PLANT TRANSFORMATION LABORATORY

PROTOCOLS

CROP SCIENCE, plant transformtaion laboratory

Nicotiana benthamiana

Plant material

Seeds of Nicotiana benthamiana seeds were obtained from the National Tobacco Germplasm Collection at the University of North Carolina. These were placed in a 1.5 ml centrifuge tube, and surface-disinfected with 70% alcohol for 1 min, 25% (v/v) Clorox bleach (6.0% NaHClO3) for 15 min, followed by four rinses with sterilized deionized water. For each sterilization or washing treatment, seeds were centrifuged at 14,000 rpm, and the liquid was decanted. Seeds were placed in petri plates containing a medium consisting of 3% sucrose and solidified with 0.8% agar (Sigma) that had been adjusted to pH 5.8 with 1N NaOH. Germinating seeds were placed in the culture chamber under 16 h of diffused cool-white light (25-30 ?mol m-2s-1) at 26°C. Germinating seedlings were later transferred to glass jars containing the same media and placed under the same light and temperature conditions to allow for further growth.

Effect of kanamycin on N. benthamiana leaf sections

Young expanding leaves of N. benthamiana were cut into 1 cm2 sections, and cultured with their abaxial surface in contact with a shoot innduction medium (SIM) (Murashige and Skoog (MS) (1962) medium (Sigma), 3% sucrose, and 1.0 mg/l indole acetic acid (IAA), and 0.1 mg/l 6-benzyladenine (BA) growth regulators (Sigma)) supplemented with seven different concentrations of kanamycin, including 10, 20, 30, 40, 50, 75, and 100 mg/l. A kanamycin free-SIM was used as control. Two replications of three plates (10 explants/plate) were used for each treatment. All treatments were kept in the dark at 26°C for 2 days, and then placed in the growth chamber under the same light and temperature regime used for seed germination as described earlier. After 2 weeks, the percentages of callus formation and shoot organogenesis per explant were recorded.

Cocultivation of explants

Leaves of N. benthamiana were excised from in vitro-grown seedlings, and placed in 108 colonies/ml of an overnight-grown culture of A. tumefaciens GV3101/cDNA. Leaves were cut into 1 cm2 sections while placed in the bacterial culture. Following a brief gentle shaking of the culture to ensure adequate coverage of the tissue with the bacterial culture, explants were blotted dry. Infected tissue was cultured with the abaxial surface in contact with the initiation medium. After 2 days of cocultivation in the dark at 26°C, infected tissue was rinsed three times in a liquid initiation medium containing 150 mg/l of cefotaxime (Sigma) to control the growth of A. tumefaciens on the surface of the tissue. The tissue was blotted dry, and placed on a regeneration medium with the leaf abaxial surface in contact with the medium.

Regeneration of transgenic plants

The regeneration medium consisted of full-strength MS medium, 3% sucrose, 1.0 mg/l IAA, and 0.1 mg/l BA (Sigma). The medium was solidified with 0.8% agar (PhytoLab Technology), and supplemented with 150 mg/l cefotaxime and 50 mg/l kanamycin to control bacterial growth, and select for transformed regenerants.

All cultures were established in 100x15 petri plates containing 10 sections per plate. Explants were incubated in a controlled growth chamber under 16 h of diffused cool white-light (25-30 ?mol m-2s-1) and at 26°C. All explants were subcultured onto a fresh regeneration/selection medium every 10 days to maintain to compensate for the degradation of kanamycin in the media and maintain a constant selection pressure. To maintain selection the pressure, regenerating plants were further subdivided to ensure direct contact of regenerated plantlets and the medium.

Rooting of regenerants

Regenerated plantlets were transferred to plates with growth regulator-free medium to promote rooting. The rooting medium consisted of full-strength MS medium, and 3% sucrose. The medium was supplemented with 150 mg/l cefotaxime to control any bacterial growth and 100 mg/l kanamycin to increase the selection pressure and lower the chance of escapes. All regenerated plantlets were incubated in a controlled growth chamber under 16 h of diffused cool-white light at 26°C, and were maintained on the rooting medium until roots developed and were 10-20 mm in length, rooting regenerants were then place individually in jars containing the same medium under the same environmental conditions to allow further the elongation of the shoots and the growth of more intensive rooting system.

Nicotiana tabacum

Plant material

The seeds of Nicotiana tabacum (f.e. cv Wisconsin*) were sterilized by subsequent submersion in 70% ethanol for 1 min and 10% bleach solution with 1-2 drops of Tween 20 for 10 min. After rinsing 3 times with sterile distilled water seeds are placed onto germination medium (1/2 strength MS medium supplemented with 0.8% agar and 3% sucrose). After 2-3 weeks seedlings roots were cut and shoots were transferred to big culture vessels containing the same media. Leaves of 5-6 week-old aseptically growing plants can be used for transformation.

Tobacco transformation

Day 1

Start a liquid culture of Agrobacterium. Use LB or YEB medium with appropriate antibiotics. Use sterile shaker flasks with 20 or 25 ml of medium. Inoculate the liquid culture from the Agrobacterium plate or glycerol stock. Grow the culture overnight on shaker at 280C.

Day 2

Measure the OD of bacterial suspension. Dilute the agro suspension with liquid MS media. A final suspension of OD600~0.5 is used for inoculation.Take young healthy green leaves, cut into pieces 0.5-0.7 cm (10-20 pieces from each leaf) and put in suspension of Agrobacterium (OD600-0.5) for 5-10 min. Then leaf segments should be blotted dry with sterile filter paper and placed on the surface of MS media supplemented with 0.8% agar and 3% sucrose and 100 ?M acetosyringone in sterile Petri dishes (10-15 explants pet plate). Plates should be incubated for 2 days at 250C in the dark.

Day 4

Transfer segments of leaves to regeneration selection media (6-8 explants per 100x25 mm Petri dish contained 25 ml media). This is MS media supplemented with 2 mg/l BAP, 0.1 mg/l NAA, 100 mg/l kanamycin and 300 mg/l timentin, 0.8% agar and 3% sucrose. Place at light, 250C. After 2-3 weeks cut segments of leaves and transfer to the fresh selection media (with the same hormone/antibiotic concentration, but without NAA). Putative transgenic shoots appears after 4-5 weeks. Separate shoots from explants and transfer to Magenta boxes containing MS media with 100 mg/l kanamycin, 300 mg/l timentin, 0.8% agar and 3% sucrose to test the root induction of putative transformants. Place at light, 250C. Roots become visible after 1-2 weeks. Wild type (non-transgenic) plants don’t form roots in presence of this concentration of kanamycin.

Transformation Protocols Nicotiana benthamiana

Plant material

Effect of kanamycin on N. benthamiana leaf sections

Cocultivation of explants

Regeneration of transgenic plants

Rooting of regenerants

Acclimatization of rooted plants

Greenhouse treatment

Acclimatized plants were transferred to the greenhouse. The Jiffy pots containing plants were individually placed in plastic pots containing the same soil mixture as within the Jiffy pots and watered thoroughly. Each pot was tagged with the corresponding cDNA clone, plant number, and date. Plants were allowed to grow under 16 h light-regime at 25°C. Upon flowering, unopened flowers were covered with small bags to prevent cross-pollination. Upon maturation, seeds from each plant were collected separately.

Tomato

Plant material

Seeds of tomato (Sweet Chelsea TMV, Cobra Hybrid) obtained from Stokes Seeds, Inc. Seeds are sterilized with 20% Clorox?commercial bleach (0.105% sodium hypochlorite) for 15 min, rinsed three times in sterilized distilled water (5 min per rinse), and then allowed to germinate in glass jars containing full-strength MS salts (Murashige and Skoog, 1962), Gamborg's B5 vitamins, and 1% sucrose. The medium was solidified with 12 g/L agar (Phytolab), and the pH was adjusted to 5.7 prior to autoclaving with 0.1 N KaOH. Eight-day-old seedlings are used as sources of cotyledon explants.

Transformation

Day 1

At about 9-10:00am from aliquoted frozen stock of GV3101 Agrobacterium strain cell clumps are placed into 0.5 ml liquid YEP or LB+100 mg/l kanamycin+ 50 mg/l rifampicin+50 mg/l gentamicin. At about 5:00pm 0.5 ml of bacterial culture placed into 25 ml of YEP+100 mg/l kanamycin+50 mg/l rifampicin+50 mg/l gentamicin for overnight. Agrobacterium cells are grown overnight at 28oC in a YEP medium (Bacto-peptone 10 g/L, Bacto-Yeast 10 g/L, NaCl 10 g/L, pH=7.0) or LB containing 100 mg/L of kanamycin, 50 mg/L rifampicin, and 50 mg/L gentamycin.

Day2

Next day at 10:00 am OD600 readings are taken (OD600~1.5-2.0). After that 25 ml of Agrobacterium suspension is placed into 50 ml sterile tube (orange cap) and centrifuged for 15 min at max speed of 3000 rpm. Pellet is resuspended into 25 ml of TRM tomato re-suspension medium (MS + MS vit, sucrose 30g/l, glucose 2.0 g/L, pH=5.6). Agro suspension is diluted with TRM medium to achieve approximately OD600 ~ 0.1: 40 ml x 0.1 : OD600 (40x0.1:1.9 = 2.0 ml of Agrobacterium + 28 ml of TRM ).

Well shaped (not curly) 8 day old cotyledons are collected and placed on TCM tomato co-cultivation medium (MS+B5 vit, 0.5 mg/L IAA, 1.0 mg/L BA, 30 g/L sucrose, 2.0 g/L glucose, 12 g/l agar, 100 mM acetosyringone) and wounded with needles. Wounded cotyledons are placed into Agrobacterium suspension, submerged into liquid and placed for 15-20 min on slow shaker. Following inoculation, cotyledons are blotted dry on a sterile filter paper and placed on fresh TCM medium for 48 h under 16 photoperiod (provided by 20 watt cool-white fluorescent tubes yielding a light intensity of 30 ?mol/m2/sec) and 25 0C.

Day4

Following cocultivation, cotyledons are washed twice with liquid TR-C medium containing 500 mg/L carbenicillin or 300 mg/L timentin, blotted dry, and 20 explants per plate placed on TR-8 tomato regeneration/selection medium (MS + B5 vit, sucrose 30 g/L, MES 700 mg/L, kinetin 1 mg/ml, IAA 0.5 mg/ml, TDZ 1 mg/ml, 100 mg/L kanamycin and carbenicillin 500 mg/L or timentin 300 mg/L, agar 8 g/L, pH 5.8). Explants are cultured for five to six weeks under the same environmental conditions described (subcultured once after first three weeks).

After 5-6 weeks

Organogenic calli with shoot primordia and small shoots are transferred to TSPM tomato shoot proliferation medium (MS salts, B5 vitamins, 1 mg/L zeatin, 0.1 mg/L indole-3-acetic acid, 100 mg/L kanamycin, 500 mg/L carbenicillin or 300 mg/L timentin, and solidified with 8 g/L agar; pH=5.7). Shoots 2-3 cm long are excised, and placed on a TR-R tomato rooting medium (MS salts, B5 vitamins, 30 g/L sucrose, 8 g/L agar, and pH adjusted to 5.7) containing 100 mg/L kanamycin for secondary selection. After acclimatization plants with well developed roots are transferred to the greenhouse for further growth and fruiting.