Plasmid construction, genetic transformation, and phenotypic analysis
The plant overexpression vector was generated using pCAMBIA1301 harboring the AA6 promoter and the tAA6 terminator. Thebra-MIR1885 genomic DNA fragment was isolated from ‘Bre’ seedlings and cloned into the pCAMBIA1301 binary vector. The binary construct was introduced into Agrobacterium tumefaciens strain GV3101. Genetic transformation of the bra-MIR1885 gene construct into B. napus was performed as described elsewhere (Bhalla and Singh, 2008). Seedlings exhibiting resistance to hygromycin were transplanted and grown in a greenhouse at 22 °C under a 16-h/8-h light/dark photoperiod. The genomic DNA extracted from T0 plants was used for PCR amplification with specific primers. The seeds (T1) were harvested separately from positive plants for further analyses.
The wild-type and transgenic lines (T2~T3 generations homozygote lines) were grown in the field, and the genomic DNA extracted from plants was used for PCR amplification with specific primers. Each transgenic line was grown in a row of 10 plants, with 30-cm spacing between plants and 40 cm spacing between rows. The phenotypes of transgenic and wild-type plants were measured in the field.