Cryopreservation of Pyramimonas mucifera.

BACKGROUND: It is important to appreciate microalgal diversity, better understand their ecosystem functioning and therefore implement conservation measures. The National Biodiversity Act of South Africa has a marine and coastal component which promotes such investigations. OBJECTIVE: To develop a cryostorage method for the marine unicellular algal species Pyramimonas mucifera. MATERIALS AND METHODS: Cell viability, measured by propidium iodide, was used to determine both optimal exposure time to 10 % DMSO and survival following thawing of cryopreserved cells. Cryopreservation was achieved by a two-step cooling method. RESULTS AND DISCUSSION: A 30-min DMSO exposure was selected for P. mucifera, as cells following such treatment retained cell shape and integrity. Although density was significantly reduced after cryopreservation, the surviving cells were capable of returning to viability levels equal to those of the untreated control (> 90%). CONCLUSION: Cultures of P. mucifera can be successfully cryopreserved and propidium iodide provides a useful indication of culture vitality.

Responses of the Buds of Three South African Sweet Potato (Ipomoea batatas) Accessions to Different Cryoprotectants.

BACKGROUND: Plant Vitrification Solution 2 (PVS2) was reported to be a successful cryoprotectant solution in regenerating plantlets from sweet potato shoot tips after cryostorage. However, this was not the case for the South African sweet potato accessions. OBJECTIVE: This study assessed the responses of buds of sweet potato to different concentrations of the components of PVS2 both individually and in combination. MATERIALS AND METHODS: The percentage material that regenerated into plantlets was recorded against the total that was evaluated. RESULTS: Regeneration of buds after treatment with 5% dimethyl sulphoxide was significantly better (p<0.001) than 10-15% dimethyl sulphoxide while exposure to ethylene glycol concentrations of 5-10% were better than 15% for all but one accession. Significant difference (p<0.001) was observed in treatment with 30-40% glycerol. Combination of the components at 5% (PVS5%) was better than 10-15% dimethyl sulphoxide and ethylene glycol and 20% glycerol. CONCLUSION: It is recommended that PVS5% be used for further development of the cryopreservation protocol for the South African accessions.

Programmed cell death and necrosis during cryopreparative drying of in vitro Eucalyptus grandis axillary buds.

In preparation for cryopreservation, Eucalyptus grandis in vitro axillary buds were dried over silica gel. Pretreatment of the buds with 5 mg per L ABA resulted in partial resistance to water loss (0.76 to 0.45 g per g fresh mass basis) as compared with untreated buds (0.76 to 0.33 g per g) and was associated with the retention of viability (70 vs. 55%). The loss of viability of the dried buds was protracted over several days. Ultrastructural examination and vital staining demonstrated cellular and tissue responses to drying. The meristem appeared to withstand drying and 72 h of rehydration whilst the leaf primordia were destroyed immediately after drying. High reactive oxygen species (ROS) activity was associated with bud excision and drying. Caspase-3-like protease activity was detected after rehydration, thereby providing evidence that the dried buds, that had ultimately died, had undergone programmed cell death. ROS production is considered to be the trigger for programmed cell death.

Effect of medium salt concentration on differentiation and maturation of somatic embryos of cassava (Manihot esculenta Crantz).

Culture of cassava somatic embryos on media with an altered macro- and micro-nutrient salt concentration affected embryo development and germination capability. In the tests, quarter-, half-, full- or double-strength Murashige and Skoog (MS) media were compared. The maximum number of somatic embryos differentiated from a proliferative nodular embryogenic callus (NEC) on either half- or full-strength MS medium, and the greatest numbers of cotyledonary stage embryos were formed on full-strength MS medium. Developed somatic embryos were then desiccated above a saturated K2SO4 solution for 10 d. After transfer to germination medium, embryos that had developed on half- and full-strength MS medium yielded 8.3 and 8.6 germinants g(-1) NEC tissue, respectively. For this important but often disregarded culture factor, either half- or full-strength MS medium is recommended for both the differentiation and development of cassava somatic embryos that are capable of germination.

The sequence of the preparative procedures affects the success of cryostorage of cassava somatic embryos.

Cassava primary somatic embryos were prepared for cryostorage using both physical drying and cryoprotectant application stages. The effects of the sequence by which these preparative events were applied to the material was investigated. The drying of the somatic embryos before the application of the cryoprotectants resulted in a significantly higher viability after cryostorage than when the material was first exposed to the cryoprotectants and then dried. The impact of these preparative procedures on sub-cellular detail was monitored by means of transmission electron microscopy.

In defence of aldehyde-osmium fixation and critical-point drying for characterization of seed-storage fungi by scanning electron microscopy.

Low-temperature scanning electron microscopy cannot be used as a general approach when resolution of definitive surface features is required for fungal characterization, because of the production of a pervading exudate by some of the species, strains or varieties. As the resolution of the light microscope is obviously limiting, scanning electron microscopy remains the best microscopical mode. Results are presented for four species within the Aspergillus flavus group which indicate that minimal (if any) distortion or collapse can be achieved with concomitant preservation of surface morphology, when particular precautions are taken during fixation and critical-point drying. Details of the steps pertaining to preparation for scanning electron microscopy following critical-point drying that are considered collectively to contribute to the success of the approach are discussed.