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- Author or Editor: Grace Q. Chen x
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.
The morphological, physiological, and biochemical changes during seed development of Lesquerella fendleri were investigated from 7 days after pollination (DAP) to desiccation. The entire course of seed development lasted ≈49 days and it can be divided into seven sequential stages (I to VII). During the early stages (I to III, 7 to 21 DAP), seed grew rapidly, showing a dramatic increase in size and fresh weight. They contained ≈75% water. During midmaturation stages (IV to V, 28 to 35 DAP), storage lipids, proteins, and other components of dry weights accumulated at maximum rates. The accumulation curves followed a sigmoidal pattern during seed development. As a result of water loss, fresh weight dropped significantly when seed progressed to late-maturation/desiccation stages (VI to VII, 42 to 49 DAP). The size of the seed decreased slightly and the color changed from green to orange–brown. Seed proteins were also analyzed using SDS-PAGE. Proteins with high molecular weights were prominent in developing seed at early stages (I to III). At Stage IV (28 DAP), proteins with low molecular weight appeared, whereas the high-molecular-weight proteins decreased in proportion. These low-molecular-weight proteins became predominant throughout the remaining stages of seed development. Forty-seven percent of freshly harvested seed at 35 DAP were able to germinate after 7 days incubation. The germination percentage increased to a maximum of 95% at 42 DAP after 7 days incubation. The relationships among seed morphology, reserve synthesis, and germination are discussed.