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Abstract
The goal of this work was to establish a transformation pipeline for upland Curinga rice (Oryza sativa L. ssp. japonica) with bar gene selection employing bialaphos and phosphinothricin as selection agents. The following genes of interest: AtNCED3, Lsi1, GLU2, LEW2, PLD-alpha, DA1, TOR, AVP1, and Rubisco were cloned into the binary vector p7i2x-Ubi and were transferred into Agrobacterium strain EHA 105. Embryogenic calli derived from the mature embryos were transformed, and transgenic cells and shoots were selected on the medium supplemented with bialaphos or phosphinothricin (PPT) using a stepwise selection scheme. Molecular analyses were established using polymerase chain reaction and Southern blot for the bar gene and the NOS terminator. Overall, 273 putative transgenic plants were analyzed by Southern blot with 134 events identified. In total, 77 events had a single copy of the transgene integrated in the plant genome while 29 events had two copies. We tested backbone integration in 101 transgenic plants from all constructs and found 60 transgenic plants having no additional sequence integrated in the plant genome. The bar gene activity was evaluated by the chlorophenol red test and the leaf painting test using phosphinothricin with several transgenic plants. The majority of T0 plants carrying the single copy of transgene produced T1 seeds in the screen house.
Highlights
Rice equates to life for thousands of millions people in Asia alone where more than 2000 million people obtain 60 to 70 % of their calories from rice and its products (FAO 2013)
This study focused on following genes linked to plant stress resistance, plant growth, and yield: The AtNCED3 encodes the key enzyme in the abscisic acid (ABA) biosynthesis via overexpression of 9-cis-epoxycarotenoid dioxygenase in Arabidopsis (Iuchi et al 2001)
The bar gene isolated from Streptomyces hygroscopicus encoding the phosphinotricine acetyltransferase (PAT) enzyme which allows resistance to herbicides containing PPT (Thompson et al 1987, Schomburg and Schomburg 2009) can be used for selection of transgenic plants as initially demonstrated by De Block et al (1989) for tobacco, potato, and tomato plants
Summary
Rice equates to life for thousands of millions people in Asia alone where more than 2000 million people obtain 60 to 70 % of their calories from rice and its products (FAO 2013). In the central part of Brazil (Cerrados), upland rice plays an important role in the cropping system whereby it is first grown after clearing land for pasture (Fageria 2010). The main limiting factors for adopting high-yielding rice varieties is drought and access to nitrogen in drought-prone rainfed rice environments (Fageria 2009). These two traits, drought tolerance and nitrogen-use efficiency, have been of high interest in past experimental research (Campbell et al 1995; Umezawa et al 2006; Serraj et al 2008) and have been reviewed several times in recent years (Hadiarto and Tran L-S 2011; Lawlor 2013). Dozens of genes with different functions and modes of action have been identified (Shinozaki and Yamaguchi-Shinozaki 2007; Yang et al 2010; Jeong et al 2013) with several going through confined field testing (Deikman et al 2011; Gaudin et al 2013)
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