CA 02469139 2004-06-22
Potato Regeneration System and Use Thereof
Field of the Invention:
This invention relates to the regeneration of plants from selected tissue from
a donor
plant. More specifically, it is a one step shoot regeneration system for
potatoes.
Background of the Invention:
Regeneration systems are used routinely in the production of seed potatoes and
the
production of transgenic potatoes. With regard to transgenic plant production,
the most limiting
factor in their production is the establishment of an efficient protocol for
plant regeneration from
transgenic tissue. Shoot regeneration in potato (Solanum tuberosum L.) depends
primarily on the
genotype, medium composition, explant tissue source and cultivation condition.
Examples of
media compositions can be found in DeBlock, 1988; Sheerman and Bevan, 1988;
Stiekema et al.,
1988; Wenzler et al., 1989. While one with no knowledge of the art would be
unaware that
subtle changes to a medium result in unpredictable outcomes, one skilled in
the art would be very
aware of this fact. Hence, they would recognize that changing a known medium
even if slightly
2o could result in a non-obvious and unpredictable result.
In general, stem sections are used as the explant source. As regeneration
efficiency is
partially dependent upon explant source, one generally is required to develop
specific media for
specific explant tissue sources. This therefore restricts the amount of
starting tissue that can be
obtained from a plant for use in a given protocol. While this may be of little
significance for
seed potato production of standard cultivars, it could be very significant for
scale up of transgenic
and rare plant materials.
In general, there is a strong interaction between the various factors
controlling
3o regeneration efficiency. This therefore requires that specific media be
developed for each set of
factors. As there are at least three factors that interact with the medium to
determine
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CA 02469139 2004-06-22
regeneration efficiency, it is clear that there is a need for many different
media for each step in
the regeneration process.
Most procedures for regeneration are mufti-step, wherein there are specific
media for, as
an example, callus formation and shoot formation. This requires that the
tissue be handled a
number of times, thereby increasing the chance of contamination. More
significantly, the cost of
multiple steps is high - each medium must be prepared from stock solutions
that frequently
contain costly growth hormones and of course, the preparation of medium, and
transfer of tissue
increases the manpower expenses tremendously. Hence, it is an objective of the
invention to
overcome the deficiencies in the prior art.
Summary of the Invention:
The present invention provides an efficient multipurpose, regeneration medium
and
protocol for using the medium to regenerate potato plants from explants. The
medium and
protocol have advantages over others including the fact that they can take
advantage of the
morphogenetic capacity of potato petioles, there is significantly increased
regeneration
efficiency, there is reduced time between placing explants onto regeneration
medium and shoot
formation, there is a low level of somaclonal variations, and there is a high
recovery rate after
2o transferring shoots to a rooting medium. In addition, it is a one-step
regeneration protocol in
contrast to classic DeBlock's [ 11 ] protocol for potato regeneration where
different media are used
at certain stages of potato cultivation. Further the protocol is relatively
genotype independent.
In one embodiment of the invention, a regeneration medium for potato is
provided comprising:
MS salts;
one of MS vitamins or BS vitamins;
a concentration of 30-50 mg/1 of adenine' 504;
a concentration of 18-22 g/1 of glucose;
a concentration of 18-22 g/1 of mannitol;
3o a concentration of 700-1500 mg/1 of MES;
a concentration of 0.03-0.05 mg/1 of gibberelic acid (GA3);
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CA 02469139 2004-06-22
a concentration of 0.015-0.03 mg/1 of NAA;
and a concentration of 1.5-5 mg/1 of zeatin riboside;
and the medium is adjusted to a pH of 5.6-5.8 with 1N KOH.
In one aspect of the invention, the medium further comprises solidified
agarose, and the agarose
concentration ranges from 3.3 to 4.2 g/1.
In another aspect of the medium of the invention, the agarose concentration is
about 4 g/l.
1o In another aspect of the medium of the invention, the glucose concentration
is about 20 g/l.
In another aspect of the medium of the invention, the mannitol concentration
is about 20 g/1.
In another aspect of the medium of the invention, the GA3 concentration is
about 0.04 mg/l.
In another aspect of the medium of the invention, the NAA concentration is
about 0.02 mg/l.
In another aspect of the medium of the invention, the zeatin riboside
concentration is about 2
mg/l.
In another embodiment of the invention a combination for regenerating potato
plants is provided.
The combination comprises a medium, and an axenic explant. The medium
comprises:
MS salts;
one of MS vitamins or BS vitamins;
a concentration of about 30-50 mg/1 of adenine' S04;
a concentration of about 18-22 g/1 of glucose;
a concentration of about 18-22 g/1 of mannitol;
a concentration of about 700-1500 mg/1 of MES;
a concentration of about 0.03-0.05 mg/1 of gibberelic acid (GA3);
3o a concentration of about 0.015-0.03 mg/1 of NAA;
a concentration of about 1.5-5 mg/1 of zeatin riboside;
CA 02469139 2004-06-22
a concentration of about 3.8-4.5 g/1 agarose;
the medium adjusted to a pH of about 5.6-5.8 with 1N KOH; and
the explant comprises one of shoots, petioles and leaves.
In one aspect of the combination of the invention, the explant is a petiole.
In another embodiment of the invention, a one-step regeneration protocol for
potato plants is
provided. The protocol comprises placing an axenic explant in an axenic medium
and culturing
the axenic explant under axenic culture conditions. The axenic explant
comprises one of leaves,
l0 stems and petioles. The axenic medium comprises:
MS salts;
one of MS vitamins or BS vitamins;
a concentration of about 40 mg/1 of adenine' 504;
a concentration of about 20 g/1 of glucose;
a concentration of about 20 g/1 of mannitol;
a concentration of about 900 mg/1 of MES;
a concentration of about 0.04 mg/1 of gibberelic acid (GA3);
a concentration of about 0.02 mg/1 of NAA;
a concentration of about 2 mg/1 of zeatin riboside;
a concentration of about 4 g/1 agarose;
the medium adjusted to a pH of about 5.7 with 1N KOH.
The axenic culture conditions comprising a temperature ranging from 22°
to 26°C with
photoperiod ranging from 14 to 18 h light and a light intensity ranging from
2500 to 3200 lux.
In one aspect of the regeneration protocol of the invention, the axenic
explant is a petiole.
In another embodiment of the invention, a use of a regeneration medium for the
one step
regeneration of potato plants from an axenic explant is provided. The
regeneration medium
comprises:
3o MS salts;
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CA 02469139 2004-06-22
one of MS vitamins or BS vitamins;
a concentration of about 40 mg/1 of adenine' 504;
a concentration of about 20 g/1 of glucose;
a concentration of about 20 g/1 of mannitol;
a concentration of about 900 mg/1 of MES;
a concentration of about 0.04 mg/1 of gibberelic acid (GA3);
a concentration of about 0.02 mg/1 of NAA;
a concentration of about 2 mg/1 of zeatin riboside; and
a concentration of about 4 g/1 agarose;
1o and the medium is adjusted to a pH of about 5.7 with 1N KOH.
In one aspect of the invention, the use further comprises transforming said
axenic plant by
Agrobacterium-mediated transformation.
Figure Descriptions:
Figure 1. Multiple shoot regeneration from potato petioles after 30 (A) and 40
days (B) of
cultivation on PetM medium. Bar=t cm.
Detailed Description of the Invention:
Example 1:
Developing regeneration protocols:
We developed and tested many different media in order to establish a high
frequency
regeneration protocol for Solanum tuberosum L. As a source of plant tissue we
used 4-5-week-
old axenically grown potato plants cvs Desiree Zarevo, Lvivyanka, Lugivskiy,
and Svitanok
kyivskiy. The range of media components included MS salts (Murashige and
Skoog, 1962), MS
or Gamborg's vitamins (Gamborg et al., 1968), 0-100 rng L-1 of adenine . 504,
5-30 g L-1 of
3o glucose, 5-30 g L-1 of sucrose, 0-60 g L-1 of mannitol, 0-2000 mg L-1 of 2-
[N-
MorpholinoJethanesulfonic acid (MES), 0-5 mg L-1 of GA3, 0-5 mg L-1 of 3-
indoleacetic acid
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(IAA), 0-5 mg L-1 of naphthaleneacetic acid (NAA), 0-10 mg L-1 of 6-
benzylaminopurine (BA),
0-10 mg L-1 of zeatin riboside (ZR), and 3-7 g L-1 of agarose at pH5.6-5.8.
Growth regulators
GA3 and ZR were filter sterilized and added to the autoclaved medium cooled to
400C. The
following explants were tested: stem segments; petioles; and leaves. The whole
plants were
placed into liquid regeneration medium, cut into 5-15 mm full-length pieces of
internodular stem
segments or petioles, and then cultivated horizontally onto the same, but
solid, medium. Leaves
(8-12 mm) were cut at the base and placed on the medium upside up. The upper
surface of some
leaves was gently wounded with sandpaper to stimulate callus and regeneration
response.
Cutting the plants submerged in liquid medium prevented excessive drying of
the excised tissue
1o and increased the viability of the explants. Callus formation and
subsequent shoot regeneration
occurred mainly on the cut/wounded parts of plant tissue. This is important
for successful
Agrobacterium-mediated transformation.
Among the carbohydrates tested, glucose at concentration of 20 gl 1 was the
best
carbohydrate source for inducing th~ formation of compact green callus. This
type of callus is
most favorable for the induction of shoot organogenesis in potato. In the
presence of sucrose, the
explants formed green and white callus, often friable, with significantly
lower regeneration rate
comparing to the medium with glucose. Adding mannitol (20 gl 1 ) to the
regeneration medium
with glucose had positive effect on forming dense, favorable for shoot
regeneration cell colonies.
2o Among the cytokinins tested, the best regeneration response from all types
of explants
was obtained on the medium with zeatin riboside. Interestingly, leaf explants
required higher
concentration of zeatin riboside to achieve maximum rate of plantlet
production (5 mgl-1 vs 2
mgl 1 for stem segments and petioles), although some regenerated shoots looked
vitrificated.
This could be related to different levels of exogenous hormones in the
explants. The medium
with zeatin riboside provided 100% regeneration efficiency with multiple
shoots per each
explant.
When benzyl adenine phosphate (BAP) was used, all explants produced callus and
regenerated shoots as well, but induction of shoot development occurred I-2
weeks later
3o comparing to the medium with the same concentration of zeatin riboside;.
Also the average
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number of shoots per explant was a bit lower. Further, the shoots regenerated
on the medium
with BAP were smaller in diameter, and their viability and survival rate after
transferring to the
rooting medium was lower in contrast to those ones regenerated on the medium
with zeatin
riboside.
Addition of gibberrelic acid to regeneration medium (0.04 mgl 1 ) stimulated
shoot
elongation both in the medium with BAP and zeatin riboside.
Among the auxins tested, napthalene acetic acid (NAA) was superior to indole
acetic acid
(IA.A) with optimum concentration of 0.02 mgl 1. This is several times lower
than normally
used in other protocols on potato regeneration.
Summarizing the data from different media compositions, the best shoot
regeneration
response from all types of explants was obtained on the Medium C (Fig. l ). ).
Other regeneration
1s media that produced some regeneration included the following:
MS salts, Gamborgs vitamins, 30 g/1 sucrose, 900 mg/1 MES, 0.1 mg/1 NAA, 5
mg/1 ZR,
pH5.7;
MS salts, Gamborgs vitamins, 20 g/1 glucose, 20 g/1 mannitol, 900 mg/1 MES,
0.04 mg/1
GA3, 0.1 mg/1 NAA, S mg/1 ZR, pH5.7; and
2o MS salts, Gamborgs vitamins, 20 g/1 glucose, 20 g/1 mannitol, 900 mg/1 MES,
0.04 mg/1
GA3, 0.02mg/1 NAA, 2 mg/1 BA, pH5.7.
The content of this medium is presented in below in "Experimental". The first
regenerated shoots were identified after 12 days of cultivation. All explants
on this medium
produced multiple shoots (100% regeneration efficiency) within 2-3 weeks.
Petioles were found
25 to have the best regenerative capacity, following by stem segments and
leaves, respectively.
Example 2:
Plant transformation, selection and regeneration
Plasmid construction
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The following three binary vectors were used for potato transformation: pBI121
(Jefferson et al.,
1987), pWIN3.12-GUS and pPIN-GUS. All of them are based on plasmid pBI121 and
designed
to express Li-D-glucuronidase (GUS) gene from different promoters. Bp I121
contains the strong
constitutive 35S promoter of the cauliflower mosaic virus (CaMV).
pWIN3.12-GUS contains the 823 by fragment of wound-inducible promoter WIN3.12
from a
hybrid poplar tree (Rollick and Gordon, 1993). To make this construct, the
promoter part of a
proteinase inhibitor-like gene WIN3.12 (GenBank accession # L11233) was
amplified in a PCR
reaction from a plasmid pWIN3.12. The plasmid pWIN3.12, which has 1.5 kb
HindIII genomic
io poplar clone of the WIN3.12 gene in the pBluescript BS+ vector (Stratagene,
USA), was kindly
given by Prof. M.P.Gordon (University of Washington, Seattle, USA). PCR
amplification was
carried out in 100 ~1 reaction mix containing 70 ng of pWIN3.12, 2 Units of
Deep Vent DNA
polymerise (New England Biolabs) and standard concentrations of primers, MgCl2
and dNTPs.
Deep Vent DNA polymerise was chosen to minimize incorrect incorporation of
nucleotides.
The sequences of the primers used for WIN3.12 promoter amplification were:
forward primer
WINS (5'-AACTGCAGAAGCTTCCAACATCAATGAT-3', 28-mer), reverse primer WIN3 (5'-
CGGGATCCTCTAGAATTTGTTGAATATGAG-3', 30-mer). The underlined parts of the
primers correspond to the WIN3.12 promoter sequence in GenBank Database,
HindIII (forward
primer) and engineered XbaI (reverse primer) sites are shown in bold and were
used in
2o subsequent cloning.. PCR amplification of WIN3.12 promoter was performed
with manual hot-
start and denaturing the template DNA at 94°C for 3 min, following by
30 repeat cycles of 30s
at 94°C, 30s at 55°C, and 1 min at 72°C, with a 10 min
extension at 72°C for the last cycle prior
to halting the reaction at 4°C. After electrophoresis in a 1% agarose
gel, a 846 by PCR product
corresponding to the desired length of the WIN3.12 promoter was excised,
purified with
NucleoSpin Extraction Kit (Clontech, USA), digested with HindIII and XbaI
enzymes, and the
823 by HindIII/XbaI DNA fragment was ligated into the corresponding sites of
pBI121
(Clontech, USA) in place of preliminary deleted CaMV 35S promoter. The result
was a
transcriptional fusion between the WIN3.12 promoter, the GUS coding region and
the nopaline
synthase polyadenylation signal. The correct insertion and full nucleotide
sequence of the
3o amplified WIN3.12 promoter was confirmed by mapping with SacI restriction
enzyme and by
s
CA 02469139 2004-06-22
DNA sequence analysis.
pPIN-GUS contains ~1 kb promoter sequence of wound-inducible potato proteinase
inhibitor II
gene (Thornburg et al., 1987). This construct was made by replacing the CaMV
35S promoter in
pBI121 with the HindIII/XbaI fragment from plasmid pRT210 (kindly provided by
Prof. R.
Thornburg, Iowa State University, Ames, YSA) containing 5'flanking sequence of
the wound-
inducible potato proteinase inhibitor IIK gene.
All plasmid constructs were maintained in DHSa E.coli strain and introduced
into PM90
Agrobacterium tuinefaciens strain via direct transformation. The presence of
the engineered
vectors in the antibiotic-selected A.tumefaciens clones was confirmed by PCR
analysis using
1 o prornotor-specific primers.
Bacterial culture
A.tumefaciens cultures were grown at 26-28°C on a shaker at 220 rpm in
liquid Luria-Bertani
(LB) medium (Sambrook et al., 1989) containing 100 mg/1 kanamycin (SigmaTM,
USA), 100
mg/1 rifampicin (Sigma, USA) and 10 mg/1 gentamicin (Sigma, USA) to mid-log
phase
(OD600~.5). The bacterial cells were collected by centifugation at 1500 rpm
for 10 min and
resuspended in liquid plant medium C to a final cell density of 1.0 (OD600)
immediately before
cocultivation.
The following explants from 4- to 5-week-old potato plants were used for plant
transformation:
internodal stem segments, petioles and leaves. The whole plants were placed in
the liquid
regeneration medium C, cut with sterile blade into 5-15 mm full-length
internodal stem segments
or petioles, and precultured by floating on 15 ml of liquid medium C in a 9 cm
Petri dish. The
leaves (8-12 mm) were cut at the base as described by DeBlock (1988) and
placed on the
medium upside up. Some of the leaves were gently wounded by sandpaper. After 2
days of pre-
cultivation at 24°C under low light intensity (500 lux), the potato
explants were immersed in
Agrobacterium suspension and incubated for 30 min with slow shaking, then
blotted with sterile
filter paper to remove the excess bacteria, placed horizontally on antibiotic-
free medium C
3o solidified with 0.4%(w/v) agarose and cocultivated at 24°C under low
light for 3-4 days. After
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CA 02469139 2004-06-22
this period, the infected explants were washed for 30 min with sterile medium
C containing 1 g/1
cefotaxime, blotted dry and transferred to selective regeneration medium C
containing 50-100
mg/1 kanamycin and either S00 mg/1 carbenicillin or 500 mg/1 cefotaxime. The
explants were
cultivated at 24°C with 16 h light photoperiod (3000 lux light
intensity) and transferred to fresh
antibiotic-containing medium C every 2 weeks. Regenerated shoots (1-1.5 cm
high) were
excised and rooted in the hormone-free medium used for potato micropropagation
(see Plant
material) and supplemented with 25 mg/1 kanamycin. Transformation frequency
was determined
as the percentage of inoculated explants that produced kanamycin-resistant
plantlets.
to Polymerase chain reaction (PCR) analysis of transgenic plants
Potato genomic DNA was isolated from 100 mg of leaf tissue of kanamycin-
resistant
transfonmants and a control plant using Sigma GenEluteTM Plant Genomic DNA Kit
(Sigma,
USA). 200-300 ng of plant DNA (5 ~1 out of 100 ~1 of extracted DNA) were
analysed in 50 ~1
of PCR mix containing Taq PCR Master Mix (Qiagen, USA) and specific primers.
PCR
amplifications were carned out with the following parameters: 94°C for
3 min, then thirty
cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 1
min 30 s, followed by a final 10 min
incubation at 72°C. Specific primers for the plants containing pBI121,
pWIN3.12-GUS and
pPIN-GUS constructs had 5'- and 3'-end nucleotide sequence of the GUS gene and
were
designed to amplify 1812 by full-length GUS: forward primer 5'GUS (5'-ATG TTA
CGT CCT
GTA GAA ACC-3', 21-mer) and reverse primer 3'GUS (5'- TCA TTG TTT GCC TCC CTG
CTG - 3', 21-mer). Another set of specific primers for the plants with
pWIN3.12-GUS
construct was used to confirm correct promoter+gene fusion (984 by amplified
DNA fragment):
forward primer 5'WIN (see "Plasmid construction") and reverse primer 3'M/GUS
with
sequence complimentary to nucleotides +89 to +109 of the GUS gene upstream
region (5'- CTT
TCC CAC CAA CGC TGA TCA - 3', 21-mer). Amplified products were separated in a
1%
(w/v) agarose gels and visualized by staining with ethidium bromide.
Southern analysis
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CA 02469139 2004-06-22
Genomic DNA was isolated as described above. Five wg of potato DNA from each
clone were
digested with 200 units of XbaI restriction enzyme in 200 p,l of reaction mix.
Complete
digestion of DNA samples was verified by running mini-gel with small aliquots
(10 pl) of the
reaction mix. Before loading the gel for Southern, the DNA was concentrated
using SpeedVacTM
Concentrator (Savant USA) to reduce volume of incubation mix to well's size.
The DNA
samples were electrophoresed in a 1% agarose gel using lxTBE buffer (Sambrook
et al., 1989),
then transferred to a Biodyne B nylon membrane (PALL) by capillary blotting
following the
manufacturer's instructions for the improved alkaline DNA transfer. Pre-
hybridization (3 h) and
hybridization (overnight) were carried out at 65°C in 10 ml of
PerfectHyb PIusTM hybridization
to buffer (Sigma, USA) supplemented by 100 pg/ml of sheared, denatured salmon
testis DNA
(Sigma, USA). A 1812 by full-length GUS nucleotide sequence was amplified by
PCR from
pBI121 plasmid, fractionated by electrophoresis, excised from the gel and
purified with
NucleoSpin Extraction KitTM (Clontech, USA) for use as a hybridization probe.
The probe was
labeled with [a-32P]dCTP to a specific activity of >109 cpm/pg using the
random priming
1 s method, and used at concentration of 4x 106 cpm of purified probe in 1 ml
of hybridization
solution. After hybridization, the membrane was washed with constant agitation
two times in
2xSSC, 0.1% SDS at room temperature for 5 min, two times in lxSSC, 0.1% SDS at
50°C for 10
min, and then exposed to X-ray film (Kodak BioMaxTM) overnight at -80°C
using intensifying
screen. Also, the membrane was exposed to a Phosphor Screen (Molecular
Dynamics, USA) and
20 image was scanned using PhosphorImagerTM and Image QuantTM software
(Molecular Dynamics,
USA).
Transgenic plant wounding and tissue sampling
25 Leaf tissue samples for protein extraction were collected from the upper
part of fully developed
young potato leaves on one side of the midrib. After the first samples had
been removed, the
lower part and the periphery of the sampled leaf (~50% of the leaf) were
wounded with lab
forceps. After 18 hours, the upper part of the same leaf was sampled as before
on the opposite
side of the midrib.
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CA 02469139 2004-06-22
GUS assay
Potato leaf samples harvested as described above, both prior to and 18 hours
after mechanical
wounding, were homogenized in lysis buffer containing SO mM sodium phosphate
pH7.0, 10
mM EDTA, 0.1 % Triton X-100, 0.1 % sarcosyl and 10 mM 2-mercaptoethanol.
Protein content
was measured with a standardized Bradford assay (Bradford, 1976). Quantitative
fluorometric
assay of GUS activity was performed as described earlier (Jefferson et al.,
1987) by incubation of
the extracts (20 pg protein) with 1mM 4-methyl-umbelliferyl-~i-D-glucuronide
(MUG) in lysis
buffer for 60 min at 37°C. GUS activity was calculated as pmol of 4-
methyl-umbelliferone (4-
1 o MU) produced per minute per mg of soluble protein.
Experimental:
Plant material
Solarium tuberosum L. cv Desiree plants were grown axenically in culture tubes
(Sigma, USA)
on the medium containing MS salt mixture (Murashige and Skoog, 1962),
Gamborg's vitamins
(Gamborg et al., 1968), 20g/1 of sucrose and 7 g/1 of DifcoTM agar with a
final pH5.8, under a 16
h light (3000 lux)/8 h dark photoperiod at 240C.
Medium for potato regeneration (Medium C)
One-step shoot regeneration medium C was used for shoot induction and plant
regeneration from
shoot, petiole and leaf explants of potato. It contained MS salts (Murashige &
Skoog, 1962), MS
vitamins or BS vitamins (Gamborg et al., 1968), 40 mg/1 of adenine ' S04~ 20
g/1 of glucose, 20
g/1 of mannitol, 900 mg/1 of MES, 0.04 mg/1 of gibberelic acid (GA3), 0.02
mg/1 of NAA, 2 mg/1
of zeatin riboside, 4 g/1 of agarose. The pH was adjusted to 5.7 with 1N KOH.
Growth
regulators GA3 and zeatin riboside were filter sterilized and added to the
autoclaved medium
cooled to 40-SOOC.
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Culture conditions
The explants were cultured at 24°C with 16 h light photoperiod (3000
lux light intensity).
Results:
1. M.A. Matzke, A.J.M. Matzke, How and why do plants inactivate homologous
(trans)genes?
Plant Physiol. 107 (1995) 679-685.
2. D.J.Bowles, Defense related proteins in higher plants, Annu. Rev. Biochem.
59 (1990) 873-
907.
3. H.R. Clarke, J.M. Davis, S.M. Wilbert, H.D.Jr. Bradshaw, M.P. Gordon, Wound-
induced and
developmental activation of a poplar tree chitinase gene promoter in
transgenic tobacco, Plant
Mol. Biol. 25 (1994) 799-815.
4. J.B. Hollick, M.P. Gordon, A poplar tree proteinase inhibitor-like gene
promoter is responsive
to wounding in transgenic tobacco, Plant Mol. Biol. 22 (1993) 561-572.
5. M. Osusky, G. Zhou, L. Osuska, R.E. Hancock, W.W. Kay, S. Misra, Transgenic
plants
expressing cationic peptide chimeras exhibit broad-spectrum resistance to
phytopathogens, Nat.
Biotechnol. 18 (2000) 1162-1166.
6. R.M. Broadway, S.S. Duffey, Plant proteinase inhibitors: mechanism of
action and effect on
2o the growth and digestive physiology of larval Heliothis zea and Spodoptera
exiqua, J. Insect
Physiol. 32 (1986) 827-833.
7. T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays
with tobacco tissue
cultures, Physiol. Plant. 15 (1962) 473-497.
8. J.E.Bourque, J.C.Miller, W.D.Park, Use of an in vitro tuberization system
to study tuber
protein gene expression, In Vitro Cell. Dev. Biol. Plant 23 (1987) 381-386.
9. RA. Jefferson, T.A. Kavanagh, M.W. Bevan, GUS fusions: 0-glucuronidase as a
sensitive
and versatile gene fusion marker in higher plants, EMBO J. 6 (1987) 3901-3907.
10. O.L. Gamborg, R.A. Miller, K. Ojima, Nutrient requirements of suspension
cultures of
soybean root cells, Exp. Cell Res. 50 (1968) 151-158.
11. M. De Block, Genotype-independent leaf disc transformation of potato
(Solanum tuberosum)
using Agrobacterium tumefaciens, Theor. Appl. Genet. 76 (1988) 767-774.
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CA 02469139 2004-06-22
12. S. Sheerman, M.W. Bevan, A rapid transformation method for Solarium
tuberosum using
binary Agrobacterium tumefaciens vectors, Plant Cell Rep. 7 (1988) 13-I6.
13. W.J. Stiekema, F. Heidekamp, J.D. Louwerse, H.A. Verhoeven, P. Dijkhuis,
Introduction of
foreign genes into potato cultivars Bintje and Desire using an Agrobacterium
tumefaciens
binary vector, Plant Cell Rep. 7 (1988) 47-50.
14. H. Wenzler, G. Mignery, G. May, W. Park, A rapid and efficient
transformation method for
the production of large numbers of transgenic potato plants, Plant Sci. 63
(1989) 79-85.
15. J.M. Davis, M.P. Gordon, B.A. Smit, Assimilate movement dictates remote
sites of wound-
induced gene expression in poplar leaves, Proc. Natl. Acad. Sci. USA, 88
(1991) 2393-2396.
1o 16. J.B. Hollick, M.P. Gordon, Transgenic analysis of a hybrid poplar wound-
inducible promoter
reveals developmental patterns of expression similar to that of storage
protein genes, Plant
Physiol. 109 (1995) 73-85.
17. R.W. Thornburg, G. An, T.E. Cleveland, R. Johnson, C.A. Ryan, Wound-
inducible
expression of a potato inhibitor II-chloramphenicol acetyltransferase gene
fusion in transgenic
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14
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