**4. Materials and Methods**

### *4.1. Plant Material and Experimental Treatments*

Two rice (*Oryza sativa* L. spp. *xian*) genotypes, SDWG005 and 9311, were used in this study. SDWG005 is an African landrace and 9311 is a widely used restorer line in China. The seeds were soaked in water at 28 ◦C for 24 h and subsequently incubated at 28 ◦C for 48 h for germination. The germinated seeds were transferred to 96-well plates filled with vermiculite with one seedling per well. The seedlings were irrigated with distilled water until the two-leaf stage and Yoshida solution thereafter [39] (pp. 53–57). The 30-day-old seedlings were transplanted into plastic barrels (30 cm diameter) filled with paddy soil. Three plants were planted in each barrel, with five tillers retained for each plant. The seedlings and plants were grown in a growth chamber with a relative humidity of 75% and a natural photoperiod.

There were two temperature treatments: (1) control, 28 ◦C during the day (6:00 am to 6:00 pm) and 22 ◦C at night (6:00 pm to 6:00 am); and (2) heat stress (HS), 38 ◦C during the day (6:00 am to 6:00 pm) and 28 ◦C at night (6:00 pm to 6:00 am).

#### *4.2. Microscopic Observations of Rice Anthers*

Florets of each genotype in the control and heat stress treatment were subjected to microscopic observation. Anthers which ascending to the top of glume from the middle part of the first branch in every genotype under different treatments were carefully collected using tweezers. Some of these anthers were morphologically observed under a VHX-2000 digital microscope (Keyence, Osaka, Japan), with the remainder fixed in a 50% FAA solution (40% formalin:glacial acetic acid:50% ethanol = 1:1:18) and then dehydrated with 100% ethanol, followed by washing with xylene. The anthers were embedded in paraffin before being sectioned (thickness, 10 microns) and stained with safranine O-Fast Green to produce permanent slices to examine the microstructure of anther sacs under a NIKON ECLIPSE E100 positive feedback microscope (Nikon, Tokyo, Japan).
