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United States Department of Agriculture

Agricultural Research Service

Research Project: Improvement and Utilization of Natural Rubber- and Castor Oil-producing Industrial Crops

Location: Bioproducts Research

Title: Effective elimination of chimeric tissue in transgenics for the stable genetic transformation of lesquerella fendleri

Author
item Chen, Grace

Submitted to: HortScience
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 2, 2010
Publication Date: January 2, 2011
Citation: Chen, G.Q. 2011. Effective Elimination of Chimeric Tissue in Transgenics for the Stable Genetic Transformation of Lesquerella fendleri. HortScience. 46(1):86-90.

Interpretive Summary: Lesquerella fendleri (Brassicaceae), under development as a new industrial oilseed crop in the southwestern region of the United States, is valued for its unusual HFA in seed. Considerable efforts have been made to improve the agronomics of L. fendleri through plant breeding. Genetic transformation, on the other hand, provides an alternative means to improve this crop. This manuscript describes a transformation system for generation of stable transgenic plants. The author utilized a ß-glucuronidase gene that confers blue color in transgenic cells for visual screening and documenting of chimeras. The results provide a general picture of the actual frequency and causes of chimera generation. To eliminate the chimeras, an efficient protocol was developed for purifying isogenic transformants through multiple rounds of shoot regeneration processes. Based on the fact that the transgenes were inherited to the second generation of the transgenic lines, the transformation system reported provides a useful means to achieving stable genetic transformation in L. fendleri.

Technical Abstract: In order to improve the potential of Lesquerella fendleri as a valuable industrial oilseed crop, a stable genetic transformation system was developed. Genetic transformation was performed by inoculating leaf segments with an Agrobacterium tumefaciens strain AGL1 carrying binary vector pCAMBIA 1301.1, which contains a ß-glucuronidase gene as a reporter gene and a hygromycine phosphotransferase II as a selection marker gene. Primary shoots were regenerated from the leaf segments on the half strength Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine, 1-naphthaleneacetic acid and hygromycin. The frequency of primary shoot generation was between 22.5% and 60%, and 81.1 - 89.3% of them were chimeras. The high frequency of chimeras was probably due to efficient protection from the hygromycin of non-transformed cells by adjacent transformed ones. The non-transformed cells were removed by multiple rounds of successive shoot regenerations. The purified isogenic shoots were sub-cultured and roots were induced on the MS medium plus indole-3-butyric acid. Most of the plantlets were able to establish roots and acclimate successfully in the greenhouse. The insertion of the hptII gene was confirmed by segregation analysis in T1 seeds, and the stable inheritance of the transgenes was demonstrated by characterization transgenic lines up to T2 generation. This transformation system can be used to obtain stable transgenic lines for genetic engineering of L. fendleri.

Last Modified: 12/22/2014
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