2.2 Imposition of the drought and heat stress
The Exp. 1 was arranged in a completely randomized block design with four replications. The ponds were kept from tasseling to ovaries fertilization under different stress treatments as follows: (I) Well-irrigated in field (CK), the soil potential was -0.12 MPa (Ⅱ) Drought stress (DS), introducing a water deficit by no irrigation about 7 d before tasseling, the soil water potential was -0.51MPa. (Ⅲ) Heat stress (HS), which was according to the method of Savin, Stone, & Nicolas (1996). The polyethylene film (100 μm thickness) was mounted on steel structures of 3.5 m height (as showed in Figure 1a), but leaving the bottom 50 cm of the four sides of each structure open and the top 30 cm open, in order to facilitate free gas exchange from top to bottom. The magnitude of the elevated canopy air temperature (about 3-5℃ higher than field) was the consequence of the greenhouse effect of the polyethylene enclosure (Cicchino, Edreira, & Otegui, 2010). (Ⅳ) Combined drought and heat stress (DHS) were realized by the both methods of DS and HS. The soil water potential (depth 40 cm) and air temperature (ear position) were recorded by Em50 (Decagon, America) and GSP-6 (Elitech, China), respectively. These sensors of Em50 were buried 40 cm underground of plot. The effects of stresses in experiment were obvious (Figure 2).
In Exp. 2, the fertilized ears were sampled. According to the methods of Burle, Gengenbach, Robert (1994) and Zhang et al. (2017), husk tissues and silks were gently removed with a fine scalpel blade, taking care to avoid breaking the ovary. Then the superior kernels (basal 18-20th line) and inferior kernels (top 3 cm) of the ear were sectioned into blocks of 4×4 (16 grains) and seated into culture media (adequate source supply) in 25×100 mm Petri dishes such that the media contacted only the cob tissue (Figure. 1e). Kernels were incubated in thermostatic incubator (YIHENG TECH-NICAL.CO., LTD) with 25℃, relative humidity 75%. To sterilize for 20 minutes with ultraviolet lamp and wipe the inner wall with 75% ethanol to protect the culture materials from contamination before using thermostatic incubator.
In Exp. 3, the soil moisture regime of 120 pots was 60±5% (well-irrigated) of soil water holding capacity before stress treatment. Then the pots were kept from tasseling to ovaries fertilization under different stress treatments as follows: (I) Well-irrigated in field (CK). (Ⅱ) Drought stress (DS), introducing a water deficit (30±5%) by no irrigation about 7 d before tasseling. (Ⅲ) Heat stress (HS), the 30 pots were 60±5% (well-water) of soil water holding capacity also transferred into greenhouse covered with polyethylene film. The elevated canopy air temperature (about 3-5℃ higher than field) was also the consequence of the greenhouse effect of the polyethylene enclosure. (Ⅳ) Combined drought and heat stress (DHS) were realized by the both methods of DS and HS. The soil moisture and temperature were recorded by HD2 (IMKO Micromodultechnic GmbH, Germany) and GSP-6 (Elitech, China) during the stress treatment (Figure 2). When the ears were fertilized, the grain was cultured in vitro by the methods of Exp. 2.