Download Binding of ColEl-kan Plasmid DNA by Tobacco

Plant Physiol. (1979) 63, 683-686
0032-0889/79/63/0683/04/$00.50/0
Binding of ColEl-kan Plasmid DNA by Tobacco Protoplasts
NONEXPRESSION OF PLASMID GENE
Received for publication September 13, 1978 and in revised form December 6, 1978
LOWELL D. OWENS
United States Department of Agriculture, Science and Education Administration, Agricultural Research, Plant
Physiology Institute, Beltsville, Maryland 20705
ColEI-kan plasmid DNA. ColEI-kan plasmid (pML2) is a 9megadalton, nonconjugative hybrid of ColEI and pSC105 plasmids and bears a gene for detoxifying kanamycin (5).
ABSTRACT
Protoplasts prepared from cultured tobacco cells were treated with
ColEl-kan plasmid DNA, a hybrid of ColEl and pSC105 plasmids bearing
a gene for kanamycin resistance. The conditions employed permitted the
uptake or irreversible binding of 2.9% of the added DNA in acid-insoluble
form. Upon commencement of division, the treated cells were plated in
agar medium containing kanamycin and differentiating hormones. Plantlets
or shoots obtained as presumptive transformants were further tested on
kanamycin medium by subculturing small leaf pieces. No evidence was
obtained for expression of the kanamycin resistance gene of ColEl-kan in
tobacco tissue.
MATERIALS AND METHODS
Protoplast Isolation and Culture. Protoplasts of Nicotiana tabacum L. "Bright Yellow" were isolated from cell suspension
cultures mainly by the methods of Uchimiya and Murashige (14,
15) but with the following changes. One g (fresh weight) of cells
was incubated for 2.5 h in 40 ml of enzyme solution (pH 5.7)
containing 9.25 mm CaCl2, 0.44 mm Ca(H2PO4)2, 0.85% Cellulysin
(Calbiochem),2 0.5% Driselase (Kyowa Hakko), 1% Rhozyme HP150 (Rohm and Haas), and 0.7 M mannitol. The Rhozyme used
had been desalted on Sephadex G-25 and lyophilized. The cell
suspensions were gently agitated manually at about 15-min intervals. The protoplast wash solution was 0.7 M mannitol, 8.5 mi
The demonstration by Aoki and Takebe (1) in 1969 that purified CaCl2, and 0.5 mm Ca(H2PO4)2 (filter-sterilized separately and
RNA from TMV' could infect protoplasts of tobacco suggested added after autoclaving) (pH 5.8).
Protoplasts were counted in a hemocytometer using the excluthe possibility that protoplasts of higher plants might be genetision
of Evan's blue dye (2 ,lI/chamber of 0.1% in 0.7 M mannitol)
cally altered by uptake of foreign nucleic acids. Ohyama et al. (11)
provided the first evidence that protoplasts could take up DNA, to indicate viability. Washed protoplasts were treated with DNA
and a number of investigators subsequently have sought to deter- (see below) and cultured at a density of 105/ml in protoplast
mine the physical and biological fate of exogenous DNA within culture medium (14) with 0.045 M glucose and 0.6 M mannitol.
protoplasts (3, 6, 8-10, 13). Although methods developed for After 4 days of incubation, fresh medium was added (0.5 of
infecting protoplasts with viral RNA have proven highly efficient, original volume), and at 8 days the cells were embedded in a 1nearly 100%o infection rates with production of over 106 viral mm-thick layer of agar medium (15) in plastic Rodac plates (65
particles per protoplast within 48 h (12), parallel results have not x 55 mm) (Falcon). Microcalli were counted by inverting the
been achieved with either viral or nonviral DNA. Uchimiya and plate on the stage of a microscope.
Screen for Kanamycin Resistance after Treatment with DNA.
Murashige (15) regenerated some 500 tobacco plants from protoplasts that had been treated with DNA isolated from plants Thirteen days after embedding in agar, the microcalli were transresistant to TMV, but none of the regenerants proved resistant to ferred en masse in slabs of agar (1.7 x 2.4 cm) to the surface of
the virus. Likewise, a preliminary report by Carlson (2) recounted shoot-inducing agar (25 ml in plastic Petri dishes, 95 x 15 mm)
an unsuccessful attempt to transform protoplasts from an auxo- medium (14) with tyrosine deleted, 5 ,UM 2-isopentenyl adenine
trophic haploid tobacco plant with DNA isolated from a wild type (Sigma) substituted for kinetic, IAA lowered to I t,M, and 10 tIM
kanamycin sulfate (Sigma) added. Each dish contained six slabs
tobacco plant.
with
a total of about 1,500 microcalli. In this initial screen for
An inherent problem of using eukaryotic chromosomal DNA
for transformation experiments is its high mol wt (equivalent to kanamycin resistance, most microcalli turned brown and grew
about 6 x 1012 daltons of DNA in diploid tobacco) and the very slowly or not at all. Calli which turned green and differencorrespondingly low concentration of DNA specifying a particular tiated buds or small shoots (2-3 mm) were transferred to fresh
gene. Given the DNA uptake rates commonly obtained with medium containing 10 AM kanamycin. Microcalli which tumed
protoplasts (15), the probability of a cell taking up one copy of a green but exhibited no shoot differentiation at the 1-mm-diameter
stage were transferred to shoot-induced medium (14) having 9.3
gene occurring once in the genome is about 1 in 200 cells.
Cloning genes in small plasmid vectors provides a way of ylM kinetin and 11.4 uM IAA but with tyrosine deleted and 10 tIM
obtaining DNA that is highly concentrated in a particular gene. kanamysin sulfate added. In parallel experiments without kanaThe probability of obtaining transformation with such molecules mycin, all microcalli turned green and grew very rapidly on the
first medium, and some shoot regeneration occurred. When unmay therefore be enhanced (7).
In this report we describe an attempt to detect expression of the
2 Mention of a trademark, proprietary product, or vendor does not
kanamycin resistance gene in tobacco protoplasts treated with
constitute a guarantee or warranty of the product by the U.S. Department
of Agriculture and does not imply its approval to the exclusion of other
'Abbreviation: TMV: tobacco mosaic virus.
products or vendors that may also be suitable.
683
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684
Plant Physiol. Vol. 63, 1979
OWENS
differentiated calli 1 mm in diameter were then transferred to the
second medium, over 90o regenerated shoots. Upon reaching a
height of 1 to 2 cm, the shoots were transferred to rooting medium
(15) with IAA omitted.
Plantlets surviving the first kanamycin resistance screen were
tested further by excising 10 replicate leaf pieces (2 x 3 mm) and
placing them on the first shoot-inducing medium described above
containing 0 to 16 yIM kanamycin. Each plastic Petri dish (90 x 24
mm) contained five leaf pieces and 25 ml of medium. The dishes
were incubated in continuous light (500 lux) at 27 C for 30 days
at which time the tissue, consisting almost entirely of differentiated
shoot growth, was harvested, dried, and weighed.
Plasmid Isolation. The two strains of bacteria used, JC41 1 thypro-IColE I and C600/ColE I-kan, were derivatives of Escherichia
coli K12 (5) and were provided by Dr. Donald Helinski. Stock
cultures were maintained on L-broth agar (5). Covalently closed
circular plasmid DNA was prepared by growing the bacteria in
500 ml of a modified M9 medium with thymine (2 ,ig/ml) added,
where required, as described (5). When the cells reached a density
of about 5 x 108/ml, chloramphenicol (300 ,ug/ml) was added,
and cells were harvested 16 h later. To prepare labeled plasmid,
2 mCi of [methyl-3H]thymidine (New England Nuclear) were
added I h after the chloramphenicol addition. A cleared lysate
was prepared and centrifuged for 40 h at 36,000 rpm at 20 C in a
dye-CsCl gradient in a Beckman type 65 rotor. The lower DNA
band was removed, extracted of dye, and concentrated by ethanol
precipitation as described (5). The DNA was stored at -20 C in
SSC (0.15 M NaCl, 0.015 M sodium citrate, pH 7.2) containing 5
mM EDTA. Agarose slab gel electrophoresis of purified DNA was
performed as described (5). Radioactivity in gel slices was determined by melting the agarose at 100 C in 0.3 ml of water and
dissolving in 10 ml of Aquasol-2 liquid scintillation cocktail (New
England Nuclear). The various plasmid preparations were shown
by agarose gel electrophoresis to be predominantly (75% or more)
the covalently closed circular form.
Plasmid DNA Uptake. Plasmid DNA was diluted in 0.5 ml of
0.1 x SSC, filter-sterilized (0.2-,um pore), and added to a sterile
polystyrene tissue culture tube (16 x 125 mm) containing protoplasts suspended in 1.5 ml of the protoplast culture medium
described above except with 0.8 M glucose as the osmoticum and
with 0.66 [Lg/ml poly-L-ornithine (mol wt 122,000, Sigma) added.
After incubation for indicated times on a tube roller (1 rpm) at
25 C, 13 ml of protoplast wash solution was added, and the
suspension was centrifuged (100g, 3 min). The pelleted protoplasts
were treated with DNase by additions of 0.9 ml of 0.7 M mannitol
containing 66 mt KH2PO4 (pH 5.8) and 10 mM MgCl2; 15 ,lI (10
units) of EcoRI endonuclease (Miles Laboratories, Inc.); and 0.1
ml DNase I (Sigma, bovine pancreas, 1 mg/ml in 30 mm MgCl2).
The resuspended protoplasts were incubated at 37 C 10 min.
Protoplasts were washed three times by centrifugation, lysed by
addition of 30 tdl of 10 x SSC and 30 ,ul of 10%1o sodium lauryl
sulfate to the pellet, and heated at 65 C for 10 min. Aseptic
conditions were maintained to this point. Acid-insoluble radioactivity was determined by addition of 0.5 mg of BSA followed by
precipitation with an equal volume of cold 10%7o trichloroacetic
acid. After 20 min on ice, the sample was centrifuged at 10,000
rpm for 10 min, washed with cold 5% trichloroacetic acid, and
centrifuged. The pellet was dissolved in 0.4 ml of 0.1 x SSC and
counted in 5 ml of Aquasol-2.
Where protoplasts were being tested for expression of kanamycin resistance, the DNase treatment was omitted, and the
washed (3 X) protoplasts were cultured as described above.3
3Biohazard containment. The work involving the recombinant plasmid
ColE 1 -kan was performed under P2 laboratory conditions as specified by
the NIH Guidelines for Recombinant DNA Research. All plants regenerated from protoplasts treated with ColEl-kan DNA were cultured in
vitro and, along with bacteria and DNA preparations, destroyed by
autoclaving before disposal.
RESULTS
Binding of 3H-labeled DNA. The effect of pH on binding of 3Hlabeled ColEl-kan DNA by tobacco protoplasts was examined.
The amount of radioactivity associated with the protoplasts following DNase treatment, ie. the "irreversibly bound" fraction,
was about five times greater at pH 5.7 than at pH 7.3. Likewise,
the portion of irreversibly bound radioactivity that was polymeric
(trichloroacetic acid-insoluble) was about 20 times greater at the
lower pH.
The kinetics of [3H]ColEl-kan DNA binding by protoplasts at
pH 5.7 is presented in Figure 1. The initial binding was very rapid,
constituting 70%o of the input DNA by 12 min. The accumulation
of irreversibly bound polymeric material was much slower,
amounting to about 3% of input DNA in 2 h.
Test for Expression of Kanamycin Resistance. A summary of
two experiments in which tobacco protoplasts were treated with
ColE1 or ColEl-kan DNA, cultured to the microcalli stage and
then screened for kanamycin resistance, is given in Table I.
Seven hundred-thousand microcalli were screened en masse on
a shoot-differentiating medium containing kanamycin at a conI
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Kinetics of [3H]ColEl-kan DNA bindi.:- by tobacco protoFIG.
plasts. Protoplasts (6 x 106) were suspended in 2 ml of protoplasts culture
medium (pH 5.7) containing 3.75 ytg [3H]ColEl DNA (74,000 cpm/,ug)
and 0.5 .tg/ml poly-L-ornithine and incubated on a tube roller (I rpm) at
25 C. Aliquots removed at 0.2, 2, and 20 h contained 1.4-, 1.1-, and 0.2 x
106 viable protoplasts, respectively, and were processed as described under
"Materials and Methods." "Total bound" radioactivity represents total
cpm associated with protoplasts prior to treatment with DNase, and
"irreversibly bound" that remaining associated after DNAase treatment.
I.
Table I, Summary of two experiments testing for
expression of the ColEl-kan gene for kanamycin
resistance in tobacco cells
DNA Treatment
ColEl (Control)
ColEl-kan
8.2 x 10
7.3 x 10
326,000
32
272,000
52
22
47
No. protoplasts treated
No. microcalli screened
on kanamycin medium
No. plantlets obtained
No. plantlets tested further1
for kanamycin resistance
1Plantlets
exhibiting gros-s morphological abnormalities
or severe stunting were excluded from further testing.
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Plant Physiol. Vol. 63, 1979
NONEXPRESSION OF ColEl-kan IN TOBACCO
centration (10 ytM) previously determined to exceed by 10%o that
required to prevent the growth, greening, and differentiation of
microcalli. From this initial screen, 32 plantlets or shoots were
obtained from cells receiving the control treatment, ColEl DNA,
and 52 from cells treated with ColEl-kan DNA (Table I).
Further testing of the presumptive transformants for resistance
to kanamycin gave the results presented in Figure 2. The plantlet
leaf pieces derived from the DNA-treated protoplasts exhibited
varying degrees of tolerance to 16 jM kanamycin relative to their
performance on medium lacking kanamycin. The distribution of
tolerance among plantlets regenerated from protoplasts treated
with ColE 1-kan DNA does not appear to differ significantly from
that exhibited by plantlets from protoplasts treated with ColEl
DNA.
DISCUSSION
The "uptake" of [3H]ColEl-kan DNA by tobacco protoplasts
was characterized by an initial rapid binding of DNA to the cell
membrane where it remained largely accessible to DNase. This
reversible binding was followed by a slower, irreversible binding
in which the protoplast-associated radioactivity was no longer
accessible to exogenous DNase. The irreversible binding or uptake
was approximately linear over a 20-h period and amounted to
about 3% (7.5 x 10-14g DNA/protoplast) of input DNA at 2.5 h,
the incubation time used in experiments where expression of the
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685
plasmid-borne gene was tested. These results are quantitatively
similar to those obtained by Uchimiya and Murashige (15) using
Nicotiana glutinosa ?rotoplasts and [3H]DNA isolated from N.
glutinosa (5.08 x 10- 4g DNA/protoplast). However, the numbers
of genome equivalents represented by these two similar amounts
of DNA are vastly different. In our experiment it represented
about 5,000 genome equivalents per protoplast; in the latter experiments, it represented about 0.005 per protoplast.
Despite the large number of genome equivalents irreversibly
bound by the protoplasts in our experiments, none of the 272,000
microcalli regenerated from protoplasts treated with ColEl-kan
DNA gave rise to plantlets that exhibited a greater range of
tolerance to kanamycin than did plantlets from protoplasts treated
with ColEl DNA. We conclude that the range of kanamycin
tolerance exhibited by plantlets from protoplasts receiving ColE 1kan DNA is a manifestation of the natural variance existing in
the cell population and not of expression of the gene specifying
kanamycin resistance carried by ColEl-kan plasmid.
This failure to obtain expression of the plasmid-borne gene in
tobacco could be due to any of several reasons. First, the transformation frequency simply may be lower than 2.7 x 10-'. Second,
the screening procedure employed required that the kanamycin
resistance gene be maintained within the plant cell for many
generations. Maintenance via autonomous replication of the plasmid would require that the plasmid be taken up physically intact
by the protoplasts. This may not have occurred. The polymeric
radioactivity recovered from the protoplasts may have represented
DNA that was partially degraded by nucleases. Complete integrity
of the plasmid DNA would not be required for maintenance of
the kanamycin resistance gene if that DNA segment was integrated
into the plant cell genome. Third, molecular barriers at the transcription or translation levels may exist which greatly reduce or
prevent expression of cloned prokaryotic (bacterial) genes in
eukaryotic cells (4). These might include transcription barriers
involving promoter and termination specificities or translation
barriers concerning specificities of ribosome binding to mRNA,
initiation factors, and termination signals (4).
From our results, we conclude that although it may be necessary
to have many gene copies taken up by a protoplast, it is not alone
a sufficient condition for causing genetic transformation. Careful
attention needs to be paid to the design of the transformation
molecule. Although it may be permissible to employ a prokaryotic
gene vehicle for cloning purposes, it is probably necessary that
some portion of the DNA molecule possess segments homologous
to one of the DNA species of the recipient plant cell.
Acknowledgments-We thankf Dr. Donald Helinski for the two plasmids and for helpful
suggestions regarding their isolation. The capable technical assistance of Jean Bellows is gratefully
2 10
acknowledged.
0~
J~~~~~~~~
1
%J
0-24
25-49
LITERATURE CITED
50- 74
75- 100
Growth of tissue
(%
of growth
on
on 16pM kanamycin
medium lacking kanamycin)
FIG. 2. Response to kanamycin of plantlets regenerated from protoplasts treated with ColEl or ColEl-kan DNA and which survived the
initial screen for transformants. Ten replicate leaf pieces (2 x 3 mm) were
placed on a differentiating agar medium containing 0 or 16 AEM kanamycin,
and the callus and shoot tissue developing therefrom were harvested 30
days later. Bars represent the proportion (per cent) of plantlets tested from
each treatment which had tissue dry weights within the range indicated
(expressed as per cent of dry weight obtained on medium lacking kanamycin). Average dry weight of the latter was 46 mg. Data are means of 10
replicates.
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ribonucleic acid. Virology 39: 439-448
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323-343
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3455-3459
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686
OWENS
10. LURQUIN PF, CL KADO 1977 Escherichia coli plasmid pBR313 insertion into plant protoplasts
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