Chloroplast transformation of rapeseed (Brassica napus) by particle bombardment of cotyledons

Abstract

A protocol for chloroplast transformation of an elite rapeseed cultivar (Brassica napus L.) was developed based on optimized conditions for callus induction and regeneration from cotyledonary tissues. Comparison of six different media with three elite cultivars showed that B5 medium plus 3 mg/l AgNO(3) supplemented with 0.6 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l 6-furfurylaminopurine was optimal for callus formation and maintenance without differentiation, while the medium suitable for regeneration was B5 medium supplemented with 1 mg/l 6-benzylaminopurine, 1 mg/l 6-furfurylaminopurine and 0.5 mg/l alpha-naphthaleneacetic acid. A rapeseed-specific chloroplast transformation vector was constructed with the trnI and trnA sequences amplified from the rapeseed chloroplast genome using two primers designed according to Arabidopsis homologs. The aadA gene was used as a selection marker regulated by the ribosome-binding site from the bacteriophage T7 gene 10L, the tobacco 16S rRNA promoter and the psbA terminator. After bombardment, cotyledonary segments were cultured for callus formation on media containing 10 mg/l spectinomycin and regeneration was carried out on medium with 20 mg/l spectinomycin. Heteroplasmic plastid transformants were isolated. An overall efficiency for the chloroplast transformation was one transplastomic plant per four bombarded plates. Southern blot analyses demonstrated proper integration of the target sequence into the rapeseed chloroplast genome via homologous recombination. The expression of the aadA gene was confirmed by Northern blot analysis. Analysis of T1 transplastomic plants revealed that the transgenes integrated into the chloroplast were inheritable with a ratio of about 8%. These results suggest that rapeseed may be a suitable crop for chloroplast transformation with cotyledons as explants under appropriate conditions.

In vitro transformation of pearl millet (Pennisetum glaucum (L). R. BR.): Selection of chlorsulfuron-resistant plants and long term expression of the gus gene under the control of the emu promoter

“…These results indicate that, at least up to three months after bombardment, some plasmids remained free, that is, not integrated in the genomic DNA of the transformed cells. Free plasmid DNA has also been observed in transformed cells of plants such as wheat, barley, maize and Colza (Langridge et al, 1992;Cheng et al, 2004Cheng et al, , 2010. Langridge et al (1992) stated, first, that endophytic microorganisms, such as mycoplasma associated with cereals cells, are responsible for the free plasmid-DNA observed in transgenic cells.…”

“…Langridge et al (1992) stated, first, that endophytic microorganisms, such as mycoplasma associated with cereals cells, are responsible for the free plasmid-DNA observed in transgenic cells. Then, Cheng et al (2004Cheng et al ( , 2010 indicated that the presence of free plasmid DNA may be due to the fact that the stable transformation of plant cells is progressive and, cell organelles such as chloroplasts and mitochondria are also transformed. So, in these organelles which are very close to the prokaryotes, the plasmid DNA can be free for a long time before being digested by plant endonucleases.…”

Recent achievements obtained by chloroplast transformation

“…Tobacco was the first higher plant in which chloroplast transformation was successfully performed [31, 32]. Similarly, a protocol for plastid transformation of an elite rapeseed cultivar ( Brassica napus L.) has been developed [33]. …”

Efficient Plastid Transformation in Arabidopsis

“…Plastid transformation in cauliflower (Brassica oleracea var botrytis) has been obtained at a very low frequency (Nugent et al, 2006). Plastid transformation in B. napus also has been obtained, but no homoplastomic plants could be obtained (Hou et al, 2003;Cheng et al, 2010). Plastid transformation in Lesquerella fendleri, another oilseed crop in the Brassicaceae, was feasible but inefficient (Skarjinskaia et al, 2003).…”