2.1 Plant materials, growth conditions, and cold treatment
Cassava cultivar TMS60444 was used in this study. The uniform stem cuttings were grown on Murashige and Skoog (MS) medium containing 3.0 g/L Gelrite, 20 g/L sucrose (Fisher), and 2 μmol/L CuSO4in a growth room at 26°C under 16 h light/8 h dark. For cold treatment, 4-week-old seedlings were transplanted into pots and grown in a greenhouse (16 h days, 26 ± 2 °C). After two weeks, seedlings with a uniform growth status were transferred to a chamber for cold treatment at 4°C under light, followed by one week of recovery at 26 °C. More than 5 plants were treated in each treatment.
2.2 Plasmid construction and cassava transformation
The cDNA sequences of the sense and antisense CRIR1 were cloned by PCR using primers covering the full length of transcripts. The PCR fragment was sequenced and inserted into the binary vector pCAMBIA1301 under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter to generate the binary vector pC35S ::CRIR1 and pC35S ::anti-CRIR1 , respectively. The plasmid was mobilized into Agrobacterium tumefaciens LBA4404 for cassava transformation using a friable embryogenic callus of cultivar TMS60444. Cassava embryogenic callus induction, Agrobacterium -mediated genetic transformation, and hygromycin-resistant calli regeneration were performed by the methods described previously (Zhang, Potrykus & Puonti-Kaerlas, 2000).