Regeneration and transformation of Crambe abyssinica

BMC Plant Biology

Methodology Article

Bangquan Huang¹  Jan G Schaart²  Richard GF Visser²  Frans A Krens²  Eibertus N Van Loo²  Iris EM Tinnenbroek-Capel²  Weicong Qi³  Jihua Cheng⁴ 

[1] College of Life Science, Hubei University, 430062, Wuhan City, China;Wageningen UR Plant Breeding, Wageningen University and Research Centre, 6700, Wageningen, AJ, the Netherlands;Wageningen UR Plant Breeding, Wageningen University and Research Centre, 6700, Wageningen, AJ, the Netherlands;Biotechnology, Jiangsu Academy of Agriculture Science, 210014, Nanjing City, China;Wageningen UR Plant Breeding, Wageningen University and Research Centre, 6700, Wageningen, AJ, the Netherlands;Wuhan Botanical Garden, Chinese Academy of Sciences, Moshan, 430074, Wuhan, Wuchang, China;

关键词: Crambe;    Oilseed crop;    Propagation;    Regeneration;    Genetic modification;    Meristematic tissue;    Efficiency;   

DOI  :  10.1186/s12870-014-0235-1

 received in 2013-12-13, accepted in 2014-08-28,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundCrambe abyssinica (crambe) is a non-food oil seed crop. Its seed oil is widely used in the chemical industry because of the high erucic acid content. Furthermore, it is a potential platform for various feedstock oils for industrial uses based on genetic modification. Here, we describe the development of a series of protocols for all steps required in the process of generating genetically modified crambe.ResultsDifferent explant types from crambe seedlings were tested for shoot regeneration using different hormone-combinations. Cotyledonary nodes on basic medium with 0.5 μM NAA and 2.2 μM BAP gave the highest regeneration percentages. For propagation by tissue culture, explants of stems, petioles, leaves and axillary buds of in vitro plantlets were tested using the optimized medium. Axillary buds showed the highest shoot proliferation efficiency. Cotyledonary nodes were used to test the proper concentration of kanamycin for selection of transformation events, and 10 to 25 mg · L-1 were identified as effective. The cotyledonary nodes and cotyledons from 7-day-old seedlings were used in Agrobacterium-mediated transformations with two kinds of selection strategies, shifting or consistent. Using the shifting selection method (10 mg · L-1 kanamycin, 25 mg · L-1, then back to 10 mg · L-1) cotyledonary nodes gave 10% transformation frequency, and cotyledons 4%, while with the consistent method (25 mg · L-1) lower frequencies were found, 1% for cotyledonary nodes and 0% for cotyledons). Later, in vitro plant axillary buds were tried as explants for transformation, however, transformation frequency was low ranging from 0.5 to 2%. Overall, testing six different vectors and two kinds of Agrobacterium strains, the average transformation frequency using the shifting method was 4.4%. Determining T-DNA insertion numbers by Southern blotting showed that approximately 50% of the transgenic lines had a single-copy insertion.ConclusionsPresent research revealed the potential of using crambe meristematic tissue for genetic transformation and in vitro propagation. The most efficient method of transformation used cotyledonary node explants from 7-days-old seedlings with a shifting kanamycin selection. Meristematic tissues (cotyledonary node or axillary bud) had the highest ability for shoot proliferation. Single-copy T-DNA insert lines could be efficiently and reproducibly generated.

【 授权许可】

Unknown   
© Qi et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.