Browsing by Author "Kioko, Joseph Ivala."

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Aspects of post-harvest seed physiology and cryopreservation of the germplasm of three medicinal plants indigenous to Kenya and South Africa.
(2002) Kioko, Joseph Ivala.; Berjak, Patricia.; Pammenter, Norman W.
The current state of global biodiversity is one of sustained and increasing decline especially in developing countries such as South Africa, where, medicinal plants face a particular threat due the herbal medicine trade, and because in situ conservation measures have not stemmed the exploitation of these plants (Chapter 1). Furthermore, seed storage, which offers an efficient ex situ conservation technique, cannot presently be applied to many medicinal plants, either because these species produce short-lived, recalcitrant seeds, or the post-shedding behaviour of the seeds is altogether unknown. This study investigated three medicinal plant species indigenous to Kenya and South Africa: Trichilia dregeana and T. emetica, of which no population inventories exist and no wild populations were encountered locally during the course of this study; and Warburgia salutaris, one of the most highly-utilised medicinal plants in Africa, and which is currently endangered and virtually extinct in the wild in some countries such as South Africa. Aspects of post-shedding seed physiology (Chapter 2) and the responses of the germplasm of the three species to cryopreservation (Chapter 3) were studied using viability and ultrastructural assessment, with the aim of establishing methods for both short-term and the long-term preservation, via appropriate seed storage and cryopreservation, respectively. The effect of cryopreservation on genetic fidelity, a crucial aspect of germplasm conservation, was assessed by polymerase chain reaction (PCR) based random amplified polymorphic DNA (RAPDs), using W. salutaris as a case-study (Chapter 4). The seeds of all three species were found to exhibit non-orthodox behaviour. On relatively slow-drying, seeds of T. dregeana and T. emetica lost viability and ultrastructural integrity at axis water contents of 0.55 g g-l (achieved over 6 d) and 0.42 g g-l (after 3 d) respectively, while flash-drying of embryonic axes facilitated their tolerance of water contents as low as 0.16 g g-l (T. dregeana, flash-dried for 4 h) and 0.26 (T. emetica, flash-dried for 90 min). Seeds of W. salutaris were relatively more tolerant to desiccation, remaining viable at axis water contents below 0.1 g g-l when desiccated for 6 d in activated silica gel. However, excised embryonic axes flash-dried to similar water contents over 90 min lost viability and were ultrastructurally damaged beyond functionality. In terms of storability of the seeds, those of T. dregeana could be stored in the fully hydrated state for at least 5 months, provided that the quality was high and microbial contamination was curtailed at onset of storage, while those T. emetica remained in hydrated storage for about 60 d, before all seeds germinated in storage. Seeds of W salutaris, even though relatively tolerant to desiccation, were not practically storable at reduced water content, losing viability within 49 d when stored at an axis water content of 0.1 g g-l. The seeds of all three species were sensitive to chilling, suffering extensive subcellular derangement, accompanied by loss of viability, when stored at 6 °C. Thus, T. dregeana and T. emetica are typically recalcitrant, while those of W. salutaris are suggested to fit within the intermediate category of seed behaviour. For either recalcitrant or intermediate seeds, the only feasible method of long-term germpalsm preservation may be cryopreservation. Subsequent studies established that whole seeds of W. salutaris could be successfully cryopreserved following dehydration in activated silica gel. However, whole seeds of T. dregeana and T. emetica were unsuitable for cryopreservation, and excised embryonic axes were utilised. For these, in vitro germination methods, as well as cryoprotection, dehydration, freezing and thawing protocols were established. Post-thaw survival of the axes of both species was shown to depend on cryoprotection, rapid dehydration and cooling (freezing) in cryovials. Embryonic axes of T. dregeana regenerated only as callus after cryopreservation, while those of T. emetica generated into apparently normal plantlets. Thawing/rehydration in a 1:1 solution of 1 µM CaC12.2H2O and 1 mM MgC12.6H2O increased the percentage of axes surviving freezing, and that of T. emetica axes developing shoots. The effect of the extent of seed/axis development on onward growth after cryopreservation was apparent for seeds of W. salutaris and excised axes of T. emetica. The seeds of W. salutaris surviving after cryopreservation germinated into seedlings which appeared similar to those from non-cryopreserved seeds, both morphologically and in terms of growth rate. Analysis using PCR-RAPDs revealed that there were no differences in both nucleotide diversity or divergence, among populations of seedlings from seeds which had been sown fresh, or those which had either been dehydrated only, or dehydrated and cryopreserved. Thus, neither dehydration alone, nor dehydration followed by cryopreservation, was associated with any discernible genomic change. The above results are reported and discussed in detail in Chapters 2 to 4, and recommendations and future prospects outlined in Chapter 5.
Post-Harvest seed physiology and conservation of the germplasm of syzgium cordatum hochst.
(2013) Cheruiyot, Anastacia Chepkorir.; Kioko, Joseph Ivala.
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.
The effect of developmental status and excision injury on the success of cryopreservation of germplasm from non-orthodox seeds.
(2007) Goveia, Meagan Jayne Theresa.; Kioko, Joseph Ivala.; Berjak, Patricia.; Pammenter, Norman William.
The zygotic germplasm of plant species producing desiccation-sensitive seeds can be conserved in the long-term only by cryopreservation. Usually the embryonic axis is excised from the cotyledons and is used as the explant for cryopreservation as it is small and provides a large surface area:volume ratio. However the shoot of the axis of most species studied does not develop after excision, with the result that survival after cryopreservation is often recorded as callus production or simply explant enlargement and/or greening. Thus, besides explant size, factors such as in vitro regeneration techniques, physical injury induced upon excision and developmental status of the seed could compromise the success of cryopreservation. This study investigated the effect of the factors mentioned above, with particular attention to the developmental status of the seeds on explant in vitro development (section 3.1), response to dehydration (section 3.2) and cryopreservation of the desiccation-sensitive embryonic axes (section 3.3) of two species: Trichilia dregeana, T. emetica and embryos of a third, Strychnos gerrardii. For all three species, investigations were conducted on the embryonic axes/embryos excised from mature seeds immediately after fruit harvesting and from mature seeds stored under hydrated conditions for different periods (in order to achieve different degrees of development). In addition, preliminary studies were carried out on axes of T. dregeana to assess whether generation of reactive oxygen species (ROS) occurs in response to wounding upon axis excision (section 3.4). Excised embryonic axes of T. dregeana and T. emetica did not develop shoots in vitro unless the explants included attached cotyledonary segments. Following the development associated with short-term storage, however, the excised axes could develop shoots after complete cotyledon excision. The embryos from the (endospermous) seeds of S. gerrardii which included the paper-thin cotyledons, developed normally in vitro, with percentage germination increasing with seed storage time. For all three species, in vitro axis germination was promoted when activated charcoal was included in the germination medium, regardless of the developmental stage of the seeds. When dehydrated to approximately 0.3 g H2O g-1 dry mass (g g-1), embryonic axes from all three species failed to develop shoots even though a minimum of 50% produced roots in all cases. Hence, shoot production was shown to be more sensitive to desiccation than was root production. Furthermore, the sensitivity of the shoot apical meristem to desiccation was not ameliorated with seed storage for T. dregeana and T. emetica, but did decrease for S. gerrardii when seeds were stored for 6 – 8 weeks. The application of certain cryoprotectants did facilitate production of shoots after dehydration by a few axes of both Trichilia spp. For T. dregeana explants, combination of glycerol and sucrose allowed for 10% of the axes to retain the ability for shoot production after dehydration while for T. emetica explants, the combination of DMSO and glycerol (10 - 20% shoot production after dehydration) was best. The efficacy of the cryoprotectants was not influenced by storage period. The provision of cryoprotectants still needs to be tested for S. gerrardii. Survival of subsequent cryopreservation of T. dregeana and S. gerrardii explants was best achieved with rapid cooling in nitrogen slush, with the cooling procedure for T. emetica explants still to be optimized. The highest post-cryopreservation survival of T. dregeana axes was achieved when seeds had been stored for three months, while the seed storage period did not affect post-thaw survival of the axes of T. emetica or S. gerrardii. A small proportion of S. gerrardii explants only, produced shoots after cryopreservation, whereas the surviving embryonic axes of T. dregeana and T. emetica regenerated only as non-embryogenic callus. Although callus production is less desirable than successful seedling establishment, it has the potential for micropropagation if embryogenicity can be induced. Ultrastructural examination of the shoot apical meristem of T. dregeana after a 3-d recovery period, following excision, revealed considerable cellular derangement, although damage of individual organelles could not be resolved microscopically. Preliminary studies on T. dregeana involving a colorimetric assay using epinephrine, confirmed the generation of ROS in response to wounding associated with axis excision. Reactive oxygen species generated appeared to persist over prolonged periods rather than occurring only as a single oxidative burst. Hence, ROS production at the wound site could be the primary factor contributing to lack of shoot development. Axes immersed in the anti-oxidant, ascorbic acid (AsA) immediately after excision, showed lower ROS production and 10% shoot development when cultured in vitro, indicating that the oxidative burst coincident with, and after excision might be counteracted if immediate ROS production can be adequately quenched. Future investigations should aim to identify the specific ROS associated with wounding and optimize an anti-oxidant treatment(s) that will facilitate shoot development. Thus, the successful cryopreservation of the germplasm of the species tested, and others producing recalcitrant seeds, depends on a spectrum of species-specific factors, some still to be elucidated.