Effective Reduction of Chimeric Tissue in Transgenics for the Stable Genetic Transformation of Lesquerella fendleri

Abstract

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 containing binary vector pCAMBIA 1301.1, which contains a β-glucuronidase gene as a reporter gene and 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% to 89.3% of these shoots were chimeras. The high frequency of chimeras was probably the result of 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 subcultured 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 the characterization transgenic lines through T2 generation. This transformation system can be used to obtain stable transgenic lines for genetic engineering of L. fendleri.