General materials and methods

2.1.1 Plant material

The wild type (WT) Arabidopsis plants used in this study were Wassilewskija-4 (Ws-4),Landsberg erecta (Ler),Columbia-0/2. The mutant Arabidopsis plants used in this study werednf.

The background and stock number of each of the WT and mutant plants used in this study is listed in Table 1.

Germplasm Background Reference

dnf Ws

Ler Landsbergerecta

ABO (Wagneret al.,1991)

Noss Nossen (Maglianoet al.,2005)

Col-0 Columbia

Col-2 Columbia

phyA Ws-4 (Whitelamet al.,1993)

phyB-1 Columbia (Reedet al.,1993)

co-2 Ler (Robsonet al.,2001)

cry1 cry2 Col-4 (Mockleret al.,1999)

fha-1 Ler (Guoet al.,1998)

Table 1. Arabidopsis germplasm information.Mutants are described as specific mutation alleles in corresponding loci. The Background is the ecotype in which the mutation is found. The stock No. is the unique number assigned to the germplasm by the Arabidopsis Biological Resource Centre (ABRC). Thecry1 cry2double mutant was obtained from Chentao Lin (University of California-Los Angeles). ThephyAmonogenic mutant was obtained from Gary Whitelam (University of Leicester).

2.1.2 Plant growth conditions

Unless otherwise stated plants were grown in Levingtons F2 compost containing 6 parts compost, 1 part sand and 1 part vermiculite. Seeds were stratified in the dark at 4 C for 4 days, to achieve uniform germination. Plants were then transferred to Sanyo growth chambers and grown at 22 C in either SDs, or LDs. SDs consisted of 8 hrs WL (100mols-1m-2) followed by 16 hrs darkness supplied by BriteGro F36WT8 lamps (Sylvania, Germany). LDs consisted of 16 hrs of white light (WL, 100mols-1m-2) followed by 8 hrs darkness.

2.1.3 Hypocotyl Elongation Assay

Seeds were sterilised and pipetted on to 3 % MS agar, supplemented with 1 % glucose. Seeds were stratified for 3 days in the dark at 4C. The seeds were then exposed to white light (100 µmol m-2s-1) for 3 hours and then placed back into the dark for a further 24 hr. Finally seeds were exposed to the appropriate light quality and fluence rate in a Percivil CLF plant Climatics growth cabinet (model 1-3LEDDLL3) for 4 days after which the length of each hypocotyl was measured with a ruler. Far-red LED’s 0.1uMol m-² s-¹ , Blue LED 0.4uMol m-² s-¹ , Red LED 2.5uMol m-² s-¹. Light source was measured using Stellar Net (EPP2000C) Spectroradiometer.

2.1.4 Flowering time analysis

Flowering time was measured by either, the number days to flower from

experiments the flowering time analysis was performed as stated above before being transferred to the different ambient temperatures of 16C, 18C, 20C, 22C and 27C. Again flowering time was measured by either number of days to flower or number of rosette leaves when the bolt is 1 cm high.

2.1.5 Arabidopsis crosses

Sepals, petals and immature anthers were removed from the flower buds of the female parent plant using watchmaker’s forceps leaving an unfertilized stigma. Stamens bearing dehiscent pollen were taken from the male parent plant and its anther was rubbed directly onto the stigma of the female parent. Stigmas were labelled and wrapped in clingfilm for two days. Mature siliques were collected approximately four weeks after fertilisation.

2.1.6 Arabidopsis Transformation

Arabidopsis plants were transformed using the floral dip method (Clough

et al., 1998). 200 ml of LB media containing the appropriate antibiotics was inoculated withAgrobacterium tumefaciensStrain GV3101 transformed with the appropriate construct and grown for 2 days at 28 ºC. The culture was centrifuged and the pellet resuspend in 5 % sucrose to an OD6000.8. Silwet L-77 was added to a concentration of 0.05 % and mixed well. Flowering Arabidopsis plants were dipped into the solution for 5 seconds. Plants were then returned to normal growth conditions and covered by a dome for 1 day. Arabidopsis plants were dipped again as above one week after the initial dipping.

2.1.7 Seed Sterilisation

Seeds (approximately 20mgs) were sterilised in 1 ml 95% ethanol for 5 mins. The ethanol was removed by pipetting and 1 ml of 20% bleach with 0.1% Tween 20 is added. Samples are left in the bleach solution for 5 mins before removing then washing the seeds 5 times with sterile water. The seeds were then suspended in 1ml top media before pipetting onto Murashige and Skoog (MS) agar plates (Murashige et al., 1962) containing sucrose and the appropriate concentration of hygromycin (see below).

2.1.8 Hygromycin selection

Hygromycin is extremely toxic to mammalian cells and great care was undertaken when using. All work with hygromycin was carried out in a laminar flow bench and all waste disposed of as described by COSHH. 2XMS was prepared by the following method: 2XMS4.3g/ MS salts (GIBCO\BRL) with 10g/L sucrose, pH 5.7 (1M KOH), autoclaved before adding 7g/L Agar. Hygromycin was added to the MS media at a working concentration of 20mgs/ ml (1ml of 20mgs/ml hygromycin/L 2XMS). Chalforan cefotaxime (1g powder) + 4mls filter sterilized water was also added to the media to help prevent agrobacterium growth on the plates.

1 L of media was sufficient for 10 large (14cm) plates (approx 80 mls each), two small l (8cm) for positive and negative controls leaving 100mls for top media (without antibiotic selection)

The sterilised seeds were added to 1ml of top media and pipetted onto the plates before placing in a cold room 4ºC in the dark for 2-3 days. The plates were

Next the plates were wrapped up in tin foil and placed in the dark for 5 days starting from the beginning of the light period; longer growth in the dark will reduce recovery in the light.

After 5 days the hygromycin sensitive seedlings will be lying on the media with short hypocotyls and hygromycin resistant with long hypocotyls and closed cotyledons. The plates were then placed in a weak light (lab bench) 2-3 days to allow hygromycin resistant seedlings to green up. The transformants were then transferred to soil for growth in appropriate conditions.