Effects of antioxidants on Agrobacterium-mediated transformation and accelerated production of transgenic plants of Mexican lime (Citrus aurantifolia Swingle)

Four antioxidants including glycine betaine, glutathione, lipoic acid, and polyvinylpyrrolidone were evaluated to improve transformation efficiency of Mexican lime, a precocious but recalcitrant citrus cultivar to Agrobacterium mediated transformation. Lipoic acid substantially improved the transformation efficiency of Mexican lime by aiding in callus development and improving shoot growth from cut ends of epicotyl segments co-cultivated with Agrobacterium. Glycine betaine was moderately beneficial while glutathione and polyvinylpyrrolidone did not have the ability to improve the transformation efficiency. A bi-functional gus-egfp fusion gene used in the study enabled visual identification of transformants and quantitative analysis of gene expression. We describe an improved protocol that allows regenerated transgenic plants to flower within 20-22 months after in vitro regeneration. This enabled the rapid evaluation of transgenic flowers and fruits. Gene expression levels could not be correlated to copy number as determined using Southern blot analysis. Our improved transformation method facilitates the rapid production and evaluation of transgenic plants, especially regarding the functional analysis of transgenes in citrus.