Plant Physiol.

(1985) 79, 568-570

0032-0889/85/79/0568/03/$0l1.00/0

Short Communication

High Efficiency Transformation of Cultured Tobacco Cells'

Received for publication June 17, 1985

GYNHEUNG AN
Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340
region of Tn5 as a selectable marker. Here I report that rapidly
growing tobacco suspension-cultured cells can be transformed at
Tobacco calli were transformed at levels up to 50% by cocultivation of a high frequency with Agrobacterium containing our Ti vectors.
tobacco cultured cells with Agrobacterium tumefaciens harboring the
binary transfer-DNA vector, pGA472, containing a kanamycin resistance
MATERIALS AND METHODS
marker. Transformation frequency was dependent on the physiological
state of the tobacco cells, the nature of Agrobacterium strain and, less
Tissue Culture. Tobacco (Nicotiana tabacum L. cv bright
so, on the expression of the vir genes of the tumor-inducing plasmid. yellow 2) cells were maintained in Linsmaier and Skoog liquid
Maximum transformation frequency was obtained with exponentially medium (14) containing 0.2 ,g/ml of 2,4-D at 28C with shaking
growing plant cells, suggesting that rapid growth of plant cells is an at 150 rpm on a gyrotary shaker and subcultured every week
essental factor for efficient transformation of higher plants.
with a 4% inoculum.
Transformation. Three d after subculture, 4 ml of the plant
cells were transferred to 100-mm Petri plates and incubated at
ABSTRACT

The efficient transfer of cloned genes into plants is a prerequisite to understand the regulation of plant gene expression. In
recent years several studies have demonstrated the utility of
naturally occurring Ti2 plasmids as transformation vectors for
higher plants (5, 17). In this technique, exogenous DNA is
inserted into the T-DNA segment of the Ti plasmid by homologous recombination using the intermediate vector system (2, 21),
or directly into one of the recently designed binary vectors (1,
4). In the binary vector system, the vector contains a selectable
marker and the minimum cis-acting sequences required for
transformation, i.e. the T-DNA border sequences (18, 20). The
vir genes, which are also necessary for T-DNA transfer (8, 10,
13, 15), are provided in trans by a helper Ti plasmid. The
development of plant drug resistance markers such as that for
kanamycin has allowed direct selection of transformants without
induction of tumorigenic growth and suppression of cellular
totipotency (1, 3, 7, 9). Regenerating mesophyll protoplasts have
been used for many transformation studies (1, 3, 7, 9), but
because plants of many species are refractive toward regeneration
(6), and/or protoplasts are killed during the treatment with A.
tumefaciens (G. An, unpublished data), alternative transformation procedures were developed, e.g. co-cultivating leaf tissue
slices with Agrobacterium (1, 12). I have previously demonstrated
that tobacco mesophyll protoplasts can be transformed by A.
tumefaciens containing the binary vector pGA472 and a helper
Ti plasmic (1). pGA472 consists of the T-DNA borders, a wide
host range replicon, and a chimeric fusion of the nopaline
synthase promoter to the neomycin phosphotransferase coding

28C with 100 gl of fresh overnight culture of A. tumefaciens

containing the binary vector pGA472 (1) and a helper Ti plasmid
pTiBo542 (16). After 48 h of co-cultivation, the bacterial cells
were washed off and the plant cells were plated on the LS agar
medium containing 0.2 mg/ml of 2,4-D, 500 ,ug/ml of carbenicillin and various amounts of kanamycin. The kanamycin-resistant transformants can be detected 2 to 3 weeks after selection.
O-Galactosidase Assay. One hundred Ml of fresh overnight
Agrobacterium was incubated with 4 ml of plant cells in the
Linsmaier and Skoog medium for 24 h. The plant cells were
removed by a brief centrifugation and the bacterial cells were
assayed for f-galactosidase activity as described previously (19).

RESULTS AND DISCUSSION

Suspension-cultured tobacco cells were transformed by cocultivating with A. tumefaciens containing the binary vector
pGA472 and a helper Ti plasmid Bo542 as described in "Materials and Methods". The kanamycin-resistant transformants
form calli of about 1 to 2 mm diameter within 20 d on agar
medium containing up to 500 gg/ml of kanamycin, whereas
untreated tobacco cells did not grow on the antibiotic plates (Fig.
1). The kanamycin-resistant calli continued to grow on fresh
kanamycin plates for several months and were shown to be
genuine transformants by the presence of NPIT activity as measured in crude extracts (Table I). All the resistant calli contained
high levels of NPT activity compared to untransformed calli,
although the amount of enzyme activity varied depending on
the isolate. I am currently analyzing the transformed tissues in
order to determine whether this variation is due to the copy
number of the kanamycin-resistant gene per genome and/or the
location of the transferred DNA in the chromosome. To obtain
the frequency of transformation, cocultivated tobacco calli were
' Supported in part by grants from the National Science Foundation
grown on kanamycin-free agar medium and then individually
(DCB-8417721) to G. A. and the United States Department of Agricul- picked and transferred to fresh agar plates containing 500 yg/ml
ture (82-CRCR-1-1027) to Eugene W. Nester. This is Scientific Paper kanamycin. Although the transformation frequency varied de7148, Project 0692, College of Agriculture Research Center, Washington pending on the experiment, 10 to 50% of the cocultivated calli
State University, Pullman, WA 99164.
grew in the presence of kanamycin.
The microcalli in the suspension culture contain an average
2Abbreviations: Ti, tumor inducing; NPT, neomycin phosphotransof 5 to 10 cells during the growth cycle. Therefore, to obtain the
ferase; T-DNA, transfer DNA.
568

TRANSFORMATION OF CULTURED CELLS

569

FIG. 1. Transformation oftobacco-cultured cells with a kanamycin resistance gene. Three-d-old tobacco cells were cocultivated with A. tumefaciens
containing pGA472 and the transformed calli were selected on the Linsmaier and Skoog agar medium containing various amounts (,Ug/ml) of
kanamycin. Upper lane, untreated tobacco cells; lower lane, cocultivated tobacco cells.

Table I. Neomycin Phosphotransferase Activity of the Transformed

Tobacco Calli
The NPT activity was measured as described previously (1).
NPT Activity
Isolate

is dependent on the specific Agrobacterium strain used (1). To

test whether the same was true for the suspension-cultured cells,
two different Agrobacterium strains, A281 and PC2760, were
used for the transformation experiments. A281 contains pTiBo542 that is a supervirulent Ti plasmid in nature (16) and
transforms
tobacco mesophyll protoplasts at least 10-fold better
mg
oftransformed
cpm/1O
No.
tobacco cells
than other Ti plasmids (1). PC2760 contains pAL4404 that is an
avirulent derivative of the octopine Ti plasmid Ach5 obtained
1
950
by deletion of the entire T-DNA and the octopine catabolism
2
670
(occ) genes (11). Figure 2a shows the growth curve of the cultured
880
3
cells and the transformation frequency obtained by cocultivating
4
430
Agrobacterium with the tobacco suspension calli at different
910
5
growth stages. As observed previously (1), the transformation
460
6
frequency was higher when pTiBo542 was used as a helper Ti
7
170
plasmid. It is, however, more difficult to regenerate plants when
NP
10
pTiBo542 is used as a helper since this plasmid contains a
a Untransformed tobacco cell.
tumorous T-DNA which is often transferred along with the
transformation frequency of individual cells, cocultivated to- kanamycin-resistance gene. Optimum transformation frequency
bacco calli were maintained in liquid medium for 2 weeks with with either helper Ti plasmic was obtained when 3- or 4-d-old
500 ug/ml carbenicillin (to prevent growth of Agrobacterium) exponentially growing cells were used for cocultivation; the transbefore selection of kanamycin-resistant transformants. The trans- formation frequency on the other days was much lower. This
formation frequency obtained by this method was about five correlation between plant cell growth and transformation fretimes lower than the frequency obtained by direct plating of quency suggests that rapid cell division may be essential for
cocultivated cells on kanamycin media, suggesting that only one efficient transformation. It is not clear whether the transfer
mechanism of Ti-plasmids is dependent on DNA replication in
or a few cells in each microcalli were initially transformed.
Closer examination of the physiological state of the cultured the host plant cells, or whether factors necessary for bacteriacell suspension and Agrobacterium strains indicates that these plant recognition and interaction are produced only when the
parameters have a pronounced effect on the transformation plant cells are dividing rapidly. Tumor induction in nature is
frequency. I have previously observed that the transformation known to require wounding. In addition to breaking the protecfrequency of tobacco mesophyll protoplasts during cocultivation tive barrier, such wounding may trigger rapid cell division at the

570

AN
(%)

Plant Physiol. Vol. 79, 1985

taming the lacZ gene fused to different vir genes as described

previously (19). Suspension-cultured tobacco cells were co-cultivated with Agrobacterium for 24 h and the expression of the
bacterial genes was measured by assaying (l-galactosidase activity
in the cocultured bacteria (Fig. 2b). The virB and virE genes
were expressed at high levels when 2- to 4-d old plants cells were
used, whereas the virD gene was induced maximally on d 1 and
2. Expression of the occ gene, which is located outside of the vir
region, was not induced under these conditions in agreement
with previous finding (19). Highest expression of the measured
vir genes did not coincide with the period where the yield of
transformed plants was high, suggestig that vir gene expression
alone is not sufficient for plant transformation.

growth
Bo542

Acknowledgments-I thank T. Nagata, P. R. Ebert, and S. E. Stachel for helpful

discussion, and T. W. Okita and C. Reeves for critical reading of the manuscript. I
also thank B. D. Watson for technical assistance. The early part of this work was
performed in Dr. Eugene W. Nester's laboratory at the Department of Microbiology
and Immunology, University of Washington, Seattle, WA 98195.
1

TIME (DAYS)

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~~~virB
w

co

<
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-J

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0CC

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FIG. 2. The growth curve and transformation efficiency of the cultured tobacco cells. Tobacco-cultured cells were grown and cocultivated
as described in "Materials and Methods". Growth was measured as fresh
weight (g) of plant cells per volume (ml). The transformation frequency
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