The use of biotechnology for the genetic improvement of wheat (Triticum aestivum L.) has been hampered by its recalcitrance to standard transformation and regeneration protocols. Male gametes present a potentially useful option for introducing gene edits via clustered regularly interspaced short palindromic repeats (CRISPR). However, the utility of male gametes for introducing genetic improvements would be dependent on the retention of viability after treatment to introduce the CRISPR components. We have studied wheat pollen morphology and its viability in a range of germination media to identify conditions that optimize the viability of in vitro hydrated pollen. The size, shape, and aperture from seven different wheat genotypes were compared using scanning electron microscope (SEM). The SEM results revealed that the pollen of all of the wheat genotypes examined in this study were monoporate; however, a significant variation in the size of the mature pollen grains was observed. The hydrated pollen of the wheat genotypes remained viable for up to five hours at 20 ± 2 °C. Of all of the germination media tested, the medium containing 5% sucrose, 10% PEG4000, 100 mg/L boric acid, 200 mg/L calcium nitrate, 100 mg/L potassium nitrate, and 100 mg/L magnesium sulphate at pH 6.5 achieved the highest percentage of pollen germination after 5 h of hydration. Impedance Flow Cytometry (IFC) provided similar results to the in vitro germination study. This work elucidates important factors that can form the basis for a pollen-based non-genetically modified system for gene editing in wheat.

Okra is a vegetable crop adapted to summer temperatures, but heat stress has been shown to reduce its growth and productivity. We measured physiological traits of 104 okra genotypes in response to high temperature, augmented by the molecular characterization of selected genotypes to identify parents for crossing. Genotypes were exposed to a short heat shock (45 °C, 4 h) in a controlled environment, followed by the assessment of chlorophyll fluorescence (F_v/F_m, F_v′/F_m′) and stomatal conductance (g_s). DNA was isolated from all genotypes using a modified CTAB method with additional PVP and RNase, and the amplification of 8 polymorphic SSR markers was used to generate a dendrogram. This preliminary screening identified 33 polymorphic genotypes with less than 50% genetic similarity and contrasting F_v′/F_m′ and g_s responses. More detailed physiological measurements (F_v/F_mF_v′/F_m′, g_s, photosynthesis (A), efficiency of the open reaction centre (Φ_PSII), and electrolyte leakage (EL)) were conducted after exposure to 45 °C for 6 h and compared to the control (30 °C). EL did not significantly increase in the heat treatment; in contrast, there were significant genotype and treatment effects observed for fluorescence (F_v/F_m, F_v′/F_m′) and photosynthetic parameters (A, Φ_PSII, g_s). In conclusion, cell membranes in okra remained unaffected after short periods of heat stress, whereas the ranking of differences of measured physiological traits (∆) between control and heat-treated plants (∆F_v′/F_m′, ∆F_v′/F_m′, ∆A, ∆Φ_PSII, ∆g_s) was indicative of genotype sensitivity to heat.