ISSN 00036838, Applied Biochemistry and Microbiology, 2012, Vol. 48, No. 8, pp. 657–666. © Pleiades Publishing, Inc.

, 2012.
Original Russian Text © M.I. Chumakov, E.M. Moiseeva, 2012, published in Biotekhnologiya, 2012, No. 1, pp. 8–20.

PROBLEMS,
PERSPECTIVES

Technologies of Agrobacterium Plant Transformation In planta

M. I. Chumakova,b and E. M. Moiseevaa
a
Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Science, Saratov, 410049 Russia
b
Chernyshevskii State University, Saratov, 410012 Russia
email: chumakov@ibppm.sgu.ru
Received August 9, 2011

Abstract—In this review, methods of Agrobacterium TDNA transfer into plant cells in planta are discussed.
The main focus is on the technologies of gene transfer into generative plant cells as a part of Agrobacterium
TDNA. The influence of the plant genotype, bacterial strain, vector construction type, inoculation medium
composition, and the conditions of plant treatment with Agrobacterium on the efficiency of Agrobacterium
transformation in planta is analysed. Based on literature and personal experimental data, the possible mech
anism of Agrobacterium transformation of generative plant cells in planta is discussed.

Keywords: genetic transformation in planta, transgenic plants, Agrobacterium tumefaciens

DOI: 10.1134/S0003683812080017

Horizontal gene transfer within a TDNA (DNA
transferred by Agrobacterium into a plant genome)
from Agrobacterium to a plant genome has been used
widely as a method of transgenic plant creation. The
big advantage of this method is an ability to insert
almost any genes into the TDNA region. There are
two types of Agrobacterium transformation methods:
in vitro, involving the cultivation of plant cells and tis
sues with the subsequent regeneration of the plant, and
in planta, where these steps are missing.
During in vitro Agrobacterium transformation,
plant tissues or cell suspension are used as recipients
for Agrobacterium inoculation. A selective agent is
added to the medium after the transformation, mor
phogenesis of transformed cells is induced, and a
whole plant is generated. One of the problems of
in vitro transformation is the chimerism of produced
transformants and somaclonal variations. For mono
cotyledonous plants, an additional complication is the
low morphogenetic potential, which in some cases
does not allow for the generation of a fertile plant; for
example, some economically important agricultural
crops can be considered. Those limitations make the
problem of agricultural crop regeneration (especially,
monocotyledonous) the most acute; it should also be
mentioned that the regeneration stage is expensive and
timeconsuming and requires specialized equipment
and highly qualified personnel. This stage can be
avoided if transgenic plants are produced by trans
forming intact plant cells in planta.
The first Agrobacterium transformation in planta
was performed on Arabidopsis seeds in 1987 [1]. Dif
ferent parts of a plant (depending on the method used)
are inoculated by Agrobacterium (T0 generation).
Plants usually do not undergo selection and are growth
in soil until flowering and seed ripening. The seeds are
then collected and transformants are selected (T1 gen
1
eration) by germination on a medium with a selecting
agent.
This review discusses methods of gene transfer
within Agrobacterium TDNA into plant cells by
in planta method, its efficiency, and factors effecting
to this process.
METHODS AND OBJECTS
OF AGROBACTERIUM
TRANSFORMATION IN PLANTA
A typology of in planta methods is not yet devel
oped; occasionally, there is not enough understanding
of what cell and tissue types can be targeted by Agro
bacterium TDNA. Most commonly, a method’s name
reflects the plant part or organ that is inoculated by
Agrobacterium: method of floral bud submersion (flo
ral dip or floral spray), application of Agrobacterium to
pistil strands (pistil drip, pollen tube pathway), seed
transformation, etc. The efficiencies of different
transformation methods in planta are listed in the table
[2–33].
The seeds transformation by in planta methods
includes the seeds incubation with Agrobacterium cells
and growing plants under natural conditions until har
vesting seeds of the next generation, which are then
placed on the medium with a selecting agent [1]. The
1 In
other publications, the symbols can be used differently; thus,
treated plans can be referred as T1, and their seeds can be
referred as T2. In this manuscript, the following symbols are
used: treated plants, T0; seeds collected from them, T1

657
658 CHUMAKOV, MOISEEVA

Efficiency of Agrobacterium plant transformation (% of treated plants) using an in planta method

A. tumefaciens
Efficiency,
Method Modification Plant species strain Source
%1
(construct)
1 2 3 4 5 6
Inoculation during germination C58Clrif(3850:1003) 0,3 [1]
Treatment with ultrasound Thale cress
(Arabidopsis thaliana) A281(pLD3),
in the presence of aluminium 0.6–0.8 [2. 3]
A281 (pPCVRN4)
oxide prior to seed inoculation
Common wheat
Seed inoculation (pPGV3850) 3.3–9.62
(Triticium aestivum L.) [4]
under vacuum conditions
Red fescue (Festuca rubra) (pPGV3850) 2.02
Seed
inoculation EHA101
Scalpel cut in the apical meristem area Maize (Zea mays) 0.62 [5]
(pWM101S6)
LBA4404
Asian rice (Oryza sativa) (pIG121Hm, 40–133 [6]
pBires), M21
Puncturing of the germination spot
LBA4404
Common wheat
(pIG121Hm, 29–703 [7]
(Triticium aestivum L.)
pBires), M21
Puncturing of the meristematic tissues White mulberry
M21 1004 [8]
of axillary buds (Morus alba L.)
Kenaf LBA4404 (pBires),
Puncturing of lateral buds n/a [9]
(Hibiscus cannabinus) M21
Common buckwheat LBA4404 (pBI121),
Puncturing of the sprout hypocotyl 36–703 [10]
(Fagopyrum esculentum) M21
Meristem LBA4404 (pBI121) 5.55 [11]
tissue Thale cress
inoculation Inoculation of the area with a cut shoot (Arabidopsis thaliana) GV3101 (pBI121,
3.6–9.66 [12]
pRD410, pGH8)
EHA105
(pBI121bar,
Barrel medic PKYLX7Gus,
Vacuum infiltration of sprouts 2.9–26.7 [13]
(Medicago truncatula) pBINmgfpERbar,
pGA482bar),
GV3101 (pSKL015)
MP51 (pGKB5) 1.0 [14,15]
GV3101
(pCAMBIA2301, 1.4–1.6 [16]
Thale cress pCAMBIA 1305)
(Arabidopsis thaliana)
GV3101 (pBI121C) 0.8–1.8 [17]
GV3101 (pBINm
0.5 [18]
gfp5ER)
Flower
bud Vacuum infiltration of inflorescence Rockcress LBA4404 (pBIN
0.02–0.7 [19]
inoculation (Arabidopsis lasiocarpa) mgfp5ER)
ASE1
Barrel medic (pSLJ525, pSKL006),
12.6–76.4 [13]
(Medicago truncatula) EHA105
(pBINmgfpERbar)
Pakchoi (Brassica rapa L. C58/pMP90
0.01 [20]
ssp. Chinensis) (pDHBNIa1)
Rape (Brassica napus) C58CIRifR (pNOV264) 0.2 [21]

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 TECHNOLOGIES OF AGROBACTERIUM PLANT TRANSFORMATION in planta 659

Table. (Contd.)
A. tumefaciens
Efficiency,
Method Modification Plant species strain Source
%
(construct)
1 2 3 4 5 6
Flower GV3101 [18,22]
0.1–0.3
bud (pBINmgfp5ER)
inoculation
GV310 [16]
Thale cress (pCAMBIA2301, 1.7–2.0
(Arabidopsis thaliana) pCAMBIA 1305)
ABI (CCR2:LUC), [23]
0.1–0.7
GV3101 (CCR2:LUC)
Inflorescence submersion into an GV3101 (pBI121C) 0.9–2.0 [17]
Agrobacterium suspension
C58C1 [24]
Common wheat
(pDs(Hyg)35S), 6.8
(Triticium aestivum L.)
AGL1 (pBECKSred)
Radish (Raphanus sativus AGL1 [25]
L. var. Longipinnatus (pCAMBIA3301) 1.4
Bailey)
Rockcress LBA4404 [19]
0.03–0.5
(Arabidopsis lasiocarpa) (pBINmgfp5ER)
Application of a suspension drop Thale cress pBI101, pGWB1 [26]
0.3–1.0
on a flower bud (Arabidopsis thaliana)
Spraying inflorescence with Agrobacte Thale cress GV3101 (pBI121C) [17]
1.3–2.4
rium suspension in the form of aerosols (Arabidopsis thaliana)
Sorghum (Sorghum [27]
GV3101 (pTd33) 1.4–2.2
bicolor L. Moench)
Maize (Zea mays) GV3101 (pTd33) 6.8 [28]
Common wheat C158 [29]
Application of an Agrobacterium 2.7
(Triticium aestivum L.) (pCV2260, pSIR42)
suspension to the pistil during or
after artificial pollination Petunia (Petunia hybrida) AGLO (pCGP1258) 7.5 [30]
Flower
inoculation Upland cotton [31]
EHA105 (pKF111) 0.04–0.9
(Grossipium hirsutum L.)
Common wheat [32]
n/a up to 2.6
(Triticium aestivum L.)
Incubating an Agrobacterium suspen Upland cotton GV3101 [33]
0.8
sion with pollen under vacuum condi (Grossipium hirsutum L.) (pCAMBIA 1301)
tions prior to its application to the pistil Petunia (Petunia hybrida) AGLO (pCGP1258) 9 [30]
Note: 1, transformants T1/seeds T0; 2, transformants T0/seeds T0; 3, transformants T1/sprouts T1; 4, 4 transformants T0/inoculated
plants T0; 5, transformants T1/regenerated shoots T0; 6, transformants T1/treated plants T0; n/a, data not available.
Transformants T0/seeds T0 is the transformation efficiency expressed as a ratio between the number of plants successfully trans
formed and treated with Agrobacterium (transformants T0) and the total number of seeds that were germinated and subsequently
treated with an Agrobacterium suspension (seeds T0), %.

efficiency of Arabidopsis transformation under these
conditions was moderate (0.32%); however, this
method became useful to obtain the mutants of this
plant [2, 18, 34], which can be used to analyze gene
functions. Gradually, techniques increasing the effi
ciency of seed transformation were developed and the
variety of transformed plants was increased. For exam
ple, the efficiency can be increased if seeds are treated
with ultrasound in the presence of aluminium oxide
during Agrobacterium incubation [3] or if inoculation
is carried out with vacuum assistance [4]. Apart from
these techniques, researchers use mechanical damag
ing of seeds during transformation, for example, dam
aging corn seeds with a scalpel before incubation with

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 48 No. 8 2012

660 CHUMAKOV, MOISEEVA
an Agrobacterium suspension [5] or puncturing two
holes in the surface of a wheat or rice seed in the
expected area of germination [6, 7] and subsequently
submerging the seeds in an inoculation medium with
Agrobacterium.
In other studies, meristematic tissues were inocu
lated with an Agrobacterium suspension [8–10] (see
table). Meristematic cells–containing tissues were
punctured with a fine needle and wrapped in a thin
cotton applicator saturated with Agrobacterium sus
pension. For the first time, this technique was used on
buckwheat by puncturing a hypocotyl of a sprout [10].
For the transformation of mulberry [8] and kenaf [9],
meristematic tissues of axillary buds were used.
One of the interesting modifications of Agrobacte
rium transformation of the meristematic tissue
in planta is a method offered by S. Chang et al., sug
gesting shoot regeneration on a plant. A shoot was
removed from an Arabidopsis plant, a suspension of
Agrobacterium was applied to the cut, and a new shoot
was regenerated (see table) [11]. This method was also
used by V. Katavic et al. [12].
A new approach to Agrobacterium transformation
in planta was the development of methods for the
transformation of generative tissue cells. One of the
first works in this area was published by N. Bechtold
et al. [14], suggesting to apply vacuum infiltration of
an Agrobacterium suspension to the tissues of a devel
oping flower. Arabidopsis plants (A. thaliana) were
grown until flower buds appeared; removed from the
soil; placed in an Agrobacterium suspension under a
bell, where vacuum conditions were created for a few
minutes; and then replanted into the soil until seed
ripening [15]. A similar approach was successfully
used for other types of plants: pakchoi (B. rapa L. ssp.
Chinensis) [20, 35, 36], Barrel medic (M. truncatula)
[13], and rape (B. napus) [21] (see table). However, there
were also some unsuccessful attempts of transformation
using this method, for example, for blindweed (C. bursa–
pastoris) and two types of Arabidopsis (A. petraea,
A. griffithiana) [19].
For the transformation of Arabidopsis generative
tissues, a simpler method, named floral dip, was also
developed [18]. This method suggests simply dipping
flowers (before opening) in an Agrobacterium cell sus
pension without vacuum assistance. There are varia
tions of the floral dip method: for inoculation, an
Agrobacterium suspension could be applied to flower
buds with a pipette [26] or sprayed over inflorescences
(floral spray) [17]. Inoculation of flower buds was
mainly used for dicotyledons—A. thaliana [18] and
Raphanus sativus [25]; however, there are reports of a suc
cessful transformation of monocotyledons (wheat) [24].
Authors comparing the vacuum infiltration and
floral dip methods contained in their works report a
similar efficiency of these methods for A. thaliana [16,
18]. However, vacuum infiltration turned out to be
more efficient for A. lasciocarpa [19], and there were
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
no transformants obtained when the floral dip tech
nique was used for Brassica napus [21].
Another transformation method is the treatment of
generative plant organs with Agrobacterium directly
during pollination–fertilization. D. Hess et al. [32]
applied an Agrobacterium suspension to an inoculation
medium with a surfactant (Tween) and acetosyringone
to wheat spikes during the anther formation period
using a pipette. A similar approach was used by V.A.
Pukhal’skii [29]. An Agrobacterium suspension was
applied with a brush to pistils of emasculated wheat
flowers after artificial pollination. A similar method
was used for other types of plants. Flowering sorghum
panicles were inoculated with an Agrobacterium sus
pension after pollination [27]. M.I. Chumakov et al.
[28] suggested a method of TDNA integration into
the corn genome by treating pistils with A. tumefaciens
cells and subsequently performing pollination.
C. Tianzi et al. [31] used a method, which they named
pistil drip. The authors applied an Agrobacterium sus
pension to cotton plant (Grossipium hirsutum) pistils
on the first or second days of florescence.
A pollen grain and a pollen tube, growing through
the stigma, are considered to be the most probable tar
gets for TDNA during the pollination process; there
fore, some studies were aiming to transform pollen
directly. Pollen was collected from blossoming plants
and mixed with a pollen germination medium, Agro
bacterium was added to the suspension, and vacuum was
applied. The pollen treated in this way was then applied to
pistils of emasculated flowers. A successful transforma
tion by this method was achieved for a cotton plant
(G. hirsutum)[33] and petunia (P. hybrid) [30].
It has to be mentioned that there are critical publi
cations regarding the Agrobacterium transformation of
monocotyledonous plants by the application of pollen
treated with an Agrobacterium suspension [37]. The
authors report that there were no positive results asso
ciated with Southern hybridization in the T1 genera
tion, which indicates, in their opinion, the instability
of nuclear genome integration or a possibility of T
DNA integration into the plastid genome. The authors
explain the positive results of Southern hybridization
in the T0 generation by the TDNA transfer into endo
phytic microflora during cultivation of seeds in the soil
without selection factors [37]. The authors also men
tion that different levels of plant resistance to antibiot
ics should be taken into account during the prelimi
nary selection of transformed plants.
FACTORS INFLUENCING THE EFFICIENCY
OF AGROBACTERIUM TRANSFORMATION
Temperature, composition of the inoculation
medium, bacterial cell concentration, use of virulence
gene inductors, Agrobacterium strain, type of vector
construct, and the plant genome are important factors
for any type of transformation technique [38]. The
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 TECHNOLOGIES OF AGROBACTERIUM PLANT TRANSFORMATION in planta
plant development stage at the moment of inocula
tion, the specifics of the flower structure and develop
ment, and length of plant tissue contact with Agrobac
terium become especially important for in planta
methods. The factors significantly influencing the effi
ciency of in planta transformation procedures are
reviewed below.
Temperature influence. The temperature interval
suitable for Agrobacterium transformation is limited by
the temperature tolerance of the proteins, participat
ing in the TDNA transfer. The effect of temperature
on the Agrobacterium transformation was studied on a
model of plant cell culture. Temperatures below 15°C
and above 29°C are critical for transformation in vitro
[39]. A temperature of 19°C is considered to be the
most efficient for a virdependent transfer, although
25°C is an optimal temperature for vir gene expression
[40]. At 28°C the efficiency of transformation
decreases, possibly due to the negative influence of the
increased temperature on the membranebound
VirB–VirD4 proteins [40] that take part in TDNA
and protein transfer through the cell membrane. The
expression of vir genes is completely suppressed at a
temperature increase to 32°C because the VirA recep
tor protein may be inactive at this temperature [41,
42]. In the studies by E.M. Lai and C.I. Kado [43],
the VirB protein, which is crucial for the TDNA
transfer process, lost its functioning at 28°C. In addi
tion, the functioning of the VirD2 protein, which is
responsible for TDNA excision and Tstrand pilot
ing, also decreased at 28°C and was completely sup
pressed at 30°C and above [44].
The question regarding temperature influence on
the efficiency of in planta transformation is interest
ing, since it is often impossible to create favorable
temperature conditions when using this method. For
the majority of monocotyledonous plants, the temper
ature of cultivation with Agrobacterium is 24–28°C
[38]. The inoculation temperature usually varies
between 22 and 26°C [1, 7, 16, 18, 25]. The transfor
mation of tobacco plant sprouts with an Agrobacterium
suspension is completely blocked at 31°C [45]. Differ
ent results were obtained in two independent studies of
Arabidopsis inoculation with Agrobacterium at 16 and
22°C: in one study, the transformation efficiency
increased at a higher temperature; in the other study, it
remained the same [12]. In corn transformation
experiments, the in planta treatment of corn pistil
strands with Agrobacterium was more efficient at a
temperature of 22–25°C compared to 18–20°C [28].
It has to be mentioned that HeLa animal cells were
transformed with Agrobacterium even at 37° [46].
Agrobacterium strain and plant genotype (line)
influence. Supervirulent strains of A. tumefaciens, car
rying standard binary or superbinary vectors, are fre
quently used for plant transformation. These combi
nations are not a prerequisite for a successful transfor
mation; however, they are very common.
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 48
661
In our corn transformation studies, it was shown
that the LBA4404 strain, carrying a vector construct
with the gus and nptII genes, on average achieved a
higher amount of transformations compared to the
other strains used (AGL0). In a study by A. Viktorek
Smagur et al. [16], the efficiency of Arabidopsis trans
formation with Agrobacterium strain GV3101, con
taining various plasmids, differed by 2–14 times in
several of the experiments.
The plant line can also significantly influence the
success of a transformation, which may be explained
by the physiological and genetic characteristics of
plants. In a study by S.J. Clough and A.F. Bent [18],
several ecotypes of Arabidopsis were transformed
in planta; for three of these ecotypes, the transforma
tion was equally efficient, whereas for the other three
a 10 to 100fold decrease in efficiency and even the
absence of transformants were observed. In a study by
V. Katavic et al. [12], the inoculation of the same
amount of plants for each of the three Arabidopsis
ecotypes with the same Agrobacterium strain showed
that two ecotypes had a similar efficiency of transfor
mation, whereas for the third one transformants could
not be obtained.
Our studies have shown that various corn lines are
transformed with different efficiency (unpublished
data). For example, the use of the Saratov Embryo
Marker/Saratov Brown Marker hybrid combination
on average had a three times higher transformation
efficiency compared to the AT3/ZMSP hybrid com
bination. The difference in the transformation effi
ciency may be due to the genetic and physiological
characteristics (the amount of flowers that are ready
for pollination, flower development stage, and the
condition of pollen) of maternal and parental plants.
Agrobacterium cell cultivation conditions and inoc
ulation medium. Usually, young bacterial cultures,
obtained by cultivation in a liquid medium until the
stationary phase (for 8–12 hours with aeration) at a
temperature of 28°C, are used for transformation [15,
16, 23, 47]. In a study by Clough and Bent [18], Agro
bacterium cells cultivated until the late stationary
phase (over 84 hours of growth) were used, and no
change in the Arabidopsis transformation efficiency by
the floral dip method was reported. It was shown that
cultivation of bacterial cells on solid media does not
have a significant effect on the efficiency of transfor
mation, even if the cultures were stored for a week at a
temperature of 4°C [47].
Some authors use YEBS or LB media to prepare
bacterial suspensions [12, 23, 47]. Sometimes, Agro
bacterium grown on a nutrientrich medium can be
resuspended in plain water [6–10]. After the incuba
tion on a nutrientrich medium, bacteria are collected
by centrifugation and then resuspended in an inocula
tion medium until the suspension density reached
OD600 = 0.8 [18]. The use of higher or lower density
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662 CHUMAKOV, MOISEEVA
suspensions (OD600 from 50.15 to 1.75) practically
does not affect the transformation efficiency [18].
At the same time, media compatible for plant
growth are often used for inoculation with Agrobacte
rium in planta, for example, Murashige and Skoog
(MS) media, which can sometimes be supplemented
with other components, including sucrose and surfac
tants [1, 4, 11, 15, 16].
For the vacuum infiltration transformation
method, initially an MS medium for plant growth sup
plemented with vitamins, cytokinins, and sucrose was
used [14]. However, it was later found that the pres
ence of salts in MS media is not crucial and almost
does not influence the transformation efficiency [18].
The critical components of inoculation media dur
ing the floral dip transformation method are sucrose
and surfactant (silwet L77, pluronic acid F68, and
Tween20) [13, 18, 25]. The transformation frequency
was increased when sucrose was added to the inocula
tion medium (0.5–10%) [13, 18]. A lack of sucrose in
inoculation media led to the absence of transformants
or a significant reduction in the transformation effi
ciency [18]. Silwet L77 is the most widely used surfac
tant, added to pesticides and fertilizers to increase
their efficiency by promoting the absorption of chem
icals through stomata. The use of silwet L77free inoc
ulation media often results in the absence of transfor
mants or in a low efficiency of the process [13, 16, 18,
25]. The optimal concentration of this surfactant is
established to be between 0.02 and 0.05% [16, 18, 25];
a further increase in the concentration reduces the
transformation efficiency [16, 25], and a 0.1% con
centration causes necrosis of plant tissues [18]. The
use of other surfactants (pluronic acid F68, Tween20)
has a smaller effect on the transformation efficiency
[25]. A lower concentration of silwet L77 (0.005%) is
used during vacuum transformation [48], although a
fourfold higher concentration leads to a twofold
increase in the transformation efficiency [18]. It is
possible that a surfactant acts similarly to a vacuum,
promoting the penetration of plant tissues into bacte
rial cells.
Plant development stage. For in planta transforma
tion methods, such as floral dip or vacuum infiltration,
the development stage of the plant generative organs at
the time of inoculation is highly important. For Arabi
dopsis transformations, the most favorable stage is
when a plant has a high amount of unopened flower
buds [18], that may be related to the specifics of the
plant gynecium formation. During its development,
an Arabidopsis ovary forms the open, vaseshaped
structure, which closes for three days before pollina
tion. At this particular time, the penetration of Agro
bacterium cells inside a developing pistil is possible.
The generative tissue development stage is impor
tant for other transformation methods as well, which
may be due to not only anatomical, but also physiolog
ical characteristics. Quite often, however, authors only
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
mention the most favorable stage for transformant cre
ation, without explaining the nature of the observed
phenomenon. For example, for wheat transformation,
transformants were only obtained if spikes were sub
merged into an Agrobacterium suspension for no more
than four to seven days prior to anther formations
(during microspore formation) [24]. The optimal
stage was when a spike reached 6–7 cm in length and
was not visible from the glume. Transformants were
not obtained if the spikes submerged into an Agrobac
terium suspension were less than 4 cm in length [24].
When using Arabidopsis seeds, transformants were
only obtained if the seeds were germinated 12 hours (but
not 6 or 24 hours) prior to inoculation. The authors sug
gested that it is related to seed damage; a seed becomes
susceptible to transformation at the moment of germina
tion when the seed cover is broken [1].
Conditions of plant inoculation with Agrobacterium.
The time of contact between an Agrobacterium suspen
sion and plant tissues in the case of the floral dip
method and the duration of vacuum assistance are also
important factors for transformation. During inocula
tion, Agrobacterium cells penetrate under the covers of
a flower bud and possibly into the intercellular space
after that; therefore, a prolonged inoculation time
increases the transformation efficiency. An increase in
the submersion time of an Arabidopsis flower bud in an
Agrobacterium suspension by 1 to 2 min was shown to
cause an almost twofold increase in efficiency [16].
The same effect was observed when increasing the sub
mersion time from 5 s to 5 min [17]. The time of floral
bud incubation in a vacuum is usually determined
empirically and can vary from 2 [16, 17] to 20–25 min
[15, 20]. In a study by ViktorekSmagur et al. [16],
a 4min incubation in a vacuum was proven to be more
efficient compared to 2, 6, and 8 min. In a study by
W.C. Wang et al. [21], transformants were only
obtained if vacuum incubation reached 5 min. How
ever, prolonged exposure to vacuum conditions can
damage the plant or decrease the transformation effi
ciency [17]. The transformation efficiency increases if
a floral bud is inoculated 2–3 times at intervals of a few
days [17, 18, 26].
Obviously, bacterial cells remain on the plant sur
face for some time after inoculation and can still trans
form the plant cells. For pakchoi, it was determined
that Agrobacterium cells can remain on the plant sur
face and in the intercellular space after vacuum infil
tration: it requires two weeks to clear the leaves and
shoots from Agrobacterium, and in inflorescences they
can remain for 19 days [36]. This is why it is important
to maintain a moist environment around an inocu
lated plant for the first 1–3 days. For this purpose,
plant pots are usually covered with glass or plastic
bells. Prolonged moist conditions (two days instead of
one) led to a twofold increase in the transformation
rate [18].
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 TECHNOLOGIES OF AGROBACTERIUM PLANT TRANSFORMATION in planta
POSSIBLE MECHANISM
OF AGROBACTERIUM
TRANSFORMATION in planta
At various development stages (from a seed to a
flower), different plant tissues can undergo Agrobacte
rium inoculation using in planta transformation meth
ods. It implies that different tissues and cells can be
targeted by TDNA. The inheritance of TDNA in the
T1 generation indicates the incorporation of TDNA
into DNA of the generative cells of the T0 plants
treated with Agrobacterium; however, the incorpora
tion into DNA of other tissues is also possible. The
inheritance of TDNA in the T2 generation was
explored in a number of in planta transformation stud
ies, and, in most cases, Mendelian inheritance of an
insert was shown [1, 12, 13, 20, 24, 25, 49]. The num
ber of inserts can vary from one to several.
An investigation of seed transformation methods
and an analysis of transgenes generated a hypothesis
that Agrobacterium cells reach the meristem through a
micropyle, enter the intercellular space of the embryo,
and remain there during the plant development.
Sometimes, Agrobacterium can transform generative
cells, forming a zygote, or a zygote itself during sporo
genesis, gametogenesis, or fertilization [34]. V. Kata
vic et al. [12] made similar conclusions in their study,
although the technique used was different from the
seed transformation method.
During in planta transformation of developing
generative tissues (floral dip, vacuum infiltration),
TDNA is only present in one homologous chromo
somes of a transgenic plant [14, 34]. This indicates
that TDNA integration occurs at the flower develop
ment stage when male and female tissue cells are
already formed. Arabidopsis is a selfpollinating plant,
and if the transformation occurred at the development
stage prior to the androecium and gynecium forma
tion, then after selfpollination some T1 transformants
would be homozygous for the transferred gene; how
ever, most studies confirm that transformants are het
erozygous [14]. On the other hand, all parts of a trans
genic plant contain the TDNA insert, which means
that the zygote contained the gene prior to its first divi
sion. It is proven that Arabidopsis transformants grown
from one pod are independent. Therefore, transfor
mation occurs at the time when separate seed buds
have already formed in the ovary [22, 49].
Developing female reproductive tissues are a sug
gested target for Agrobacterium transformation using
the floral dip and vacuum infiltration methods [22,
49]. The success of transformation depends on the
flower development stage. The most favorable stage for
Arabidopsis Agrobacterium transformation is when a
plant carries a large amount of unopened flower buds
[18]. This implies that reproductive tissues are accessi
ble for Agrobacterium cells at this stage. It is possible
that female tissue transformation occurs when the
gynecium of Arabidopsis is opened, but closed locules
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
663
are not formed [22]. Transformants are only obtained
if flower buds are inoculated between the 6th and 11th
days prior to florescence, when pistils are opened [22].
Probably, this is when bacteria penetrate the ovary and
TDNA is transferred to female reproductive tissues.
A sixfold increase in the transformation efficiency was
observed when mutated Arabidopsis with not fully
accreted carpels and a partly opened gynecium was
used [22].
To determine the tissue targets for TDNA, hys
tochemical tissue staining was used after inoculation
with Agrobacterium carrying vector constructs with the
betaglucuronidase gene [22, 49]. Expression of the
uidA (gus) gene was observed in female tissues and pol
len grains after vacuum infiltration [49] or submersion
of Arabidopsis floral buds [22] into an Agrobacterium
suspension. However, no staining of pollen grains was
observed when using a vector with the gus gene under
the control of a promoter that only mediates expres
sion in pollen [22]. In addition, in these studies, no
transgenic offspring was received from plants polli
nated with pollen from Agrobacteriuminoculated
plants with removed pistils. On the contrary, transfor
mants were obtained during pollination of emascu
lated plants with removed anthers, treated with Agro
bacterium and pollen of untreated plants [22, 49].
Nevertheless, it cannot be excluded that the male
gametophyte is transformed during germination on a
stigma or during fertilization. The discussion is still
open on whether the expression of gus genes in these
experiments is an indicator of the stable transforma
tion of plant tissues, which then pass this gene onto the
next generation. It cannot be completely excluded that
gene expression is simply a consequence of plant tissue
contamination with bacteria. N. Bechtold et al. [50]
did not observe gus gene expression in pollen grains or
the embryo sac after vacuum infiltration of Arabidopsis
when the gene was under the control of a gametophyte
(male and female) promoter. On the example of
mutated lines of Arabidopsis, it was show that the
female gametophyte is a target for TDNA. Later, the
possibility of Arabidopsis endosperm transformation
was also shown [51]; during this process, the transfor
mation of the ovary and endosperm happen indepen
dently. The above information indicates that transfor
mation occurs in an already formed embryo sac, con
taining an ovary and a central cell.
According to some authors, the floral dip method is
only efficient for some species of the Brassicaceae
family [51, 52]. It is possible that the mechanism of
transformation during pollination–fertilization is dif
ferent from the one described above, since the
gynecium of other species may not have any contact
with inoculated surface tissues of a plant either during
development or during pollination. In this case, the
male gametophyte—a pollen grain or a pollen tube
germinating on a stigma—is a target for Agrobacterium
during the pollination process [29, 31–33, 53].
Vol. 48 No. 8 2012
664 CHUMAKOV, MOISEEVA
The possibility of pollen transformation by Agro
bacterium is still being discussed. Pollen grains are pro
tected by a thick exine, and the penetration of Agro
bacterium through it is unlikely; however, they do have
a pore for future pollen tube growth. The leading edge
of a pollen tube does not have a rigid cellular wall,
which may benefit the contact of bacteria with a pollen
tube. A pollen tube is also able to perform exocytosis,
and thus may in theory capture exogenous DNA [54,
55]. Vacuum conditions during the incubation of pol
len with Agrobacterium may promote a closer contact
of bacteria with pollen grains [30, 33].
According to some data, a pollen transformation
with Agrobacterium in vitro on a nutrient medium was
successful and expression of the betaglucuronidase
gene transferred withinTDNA was observed [56, 57].
However, in a study by E. Stoger et al. [58], the pos
sibility of Agrobacterium transformation of pollen was
questioned. Tobacco plant pollen was incubated with
suspensions of different Agrobacterium strains, carry
ing binary vectors, including constructs with the gus
gene. Blue staining of pollen grains was observed for
constructs effectively expressed in bacteria. However,
if pollen was cultivated with Agrobacterium strains,
carrying TDNA genes expressed only in plants (the
gus gene with an intron), no staining was observed.
The authors suggested that a stained product can dif
fuse from Agrobacterium cells to pollen grains or
stained Agrobacterium cells can be absorbed on the
surface of pollen grains, which would explain the false
positive staining of pollen [58].
One of the suggested mechanisms for TDNA pen
etration into the generative tissues of corn after the
treatment of pistil strands is Agrobacterium cell move
ment towards the ovary together with the germinating
pollen tube and subsequent absorption of TDNA (as
part of bacteria or a Tcomplex) during fusion between
a pollen tube and an ovary [53, 59]. It was shown that
six micron pieces of latex (the size of Agrobacterium is
(0.6–1.0) × (1.5–3.0) micron) can reach an embryo
sac together with a pollen tube [60].
Haploid inducer lines of corn were used to explore
the mechanism of TDNA penetration into an embryo
sac during in planta transformation [53]. The develop
ment of matroclinous haploids (plants that only carry
a maternal set of chromosomes) in the next generation
is then possible; a pollen tube reaches an embryo sac,
but the sperm of haploid inducer line pollen grains is
unable to fuse normally with an ovule [61]. The pres
ence of transformed haploid corn plants indicate the
possibility of Tcomplex (or Agrobacterium cells)
delivery within the cytoplasm of a pollen tube to an
embryo sac [53].
CONCLUSIONS
Thus, the technology of Agrobacterium plant cell
transformation in planta allows one to bypass the com
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
plications that accompany, for example, the transfor
mation of agricultural monocotyledons with a low
morphogenetic potential, and to simplify the expen
sive and timeconsuming stage of plant regeneration,
requiring special equipment and highly qualified per
sonnel. The development of in planta techniques is a
promising task, provided that some difficulties can be
overcome. For example, transformation via inflores
cence inoculation—one of the most developed and
repeatable methods—is only efficient for some spe
cies, mainly for the cruciferous family, which must be
due to the characteristics of their generative organs.
Therefore, when working with new species, the
methods of in planta transformation have to be
adapted and conditions (the plant development stage,
inoculation media, and the technical process) have to
be optimized. For example, a positive effect can be
obtained through actions promoting the Agrobacte
rium penetration—vacuum conditions and mechani
cal damaging of covers (ultrasound, treatment with
aluminium oxide particles, puncturing, scalpel cut,
etc.). For the floral bud inoculation methods, impor
tant factors are the addition of sucrose and surfactant
into the inoculation medium and selection of an opti
mal stage of flower bud development; for transforma
tion using the pollination–fertilization process, selec
tion of an inoculation time (before, during, or after
pollination), pollen treatment (vacuum), etc. The
effects of plant genotypes, type of vector constructs,
composition of inoculation media, and the conditions
of treatment with Agrobacterium on the efficiency of
Agrobacterium transformation in planta also have to be
taken into account.
An analysis of studies exploring the mechanisms of
TDNA transfer into generative plant cells in planta
has shown that in a number of cases the ovule is proven to
be the target for TDNA, whereas the possibility of male
gametophyte transformation is more controversial.
ACKNOWLEDGMENTS
The study was supported by the Russian Founda
tion for Basic Research, grant no. 110401331.
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