In planta transformation of Arabidopsis
Vacuum infiltration method
Floral-dip method
Advantages:
1. Simple, short
2. No somaclonal variation
Disadvantages:
1. Limited to Arabidopsis so far
2. Some success with Medicago (Harrison et al. 1999)
and Brassica (Liu et al. 1996)
3. Will probably be useful only with those species, which
produce large number of seeds per plant.
Several in planta methods of transformation have been described in past
30 years. Most of them were not reproducible and the apparent positive
results obtained were generally the results of artifacts or ambiguity.
The first experiments were based on the assumptions that similar to what
was observed with some bacteria (Avery et al. 1944), the incubation in
defined conditions of plant organs or cells with extracted DNA could allow
this DNA to enter cell and finally nucleus leading to a heritable
modification.
Seed imbibitions-germination or pollination-fertilization were the two
preferred processes during which purified DNA was applied.
All attempts were futile till Agrobacterium was discovered as the genetic
engineer.
Pollen as vector for plant transformation
Pollen were treated with DNA prior to or during pollination.
In 1980s a few groups reported using wheat that by depositing DNA
containing npt gene on stigma or injecting into style transgenic
progenies were obtained. Transformation frequency 1-20% were
reported. But this method was not reproduced by others.
Germinating seeds as targets for DNA transfer
In the first report (1969) seedlings of a white flowering Petunia hybrida
mutant were incubated with the DNA extracted from young leaves of red
flower. A high percent (27% vs 9%) of plants derived from the treated
seeds showed red flowers as compared to the control seeds treated with
their own DNA. Some genetic analysis was done to show that new
anthocyanin synthesis loci were present. These results were never
confirmed or repeated.
In a case study of Arabidopsis (1971) experiments suggested that
exogenous DNA could be absorbed by germinating seedlings,
translocated towards floral organs, and recovered in the progeny.
Thiamineless mutants of Arabidopsis were treated with bacterial DNA
(1974). Correction of the mutation was relatively high (10-2 vs 10-4).
Using Agrobacterium, first unambiguous but inefficient (therefore nearly
irreproducible) report was published in 1987. Feldmann and Marks imbibed
Arabidopsis seeds in a suspension of Agrobacterium tumefaciens bearing
npt gene on T-DNA. They used MS media with 4% sucrose. Imbibition
occurred for 24 h at 28oC. The imbibed seeds were grown normally and
allowed to produce seeds by self-pollination. Among these seeds some
gave rise to entirely transformed plants which could be selected on antibiotic
selection media. The transformation frequency average to 1 transformant in
the progeny of 100 plants derived from treated seeds.
Feldmann group generated more than 17000 T-DNA lines using this
method. This collection called as “Feldmann lines” served as first resource
for Forward genetics in Arabidopsis genomics era.
Feldmann KA & Marks MD (1987) Mol. Gen. Genet. 208:19
Vacuum infiltration method
•Grow Arabidopsis to flowering
stage
•Uproot plants
•Application of Agrobacterium in
vacuum condition in sucrose
containing growth media.
• Re-planting
•Seed collection
Bechtold, N., Ellis, J., Pelletier, G. 1993 C. R. Acad. Sci. Paris Life Sciences 316:1194-1199.
Floral-dip method
Procedure:
Dip a bunch of flowering plants of Arabidopsis
in Agrobacterium suspension prepared by
suspending fully grown culture of bacteria in
5% sucrose supplemented with surfactant
(Silwet L-77).
Clough, SJ & Bent, AF (1998) The Plant Journal 16: 735-743.
Flowering stage
Modified solutions
Effects of inflorescence developmental stage and inoculation medium
composition on the rate of transformation.
(a) Transformation of plants of different height/developmental stage. (b) Effect of
modified inoculation media on transformation rate (standard inoculation medium
contained MS medium, pH 5.7, 44 um BAP, 5% sucrose, 0.005% Silwet L-77).
Effect of concentration of Silwet L-77 on transformation rates following dip
inoculation.
Effect of repetitive dip inoculations on transformation:
(a) Plants dip-inoculated only once during the same growth period as the
plants that were dipped multiple times.
(b) Plants that were dip-inoculated at the indicated day intervals during a 15
day period commencing the day after primary inflorescences were
clipped.
Effect of various sugars on transformation
Sugar
% Transformation
No sugar
0.04 ± 0.01
Sucrose, 0.5%
0.40 ± 0.13
Sucrose, 1.25%
0.34 ± 0.03
Sucrose, 2.5%
0.47 ± 0.13
Sucrose, 5%
0.36 ± 0.08
Sucrose, 10%
1.42 ± 0.25
Glucose, 0.5%
0.14 ± 0.08
Glucose, 1.25%
0.11 ± 0.08
Glucose, 5%
0.76 ± 0.29
Glucose, 10%
0.33 ± 0.12
Mannitol, 5% a
death
5% food-grade sucrose
0.48 ± 0.27
Plants dipped in A. tumefaciens resuspended to OD600 = 0.8 in aqueous
Silwet L-77 (0.05%) with sugar as noted. Values are mean ± SE.
aSilwet
L-77 0.005% for mannitol treatment.
Effect of inoculum density on rate of transformation
Inoculum OD600
% Transformation
0.15
0.50 ± 0.02
0.42
0.21 ± 0.05
0.80
0.51 ± 0.14
1.10
0.51 ± 0.09
1.75
0.57 ± 0.15
0.8 (84 h)
0.50 ± 0.05
Plants inoculated by vacuum infiltration with A.
tumefaciens in MS Medium with BAP, 5% sucrose and
0.005% L-77. All bacteria resuspended from a fresh
overnight liquid culture, except '84 h' from culture grown
for 84 h. Values are mean ± SE.
Different ecotypes and Agrobacterium strains
Ecotypes Ws-O, Nd-O, No-O were transformed at rates similar to Col-O. In
contrast, Ler-O, Dijon-G and Bla-2 transformed at 10- to 100-fold lower rates. In
one of the experiments, zero transformants were obtained with Ler-0.
In experiments that examined the use of other Agrobacterium strains, LBA4404,
GV3101, EHA105 and Chry105 were used successfully to transform ecotype
Col-0 by the floral dip method.
Transgenic plants obtained by in planta transformation
methods are hemizygous therefore transformation in flower
must be occurring after the divergence of male and female
germline. Only one of them gets transformed as a result
generates hemizygous transgenic plants after selffertilization.
Male or female?
These studies addressed it
1. Ye et al. (1999) Plant J. 19: 249-257.
2. Bechtold et al. (2000) Genetics 155:1875-1887.
3. Desfeux et al. (2000) Plant Physiol 123: 895-904.
Ye et al. (1999)
GUS expression seen in floral buds of infiltrated Arabidopsis plant 3-5 days post
vacuum-infiltration transformation, or in immature seeds 3 weeks postinfiltration.
Plant infiltrated with
Agrobacterium containing
pMON15726 (GUS gene)
Plant infiltrated with
Agrobacterium
containing no GUS
gene.
Immature seeds
in siliques
expressing
GUS.
Ye et al. (1999)
Distribution of transgenic
seeds on a single plant.
The small insert represents
the plant at the time of
vacuum infiltration. The
secondary bolts were
numbered and their lengths
were measured and shown in
centimetres. The green bars
on the plant represent
siliques bearing transgenic
seeds, whereas the black
ones represent siliques
bearing no transgenic seeds.
The numbers next to the
green bars show the number
of transgenic seeds
recovered from each silique.
Ye et al. (1999)
Target of transformation as revealed by crosses
No.
attempted
No.
successful
c
Wt X Wt
30
13
43.3
Inf X Wt
187
87
46.5
15
Wt X Inf
138
88
63.7
0
Emasc.
ctrl
30
0
0
0
b
Cross
Percentage
efficiency
No. of transgenic
seeds
bCrosses:
Wt X Wt where pollen donors and recipients were both wild-type plants;
Inf X Wt where the infiltrated plants served as the pollen recipients and the wild-type
plants served as the pollen donors; Wt X Inf where the wild-type plants served as the
pollen recipients and the infiltrated plants served as the pollen donors; Emasc. Ctrl
where the infiltrated plants were hand emasculated and allowed to grow to maturity.
cPercentage
efficiency was expressed as the number of successful crosses divided by
the number of crosses attempted (100).
Bechtold et al. (2000) Genetics 155:1875-1887
Desfeux et al. (2000) Plant Physiol. 123: 895-904.
Used a mutant deficient in male
gametogenesis. Success of transformation
of such plant and transmittance of transgene
to next generation suggested that female
gametophyte is the target of transformation.
GUS expression in ovules/developing seeds of
flowers from previously non-transformed plants
dipped in Agrobacterium carrying ACT11-gusA T-DNA
constructs. A, B, and D, ACT11-gusA (no intron)
construct. C and E, ACT11-gusA-intron construct. A,
Staining of an entire locule cavity, likely due to
bacterial GUS expression from Agrobacterium
colonizing the locule interior. B, Elongating seed pod
from fertilized flower. C, Entire flower with staining of
ovules only. D, Close-up of ovules in a segment of a
dissected flower showing no staining, localized
staining, or complete staining of individual ovules. E,
Close-up of two ovules (partially overlapping in photo)
showing staining of embryo/embryo sac rather than
entire ovules.
Target of transformation revealed by crosses
Desfeux et al. (2000) Plant Physiol. 123: 895-904.