Plant Transformations

2.3.1 Tomato transformation

All procedures were performed in a sterilized environment and all Petri dishes were sealed with parafilm before moving to the tissue culture room. Tomato seeds were surface sterilized prior to germination by soaking for 5 min in 75% (v/v) ethanol, 20 min in saturated tri-sodium orthophosphate solution and 10 min in 50% (v/v) bleach (locally purchased from Tesco, UK). This was followed by four washes with sterile distilled water (SDW) to remove the bleach solution. Seeds were germinated in sterilized pots containing autoclaved solid MS medium (4.2 g/l MS salt, 3% (w/v) sucrose, 1 ml/l R3 vitamin (1 g/l thiamine, 0.5 g/l nicotinic acid, 0.5 g/l pyridoxine) and 1% (w/v) agar; pH 5.5-5.8 by KOH) in the growth rooms (16 h of light at 25oC and 8 h of darkness at 18oC). Cotyledons of the two-week old tomato seedlings were excised in a flow hood and rinsed briefly in liquid MS24D medium (liquid MS medium supplemented with 0.1 mg/l kinetin and 0.2 mg/l 2-4D) before placing at high density on Petri dishes with solid M1 medium (MS supplemented with 1.75 mg/l zeatin and 0.87 mg/l IAA). The Petri dishes were then sealed with parafilm and incubated overnight under low light conditions (covered by a layer of muslin) in the inverted position to prevent water condensation.

On the next day, a 10 ml overnight liquid culture of Agrobacterium (LBA4404: pAL4404) carrying the appropriate construct was spun down (3,000 x g, 10 min, RT) and suspended in 100 ml of liquid MS medium to about 0.1-0.2 OD600 in a sterilized

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flow hood. The overnight cultured cotyledons were then transferred from the M1 medium to the Agrobacterium suspension and incubated for 15 min with occasional shakings. The cotyledons were then briefly rinsed in sterilized liquid MS24D medium to remove the excessive Agrobacterium. The cotyledons were placed back on to the same Petri dishes with M1 medium, covered with muslin and co-incubated with Agrobacteriumfor two days.

The infected cotyledons were then transferred to plates with M13 medium (M1 supplemented with 75 mg/l kanamycin and 200 mg/l augmentin) at a density of 15 cotyledons per Petri dish (Sterlin, 9 cm). Plates were covered with muslin and incubated in the growth room in the inversed position to induce callus formation. The muslin cover was removed after the first week incubation and from this point the explants were sub-cultured to freshly prepared M13 media every 2 weeks to maintain the antibiotic selection pressure.

The explants developing calluses of 1 to 2 mm in diameter were transferred to sterilized plastic pots containing shoot inducing medium M4 (M1 with 50 mg/l kanamycin and 200 mg/l augmentin). Once the shoots were regenerated from the calluses, they were excised and placed into pots with root inducing medium M16 (MS medium with 50 mg/l kanamycin and 200 mg/l augmentin). When a branching root system had been established, the whole plant was transferred to compost in a 9 cm pot and covered with a transparent plastic bag to maintain humidity. The small plants were then allowed to

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recover for one to two weeks before removing the plastic bag cover. The transgenic tomato plants were re-potted and maintained in the greenhouse under general conditions as previously described (Section 2.2).

2.3.2 Tobacco transformation

The surface sterilized tobacco seeds were first germinated and grown in sterilized plastic pots with MS medium for 4-5 weeks. The leaves were cut into 1 cm diameter leaf disks in a sterilized flow hood and dipped into the Agrobacterium suspension (as prepared in section 2.3.1). The leaf disks were then blotted on an autoclaved filter paper (Whatman, UK) to remove the excessive Agrobacterium before incubating overnight on Petri dishes containing callus inducing medium (MS with 1 mg/l BA and 0.1mg/l NAA). The leaf disks were transferred to fresh callus inducing medium (MS with 1 mg/l BA, 0.1mg/l NAA, 100 mg/l kanamycin and 200 mg/l augmentin) and sub-cultured to freshly prepared medium every two weeks. The shoots regenerated from the calluses were transferred to root inducing medium (MS with 100 mg/l kanamycin) and the following procedures were the same as for the tomato transformation (see section 2.3.1 for details).

2.3.3 Arabidopsis transformation

The floral dip method (Clough and Bent, 1998) was used to transform Arabidopsis using Agrobacterium tumefaciens C58C1 (pCH32). 100 ml of the Agrobacterium

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overnight culture was spun down (3,000 x g, RT) and suspended in 200-300 ml 5% (w/v) sucrose solution. Silwet-L77 was added to the Agrobacterium solution to a final concentration of 0.05% (v/v) prior to dipping. The aerial parts of flowering Arabidopsis plants grown in 9 cm pots were dipped into the Agrobacterium solution for 15 s with gentle agitation. The dipped plants were covered with folded plastic sleeves for 24 h to maintain humidity. After transformation, plants were watered and grown normally for 3-4 weeks; the seeds of the transformed plants were then harvested and screened on MS media with appropriate antibiotics according to the selection marker present in the transgene construct. The collected seeds were first surface sterilized by soaking in 50% (v/v) bleach with a drop of Triton X-100 for 5 min and washed 5 times with SDW. This was followed by suspending the seeds in autoclaved 0.7% (w/v) agar solution (40 oC) and pouring them onto Petri dishes with solid MS medium (1% (w/v) agar) with suitable antibiotics. Petri dishes were dried for 30 min in the flow hood and sealed with parafilm. The dishes were then kept in the cold room (4 oC) without light for 2-3 days before being transferred to the tissue culture room (18 h photoperiod, 23-25oC). After 2 to 3 weeks, Arabidopsis seedlings that developed dark green true leaves and an extending root system were transferred to compost in the 9 cm pots and grown in the glasshouse under general conditions as previously mentioned (Section 2.2).