Post-Harvest seed physiology and conservation of the germplasm of syzgium cordatum hochst.
Cheruiyot, Anastacia Chepkorir
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There is global concern about the ex situ conservation of the germplasm/genetic resources of recalcitrant-seeded species. While orthodox (desiccation tolerant) seeds afford an ideal means for ex situ conservation, this is impossible for recalcitrant seeds which are shed at high water contents, are metabolically active, and are desiccation sensitive, with those of many species losing viablity when only a small proportion of tissue water has been removed. Storing such seeds in the short- to medium-term is possible when parameters to be optimised include the means to obviate dehydration and the most equitable storage temperature – and, if necessary – the best way to curb the activity of seed-associated micro-organisms (usually fungi) during such hydrated storage. Presently, it is generally agreed that the only option for longterm ex situ conservation of the germplasm of recalcitrant-seeded species is by cryopreservation (usually in liquid nitrogen) of explants representing the same genetic diversity as do the seeds. To achieve this, the explants of choice are embryonic axes excised from the seeds. However, there are still many problems impeding progress particularly for tropical/sub-tropical species: presently, these need to be resolved on a species-specific basis. To this end, the current investigation was focused on germplasm of the tropical/sub-tropical recalcitrant-seeded species, Syzygium cordatum Hoechst. There were two major aspects to the study, viz. optimisation of the ‘shelf-life’ of intact seeds in the interest of almost immediate planting programmes, and attempting to develop a protocol which would result in successful cryopreservation of zygotic axes excised from the seeds. Chapter One of this Thesis provides an overview of the theoretical basis underlying these two approaches to conservation, as well as a description and significance of the species under study. Chapter Two describes the study seeking to establish optimal short-term storage conditions for the recalcitrant seeds of S. cordatum. Seeds were stored at various relative humidities at three different temperatures (6 ºC, 16 ºC and 25 ºC) for differing periods. Seeds stored at all these temperatures maintained stable water contents. The most mature seeds that were stored in a saturated atmosphere at both 16 ºC and 25 ºC reached their root protrusion stage after three weeks. This, however, occurred in only a small percentage of the seed batches. The majority of the seeds that were stored under saturated atmospheric conditions at 16 ºC and 25 ºC had not reached the stage of radicle elongation before the sixth week of storage, but after this time there was evidence of damage associated with both fungal proliferation and desiccation sensitivity. Seeds stored at 6 ºC and 25 ºC for the longest period had also lost vigour. For seeds stored at 6 ºC and 25 ºC (whether under hydrated or nonhydrated conditions), those stored for the shortest and longest periods produced the smallest seedlings. The seeds stored at 16 ºC appeared to have maintained vigour and seedling size did not change with the period of seed storage prior to sowing. Storage at 6 ºC may have caused stress associated with chilling, while at 25 ºC, seed storage was compromised by fungal proliferation. Those seeds stored in unsaturated atmospheric conditions at 16 ºC exhibited an increase in their germinative index and germination rate after six weeks. This is possibly associated with the ability of seeds, where vigour was not compromised, to counteract fungal proliferation because there was a decrease in the number of seeds showing fungal proliferation. In contaminated seeds, the fungus appeared to proliferate from the surface of the coat, to the cotyledons and eventually to the axes. Seeds generally did harbour fungal inoculum at harvest, but proliferation, was reduced at cool temperatures.Based on the above observations, storage in sealed plastic bag (non-saturated atmospheric conditions) at 16 ºC was chosen for the short-term maintenance of seeds of S. cordatum. The studies described in Chapter Three sought to establish a protocol for the cryopreservation of embryonic axes of S. cordatum. These studies involved the stepwise optimisation of decontamination, regeneration and growth, dehydration, cryoprotection and cooling (freezing) conditions. The most suitable combination of biotechnological manipulations for the preparation of embryonic axes of S. cordatum for cryopreservation were: decontamination by exposure to 1% (v/v) Ca(OCl)2 for 5 min; cryoprotection using a 5% solution of dextran and DMSO for 1 h followed by exposure to a 10% solution of these cryoprotectants for another hour; then dehydration in a flash dryer for 75 min; and regeneration in agitated liquid medium containing woody plant medium, 10 g l-1 polyvinylpyrrolidone and 75 mg l-1 citric acid. A major achievement following this procedure, was the prevention of excessive exudation of phenolic compounds from the explants. Nevertheless, despite optimisation of all these procedures, axes did not survive cryogenic exposure. One of the objectives of the present study was to develop the means for visualisation of intracellular detail of axis cells of S. cordatum. An experiment was thus entrained to investigate the effects of exposing shoot tips to 75 mg l-1 citric acid for 10 min before fixation during preparation for transmission electron microscopy. In the absence of any ameliorative treatments, large electron dense polyphenolic precipitates were observed mainly inside vacuoles closely associated with the tonoplast. Less dense, small precipitates were located between the plasmalemma and the cell wall, and organelles were generally not clearly visible, probably because of leaching of phenolics into the cytoplasm. Thus the effects of various treatments on organelles and the entire cell ultrastructure could not be conclusively determined. When treated with citric acid, cells had no visible polyphenolic precipitates and the apparently intact organelles were clearly visible, so paving the way for electron microscopical examination of this – and perhaps any other – plant tissue containing substantial amounts of phenolic substances.