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.

Keywords: Crambe abyssinica; Southern blotting; genetic transformation; hygromycin selection; qRT-PCR.

Figure 1

Schematic map of the vector used in this study. Horizontal bars under genes or promoters represent the sites of restriction enzymes and the hpt probe used for Southern blot analysis. 1: XhoI; 2: hpt probe; 3: SmaI, XhoI, EcoRV; 4: EcoRI, SmaI, BamHI, XbaI, SalI, BamHI, EcoRV; 5: EcoRV, EcoRI; 6: BamHI; 7: XbaI, EcoRV; 8: EcoRV, EcoRI, HindIII; 9: HindIII, SalI, PstI. LB, left border; hpt, hygromycin gene; NP, napin promoter; RB, right border.

Figure 2

Results of shoot regeneration from hypocotyl explants grown on the regeneration medium supplemented with hygromycin at concentrations of 0 mg L⁻¹ (A), 5 mg L⁻¹ (B), 10 mg L⁻¹ (C), and 20 mg L⁻¹ (D).

Figure 3

PCR and Southern blot results of the putative transgenic lines of crambe. (A) The PCR product size of the hpt gene is 579 bp. Lane 1–10, putative transgenic lines. Lane 11, wild type. M, molecular markers (MassRuler “Express DNA ladder Mix” from Fermentas). (B) Southern blot analysis of genomic DNA digested with BglII and hybridized with the hpt probe. M, DIG labeled molecular markers. Lane 1, wild type. Lane 2–9, transgenic lines.

Figure 4

Southern blot analysis showing different amounts of DNA used for digestion with HindIII and hybridized with the nptII probe according to Li et al. (2012). (A) DNA from transgenic Arabidopsis with the nptII gene, lane 1, 5 μg; lane 2, 10 μg; lane 3, 20 μg; lane 4, 50 μg. (B) DNA from transgenic crambe with the nptII gene, lane 1, 5 μg; lane 2, 10 μg; lane 3, 20 μg; lane 4, 50 μg; lane5, 80 μg; lane 6, 100 μg. M, DIG labeled molecular markers.

Figure 5

Southern blot analysis on DNAs from two transgenic lines with 2 (A) and multiple copies (B) of transgenes respectively, digested with 13 different restriction enzymes and hybridized with the hpt probe. (A,B) From lane 1 to 13: BglII, KpnI, SacI, SalI, SmaI, StuI, HindIII, XhoI, XbaI, PstI, EcoRV, DraI, and BamHI. M, DIG labeled molecular markers. DNA, 80 μg. Enzyme, 20 μl.

Figure 6

Southern blot analysis on DNAs from 3 different transgenic lines (one transgenic line for one enzyme), digested with different amounts of the enzymes. M, DIG labeled molecular markers. Lane 1: PstI, 20 μl; Lane 2: KpnI, 20 μl; Lane 3: PstI, 10 μl; Lane 4: KpnI, 10 μl; Lane 5: BglII, 20 μl; Lane 6: BglII, 10 μl. DNA, 80 μg.

Figure 7

The qRT-PCR results showing the relative expression levels of CaFAE1 And CaFAD2 mRNA during the seed development. D, days after flowering. Data is the average of three biological replicates for each time point.

Figure 8

Crambe seeds at different development stages. The upper panel is the whole seed and lower panel is the cross section of the seeds, but the whole seed with half silique removed at the last two stages. Figures represent days after flowering.