Leaf structural traits mediating pre-existing physical innate resistance to sorghum aphid in sorghum under uninfested conditions.

KEY MESSAGE: We found four indicative traits of innate immunity. Sorghum-resistant varieties had a greater trichome, stomatal and chloroplast density, and smaller mesophyll intercellular width than susceptible varieties. The sorghum aphid (SA), Melanaphis sorghi (Theobald), can severely reduce sorghum yield. The contribution of structural traits to SA resistance has not been extensively studied. Moreover, the current screening method for resistance is inherently subjective for resistance and requires infestation in plants. Quantifying the microanatomical basis of innate SA resistance is crucial for developing reliable screening tools requiring no infestation. The goal of this study was to identify structural traits linked to physical innate SA resistance in sorghum. We conducted controlled environment and field experiments under no SA infestation conditions, with two resistant (R. LBK1 and R. Tx2783) and two susceptible (R. Tx7000 and R. Tx430) varieties. Leaf tissues collected at the fifth leaf stage in the controlled environment experiment were analyzed for the epidermal and mesophyll traits using light and transmission electron microscopy. Leaf tissues collected at physiological maturity in the field experiment were analyzed for surface traits using scanning electron microscopy. Our results showed that stomatal density, trichome density, trichome length, and chloroplast density are key leaf structural traits indicative of physical innate SA resistance. We found that resistant varieties had a greater density of trichomes (39%), stomata (31%), and chloroplast (42%), and smaller mesophyll intercellular width (- 52%) than susceptible varieties. However, the chloroplast, mitochondria, and epidermal cell ultrastructural traits were ineffective indicators of SA resistance. Our findings provide the foundation for developing an objective high-throughput method for SA resistance screening. We suggest a follow-up validation experiment to confirm our outcomes under SA infestation conditions.

Morphological analysis and stage determination of anther development in Sorghum [Sorghum bicolor (L.) Moench].

MAIN CONCLUSION: The characteristics of sorghum anthers at 18 classified developmental stages provide an important reference for future studies on sorghum reproductive biology and abiotic stress tolerance of sorghum pollen. Sorghum (Sorghum bicolor L. Moench) is the fifth-most important cereal crop in the world. It has relatively high resilience to drought and high temperature stresses during vegetative growing stages comparing to other major cereal crops. However, like other cereal crops, the sensitivity of male organ to heat and drought can severely depress sorghum yield due to reduced fertility and pollination efficiency if the stress occurs at the reproductive stage. Identification of the most vulnerable stages and the genes and genetic networks that differentially regulate the abiotic stress responses during anther development are two critical prerequisites for targeted molecular trait selection and for enhanced environmentally resilient sorghum in breeding using a variety of genetic modification strategies. However, in sorghum, anther developmental stages have not been determined. The distinctive cellular characteristics associated with anther development have not been well examined. Lack of such critical information is a major obstacle in the studies of anther and pollen development in sorghum. In this study, we examined the morphological changes of sorghum anthers at cellular level during entire male organ development processes using a modified high-throughput imaging variable pressure scanning electron microscopy and traditional light microscopy methods. We divided sorghum anther development into 18 distinctive stages and provided detailed description of the morphological changes in sorghum anthers for each stage. The findings of this study will serve as an important reference for future studies focusing on sorghum physiology, reproductive biology, genetics, and genomics.

High-throughput imaging of fresh-frozen plant reproductive samples in a variable pressure SEM.

Conventional light and electron microscopy are the most widely used techniques for examining plant reproductive tissues; however, they are time-consuming or expensive. The anther is the male part of the plant reproductive system. Structural changes drive development, and any structural defect may lead to an increase in fertility or cause sterility; thus, quick detection of structural changes is crucial in reproductive biology. We optimized an existing low-temperature SEM alternative to examine the internal structure of hydrated, fresh-frozen anthers. In contrast with the original technique, our method does not require precooling adhesion (ethanol to fix the specimen), and the cryo-sectioning can be conducted at atmospheric pressure. In addition to enabling the differentiation between aerial and liquid-filled intercellular spaces, this method is expected to facilitate the detection of quick (during a day) developmental changes in plant reproductive tissues, which is a current challenge using conventional approaches.•This method allows the high-throughput imaging of fresh-frozen plant reproductive samples collected every 10 min, which is important for developmental studies.•The cryo-images of samples with thickness ranging from 0.2 to 3 mm can be well-preserved at 800X magnification.•This method does not require chemical processing, critical point drying, customized cryo-accessories, controlled temperature cold stages, or metal coating. This simplified method does not require highly skilled personnel, and it is suitable in most microscopy laboratories.