Knock down Os1bglu1 β-glucosidase in rice by Agrobacterium-mediated transformation
Wipaporn Wanthanalert and Mariena Ketudat-Cairns
pp. 23 - 32
Abstract
This research attempted to study the function of Os1bglu1 by RNAi technique. The suppression of Os1bglu1gene was done using the 3’UTR region. The target gene fragment was cloned into the pHELLSGATE8 vector. The high percentages of effective callus induction of 93% were obtained when the seeds were cultured on N6D medium for 4-6 weeks at 28°C. The suitable transformation conditions were to incubate the calli with Agrobacterium (OD600 = 0.02) and blot dry to remove excess bacteria cells, then transferred to co-cultivation medium (pH 5.2) with 200 μM acetosyringone and incubate for three days at 25°C. The 20% transformation efficiency was obtained from the transformed calli with control plasmid, while transformation efficiency of only 15% was obtained from pHELLSGATE8 Os1bglu1 constructs. The transformed calli with control construct showed higher growth rate than the transformed calli with pHELLSGATE8 Os1bglu1construct. The expression of Os1bglu1 mRNA was not found in the transformed calli and siRNAs were found in the transformed calli. However no siRNAs were detected in the control transformed calli. The regeneration efficiencies of 6% were obtained from only the calli transformed with the control construct. The calli transformed with the knock down Os1bglu1 constructs were not able to regenerate. This may indicated that Os1bglu1 is involved in regeneration of rice from callus tissue.
