The experimental material comprised two parental maize inbred lines viz. LM 11, heat stress susceptible (HS), and CML 25, heat stress-tolerant (HT). The seedlings of two inbreds were raised during the second week of March in glasshouse conditions at 28°C/23°C and 16 h light (Day)/8 h dark (Night) photoperiod at the School of Agricultural Biotechnology, Punjab Agricultural University, India, during spring 2016. The plants were grown till they reached the reproductive stage (Figure 1). The reproductive phase of maize is categorized into six stages, with the emergence of tasselling and silking; followed by a blister where kernels with clear liquid get secreted and filled with milky fluid; accompanied by doughy consistency and extended kernels and milk line progression towards the kernel tip and finally, a black layer formed at the base of grains. At the reproductive growth stage, from tassel emergence to early grain-filling (lag-phase), maize plants were exposed to natural heat stress and experienced 42°C during the daytime and 35°C during the nighttime. Drought stress is confounded naturally during heat stress. To maintain the microclimate conditions with low RH (<40%) and to avoid the compound effect of drought and heat stress, regular irrigation was applied for at least two weeks during tassel emergence until one week after pollination, which increases the probability of irreversible damage due to heat stress. Both inbreds showed differential responses to heat stress for phenological attributes like top leaf firing, tassel blast, pollen viability and shedding duration, kernel number and weight, and yield (Jagtap, 2020 ). Three different tissue samples, viz. flag leaf, pollen, and ovule from the inbreds, LM 11 and CML 25, were collected in 5 replicates after five days of pollination. Tissue purity was maintained by bagging the tassel and cob. Ovules were isolated from ear florets with a silk length of ~10 cm by removing the silk and ovary wall with forceps and cutting the ovule at its base from the floret under the microscope. Each tissue was pooled (pool of five plants) for each inbred to reduce biological sampling error. A total of six samples (3 tissues x 2 inbreds) were immediately frozen in liquid nitrogen and stored at -80°C until processed for RNA isolations.
Free full text: Click here