Effect of elevated temperature after PM on seed quality

During the post-PM stage, after attaining maximum weight, seeds begin to enter into the maturation drying phase, and the connection between the mother plant is diminished (Bewley et al., 2012). An association between post-PM environmental conditions and seed germination and seed vigour loss has been reported by many researchers (Castillo et al., 1993; Castillo et al., 1994; Siddique & Wright, 2004; TeKrony & Hunter, 1995; Wang et al., 2012). Results of the present field study showed a linear decrease in both seed germination and vigour with increasing number of hours above 25 ˚C and increasing HTT; the incidence of abnormal seedlings increased when daily mean maximum temperature increased from 25 ˚C to 34˚C (range of increase during phase-II, was 33.4 -34 ˚C over the two seasons (Figure 4.2 (c) and 4.3 (c)). The elevated temperature during phase-II (PM →HM) in this study did not have a significant effect on seed mass because seeds had reached their maximum mass by PM.

Elevated temperature during phase-II (PM →HM), thus significantly reduced seed germination and vigour (AA) in both seasons. However, the effect was more prominent in the second season because of the exposure to higher temperature (HTT) during this season than in the 2011-12 season (605 cf. 340 ˚Ch). In contrast, seeds of commercially acceptable quality (>90%) levels of seed germination and seed vigour (AA) were harvested from control plants exposed to ambient phase-II (PM →HM) field temperature during this time. Hourly thermal time (HTT) (Tb= 25 ˚C) was significantly correlated with germination and vigour (AA) (Figure 4.5 & 4.6) and the correlation for two seasons indicated a consistent negative effect (Figure 4.5 & 4.6). Similarly, increasing number of hours above 25 ˚C also showed a significant correlation with seed germination and vigour (Figure 4.9 and 4.10). The decrease in seed vigour with temperature above 25 ˚C during phase-II (PM →HM) was also characterized by high seed conductivity, resulting in a significant linear negative relationship between (HTT) (Tb= 25 ˚C) and seed conductivity (Figure 4.7). A negative correlation between the two seed vigour tests (AA and conductivity test) indicated that either could be used for vigour testing of forage rape (Figure 4.4). The temperature inside the plastic sheet cages was not controlled and it is acknowledged that by manipulating the ambient field temperature in this way, the duration, intensity and diurnal difference were substantially greater than normal field conditions. However, the response to heat stress during phase-II(PM →HM), was well described by linear regression, suggesting that around 300 ˚C h between PM and HM were required to produce a commercially unacceptable seed lot (seed vigour < 80%, as assessed by the AA test). It has been reported that seed vigour (AA-germination) of 80% can be taken as a minimum acceptable standard for planting seed in a wide range of field environment for acceptable emergence (Egli & TeKrony, 1995, 1996). Further, the significant correlation between numbers of hours above 25 ˚C and seed vigour also indicated that at least 100 hours above 25 ˚C were required to produce low vigour seed.

The present study demonstrated that seed conductivity increased as the degree hours (°Ch) increased, indicating that membrane integrity has been compromised due to this higher post-PM temperature. Signs of membrane destabilization as indicated by higher electrolyte leakage have been associated with the incidence of abnormal seedlings and attributed to the damage or death of some tissues in the seed parts, especially in the meristematic tissues (Marcos Filho, 2015).This relationship between conductivity and abnormal seedlings was also found in the present study; there was a significant relationship between conductivity and AA, with the reduction in AA-germination being caused by abnormal seedling production, not seed death. The conductivity test was able to detect this early seed deterioration (cellular membrane damage).In the present study, conductivity of non-stressed forage rape seeds was similar to the conductivity ranges proposed by Elias and Copeland (1997) for canola (Brassica napus). The integrity and functions of the membranes are sensitive to high temperature and as has been reported in earlier studies, heat stress disrupts cellular membrane stability and functions, thus enhancing the permeability of membranes to ions (Bailly et al., 2002b; Wahid & Shabbir, 2005). This phenomenon is the most cited cause of seed deterioration (McDonald, 1999). The changes in structure and functions of membranes are mostly because of an alteration in phospholipids’ fatty acid composition (Larkindale & Huang, 2004).High temperature modifies the phospholipid compositions resulting in loss of membrane integrity due to changes in membrane configuration or structure and/or changes in properties of membrane-bound enzymes which lead to the leakage of ions in incubation water (Ren et al., 2009; Taiz & Zeiger, 1998).Heat stress after PM has also been reported to increase seed conductivity due to its effect on seed metabolism and the ability to maintain optimum metabolic activity, which leads to physiological changes (Dornbos et al., 1989; Grass & Burris, 1995b). Therefore, results of this study are consistent with these reports suggesting that heat stress may have modified the compositions of fatty acids in phospholipids leading to the increased electrolyte leakage and a reduction in seed vigour. Additionally, oxidative stress and overproduction of reactive oxygen species (ROS) by heat stress may also lead to lipid peroxidation of the cellular membranes and thus increase electrolyte leakage leading to loss of vigour and viability (Camejo et al., 2006; Rodríguez et al., 2005).This is confirmed in Chapter 5, where a reduction in seed germination and vigour due to heat stress was related with ROS (H2O2) accumulation and lipid peroxidation. The mechanism of H2O2

accumulation and lipid peroxidation is presented in Chapter 5.

A further, possible reason for reduction in seed vigour during this phase might be due to inactivation of membrane bound antioxidant enzymes which are necessary to detoxify excessive ROS production to minimize their deleterious effects on the cells and membrane lipid peroxidation; and/or reduced respiratory activity by reduction in ATP activity due to alteration in mitochondrial structure or functions as reported by Grass and Burris (1995b). They suggested that heat stress post PM reduced mitochondrial activity through ultrastructural changes in mitochondria and their functions. This is

confirmed in Chapter 6, where reduced activity of antioxidant enzymes and low ATP energy levels were recorded in low vigour heat stressed seeds.

In this study, the results confirmed those presented in Chapter 3, is that seeds from the top of the raceme position had slightly lower germination and vigour than those from the middle and basal raceme positions and this effect was more pronounced during post-PM heat stress. This raceme position effect was more profound with respect to seed vigour in both seasons. Reason for this have been discussed in Chapter 3.

Climatic conditions in Canterbury, New Zealand during December and the following January are very important for high quality forage rape seed production. In the present study, most of the temperature increase above 25 ˚C occurred from the last week of December until the middle of January in both seasons (Figure 4.2 and 4.3). Thus, a forage rape seed grower may avoid this situation of adverse climatic conditions during forage rape seed maturation by carefully selecting a region or area of production where temperature seldom rises above 25 ˚C during the last three weeks of seed maturation to avoid seed vigour loss.

Another possible option is to change sowing date to avoid the heat stress at this critical stage of seed development, and/or seeds may be harvested before they have reached HM and dried under controlled conditions to maintain high quality seeds, thereby reducing pre-HM seed deterioration.

4.6

Conclusions

• Exposure to elevated temperature of more than 100 ˚C h (Tb= 25 ˚C) during phase-I (before

PM) when SMC was between 80-50% significantly decreased seed germination and seed vigour in both seasons and was correlated with increasing hourly thermal time (HTT) and the number of hours exceeding 25 ˚C.

• Seed mass was also decreased with higher temperature before PM but was not decreased

after PM.

• A greater reduction in seed quality (seed germination and vigour) was found for exposure to elevated temperature after PM in both seasons, when SMC was between 50-14 % SMC (phase- II).

• Assuming an AA test result of 80% or greater is commercially acceptable, around 300 ˚Ch after PM was required to reduce seed vigour to an unacceptable level.

• Seeds harvested from the top of the raceme position were more sensitive to heat stress. Heat stress decreased seed germination, vigour and seeds mass from all three raceme positions but the largest decrease was found from the seeds harvested from the top of the raceme rather than those from the middle and basal raceme positions.

Chapter 5