The effects of detergent action on several photochemical and physical properties of chloroplast lamellae have been investigated. The general observations made by other authors who have employed detergents in previous reports have been largely confirmed. It has not however, been found possible to ascribe any of the particular effects which have been observed on the properties of lamellae, to any characteristic of molecular structure and/or polarity of the detergents studied. One detergent of particular interest has emerged from this survey, in the form of G. 711. No other instance is known of any detergent which is an efficient solubiliser of chloroplast lamellae and yet which does not inhibit System II reactions to a high degree at low concentrations of detergent. Of the two photosynthetic photoacts, least is known of the short wavelength System II. The detergent may be of considerable use in future work on the elucidation of the mechanism of the photosynthetic photoevolution of oxygen from water.

Proteolytic enzymes are common in plants but are usually specifi to endogenous protein. Plant proteases with specificities applicable to the food industry include papain, ficin and bromelain. Other plants have been used in traditional methods of food preparation for their proteolytic action on food components. The following species were investigated for propagation in tissue culture: Carica papaya, Ficus carica, Cynara cardunculus, Galium verum, Circium arvense, Dieffenbachia amoena, D. picta and Ananas comosus. Tissues of the first five of these demonstrated proteolytic activity by clearing of milk turbidity in agar medium. Commercial papain and ficin preparations are currently obtained from latex of immature papaya and fig fruit, respectively. This investigation was conducted, in part, to determine the feasibility of producing these two enzymes by the in vitro cell culture technique. Standard method of aseptic seed germination and leaf tissue excision were employed for callus initiation. Cell suspension cultures derived from callus were maintained in B5 medium at 28 °C in darkness. Proteolytic activity was determined by a modification of the Food Chemicals Codex method for papain and protein content was determined by Bradford's dye-binding, method. Production of protein and protease varied among cell cultures, but could be influenced by changes to some nutritional factors. Fig cells were grown in medium supplemented with single amino acids in the presence of either nitrate or ammonia as a source of inorganic nitrogen. All nitrate-based media produced higher yields of cell dry weight than ammonia-based media. Glutamic and aspartic acids were most stimulatory growth, protein accumulation and protease activity of fig cells. Skimmed milk, added at 3% (v/v), was a highly effective growth stimulant, and also resulted in higher protein and protease levels than the amino acids. Fresh casein and whey, added individually, produced similar results to skimmed milk. Citric acid, added at the level found in the 3% milk supplement, also caused stimulation of fig cell growth, protein synthesis and protease activity not significantly different from skimmed milk. It appears that nitrogen accumulation and reduction in fig cells may have been limited by an energy requirement which could be satisfied with the addition of citric acid or milk whey to the basal medium. / Land and Food Systems, Faculty of / Graduate

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Vita. / Digitized by Kansas Correctional Industries

Call number: LD2668 .T4 1957 C37 / Master of Science

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Dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, Johannesburg, South Africa / In the forestry industry the requirement for the maintenance of a broad genetic base is integral to the success of breeding programmes such as those for Eucalyptus grandis, an important species to the South African forestry industry. Plant cryopreservation is an economical option to maintain such a genetic base, as it allows storage of vegetative materials at sub-zero temperatures, while maintaining juvenility. However, successful cryopreservation of this sub-tropical species has been restricted by its sensitivity to cryopreparative drying. As a consequence, the viability of material is reduced even before reaching the freezing stage. Despite this abiotic stress restriction, evidence of upstream ‘cross-talk’ implying downstream ‘cross-tolerance’ has suggested the possibility that cold acclimation may improve the tolerance responses towards dehydration stress by means of ‘cross-acclimation’. It was therefore the aim of the study to understand some of the physiological and biochemical responses of in vitro E. grandis shoots to different non-freezing low (chilling) temperatures and exposure periods, and to establish an appropriate ‘cross-acclimation’ regime for the physical drying pre-treatment. E. grandis shoot clusters (4-8 leaves and 2-5 axillary buds) were exposed to the chilling temperatures of 5°C, 10°C or 15°C for 1 or 3 days. The physiological and biochemical responses were evaluated, and thereafter the appropriate cold acclimation (or ‘cross-acclimation’) regime selected. The appropriate physical drying time was also selected for shoot clusters according to their physiological responses. When the appropriate regimes had been determined, the physiological and biochemical responses of shoot clusters treated consecutively to cold acclimation and then physical drying were evaluated. The physiological responses evaluated were water content, viability, and vigour (i.e. the number of visible axillary buds and shoots produced over 2 weeks). The biochemical responses measured were the concentrations of: 1) total soluble sugars, 2) starch, 3) phenolic acid, and 4) superoxide. The data suggested that the appropriate cold acclimation regime was treatment at 10°C for 3 days. This was based on the accumulation of the high levels of phenolic acid (3.05 ± 0.09 mg GAE.g-1 FWS) and positive vigour responses (11.90 ± 0.60 visible axillary buds/week and 3.10 ± 0.20 visible shoots/week), compared with the other chilling temperature treatments. The appropriate drying time selected for shoot clusters was 80 min over activated silica gel to achieve a water content of 0.32 ± 0.04 g water.g-1 FWS. In the dried material there were high levels of soluble sugars (47.65 ± 1.90% of the fresh weight of shoots) and unknown components that accounted for 48.10 ± 1.86% of the fresh weight, followed by phenolic acid (3.09 ± 0.05%) and proline (0.490 ± 0.011%). Despite these measured responses, viability of the shoots was impacted by drying, dropping to 88.9 ± 3.9%. When shoot clusters were pre-treated at 10°C for 3 days and then physically dried, viability of all (100%) the material was retained and the water content did not drop as low as with physical drying alone, dropping to 0.52 ± 0.05 g water.g-1 FWS. The biochemical responses showed that tolerance was strongly dependent on a high proportion of soluble sugars (83.66 ± 1.48% of the fresh weight of shoots) and phenolic acid (3.77 ± 0.12%), followed by proline (0.406 ± 0.018%). The study had confirmed that ‘cross-acclimation’ through means of cold acclimation (chilling pre-treatment at 10°C for 3 days) can induce ‘cross-tolerance’ towards physical drying, where osmotic adjustments and osmoprotection appeared to have been improved. It is therefore possible that this may have the potential to improve survival during the latter stages of the cryopreservation procedure, despite the higher retention of water in shoot clusters after drying.