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Morphometric and biochemical characterization of red beet (Beta vulgaris L.) hairy roots obtained after single and double transformations

  • Genetic Transformation and Hybridization
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Abstract

It is known that T-DNA of Agrobacterium rhizogenes affects processes of plant development and activates the synthesis of secondary metabolites in transformed plant cells. In the present investigation, we provide evidence that different strains of A. rhizogenes significantly affect morphometric, morphological and functional characteristics of hairy roots of red beet (Beta vulgaris L.). Infection with four strains of A. rhizogenes (A4, A 2/83, A 20/83 and LMG-150) resulted in ten clones of hairy roots, which were named accordingly as A4(1), A4(2), A4(3), A 2/83(1), A 2/83(2), A 2/83(3), A 20/83(1), A 20/83(2), A 20/83(3) and LMG-150. Their growth characteristics, pigment content, levels of endogenous auxin and T-DNA copy number showed significant differences probably due to the physiological status of the host cell rather than the T-DNA copy number. Although A 2/83 showed highest hairy root induction capacity, the best hairy root clone was obtained with strain LMG-150 that produced highest biomass and pigments. In this root clone, the enzyme peroxidase was found involved in altering the endogenous auxin pool. When root clone LMG-150 was re-transformed to insert additional individual rol genes, two double transformed clones were obtained, one for rolABC and the other for rolC gene where the former produced higher biomass and betalaine than the latter. Despite the established fact that rol genes of T-DNA influence endogenous phytohormones, no direct correlation among the single transformants and the double transformants was found. This is the first report, in our knowledge, where a hairy root clone has been used to obtain double transformants.

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References

  • Abeysekere M, Sampathu SR, Shankaracharya ML (1990) Studies on different methods of extraction of betalaines from red beet (Beta vulgaris). J Food Sci Technol 27:336–339

    CAS  Google Scholar 

  • Agostini E, Milrad de Forchetti S, Tigier HA (1997) Production of peroxidases by hairy roots of Brassica napus. Plant Cell Tissue Organ Cult 47:177–182

    Article  Google Scholar 

  • Agostini E, Hernández-Ruiz J, Arnao MB, Milrad SR, Tigier HA, Acosta MA (2002) Peroxidase isoenzyme secreted by turnip (Brassica napus) hairy-root cultures: inactivation by hydrogen peroxide and application in diagnostic kits. Biotechnol Appl Biochem 35:1–7

    Article  PubMed  CAS  Google Scholar 

  • Bais HP, George J, Ravishankar GA (1999) Influence of polyamines on growth and production of coumarins in hairy root cultures of Cichorium intybus L. cv Lucknow local (Witloof chicory). J Plant Growth Reg 18:33–37

    Article  CAS  Google Scholar 

  • Bais HP, Sudha G, George J, Ravishankar GA (2001) Influence of exogenous hormones on growth and secondary metabolite production in hairy root cultures of Cichorium intybus L. cv. Lucknow local. In Vitro Cell Dev Biol Plant 37:293–299

    CAS  Google Scholar 

  • Barbier-Brygoo H, Maurel C, Shen WH, Ephritikhine G, Delbarre A, Guern J (1990) Use of mutants and transformed plants to study the action of auxins. In: Roberts JA, Kirk C, Venis M (eds) Hormone perception and signal transduction in animals and plants. Company of Biologists, Cambridge, pp 66–77

    Google Scholar 

  • Bhagyalakshmi N, Thimmaraju R, Narayan MS (2004) Various hexoses and di-hexoses differently influence growth, morphology and pigment synthesis in transformed root cultures of red beet (Beta vulgaris). Plant Cell Tiss Org Cult 78:183–195

    Article  CAS  Google Scholar 

  • Bonhomme V, Laurain Mattar D, Fliniaux MA (2000) Effects of the rolC gene on hairy root: induction development and tropane alkaloid production by Atropa belladonna. J Nat Prod 63:1249–1252

    Article  PubMed  CAS  Google Scholar 

  • Bulgakov VP, Khodakovskaya MV, Labetskaya NV, Tchernoded GK, Zhuravlev YN (1998) The impact of plant rolC oncogene on ginsenoside production by ginseng hairy root cultures. Phytochemistry 49:1929–1934

    Article  CAS  Google Scholar 

  • Bulgakov VP, Tchernoded GK, Mischenko NP, Shkryl Yu N, Fedoreyev SA, Zhuravlev Yu N (2004) The rolB and rolC genes activate synthesis of anthraquinones in Rubia cordifolia cells by mechanism independent of octadecanoid signaling pathway. Plant Sci 166:1069–1075

    Article  CAS  Google Scholar 

  • Camilleri C, Jouanin L (1991) The Tr-DNA region carrying the auxin synthesis genes of the Agrobacterium rhizogenes agropine -type plasmid pRiA4: nucleotide sequence analysis and introduction in to tobacco plants. Mol Plant Microbe Interact 4:155–162

    PubMed  CAS  Google Scholar 

  • Cardarelli M, Mariotti D, Pomponi M, Spano L, Capone I, Constantino P (1987) Agrobacterium rhizogenes T-DNA genes capable of inducing hairy root phenotype. Mol Gen Genet 209:475–480

    Article  PubMed  CAS  Google Scholar 

  • Carrizo CN, Pitta-Alvarez SI, Kogan MJ, Giulietti AM, Tomaro ML (2001) Occurrence of cadaverine in hairy roots of Brugmansia candida. Phytochemistry 57:759–763

    Article  PubMed  CAS  Google Scholar 

  • Delbarre P, Muller V, Imhoff H, Barbier-Brygoo C, Maurel N, Lebanc C, Perrot-Rechenmann GJ (1994) The rolB gene of Agrobacterium rhizogenes does not increase the auxin sensitivity of tobacco protoplast by modifying the intracellular auxin concentration. Plant Physiol 105:563–569

    PubMed  CAS  Google Scholar 

  • Doran PM (2002) Properties and applications of hairy root cultures. In: Oksman- Caldentey KM, Barz WH (eds) Plant biotechnology and transgenic plants. Mercel Dekker Inc, New York, pp 143–162

    Google Scholar 

  • Estruch JJ, Chriqui D, Grossman K, Schell J, Spena A (1991a) The plant oncogene rolC is responsible for the release of cytokinins from glucoside conjugates. EMBO J 10:2889–2895

    PubMed  CAS  Google Scholar 

  • Estruch JJ, Schell J, Spena A (1991b) The protein encoded by the rolB plant oncogene hydrolyses indole glucosides. EMBO J 10:3125–3128

    PubMed  CAS  Google Scholar 

  • Faiss M, Strnad M, Redig P, Doležal K, Hanuš J, Van Onckelen H, Schmülling T (1996) Chemically induced expression of the rolC-encoded β-glucosidase in transgenic tobacco plants and analysis of cytokinin metabolism: rolC does not hydrolyze endogenous cytokinin glucosides in planta. Plant J 10:33–46

    Article  CAS  Google Scholar 

  • Harter LN (1960) Critical values for Duncan’s new multiple range test. Biometrics 16:671–685

    Article  Google Scholar 

  • Inze D, Follin A, van Lijsebettens M (1984) Genetic analysis of individual T-DNA genes of Agrobacterium tumefaciens; further evidence that two genes are involved in indole-3-acetic acid synthesis. Mol Gen Genet 194:265–274

    Article  CAS  Google Scholar 

  • Knegt E, Bruinsma J (1973) Rapid sensitive and accurate determination of indolyl -3-acetic acid. Phytochemistry 12:753

    Article  CAS  Google Scholar 

  • Lacroix B, Tzfira T, Vainstein A, Citovsky V (2006) A case of promiscuity: Agrobacterium’s endless hunt for new partners. Trends Genet 22:29–37

    Article  PubMed  CAS  Google Scholar 

  • Lagrimini LM (1996) The role of the tobacco anionic peroxidase in growth and development. In: Obinger C, Burner U, Ebermann R, Penel C, Greppin H (eds) Plant peroxidases, biochemistry and physiology. University of Geneva, Geneva, pp 235–242

    Google Scholar 

  • Lemcke K, Schmulling T (1998) Putative rolB gene homologue of Agrobacterium rhizogenes TL-DNA that alter plant morphogenesis or hormone sensitivity. Plant J 15:423–434

    Article  PubMed  CAS  Google Scholar 

  • Liu ZH, Liu HY, Wang HY (1996) Effect of light on endogenous indole-3-acetic acid, peroxidase and Indole-3-acetic acid oxidase in soybean hypocotyls. Bot Bull Acad Sin 37:113–119

    CAS  Google Scholar 

  • Maurel S, Barbier-Bryqoo H, Spena A, Tempe G, Guern G (1991) Single rol genes from the Agrobacterium rhizogenes T(L)-DNA alter some of the cellular responses to auxin in Nicotiana tobacum. Plant Physiol 97:212–216

    PubMed  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Nilsson T (1970) Studies in to the pigments in beet root (Beta vulgaris L. sp. vulgaris var. rubra L.). Lantbrukshogsk Ann 36:179–219

    CAS  Google Scholar 

  • Palazón J, Cusidó RM, Roig C, Piñol MT (1998a) Expression of the rolC gene and nicotine production in transgenic roots and their regenerated plants. Plant Cell Rep 17:384–390

    Article  Google Scholar 

  • Palazón J, Cusidó RM, Gonzalo J, Bonfill M, Morales S, Piñol MT (1998b) Relation between the amount the rolC gene product and indole alkaloid accumulation in Catharantus roseus transformed root cultures. J Plant Physiol 153:712–718

    Google Scholar 

  • Prabha TN, Bhagyalakshmi N (1998) Carbohydrate metabolism in ripening banana fruit. Phytochemistry 48:915–919

    Article  CAS  Google Scholar 

  • Pressey R (1990) Anions activate the oxidation of indoleacetic acid by peroxidases from tomato and other sources. Plant Physiol 93:798–804

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. In: Ford N, Nolan C, Ferguson M, Ockler M (eds) Cold Spring Harbor laboratory Press, New York, pp 1.3–7.8

  • Savitha BC, Thimmaraju R, Bhagyalakshmi N, Ravishankar GA (2006) Different biotic and abiotic elicitors influence betalaine production in hairy root cultures of Beta vulgaris in shake flask and bioreactor. Process Biochem 41:50–60

    Article  CAS  Google Scholar 

  • Schmulling T, Schell J, Spena A (1988) Single genes from Agrobacterium rhizogenes influence plant development. EMBO J 7:2621–2629

    PubMed  CAS  Google Scholar 

  • Schmulling T, Schell J, Spena A (1989) Promoters of the rolA, B and C genes of Agrobacterium rhizogenes are differentially regulated in transgenic plants. Plant Cell 1:665–670

    Article  PubMed  CAS  Google Scholar 

  • Schmulling T, Fladung M, Grossman K, Shell J (1993) Hormonal content and sensitivity of transgenic tobacco and potato plants expressing single rol genes of A. rhizogenes T-DNA. Plant J 3:371–382

    Google Scholar 

  • Sedira M, Butler E, Gallagherb T, Welandera M (2005) Verification of auxin-induced gene expression during adventitious rooting in rolB-transformed and untransformed apple. Plant Sci 168:1193–1198

    Article  CAS  Google Scholar 

  • Shanks JV, Bhadra R (1997) Characteristics of selected hairy root lines of Catharanthus roseus. In: Doran PM (ed) Hairy roots: culture and applications. Harwood academic Publishers, pp 51–65

  • Shkryl YN, Veremeichik1 NG, Bulgakov PV, Tchernoded GK, Mischenko NP, Fedoreyev SA, Zhuravlev YN (2008) Individual and combined effects of the rolA, B and C genes on anthraquinone production in Rubia cordifolia transformed calli. Biotechnol Bioeng. doi:10.1002/bit. 21727

  • Stossel A, Venis MA (1970) Determination of sub-microgram levels of indole-3-acetic acid: a new highly specific method. Anal Biochem 34:344

    Article  Google Scholar 

  • Spena A, Schmülling T, Koncz C, Schell JS (1987) Independent and synergistic activity of rol A, B and C loci in stimulating abnormal growth in plants. EMBO J 6:3891–3899

    PubMed  CAS  Google Scholar 

  • Sudha CG, Obul Reddy B, Ravishankar GA, Seeni S (2003) Production of ajmalicine and ajmaline in hairy root cultures of Rauvolfia micrantha Hook, a rare and endemic medicinal plant. Biotechnol Lett 25:631–636

    Article  PubMed  CAS  Google Scholar 

  • Tanaka N, Fuzikawa Y, Aly MAM, Sanoeka H, Fujita K, Yamashita I (2001) Proliferation and rol gene expression in hairy root lines of Egyptian clover. Plant Cell Tissue Organ Cult 66:175–182

    Article  CAS  Google Scholar 

  • Thimmaraju R, Bhagyalakshmi N, Ravishankar GA (2004) In situ and ex situ adsorption and recovery of betalaines from hairy root cultures of Beta vulgaris. Biotechnol Prog 20:777–785

    Article  CAS  Google Scholar 

  • Thimmaraju R, Bhagyalaskshmi N, Narayan MS, Ravishankar GA (2003a) Kinetics of pigment release from hairy root cultures of red beet under the influence of pH, sonication, temperature and oxygen stress. Process Biochem 38:1067–1074

    Article  CAS  Google Scholar 

  • Thimmaraju R, Bhagyalaskshmi N, Narayan MS, Ravishankar GA (2003b) Food grade chemical and biological agents permeabilize red beet hairy roots assisting the release of betalaines. Biotechnol Prog 19:1274–1282

    Article  PubMed  CAS  Google Scholar 

  • Thimmaraju R, Vinod Kumar, Venkatachalam L, Bhagyalakshmi N, Ravishankar GA (2005) Peroxidase production from hairy root cultures of red beet (Beta vulgaris). Electron J Biotechnol 8:185–196

    Article  Google Scholar 

  • Wang JW, Tan RX (2002) Artemisin production in Artemisia annua hairy root cultures with improved growth by altering the nitrogen source in the medium. Biotechnol Lett 24:1153–1156

    Article  CAS  Google Scholar 

  • Zhang L, Ding R, Chai Y, Bonfill M, Moyano E, Oksman-Caldentey KM, Xu T, Pi Y, Wang Z, Zhang H, Kai G, Liao Z, Sun X, Tang K (2004) Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures. Proc Natl Acad Sci USA 101:6786–6791

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

First and the second author are grateful to CSIR, India for financial support in terms of Senior Research Fellowships. The authors thankfully acknowledge the Director, CFTRI and the Head, Plant Cell Biotechnology Department for their constant support and encouragement during the research work.

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  1. R. Thimmaraju

    Present address: Department of Plant and Soil Sciences, University of Delaware, Newark, DE, 19711, USA

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  1. Plant Cell Biotechnology Department, Central Food Technological Research Institute, Mysore, 570 020, India

    R. Thimmaraju, L. Venkatachalam & N. Bhagyalakshmi

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  1. R. Thimmaraju
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Correspondence to N. Bhagyalakshmi.

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Communicated by P. Kumar.

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Thimmaraju, R., Venkatachalam, L. & Bhagyalakshmi, N. Morphometric and biochemical characterization of red beet (Beta vulgaris L.) hairy roots obtained after single and double transformations. Plant Cell Rep 27, 1039–1052 (2008). https://doi.org/10.1007/s00299-008-0527-8

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