U.S. patent application number 10/291039 was filed with the patent office on 2003-12-18 for pre- and post-harvest inhibition of remobilisation of storage compounds.
This patent application is currently assigned to Mogen International N.V.. Invention is credited to Goddijn, Oscar Johannes Maria, Krause, Klaus-Peter, Tigelaar, Hendrik, Van Dun, Cornelis Maria Petrus.
Application Number | 20030233678 10/291039 |
Document ID | / |
Family ID | 8228886 |
Filed Date | 2003-12-18 |
United States Patent
Application |
20030233678 |
Kind Code |
A1 |
Goddijn, Oscar Johannes Maria ;
et al. |
December 18, 2003 |
Pre- and post-harvest inhibition of remobilisation of storage
compounds
Abstract
This invention describes a method to prevent sprouting in
vegetatively propagated plants such as potato, strawberry, banana
and bulbous plants such as onion and bulbous flowers, by
transforming a plant or a plant from one of its parental lines with
a gene coding for trehalose phosphate synthase. Restoration of
sprouting is also provided for.
Inventors: |
Goddijn, Oscar Johannes Maria;
(Leiden, NL) ; Tigelaar, Hendrik; (Utrecht,
NL) ; Krause, Klaus-Peter; (Hanhofen, DE) ;
Van Dun, Cornelis Maria Petrus; (Roosendall, NL) |
Correspondence
Address: |
SYNGENTA BIOTECHNOLOGY, INC.
PATENT DEPARTMENT
3054 CORNWALLIS ROAD
P.O. BOX 12257
RESEARCH TRIANGLE PARK
NC
27709-2257
US
|
Assignee: |
Mogen International N.V.
Einsteinweg 97
Leiden
NL
2333CB
|
Family ID: |
8228886 |
Appl. No.: |
10/291039 |
Filed: |
November 8, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10291039 |
Nov 8, 2002 |
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09529993 |
Jul 21, 2000 |
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6559364 |
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09529993 |
Jul 21, 2000 |
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PCT/EP98/07010 |
Oct 30, 1998 |
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Current U.S.
Class: |
800/284 ;
435/196; 536/23.2 |
Current CPC
Class: |
C12N 15/8261 20130101;
C12N 9/16 20130101; C12N 9/1051 20130101; Y02A 40/146 20180101;
C12N 15/8245 20130101; C12N 15/8267 20130101 |
Class at
Publication: |
800/284 ;
536/23.2; 435/196 |
International
Class: |
A01H 001/00; C12N
015/82; C07H 021/04; C12N 009/16 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 30, 1997 |
EP |
97203371.6 |
Claims
1. A method to inhibit pre- and/or postharvest remobilisation of
storage compounds in plants by transforming a plant or a plant from
its parental line with a recombinant DNA capable of expression of a
protein, characterized in that the protein is trehalose phosphate
synthase (TPS).
2. A method to prevent sprouting of a plant part by transforming
the plant or a plant from its parental line with a recombinant DNA
capable of expression of a protein, characterized in that the
protein is trehalose phosphate synthase (TPS).
3. A method according to claim 2, characterized in that the
recombinant DNA comprising the gene coding for TPS is of bacterial,
fungal, animal, plant or human origin, preferably derived from
Escherichia coli.
4. Method to induce sprouting in a plant which is made
non-sprouting according to the method of claim 2 or 3 characterized
in that said plant is provided with recombinant DNA coding for TPS
flanked by target sites of a site-specific recombinase and that the
recombinant DNA coding for TPS is removed by providing said plant
either through tranformation with a gene coding for the
corresponding recombinase or through crossing with a plant
harbouring a recombinant DNA capable of expressing said
recombinase.
5. Method to induce sprouting in a plant which is made
non-sprouting according to the method of claim 2 or 3 by
transforming it with a recombinant DNA which comprises a gene
coding for a compound which is capable of neutralising the effects
of TPS under control of and inducible promoter and forcing
expression of the TPS neutralizing compound by induction of the
inducible promoter.
6. Method according to claim 5, characterized in that the
neutralising compound is trehalose phosphate (TPP).
7. Method according to claim 5, characterized in that the
neutralising compound is antisense trehalose phosphate
synthase.
8. Method according to claim 5, characterized in that the
neutralizing compound is trehalose phosphate hydrolase (TreC).
9. Method according to claim 5 characterized in that the
neutralising factor is a suppressor which is capable of suppressing
expression of the TPS.
10. A method to release the inhibition of pre- and/or postharvest
remobilisation of storage compounds in plants caused by the
expression of trehalose phosphate synthase, by treating the storage
organ of the plant with gibberellic acid.
11. Method to induce sprouting in a plant which is made
non-sprouting according to the method of claim 2 or 3 by treating
the plant with gibberellic acid.
12. Method to induce sprouting in a plant which is made
non-sprouting according to the method of claim 2 or 3 by treating
wounding the plant.
13. Method according to claim 1, characterized in that the storage
compound is inulin and the plant is chicory.
14. Method according to claim 10, characterized in that the storage
compound is sucrose and the plant is chicory.
15. Method according to claim 1, characterized in that the storage
compound is sucrose and the plant is sugarbeet.
16. Method according to claim 10, characterized in that the storage
compound is sucrose and the plant is sugarbeet.
Description
FIELD OF THE INVENTION
[0001] This application is concerned with the pre- and postharvest
inhibition of remobilisation of storage compounds. Especially, the
application describes the prevention of sprouting, especially in
vegetatively propagated plants by transforming them with
recombinant DNA and a method to restore sprouting in these
lines.
BACKGROUND ART
[0002] In traditional breeding as well as in agricultural genetic
engineering the major goal is to obtain crops with a high yield,
which generally means that the goal has been to increase storage of
the plant in the organs of the plant that are used for storage,
such as the tubers in potato, the taproot in sugarbeet, and the
leaves in leafy crops such as lettuce. However, other processes in
plants, such as flowering and or sprouting, often give a yield
penalty.
[0003] Sprouting normally can be inhibited by cold storage at very
low temperatures (slightly above freezing). Cold storage is not
only expensive, but also inflicts deleterious effects upon storage
organs, which render them unsuitable for further processing or
result in yield losses of commercial products as starch For example
when potato tubers are subjected to cold temperatures, they convert
starch to reducing sugars, a phenomenon known as `cold sweetening`.
The development of reducing sugars is very undesirable because
during baking and frying e.g. the Maillard reaction occurs that
results in undesired browning.
[0004] To prevent cold sweetening potatoes can be stored at higher
temperatures, but this results in undesired sprouting. Amongst
others, chlorpropham (CIPC) is used by the industry to control
tuber sprouting. Although CIPC has been used effectively, it still
is considered as an undesirable chemical treatment. All around the
world, there is an increasing emphasis on replacing chemical
control agents with biological control mechanisms that are safe and
more environmentally acceptable.
[0005] When considering a genetic approach to inhibit sprouting, it
must also be considered that for the development of see-potatoes
sprouting is a desired property, and that thus a mechanism should
be at hand which enables seed-potato production but which prevents
sprouting in potatoes cultured for consumption or further
processing.
SUMMARY OF THE INVENTION
[0006] This invention comprises a method to inhibit pre- and
postharvest remobilisation of storage compounds. More specifically,
the invention comprises a method to prevent sprouting of a plant
part by transforming the plant or its ancestor with a recombinant
DNA capable of expression of a protein, characterized in that the
protein is trehalose phosphate synthase (TPS). More specifically
the recombinant DNA comprising the gene coding for TPS is of
bacterial, fungal, animal, plant or human origin, preferably
derived from Escherichia coli.
[0007] In another embodiment the invention comprises a method to
induce sprouting in a plant by providing said plant with
recombinant DNA coding for TPS flanked by target sites of a
site-specific recombinase and removing the recombinant DNA coding
for TPS by providing said plant either through transformation with
a gene coding for the corresponding recombinase or through crossing
with a plant capable of expressing said recombinase.
[0008] Still another embodiment of the invention comprises a method
to induce sprouting in a plant by providing a plant with
recombinant DNA coding for TPS and subsequently or simultaneously
transforming it with a recombinant DNA which comprises a gene
coding for a molecule that can neutralize the effect of TPS under
control of an inducible promoter and forcing expression of the
neutralizing molecule by induction of the inducible promoter. An
example of such a neutralizing molecule is trehalose phosphate
phosphatase (TPP) or the product of the antisense TPS gene.
[0009] Another embodiment of the invention is formed by removing
the inhibition of pre- and post-harvest mobilisation of storage
compounds by external treatment with compounds that neutralize the
inhibitory effect of the expression of the TPS gene. Preferably
this is accomplished by applying gibberellic acid. Still another
embodiment of the invention is to restore sprouting by
wounding.
[0010] A further object of the invention is a method to induce
sprouting in a plant by providing a plant with recombinant DNA
coding for TPS and subsequently or simultaneously transforming it
with a recombinant DNA which comprises a gene coding a suppressor
under control of an inducible promoter, said suppressor capable of
suppressing expression of the TPS and forcing expression of the
suppress or by induction of the inducible promoter.
[0011] Also the invention provides for plants made by any of the
above mentioned methods, specifically vegetatively propagated
plants and more specifically potato and onion.
[0012] Further the gene coding for TPS can be placed under control
of a specific promoter, such as the patatin promoter, which
specifically gives expression in the tuber of the potato plant.
[0013] Another embodiment of the invention is the inhibition of the
catabolism of inulin in chicory, the inhibition of sucrose
catabolism in sugarbeet and the inhibition of starch degradation in
potato.
DESCRIPTION OF THE FIGURES
[0014] FIG. 1. Sprouting behaviour of patatin-TPS tubers with or
without treatment with gibberellic acid (GA) after 14 days (FIG.
5A) and after 25 days (FIG. 5B).
DETAILED DESCRIPTION OF THE INVENTION
[0015] For definition purposes only the general term of a
transformed plant is a plant totality or a plant grouping. This
term is meant to cover a broad spectrum of plants and is not
confined to one specific variety.
[0016] The invention is concerned with a met hod for the pre-
and/or postharvest inhibition of remobilisation of storage
compounds. The remobilisation of storage compounds is the process
that plants undertake to utilise the compounds that have been
stored, generally in specialised storage organs. A typical example
of such a mobilisation is the process of sprouting from storage
organs such as tubers, bulbs or seeds.
[0017] Specifically, provided are methods for the inhibition of
sprouting, preferably in vegetatively propagated plants and methods
to restore sprouting capabilities again in plants that have been
inhibited. Sprouting in this sense is defined as the formation of
shoots, runners, stolons or suckers, especially from storage
tissue
[0018] The basis of this invention is found in the fact that it has
been surprisingly found that expression of TPS inhibits sprouting.
TPS is an enzyme which is active in the trehalose synthesis
pathway, which is not presently known to play a role in sprouting
tissue. However, it has been recently found (WO 97/42326) that the
enzymes TPS and TPP are able to change dramatically the
carbohydrate metabolic and photosynthetic capacity of tissues in
which they are expressed. It has furthermore been found that the
effects of TPP and TPS are opposite, i.e. by simultaneous
expression no major effects on the plant physiology and phenotype
can be observed. In said application it has additionally been found
that by expressing TPS in the tuber also the effects of the `cold
sweetening` process can be diminished, because the proportion of
reducing sugars is decreased at harvesting and after storage. Thus,
taking also into regard the present invention, expression of TPS
may improve the storage of potatoes in two ways: for cold storage
the effect of diminishing the cold sweetening process is important,
while for storage under more moderate temperature the prevention of
sprouting prevails.
[0019] Thus, TPS is capable to prevent remobilisation of storage
compounds. This is also applicable in other crops, such as chicory,
which is subject to degradation of the inulin into other
carbohydrates. Expression of TPS in the storage organs of chicory
prevents catabolic degradation of the inulin. Similarly, sucrose
breakdown in sugarbeet can be prevented. Thus, expression of TPS in
the taproots of sugarbeet prevents the loss of sucrose during
storage of the sugarbeets.
[0020] Generally, the anti-sprouting effect is obtained by the
expression of the TPS gene preferably in the tissues which are
prone to sprouting, such as the potato tuber. For specific
expression in the potato tuber the patatin promoter or any other
tuber-specific promoter may be used to drive the expression of the
TPS gene. We have, however, noted that it is most important that
the promoter is active at the end of the filling phase of the tuber
and during storage of the tuber. If the tuber-specific promoter is
not very active anymore at that point, the inhibitory effects of
the expression of TPS will wane off, and a delay in sprouting in
stead of a complete inhibition of sprouting will be the result.
[0021] The TPS gene is encoding a trehalose phosphate synthase.
Several genes coding for this enzyme are known and can be found in
all kind of organisms (WO 97/42326). In the experiments sustaining
the invention the gene derived from Escherichia coli is used, but
also other genes coding for TPS, e.g. derived from yeast or plants,
are equally useful. In other embodiments of the invention compounds
neutralizing the effect of TPS such as trehalose phosphate
phosphatase (TPP) are used. Also the gene coding for TPP is derived
from E. coli, but it can equally well be derived from other
organisms such as yeast, plants or even humans (WO 97/42326). Not
only the TPP is useful to restore the effects of TPS but any enzyme
capable of degrading trehalose-6-phosphate can be used. A further
example of such an enzyme is trehalose-6-phosphate hydrolase
(TreC). A gene coding for this enzyme can be drived from E. coli
(Rimmele, M., and Boos, W., Trehalose-6-phosphate hydrolase of
Escherichia coli. J. Bacteriol. 176, 5654-5664, 1994).
[0022] In its simplest form the invention is directed to inhibit
pre- and postharvest remobilisation of storage compounds in a
transgenic plant by transforming plant with a recombinant DNA
cassette which comprises the gene coding TPS and optionally a
selectable marker gene. More specifically such a method prevents
sprouting. Restoration of sprouting can be obtained by neutralizing
the effect of TPS. This can be achieved in a number of ways. The
following are given by example but methods to inhibit the effect of
TPS are not limited to these examples.
[0023] A first system of restoration of sprouting is to introduce
next to the TPS gene a gene coding for TPP, which is able to
overcome the anti-sprouting effects caused by the TPS. To prevent
the constitutive expression of TPP it is envisaged to bring
expression of TPP under control of an inducible promoter. Inducible
promoters include any promoter capable of increasing the amount of
gene product produced by a given gene, in response to exposure to
an inducer. In the absence of an inducer the DNA sequence will not
be transcribed. Typically, the factor that binds specifically to an
inducible promoter to activate transcription is present in an
inactive form which is then directly or indirectly converted to the
active form by the inducer. The inducer may be a chemical agent
such as protein, metabolite (sugar, alcohol, etc.), a growth
regulator, herbicide, or a phenolic compound or a physiological
stress imposed directly by heat, salt, wounding, toxic elements
etc., or indirectly through the action of a pathogen or disease
agent such as a virus. A plant cell containing an inducible
promoter may be exposed to an inducer by externally applying the
inducer to the cell such as by spraying, watering, heating, or
similar methods. Inducible promoters are known to those familiar
with the art and several exist that could conceivably be used to
drive expression of the TPP gene. Inducible promoters suitable for
use in accordance with the present invention include, but are not
limited to, the heat shock promoter, the mammalian steroid receptor
system and any chemically inducible promoter. Examples of inducible
promoters include the inducible 70 kD heat shock promoter of
Drosophila melanogaster (Freeling, M. et al., Ann. Rev. Genet. 19,
297-323) and the alcohol dehydrogenase promoter which is induced by
ethanol (Nagao, R. T. et al., in: Miflin, B. J. (ed.) Oxford
Surveys of Plant Molecular and Cell Biology, Vol. 3., pp. 384-438,
Oxford Univ. Press, 1986). A promoter that is inducible by a simple
chemical is particularly useful. Examples for the last category are
the promoters described in WO 90/08826, WO 93/21334, WO 93/031294
and WO 96/37609.
[0024] Thus, the anti-sprouting effect can be restored by treatment
with the inducer, and these restored sprouting lines can be used to
propagate the seeding material, such as seed-potatoes. Without the
presence of the inducer, sprouting of the offspring is still
inhibited by the expression of TPS. This thus also functions as a
way to produce germplasm protection.
[0025] A further method to restore the original sprouting phenotype
again is to provide the plant with a recombinant DNA cassette which
comprises next to the TPS gene an antisense TPS gene, said
antisense gene being under control of an inducible promoter As with
the above-mentioned example on the induction of TPP also the
antisense TPS is capable of negating the effect of the (sense) TPS
expression because by annealing with the TPS mRNA it prevents
successful translation of the TPS and thus inhibits the
anti-sprouting effect.
[0026] A third system of restoration of the original sprouting
phenotype is by introducing the DNA coding for a suppressor
protein, said suppressor capable of suppressing the expression of
TPS, while the expression of the suppressor is under control of and
inducible promoter. Such a suppression can for instance be
accomplished by use of the tet-repressor system, where a specific
binding site, which can be recognized by the repressor, is
introduced near the RNA-polymerase binding site of the gene which
expression needs to be suppressed. If the tet-repressor is
available then this repressor will bind to the specific sequence
and thus, by steric hindrance, prevents the RNA-polymerase to
initiate transcription. The gene coding for the tet-repressor can
be adjacent the gene which expression should be controlled, but
this is not necessary.
[0027] When the gene for the repressor is put under control of an
inducible promoter the expression of the suppressor-molecule and
thus the suppression of the TPS gene can be induced by applying an
external inducer. Then, the TPS effect will not be established and
normal sprouting will be the result.
[0028] A further system to restore the normal phenotype is to
provide the gene coding for TPS or the expression cassette
comprising said gene flanked by two site-specific recombination
sites, which can be recognized by the corresponding
recombinase.
[0029] A number of different site-specific recombinase systems can
be utilized in accordance the present invention, including but not
limited to the Cre/lox system of bacteriophage P1, the FLP/FRT
system of yeast, the Gin recombinase of phage Mu, the Pin
recombinase of E. coli, and the R/RS system of the pSR1 plasmid.
The two most used site-specific recombinase systems are the
bacteriophage P1 cre/lox and the yeast F(P/FRT systems. In these
systems a recombinase (Cre or FLP) interacts specifically with its
respective site-specific recombination sequence (lox or FRT,
respectively) to invert or excise the intervening sequences. The
site-specific recombination sequence for each of those two systems
is relatively short (34 bp for lox and 34-47 bp for FRT). Use of
such a site-specific recombinase in plants is for instance
described in U.S. Pat. No. 5,527,695. The DNA to be excised can be
flanked by direct repeats of the site-specific recombination site,
and subsequent introduction of the recombinase activity excises the
DNA (and thus restores the original phenotype). The FLP/FRT
recombinase system has been demostrated to function efficiently in
plant cells. Although the site-specific recombination sequences
must be linked to the ends of the DNA sequence to be excised for
inverted, the gene encoding the site-specific recombinase may be
located elsewhere and thus can be separately introduced into the
plant cells through standard transformation procedure, or through
cross-pollination with a plant that already is capable of
expressing the recombinase gene.
[0030] However, upon this last method of restoration the TPS gene
is lost from he transgenic plants.
[0031] Other ways to remove the inhibitory effects of the
expression of the TPS gene on the remobilisation of storage
compounds are external treatments of the storage organs with
compounds that are capable of neutralizing the effects of the
expression of the TPS gene. Surprisingly, we have found that
treatment with gibberellic acid (GA) was able to induce sprouting
in potato tubers contain the TPS gene. This was accomplished by
incubation of whole tubers or cut pieces in a solution of
commercially available GA. It is, however, envisaged that the
method of treatment can be varied and that for instance spraying of
tubers with a GA solution would yield comparable results. Depending
on the way of application the concentration of GA in the solution
should be in the range of 0.1 to 10,000 ppm. It is further believed
that the effect of GA is a neutralization of the effects of
expression of the TPS gene. Therefor, it is envisaged that also in
other examples of inhibition of remobilisation of storage
compounds, treatment with GA will be able to restore the inhibitory
effects of the expression TPS.
[0032] Also surprisingly, we have found that wounding of potato
tubers (through cutting off pieces containing at least one active
meristem) alone was sufficient to induce sprouting of those
pieces.
[0033] The recombinant DNA constructs of the present invention can
be constructed using recombinant DNA technology known to those
skilled in the art. The recombinant gene constructs can be inserted
into vectors, which can be commercially available, specifically
suited for transformation to plants and to express the gene product
in the transformed cells. Transformed cells (those containing the
recombinant DNA inserted into the host cell's DNA) are selected
from untransformed cells through the use of a selectable marker
included as part of the introduced recombinant DNA. Selectable
markers include genes that provide antibiotic or herbicide
resistance. Those cells containing the recombinant DNA are capable
of surviving in the presence of antibiotic or herbicide
concentrations that kill untransformed cells. Examples of
selectable marker genes include the bar gene which provides
resistance to the herbicide Basta, the nptII gene which confers
kanamycin resistance, the hpt gene which confers hygromycin
resistance and the cah gene which gives resistance to cyanamid. An
entire plant can be generated from a single transformed plant cell
through cell culturing techniques known to those skilled in the
art.
[0034] With regard to the applicability of the invention in
different plant species, it has to be mentioned that one particular
embodiment of the invention is merely illustrated with transgenic
potato plants as an example, the actual applicability being in fact
not limited to this plant species. Any plant species can be
provided with a recombinant DNA sequence according to the
invention, but preferably plant species which are normally
vegetatively propagated are especially useful.
[0035] Although some of the embodiments of the invention may not be
practicable at present, e.g. because some plant species are as yet
recalcitrant to genetic transformation, the practicing of the
invention in such plant species is merely a matter of time and not
a matter of principle, because the amenability to genetic
transformation as such is of no relevance to the underlying
embodiment of the invention.
[0036] Transformation of plant species is now routine for an
impressive number of plant speicies, including both the
Dicotyledoneae as well as the Monocotyledoneae. In principle any
transformation method may be used to introduce recombinant DNA
according to the invention into a suitable ancestor cell, as long
as the cells are capable of being regenerated into whole plants.
Methods may suitably be selected from the calcium/polyethylene
glycol method for protoplasts (Krens, F. A. et al., 1982, Nature
296, 72-74; Negrutiu I. et al, June 1987, Plant Mol. Biol. 8,
363-373), electroporation of protoplasta (Shillito R. D. et al.,
1985 Biol/Technol. 3, 1099-1102), microinjection into plant
material (Crossway A. et al., 1986, Mol. Gen. Genet. 202,
1719-185), (DNA or RNA-coated particle bombardment of various plant
material (Klein T. M. et al., 1987, Nature 327, 70), infection with
(non-integrative) viruses and the like. A preferred method
according to the invention comprises Agrobacterium-mediated DNA
transfer. Especially preferred is the use of the so-called binary
vector technology as disclosed in EP A 120 516 and U.S. Pat. No.
4,940,838). Tomato transformation can be preferably done
essentially as described by Van Roekel et al. (Van Roekel, J. S.
C., Damm, B., Melchers, L. S., Hoekema, A. (1993). Factors
influencing transformation frequency of tomato (Lycopexsicon
esculentum). Plant Cell Reports, 12, 644-647). Potato
transformation can be preferably done essentially as described by
Hoekema et al. (Hoekema, A., Huisman, M. J., Molendijk, L., van den
Elzen, P. J. M., and Cornelissen, B. J. C. (1989). The genetic
engineering of two commercial potato cultivars for resistance to
potato virus X. Bio/Technology 7, 273-278). Generally, after
transformation plant cells or cell groupings are selected for the
presence of one or more markers which are encoded by plant
expressible genes co-transferred with the nucleic acid sequence
encoding the protein according to the invention, whereafter the
transformed material is regenerated into a whole plant.
[0037] Although considered somewhat more recalcitrant towards
genetic transformation, monocotyledonous plants are amenable to
transformation and fertile transgenic plants can be regenerated
from transformed cells or embryos, or other plant material.
Presently, preferred methods for transformation of monocots are
microprojectile bombardment of embryos, explants or suspension
cells, and direct DNA uptake or electroporation (Shimamoto, et al,
1989, Nature 338, 274-276). Transgenic maize plants have been
obtained by introducing the Streptomyces hygroscopicus bar-gene,
which encodes phosphinothricin acetyltransferase (an enzyme which
inactivates the herbicide phosphinothricin), into embryogenic cells
of a maize suspension culture by microprojectile bombardment
(Gordon-Kamm, 1990, Plant Cell, 2, 603-618). The introduction of
genetic material into aleurone protoplasts of other monocot crops
such as wheat and barley has been reported (Lee, 1989, Plant Mol.
Biol. 13, 21-30). Wheat plants have been regenerated from
embryogenic suspension culture by selecting only the aged compact
and nodular embryogenic callus tissues for the establishment of the
embryogenic suspension cultures (Vasil, 1990 Bio/Technol. 8,
429-434). The combination with transformation systems for these
crops enables the application of the present invention to
monocots.
[0038] Monocotyledonous plants, including commercially important
crops such as rice, banana and corn are also amenable to DNA
transfer by Agrobacterium strains (vide WO 94/0097; EP 0 159 418
B1; Gould J, Michael D, Hasegawa O, Ulian E C, Peterson G, Smith R
H, (1991) Plant. Physiol. 95, 426-434).
[0039] Following DNA transfer and regeneration, putatively
transformed plants may be evaluated, for instance using Southern
analysis, for the presence of the recombinant DNA according to the
invention, copy number and/or genomic organization. In addition, or
alternatively, expression levels of the newly introduced DNA may be
undertaken, using Northern and/or Western analysis, techniques well
known to persons having ordinary skill in the art. After the
initial analysis, which is optional, transformed plants showing the
desired copy number and expression level of the newly introduced
recombinant DNA according to the invention may be tested for their
male sterility or restoration to fertility. Alternatively, the
selected plants may be subjected to another round of
transformation, for instance to introduce further genes, such as
the antisense TPS gene, the TPP gene or the suppressor gene.
[0040] To obtain transgenic plants capable of constitutively
expressing more than one chimeric gene, a number of alternatives
are available including the following:
[0041] A. The use of DNA, e.g a T-DNA on a binary plasmid, with a
number of modified genes physically coupled to a selectable marker
gene. The advantage of this method is that the chimeric genes are
physically coupled and therfore migrate as a single Mendelian
locus.
[0042] B. Cross-pollination of transgenic plants each already
capable of expressing one or more chimeric genes, preferably
coupled to a selectable marker gene, with pollen from a transgenic
plant which contains one or more chimeric genes coupled to another
selectable marker. Afterwards the seed, which is obtained by this
crossing, maybe selected on the basis of the presence of the two
selectable markers, or on the basis of the presence of the chimeric
genes themselves. The plants obtained from the selected seeds can
afterwards be used for further crossing. In principle the chimeric
genes are not on a single locus and the genes may therfore
segregate as independent loci.
[0043] C. The use of a number of a plurality chimeric DNA
molecules, e.g. plasmids, each having one or more chimeric genes
and a selectable marker. If the frequency of co-transformation is
high, then selection on the basis of only one marker is sufficient.
In other cases, the selection on the basis of more than one marker
is preferred.
[0044] D. Consecutive transformation of transgenic plants already
containing a first, second, (etc.), chimeric gene with new chimeric
DNA, optionally comprising a selectable marker gene. As in method
B. the chimeric genes are in principle not on a single locus and
the chimeric genes may therefore segregate as independent loci.
[0045] E. Combinations of the above mentioned strategies.
[0046] Plants, in which this invention is particularly useful, are
plants which are able to propagate vegetatively and in which
sprouting at a certain moment is an undesired property. The most
outstanding examples are potato and onion, but the invention can
also be used in flower bulbs, strawberries and banana. Next to the
complete inhibition of sprouting and an inducible restoration
mechanism, it is also envisaged that the inhibition can be made
inducible. This, for instance, would be useful in strawberry and
banana, where sprouting is a desired property for the
multiplication of plants, but where sprouting can be competitive
with regard to other processes such as fruit ripening. If the TPS
gene is placed under control of an inducible promoter it is
possible to inhibit sprouting at any time during the growing of the
crops, for instance during the period of seed setting or fruit
ripening. Preferably such an induction of expression of the TPS
gene is performed by a chemical inducible promoter which reacts on
the (external) application of a chemical substance. Furthermore, in
this embodiment of the invention it would be preferable also to
make the expression of TPS tissue specific for meristematic tissue.
Promoters, which are specific for meristematic tissue are readily
available in the art (for instance the HMG2 promoter from Enjuto et
al., Plant Cell 7, 517, 1995 and the rice PCNA promoter from Kosugi
et al., Plant J. 7, 877, 1995).
[0047] Next to the sprouting the mechanism of inhibition of pre-
and postharvest remobilisation of storage compounds is also of use
in chicory to prevent degradation of inulin and in sugarbeet to
prevent degradation of sucrose.
[0048] The following examples are further provided for illustrative
purposes only and are in no way intended to limit the scope of the
present invention.
[0049] Standard methods for the isolation, manipulation and
amplification of DNA, as well as suitable vectors for replication
of recombinant DNA, suitable bacterium strains, selection markers,
media and the like are described for instance in Sambrook, J.,
Fritsch, E. P., and Maniatis, T. (1989) Molecular cloning; a
laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; DNA Cloning: Volumes I and II (D. N. Glover ed.
1985); and in: From Genes To Clones (E.-L. Winnacker ed, 1987).
[0050] DNA Manipulations
[0051] All DNA procedures (DNA isolation from E. coli, restriction,
ligation, transformation, etc.) are performed according to standard
protocols (Sambrook et al. (1989) Molecular Cloning: a laboratory
manual, 2nd ed. Cold Spring Harbor Laboratory Press, CSH, New
York).
[0052] Strains
[0053] In all examples E. coli K-12 strain DH5.alpha. is used for
cloning. The Agrobacterium tumefaciens strains used for plant
transformation experiments are EHA 105 and MOG 101 (Hood et al.,
Trans. Research 2, 208-218, 1993)
[0054] Generation of Potato Plants Transgenic for Pat-TPS.
[0055] Construction of pMOG845 harboring the E. coli tps gene under
control of the tuber-specific patatin promoter, triparental mating
to Agrobacterium and the generation of transgenic potato plants,
Solanum tuberosum cv. kardal, are described in WO 97/42326.
[0056] Experimental Part
EXAMPLE 1
[0057] In one part of the experiment, tuber material was produced
from in vitro potato plants transgenic for pMOG845 (patatin-tps). A
field trial experiment was set-up using tubers of 9 independent
transgenic lines, 3 plots per line, 5 tubers per plot. Tubers were
transferred to the field at the beginning of May and the sprouting
process was monitored on a regular basis. Results are depicted in
table 1. In the second part of the experiment pat-TPS plants (var
Kardal) derived from tissue culture plants were grown in the
phytochamber under 500 .mu.mol quanta m-2 s-1 (16 h light, 20_C; 8
h dark (15_C) ). Tubers were harvested after three months and
stored in the cold (4_C) for 2 months. Then they were transferred
to room temperature (RT) and sprouting was assessed during a period
of four weeks.
1 TABLE 1 Sprouting Plant-line Field Phytochamber Kardal all tubers
all tubers 845-17 all tubers* delayed 845-13 all tubers all tubers
845-28 none none 845-4 all tubers all tubers 845-11 none none
845-22 2/15 tubers none 845-2 all tubers* delayed 845-1 all tubers*
delayed 845-25 all tubers all tubers *means that the plants in that
plot were significantly smaller compared to wild-type indicating a
delay in sprouting Delayed means that no sprouts were visible after
2 week transfer to RT after a 2 month cold period None means
sprouting does not occur within 4 weeks.
[0058] Tubers revealing the complete absence of sprouting have been
shown to have a high expression level of the transgene. A reduction
of cold-sweetening as described in WO 97/42326 is observed in the
non-sprouting lines and to a lesser extent in the tubers delayed in
sprouting or normal sprouting tubers.
EXPERIMENT 2
[0059] Gibberellic Acid Reverts Anti-Sprouting Phenotype
[0060] Whole tubers obtained from the plants of Example 1 grown
under phytochamber conditions were taken. Approximately 1 week
after transfer to RT they were incubated for 24 h in a solution
containing 0.17% (w/v) gibberellic acid (GA 4 and GA 7; formulation
commercially available as Berelex.RTM., Zeneca, Ridderkerk,
Netherlands) Control tubers were not incubated. Further storage was
done at RT. The induction of sprouting occurred in GA-treated and
non-treated wildtype tubers after 8 days. After 14 days, 95% of the
14 non-treated wildtype tubers sprouted, while none of the
transgenic lines did (FIG. 1A). In contrast, all tubers (5) from
GA-treated wildtype tubers and 80%, 50%, 100% and 17% of the
GA-treated transgenic tubers from lines 845-1, -17, -22, -28 form
sprouts, respectively, All non-treated transgenic tubers did not
sprout. After 25 days it can be seen that lines 845-1 and 845-17
show delayed sprouting in the non-treated tubers (FIG. 1B).
EXPERIMENT 3
[0061] Wounding Reverts Anti-Sprouting Phenotype
[0062] Pat-TPS plants (Var. Kardal) derived from tissue culture
plants were grown in the phytochamber under 500 .mu.mol quanta m-2
s-1 (16 h light, 20.degree. C.; 8 h dark (15.degree. C.)). Tubers
were harvested after three months and stored in the cold (4.degree.
C.) for 2 months.
[0063] Three days after transfer to room temperature (RT), tuber
pieces were cut with a knife containing at least one active
meristem (eye). Cut pieces originating from 3-10 tubers per line
were washed for 15 min in tap water. Approximately 6-10 pieces were
subsequently incubated for 10 min on either water or on a 1, 10 or
1000 ppm solution of gibberellic acid (GA3, SIGMA, Zwijndrecht,
Netherlands). All pieces from one treatment were transferred to
containers onto wet paper tissue and covered with a plastic top to
prevent drying out. Sprouting of wild-type and tps tuber pieces
occurred within 4 days incubated either on water or on the
different gibberellic acid solutions, indicating that wounding per
se is sufficient to restore sprouting.
* * * * *