Stress, storage and survival of neem seed

Neem ( Azadirachta indica ) is an important multipurpose tropical tree species, frequently used in planting programmes in the arid tropics. However, its seeds are difficult to store for extended periods of time, as are those of many other tropical species which display intermediate or recalcitrant storage behaviour. This thesis describes the results of our investigations concerning factors involved in the rapid loss of germinability of neem seeds. Stress and survival under different conditions were studied in an attempt to understand the mechanisms associated with this loss of viability and to improve the storage longevity of neem seeds.When neem seeds were stored at 75% RH at 20°C, they almost completely lost germinability within six months, whereas viability only decreased slightly at 32% RH. Early experiments showed that at 20°C, the best survival could be obtained when seeds were equilibrated to 50% RH. Later, it became clear that further drying below an equilibrium RH of 50% renders neem seeds sensitive to imbibitional stress, which could be partially alleviated by soaking the seeds at elevated temperature. The sensitivity to imbibitional stress increased with seed ageing. The fact that controlled rehydration at high temperatures (up to 40°C) could considerably improve the percentage of germination is evidence that seeds can withstand considerable dehydration. This indicates that neem seeds can be considered as orthodox, although the ability to easily survive rehydration is part of desiccation tolerance. Yet, the critical water content below which the seeds will not germinate was difficult to determine, because such dry seeds are extremely sensitive to imbibitional stress. Our data show that this water content is below 4%.Results of storage in the cold indicate that neem seed is sensitive to temperatures of≤10° C. At MCs of 4-8%, seeds were considerably more tolerant of low temperature storage and had an overall viability of 50% on average after 2 years of storage at temperatures between -20 and 20° C.The study of neem seed longevity under different conditions of water content and temperature revealed that there were hardly any differences in storage behaviour between seed batches and between seed lots, whatever their provenance (African Sahel or Asia). However, the seeds originating from mature yellow fruits lived longer than the seeds from younger green or older brown fruits.High cell cycle activity has been associated with recalcitrance in seeds at maturity. This activity was reduced in neem seed at seed maturity, as it is in orthodox seeds. During dehydration of fresh seeds, total glutathione, oligosaccharides and phospholipids accumulated and remained stable, particularly in dry seeds. The ability to accumulate protective compounds during slow drying led us again to conclude that neem seed has features of orthodox rather than recalcitrant storage behaviour.The fact that there was an apparent optimum moisture level for storage of neem seed led us to investigate the properties of water in the seed tissues. The water contents below which only non-frozen water was present and below which the cytoplasm was in the glassy state were determined. This information allowed the construction of state-phase diagrams, which showed that during drying at room temperature the formation of cytoplasmic glasses occurred at approximately 7% MC, - slightly higher in axes than in cotyledons. Lowering the MC improved storage longevity, coinciding with the seeds being in the glassy state (below T g ). Seeds in the liquid phase (above T g ) were shorter lived. All of the reported cases of successful storage of neem seeds may therefore have been with seeds in the glassy state. The state-phase diagrams of neem seeds suggest that glasses are involved in the prolonged storage stability. In addition, the loss of chilling sensitivity in seeds having MCs≤8% can also be attributed to the presence of a glassy state.Part of the storage problems of neem seeds could be ascribed to injuries to membranes during rehydration; part could be put down to chilling sensitivity. This led to the hypothesis that difficulties with storage stem from phenomena associated with the intrinsically elevated T m (10°C) of membranes in neem seeds. Support for a role for the plasma membranes in chilling sensitivity came from results that indicated increased permeability at chilling temperatures. Freeze-fracture replicas of plasma membranes from chilled hydrated axes showed lateral phase separation and signs of inverted hexagonal phase. Imbibitional injury, which occurred when dry seeds were rehydrated below a critical temperature, is also associated with extensive plasma membrane damage. Low temperature scanning electron microscopy revealed that axis cells did not become turgid after soaking of dry seeds at 5°C and that the internal cytoplasmic structure was disorganised. Soaking at 35°C prevented this damage.This work offers new insights into the biology of neem seed. It explored the causes of the loss of seed viability and provided explanations for the complex storage behaviour of neem seeds. The significant loss of viability upon drying and cold storage indicates that neem seeds still pose more problems than do orthodox seed types originating from temperate climates. Understanding the role played by water in seeds can help develop efficient methods for long term storage of tropical seeds displaying intermediate (or recalcitrant) storage behaviour. The results of the present research may assist projects and programmes aimed at improving the handling and storage of such seeds.