Improved Agrobacterium-mediated transformation of cowpea via sonication and vacuum infiltration

Bakshi, S. K.; Sadhukhan, Ayan; Mishra, Sagarika; Sahoo, Lingaraj

doi:10.1007/s00299-011-1133-8

Cited by 87 publications

(58 citation statements)

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“…Heiankogiku). Vacuum infiltration has been used in other refractory plants, such as cowpea (Bakshi et al 2011), pine (Charity et al 2002), and radish (Park et al 2005).…”

Section: Effect Of Vacuum Infiltration On Agrobacterium After Woundingmentioning

confidence: 99%

Improving the transformation efficiency of Cucurbita species: factors and strategy for practical application

Nanasato

Okuzaki

Tabei

2013

Plant Biotechnology

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Cucurbita species are refractory to transformation. Hence, the only 2 reports published regarding the transformation of Cucurbita species until our successful transformation of C. moschata (cv. Heiankogiku) in 2011. The efficiency was 2.7±1.3% using wounded explants vortexed with whisker suspension. To improve transformation efficiency, transformation experiments were carried out with various ages of cotyledonary explants. The highest efficiency was obtained with 1-day-old (3.2±0.9%) and 2-day-old (3.3±0.8%) explants after germination. Histochemical analysis of GUS activity revealed that wounding allowed Agrobacterium access to the deeper layer of explants. These results suggested that cells having a high ability of shoot organogenesis exist not on the surface but in the deeper layers of explants. We applied vacuum infiltration to wounded explants to enhance Agrobacterium access to the deeper layers, which resulted in improvement of transformation efficiency by 3-fold (9.2±2.9%). To verify the efficacy of our transformation procedure for other Cucurbita species, we attempted to obtain transformants with 3 Cucurbita species: C. maxima (cv. Ebisu), C. pepo (cv. Black Tosca), and C. ficifolia (cv. Kurotanenankin). The average regeneration efficiencies were 54.2±7.2%, 62.5±19.1%, and 72.2±13.4% under our regeneration system. Although the efficiency for production of tansgenic plants was very low (ca. 0.2-0.3%), a transgenic line was obtained from C. maxima and C. pepo, respectively. We discuss recent advances that may help in the development of beneficial applications for the transformation of Cucurbita species.

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“…Heiankogiku). Vacuum infiltration has been used in other refractory plants, such as cowpea (Bakshi et al 2011), pine (Charity et al 2002), and radish (Park et al 2005).…”

Section: Effect Of Vacuum Infiltration On Agrobacterium After Woundingmentioning

confidence: 99%

Improving the transformation efficiency of Cucurbita species: factors and strategy for practical application

Nanasato

Okuzaki

Tabei

2013

Plant Biotechnology

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“…The transformation procedure described in a recently published protocol by our laboratory (Bakshi et al 2011) was used with few modifications. Cowpea cotyledonary node explants were subjected to a combination of 20 s sonication followed by 5 min vacuum infiltration prior to inoculation in bacterial suspension for 30 min with occasional shaking.…”

Section: Determination Of Mannose Concentration For Efficient Selectionmentioning

confidence: 99%

“…The possibility of recovering stable transgenic plants of cowpea has been reported through Agrobacterium-mediated transformation of cotyledonary explants (Muthukumar et al 1996;Popelka et al 2006;Chaudhury et al 2007;Solleti et al 2008a, b;Bakshi et al 2011) and direct DNA delivery to shoot apices by particle bombardment (Ivo et al 2008). Using Agrobacterium-mediated transformation of cotyledonary node explants, we recently reported the development of transgenic cowpea expressing the common bean a-amylase inhibitor (a-ai1) gene for storage pest resistance (Solleti et al 2008b) and the Btcry1Ac gene (Bakshi et al 2011) for insect resistance.…”

Section: Introductionmentioning

confidence: 98%

“…Although promising results have been obtained with the negative selection protocols, cowpea genetic transformation still remains far from routine due to the poor selection of transformed shoots on antibiotic-or herbicide-supplemented medium leading to regeneration of escapes, growth retardation of selected transformed shoots owing to longtime exposure to stringent selection and consequently low rooting efficiency of the transgenic plants (Bakshi et al 2011). During such negative selection, the majority of cells in the culture tissue die.…”

Section: Introductionmentioning

confidence: 98%

“…Using Agrobacterium-mediated transformation of cotyledonary node explants, we recently reported the development of transgenic cowpea expressing the common bean a-amylase inhibitor (a-ai1) gene for storage pest resistance (Solleti et al 2008b) and the Btcry1Ac gene (Bakshi et al 2011) for insect resistance. Previously published protocols for cowpea transformation are all based on negative selection using either the hygromycin phosphotransferase (hpt) gene, which confers resistance to the aminocyclitol antibiotic hygromycin (Muthukumar et al 1996), the neomycin phosphotransferase II (nptII) gene, which confers resistance to aminoglycoside antibiotics, such as kanamycin (Chaudhury et al 2007;Solleti et al 2008b;Bakshi et al 2011), and geneticin (Solleti et al 2008a), the bar gene, which encodes for phosphinothricin acetyltransferase and confers resistance to the herbicide l-phosphinotricin (Popelka et al 2006) or the mutated ahas gene, which encodes a mutated acetohydroxyacid synthase and confers resistance to the herbicide imazapyr (Ivo et al 2008).…”

Section: Introductionmentioning

confidence: 99%

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Successful recovery of transgenic cowpea (Vigna unguiculata) using the 6-phosphomannose isomerase gene as the selectable marker

et al. 2012

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A new method for obtaining transgenic cowpea was developed using positive selection based on the Escherichia coli 6-phosphomannose isomerase gene as the selectable marker and mannose as the selective agent. Only transformed cells were capable of utilizing mannose as a carbon source. Cotyledonary node explants from 4-day-old in vitro-germinated seedlings of cultivar Pusa Komal were inoculated with Agrobacterium tumefaciens strain EHA105 carrying the vector pNOV2819. Regenerating transformed shoots were selected on medium supplemented with a combination of 20 g/l mannose and 5 g/l sucrose as carbon source. The transformed shoots were rooted on medium devoid of mannose. Transformation efficiency based on PCR analysis of individual putative transformed shoots was 3.6%. Southern blot analysis on five randomly chosen PCR-positive plants confirmed the integration of the pmi transgene. Qualitative reverse transcription (qRT-PCR) analysis demonstrated the expression of pmi in T₀ transgenic plants. Chlorophenol red (CPR) assays confirmed the activity of PMI in transgenic plants, and the gene was transmitted to progeny in a Mendelian fashion. The transformation method presented here for cowpea using mannose selection is efficient and reproducible, and could be used to introduce a desirable gene(s) into cowpea for biotic and abiotic stress tolerance.

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Prunella vulgaris L. hairy roots: Culture, growth, and elicitation by ethephon and salicylic acid

Wang

et al. 2016

Engineering in Life Sciences

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Prunella vulgaris L. hairy roots: Culture, growth, and elicitation by ethephon and salicylic acidHairy roots have gained popularity as an alternative source for secondary metabolites not only because of their biosynthetic and bioactive potentials, but also because of studies of experimental systems aiming to understand secondary metabolic pathways. While many secondary metabolites have been identified in Prunella vulgaris, a comprehensive understanding of the metabolic pathways has remained elusive. Here, Agrobacterium rhizogenes (ATCC15834)-mediated hairy root induction of P. vulgaris resulted in 15-30 times more rosmarinic acid (RA) than in the intact plant. After the growth and RA biosynthesis of P. vulgaris, hairy roots were investigated over a 1-month period, we studied elicitation by ethephon (Eth) and salicylic acid (SA) and found 200 μg L −1 Eth and 6.9 mg L −1 SA exhibited the optimum induction efficiency. These concentrations were further selected for a time-course assay of hairy root cultures to assess exposure to these compounds over 8 days. During that period, RA reached maximum accumulations of 1.66-fold 8 days after Eth elicitation and 1.48-fold 2 days post-SA addition. We also examined the transcripts of genes involved in the RA biosynthesis pathway, and found the expression of tyrosine aminotransferase (TAT) and phenylalanine ammonia-lyase (PAL) were increased by Eth, and SA heightened the expression of TAT, 4-hydroxyphenylpyruvate reductase (HPPR), PAL, 4-coumaric acid CoA-ligase 1 (4CL1), and cytochrome P450-dependent monooxygenase (CYP). Altogether, our results indicate that the convenient hairy root culture system of P. vulgaris can provide an alternate source for RA production and would be conducive for the engineering of secondary metabolic pathways in P. vulgaris.

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Improved Agrobacterium-mediated transformation of cowpea via sonication and vacuum infiltration

Cited by 87 publications

References 50 publications

Improving the transformation efficiency of Cucurbita species: factors and strategy for practical application

Improving the transformation efficiency of Cucurbita species: factors and strategy for practical application

Successful recovery of transgenic cowpea (Vigna unguiculata) using the 6-phosphomannose isomerase gene as the selectable marker

Prunella vulgaris L. hairy roots: Culture, growth, and elicitation by ethephon and salicylic acid

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