Abstract
Crambe abyssinica is a dedicated oilseed crop suitable for production of industrial feedstocks. Genetic modification of crambe has progressed substantially in the last few years, but the transformation efficiency needs to be further improved. Meanwhile, developing a reliable molecular system including Southern blot and qRT-PCR analyses is desired for effectively evaluating transgenic lines and gene expression levels of both endogenous and transgenes. In this study, we have developed an efficient transformation protocol with hygromycin as the selective agent for crambe transformation. In the regeneration test, addition of hygromycin at concentration of 5 mg L−1 resulted in 18% of shoot regeneration using crambe hypocotyls as explants, while no regeneration occurred when the hygromycin concentration reached 10 mg L−1. Based on this result, the hygromycin concentration up to 10 mg L−1 was used in the subsequent transformations. The results showed that the transformation efficiency under constant low selection pressure (H3-H3) was similar to that under higher selection pressure first, followed by transfer to lower selection pressure (H10-H3). The PCR, Southern blot and fatty acid composition analyses confirmed the integration of transgenes in the crambe genome. We have also optimized the Southern and qRT-PCR methods for future studies on crambe or related species. For Southern blot analysis on crambe, more than 50 μg DNA is required for a clear band. The choice of enzymes for DNA digestion was not rigid for confirmation of the T-DNA integration, while for determining the copy number of transgenes, suitable enzymes should be chosen. Increasing the enzyme concentration could improve the digestion and 20 μl enzyme was recomended for a complete digestion of up to 80 μg crambe DNA. For qRT-PCR analysis, around 20 days after flowering was observed to be the suitable sampling time for expresseion analysis of genes invovled in the seed oil biosynthesis.
Highlights
Crambe abyssinica is a dedicated oilseed crop for producing industrial feedstocks as it does not outcross with any food oil crops that are currently in commercial production, and eliminating the problem of gene flow (Li et al, 2012)
A wide range of hygromycin concentrations have been used for transformation of various plant species, ranging from 3 mg L−1 for rapeseed (Liu et al, 2011; Zhang et al, 2011) to 100 mg L−1 for onion (Eady and Lister, 1998)
Hygromycin at 5 mg L−1 could already significantly reduce the regeneration frequency (18%) of crambe hypocotyls compared to 94% when no hygromycin was added in the medium
Summary
Crambe abyssinica is a dedicated oilseed crop for producing industrial feedstocks as it does not outcross with any food oil crops that are currently in commercial production, and eliminating the problem of gene flow (Li et al, 2012). Li et al (2010, 2011) firstly reported a regeneration protocol with over 95% of regeneration frequency and a transformation protocol with 2.1% of transformation frequency using hypocotyls as explants and kanamycin as selective agent Using this transformation protocol, Li et al (2012) successfully transformed three target genes into crambe for increasing erucic acid levels in the seed oil and obtained a transgenic line with 73% of erucic acid content in average compared with 60% in the wild type. Chhikara et al (2012) reported another protocol with 50–70% regeneration and 6.7–8.3% transformation frequency using hypocotyls as explants but hygromycin as selective agent These achievements have demonstrated the feasibility of developing crambe into a bio-platform for industrial feedstock production. Further improvement of transformation efficiency and optimization of molecular methods for efficient evaluation of transgenic lines are highly desirable
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