Transgenic tomato plants

Agropbacterium-mediated tomato transformation using etiolated cotyledon explants

This protocol includes (I) a step-by-step decription of the procedure, (II) recipes for growth/selection media, (II) and a time schedule to facilitate planning of the epxeriment. 

I. Step-by-step protocol

Sterilisation of seeds and elimination of viruses: 

- make sure seeds are dry

- incubate over night at 70 °C 

- incubate for 5 min in 70 % ethanol, rinse with excess of water

- incubate in 10 % trisodiumhosphate for 3 hrs; agitate gently        

- rinse 3 x 5 min with excess of water

- incubate 15 min in 1.5 % sodium hypochlorite (made from von 15 % stock), containing 5 drops/100 ml Tween 20

- rinse 5 x in approx. 100 ml sterile water

Growth of seedlings: 

- sow 30 seeds on sterile medium #1, in 0.5 l jars (we use glas jars meant to bottle fruits) 

- stratify for 2 days at 4°

- germinate for 10-14 days at 22 °C in the dark (make sure that seedlings are etiolated, cotyledons should be about 1 cm

   long (not fully expanded), first true leaves not developed yet).

Preconditioning of cotyledons:

-  harvest cotyledons under the sterile hood - be very careful, try to avoid unnecessary damage

- cut off the tip and the base of the cotyledons on sterile glass plate; use a sclapel blade to make little wound across the

   mid vein

- place cotyledons upside down onto conditioning medium (medium #2) 

- wrap petri dishes in aluminum foil, incubate 2-3 days at 22 °C

Cultivation of Agrobacteria:

- Inoculate a 2 ml pre-culture of Agro strain (LBA4404, GV3101), using a single colonie, medium #3 with antibiotics.

- grow for 1 or 2 days (depending on how fresh the plate is from which you took the single colony) at 28 °C, 220 rpm. 

- use the pre-culture at 1:1000 to inoculate a 10 ml culture in a 50 ml Erlenmeyer flask, medium #3 with antibiotics and

  acetosyringone; grow for 24 hrs at 28 °C, 220 rpm

- harvest cells by centrifugation, resuspend in 10 mM MgSO₄ (or medium #1) to result in OD₅₉₀ 0.5-1.0 (equals cell density   of approx. 10⁸  cells/ml). You will need about 10 ml of the agro cell suspension.

Co-cultivation with agrobacteria:

- pipet 1 to 2 drops of of the agrosuspension onto the cotyledons – it should not become confluent, but stay a drop because

  of surface tension

- incubate 2 days at 22°C in the dark

- then pipet off the agrosuspension (optinal, only if it turns out to be difficult to get rid of  the agros: rinse cotyledons in

  medium #1, blot dry on sterile filter paper)

Selection and regeneration (22-25 °C, 16 hr photoperiod):

- Place cotyledons (top-side up) onto selection medium (35 mg/l kana­mycin).

- Transfer to fresh selection medium after 3 days (35 mg/l Kanamycin). 

- In the following, transfer to fresh medium every seven days (2 x with 50 mg/l kanamycin, finally 100 mg/l). 

- When green calli or shoots are appearing, separate them from the rest of the cotyledon and bring it into direct contact with

  the selection medium. 

- Once the shoot is 2-3 cm tall (after maybe 2 months), transfer it to rooting medium.

Rooting (22-25 °C, 16 hr photoperiod):

Please note: we use Vancomycin in the rooting medium (rather than Cefotaxim oder Carbenicillin); it is much more expensive but better for root development. Use Magenta jars, or small glass jars; takes about 1 month.

Transfer to soil:

Transfer plantlets to soil in small pots (does not need to be sterile anymore). The plants should be allowed to adjust slowly to reduced humidity. Therefore, keep the pot in a plastic bag; punch a few holes into the bag, add more holes day after day, thereby allowing for increasing air exchange. 

II.: media recipes

Vitamins, hormones, and antibiotics are added after autoclaving from filter-sterilized stocks.

Medium #1: gemination, co-cultivation

Murashige & Skoog + minimal organics (MSMO, Sigma M 6899) 4.4 g/l

Alternatively, use MS basal salt mixture from Duchefa

Saccharose 30 g/l

pH 5.8 (KOH)

(6 g/l Agar, for co-cultivation plates)

Medium #2: conditioning

MSMO

1 mg/l naphtyl acetic acid (NAA)

0.1 mg/l 6-benzylaminopurine (BAP)

30 g/l Saccharose

0.6 % Agar

pH 5.8 (KOH)

Medium #3: bacterial culture

10 g/l Yeast extract

10 g/l bacto peptone

5 g/l NaCl

0.2 mM acetosyringone (400 mM stock in DMSO)

add antibiotics according to requirements of Agro strain used

Medium #4: selection                                                            

MSMO                                                                                                                                  

Thiamin 9.6 mg/l

Nicotinic acid 1 mg/l   

Pyridoxine 1 mg/l 

1 mg/l trans-Zeatin

35 (50, 100) mg/l Kanamycin  

250 mg/l Tricarcillin 

0.6 % Agar      

30 g/l Saccharose         

pH 5.8  (KOH)

Medium #5: rooting

MSMO

Thiamin 9.6 mg/l

Nicotinic acid 1 mg/l

Pyridoxine 1mg/l

0.1 mg/l IAA

20 mg/l Kanamycin

500 mg/l Vancomycin

0.6 % Agar

30 g/l Saccharose

pH 5.8 (KOH)         

III.: Time schedule

this protocol was modified from Biotechnology (1987), Vol 5: 726-730