Seeds Forming A Gradient Biology Essay

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All seed was obtained from Dr. Ian Taylor The University of Nottingham and Rachel Smeeton The University of Nottingham. Tomato seeds cv. Alisa Craig (Tm2a) were included as a wild type (WT) control. Other seed types include BCH12, sp5, G29 and TRIPLE transgenic lines. It is important to note that the level of ABA over-expression in these genotypes increases in the following order: BCH12 < sp5 < G29 < TRIPLE.

3.1.2 Seeds of the rbcS-17 and ZEP transgenic genotypes

New seed stocks were obtained from Dr. Ian Taylor (The University of Nottingham) and Rachel Smeeton (The University of Nottingham). Including rbcS-17 (Tung et al., 2008), ZEP-6, ZEP-10A and ZEP-11. The constructs in the ZEP genotypes were based on the tomato NCED1 coding sequence driven by the tomato ZEP promoter and were built by E. Harrison and A.J. Thompson at the University of Warwick. The rbcS-17 genotype (Tung et al., 2008) was selected for comparison with the ZEP genotypes on the basis that it had also been transformed with a construct in which a light sensitive/circadian promoter drove the tomato NCED1 coding sequence.

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3.2 Germination protocols - Petri dish germination and norflurazon application

3.2.1 Germinating seeds forming a gradient of ABA over-production (TRIPLE>G29>sp5>BCH12>WT)

100 seeds from each of the 5 genotypes (WT, BCH12, sp5, G29, TRIPLE) were germinated on sterile filter paper in 9 cm Petri dishes at room temperature. 20 seeds from each genotype were germinated in distilled water. 80 seeds from each genotype were subject to treatment with norflurazon. This involved applying 0.5 or 1.0 mg l-1 solutions for 2 or 4 days. A total of 20 replicate seeds from each genotype were sown for each combination of concentration - duration treatment. The seeds were removed from the norflurazon solution after the required duration of treatment had expired. The seeds were vigorously washed for 1 minute under distilled water, to remove any residual norflurazon. The washed seeds were then placed on sterile filter paper in 9cm Petri dishes containing distilled water. All seeds were stored in a light room until the time for imbibition. All 100 seeds were planted simultaneously the day after the 4-day treatments with norflurazon were complete.

3.2.2 Germinating seeds of the rbcS-17 and ZEP genotypes

60 seeds from each genotype (rbcS-17, ZEP-6, ZEP-10A, ZEP-11), together with the control seeds, were germinated on sterile filter paper in 9 cm Petri dishes at room temperature. 20 seeds from each genotype were germinated in distilled water. 40 seeds from each genotype were subject to treatment with norflurazon. This involved applying 0.5 mg l-1 solutions for 2 or 4 days. A total of 20 replicate seeds from each genotype were sown for each combination of concentration - duration treatment. The seeds were removed from the norflurazon solution after the required duration of treatment had expired. The seeds were vigorously washed for 1 minute each under distilled water, to remove any residual norflurazon. The washed seeds were then placed on sterile filter paper in 9cm Petri dishes containing distilled water. All seeds were stored in a light room until the time for imbibition. All 60 seeds were planted simultaneously the day after the 4-day norflurazon treatments were complete.

3.3 Imbibition of seed

The experiments were conducted within a partially environmentally controlled glasshouse. The temperature never fell below 18oC or exceeded 24oC (±0.5 oC). Artificial light was used to supplement natural light where necessary. Plants were irrigated with water that contained a tomato feed (1:1:3 NPK).

3.3.1 Gradient of ABA over-production (TRIPLE>G29>sp5>BCH12>WT)

A total of 500 seeds were planted in Levington F2 compost at a depth of 1 cm in individual 9 cm pots. All the seeds received a thorough watering in the 9 cm pots. In order to maintain high humidity in the air spaces above and within the soil, the 9 cm pots were all capped with 9 cm Petri dish lids.

3.3.2 Comparison of rbcS-17 and ZEP genotypes together with WT, sp5 and G29 controls

A total of 420 seeds were planted in Levington F2 compost at a depth of 1 cm in individual 9 cm pots. All the seeds received a thorough watering in the 9 cm pots. In order to maintain high humidity in the air spaces above and within the soil, the 9 cm pots were again all capped with 9 cm Petri dish lids.

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3.4 Scoring germination and photobleaching

The seeds in both experiments were kept under standard glasshouse conditions (section 3.3). Germination was recorded daily. For this investigation germination was judged to be complete at the time of hypocotyl hook emergence. After germination was confirmed the Petri dish lids were removed to avoid subjecting seedlings to a high humidity environment. 7 days after the last seed had germinated the plants were scored for bleaching. The classification system assigned the plants to one of four categories: no bleaching, mild-bleaching, severe-bleaching and death.

3.5 The 'lollipop' effect

In the experiment to compare the rbcS-17 and ZEP genotypes, the plants that germinated were monitored for the 'lollipop' effect. Tung et al., (2008) first reported and named the 'lollipop' phenotype, defining it as, ' a reduced ability of the expanding cotyledons to escape from the testa'. Based on this description the plants were scored visually for the presence/absence of the 'lollipop' effect.

3.6 Leaf chlorophyll content

In the experiment designed to compare the rbcS-17 and ZEP genotypes, leaf chlorophyll content was measured using a portable Minolta SPAD-502 meter. This is a non-destructive method of measuring leaf chlorophyll content. The measurements were taken from three expanded leaves at the top of the plant and an average was recorded. It is known that SPAD readings can be specifically correlated with tomato leaf chlorophyll content measured destructively by chlorophyll extraction. The SPAD readings have a near linear relationship with the extractable leaf chlorophyll content of tomato plants (Smeeton, 2010).

3.7 Destructive harvest, plant growth measurements and dry weight

At the end of each experiment the plants were transferred for a destructive harvest and various plant growth measurements. The gradient of ABA over-production (TRIPLE>G29>sp5>BCH12>WT) experiment had a total of 2 harvests. The first harvest was carried out 28 days after transfer. The second harvest was carried out 42 days after transfer. In both harvests there were 8 different batches. Each batch contained 1 plant of each genotype.

The experiment designed to compare the rbcS-17 and ZEP lines only had a single harvest. This harvest was carried out 35 days after transfer to the glasshouse. In this harvest there were 8 batches. Each batch contained 1 plant of each genotype. It is important to note that only 3 plants of the ZEP-11 genotype were available to be selected for destructive harvest; 8 plants represented all other genotypes.

The plant height (cm), cotyledon length (cm) and length of the first three sideshoots (cm) were determined. The plant height was measured from the point where the stem emerged from the soil to the point of the last internode. Cotyledons were measured individually and averaged. The first three sideshoots were removed from the leaf axils. Their measurements were taken from the point at which they were severed to the tip of the terminal leaf.

In both experiments all plant biomass from each plant selected for destructive harvest was placed individually into paper bags. The bags were placed in a Gannet Model L6499 oven, where they dried for 72 hours at 80oC. The bags were removed from the oven and the dry matter content was weighed (g) using precision balance scales.

3.8 Data analysis

Data analysis was performed by an analysis of variance (ANOVA) using the program Genstat (12th Edition).