Development of a haploid transformation system and overexpression of Phytochrome B gene in Brassica napus L.

Abstract

Oilseed rape (Brassica napus L.) is a renowned crop worldwide for its edible oil as well as a source of protein animal feed. Agrobacterium-mediated transformation has proved to work efficient for genetic engineering of this crop. The majority of successful transformation systems hitherto described for B. napus use diploid plant materials such as hypocotyls, cotyledons, and cotyledonary petioles. When diploid plant materials were used, first generation transgenic plants (T1) would be hemizygous for the transformed gene(s). Consequently, much time and labour has to be invested to produce homozygous transgenic plants by repeated selfing or by applying the doubled haploid technology. The use of haploid cells and tissue explants could provide an alternative approach for efficient transformation of oilseed rape, since haploid transgenic plants will become homozygous in one step after colchicine treatment. This study reports the potential of using leaf and petiole explants of haploid plants propagated in vitro for an efficient Agrobacterium-mediated gene transfer. Compared to a standard protocol for hypocotyl segments, significantly higher callus production (P=0.05) with leaf explants was achieved when cultured in callus induction medium CIM-L (MS basal medium supplemented with 1 mg/l BAP, 0.1 mg/l NAA, 0.1 mg/l GA3, 5 mg/l AgNO3 and solidified with 5 g/l Agarose). For petiole explants CIM-P medium (MS basal supplemented with 2 mg/l BAP, 0.01 mg/l picloram, 5 mg/l AgNO3 and solidified with 5 g/l Agarose) had a significant positive effect (P=0.05) on callus production. Highest shoot regeneration was achieved in DKW medium supplemented with 1 mg/l BAP, 0.1 mg/l NAA, 0.1 mg/l GA3, and 5 mg/l AgNO3 and solidified with 5 g/l Agarose. Analysis of the ploidy showed that 96% of plantlets regenerated from leaf and 92% from petiole explants remained haploid. Using Agrobacterium tumefaciens strain AGL0, which carried the binary plasmid pAK-UGI 9-1, a transformation efficiency of 1.8% and 2.9% was achieved for leaf and petiole explants, respectively. Presence of GUS gene in regenerated plantlets was confirmed by PCR. Results indicate that transformation of leaf and petiole explants derived from in vitro propagated haploid plants provides a suitable alternative for generation of homozygous transgenic plants in rapeseed. Lodging could be a serious problem in oilseed Brassica napus. Lodging could be decreased by reducing plant height. This study investigated the possibility of reducing plant height by overexpression of A. thaliana PHYB gene in B. napus plants. Ectopic expression of Arabidopsis PHYB cDNA under the control of constitutive CaMV35S promoter in Brassica napus caused pleiotropic effects such as decreased apical dominance, a higher number of small leaves and increased leaf chlorophyll content. A strictly light-dependent short hypocotyl phenotype was observed with transgenic seedlings under in vitro culture conditions. Supposed homozygous transgenic plants in the segregating population (T2) exhibited substantially modified plant architecture with reduced apical dominance. Beginning of flowering in these plants was significantly delayed when compared with control plants. At maturity, supposed homozygous transgenic plants remained 28% shorter than the corresponding controls. Even though PHYB overexpressing plants resulted in reduced plant height, which could reduce lodging under field conditions, extremely delayed flowering make these plants difficult to fit into a rapeseed breeding program.