Plant material

2.1.1 Plant propagation

Seeds to be sown straight to soil were first stratified over 48 hours in water at 4 oC in a plastic microfuge tube before being shaken onto damp seed-raising mix in plastic pots. The pots and their contents were then thoroughly watered, placed under plastic to maintain high humidity, and transferred to a glasshouse. The plastic was removed once the seeds had germinated. The glasshouse operated a 16 hour day regime with a daytime temperature of 22 oC and a night time temperature of 17 oC.

Seeds planted to be grown in a growth room were first surface-sterilised (70 % (v/v) ethanol for one minute, 50 % (v/v) sodium hypochlorite for one minute, and three washes of sterilised water) before being transferred to plates containing Murashige and Skoog (MS) (Murashige and Skoog, 1962) medium and antibiotic selection (if required) and stratified at 4 oC for 48 hours. Growth rooms operated a 16 hour day regime with a daytime temperature of 22 oC and a night time temperature of 17 oC with light levels at 100-150 µE / m2/ s. Once the plants had been characterised as containing the T-DNA insert and had reached a suitable transplanting size (generally four true leaves) the plants were potted in the glasshouse. The plants were transferred from the tissue culture plate to a small hole in pre-wetted seed-raising potting mix, making sure the roots went into the hole. The potting mix was then squeezed together around the roots and the pots and the contents watered thoroughly. The plants were then placed under plastic for 48 – 72 hours in continuous light and ambient

temperature. After 48 – 72 hours, the plastic was removed and the plants transferred to 16 hour days in the glasshouse under PC2 containment under ERMA development approval number #GMO 2001/AGPN007 and import approval number GMC99006; MAF #20022015068.

2.1.2 Generation of out-crossed progeny

Healthy plants were selected and the sepals and petals of unopened flowers were gently prised apart using size four ultra fine antistatic antimagnetic tweezers (Geneva Importers Ltd, Wanaka, New Zealand) to reveal anthers and style. The anthers were removed with tweezers taking care not to damage the style or stigma and the stigma was brushed with pollen from anthers of the pollen donor. The pollen donor was selected based on its genotype and pollen quality. Anthers with copious amounts of white fluffy pollen were considered ideal paternal parents.

2.1.3 Root phenotype analysis

Lines carrying T-DNA insertions were sown on large plastic plates (22 x 22cm). The plates were sterilised with 50 % (v/v) sodium hypochlorite overnight and rinsed three times with sterile water, once with 70 % (v/v) ethanol and finally 100 % ethanol. The plates were left to dry completely in a laminar flow cabinet before MS medium with 2 % (w/v) sucrose and 1 % (w/v) agar was poured into them and allowed to set. Seeds were surface sterilised (70 % (v/v) ethanol for one minute, 50 % (w/v) sodium hypochlorite for one minute, and three washes of sterilised water) and placed along one edge of the plate, 1.3 cm apart. The plates were placed at 4 oC for 48-72 hours before being transferred to the growth room and maintained under 16 hour day regime with a daytime temperature of 22 oC and a night time temperature of 17 oC with light levels at 100-150 µE / m2/ s. The plates were initially placed horizontally to

encourage the roots to grow into the medium. Once the roots had penetrated the medium the plates were inclined to the vertical (approximately two days after germination). Roots were scored nine days later. Plates were either scanned or photographed with a digital camera. The Image Processing and Analysis in Java (ImageJ, http://rsb.info.nih.gov/ij) programme was used to determine root length, alpha angle (Figure 2-1 A), beta angle (Figure 2-1 B) and the number of lateral roots.

Figure 2-1: Root angle measurements. A: α angle, the angle roots deviate from the vertical, B: β angle, the angle between one root bend and another.

2.1.4 Pollen viability

Alexander’s stain was used to test for pollen viability. Alexander’s stain stock solution was stored in the dark at room temperature until use (Table 2).

Table 2: Formulation of Alexander’s (1969) stain. The reagents were used to make a 50 x stock solution. (Johnson-Brousseau and McCormick, 2004)

Reagent Amount

95 % (v/v) ethanol 10 ml

1 % (w/v) malachite green in 95 % (v/v) ethanol 5 ml

phenol 5 g

1 % (w/v) acid fuschin in water 5 ml

1 % (w/v) orangeG in water 0.5 ml

glacial acetic acid 2 ml

glycerol 25 ml

water 50 ml

A modified Alexander’s stain (Alexander, 1969, 1980) stock solution without Chloral hydrate (50 x) was diluted (1:50) with water before use. Pollen collected from plants grown in the glasshouse was placed in a microfuge tube containing 1 ml of a working concentration Alexander’s stain and allowed to sit for two minutes. Pollen was then removed from the microfuge tube and placed on a slide under a coverslip and assessed with a light microscope for colour. Pollen scored as red was deemed viable. Pollen

2.1.5 Reporter gene visualisation

Apical root tips of plants grown on MS medium with 1 % (w/v) sucrose in a 16 hour day growth room with a daytime temperature of 22 oC and a night time temperature of 17 oC with light levels at 100-150 µE / m2/ s were assessed for GFP expression 7-10 days after germination (DAG). The seedlings were pulled gently from the plate and the root tip and lateral roots were stained with 10 µg/ml propidium iodide for one minute at room temperature followed by three washes of sterile water. The shoot tip and inflorescence meristem were dissected using size four tweezers (Geneva

Importers Ltd, Wanaka, New Zealand) and an ultra fine antistatic antimagnetic

dissecting knife. Siliques containing embryos of all stages were selected from healthy plants. The siliques were slit open using an ultra fine dissecting knife and placed on a slide, covered with a coverslip and gently squashed until the embryos popped out of the seed coat.

All material was assessed with a Leica DMRBE confocal microscope with a mixed argon krypton laser. The excitation wavelength used was 488 nm in conjunction with a short pass beam splitter at 510 nm and a pinhole of 90. The detector used was a short pass 580 nm beam splitter and a BPFITC barrier filter. Any remaining light was then directed via a mirror to a 590 nm long pass barrier filter.

Cotyledons and true leaves of plants containing GUS constructs were grown for 5 -10 days on MS medium in a growth room under16 hour days with a daytime temperature of 22 oC and a night time temperature of 17 oC with light levels at 100-150 µE / m2/ s. The seedlings were pulled gently from the plate and placed in wash solution (Table 3) for six hours at 37 oC. The solution was made (Table 3) as required and stored at -20

o

C for no longer than two weeks. The concentration of potassium ferricyanide and potassium ferrocyanide was adjusted to restrict GUS to site of production. The stain was removed using a pipette and replaced with 70 % (v/v) ethanol and a drop of acetic acid (0.1 %) and left over night to remove the chlorophyll and discolour the tissue. To visualise the SAM the leaves and cotyledons were carefully removed using an ultra fine dissecting knife and tweezers. Whole plants and plant sections were visualised on an Olympus BX50 light microscope and photographed with Colour View 111 soft imaging system and analysisB software (from Olympus, Singapore).

Table 3: Formulation of GUS wash solution.

Reagents Final concentration

500 mM sodium phosphate buffer (Na2HPO4 + NaH2PO4 pH7.0) 50 mM

100 mM potassium ferricyanide (K3Fe(CN)6) 4 mM – 8 mM

100 mM potassium ferrocyanide (K4Fe(CN)6.3H2O 4 mM – 8 mM

10 % (v/v) triton X 100 0.1 % (v/v)

methanol 20 % (w/v)

100 mM X-Gluc in DMF 2 mM

water to volume

2.1.5.1

Plant fixative; Formaldehyde:acetic acid (FAA)

Tissue was prefixed with 90 % (v/v) ice-cold acetone at room temp for 20 minutes then washed twice with cold water before the GUS wash solution (Table 3) was added. The tissue was then infiltrated on ice until everything sunk before being incubated at 37 ˚C overnight. The tissue was then taken through an ethanol series (30 min at room temperature of 20 % 9v/v) and 35 % (v/v) ethanol) before fixing with FAA (50 % (v/v) EtOH, 3.7 % (v/v) formaldehyde, 10 % (v/v) glacial acetic acid) for 30 minutes. The material was stored in 70 % (v/v) ethanol at room temperature.