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.