Seed Quality tests

Twenty plants from each treatment were selected randomly (5 per plot) at the following seed development stages.

i. Physiological maturity (maximum seed mass, ≈ 50% SMC)

ii. Pre-final desiccation (≈ 25% SMC) iii. Harvest maturity ( ≈ 14% SMC)

Seeds were hand harvested from the two sowing dates at the three above seed development stages to eliminate the effects of machine threshing on seed quality. All pods were removed from the plants, shelled and the seeds were mixed together to make a working sample (seed lot) for each development stage. They were then ambient air dried to between 8-10% SMC, placed in hermetically sealed zip lock bags and stored at 5 °C until quality testing was conducted.

All seed quality tests, viz. standard germination, vigour (conductivity test, accelerated aging test) and thousand seed weight (TSW) were performed according to internationally agreed methods prescribed by the International Seed Testing Association (ISTA, 2016) at the Lincoln University, Seed Research Centre laboratory.

2.3.3.1 Seed moisture content (SMC)

Seed Moisture Content (SMC) was determined by using a mechanically ventilated forced air oven (Sanyo, Model MOV-212). 5-6 g seed was taken at random from each sample and placed into an aluminium container which was covered with its lid. The container was then weighed to three decimal places and placed into the oven (with lid removed). The oven was set at 103 ± 1˚C and the container with seeds was left to dry for 17 hours (low constant temperature method; ISTA 2016). The containers were then removed from the oven and covered with their lid and then allowed to cool for 15 min in a 36

desiccator and re-weighed to determine the moisture loss. SMC percentage was calculated on a fresh weight basis using the following equation:

SMC (%) = (M2-M3) x 100 / (M2-M1) Where:

M1 = moisture tin with cover pre oven

M2 = moisture tin with cover and seed pre-oven M3 = moisture tin with cover and seed post-oven.

2.3.3.2 Thousand seed weight (TSW)

Thousand seed weight (TSW) was obtained by counting and weighing eight replicates of 100 randomly selected seeds from each plot. The average weight of the eight replicates of 100 seeds was multiplied by 10 to give 1000-seed weight. The weight was expressed to three decimal places (ISTA, 2016).

2.3.3.3 Germination test

Germination tests were carried out using the top of paper (TP) method as specified in the ISTA Rules for brassica species (ISTA, 2016). Four randomly selected replicates of 100 seeds from each seed lot were placed on a moist germination blotter and placed into a plastic sandwich box which was then covered with a lid (Figure 2.1). These sandwich boxes were placed in a germination room at 20 ±2 °C.

Figure 2.1 Four replicates of 100 seeds were placed on germination blotters placed into plastic sandwich boxes.

Figure 2.4 (a) Normal seedling; the primary root is visible.

(b), (c) & (d) Abnormal seedlings; the primary root is trapped in seed coat. (Source: ISTA, Seedling Evaluation Handbook, ISTA 2013)

(a) Normal (b)Abnormal (C)Abnormal (d)Abnormal

The un-germinated or remaining seeds were counted as either fresh un-germinated seeds or dead seeds by visual assessment or by dissecting the seed (ISTA, 2016). The variability among replicates in the germination test results was assessed using a tolerance table (ISTA, 2016). If the difference among the four replicates of 100 seeds (from lowest to highest) did not fall within the maximum range of tolerance allowed, the germination test was repeated (Appendix 2). Replicate data exceeding the tolerance indicate a problem with the test result in that the variation has exceeded that explained by random sampling variation alone (ISTA, 2016).

2.3.3.4 Vigour tests

(i) Conductivity test

A single cell electrical conductivity meter (CDM210, Radiometer, Copenhagen) was used to measure change or difference in conductivity as electrolytes leached in soaked water (Figure 2.5). Prior to the conductivity test, seed moisture content of each lot was determined by the method described in Section 2.3.3.1, as it is known that the initial SMC of seeds is source of variation in conductivity (Castillo et al., 1992). The seeds harvested at all three stages i.e. 50%, 25% and 14 % SMC were ambient air dried to 8-10% (as mentioned in Section 2.3.3) to minimize the variation between seed lots and replicates.

Four replicates of 100 seeds were randomly selected from each seed lot, weighed and each replicate soaked in 50 ml deionized water in plastic vials (ISTA, 2016) and kept in a controlled temperature room (20°±2 ˚C) for 16 hours. After 16 hours of imbibition, the seeds and steep water were stirred to mix briefly and the electrical conductivity of the leachates from imbibed seeds was measured by inserting the single electrode cell into the water. The conductivity of the water of a control (flask with deionised water at 20 C˚) was also measured and this value (background reading) was subtracted from the 39

conductivity reading of the steep water from each sample. The conductivity per gram of seed weight for each replicate was calculated after accounting for background conductivity of the original water and the average of four replicates provided the results. Thus for each replicate:

Conductivity (µS cm-1 _g-1_{) = Conductivity reading – background reading} Weight (g) of seed sample

The samples were retested, if the mean conductivity of the four replicates differed (lowest to highest) between replicates by more than the maximum tolerance values allowed for the conductivity test (Appendix 3) (ISTA, 2016). For this vigour test, the higher the conductivity readings, the lower the vigour of seed.

(ii) Accelerated ageing (AA) test:

The accelerated ageing (AA) test subjects the seed to two environmental variables (high temperature and high relative humidity) for a short period of time as described by Hampton and TeKrony (1995) and ISTA (2016). This treatment accelerates natural seed deterioration.

Accelerated ageing was performed using an inner aging chamber i.e. plastic boxes (11.0 x 11.0 x 3.5 cm) (Figure 2.6) with a suspended wire tray of 10.0 x 10.0 x 0.3 cm (length x width x depth) with a mesh screen inside with a pore size of 1.16 ±0.01 x 1.63 ±0.01 mm. A minimum of 400 seeds per sample, after being weighed, were placed on the mesh screen so as to form a single layer. 40 ml deionized water was placed in each inner chamber and the mesh screen tray with seeds was then placed in the inner chamber so that the seeds did not come into contact with the water. The inner chamber boxes were covered with a tight lid and placed in an outer chamber (oven) for 72 hours (Figure 2.7), the outer

Figure 2.5 Electrical conductivity meter used for the conductivity test.

chamber (oven) door was closed to recover the temperature to 41 ± 0.3 ˚C and the aging temperature was continuously monitored during the aging period to be certain that temperature was constant at that level. After 72 hours of aging, seeds were removed from the inner chamber and planted for a standard germination test using the top of paper method within one hour after removal as described in Section 2.3.3.3.

Germination evaluations were performed at 5, 7 and 10 days and the results expressed as mean percentage of normal seedlings for each sample. Seed vigour for this test is determined by the extent of difference in the percentage normal seedlings from the standard germination test (control) and the germination after AA; the greater the difference, the lower the vigour.

Figure 2.6 Inner chamber plastic box with a wire tray for AA, and mesh screen placed on tray with single layer of forage rape seeds.

Figure 2.7 Outer ageing chamber (Oven) with inner chamber placed on shelves, maintaining constant temperature of 41± 0.3 ˚C is used.