Alleviating delayed field curing-induced deterioration in rice seeds by cathodic water invigoration: a comparison of seed and seedling responses in two upland rice species and their interspecific hybrid.

Abstract

Rice constitutes the major source of the world’s food supply. A number of varieties are grown in many parts of the world, all which produce orthodox seeds that are usually stored between growing seasons. As in other crop species, storage-induced loss of seed quality (viability and vigour) is inevitable but considerable research effort has been invested in optimising seed bank storage protocols for rice to ensure acceptably high levels of seed germinability and seedling emergence. However, poor post-harvest practices such as delayed field curing before threshing in developing countries in the humid tropics, such as Ghana, result in curing-induced seed deterioration in the field prior to ex situ storage. Given that many rice growing regions are likely to experience significant levels of climate change, seed processing and storage induced declines in rice seed quality could exacerbate the crop losses incurred by rice farmers in these regions in the future. This motivated the present study which was designed around three aims: 1) to investigate how environmental conditions and the duration of field curing influenced seed macro-structural integrity, susceptibility to microbial infection, and seed germinability and vigour; 2) to identify macro-structural and ultrastructural indicators/ biomarkers of field curing and associated storage-induced stress/ damage; 3) to assess whether the invigoration of field-cured seeds with cathodic water (CW), an established antioxidant-based seed invigoration medium, and deionized water (DW) can alleviate the deteriorative effects of delayed field curing on rice seed cellular integrity, germination and subsequent seedling growth and biomass. The specific objectives of this comparative study, which involved an Asian (Oryza sativa L.) and African (Oryza glaberrima Steud) upland rice species and their interspecific hybrid (O. sativa × O. glaberrima), were as follows: a) to compare the impacts of delayed field curing in wet and dry environments on seed physical, physiological and pathological quality; b) to identify potential ultrastructural biomarkers of seed sensitivity to delayed field curing-induced stress/ damage in embryonic root meristematic cells using transmission electron microscopy (TEM); c) to assess whether CW invigoration alleviates cellular stress/ damage induced by delayed field curing using selected ultrastructural biomarkers of seed sensitivity to such curing-induced stress/ damage; and d) to assess whether invigoration with CW and DW improves seed germinability and emergence, and subsequent plant growth in field cured seeds, relative to non-invigorated (NI) dry seeds. For objective (a), seeds of both species and their interspecific hybrid were grown in Ghana, harvested, field cured in open (wet) and within ventilated rainproof containers (dry) for five weeks. Harvested panicles were sampled from the wet and dry cured environments at weekly intervals for 5 weeks, hand threshed, and depending on the moisture content (MC) at sampling, seed samples were further dried to 12% and stored hermetically at 4°C until used. Sub-samples of both species and the hybrid from the weekly samples were used for seed microflora studies and germination assay. Environmental parameters (temperature and relative humidity) were measured in wet (weather station) and dry (tiny tag data loggers) cured environments. Seed samples of both species and hybrid were taken daily from wet and dry cured environments at 08h00 and 15h00 during the 5 week period, equilibrated overnight at 20°C for seed MC and water activity measurements. Sub-samples of seed samples of both species and hybrid taken daily at 08h00 from the wet and dry cured environments were used to measure percentage of seeds with cracked endosperm. Results revealed that dry field curing delayed endospermic cracking and reduced crack frequency (10% in O. sativa; 40% in O. glaberrima; 36% in the hybrid), enhanced subsequent seed germinability (2%), and reduced subsequent seedling vigour (radicle length, 37%; seedling dry weight, 11%) relative to wet cured seeds across both species and hybrid. However, fungal infection was higher (22%) in dry than wet cured seeds: seeds infected with fungal species (e.g., Aspergillus flavus, Bipolaris oryzae, Curvularia lunata, Fusarium moniliforme and Phoma sp) was higher in dry cured O. sativa (22%) and the hybrid (31%) than the wet cured seeds. Seed germinability and seedling vigour declined with delayed field curing, but the decline was higher in the hybrid than in both species. For objective (b), seeds of both species and their interspecific hybrid field cured for different durations (0, 2 and 5 weeks) and hermetically stored at 4°C for 20 months were removed from storage, equilibrated overnight at 25°C and invigorated with DW for 18 h. Untreated dry (nonimbibed seeds, cured for different durations (0, 2 and 5 weeks) were used as the controls for this study. These controls were used for two reasons: 1) to establish whether ultrastructural abnormalities induced by curing are permanent or can be reversed/ repaired during imbibition; 2) to establish whether seed deterioration during curing leads to imbibitional damage. The severity of ultrastructural lesions related to cellular stress/ damage differed across species and the hybrid and progressed at a rate in agreement with the viability and vigour loss brought about by delayed field curing. These microscopy studies also served to identify useful ultrastructural biomarkers of field curing induced cellular stress/ damage in both rice species and the hybrid. These include marked abnormalities in cell wall and key organelle [nucleus (N), nucleolus (nu), mitochondria (M), lipids, vacuoles, plastids and amyloplasts] ultrastructural integrity with delayed field curing. This study suggests that these biomarkers may serve as useful screening tools for rice breeders looking to identify species/ varieties that produce seeds that are more resistant to field curing-induced deterioration. For objective (c), seeds of both species and their interspecific hybrid were field cured for different durations (0, 2 and 5 weeks), stored hermetically and removed from storage and equilibrated as in the experiment for objective (b). However, in the experiment designed to address this objective, seeds of both species and hybrid cured for different durations (0, 2 and 5 weeks) were invigorated with CW and DW for 18 h before comparing their ability to alleviate the effects of field curing induced cellular stress/ damage in terms of the ultrastructural biomarkers of such stress/ damage identified via objective (b). Seed germinability was significantly higher in CW invigorated 5 week cured seeds of both species and 2 and 5 week cured seeds of the hybrid than DW invigorated seeds suggesting that CW invigoration was more effective in alleviating curing-induced stress/ damage. Ultrastructural studies showed that CW invigoration of cured seeds of both species and hybrid appears to have enhanced the repair of damaged cellular components and/ or reduced the damage that ensues during imbibition in the deteriorated seed. More importantly, seeds subjected to CW invigoration exhibited more prominent signs [e.g., M exhibited elongated profile, homogenous matrix, well-differentiated cristae (Cr), and well-defined outer mitochondrial membrane (OM) and inner mitochondrial membrane (IM), and highly developed Golgi bodies (G)] of repair and germinative metabolism than those imbibed with DW. Interestingly, the degree of mitochondrial and G development in CW invigorated seeds was greater in the hybrid than the two species. The results obtained for these biomarkers indicated that CW invigoration can alleviate the cellular stress/ damaged induced by field curing and also point to superior restorative effects of CW on viability, vigour and cellular integrity and metabolism in cured seeds compared with DW. For objective (d), endosperm integrity (i.e., nature/severity of physical damage to endosperm, caryopsis coat thickness, and presence of seed microflora) of stored (4°C for 20 months) NI seeds of both species and their interspecific was assessed using scanning electron microscopy (SEM). Seeds of both species and the hybrid stored for the same duration as in the SEM studies were also equilibrated overnight at 25°C and invigorated for 18 h in CW and DW before being sown in individual pots under greenhouse conditions. Untreated dry seeds, cured for different durations were used as the controls for this study. The uncured seeds (0 week) of both species and the hybrid did not exhibit macro-structural damage; however, 5 week-cured seeds exhibited deep cracks which exposed the aleurone cells (grains and lipid bodies) and facilitated fungal infection and insect damage which may have contributed to viability and vigour loss in cured seeds. Cathodic water invigoration significantly enhanced seedling emergence (17% in 5 week cured O. sativa; 23% in 5 week cured O. glaberrima; 16 and 19% in 2 and 5 week cured hybrid, respectively) in field cured seeds of both species and the hybrid than DW invigoration. Also, CW invigoration significantly produced taller seedlings in CW invigorated cured (2 and 5 weeks) seeds of both species and the hybrid than DW invigorated seeds. Across both species and hybrid, CW enhanced panicle biomass, although differences between CW and DW were not always significant. The invigorative effect of CW on plant growth from cured seeds was generally not observed in terms of total biomass yield and changes in biomass allocation to stem, leaves, and roots. Overall, the study has deepened our understanding of the physical, pathological, physiological, and ultrastructural lesions that contribute to loss of rice seed quality (seed viability and vigour) during delayed field curing. It has also served to identify a number of seed physiological, ultrastructural, and macro-structural biomarkers/ indicators that can be used by breeders for screening rice varieties for sensitivity/ tolerance to field curing. The results have also provided a basis for many new opportunities for research on alleviating seed processing- and storage - induced deterioration in crop seed quality through seed soaking/ invigoration treatments (particularly those involving CW and other antioxidant-based solutions), which is going to represent an increasingly important research area as the effects of climate change and developmental challenges threaten food security in many parts of the world. However, future studies must screen a larger number of rice genotypes to assess the plasticity of the biomarkers identified to be reliable indicators of curing-induced stress and stress recovery before they are adopted by the seed industry. Additionally, future studies must use biochemical, physiological, molecular, omic and biophysical approaches to fully understand the mechanism(s) of action of CW invigoration in deteriorated rice seed in order to extend this method to other crop species that experience a decline in seed quality during seed processing and or storage.