Transformation Protocol for Tobacco

Plant material:In vitro plants of Nicotiana tabacum L. cv Wisconsin 38 Day 1 Prepare bacteria: • Streak YM plus antibiotic plates with bacteria. • Incubate plates for 2-3 days at 28°C. Day 3 Transformation:

• Measure about 20mL Minimal A medium for each bacterial strain.

• Scrape or wash bacteria from plate with sterile loop and suspend in 20mL of Minimal A.

• Adjust density to an OD600 0.9-1.0.

• Take first healthy fully expanded leaves from 4-5 week old tissue culture grown tobacco plant, cut into 0.5cm squares (or can use a cork borer, which is about 1.0cm diameter) in deep petri dish, under sterile RMOP liquid medium. Store tissue pieces in RMOP in deep petri dish.

• Transfer leaf pieces (about 20 per transformation) to deep petri dish containing bacterial suspension.

• Swirl to ensure bacteria has contacted cut edge of leaf and let stand for 5 minutes.

• Remove leaf pieces from suspension and blot dry on filter paper or on the edge of the container.

• Place leaf pieces with adaxial side (upper leaf surface) on solid RMOP, about 10 pieces per plate.

• Incubate plates in dark at 28°C for: 2-3 days A. tumefaciens 5 days S. meliloti 5 days M. loti 5-11 days Rhizobium sp. NGR234 Day 6-14 Selection:

• Transfer leaf pieces onto solid RMOP-TCH, with abaxial surface (lower surface of leaf) in contact with media.

• Incubate plates for 2-3 weeks in the light at 28°C, with 16 hours daylight per day.

• Subculture every 2 weeks. Plantlet formation:

• When shoots appear transfer to MST-TCH pots.

• Incubate plantlets with 16 hours daylight for 1-2 weeks.

• When roots form the plants can be transferred to soil in the glasshouse.

• Plants can be maintained in tissue culture but will need to be subcultured every two weeks. This is done by cutting the growing tip of plant, removing excess leaves and transferring this to fresh media.

Media and Solutions for Tobacco Transformation
YM Media

Mannitol                        10g

Yeast extract                0.4g

K2HPO4 (10% w/v stock)            1 ml

KH2PO4 (10% w/v stock)            4 ml

NaCl (10% w/v stock)                1 ml

MgSO4.7H2O (10% w/v stock)    2 ml

• Add Water to 1L

• pH 6.8

• Agar 15g/L

• Autoclave

• When ready to pour add antibiotic selection if required

Keep poured plates for 2 days at room temperature to visualise any contamination, then store at 4°C.

RMOP + RMOP-TCH media  (Svab, et al, 1975) Sucrose  30g    3%

Myo-inositol  100mg   0.1%

MS Macro 10x               100mL   1x

MS Micro 1000x  1mL    1x

Fe2EDTA Iron 100x  10mL    1x

Thiamine-HCl (10mg/mL stock) 100μL   1mg

NAA (1mg/mL stock)   100μL    0.1mg

BAP (1mg/mL stock)   1mL    1mg • pH 5.8

• Phytagel 2.5g/L for solid

• autoclave

• for RMOP-TCH, when ready to pour add:

• Timentin (200mg/mL stock) 1mL

• Claforan (250mg/mL stock) 1mL

• Hygromycin (50mg/mL stock) 1mL

'''BAP (1mg/ml) (6-Benzylaminopurine) ''' Add 1N KOH drop wise to 100mg BAP until dissolved. Make up to 100mL

with Milli-Q H2O

• Store 4°C

NAA (1mg/ml) (Naphthalene acetic acid)

Dissolve 100mg NAA in 1mL absolute ethanol. Add 3mL 1N KOH. Make up

to 80mL with Milli-Q H2O. Adjust pH to 6.0 with 1N HCl, make up to 100mL

with Milli-Q H2O.

• Store 4°C Cefotaxamine® (250mg/ml) Add 8ml sterile Milli-Q H2O to 2g Claforan • Store 4°C in dark Timentin® (200mg/ml) Add 15ml sterile Milli-Q H2O to 3g Timentin

• Store 4°C MST + MST-TCH media (Svab, et al, 1975) Sucrose 30g    3%

MS Macro 10x              100mL   1x

MS Micro 1000x 1mL    1x

Fe2EDTA Iron 100x 10mL    1x

• pH 5.8

• Phytagel 2.5g/L

• autoclave

• for MST-TCH, when ready to pour add:

• Timentin® (200mg/mL stock) 1mL

• Cefotaxamine® (250mg/mL stock) 1mL

• Hygromycin (50mg/mL stock) 1mL MS Macro 10x  (Murashige and Skoog, 1962)

Final concentration

10x (g/L)   1 x mM

KNO3 19.0    18.8

NH4 N03   16.5    20.6

CaCl2.2H2O 4.4    3.0

MgS04.7H2O 3.7    1.5

KH2PO4   1.7    1.25

• Store 4°C

• Substituting chemicals:

• CaCl2 3.3g/L

• MgS04 1.8g/L MS Micro 1000x   (Murashige and Skoog, 1962) Final concentration 1000x (g/L)                        1 x μM

MnS04.4H20              22.3    100

ZnS04.7H20 8.6    30

H3BO3 6.2                 100

KI 0.83    5.0

Na2MoO4.2H2O 0.25    1.0

CuSO4.5H2O 25mg    0.1

CoCl2.6H2O 25mg    0.1

• Store 4°C

• Substituting chemicals:

• MnS04.H20 16.9/L

'''FeSO4EDTA Iron 100x '''    g/1L                               1 x mM

FeS04.7H20              2.78    0.1

Na2EDTA 3.72                 0.1

• Store 4°C in dark bottle

References: Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Phys. Plant. 15: 473-497. Svab, Z., P. Hajdukiewicz and P. Maliga. 1975. Transgenic tobacco plants by co- cultivation of leaf disks with pPZP Agrobacterium binary vectors. In “Methods in Plant Molecular Biology-A Laboratory Manual”, P. Maliga, D. Klessig, A.. Cashmore, W. Gruissem and J. Varner, eds. Cold Spring Harbor Press: 55- 77.