title: Improvement in Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) by the inhibition of polyphenolics released during wounding of cotyledonary node explants.
abstract: Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) has been performed using cotyledonary node explants (CNs), which release phenolics upon excision that are detrimental to the viability of Agrobacterium tumefaciens and result in low transformation frequency. Twelve low molecular weight phenolic compounds and salicylic acid were identified in the exudates released upon excision during the preparation of cotyledonary nodes by reverse phase high-performance liquid chromatography (RP-HPLC). Zone inhibition assays performed with the explant exudates released at periodic intervals after excision showed the inhibition of A. tumefaciens. Agroinoculation of freshly excised cotyledonary nodes of chickpea showed 98-99 % inhibition of colony forming units (cfu). Osmium tetraoxide fixation of excised tissues showed enhanced accumulation of phenolics in the sub-epidermal regions causing enzymatic browning, affecting the viability and performance of A. tumefaciens for T-DNA delivery. The periodic analysis of exudates released from excised CNs showed enhanced levels of gallic acid (0.2945 ± 0.014 μg/g), chlorogenic acid (0.0978 ± 0.0046 μg/g), and quercetin (0.0971 ± 0.0046 μg/g) fresh weight, which were detrimental to A. tumefaciens. Quantitative assays and the elution profile showed the maximum leaching of phenolics, flavonoids, and salicylic acid immediately after the excision of explants and continued till 4 to 8 h post-excision. Pre-treatment of excised explants with inhibitors of polyphenol oxidase like L-cysteine, DTT, and sodium thiosulfate before co-cultivation showed the recovery of A. tumefaciens cfu, decreased the accumulation of phenolics, and improved transformation frequency. Our results show the hypersensitive response of excision stress for the expression of defense response-related genes and synthesis of metabolites in grain legume chickpea against pathogen infestation including Agrobacterium.
authors.affiliation: Plant Transgenic Lab, CSIR-National Botanical Research Institute, PO Box # 436, Rana Pratap Marg, Lucknow, 226 001, UP, India.
authors.forename: Reena
authors.initials: R
authors.lastname: Yadav
authors.affiliation: Plant Transgenic Lab, CSIR-National Botanical Research Institute, PO Box # 436, Rana Pratap Marg, Lucknow, 226 001, UP, India.
authors.forename: Meenakshi
authors.initials: M
authors.lastname: Mehrotra
authors.affiliation: Plant Transgenic Lab, CSIR-National Botanical Research Institute, PO Box # 436, Rana Pratap Marg, Lucknow, 226 001, UP, India.
authors.forename: Aditya K
authors.initials: AK
authors.lastname: Singh
authors.affiliation: Central Instrumentation Facility (CIF), CSIR-National Botanical Research Institute, PO box # 436, Rana Pratap Marg, Lucknow, 226 001, UP, India.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Niranjan
authors.affiliation: Plant Transgenic Lab, CSIR-National Botanical Research Institute, PO Box # 436, Rana Pratap Marg, Lucknow, 226 001, UP, India.
authors.forename: Rani
authors.initials: R
authors.lastname: Singh
authors.affiliation: Plant Transgenic Lab, CSIR-National Botanical Research Institute, PO Box # 436, Rana Pratap Marg, Lucknow, 226 001, UP, India.
authors.forename: Indraneel
authors.initials: I
authors.lastname: Sanyal
authors.affiliation: Central Instrumentation Facility (CIF), CSIR-National Botanical Research Institute, PO box # 436, Rana Pratap Marg, Lucknow, 226 001, UP, India.
authors.forename: Alok
authors.initials: A
authors.lastname: Lehri
authors.affiliation: Department of Biotechnology, Faculty of Science, University of Kumaun, Bhimtal Campus, Nainital, 263 001, Uttarakhand, India.
authors.forename: Veena
authors.initials: V
authors.lastname: Pande
authors.affiliation: Plant Transgenic Lab, CSIR-National Botanical Research Institute, PO Box # 436, Rana Pratap Marg, Lucknow, 226 001, UP, India. dvamla@rediffmail.com.
authors.forename: D V
authors.initials: DV
authors.lastname: Amla
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000254:growth & development; D000975:Antioxidants / Q000378:metabolism; D004156:Catechol Oxidase / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D029863:Cicer / Q000235:genetics* / Q000382:microbiology*; D015169:Colony Count, Microbial; D018548:Cotyledon / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D005419:Flavonoids / Q000378:metabolism; D005822:Genetic Vectors / Q000378:metabolism; D018551:Solanum lycopersicum / Q000235:genetics; D013058:Mass Spectrometry; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability; D030821:Plants, Genetically Modified; D059808:Polyphenols / Q000378:metabolism*; D020156:Salicylic Acid / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D000975:Antioxidants; D004269:DNA, Bacterial; D005419:Flavonoids; D059808:Polyphenols; C036483:T-DNA; D004156:Catechol Oxidase; D020156:Salicylic Acid
keywords: Agrobacterium; Cicer arietinum; Cotyledonary node; GUS; Gallic acid; L-cysteine; Polyphenol oxidase; Quercetin; Salicylic acid; uidA