Agro-bacterium-mediated transformation is a common method for inserting genes into dicotyledonous plants using the soil bacterium Agro-bacterium. The method involves using Agro-bacterium containing a T-DNA vector to transfer genes of interest into plant cells, followed by regeneration of transformed plants on selection media. Protocols are provided for transforming tobacco and chrysanthemum explants using Agro-bacterium, including co-cultivation, selection, regeneration of shoots, rooting of shoots, and verification of transformation. Transformed tobacco and chrysanthemum plants were successfully recovered using this method.

1. Agro-bacteriummediatedTransformation
M.PHOOL BADSHAH

2. Introduction
• A common method for the transfer of genes into dicotyledonous
plants is to use the plant pathogen Agro-bacterium.
• In nature, plant cells infected by this soil bacterium integrate
segments of the large tumor-inducing (Ti) plasmid of A. tumafaciens
or the root-inducing (Ri) plasmid of A. rhizogenes into their nuclear
genomes, leading to crown gall or hairy root disease, respectively.
• To utilize Agro-bacterium to genetically modify crops, it is essential
to have T-DNA vectors into which target genes can be inserted.

3. Objectives and Goals:
• To enable the user to gain expertise in the use of Agro-bacterium for
plant genetic engineering.
• Methodologies are presented for tobacco, which serves as a model
system, and for chrysanthemum, which is an example of a
horticultural useful crop amenable to transformation.
• To produce genetically modified plants by the treatment of leaf
explants with Agro-bacterium followed by regeneration of plants
from these explants in the presence of a selection agent, and to
verify transformation by biological and molecular means.

4. Biological Materials:
• Agro-bacterium tumafaciens strain LBA4404 (available from the State
University of Leiden) or EHA101 (available from Washington University)
are used.
• Equipment and Supplies:
• Tissue culture work station with Bunsen burner, ethanol burner or
electric sterilizer
• Lighted plant growth chamber or culture room, 24 DC
• Incubator, dark 28 DC, or ambient room temperature up to 32 DC
may be used
• Gyratory shaker
• Refrigerator for storage of media and bacterial cultures - Dissection
microscope.

5. hg
• Dissection microscope
• Petri dishes, sterile disposable plastic, 100 X 15mm or 100 X 20mm
• Erlenmeyer flasks, sterile 125 ml with cotton plugs
• Forceps, sharp scalpels, inoculation loop
• Pipettes, sterile 5 ml or 10 ml graduated
• Filter paper or paper towels, sterile
• Magenta GA-7 boxes - 24-well or 96-well plates, or micro
centrifuge tubes for GUS staining assay .
• Para film
• Filter sterilization units, O.22-Jlm pore size
• Carbenicillin
• Kanamycin

6. s
• Hygromycin B,
• Acetosyringone
• Preparation of Media:
• Prepare and autoclave the basal media.
• Cool media to about 50 DC.
• All antibiotic solutions should be filter-sterilized.
• Add antibiotics to the medium, swirl to mix.
• Dispense media into Petri dishes, 25-30 ml/dish, or as directed.

7. Preparation of GUS Staining Reagents:
• Prepare phosphate buffer: Start with two solutions, 100 mM each of
K phosphate, monobasic (KH2P04) and dibasic (K2HP04).
Gradually add the monobasic solution to the dibasic solution until
pH 7.0 is obtained. Keep the pH 7.0 phosphate buffer at 4 DC.
• Prepare Triton/ethanol stock. Mix 10ml Triton X-100, 40ml ethanol,
and 50 ml water. Keep at room temperature.
• Prepare X-Gluc stock: 10mg X-Gluc per ml of dimethylsulfoxide.
• Mix the above. For 100ml of GUS stain mix 94ml phosphate buffer,
1ml Triton/ethanol and 5 ml X-Gluc stock. Refrigerate the GUS
stain until needed. It is best to use fresh.

8. Treatment of Materials:
• Plant Materials:
• Grow shoots of tobacco and chrysanthemum in vitro using MS
medium in Magenta GA-7 boxes.
• If starting from seeds, surface-sterilize seeds, sow on MS medium in
petri dishes, allow to germinate in the light, and initiate shoot
cultures.
• Maintain shoot cultures at 24°C in a growth chamber with 16h
light/8h dark, 55micro-mole-m-2s-1, using plant growth lights.
• Subculture shoots by cutting tops off and placing in fresh medium
every 6 weeks.

9. Preparation of Bacterial Culture:
• Use Agro-bacterium tumefaciens strain LBA4404 or EHA101
containing a binary vector with a selectable marker such as neo
(NPTII, for kanamycin resistance in both plant systems), hph (for
hygromycin resistance in tobacco), or aadA (for spectinomycin
resistance in chrysanthemum).
• Streak and culture for 2 days in the dark at 28°C on LB agar
medium in petri dishes with appropriate tetracyclin concentration for
plasmid maintenance.

10. • Using a sterile inoculation loop, transfer a single Agro-bacterium
colony from the plate into about 25 ml of liquid LB medium without
the antibiotics in a sterile Erlenmeyer flask.
• Place the flask on a shaker overnight at 28°C, 150 - 250 rpm
• 4. Dilute the 25ml culture with 1O-15ml of fresh liquid LB medium.
Let the Agro-bacterium suspension grow another 3-4h on a shaker at
28°C. Optional: Measure Optical Density at 550nm until cells reach
a reading of 0.8-1.0 (0.8- 1 X 109 cells/ml).

11. Protocols:
• Tobacco Transformation: Preparation of Leaf Sections and Co-
cultivation :
• Prepare leaf sections (5 X 5 mm) from 4-5-week-old tobacco shoot
cultures. The top three fully expanded leaves are a good source of
explants. Place the sections on MS medium.
• When all sections are prepared, transfer them into Agro-bacterium
suspension. Make sure the sections are fully submerged for several
seconds.
• Blot the sections on a sterile filter paper and culture on tobacco co
cultivation medium SM, 30-40 sections per petri dish.
• Wrap the petri dishes with Parafilm, and incubate them In the
growth chamber for 2-3 days.

12. Selection and Regeneration of Tobacco
Transformants:
• Optional: after co cultivation, wash the leaf sections in liquid SM-C
medium.
• Transfer leaf sections to SM-CK300 medium to select for
kanamycin resistance or SM-CH20 medium to select for
hygromycin resistance, depending on the selectable marker in the
vector.
• Wrap the plates with parafilm and incubate in the lighted growth
chamber.
• Once or twice each week, examine the leaf sections for any signs of
contamination. Within 2-3 weeks, tiny shoots should appear around
the edge of sections.

13. hj
• The sections themselves will turn bleached in appearance on
kanamycin, or will appear brown and dead on hygromycin.
• Control explants in the absence of the antibiotics remain healthy and
green in appearance, and will regenerate large numbers of shoots
around the explant at the cut surface.
• When shoots are 10-15 mm in length, remove and place them on
rooting medium MS-K or MS-H in Magenta boxes, to maintain
kanamycin or hygromycin selection, respectively.

14. • Transformed shoots, but not nontransformed shoots, will develop
roots in the rooting medium. Rooting will occur in 7-14 days.
• When plantlets are at least 3 cm tall, transfer to soil in pots.
• After potting in soil, keep the plants under high humidity conditions
(i.e., under moist or in plastic bags with vent holes) and gradually
adapt them to greenhouse conditions.

15. Results:
• Transformed shoots of tobacco were recovered efficiently following
co-cultivation with Agro-bacterium tumefacien due to the
effectiveness of the selection system and the capacity of tobacco to
regenerate in vitro.
• Reporter gene expression, such as for GUS activity provided partial
evidence for the transformed nature of the recovered shoots.
• Transgenic tobacco plants developed normally to maturity.
• Recovery of transformed shoots also was efficient in
chrysanthemum . The rooting assay provided partial evidence for
transformation of chrysanthemum. Transgenic chrysanthemum
plants also developed normally.