Improvements in the efficiency of Agrobacterium rhizogenes-mediated transformation of Oroxylum indicum for induction of hairy roots

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Online ISSN : 2229-4473.
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Doi: 10.1007/s42535-022-00476-2
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Keywords: n Oroxylum indicumn , n Agrobacterium rhizogenes ATCC 15834, Hairy root, Anti-browning agents, Flavonoids


Abstract


Oroxylum indicum (Shyonak) is used as a medicinal herb in the ethnomedicinal system of Asia for prevention and treatment of diseases like respiratory disorder, gastric ulcers, diabetes, cancer, rheumatoid problems, etc. Prior studies have linked the pharmacological properties to flavonoids present in seeds, stems, and root bark. The current research aims to develop a long-term stable system of transgenic hairy roots of O. indicum using Agrobacterium rhizogenes strain ATCC 15834. The best medium, explant type, infection time, co-cultivation period, and anti-browning chemicals were optimized during the hairy root induction. The maximum transformation frequency (49.16 ± 2.44%) was documented for leaf explant after 30 days of inoculation on a semi-solid B5 medium with cefotaxime (200 mg/l), at 48-h co-cultivation period followed by a 2-h infection phase. Explant browning also hindered biomass growth. Hence, different anti-oxidants were used to control cell biomass browning. A transformation frequency up to 65.8% was obtained in B5 media with cysteine (3.71 mM). Successful transformation of the hairy root lines was confirmed using PCR amplification using A. rhizogenes virD, aux1, rolA, rolB, and rolC genes specific primers. Subsequently, in vitro production of four flavonoids viz., baicalein, hispidulin, scutellarin, and biochanin-A, were quantified in hairy root culture of O. indicum and compared with roots of two months old greenhouse cultivated plantlets and roots of in vitro regenerated plants. Higher quantities of baicalein, hispidulin, and scutellarin (viz., 11.47 ± 1.5 μg/g DW, 10.45 ± 0.27 μg/g DW, and 24.75 ± 0.85 μg/g DW, respectively) were quantified in the hairy root cultures of O. indicum.

Graphical abstract


n              Oroxylum indicumn            , n                     Agrobacterium rhizogenes ATCC 15834, Hairy root, Anti-browning agents, Flavonoids


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Acknowledgements



Author Information


Bisht Deepa
Department of Biotechnology, Kumaun University, Nainital, India
deepa.2020rbt09@mnnit.ac.in