Protocol for Stable Maize Transformation
N. Raizada and Virginia Walbot
of Biological Sciences, Stanford University, Stanford, CA 94305-5020
is a detailed procedure of the biolistic transformation of maize
embryogenic callus as first described by Fromm et al. (1990) and
Gordon-Kamm et al., (1990) and adapted by us.
– The ubiquitin promoter- bar herbicide resistance plasmid,
pAHC20, is kindly provided by Peter Quail (PGEC, Albany, CA) (Christensen
and Quail, 1996). The bar gene encodes resistance to the
herbicide Basta® or bialaphos (De Block et al., 1987).
of Embryogenic Callus Cultures
X B73 (HiII) seed (Armstrong and Green, 1985) is obtained from Monsanto
(Chesterfield, MO) and sib- or self-pollinated. Nine to thirteen
days after pollination, 1-2 mm embryos are dissected in the dark,
and callus is induced on N6 1-100-25 media (Armstrong, 1994) containing
N6 salts+ vitamins, 1 mg/L 2,4-D, 100 mg/L casamino acids, 25 mM
proline, 20 g/L sucrose, 2 g/L Phytagel (Sigma) and 10 µM
AgNO3 (Armstrong and Green, 1985; Songstad et al., 1991).
Calli are maintained in complete darkness at 27ºC, 75% RH in
100 x 25 mm Petri dishes during the entire callus induction, bombardment
and herbicide selection procedures. Following 4 weeks on induction
media with one subculture, calli are transferred to media lacking
AgNO3, and maintained for up to 3 months, with a 2 week
subculture regime, selecting for white, embryogenic, friable tissues
at each subculture. Every 3 months, new cultures are initiated.
and double-stranded M13 DNA are isolated as supercoiled DNA using
a Wizard Maxiprep Kit (Promega), then extracted once with phenol:chloroform
and ethanol precipitated. DNA is precipitated onto 1µM spherical
gold particles (Alameda Scientific Instruments, ASI) and accelerated
onto immature somatic embryoid tissue using a Helium PDS 1000HE
device (BioRad) (see Sanford et al., 1993) following the procedure
of Wan et al., 1994. Equimolar quantities of all plasmids are coprecipitated.
All DNA precipitation and bombardment steps are performed under
sterile conditions at room temperature.
precipitation for three bombardments uses 2 mg of gold resuspended
in ethanol; gold is centrifuged in a Beckman TJ-6 swinging bucket
rotor at 2000 rpm in an Eppendorf tube. The pellet is rinsed once
in distilled water, recentrifuged, and resuspended in 25 µL
of 1 µg/µL total DNA. In between the addition of each
of the following reagents, the tube is briefly vortexed: 220 µL
H20, 250 µL 2.5M CaCl2, 50µL 0.1M
spermidine, free base (Sigma or Aldrich). Fresh spermidine is found
to be the most critical reagent in this procedure. The precipitating
DNA mix is then placed on ice for 5 min. Each tube is then vortexed
for 1-2 min at room temperature, and centrifuged at 500 rpm for
5 min in a Beckman TJ-6 centrifuge. The supernatant is removed and
the pellet is resuspended in 600 µL ethanol, and centrifuged
for 1 min on a table-top microfuge at 14,000 rpm. The final pellet
is resuspended in 36 µL of ethanol and used immediately or
stored on ice for up to 4 hr prior to bombardment. All steps involving
aqueous solutions are performed efficiently to prevent agglomerization
of the DNA (Sanford et al., 1993).
bombardment, tissues are used 5-9 days following subculture. Four
to six hours prior to bombardment, the best embryogenic, friable
calli clumps growing rapidly on the surface of callus cultures are
gently removed as ~5 mm diameter clumps and crowded together (embryoids
facing up) in a ~3x3 cm area on the surface of a Baxter S/P Filter,
Grade 363 (5.5 cm) filter, and placed on osmotic induction media
(N6 1-100-25, 0.2 M mannitol, 0.2 M sorbitol [Armstrong, 1994; Kemper
et al., 1996; Vain et al., 1993]) at 27ºC in darkness. Ten
plates are prepared for each transgenic line, along with an additional
plate that is bombarded with plasmids encoding the anthocyanin regulators
pR and pC1 (Ludwig et al., 1990) as a positive control for the DNA
precipitation and bombardment procedures. Red spots are scored 16-40
hr after bombardment.
minutes prior to shooting, filters are removed from the medium and
placed onto sterile opened Petri dishes to allow the calli surface
to partially dry. Ten µL of the 36 µL gold-DNA-ethanol
solution are spread onto the surface of a macrocarrier (ASI), briefly
dried, and accelerated in a vacuum of 27 psi against a wire mesh
screen (ASI). Each plate is typically shot once with a 650 psi disc
and a second time with a 1100 psi disc that has been briefly soaked
in isopropanol to promote a good seal to the rupture disk holder.
The distance from the rupture disc to the macrocarrier is 1.0 cm
and from the mesh screen to the target, 5.9 cm. If gold residue
is not seen on the surface of the calli, they are bombarded a third
bombardment, the filters holding the calli are transferred back
onto osmotic media and incubated for 16 hr in darkness at 27ºC.
The filters are then transferred onto callus maintenance media N6
1-100-25 (no herbicide) for 1-2 days to promote tissue recovery
prior to herbicide selection.
callus is removed from the bombardment filters and placed onto herbicide
selection media: N6 2-0-0 (N6 salts + vitamins, 2 mg/mL 2,4-D, no
proline or casamino acids) containing 3 mg/mL bialaphos (Meiji Seika
Kaisha Ltd, Japan) (Spencer et al., 1990; Denney et al., 1994).
All tissues are nonselectively subcultured without breaking every
12 days for 10-12 weeks. After the first subculture, the tissue
is partially flattened on the media to promote direct contact with
the herbicide. After 6-8 weeks on herbicide, white, fast-growing
sectors can be detected growing out of the nonproliferating and
partially necrotic mother calli. These resistant sectors are permitted
to grow to a diameter of 1 cm, and then they are selected and subcultured
onto fresh herbicide media. Resistant calli are permitted to proliferate
to occupy an entire plate, and are subcultured every two weeks,
during which embryogenic sectors are aggressively chosen. If necessary,
0.4 M sorbitol are added to the media for 1-5 days to promote the
induction of embryogenic sectors. Individual resistant callus lines
(designated cA1, cA2, cA3, etc.) are checked by RNA gel blot hybridization
for the presence of transgene expression and selectively regenerated.
is performed following the procedure of Armstrong (1994). Embryogenic
calli are placed in the dark onto Regeneration 1 media for two weeks.
This media contains MS salts + vitamins (Sigma), 2% sucrose, 0.1
mg/mL 2,4-D and 0.1 µM ABA to promote the formation of somatic
embryos, and 1 mg/mL bialaphos. Embryogenic calli are then transferred
to Regeneration 2 media and maintained in the dark for 2 weeks.
Regeneration 2 media contains N6 salts + vitamins, 1 mg/mL bialaphos
and 6% sucrose to promote embryo enlargement and maturation. Differentiated,
white somatic embryoids are then placed onto Regeneration 3 (MS
salts + vitamins, 2% sucrose, 1 mg/mL bialaphos, no hormones) media
<2 weeks in 100 mm x 25 mm Petri dishes to promote seedling germination;
conditions are16 hr day (70 µMol m-2s-1)
and 8 hr night at 25-27ºC. Immature seedlings are
transferred onto the same media in baby jars, and transferred to
soil after substantial root development. Five or more plants from
each regenerated line are transferred to the greenhouse.
conditions are 16 hr day, 8 hr night, 28ºC day and 22ºC
night, with a mixture of 1000W sodium vapor and metal halide lamps.
Seedlings are transferred into 15 cm round pots into loose soil
containing peat moss and perlite (Sunshine Mix or Metromix 350)
with Nutricote 13-13-13 slow release fertilizer, and maintained
in a high humidity environment under individual 1 pint supermarket
plastic cups for 3 days at 225-500 µMol m-2 s-1,
or summer partial shade sunlight. After hardening off, the seedlings
are maintained in full summer sunlight or ~1600 µMol m-2s-1
greenhouse light and transferred to potting soil. Plants are outcrossed
to promote vigorous kernel development. The initial regenerated
plants are called T0 while the first seed belongs to
the T1 generation.
test for bialaphos resistance, a 5 cm diameter marked leaf surface
is painted with 0.75% glufosinate ammonium (Ignite 600, 50% solution,
Hoescht, Canada) with 0.1% Tween 20 using a Q-tip. The area is visually
scored for the presence or absence of necrosis 5-7 days later.
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