An efficient method for transformation of pre-androgenic, isolated Brassica napus microspores involving microprojectile bombardment and Agrobacterium-mediated transformation

Abstract

The physical barrier imposed by the thick microspore wall constitutes an obstacle for an efficient Agrobacterium-mediated transformation of vacuolate microspores prior to androgenic induction and haploid embryogenic commitment. It is thus necessary to implement additional methods to overcome this drawback. In this study, we focused on the optimization of a protocol to allow for the exogenous DNA to enter the microspore in an efficient manner. We tested different options, based on microprojectile bombardment, to be applied prior to agroinfiltration. From them, the best results were obtained through co-transformation by microspore bombardment with DNA-coated microprojectile particles, followed by Agrobacterium tumefaciens infection. This method provides an efficient means to integrate extraneous DNA into rapeseed microspores prior to androgenesis induction.

Haploids and doubled haploids in Brassica spp. for genetic and genomic research

“…Different microspore transformation methods have been attempted in Brassica including Agrobacterium tumefaciens (Pechan 1989;Dormann et al 2001), particle bombardment (Fukuoka et al 1998;Nehlin et al 2000), microinjection (Jones-Villeneuve et al 1995), and electroporation (Guerche et al 1987;Jardinaud et al 1993). A recent paper combined particle bombardment and Agrobacterium-mediated transformation in B. napus microspores (Abdollahi et al 2009a). The integrated system of bombarding DNA coated particles followed by Agrobacterium was more effective with regards to generating GUS positive microspores, however, no embryos were produced.…”

Genetic transformation of Gentiana dahurica Fisch by Agrobacterium tumefaciens using zygotic embryo-derived callus

“…Histochemical GUS assays were conducted according to Jefferson's standard protocol (Jefferson et al 1987;Abdollahi et al 2009;Yasmeen 2009). These transgenic plantlets were used for transient GUS expression assays after 3 days of co-cultivation.…”

Improved Agrobacterium-mediated transformation and high efficiency of root formation from hypocotyl meristem of spring Brassica napus ‘Precocity’ cultivar

“…Plant transformation technologies facilitate integration of a gene into the plant genome, followed by regeneration of the entire plant. Various techniques have been used for transformation in plants, including electroporation, protoplast transfection, and microspore transfection (Hu et al, 1999;Bhalla and Singh, 2008;Abdollahi et al, 2009). However, the Agrobacterium-mediated transformation approach is considered to be superior to other methods owing to its ease of application and low cost.…”