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Direct amplification of plant genomic DNA from leaf and root pieces using PCR

Article  in  Plant Molecular Biology · October 1991

DOI: 10.1007/BF00040656 · Source: PubMed

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Plant Molecular Biology 17: 555-557, 1991.
© 1991 Kluwer Academic Publishers. Printed in Belgium. 555

Update section
Short communication

Direct amplification of plant genomic DNA from leaf and root pieces
using PCR

Pierre Berthomieu and Christian Meyer*

Laboratoire de Biologie Cellulaire, INRA, route de St-Cyr, 78026 Versailles Cedex, France f'author for
correspondence)

Received 24 August 1990; accepted in revised form 24 May 1991

The screening of transformed plants for integra- intron we were able to obtain two bands of the
tion of foreign DNA is a relatively time- expected size corresponding to the two different
consuming procedure which involves genomic genes (Fig. 1). As a positive control for PCR am-
DNA preparation [ 1] and Southern blot analysis
of these preparations. In order to speed up this
process, we have developed a one-step procedure
based on PCR (polymerase chain reaction) tech-
nology. This allowed us to directly amplify a
genomic target (a resident gene or an inserted
foreign DNA) using small leaf or root pieces
placed in the PCR reaction mixture.
Blue or yellow tips were used to punch plant
leaves and roots and to deposit the resulting plant
piece in a 100/A PCR reaction mixture kept at
4 °C and made up with 200/~M of each dNTP,
30 pmol of each primer, 2.5 units of Taq poly-
merase (Promega) in PCR buffer (50 mM KC1,
10mM Tris-HC1 pH 9.0 at 25 °C, 1.5mM
MgC12, 0.01~o (w/v) gelatin, 0.1~o (v/v) Triton
X100). The mixture was then overlaid with par-
affin oil and submitted to denaturation for 5 min
at 94 °C in a Perkin-Elmer Cetus thermal cycler
and subsequently to 30 cycles of amplification
Fig. 1. Analysis of PCR products obtained after genomic am-
(1 min at 94 °C, 2 min at 60 °C, 2 min at 72 °C).
plification of the first intron of the two nitrate reductase genes
After that, the PCR reaction was directly loaded ofNicotiana tabacum. Lane a, negative control: no DNA sam-
onto a 1.5 ~o agarose gel for electrophoretic anal- ple; lane b, tobacco leaf piece; lane c, purified tobacco genomic
ysis. DNA (100 rig); lane d, DNA ladder (1 kb ladder, BRL). The
To check the efficiency and the reliability of this two bands correspond to the two homeologous nitrate reduc-
tase genes present in the amphidiploid tobacco genome. The
method we first amplified, using leaf pieces, a
size of these two bands is consistent with the position of the
resident genomic target, namely the first intron of two primers used for PCR (on both sides of the intron). The
the two homeologous nitrate reductase genes in length of the expected PCR product is indicated for each
tobacco [2]. Using a pair of primers flanking this band.
556
plification we used tobacco genomic DNA puri-
fied by the method of Dellaporta et al. [1] and
obtained the same result as with leaf pieces
(Fig. 1). This shows that, although leaf pieces gave
a lower yield in PCR experiments than purified
genomic DNA (Fig. 1), we were able to directly
use plant leaves for amplification of a single-copy
genomic target.
Fig. 2. Analysis of PCR products obtained after direct am-
plification of the B. thuringiensis toxin gene in transformed
plants. Lane a, positive control: root piece from a cabbage
plant already known to be transformed with the B. thuring-
iensis toxin gene; lane b, negative control: root piece from an
untransformed cabbage plant; lane c, analysis of a root piece
from a cabbage plant supposed to be transformed with the
B. thuringiensis toxin gene; lanes d and i, DNA ladder (1 kb
ladder BRL); lane e, positive control: leaf piece of a tobacco
plant already known to be transformed with the B. thuring-
iensis toxin gene; lane f, negative control: leaf piece of an
untransformed tobacco; lanes g, h, i and j, analysis of leaf
pieces from tobacco regenerants supposed to be transformed
with the B. thuringiensis toxin gene: only the plant correspond-
ing to the h lane is actually transformed; lane k, modified
A. tumefaciens C58 pMP90 strain carrying the B. thuringiensis
toxin gene (the lower band corresponds to the 343 bp termi-
nal fragment of the B. thuringiensis toxin gene, the upper band
corresponds to a 569 bp fragment of the virC locus of the
plasmid pTiC58); lane m, positive control: plasmid DNA
containing the B. thuringiensis toxin gene (30 pg); lane n, neg-
ative control: no DNA sample. The oligonucleotides corre-
sponding to the Agrobacterium Ti plasmid (virC locus) were
used as an internal control in each amplification to detect a
possible contamination of the tested plants with Agrobacte-
rium, which could then lead to false-positive plants.
Using this procedure, we analysed then tobacco
plants transformed with Agrobacterium tume-
faciens strain C58 pMP90 [3 ] carrying the Bacillus
thuringiensis crylC toxin gene [4] as well as cab-
bage roots (Brassica oleracea) transformed with
A. rhizogenes strain HRI [5] carrying the same
gene. We used two sets of primers for each am-
plification: one specific for the B. thuringiensis
toxin gene and the other one specific for a region
of the Agrobacterium Ti plasmid which is not
transferred to the plant (the virC locus). The re-
suits of the direct PCR amplifications performed
on these plants are shown in Fig. 2. The untrans-
formed tobacco and cabbage plants did not show
any amplified material, nor did three of the four
analysed tobacco plants in which the toxin gene
is thus supposed to be absent or rearranged. The
control transformed cabbage root and tobacco
plant as well as the analysed cabbage root and
one of the four analysed tobacco plants showed
a band of the right size resulting from an insertion
of the toxin gene into the plant genome as the
band corresponding to the Agrobacterium plasmid
is not present in these samples.
In conclusion, we must stress the fact that in
this procedure the number of target D N A mole-
cules available for amplification is certainly very
low. Since it has been observed that PCR ampli-
fication of targets of low copy number can some-
times fail [6], we cannot rule out the possibility
of appearance of false-negative transformants
with our method. Indeed, in a few cases, we could
not observe amplification of the target D N A for
some unknown reasons. But this problem can be
simply overcome by the use of an internal control,
like the amplification of a resident gene with a
couple of specific primers, for each PCR experi-
ment.
Acknowledgements
P.B. was supported by a grant from GIE Clause-
Limagrain.

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