An efficient shoot regeneration system andAgrobacterium-mediated transformation withcodAgene in a doubled haploid line of broccoli

Abstract

Broccoli is an important vegetable crop belonging to the genus of Brassica. However, it is often affected by biotic and abiotic stresses that result in large losses in yield and quality. Genetic manipulation has opened the opportunity for germplasm improvement of broccoli. In this study, an efficient shoot regeneration and Agrobacterium-mediated transformation system was established. The optimum medium for shoot induction was selected for three types of explant including hypocotyl, petiole, and peduncle, and up to 90% regeneration frequency was obtained. The transformation procedure was developed with the EHA105 strain, harboring the PUC19 vector, along with the target gene of codA and a hygromycin-resistance gene. Several factors were optimized, including hygromycin concentration for selection and the Agrobacterium density yielding the highest transformation frequency. Among the three types of explants, peduncle explants showed the highest response to this procedure, and the highest frequency of transformation (3.4%) was obtained depending on the analysis of polymerase chain reaction and Southern blot. In conclusion, the present study introduced a reliable system for plant regeneration and genetic transformation with three types of explants for a DH line of broccoli, and peduncle explant resulted in the highest transformation frequency.