Biology Reference
In-Depth Information
resistance is simple. To aid seed collection once the plants stop producing new
flowers, the flowering stems are gently grouped together with string and tied to a
bamboo skewer placed in the center of the pot. Progeny seeds are collected from
the dipped plants after the fruit are dry and light brown in color. These collected
seeds are usually known as the first transformed generation, the T1 seeds. After col-
lection, all
A. thaliana
seeds should be dried in a desiccator for at least 1 week before
sowing or the germination frequency will be low.
A. thaliana
produces large volumes of seed, and so recovering many transformed
lines from each pot of dipped plants is standard. In general, the transformation
efficiency of floral dipping ranges from 1% to 3% (Clough & Bent, 1998). The
efficiency of the transformation can be increased by treating the
A. tumefaciens
cultures with a virulence buffer containing acetosyringone for 2 h prior to plant trans-
formation. Acetosyringone is a chemical released by dicotyledonous plants upon
wounding that induces the virulence of the bacterium (
Winans, 1991
). Cotransforma-
tion with multiple transgenes can be accomplished by dipping the plants in an
A. tumefaciens
suspension containing different transgenic bacteria, as opposed to
creating a bacterial strain harboring multiple binary vectors (
Davis et al., 2009
).
15.1.1.3
Transgenic plant recovery
Transgenic lines are recovered by growingT1 seeds on selective antibioticmedia. The
antibiotics kanamycin and hygromycin and the herbicideBasta are commonly used for
selection. For selection, antibiotics are added to sterile media poured into plates; the
herbicide is applied when watering young seedlings grown on soil. T1 seeds are sur-
face sterilized and aseptically sown in Petri plates containing antibiotic media. After
10-18 days, antibiotic-resistant seedlings are visible because they have expanded
green leaves and long roots. Resistant seedlings are removed from the plate and trans-
planted to soil and then grown tomaturity for T2 seed collection (
Fig. 15.2
). In general,
experiments are carried out with T2 seedlings or the subsequent T3 plants. At least
three independent transgenic lines (lines derived from separate pots of dipped plants)
should be used in every experiment because the T-DNA insertion is random.
There are multiple ways to surface sterilize seeds to prepare them for aseptic cul-
ture. Most methods involve bleach, ethanol, hydrogen peroxide, or chlorine gas. In
our hands, the most efficient protocol is to place seed on a double layer of filter paper
in a sterile laminar flow hood and then rinse the seed with an aqueous solution of 5%
hydrogen peroxide and 75% ethanol. The hydrogen peroxide/ethanol solution is
pipetting directly onto the seeds on the filter paper. About 2 ml of solution should
be used for about 250
l of seeds on 100-mm round filter paper. Depending on
the flow rate of the laminar hood, the ethanol/peroxide solution will fully evaporate
in 5-20 min. After the seeds and filter paper are dry, the seeds are sown at high den-
sity (10-20 seeds/cm
2
) by sprinkling them from the dried filter paper onto the plates.
In addition to the selective antibiotics, 100-500
m
g/ml carbenicillin or 100
m
g/ml
vancomycin can be added to the media to halt the proliferation of any
A. tumefaciens
that escaped sterilization of the seed surface.
m
