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Phytochemical alteration and new occurring compounds in hairy root cultures of Mitracarpus hirtus L. induced by phenylurea cytokinin (CPPU)

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Abstract

Hairy root cultures of Mitracarpus hirtus L. were obtained after transforming leaf-disc explants with wild strain Agrobacterium rhizogenes A13. The root cultures of M. hirtus showed high efficiency of shoot formation in both transformed and non-transformed cultures when illuminated with light. However, transformed hairy root proliferation was approximately 3.8–5 times higher than the control in both solidified and liquid plant growth regulator free media. Putatively transformed roots were identified by the presence of the rol gene via PCR. Integration of the rol gene into the plant genome was confirmed via Southern blot analysis after 5 months with no detection in non-transformed roots. In addition, the effect of 2-chloro-4-pyridyl-N-phenylurea (CPPU), a synthetic cytokinin, when applied as an elicitor for hairy root cultures of M. hirtus was investigated. The 24-day-old hairy root cultures of high root proliferation line R107-3, were incubated for 48 h in media supplemented with 0 or 5 mg l−1 CPPU. The methanolic extracts of root tissues were subsequently analyzed for biochemical constituents using Gas Chromatography Mass Spectrometry (GC-MS). The alteration of plant secondary metabolites produced after CPPU treatment was analyzed. Compared to the control (with quality higher than 80 %), six unique compounds were found, five original compounds absent, 11 with increased, and five with decreased contents. Increased contents of two metabolites, chrysophanol and 2-methoxy-4-vinylphenol, showed pharmaceutical potential. CPPU was also found to elicit the alkaloid compound, Eseroline, 7-bromo-, methylcarbamate (ester), which could not be detected in the non-treated sample. The findings of this study demonstrate the establishment of transgenic hairy root of M. hirtus and the application of CPPU as an elicitor to induce variations in plant secondary metabolite that shows its potential to apply for bio-reactor system.

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Acknowledgments

This work was supported by Mahidol University and a partial Grant by the “Strategic Consortia for Capacity Building of University Faculties and Staff of Thailand Research Fund” from the Commission on Higher Education (CHE), Thailand. The authors are grateful for partial support of the Excellent International Student Scholarship at Chiba University Program from the Japanese government through Chiba University. This research work was conducted under the collaboration of Mahidol University and Chiba University for the double degree program of K. Pansuksan.

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Authors and Affiliations

  1. Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand

    Kanoktip Pansuksan & Kanyaratt Supaibulwatana

  2. Laboratory of Plant Cell Technology, Graduate School of Horticulture, Chiba University, Matsudo, Chiba, 271-8510, Japan

    Kanoktip Pansuksan & Masahiro Mii

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  1. Kanoktip Pansuksan
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Correspondence to Kanyaratt Supaibulwatana.

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Pansuksan, K., Mii, M. & Supaibulwatana, K. Phytochemical alteration and new occurring compounds in hairy root cultures of Mitracarpus hirtus L. induced by phenylurea cytokinin (CPPU). Plant Cell Tiss Organ Cult 119, 523–532 (2014). https://doi.org/10.1007/s11240-014-0552-y

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