Stability of transgene integration and expression in subsequent generations of doubled haploid oilseed rape transformed with chitinase and β-1,3-glucanase genes in a double-gene construct

Abstract

A double-gene construct with one chitinase and one beta-1,3-glucanase gene from barley, both driven by enhanced 35S promoters, was transformed into oilseed rape. From six primary transformants expressing both transgenes 10 doubled haploid lines were produced and studied for five generations. The number of inserted copies for both the genes was determined by Southern blotting and real-time PCR with full agreement between the two methods. When copy numbers were analysed in different generations, discrepancies were found, indicating that at least part of the inserted sequences were lost in one of the alleles of some doubled haploids. Chitinase and beta-1,3-glucanase expression was analysed by Western blotting in all five doubled haploid generations. Despite that both the genes were present on the same T-DNA and directed by the same promoter their expression pattern between generations was different. The beta-1,3-glucanase was expressed at high and stable levels in all generations, while the chitinase displayed lower expression that varied between generations. The transgenic plants did not show any major impact on fungal resistance when assayed in greenhouse, although purified beta-1,3-glucanase and chitinase caused retardment of fungal growth in vitro.

Transgenic Brassica napus L. lines carrying a two gene construct demonstrate enhanced resistance against Plutella xylostella and Sclerotinia sclerotiorum

“…The transgene crops with chitinase also have high-level resistance to fungus (Broglie et al 1991;Corrado et al 2008;Grison et al 1996;Moravčíková et al 2004;Tohidfar et al 2005). Moreover, introducing two combined genes from different families can act synergistically with improved control of fungal diseases (Melander et al 2006;Sridevi et al 2008). Different kinds of single or combinational foreign genes are now being introduced into plants to enhance their resistance against insects and fungal diseases.…”

Assessment of inheritance pattern and agronomic performance of transgenic rapeseed having harpinXooc-encoding hrf2 Gene

“…One to two weeks after selection, calluses with small shoots were observed. Shoots, 2-3 cm in length, were separated and transferred to the MS medium for rooting, and then transplanted into a field for maturation (Melander et al 2006). The T 1 generation of the transgenic rapeseed was produced in the field by self-pollination of the regenerated plants, and untransformed, seed-derived plants were grown as control plants.…”

“…The advances in genetic engineering have lead to improvement in production, quality and resistance to diseases and pests. Transgenic lines of rapeseed have been evaluated for seed quality (Ponstein et al 2002;Wu et al 2008) and resistance to fungi (Grison et al 1996;Melander et al 2006;Dong et al 2008), viruses (Lu et al 1996) and insects (Stewart et al 1996;Å hman et al 2006). Transgenic rapeseeds with herbicide resistance are grown commercially in Canada and the USA.…”

Identification and antifungal assay of a wheat β-1,3-glucanase

“…Several b-1,3-glucanases and chitinases inhibit the growth of phytopathogenic fungi in vitro (Jin et al 2007; Kirubakaran and Sakthivel 2007). Transgenic plants overexpressing chitinase and b-1,3-glucanase genes show enhanced resistance against fungal pathogens (Melander et al 2006;Mackintosh et al 2007).…”