2.2 Experimental design

This experiment used a split-plot experimental design with four replications. The main treatment factor was [CO2] and subplot treatment factor was N application rate. There were two [CO2] levels: ambient CO2 (400 μmol mol-1) and eCO2 (600 μmol mol-1). Since we have two independent growth chambers, we set one chamber for ambient CO2 and the other for eCO2 treatments, and repeated the whole experiment four times. Each time was considered one replication. The eCO2 concentration was achieved by pumping in industrial grade pure CO2 to a partial pressure of 600 μmol mol-1 (actual records range 595-640 μmol mol-1), and the ambient condition had a CO2 partial pressure range of 405 to 430 μmol mol-1 during the growth period. As we focused on the adaptation of rice plants to eCO2 at the early seedling stage, we initiated eCO2 treatment from seeding. N application rates included four levels: 0 (N0), 5 (N5), 10 (N10) and 15 (N15) kg N 666.7 m-2, which was comparable to the general range of N fertilization rate in the region at this growth phase. Half of the N fertilizer (in the form of urea, 46% N) was premixed into the soil three days prior to seeding, and the remaining portion was top-dressed in a water-solution form at true leaf-age 1, 2, 3 and 4, respectively, each at a 12.5% of the total N application rate. Together, there were eight treatments, combined from two levels of [CO2] and four levels of N application rate, and each treatment had two plastic trays comprising 192 plants.