Conservation of tree seeds from tropical dry-lands

The tropical trees, Azadirachta indica (neem), Lannea microcarpa, Sclerocarya birrea and Khaya senegalensis, are important multipurpose species. Unfortunately, difficult seed storage behaviour limits the utilization of these species in reforestation programs and agroforestry systems. This thesis presents the results of investigations aimed at a better understanding of the seed biology, particularly focussed on the improvement of seed survival after drying and subsequent dry storage. Seeds collected from several stands in Burkina Faso were studied in an attempt to elucidate the causes of viability loss and improve life span. Initially it was thought that the difficulty in storing seeds of these species is due to intolerance of desiccation and sensitivity to low (subzero 0C) storage temperatures. However, the results of the current study indicated that other factors are responsible for the difficult storage behaviour of these seeds. When the moisture content (MC) was reduced from 27 to 5%, neem seeds became sensitive to rehydration temperatures below 25°C, resulting in a noticeable decrease in germination percentage. Rehydration of these dried seeds at elevated temperatures (25-400C) for one hour prior to further incubation at 300C substantially improved germination to a level comparable to that of fresh seeds (27% MC). We concluded that neem seed is desiccation tolerant, but that during drying its germination requirements change, which is the main cause of its difficult storage behaviour.Germination capability of S. birrea seeds was low in all seed lots at harvest, but improved substantially during drying and/or dry storage. Improvement of germination with drying is completely in contrast with what would happen in a desiccation-sensitive seed. We interpreted the observed increase in germination during drying and dry storage as resulting from the slow disappearance of an initially present physiological dormancy. Seeds of S. birrea thus behave as orthodox seeds, but the irregularity in their germination behaviour has obviously contributed to discrepancies between reports on the species seed biology.Seeds of L microcarpa harvested in three successive years were extremely variable as to their germinability and storability. Some seed accessions had a generally low germination, while others had an initially high germination. Drying did not affect germination capability of the poorly germinating seed accessions, but did so in the well germinating accessions. These observations would suggest that seeds can be either tolerant or sensitive to desiccation. However, when mechanically scarified prior to germination tests, seeds from all accessions germinated at high percentage (>80%), regardless of MC and age. This indicates that seeds experienced germination constraints upon drying rather than being sensitive to desiccation. Imbibed, intact seeds that failed to germinate displayed a moderate rate of respiration with RQ values< 1, which indicates that the seed coat is permeable to water and respiratory gases. On scarification, water uptake and respiration steeply rose in narrow association with radicle emergence. Non-scarified seeds lost germination ability and cellular viability after approx 15d of moist incubation at 300C. Apparently, coat-imposed inhibition of germination does not prolong viability of seeds in the hydrated state. These results suggest that radicle emergence is inhibited mechanically and not by germination inhibitors or physiological dormancy. We conclude that L microcarpa seeds display orthodox storage behaviour, but that physical dormancy explains the difficult seed storage behaviour reported for this species.In the case of K. senegalensis, the MC of seeds at harvest was low (<0.1g H2O g DM"1) and further drying did not affect the initially high germinability (90-100%). During storage a relative!y fast viability loss was observed for seeds stored at 4°C in comparison with seeds stored at -20, 15, and 200C. The sensitivity to low above-zero (0C) temperatures might be the cause of the erratic loss of viability often observed in these seeds.We concluded that seeds of all the species investigated in this thesis display orthodox storage behaviour, but that various factors including sensitivity to imbibitional stress, physiological and physical dormancy, and sensitivity to low above-zero (0C) storage temperatures cause the reported difficulties in storing these seeds. Tropical tree seeds are thought to be particularly sensitive to low imbibition temperatures because of the relatively high gel-to-liquid crystalline phase transition temperature (Tm) of their membranes as compared to the relatively low T177 of membranes of temperate zone seeds. In relation with this, the sensitivity of the four seed species to rehydration at low temperatures was investigated. The results indicated that dried neem seeds are particularly sensitive to imbibitional stress, whereas the seeds of the other species appeared to be less (K. senegalensis) or not (L microcarpa and 5. birrea) sensitive to low imbibition temperatures. This difference in sensitivity to imbibitional stress was observed despite comparable T177 of membranes in the roots of seedlings of K. senegalensis, L microcarpa and neem (11.5, 14.5 and 9.60C, respectively). The hypothesis of tropical tree seeds being particularly sensitive to imbibitional stress because of their high membrane Tm therefore does not have general validity. LTSEM images of cryo-pianed K. senegalensis seed embryos revealed the presence of a dense peripheral cell layer, which might have slowed water uptake and reduced sensitivity to imbibitional stress. On the other hand, for the sensitive neem seeds, it was observed that the loss of germinability of imbibitional damaged seeds did not involve death of all the cells. An assessment of membrane integrity by ESR and cryo-SEM indicated that seed germination is not supported when approx. 30% or more of axis cells have died. This supposes that there are still on average 70% or less of live cells in a non-germinating seed. From this observation, we argue that up to this lethal threshold, seeds are able to repair the damage caused by imbibitional stress or survive with a number of damaged cells. We suggest that differences in tolerated number of damaged cells, in intrinsic repair capabilities, or in morphology of the outer cell layers determine the level of sensitivity of seeds to imbibitional stress.Low temperatures are expected to better maintain viability of seeds in dry storage than do high temperatures. However, we noticed that storage conditions at 4°C were more detrimental for K. senega/ensis seeds than at -200C or 15°C, and this was, to a lesser extent, true for L microcarpa seeds. Because viability loss associated with ageing is often attributed to the loss of membrane barrier function, membrane polar lipids (PLs) were analysed in several K. senegalensis seed lots differing in age and viability. The data indicated that the content of membrane PLs steadily decreased with ageing. However, it appeared that seeds can withstand the loss of a certain amount of PLs without losing germinability. Based on data of seeds stored dry at 15°C and at 4°C, it was observed that the loss of germinability coincided with a loss of between 15% and 25% of the PLs. With the decrease in PL content, the free fatty acid content rose. Because the free fatty acid also can arise from degradation of triacylglycerols, it was difficult to establish a critical content of accumulated free fatty acids associated with viability loss.Chemical analysis of membranes in 20 year-old dry-stored K. senegalensis seeds, which were already non-germinable for a long period of time, indicated a loss of 80% of the PLs. However, this did not lead to changes in the structure of the plasma membranes in the dried axis as could be observed by high resolution SEM after freeze fracturing. This was interpreted to mean that the deesterified acyl chains were still at the position, at which they originally occurred in the membranes of the fresh seed. This was also the case for the 4°C-stored dried specimens that had just become non-viable. The IMPs (transmembrane proteins) were regularly distributed in the plane of the membrane, indicating the absence of a phase separation in the dry state. The data of the current study suggest that membrane phase separation, thought to occur during viability loss, is unlikely to happen in the dry state. However, at rehydration the plasma membranes suffered considerable damage or ceased existing.The question as to how the deesterification takes place - via free radical activity or by phospholipase activity - was addressed. With free radicals being involved one would not expect a greater amount of acyl chains to be split off at 4°C than at 15°C. However, since the physical state of the membrane determines the activity of phospholipases, we focussed on the membrane phase issue. The mismatch between gel phase domains and the liquid crystalline matrix at mid-melting temperature gives better access to the site of action, thus increasing the activity of phospholipases. At either the gel or liquid crystalline phase, accessibility to the site of action is more difficult. An ESR spin labe! study indicated that membranes in dried and rehydrated axes of K. senegalensis seeds were rigid at -200C, while at 7°C they appeared to be in a mixed liquid crystalline - gel phase condition. The liquid crystalline phase prevailed at 15°C. Thus, it can be expected that during dry storage at 4°C the mixed phase can occur for extended periods of time. Therefore, we suggest that the deesterification of acyl chains leading to the loss of viability of seeds at low above zero (0C) temperatures occurs, at least part!y, via phospholipase activity. Because of their high Tm, membranes of tropical seeds are likely to reach mid me!ting condition at low above zero (0C) temperatures that are high enough to support enzyme activity. By contrast, it is expected that the lower mid-me!ting temperatures of seeds from the temperate climate zones are too low to effectively support phospholipase activity at sub-zero (0C) temperatures. This special feature of tropical seeds dismisses the use of cold rooms for short and medium term (1-3 years) storage purposes.The results obtained in this study will increase and facilitate the use of these species in reforestation and agroforestry programs in Burkina Faso and other Sahelian countries.