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1. Agro-bacterium mediated
Transformation
Presented by ‘ 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, 3',5' -dimethoxy-4'
• 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.
• 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).

10. 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.

11. 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.

12. 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.
• 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.

13. 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