Recovery of herbicide-resistant Azuki bean [ Vigna angularis (Wild.), Ohwi & Ohashi] plants via Agrobacterium -mediated transformation
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.
Abstract: Transgenic 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
Citations
More filters
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: 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.
65 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)....
[...]
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...
[...]
...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…...
[...]
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.
33 citations
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.
7 citations
References
More filters
TL;DR: The transmission of the introduced gene into the progeny of the regenerated transgenic plants was studied over two generations, and stable transmission was shown to take place.
Abstract: An analysis of the progeny of primary transgenic pea plants in terms of transmission of the transferred DNA, fertility and morphology is presented. A transformation system developed for pea that allows the regeneration of fertile transgenic pea plants from calli selected for antibiotic resistance was used. Expiants from axenic shoot cultures were co-cultivated with a nononcogenic Agrobacterium tumefaciens strain carrying a gene encoding hygromycin phosphotransferase as selectable marker, and transformed callus could be selected on callus-inducing media containing 15 mg/l hygromycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on the hygromycin resistant calli, and the regenerated shoots could subsequently be rooted and transferred to the greenhouse, where they proceeded to flower and set seed. The transmission of the introduced gene into the progeny of the regenerated transgenic plants was studied over two generations, and stable transmission was shown to take place. The transgenic nature of the calli and regenerated plants and their progeny was confirmed by DNA and RNA analysis. The DNA and ploidy levels of the progeny plants and primary regenerants were studied by chromosome analysis, and the offspring of the primary transformants were evaluated morphologically.
51 citations
Journal Article•
TL;DR: Two plasmid vectors were introduced into soybean (Glycine max (L.) Merr.
Abstract: Two plasmid vectors were introduced into soybean (Glycine max (L.) Merr.) and azuki bean (Vigna angularis Willd. Ohwi and Ohashi) using different transformation systems. Azuki bean epicotyl explants were prepared from etiolated seedlings and co-cultivated with Agrobacterium tumefaciens for 2 days. Adventitious shoots were developed from the callus of the explants on a regeneration medium containing hygromycin, and the shoots were excised and transferred to a rooting medium containing hygromycin at the same concentration. Rooting shoots were transferred to soil and grown in a glass-house to produce viable seeds. PCR analysis confirmed clearly the presence of the hpt gene in most of the azuki beans regenerated under hygromycin selection. A soybean embryogenic suspension culture was generated from immature cotyledons, and used for the introduction of plasmids by particle bombardment. Hygromycin-resistant embryogenic clones were isolated after 8 weeks of hygromycin selection, and then the green clones were matured on the differentiation medium. After desiccation, the embryos were germinated on the rooting medium, and the plants were transferred to soil in a glass-house. More than 50% of the regenerated soybean plants tolerant to hygromycin yielded the hpt fragment on PCR analysis. The azuki bean transformants were obtained more rapidly and with higher efficiency than the soybean transformants.
10 citations
"Recovery of herbicide-resistant Azu..." refers background in this paper
...Among the tribe Phaseoleae, azuki bean is not one of the cosmopolitan crops but it has a reproducible and efficient Agrobacterium-mediated transformation system (Yamada et al., 2001; El-Shemy et al., 2002)....
[...]
6 citations
"Recovery of herbicide-resistant Azu..." refers background in this paper
...…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; Schroeder et al., 1993; Shade et al., 1994; Zubko et al., 1990)....
[...]