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.