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Improvement in Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) by the inhibition of polyphenolics released during wounding of cotyledonary node explants

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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.

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Abbreviations

α1-PI:

Alpha-1-proteinase inhibitor

BAP- 6:

Benzylaminopurine

Cfu:

Colony forming units

CN:

Cotyledonary node

DMSO:

Dimethylsulfoxide

DTT:

Dithiothreitol

EDTA:

Ethylene diamine tetraacetic acid

GUS:

β-glucuronidase

MES:

2-(N-morpholino) ethane sulfonic acid

MIC:

Minimal inhibitory concentration

MS:

Murashige and Skoog

MU:

4-methylumbelliferone

MUG:

4-methylumbelliferyl-β-d-glucuronide

PPO:

Polyphenol oxidase

RP-HPLC:

Reverse phase high-performance liquid chromatography

Rfu:

Relative fluorescence units

SA:

Salicylic acid

TSP:

Total soluble protein

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Acknowledgments

We are thankful for the consistent support of Dr. C. S. Nautiyal, Director, CSIR-NBRI, Lucknow for carrying out this research work and thanks to Dr. Dheer Singh, Joint Director, CRC, Gobind Ballabh Pant University of Agriculture and Technology, Pantnagar, Uttarakhand, India for generous supply of high quality seeds of chickpea, is gratefully acknowledged. The grant of research fellowships to RY, MM, AKS by CSIR, New Delhi and by UGC, New Delhi to RS is kindly acknowledged. This work was carried out under the CSIR Network Projects NWP-03 and BSC-0204.

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Authors and Affiliations

  1. Plant Transgenic Lab, CSIR-National Botanical Research Institute, PO Box # 436, Rana Pratap Marg, Lucknow, 226 001, UP, India

    Reena Yadav, Meenakshi Mehrotra, Aditya K. Singh, Rani Singh, Indraneel Sanyal & D. V. Amla

  2. Central Instrumentation Facility (CIF), CSIR-National Botanical Research Institute, PO box # 436, Rana Pratap Marg, Lucknow, 226 001, UP, India

    Abhishek Niranjan & Alok Lehri

  3. Department of Biotechnology, Faculty of Science, University of Kumaun, Bhimtal Campus, Nainital, 263 001, Uttarakhand, India

    Veena Pande

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  1. Reena Yadav
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Supplementary Table 1

Variation in the phenolic and flavonoid compounds at different time intervals after the treatment. (DOC 42 kb)

Supplementary Table 2

Zone assay for inhibition of Agrobacterium tumefaciens. (DOC 43 kb)

Supplementary Fig. 1

Mass spectrum (MS/MS) of gallic acid, chlorogenic acid, caffeic acid, rutin, sinapic acid, coumaric acid, ferulic acid, diadzein, quercetin, genestein, kaempferol and biochanin A in negative mode. (DOC 786 kb)

Supplementary Fig. 2

Minimal inhibition zone assay performed against Agrobacterium tumefaciens with compounds of different retention time, collected by fraction collector through RP-HPLC. (a) 3 min (b) 6 min (c) 8 min (d) 9 min (e) 16 min (f) 23 min (g) 27 min (h) 40 min and (i) 52 min retention time respectively. (GIF 325 kb)

High resolution image (TIF 722 kb)

Supplementary Fig. 3

GUS fluorometric assay of co-cultivated of chickpea explants. (GIF 89 kb)

High resolution image (TIF 86 kb)

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Yadav, R., Mehrotra, M., Singh, A.K. et al. Improvement in Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) by the inhibition of polyphenolics released during wounding of cotyledonary node explants. Protoplasma 254, 253–269 (2017). https://doi.org/10.1007/s00709-015-0940-0

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