The experiment was carried out in the net house of Hunan Agricultural University, Changsha, Hunan, PR China, in the early rice growing season from April to August of 2013. The tested rice variety was a heat tolerant early indica rice ‘Shenyou 9576’. Germinated seeds of rice were pre-grown with complete Kimura B nutrient solution (Yoshida et al., 1976) in a green house until 15 days. Seedlings were then transferred to earthen pots of 30 cm in diameter and 32 cm in depth filled with 7.0 kg of sieved, dry paddy soil (the contents of soil organic matter, alkaline hydrolytic nitrogen, effective phosphorus, available potassium were 1.8%, 66.2 mg kg-1, 8.5 mg kg-1, and 8.0 mg kg-1, respectively, and soil pH was 5.4) amended with 1.0 g CO(NH2)2, 0.4 g P2O5, and 0.6 g K2O per kg soil to grow. Two seedlings were transplanted in each earthen pot. Proper management practices were provided as per requirement for proper growth. The treatment consisted of three temperature regimes which are designated as high temperature (35/28oC, 12h light/12h dark, 75-80% relative humidity), low temperature (25/20oC, 12h light/12h dark, 75-80% relative humidity) and natural condition (35/25oC- day/night) as control. The treatments were imposed after anthesis by transferring pots into different growth chambers, but for the control treatment pots were kept in the net house under natural condition. The experiment was performed according to a complete randomized design (CRD) with three replications.
Sampling method
Panicles and flag leaves from each treatment were collected at 7-day interval after anthesis i.e. 7, 14, 21, 28, 35 days after anthesis (DAA). Samples were collected on 9.00 to 11.00 am and immediately wrapped in aluminum foil and frozen in liquid nitrogen, then placed into a sealed plastic bag and stored at -60oC until use for different analysis. Rice grains were harvested at 35 DAA and then were sun dried to achieve 14% moisture content. Rough rice (paddy rice) was dehusked by a SBS-80 dehuller, then was polished by a rice polisher for 2 minutes. Milled rice samples were kept in sealed bags under refrigeration (4oC) for later analysis.
Chalkiness and head rice rate measurement
Chalkiness was measured with a system composed of a scanner and a special software Chalkiness 2.0 developed by Hunan Agricultural University (Chen et al., 2011). Head rice refers to the whole grains of milled rice and was computed by using the following equation (Gummert, 2010).
Head rice (%) = (Wt of whole grains/Wt of paddy samples) x 100
Extraction, purification and determination of indole-3-acetic acid (IAA), gibberellins (GA1 and GA4), zeatin (Z ), zeatin riboside (ZR) and abscisic acid (ABA)
Fresh plant materials (panicles or flag leaves) of 500 to 700 mg were frozen in liquid nitrogen and ground in a mortar with a pestle. After adding 800 μL of 80% methanol, samples were mixed thoroughly and kept overnight at 4°C. Then the mixtures were centrifuged at 4,800 g and 4°C for 10 min. The supernatant was transferred to a new tube and the residues were re-extracted with 800 μL of 80% methanol for 4 hours at 4°C for two times. The supernatants were combined and dried in a Jouan RCT-60 vacuum concentration system, then dissolved in 200 μL of 0.1 molL-1 buffer (pH 7.8). The aqueous phase was purified through a Waters Sep-pak C18 cartridge (Waters, USA) and followed by a wash with 800 μL of ddH2O, and then the eluted was vacuum freeze dried. The dried extract was dissolved in 40 μL of 50% methanol and used for an assay by a SHIMADJU LCMS-8030 mass spectrometer. A BEH C18 column (100 mm × 2.1 mm, 1.7 μm) was used and mass-to-charge ratio were 174/130.05, 220/136.1, 352.2/220.1, 347/259.2, 331/243.25, 263/153.2 for IAA, Z, ZR, GA1, GA4 and ABA,
respectively.
Statistical analysis
All experimental data were analyzed following analysis of variance. Mean separation of the treatments was done by using Least Significant Difference (LSD) at 5% level. All statistical analysis was performed by using a statistical software, DPS version 12.01 and Microsoft Excel 2003 (Microsoft, USA) was used to generate graphs.
Under the natural condition (control) of the experiment site, daily maximum, minimum and average temperature (oC) from flowering to harvest.