Isoflavone production in hairy root cultures and plantlets of Trifolium pratense
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The aim of this study was to develop a Trifolium pratense hairy root (HR) production protocol and select HR lines with high isoflavone yield following elicitor treatments.
We obtained 13 independent HR lines, producing approximately three times more isoflavonoids than seedlings (3.3 mg/g dry weight) and in which 27 isoflavonoids were detected. Each HR line had its own isoflavonoid profile. These lines produced as major components daidzein, genistein, formononetin and biochanin A. Sucrose, salicylic acid (SA), yeast extract (YE) and flagellin 22 (flg22) were tested as elicitors. Using SA 140 mg/L, allowed the maximum isoflavonoid production in plantlets (11.9 mg/g dry weight) but reduced root growth, possibly as a result of its toxicity. The highest isoflavone production in HR (27.9 mg/g dry weight) was obtained using sucrose 60 g/L, for 3.5 days.
This work reports the high production of various isoflavonoids with T. pratense elicited HR cultures.
KeywordsElicitors Plantlets Red clover Salicylic acid Sucrose
Thanks to the Coordination for the Improvement of Higher Education Personnel and National Council for Scientific and Technological Development/Brazil for the financial assistance provided through Ph.D. and Scientific Initiation scholarships. J.A.S.Z. thanks CNPq for the researcher fellowship, Programa Iberoamericano CYTED - proyecto BIFRENES 416RT0511 and the National Institute of Science and Technology - INCT BioNat - Grant # 465637/2014-0. Thanks also to Dr. Sandra Beatriz Rech, Dr. Miguel Dall’Agnol, Dr. Amelia Henriques and Marí Castro for the collaboration. Thanks to Dr. Marie Garmier for careful reading and editing of the manuscript. The project benefits from the support of the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS).
Supplementary Fig. 1—Colour modification in the T. pratense plantlets flavonoid extracts cultivated in presence of Sucrose plus phytagel as gelling agent. From left to right extracts of cultures grown on 10, 30, 60, 90 and 120 g/L of sucrose.
Supplementary Table 1—Root growth (mm) and isoflavone content (mg/g dry plants) on T. pratense seedlings grown in medium with elicitors at different concentrations during the first seven days after germination.
Supplementary Table 2—Isoflavones and total isoflavones content of the T. pratense HR lines 1HR, 2HR, 4HR and 6HR during the first 5 months of culture.
Supplementary Table 3—Effect of elicitors on the accumulation of major and total isoflavones (mg/g dry plant) in two T. pratense hairy roots line.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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