*4.7. Phenotypic Analysis of Transgenic Rice in a Greenhouse*

The *SiWLIM2b* complete open reading frame (ORF) was amplified by PCR usingthe primers F1 (BamH I) and R1 (Sac I). The amplified PCR fragment was cloned into vector pMWB014 (driven by the ubiquitin promoter) digested with BamH I and Sac I. The pMWB014-*SiWLIM2b* was confirmed by sequencing and transformed into the kitaake rice (WT) cultivar by Agrobacterium-mediated transformation. The transformed callus was cultured on medium with 1.0 mg/L glufosinate. Six transgenic rice lines were obtained by using primers F2 (designed according to the CDS of *SiWLIM2b* gene) and R2 (designed according to NOS terminator sequence) for PCR verification. The resulting transgenic rice was cultured to the T3 generation. We selected three transgenic lines in the T3 generation (OE1135, OE1136, and OE1144) for subsequent functional analysis. To determine whether transgenic rice seedlings could resist drought stress, we conducted a pot experiment in the greenhouse. Rice seeds were soaked with 2.5% sodium hypochlorite (NaClO) for 30 min, then rinsed with tap water 5–6 times. Sterilized seeds were germinated in a 28-degree incubator, and the water was changed every 12 h. After germination, the seeds wereplanted in nutrient soil and grown to the three-leaf stage under a 16-h/8-h photoperiod at 30 ◦C. Rice seedlings were then subjected to drought treatment for 12 days and survival was scored by dividing the number of plants alive after drought treatment for 12 days by the total number of plants surveyed, using one green leaf as the standard for survival. Relative water content [77,78] and MDA content [66] were determined in transgenic and control rice after 5 days of drought treatment. All experiments were set up with three independent biological replicates.
