Abstract
Engineering oilseed crops for industrial purposes requires a suitable crop that does not outcross to any food oilseed crop, thus eliminating problems of gene flow. Crambe abyssinica is such a dedicated crop as it does not hybridize with any existing food oilseed crops. However, lack of regeneration and transformation protocols has limited the use of C. abyssinica in genetic manipulation studies. In this study, efficient regeneration and transformation protocols for Crambe have been developed. Hypocotyls of C. abyssinica cv. Galactica were incubated on a Murashige and Skoog medium supplemented with various plant growth regulators (PGRs). Among the different PGR combinations tested, 10 μM thidiazuron and 2.7 μM α-naphthaleneacetic acid promoted highest frequency of regeneration, up to 60%. Among six Agrobacterium stains evaluated, each harbouring the cloning vector containing the neomycin phosphotransferase (nptII) and β-glucuronidase (gus) genes. EHA101 and AGL-1 yielded the highest transformation frequencies of 1.3 and 2.1%, respectively. Putative transgenic lines were recovered, and confirmed as transgenic by Southern blot analysis. Subsequently, Agrobacterium-mediated transformation of hypocotyls of cv. Galactica with constructs harbouring the wax synthase and fatty acid reductase genes have also successfully recovered confirmed transgenic plants carrying these transgenes.
Similar content being viewed by others
Abbreviations
- BA:
-
6-Benzyladnine
- GA:
-
Gibberellin
- GUS:
-
β-Glucuronidase
- IBA:
-
3-Indole-butyric acid
- NAA:
-
α-Naphthaleneacetic acid
- nptII :
-
Neymycin phosphotransferase
- TDZ:
-
Thidiazuron
References
Aldrich J, Cullis C (1993) RAPD analysis in flax: optimization of yield and reproducibility sing Klen Taq1 DNA polymerase, Chelex 100, and gel purification of genomic DNA. Plant Mol Biol Rep 11:128–141
Babic V, Datla G, Scoles GJ, Keller WA (1998) Development of an efficient Agrobacterium-mediated transformation system for Brassica carinata. Plant Cell Rep 17:183–188
Cardoza V, Stewart CN (2003) Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants. Plant Cell Rep 21:599–604
Gao HB, Wang Y, Gao F, Luo P (1998) Studies on the Plant regeneration from single cell culture of Crambe abyssinica. Hereditas (Beijing) 20(suppl):50–52
Hachey JE, Sharma KK, Moloney MM (1991) Efficient shoot regeneration of Brassica campestris using cotyledon explants cultured in vitro. Plant Cell Rep 9:549–554
Horsch RB, Fry JE, Hoffmann NL, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231
James C (2004) Global status of commercialized biotech/GM crops. ISAAA briefs no 32. ISAAA, Ithaca
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
Khan MR, Rashid H, Ansar M, Chaudry Z (2003) High frequency shoot regeneration and Agrobacterium-mediated DNA transfer in Canola (Brassica napus). Plant Cell Tissue Organ Cult 75:223–231
Lardizabal KD, Metz JG, Sakamoto T, Hutton WC, Pollard MR, Lassner MW (2000) Purification of a Jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic Arabidopsis. Plant Physiol 122:645–655
Macilwain C (2005) Stray seeds had antibiotic-resistant genes. Nature 435:548
Moloney MM, Walker JM, Sharma K (1989) High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Rep 8:238–242
Murashige F, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–492
Murphy DJ (ed) (1994) Designer oil crops. VCH press, Weinheim
Murphy DJ (1996) Engineering oil production in rapeseed and other oil crops. Trends Biotechnol 14:206–213
Murphy DJ (1999) Production of novel oils in plants. Curr Opin Biotechnol 10:175–180
Nath UK, Wilmer JA, Wallington EJ, Becker HC, Möllers C (2009) Increasing erucic acid content through combination of endogenous low polyunsaturated fatty acids alleles with Ld-LPAAT +Bn-fae 1 transgenes in rapeseed (Brassica napus L.). Theor Appl Genet 118:765–773
Poulsen GB (1996) Genetic transformation of Brassica. Plant Breed 115:209–225
Quoirin M, Lepoivre P, Boxus P (1977) Un premier bilan de dix anne′es de recherche sur les cultures de me′riste`mes et la multiplication in vitro de fruitiers ligneux (in French), Compte rendu des recherches, Station des Cultures Fruitie`res et Marrıˆche`res de Gembloux 1976–1977, pp 93–117
Schmidt R, Willmitzer L (1988) High efficiency Agrobacterium-mediated transformation of Arabidopsis leaf and cotyledon explant. Plant Cell Rep 7:583–586
Sharma KK, Bhojwani SS (1990) Histological aspects of in vitro root and shoot differentiation from cotyledon explants of Brassica juncea. Plant Sci 69:207–214
van Miltenburg R, Rüger B, Grünewald-Janho S, Leons M, Schröder C (1995) The DIG system user’s guide for filter hybridization. Boehringer Mannheim GmbH, Biochemica, Germany
Vaughan SP, James DJ, Lindsey K, Massiah AJ (2004) Characterization of FaRB7, a near root-specific gene from strawberry (Fragariaxananassa Duch.) and promoter activity analysis in homologous and heterologous hosts. J Exp Bot 57:3901–3910
Walkey DG (1972) Production of apple plantlets from axillary bud meristems. Can J Plant Sci 52:1085–1087
Wang Y, Peng P (1998) Intergeneric hybridization between Brassica species and Crambe abyssinica. Euphytica 101:1–7
Wiberg E, Edwards P, Byrne B, Stymne S, Dehesh K (2000) The distribution of caprylate, caprate and laurate in lipids from developing and mature seeds of transgenic Brassica napus L. Planta 212:33–40
Zhang S, Zhu LH, Li XY, Ahlman A, Welander M (2005) Infection by Agrobacterium tumefaciens increased the resistance of leaf explants to selective agents in carnation (Dianthus caryophyllus L. and D. chinensis). Plant Sci 168:137–144
Zhu LH, Li XY, Welander M (2008) Overexpression of the Arabidopsis gai gene in the apple genome reduces plant size. Plant Cell Rep 27:289–296
Acknowledgments
We wish to thank Dr. Robert van Loo for providing the seeds of Glactica, Dr. Ed Cahoon for providing the vector pMS9, Dr. John Dyer for providing the pKan-Wax-FAR vector, and Prof. Sten Stymne for his support to the work, SIDA (Swedish Research Link), Dr. Anders Carlsson and Prof. Bangquan Huang for providing financial support to Xiaofeng Yan. This work is part of ICON, a European Commission FP7 project and E.C. is greatly acknowledged for its financial contribution.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Li, X., Ahlman, A., Yan, X. et al. Genetic transformation of the oilseed crop Crambe abyssinica . Plant Cell Tiss Organ Cult 100, 149–156 (2010). https://doi.org/10.1007/s11240-009-9630-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-009-9630-y