Abstract
Agrobacterium rhizogenes A4M70GUS-mediated transformation of two local breeding lines of sugar beet was obtained using 4-week-old seedlings. Root formation efficiency was 61.54% for SBa genotype and 36.36% for SBb genotype. Five highly proliferated hairy root lines have been established in liquid hormone-free MS medium. Transgenic nature of the hairy root clones was evaluated by GUS assay, PCR and RT-PCR analyses. Hairy root-derived calli were induced using different plant growth regulators (PGRs): auxin, auxin/cytokinin and cytokinin. The best callus induction response was achieved on MS medium containing both 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg/l thidiazuron (TDZ). Globular embryo-like structures were observed in friable callus after its prolonged cultivation on MS medium supplemented with TDZ and giberellic acid (GA3) at 1 mg/l each, followed by growth on MS medium containing 1% glucose and 0.5 mg/l 2,3,5-triiodobenzoic acid (TIBA). Histological analysis revealed somatic embryos at different stages of development in hairy root-derived callus of sugar beet.
Similar content being viewed by others
References
Alpizar E, Dechamp E, Lapeyre-Montes F, Guilhaumon C, Bertrand B, Jourdan C, Lashermes P, Etienne H (2008) Agrobacterium rhizogenes-transformed roots of coffee (Coffea arabica): conditions for long-term proliferation, and morphological and molecular characterization. Ann Bot 101:929–940
Altamura MM (2004) Agrobacterium rhizogenes rolB and rolD genes: regulation and involvement in plant development. Plant Cell Tiss Organ Cult 77:89–101
Atanassov AI (1976) Analysis of organogenetic abilities of continuously cultivated somatic tissue of sugar beet on the basis of obtaining single-celled clones and suspension cultures. In: Novak FJ (ed) Use of tissue culture in plant breeding. Institute of Experimental Botany, Olomouc, pp 69–80
Balen B, Leljak-Levanić D, Mihaljevič S, Jelenić S, Jelaska S (2004) Formation of embryogenic callus in hairy roots of pumpkin (Cucurbita pepo L.). In Vitro Cell Dev Bio-Plant 40:182–187
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 Organ Cult 99:305–312
Cabrera-Ponce JL, Vegasgarcia A, Herreraestrella L (1996) Regeneration of transgenic papaya plants via somatic embryogenesis induced by Agrobacterium rhizogenes. In Vitro Cell Dev Biol-Plant 32:86–90
Cai D, Klein M, Kifle S, Harloff H-J, Sandal NN, Marcker KA, Klein-Lankhorst RM, Salentijn EMJ, Lange W, Stiekema WJ, Wyss U, Grundler FMW, Jung C (1997) Positional cloning of a gene for nematode resistance in sugar beet. Science 275:832–834
Christey MC (2001) Use of Ri-mediated transformation for production of transgenic plants. In Vitro Cell Dev Biol Plant 37:687–700
D’Halluin K, Bossut M, Bonne E, Mazur B, Leemans J, Botterman J (1992) Transformation of sugar beet (Beta vulgaris L) and evaluation of herbicide resistance in transgenic plants. Biotechnology 10:309–314
Dicola A, Poma A, Spano L (1997) rolB expression pattern in the early st-ages of carrot somatic embryogenesis. Cell Biol Int 21:595–600
Ehlers U, Commandeur U, Frank R, Landsmann J, Koenig R, Burgermeister W (1991) Cloning of the coat protein gene from beet necrotic yellow vein virus and its expression in sugar beet hairy roots. Theo App Gen 81:777–781
Grierson D, Covey SN (1988) Plant molecular biology, 2nd edn. Chapman and Hall, New York
Gurel E, Gurel S, Lemaux PG (2008) Biotechnology applications for sugar beet. Crit Rev in Plant Sci 27:108–140
Gutierrez-Pesce P, Taylor K, Muleo R, Rugini E (1998) Somatic embryogenesis, shoot regeneration from transgenic roots of the cherry rootstock Colt (Prunus avium, P. pseudocerasus) mediated by pRi 1855 T-DNA of Agrobacterium rhizogenes. Plant Cell Rep 17:574–580
Haccius B (1978) Question of unicellular origin of non-zygotic embryos in callus cultures. Phytomorphology 28:74–81
Hall RD, Riksen-Bruinsma T, Weyens GJ, Rosquin IJ, Denys PN, Evans IJ, Lathouwers JE, Lefebvre MP, Dunwell JM, van Tunen JK, Krens FA (1996) A high efficiency technique for the generation of transgenic sugar beets from stomatal guards cells. Nat Biotechnol 14:1133–1138
Hamill JD, Parr AJ, Robins RJ, Rhodes MJC (1986) Secondary product formation by cultures of Beta vulgaris and Nicotiana tabacum transformed with Agrobacterium rhizogenes. Plant Cell Rep 5:111–114
Hong PI, Chen JT, Chang WC (2008) Promotion of direct somatic embryogenesis of Oncidium by adjusting carbon sources. Biol Plant 52:597–600
Ishizaki T, Hoshino Y, Masuda K, Oosawa K (2002) Explants of Ri-transformed hairy roots of spinach can develop embryogenic calli in the absence of gibberellic acid, an essential growth regulator for induction of embryogenesis from non-transformed roots. Plant Sci 163:223–231
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907
Jheng FY, Do YY, Liauh YW, Chung JP, Huang PL (2006) Enhancement of growth and regeneration efficiency from embryogenic callus cultures of Oncidium “Gower Ramsey” by adjusting carbohydrate sources. Plant Sci 170:1133–1140
Kifle S, Shao M, Jung C, Cai D (1999) An improved transformation protocol for studying gene expression in hairy roots of sugar beet (Beta vulgaris L.). Plant Cell Rep 18:514–519
Kubalakova M (1990) Somatic embryogenesis and cytoplasmic sterility in Beta vulgaris L. var. Saccharifera. Biol Plant 32:414–419
Kulshreshtha S, Coutts RHA (1997) Direct somatic embryogenesis and plant regeneration from mature sugar beet (Beta vulgaris L.) zygotic cotyledons. Plant Growth Reg 22:87–92
Lou H, Kako S (1995) Role of high sugar concentrations in inducing somatic embryogenesis from cucumber cotyledons. Sci Hortic 64:11–20
Mishutkina YV, Gaponenko AK (2006) Sugar beet (Beta vulgaris L.) morphogenesis in vitro: effects of phytohormone type and concentration in the culture medium, type of explants and plant genotype on shoot regeneration frequency. Plant Genet 42:210–218
Moghaddam BE, Mesbah M, Yavari N (2000) The effect of in planta TIBA and proline treatment on somatic embryogenesis of sugar beet (Beta vulgaris L.). Euphytica 112:151–156
Mugnier J (1987) Infection by Polymyxa betae and Plasmodiophora brassicae of roots containing root-inducing transferred DNA of Agrobacterium rhizogenes. Phytopathology 77:539–542
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Paul H, Zijlstra C, Leeuwangh JE, Krens FA, Huizing HJ (1987) Reproduction of the beet cyst nematode Heterodera schachtii Schm. on transformed root cultures of Beta vulgaris L. Plant Cell Rep 6:379–381
Petit A, David C, Dahl GA, Ellis JG, Guyon P (1983) Further extension of the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation. Mol Gen Genet 190:204–214
Piispanen R, Aronen T, Chen X, Saranpaa P, Haggman H (2003) Silver birch (Betula pendula) plants with aux and rol genes show consistent changes in morphology, xylem structure and chemistry. Tree Physiol 23:721–733
Smigocki AC, Ivic-Haymes S, Campbell L, Boetel M (2006) A sugarbeet root maggot (Tetanops myopaeformis Röder) bioassay using Beta vulgaris L. seedlings and in vitro propagated transformed hairy roots. J Sug Beet Res 43:1–13
Snyder GW, Ingersoll JC, Smigocki AC, Owens LD (1999) Introduction of pathogen defense genes and a cytokinin biosynthesis gene into sugar beet (Beta vulgaris L.) by Agrobacterium or particle bombardment. Plant Cell Rep 18:829–834
Strickland GS, Nichol WJ, McCall MC, Stuart AD (1987) Effect of carbohydrate source on alfalfa somatic embryogenesis. Plant Sci 48:113–121
Taya M, Mine K, Kino-Oka M, Tone S, Ichi T (1992) Production and release of pigments by culture of transformed hairy root of red beet. J Ferment Bioeng 73:31–36
Tepfer D (1984) Transformation of several species of higher plants by Agrobacterium rhizogenes: sexual transmission of the transferred genotype and fenotype. Cell 37:959–967
Tepfer M, Casse-Delbart F (1987) Agrobacterium rhizogenes as a vector for transforming higher plants. Microbiol Sci 4:24–28
Tetu T, Sangwan RS, Sangwan-Norreel BS (1987) Hormonal control of organogenesis and somatic embryogenesis in Beta vulgaris callus. J Exp Bot 38:506–517
Thimmaraju R, Venkatachalam L, Bhagyalakshmi N (2008) Morphometric and biochemical characterization of red beet (Beta vulgaris L.) hairy roots obtained after single and double transformation. Plant Cell Rep 27:1039–1052
Tiwwari RK, Trivedi M, Guang ZC, Guo GQ, Zheng GC (2008) Agrobacterium rhizogenes mediated transformation of Scutellaria baicalensis and production of flavonids in hairy roots. Biol Plant 52:26–35
Van Larebeke N, Genetello CH, Hernalsteens JP, De Picker A, Zaenen I, Messens E, Van Montagu M, Shell J (1977) Transfer of Ti plasmid between Agrobacterium strains by mobilization with the conjugative plasmid RP4. Mol Gen Genet 152:1119–1124
Vilaine F, Casse-Delbart F (1987) Independent induction of transformed roots by TL- and TR- regions of the Ri plasmid of agropine type Agrobacterium rhizogenes. Mol Gen Genet 206:17–23
Vinterhalter B, Orbović V, Vinterhalter D (1999) Transgenic root cultures of Gentiana punctata L. Acta Soc Bot Polon 68:275–280
Vinterhalter B, Ninković S, Cingel A, Vinterhalter D (2006) Shoot and root culture of Hypericum perforatum L. transformed with Agrobacterium rhizogenes A4M70GUS. Biol Plant 50:767–770
Yang DC, Choi YE (2000) Production of transgenic plants via Agrobacterium rhizogenes-mediated transformation of Panax ginseng. Plant Cell Rep 19:491–496
Zdravković-Korać S, Ćalić D, Druart PH, Radojević Lj (2003/4) The horse chesnut lines harboring the rol genes. Biol Plant 47:487–491
Zdravković-Korać S, Muhovski Y, Druart P, Ćalić D, Lj Radojević (2004) Agrobacterium rhizogenes-mediated DNA transfer to Aesculus hippocastanum L. and regeneration of transformed plants. Plant Cell Rep 22:698–704
Zhang CL, Chen DF, Elliott MC, Slater A (2001) Thidiazuron-induced organogenesis and somatic embryogenesis in sugar beet (Beta vulgaris L.). In Vitro Cell Dev Biol-Plant 37:305–310
Zhang CL, Chen DF, Kubalakova M, Zhang J, Scott NW, Elliott MC, Slater A (2008) Efficient somatic embryogenesis in sugar beet (Beta vulgaris L.) breeding lines. Plant Cell Tiss Organ Cult 93:209–221
Zhao X, Cao G, Lin R, Sun Y, Li W (1994) A rapid and efficient DNA extraction method of genus Fagopyrum for RAPD analysis. In: Javornik B, Bohanec B, Kreft I (eds) Proceedings of impact of plant biotechnology on agriculture. Biotechnical Faculty, Ljubljana, pp 171–175
Acknowledgments
This work was supported by Ministry of Science and Technology of Serbia, grant No. 143026 B.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ninković, S., Djordjević, T., Vinterhalter, B. et al. Embryogenic responses of Beta vulgaris L. callus induced from transgenic hairy roots. Plant Cell Tiss Organ Cult 103, 81–91 (2010). https://doi.org/10.1007/s11240-010-9757-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-010-9757-x