Efficient selection and evaluation of transgenic lines of Crambe abyssinica

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.

Down‐regulation of crambe fatty acid desaturase and elongase in Arabidopsis and crambe resulted in significantly increased oleic acid content in seed oil

“…Residual genomic DNA was removed through DNase treatment using TURBO DNA-free (Ambion, Austin, TX). First-strand cDNA was synthesized according to Li et al (2013) using RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany). The cDNA was diluted 4 x, and 2 lL was used for 20-lL qRT-PCR using Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA) with Platinum SYBR Green qPCR SuperMix-UDG module (Invitrogen, Carlsbad, CA).…”

Evaluation on the effectiveness of 2-deoxyglucose-6-phosphate phosphatase (DOGR1) gene as a selectable marker for oil palm (Elaeis guineensis Jacq.) embryogenic calli transformation mediated by Agrobacterium tumefaciens

“…Later it was reported that adding various thiol compounds in the co-cultivation medium significantly increased the transformation efficiency of soybean (Olhoft and Somers, 2001; Olhoft et al, 2001; Zeng et al, 2004). In crambe, ( Crambe abyssinica ), it was reported that the transformation efficiency was similar when transformants were exposed to either constant low selection pressure (continuously on 3 mg/l hygromycin) or exposed to higher concentration and followed by lower selection concentration (started by 10 mg/l and followed by 3 mg/l of hygromycin; Li et al, 2013). This observation is in contradiction to many reports where higher transformation efficiency is obtained by gradually increasing the selection agent from low to higher concentration.…”

Selectivity of post-emergence herbicides to crambe

“…Crambe has some good agronomic properties, such as potential for phytoremediation of heavy metal contaminated soils and sediments (Chhikara et al, 2012) and tolerance to abiotic stress such as reduction of soil nematodes, which make it easily accepted by commercial production after improvement (Li et al, 2013). The weed control on crambe is important, such as in any other agricultural species, for the success of the activity.…”