As a consequence of previous lack of success in cryostoring axes excised from newly shed seeds of Trichilia dregeana , the effects of the mode of axis excision on seedling production were investigated. Although vigorous root production occurred, no shoots were produced when the cotyledons were severed as closely as possible to the axis surface (explant-type 0). In contrast, shoot production was increasingly facilitated when small to larger segments of cotyledonary tissue were left attached to the axes (explant-types 1, 2 and 4), which did not compromise axis drying rate. However, root growth of explant-types 1, 2 and 4 was negatively affected, probably by leakage into the medium of an inhibitory or toxic substance(s) from the cut surfaces of the cotyledonary tissue. Microscopical examination revealed that the cotyledons were sessile, and their insertions were contiguous with the shoot apex in axes from newly shed seeds, leading to the suggestion that failure of shoot production by type 0 explants in vitro was the direct consequence of the proximity of the wound sites to the apical meristem. When seeds were stored hydrated for 6 months, the shoot apex had elongated, positioning the apical meristem some distance from the top of the cotyledonary insertions. In contrast to axes excised as type 0 explants from newly shed seeds, the equivalent explants from the stored seeds rapidly formed shoots and leaves in vitro . This indicates that the developmental status of axes, when excised, dictates failure or success in their further development in vitro , and that this aspect needs to be resolved before any further manipulations for cryostorage are attempted.
A study was conducted to optimize the seed cryopreservation protocol to improve post-thaw recovery, and to assess, using PCR-based random amplified polymorphic DNA, the effect of cryopreservation on the genetic fidelity of plants regenerated from cryopreserved seeds. Seeds from freshly collected green hard fruits were non-germinable, while seeds from other stages attained 100% germination, even after dehydration to ∼0.1 g/g. After cryopreservation, seeds from yellow-green and green hard fruits that were stored until they were soft attained higher germination percentages than seeds from brown fruits. No morphological differences were apparent in seedlings or saplings from freshly sown, dehydrated or cryopreserved seeds. DNA concentration from different seedlings was 20-1015 ng/µl. Of the ten primers tested, two (OPA 02 and OPA 18) resulted in no amplification. The remaining primers produced 104 fragments. Each primer produced a distinct pattern, but for each primer, most samples produced identical banding patterns. These bands, consistent even over a 100-fold range in DNA concentration, were scored. Among the bands, some polymorphisms were detectable within and between the three populations (treatments): seedlings from (1) freshly collected, non-dried seeds, (2) dehydrated seeds, and (3) seeds cryopreserved in liquid nitrogen. All populations exhibited varying nucleotide diversities. Significantly, the nucleotide divergence between the seedling populations (0.08-0.17%) was much lower than the nucleotide diversity within them. There was higher variation within each treatment than between the treatments. The phenogram of individuals from all three treatments showed 95-65% similarities among individuals, and a variation among individuals from all treatments. Neither dehydration nor cryopreservation had an effect on genetic fidelity of seeds.
A study was conducted to determine the optimum methods for conditioning explants to be used in the development of a simple protocol for long-term conservation of the germplasm of Dioscorea rotundata via cryopreservation. Shoot tips from cultures maintained in vitro were exposed to high concentrations of sucrose prior to silica gel-based dehydration and vitrification solution-based cryopreservation protocols. Explant water contents were determined, and ultrastructural studies were also carried out. Initially, culturing explants on medium supplemented with 0.3 M sucrose for 3-5 d considerably reduced tissue water content from about 12.2 g/g dry mass to between 4.8 and 5.5 g/g dry mass before cryoprotection with modified PVS2 (MPVS2) or silica gel dehydration. Ultrastructural studies indicated that cells had deposits of starch in plastids following sucrose treatments. Survival for D. rotundata shoot tips treated with MPVS2 vitrification solution, unloaded with 1.0 M sucrose medium and cooled to -7 degree C, was 16 percent for 15 min treatment and 44 percent for 40 min. After the 40 min MPVS2 treatment the TTZ test indicated 88 percent viability retention of explants cooled to -70 degree C, and 44 percent at -196 degree C. Plantlet development was obtained for -70 degree C-cooled shoot tips, whereas only callus development occurred from tissues exposed to liquid nitrogen. Explant regeneration was not obtained with air-dehydration techniques. It was concluded that vitrification-solution based cryopreservation presently offers the best option for conservation of this Dioscorea species.
The seeds of Trichilia emetica, a multi-purpose tropical forest species, displayed typical recalcitrant behaviour, being shed at an average axis water concentration of 2.82 g per g dry matter (g g− 1), and losing viability when dehydrated to axis water concentrations below 0.42 and 0.26 g g− 1, when dried slowly or rapidly, respectively. The ultrastructure at shedding was indicative of active metabolism, as would be expected of mature recalcitrant seeds which grade into germinative metabolism after shedding. Rapid dehydration enabled the maintenance of ultrastructural integrity to water concentrations as low as 0.3 g g− 1, while cells of axes dried slowly to similar water concentrations displayed total subcellular destruction. In the fully hydrated state, the storage lifespan of the seeds was limited to 60 days at 16 °C, after which all the seeds had germinated in storage. Ultrastructural examination, however, indicated that prolonged mild water stress had occurred, which the seeds are suggested to have suffered as germination proceeded in storage. When stored at 6 °C, the seeds showed extensive ultrastructural derangement, which was accompanied by loss of viability after 20 days, presumably as a result of chilling injury, while storage at 25 °C resulted in all seeds germinating in storage in 35–40 days. Even though the seedcoat has been shown to inhibit germination, it did not appear to affect seed longevity or germination in storage at any of the temperatures used.
In vitro asybiotic seed germination of rare Phalaeonopsis species
and hybrids
JICSTDA: Joint International Conference on Science and Technology
for Development in Africa, Cape Town, 26-28 June 2012
<p>Concerns over the negative impacts of climate change on ecosystems and human life have entered a new phase where many hypothetical views are fast becoming realities. Presently, the rampaging effect of climate change is, in theory, causing ecological catastrophes, and it is being felt at an alarming scale worldwide. As an important ecological niche, the soil ecosystem hosts a diversity of microbiomes and macrobiomes and affords a soil-plant-microbes ecological continuum. Also, it supports essential ecological processes meant to promote life-sustaining habits. However, changes in plant diversity due to increasing greenhouse effects, anthropogenic activities, and global warming have severely impacted the stability of soil microbial communities and interactions, particularly the soil-plant-microbe interaction. A good understanding of the mechanisms underpinning the plant-soil-microbial interactions, the complexity of the soil microbiome, ecosystem adaptability to climate change-induced stresses, and niche functionality of microbiota is necessary for the empirical impact assessment of climate change on soil microbial behaviors. Moreover, the soil system parameters and the various ecological services affected need to be further studied to identify opportunities that could assist the quest to mitigate the debilitating effects of climatic change in the soil ecosystem and sustainable food security initiatives.</p>
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