CHAPTER 3: RESULTS
4.4 Responses of T. dregeana from each provenance to storage under chilling
Recalcitrant seeds are not only desiccation sensitive, some are also sensitive to chilling, especially those from tropical/subtropical origin (Nishida and Murata, 1996). Some recalcitrant species, such as mango, cocoa and Borneo camphor, are sensitive to moderate chilling temperature of 10oC to 15oC (Roberts and King, 1980), while some others, such as fluted pumpkin, will lose viability within two weeks of storage at 6oC (Ajayi et al., 2006). T. emetica also lost viability steadily with storage time at 6oC (Kioko et al., 2006).
In the present study, T. dregeana seeds from the four locations were stored at 3oC, 6oC and 16oC. One consistent observation was that the water content of the seeds’ embryonic axes increased with storage time. Additionally, the lower the storage temperature, the higher the increase in water content of the seeds stored at that temperature. One possible cause of this observation may be the storage method. The seeds were packed in brown paper bags and sealed in plastic bags. This storage method did not provide any medium for water that is given off during respiration to be absorbed; possibly, the water was reabsorbed by the seed, leading to an increase in water content. The seeds were also dusted with benomyl, which became moistened (by the water that was given off during respiration) with storage time, and in turn, kept the seeds moistened throughout the storage period. The effect of this may also contribute to the observed increase in water content. Walker et al. (1991) also reported a significant increase in water content when 4- weeks old plants of chilling-sensitive cultivated tomato were stored under chilling conditions.
The lower the storage temperature, the higher the sensitivity to that temperature and the least the survival observed in each provenance. Gualanduzzi et al. (2009) suggested that, post-harvest storage at different chilling temperatures impose different degree of chilling stress. Pammenter et al. (1994) suggested that the accumulation of damage is positively related to the duration of applied stress (in this case, chilling). Also, it was reported by Whitaker (1995) that the effect of chilling can be primary or secondary. The primary injury is a physical injury that occurs instantaneously at short-term exposure to threshold chilling temperature but it is reversible (Engelman, 1991). On the other hand the irreversible, secondary injury is at biochemical/metabolic level, which likely takes place when tissues are stored for longer period under chilling conditions. In this study, seeds stored at 3oC had the lowest viability while those stored at 16oC had the highest viability, across the four provenances. Also, for each of the storage temperatures across the four provenances, it was observed that the longer the storage period, the lower the viability.
However, the survival of the seeds from each provenance, at each storage temperature, was different. At 3oC, seeds from MTZN were the most sensitive, losing viability drastically at eight weeks and total loss from the 12th week. On the other hand, PMB seeds were the most tolerant, as they were still able to maintain some viability until the 16th week. The survival at this temperature was in the order MTZN < DBN < P.ED <
PMB. At 6oC, only seeds from MTZN and P.ED totally lost viability at some weeks, i.e.
MTZN at 18th week and P.ED at 14th and 16th week. DBN and PMB seeds maintained some viability, howbeit little, throughout the 18 weeks in storage. However, mean viability, at this storage temperature, was in the order MTZN = DBN < PMB < P.ED.
Viability was not totally lost for seeds stored at 16oC throughout the 18-week storage treatment, across the four locations. However, seeds from P.ED were the most tolerant while MTZN were the most sensitive to this storage temperature. The order of survival at this temperature was MTZN < DBN < PMB < P.ED.
The underlying causes of chilling sensitivity in recalcitrant seeds are not very clear, neither is this topic as extensively researched as desiccation sensitivity. However, past studies suggest some main effects of chilling. Chilling temperature affects the function of enzymes within the cells. Reduction in temperature below a threshold can cause
enzymatic activities and functions to be disturbed, consequently, agitating cell functions (King and Roberts, 1980a, 1980b). The effect of chilling injury on TCA cycle has been suggested by Berjak et al. (1996). Recently, decrease in the synthesis of citrate in the TCA cycle, was observed by Tsuchida et al. (2010), when cucumber fruits where stored under chilling conditions.
Adverse effects of chilling have also been reported at the ultrastructural level. Niki et al.
(1978) reported distruptions in cell organelles like the nuclei, mitochondria, ribosomes and tonoplast. When the callus tissues of Cornus stolonifera were kept at 0oC, those authors also observed ultrastructural changes in proplastids and rough endoplasmic reticulum (RER). The RER was reported to release ribosomes/microvesicles and thus were turned to smooth endoplasmic reticulum. The microvesicles in turn, developed into large vesicles and occupied a large portion of the cytoplasm. These changes were believed to be consequences of degradative processes taking place within the cells as a result of cold stress on the callus tissues. Hydrated storage of Azadirachta indica seeds under chilling conditions resulted in reduced viability which was accompanied by the degeneration of the organelles such as mitochondria, plastids, vacuoles and the plasmalemma (Berjak et al., 1995). Similarly, organelles within the cells of T. emetica seeds were seriously degraded and the plasmalemma had receded from the cell wall after 20 d at 6oC (Kioko et al., 2006).
Another damage associated with chilling sensitivity is membrane phase change (from liquid crystalline to gel state) and phase separation/redistribution of membrane components. Associated with temperature induced phase changes of membrane lipids are also changes in their permeability, fluidity and the viscosity (Yoshida and Niki, 1979;
Platt-Aloia and Thomson, 1992; Nishida and Murata, 1996; Berjak et al., 1996; Sacandé et al., 2001; Campos et al., 2003; Gualanduzzi et al., 2009). Production of reactive oxygen species (ROS) which consequently lead to lipid peroxidation and oxidative stress is associated with chilling injury as well, as it was observed in zucchini squash fruits stored under chilling conditions (Gualanduzzi et al., 2009).
Deleterious effects of chilling sensitivity are not marked in seeds only but also in fruits like in zucchini (Gualanduzzi et al., 2009), mango (Dea et al., 2010), cucumber (Tsuchida et al., 2010); photosynthetic activities as in coffee seedlings (Oliveira et al., 2002) and in leaves (Campos et al., 2003). Storage of 4-weeks old plants of Lycopersicon esculentum and rooted cutting of L. hirsutum at 2oC for 3 d reduced the uptake of photosynthetic CO2 and stomatal conductance (Walker et al., 1991). All the phenological stages of reproduction was reduced in chickpea, as well as abscission of young buds and flowers and abortion of pods at < 10oC in the field. Pollen viability was also suppressed under this condition. Chilling also caused chlorosis, necrosis and curling of the leaves of chickpea (Kumar et al., 2010)
The effects of chilling highlighted above are impacting cells either independently or collectively; but the ultimate effect is cell death, leading to lack of germination or low seedling vigour. These effects increase with increased storage period and with lower chilling temperatures.
4.5 Comparison between T. dregeana seeds harvested from Mtunzini in 2009 and