pulses are multipurpose crops for providing income, employment and food security in the underprivileged regions, notably the FAO-defined low-income food-deficit countries. Owing to their intrinsic ability to endure environmental adversities and the least input/management requirements, these crops remain central to subsistence farming. Given their pivotal role in rain-fed agriculture, substantial research has been invested to boost the productivity of these pulse crops. To this end, genomic tools and technologies have appeared as the compelling supplement to the conventional breeding. However, the progress in minor pulse crops including dry beans (Vigna spp.), lupins, lablab, lathyrus and vetches has remained unsatisfactory, hence these crops are often labeled as low profile or lesser researched. Nevertheless, recent scientific and technological breakthroughs particularly the next generation sequencing (NGS) are radically transforming the scenario of genomics and molecular breeding in these minor crops. NGS techniques have allowed de novo assembly of whole genomes in these orphan crops. Moreover, the availability of a reference genome sequence would promote re-sequencing of diverse genotypes to unlock allelic diversity at a genome-wide scale. In parallel, NGS has offered high-resolution genetic maps or more precisely, a robust genetic framework to implement whole-genome strategies for crop improvement. As has already been demonstrated in lupin, sequencing-based genotyping of the representative sample provided access to a number of functionally-relevant markers that could be deployed straight away in crop breeding programs. This article attempts to outline the recent progress made in genomics of these lesser explored pulse crops, and examines the prospects of genomics assisted integrated breeding to enhance and stabilize crop yields.

25 citations


Book ChapterDOI
01 Jan 2019
TL;DR: DNA marker analysis suggests that there are obvious genetic distinctions between different forms, but the diversity among cultivated germplasm is quite low, indicating that the wild forms could be an important genetic resource for breeding.
Abstract: Adzuki bean [Vigna angularis (Willd.) Ohwi & Ohashi], an annual pulse crop, belongs to the genus Vigna and subgenus Ceratotrapis. It provides nutritional elements for the human diet and fertilizes soil by nitrogen fixation. It has been traditionally planted and consumed in East and Southeast Asia, especially in China, Japan and Korea, so it came to be called the Asia legume. Adzuki bean was dispersed to other continents for commercial uses in recent decades. Wild adzuki bean (V. angularis var. nipponensis), considered to be the ancestor of cultivated adzuki bean, occurs in East Asia and in the Himalayan Region, which are presumed to be where the domestication of adzuki bean took place. Another wild form, V. nepalensis, called the weedy adzuki bean, is mainly found in Eastern Nepal and around. A large portion of adzuki bean germplasm has been collected and conserved in different gene banks. DNA marker analysis suggests that there are obvious genetic distinctions between different forms, but the diversity among cultivated germplasm is quite low, indicating that the wild forms could be an important genetic resource for breeding. However, the genetic and genomic studies on this species are lagging and include only low-density genetic maps and a few maps of genes. That is the reason conventional breeding of adzuki bean has achieved rapid improvement, while no modern biotechnology has yet been used in breeding.

4 citations


References
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Journal ArticleDOI
TL;DR: In vivo redox biosensing resolves the spatiotemporal dynamics of compartmental responses to local ROS generation and provide a basis for understanding how compartment-specific redox dynamics may operate in retrograde signaling and stress 67 acclimation in plants.
Abstract: In experiments with tobacco tissue cultured on White's modified medium (basal meditmi hi Tnhles 1 and 2) supplemenk'd with kiticthi and hidoleacctic acid, a slrikin^' fourlo (ive-told intTease iu yield was ohtaitu-d within a three to Tour week j^rowth period on addition of an aqtteotis exlrarl of tobacco leaves (Fi^'ures 1 and 2). Subse(iueutly it was found Ihiit this jnoniotiou oi' f^rowih was due mainly though nol entirely to inorj^auic rather than organic con.stitttenls in the extract. In the isolation of Rrowth factors from plant tissues and other sources inorj '̂anic salts are fre(|uently carried along with fhe organic fraclioits. When tissue cultures are used for bioassays, therefore, il is necessary lo lake into account increases in growth which may result from nutrient elements or other known constituents of the medium which may he present in the te.st materials. To minimize interference trom rontaminaitis of this type, an altempt has heen made to de\\eh)p a nieditmi with such adequate supplies of all re(iuired tnineral nutrients and cotntnott orgattic cottslitueitls that no apprecial»le change in growth rate or yield will result from the inlroduclion of additional amounts in the range ordinarily expected to be present in tnaterials to be assayed. As a point of referetice for this work some of the culture media in mc)st common current use will he cotisidered briefly. For ease of comparis4)n Iheir mineral compositions are listed in Tables 1 and 2. White's nutrient .solution, designed originally for excised root cultures, was based on Uspeuski and Uspetiskaia's medium for algae and Trelease and Trelease's micronutrieni solution. This medium also was employed successfully in the original cttltivation of callus from the tobacco Iiybrid Nicotiana gtauca x A', tanijadorffii, atitl as further modified by White in 194̂ ^ and by others it has been used for the

60,055 citations


"Recovery of herbicide-resistant Azu..." refers methods in this paper

  • ...Seeds were germinated at 25°C in the dark condition on MS basal medium (Murashige and Skoog, 1962), containing 30 g/l sucrose and 8 g/l agar (Wako Pure Chemical Industries) (pH 5....

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  • ...Seeds were germinated at 25°C in the dark condition on MS basal medium (Murashige and Skoog, 1962), containing 30 g/l sucrose and 8 g/l agar (Wako Pure Chemical Industries) (pH 5.8)....

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Journal ArticleDOI
TL;DR: The newpPZP Agrobacterium binary vectors are versatile, relatively small, stable and fully sequenced, allowing their use inAgrobacteria strains with different drug resistance markers.
Abstract: The newpPZP Agrobacterium binary vectors are versatile, relatively small, stable and are fully sequenced. The vectors utilize the pTiT37 T-DNA border regions, the pBR322bom site for mobilization fromEscherichia coli toAgrobacterium, and the ColE1 and pVS1 plasmid origins for replication inE. coli and inAgrobacterium, respectively. Bacterial marker genes in the vectors confer resistance to chloramphenicol (pPZP100 series) or spectinomycin (pPZP200 series), allowing their use inAgrobacterium strains with different drug resistance markers. Plant marker genes in the binary vectors confer resistance to kanamycin or to gentamycin, and are adjacent to the left border (LB) of the transferred region. A lacZ α-peptide, with the pUC18 multiple cloning site (MCS), lies between the plant marker gene and the right border (RB). Since the RB is transferred first, drug resistance is obtained only if the passenger gene is present in the transgenic plants.

1,535 citations


"Recovery of herbicide-resistant Azu..." refers methods in this paper

  • ...A dual marker binary plasmid pZHG comprising the reporter gene sgfp (green fluorescent protein) and hpt gene (hygromycin phosphotransferase) as a selective marker with a multiple cloning site between them was constructed based on pZH2 vector which is a derivative of pPZP202 (Hajdukiewicz et al., 1994)....

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  • ...…marker binary plasmid pZHG comprising the reporter gene sgfp (green fluorescent protein) and hpt gene (hygromycin phosphotransferase) as a selective marker with a multiple cloning site between them was constructed based on pZH2 vector which is a derivative of pPZP202 (Hajdukiewicz et al., 1994)....

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Journal ArticleDOI
TL;DR: It is reported that an extensively modified GFP is a versatile and sensitive reporter in a variety of living plant cells and in transgenic plants, and the codon usage effect might be universal, allowing the design of recombinant proteins with high expression efficiency in evolutionarily distant species such as humans and maize.
Abstract: Background: The green-fluorescent protein (GFP) of the jellyfish Aequorea victoria has recently been used as a universal reporter in a broad range of heterologous living cells and organisms. Although successful in some plant transient expression assays based on strong promoters or high copy number viral vectors, further improvement of expression efficiency and fluorescent intensity are required for GFP to be useful as a marker in intact plants. Here, we report that an extensively modified GFP is a versatile and sensitive reporter in a variety of living plant cells and in transgenic plants. Results We show that a re-engineered GFP gene sequence, with the favored codons of highly expressed human proteins, gives 20-fold higher GFP expression in maize leaf cells than the original jellyfish GFP sequence. When combined with a mutation in the chromophore, the replacement of the serine at position 65 with a threonine, the new GFP sequence gives more than 100-fold brighter fluorescent signals upon excitation with 490 nm (blue) light, and swifter chromophore formation. We also show that this modified GFP has a broad use in various transient expression systems, and allows the easy detection of weak promoter activity, visualization of protein targeting into the nucleus and various plastids, and analysis of signal transduction pathways in living single cells and in transgenic plants. Conclusion The modified GFP is a simple and economical new tool for the direct visualization of promoter activities with a broad range of strength and cell specificity. It can be used to measure dynamic responses of signal transduction pathways, transfection efficiency, and subcellular localization of chimeric proteins, and should be suitable for many other applications in genetically modified living cells and tissues of higher plants. The data also suggest that the codon usage effect might be universal, allowing the design of recombinant proteins with high expression efficiency in evolutionarily distant species such as humans and maize.

1,403 citations


"Recovery of herbicide-resistant Azu..." refers background in this paper

  • ...The presence of sgfp (S65T) was detected by blue light excitation (Chiu et al., 1996)....

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Journal ArticleDOI
TL;DR: The construction of new helper Ti plasmids for Agrobacterium-mediated plant transformation using T-DNA regions deleted using site-directed mutagenesis to yield replicons carrying thevir genes that will complement binary vectorsin trans.
Abstract: We describe the construction of new helper Ti plasmids forAgrobacterium-mediated plant transformation. These plasmids are derived from three differentAgrobacterium tumefaciens Ti plasmids, the octopine plasmid pTiB6, the nopaline plasmid pTiC58, and the L,L-succinamopine plasmid pTiBo542. The T-DNA regions of these plasmids were deleted using site-directed mutagenesis to yield replicons carrying thevir genes that will complement binary vectorsin trans. Data are included that demonstrate strain utility. The advantages ofAgrobacterium strains harbouring these ‘disamed’ Ti plasmids for plant transformation viaAgrobacterium are discussed.

1,302 citations


"Recovery of herbicide-resistant Azu..." refers methods in this paper

  • ...Agrobacterium strain and plasmid A. tumefaciens strain EHA105 containing the disarmed hypervirulent plasmid pTiBo542 in the C58 chromosomal background (Hood et al., 1993) was used for transformation....

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  • ...tumefaciens strain EHA105 containing the disarmed hypervirulent plasmid pTiBo542 in the C58 chromosomal background (Hood et al., 1993) was used for transformation....

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Journal ArticleDOI
TL;DR: Progeny from two transgenic soybean plants demonstrated co-segregation of kanamycin resistance and either GUS expression or glyphosate tolerance in a 3:1 ratio indicating a single insert inherited in a Mendelian fashion.
Abstract: Transgenic soybean plants have been produced using an Agrobacterium-mediated gene transfer system. This procedure relied on a regeneration protocol in which shoot organogenesis was induced on cotyledons of soybean genotypes selected for susceptibility to Agrobacterium. Cotyledon explants were inoculated with Agrobacterium tumefaciens pTiT37-SE harboring pMON9749 (conferring kanamycin resistance and β-glucuronidase “GUS” activity) or pTiT37-SE∷pMON894 (conferring kanamycin resistance and glyphosate tolerance) and cultured on shoot induction medium containing kanamycin. Plantlets were tested for gene insertion 3–4 months post-inoculation. Approximately 6% of the shoots (8 plants to date) produced on the kanamycin-selected cotyledons were transgenic based on assays for GUS expression, kanamycin resistance or glyphosate tolerance. Progeny from two of these plants demonstrated co-segregation of kanamycin resistance and either GUS expression or glyphosate tolerance in a 3:1 ratio indicating a single insert inherited in a Mendelian fashion.

561 citations


"Recovery of herbicide-resistant Azu..." refers background in this paper

  • ...generate transgenic soybeans (Hinchee et al., 1988; Chee et al., 1989), chickpeas (Fontana et al....

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  • ...The majority of legume transformation studies have favored the use of Agrobacterium tumefaciens to generate transgenic soybeans (Hinchee et al., 1988; Chee et al., 1989), chickpeas (Fontana et al., 1993) and pea (Puonti-Kaerlas et al., 1990, 1992; De Kathen and Jacobsen 1990; Davies et al., 1993;…...

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