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Enhanced trans-resveratrol production in genetically transformed root cultures of Peanut (Arachis hypogaea L.)

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Abstract

In the present study, 30 Ri-transformed root lines of peanut (Arachis hypogaea) cv. JL-24, a popular Indian cultivar, obtained following infection with Agrobacterium rhizogenes strains LBA9402, A4 and R1000 were selected on the basis of growth index and maintained in vitro for 3 years. The root lines showed high degree of branching and rapid, plagiotropic growth on phytohormone free solid N/5 medium but were devoid of root hairs. Trans-resveratrol was isolated by preparative HPLC from Ri-transformed roots and identified by ESI–MS/MS. Strain independent variability was observed among 30 Ri-transformed root lines on the basis of lateral root density per cm (7.60 ± 0.30 to 4.5 ± 0.5), relative thickness (0.54 ± 0.07 to 1.54 ± 0.1 mm), growth index (9.16 ± 1.1 to 17.79 ± 1.35 FW basis and 10.77 ± 0.95 to 19.46 ± 1.78 DW basis) and trans-resveratrol content 0.27 ± 0.03 (root line R1000-1) to 0.969 ± 0.141 mg g DW−1 (root line RIX-19) in solid N/5 medium, which was 4.1–14 fold greater than in excised non-transformed root cultures (0.06 ± 0.01 mg g DW−1). Optimum growth and productivity in liquid culture was achieved in N/5 medium supplemented with 0.01 % activated charcoal. Root line RIX-19 showed maximum trans-resveratrol accumulation (1.21 ± 0.09 mg g DW−1) and productivity (0.37 ± 0.08 mg per flask), which was 19 fold higher than non-transformed root cultures. This optimized protocol can be utilized for large scale cultivation of transformed root cultures in industrial bioreactors for mass synthesis of trans-resveratrol.

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Acknowledgments

MH is grateful to the University Grants Commission, New Delhi, for the award of Junior (Sanction No.—UGC/1015/Jr. Fellow (Sc.); date-15.10.09) and Senior Research Fellowship (sanction No.—UGC/430/Jr. Fellow; date-16.04.12) and expresses his sincere gratitude to Prof M. Dasgupta, Department of Biochemistry, C.U., for her guidance and encouragement. Thanks are due to Mr. Sudip Saha for his help in HPLC analysis. We also thank the Coordinator, DBT-IPLS, University of Calcutta for providing ESI–MS/MS facilities.

Author contribution

MH and SJ conceived and designed research.MH did all the experiments and analyzed the data. SJ and MH wrote the final manuscript. All authors read and approved the manuscript.

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  1. Department of Botany, Center of Advanced Study, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, West Bengal, 700 019, India

    Mihir Halder & Sumita Jha

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Fig. S1

Expression of Ri T-DNA genes at the transcription level in Ri-transformed root lines by RT- PCR analysis using rolA, rolB, rolC and rolD primers. Lane 1: molecular marker (1000 bp plus DNA ladder), lane 2: positive control (pLJ1), lane 3: negative control (genomic DNA of NTH) lanes 4-17: genomic DNA of Ri-transformed root lines (TIFF 1493 kb)

Fig. S2

Variation in growth of 30 Ri-transformed root lines after 28 days on solid N/5 medium (a) Growth index based on fresh weight; (b) Growth index based on dry weight. Bars with the same letters are not significantly different at p ≤ 0.05 according to ANOVA and DMRT for each data point (TIFF 1454 kb)

Fig. S3

Variation in primary root elongation of 30 Ri-transformed root lines after 28 days. Bars with the same letters are not significantly different at p ≤ 0.05 according to ANOVA and DMRT for each data point (TIFF 1994 kb)

Fig. S4

Growth kinetics of two randomly selected Ri-transformed root lines RIX-23 and RIX-33 cultured on solid N/5 medium showing root tip elongation (mm) per day. All values are the means ± standard deviation of three independent experiments (n = 15) (TIFF 566 kb)

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Halder, M., Jha, S. Enhanced trans-resveratrol production in genetically transformed root cultures of Peanut (Arachis hypogaea L.). Plant Cell Tiss Organ Cult 124, 555–572 (2016). https://doi.org/10.1007/s11240-015-0914-0

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