U.S. patent application number 11/243080 was filed with the patent office on 2006-02-16 for pathogen-resistant grape plants.
Invention is credited to Dennis J. Gray, Zhijian Li, Jayasankar Subramanian.
Application Number | 20060035377 11/243080 |
Document ID | / |
Family ID | 26832159 |
Filed Date | 2006-02-16 |
United States Patent
Application |
20060035377 |
Kind Code |
A1 |
Subramanian; Jayasankar ; et
al. |
February 16, 2006 |
Pathogen-resistant grape plants
Abstract
The invention features a method of producing a grape somatic
embryo having resistance to a plant pathogen, the method including
the steps of (a) culturing a grape somatic embryo in a first liquid
culture medium that includes a plant growth regulator and a
phytotoxin from a plant pathogen; (b) exchanging the first liquid
culture medium for a second liquid culture medium not including the
phytotoxin; (c) recovering a living grape cell or grape cell
cluster from the second liquid culture, the living cell or cell
cluster being resistant to the pathogen; and (d) culturing the
grape cell or grape cell cluster in a third culture medium to
produce a grape somatic embryo.
Inventors: |
Subramanian; Jayasankar;
(Tavares, FL) ; Li; Zhijian; (Altamonde Springs,
FL) ; Gray; Dennis J.; (Howey-in-the-Hills,
FL) |
Correspondence
Address: |
BEUSSE BROWNLEE WOLTER MORA & MAIRE
390 N. ORANGE AVENUE
SUITE 2500
ORLANDO
FL
32801
US
|
Family ID: |
26832159 |
Appl. No.: |
11/243080 |
Filed: |
October 4, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09570217 |
May 12, 2000 |
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11243080 |
Oct 4, 2005 |
|
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60134275 |
May 14, 1999 |
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60148251 |
Aug 11, 1999 |
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Current U.S.
Class: |
435/430.1 ;
800/279 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/8282 20130101 |
Class at
Publication: |
435/430.1 ;
800/279 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C12N 5/00 20060101 C12N005/00; C12N 15/87 20060101
C12N015/87; A01H 1/00 20060101 A01H001/00 |
Claims
1. A method of identifying a protein that provides pathogen
resistance to a grape plant, said method comprising the steps of:
(a) culturing a grape somatic embryo in a first liquid culture
medium comprising a plant growth regulator and a phytotoxin from a
plant pathogen; (b) exchanging said first liquid culture medium for
a second liquid culture medium not comprising said phytotoxin; (c)
recovering a grape cell or grape cell cluster from said second
liquid culture; (d) culturing said grape cell or grape cell cluster
in a third culture medium to produce a grape somatic embryo having
resistance to said plant pathogen; (e) recovering said grape
somatic embryo having resistance to said plant pathogen; and (f)
identifying a protein that is expressed in said grape somatic
embryo and that is not expressed in a grape somatic embryo not
cultured in a culture medium comprising said phytotoxin from said
plant pathogen, wherein said identified protein is a protein that
confers on a plant pathogen resistance.
2. A method for identifying a protein that, when expressed in a
grape plant, confers on said plant pathogen resistance, said method
comprising the steps of: (a) contacting an embryogenic cell,
embryogenic culture, or somatic embryo, with a plant pathogen; and
(b) measuring the level of expression of a protein, wherein an
increased level of expression of the protein by said embrogenic
cell, embryogenic culture, or somatic embryo, relative to an
embryogenic cell, embryogenic culture, or somatic embryo not
contacted with said plant pathogen, identifies said protein as one
that, when expressed in a plant, confers on said plant pathogen
resistance.
3. A method of identifying protein candidates for conferring
pathogen resistance activity in a grape plant, said method
comprising the steps of: (a) culturing a grape somatic embryo in a
first liquid culture medium comprising a plant growth regulator and
a phytotoxin from a plant pathogen; (b) exchanging said first
liquid culture medium for a second liquid culture medium not
comprising said phytotoxin; (c) recovering a grape cell or grape
cell cluster from said second liquid culture medium; (d) culturing
said recovered grape cell or grape cell cluster in a third culture
medium; (e) recovering a grape cell or grape cell cluster from said
third culture medium; and (f) identifying a protein that is
expressed in said grape somatic embryo and that is not expressed,
or less expressed, in a grape somatic embryo not cultured in a
culture medium comprising said phytotoxin from said plant
pathogen.
4. A method of identifying protein candidates for conferring
pathogen resistance activity in a grape plant, said method
comprising the steps of: (a) contacting an embryogenic cell,
embryogenic culture or somatic embryo with a plant pathogen; and
(b) identifying a protein exhibiting an increased level of
expression in said embryogenic cell, embryogenic culture or somatic
embryo relative to an embryogenic cell, embryogenic culture, or
somatic embryo not contacted with said plant pathogen.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
09/570,217 filed May 12, 2000, which claims the benefit to U.S.
Provisional Application Nos. 60/134,275, filed May 14, 1999 and
60/148,251, filed Aug. 11, 1999, each of which is hereby
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] This invention relates to plants having an increased level
of resistance to a pathogen and to methods for producing them.
[0003] Grapevines are a deciduous temperate fruit crop of ancient
origin. Grape production (65.times.10.sup.6 metric tons) exceeds
that of any other temperate fruit crop, and ranks third after
Citrus and banana production. In addition, due to its uses for
fresh fruit, juice, jelly, raisins, and wine, grapes surpass all
other fruit crops in value. Therefore, successful efforts to
improve grapevines are likely to have a major impact on commercial
viticulture.
[0004] Current methods for improving grapevines are time-consuming
and labor intensive. For example, genetic improvement in grapes
through conventional breeding is severely limited by a number of
factors such as long pre-bearing age and varying ploidy levels.
Cultivated gapes are also highly heterozygous and do not generally
breed true from seeds. Moreover, grape breeding programs are
expensive, long-term products. Although plant biotechnology is an
attractive alternative for genetic improvement in grapes (Kuksova
et al., Plant Cell Tiss. Org. Cult. 49:17-27, 1997), in vitro
genetic manipulation can be addressed only if there is an effective
regeneration system. Accordingly, methods that reduce any of these
problems would represent a significant advancement in the art.
SUMMARY OF THE INVENTION
[0005] We have discovered methods for growing perennial grape
embryogenic cultures and for growing large quantities of somatic
grape embryos from such perennial embryogenic cultures in a
relatively short period using a liquid suspension culture. Several
advantages are provided by the present methods. These approaches,
for example, facilitate an extraordinarily high frequency of
somatic embryo formation and plant regeneration. Such frequencies
have not been previously reported for grapevine regeneration of any
known cultivar, and render the method useful for large-scale
production of clonal planting stock of grape plants. In addition,
the methods produce embryos free of such common abnormalities as
fusion and fasciations of somatic embryos. The methods of the
invention also result in enhanced embryogenic culture initiation
frequency, allowing for the production of highly embryogenic
cultures that can then be successfully carried through the
subsequent stages of the regeneration process to the whole plant
level. Because of these advantages, the methods of the invention
are especially useful in the application of biotechnology for the
genetic improvement of this crop.
[0006] Embryogenic cells that are resistant to a plant pathogen can
be selected in vitro using methods of the present invention. From
these cells, or from the culture medium, proteins whose expression
is upregulated in response to a pathogen (and the nucleic acid
molecules encoding them) are identified. The proteins and nucleic
acid molecules can then be used to produce pathogen-resistant
plants (i.e., a transgenic or non-transgenic plant expressing such
a protein) or to increase plant resistance to a pathogen (e.g., by
applying recombinant protein to the surface of a plant.
[0007] Accordingly, in a first aspect, the invention features a
method of producing a grape somatic embryo having resistance to a
plant pathogen, the method including the steps of (a) culturing a
grape somatic embryo in a first liquid culture medium that includes
a plant growth regulator and a phytotoxin from a culture of the
plant pathogen; (b) exchanging the first liquid culture medium for
a second liquid culture medium not including the phytotoxin; (c)
recovering a living grape cell or grape cell cluster from the
second liquid culture, the living cell or cell cluster being
resistant to the pathogen; and (d) culturing the grape cell or
grape cell cluster in a third culture medium to produce a grape
somatic embryo.
[0008] In a second aspect, the invention features a method for
producing a grape plant having resistance to a plant pathogen, the
method including the steps of (a) culturing a grape somatic embryo
in a first liquid culture medium that includes a plant growth
regulator and a phytotoxin from a culture of the plant pathogen;
(b) exchanging the first liquid culture medium for a second liquid
culture medium not including the phytotoxin; (c) recovering a
living grape cell or grape cell cluster from the second liquid
culture, the living cell or cell cluster being resistant to the
pathogen; (d) culturing the grape cell or grape cell cluster in a
third culture medium to produce a grape somatic embryo; and (e)
growing a plant from the grape somatic embryo.
[0009] In the methods of the first and second aspects, the
phytotoxin may be obtained, for example, from a bacterium or
fungus. A preferred plant growth regulator in step (a) is an auxin
(e.g., 2,4-D, NAA, NOA, or picloram). If desired, the second
culture medium may also include a plant growth regulator. In other
preferred embodiments, steps (a)-(d) of the method are repeated at
least two time, more preferably at least three times, and most
preferably at least four or five times. The culture step (a) can be
for a day or two, but is preferably for at least four days, six
days, or more. In preferred embodiments, the culture step (a) is
for at least nine or ten days.
[0010] In a third aspect, the invention features a grape plant
regenerated from a cell or cell cluster that has been selected in
the presence of a phytotoxin from a plant pathogen, wherein the
plant has an increased level of resistance to the pathogen relative
to a control grape plant regenerated from a cell or cell cluster
not selected in the presence of the phytotoxin. The grape plant is
preferably expressing a protein at a level that is at least 25%
greater than the level of the protein in the control plant, wherein
the protein is selected from the group consisting of (i) a protein
having a molecular weight of about 8 kDa and including the
polypeptide of SEQ ID NO: 1; (ii) a protein having a molecular
weight of about 22 kDa and including the polypeptide of SEQ ID NO:
2; (iii) a protein having a molecular weight of about 22 kDa and
including the polypeptide of SEQ ID NO: 3; and (iv) a protein
having a molecular weight of about 33 kDa and including the
polypeptide of SEQ ID NO: 4. More preferably, the grape plant is
expressing a protein at a level that is at least 50%, 100%, 200%,
300%, or even 500% greater than the level of the protein in the
control plant.
[0011] In a fourth aspect, the invention features a transgenic
grape plant containing a transgene encoding a polypeptide
substantially identical to the polypeptide having the amino acid
sequence of SEQ ID NO: 5, wherein the transgene is operably linked
to a promoter. In preferred embodiments, the nucleic acid molecule
has the nucleotide sequence of SEQ ID NO: 6, and the promoter is a
constitutive promoter, an inducible promoter, or a tissue-specific
promoter.
[0012] In a fifth aspect, the invention features a transgenic grape
plant containing a transgene encoding a PR-5 protein that confers
on the plant resistance to a pathogen, wherein the nucleic acid
molecule is operably linked to a constitutive promoter.
[0013] In a sixth aspect, the invention features a transgenic grape
plant containing a transgene encoding a thaumatin-like protein that
confers on the plant resistance to a pathogen, wherein the nucleic
acid molecule is operably linked to a constitutive promoter.
[0014] In a seventh aspect, the invention features a transgenic
grape plant containing a transgene encoding a lipid transfer
protein that confers on the plant resistance to a pathogen, wherein
the nucleic acid molecule is operably linked to a constitutive
promoter. In a preferred embodiment, the lipid transfer protein is
substantially identical to the amino acid of SEQ ID NO: 5.
[0015] In an eighth aspect, the invention features a plant
component from the plant of the third, fourth, fifth, sixth, or
seventh aspect.
[0016] In a ninth aspect, the invention features a method of
selecting a plant having pathogen resistance. The method includes
determining the levels of a protein in the plant, wherein the
protein includes an amino acid sequence selected from the group
consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID
NO: 4, and wherein the levels of the protein are directly
proportional to the levels of pathogen resistance in the plant. The
pathogen may be, for example, a bacterium or fungus.
[0017] In a tenth aspect, the invention features a substantially
pure polypeptide substantially identical to the sequence of SEQ ID
NO: 5. Preferably, the polypeptide, when expressed in a grape
plant, confers on the plant increased pathogen resistance relative
to the plant not expressing the polypeptide.
[0018] In an eleventh aspect, the invention features a
substantially pure nucleic acid molecule encoding a polypeptide
substantially identical to the sequence of SEQ ID NO: 5.
Preferably, the polypeptide, when expressed in a grape plant,
confers on the plant increased pathogen resistance relative to the
plant not expressing the polypeptide. In one preferred embodiment,
the nucleic acid molecule has the sequence of SEQ ID NO: 6.
[0019] In a twelfth aspect, the invention features a method of
identifying a protein that confers on a plant pathogen resistance.
The method includes the steps of (a) culturing a grape somatic
embryo in a first liquid culture medium including a plant growth
regulator and a phytotoxin from a plant pathogen culture; (b)
exchanging the first liquid culture medium for a second liquid
culture medium not including the phytotoxin; (c) recovering a grape
cell or grape cell cluster from the second liquid culture; (d)
culturing the grape cell or grape cell cluster in a third culture
medium to produce a grape somatic embryo having resistance to the
plant pathogen; (e) recovering the grape somatic embryo having
resistance to the plant pathogen; and (f) identifying a protein
that is expressed in the grape somatic embryo and that is not
expressed in a grape somatic embryo not cultured in a culture
medium including the phytotoxin from the plant pathogen culture,
wherein the identified protein is a protein that confers on a plant
pathogen resistance.
[0020] In a thirteenth aspect, the invention features another
method for identifying a protein that, when expressed in a grape
plant, confers on the plant pathogen resistance, the method
including the steps of (a) contacting an embryogenic cell,
embryogenic culture, or somatic embryo, with a plant pathogen; and
(b) measuring the level of expression of a protein, wherein an
increased level of expression of the protein by the embryogenic
cell, embryogenic culture, or somatic embryo, relative to an
embryogenic cell, embryogenic culture, or somatic embryo not
contacted with the plant pathogen, identifies the protein as one
that, when expressed in a plant, confers on the plant pathogen
resistance. The level of expression may be measured, for example,
using SDS-PAGE, ELISA, or Western Blot analysis. Protein level is
preferably standardized in comparison to total protein level.
[0021] In a fourteenth aspect, the invention features a method for
producing a plant having increased resistance to a plant pathogen,
the method including overexpressing a protein identified by the
method of twelfth aspect or the method of the thirteenth
aspect.
[0022] In a fifteenth aspect, the invention features a method for
decreasing pathogen-mediated damage to a plant, the method
including contacting the plant with a recombinant form of a protein
that exhibits increased level of expression following contact with
a pathogen.
[0023] In a sixteenth aspect, the invention features a method for
identifying a cell that is expressing a protein that confers
pathogen resistance. The method including the steps of (a)
contacting a cell with a phytotoxin from a pathogen culture; and
(b) monitoring disease resistance of the cell, wherein increased
pathogen resistance, relative to a control cell, identifies the
cell as a cell that is expressing a protein that confers on the
plant resistance to a pathogen.
[0024] In a seventeenth aspect, the invention features a
substantially pure polypeptide including the amino acid of SEQ ID
NO: 1 and having a molecular weight of about 8 kDa as determined by
reducing SDS-PAGE, wherein the polypeptide is expressed at an
increased level in a grape plant in response to contact with a
filtrate of a culture of Elsino.delta. ampelina.
[0025] In an eighteenth aspect, the invention features a
substantially pure polypeptide including the amino acid of SEQ ID
NO: 3 and having a molecular weight of about 22 kDa as determined
by reducing SDS-PAGE, wherein the polypeptide is expressed at an
increased level in a grape plant in response to contact with a
filtrate of a culture of Elsino.delta. ampelina.
[0026] In a nineteenth aspect, the invention features a DNA
molecule that hybridizes to the DNA of SEQ ID NO: 6.
[0027] In a twentieth aspect, the invention features a transgenic
plant containing a transgene that hybridizes to the DNA of SEQ ID
NO: 6 under high stringency conditions.
[0028] In a twenty-first aspect, the invention features a
regenerated grape plant that is expressing a protein at a level
that is at least 25% greater than the level of the protein in a
control grape plant regenerated from a cell or cell cluster not
selected in the presence of a phytotoxin from a plant pathogen
culture, wherein the protein is selected from the group consisting
of: (i) a protein having a molecular weight of about 8 kDa and
comprising the polypeptide of SEQ ID NO: 1; (ii) a protein having a
molecular weight of about 22 kDa and comprising the polypeptide of
SEQ ID NO: 2; (iii) a protein having a molecular weight of about 22
kDa and comprising the polypeptide of SEQ ID NO: 3; and (iv) a
protein having a molecular weight of about 33 kDa and comprising
the polypeptide of SEQ ID NO: 4. Preferably the plant is expressing
at least two proteins (and more preferably at least three or even
all four) at levels that are at least 25% greater than the level of
the protein in a control grape plant.
[0029] In a twenty-second aspect, invention features a
substantially pure polypeptide comprising the amino acid sequence
of SEQ ID NO: 4.
[0030] Terms used herein are defined as follows:
[0031] By "perennial grape embryogenic culture" is meant an
embryogenic culture in which embryogenic cells or cell masses have
been repeatedly selected, subcultured, and maintained as an in
vitro culture. Such perennial grape embryogenic cultures can be
maintained for at least half a year, preferably three years, and
most preferably four or more years.
[0032] By "embryogenic cell," "embryogenic cell mass," or
"embryogenic cultures" is meant a cell or collection of cells
having the inherent potential to develop into a somatic embryo and,
ultimately, into a plant. Typically such cells have large nuclei
and dense cytoplasm. Additionally, such cells are usually
totipotent in that they typically possess all of the genetic and
structural potential to ultimately become a whole plant.
[0033] By "increased level of embryogenesis" is meant a greater
capacity to produce an embryogenic cell or embryogenic cell mass in
a perennial grape embryogenic culture than the level of a control
non-perennial grape embryogenic culture. In general, such an
increased level of embryogenesis is at least 20%, preferably at
least 50%, more preferably at least 100% and most preferably at
least 250% or greater than the level of a control embryogenic
culture. The level of embryogenesis is measured using conventional
methods.
[0034] By "explant" is meant an organ, tissue, or cell derived from
a plant and cultured in vitro for the purpose of initiating a plant
cell culture or a plant tissue culture. For example, explant grape
tissue may be obtained from virtually any part of the plant
including, without limitation, anthers, ovaries, ovules, floral
tissue, vegetative tissue, tendrils, leaves, roots, nucellar
tissue, stems, seeds, protoplasts, pericycle, apical meristem
tissue, embryogenic tissue, somatic embryos, and zygotic
embryos.
[0035] By "plant growth regulator" is meant a compound that affects
plant cell growth and division. Preferred plant growth regulators
include natural or synthetic auxins or cytokinins. Exemplary auxins
include, but are not limited to, NOA, 2,4-D, NAA, IAA, dicamba, and
picloram. Exemplary cytokinins include, but are not limited to, BA
and zeatin.
[0036] By "somatic embryogenesis" is meant the process of
initiation and development of embryos in vitro from plant cells and
tissues absent sexual reproduction.
[0037] By "somatic embryo" is meant an embryo formed in vitro from
somatic cells or embryogenic cells by mitotic cell division.
[0038] By "mature somatic embryo" is meant a fully-developed embryo
with evidence of root and shoot apices and exhibiting a bipolar
structure. Preferred mature somatic embryos are those with
well-defined cotyledons.
[0039] By "plantlet" is meant a small germinating plant derived
from a somatic embryo.
[0040] By "regeneration" is meant the production of an organ,
embryo, or whole plant in plant tissue culture.
[0041] By "plant cell" is meant any cell containing a plastid. A
plant cell, as used herein, is obtained from, without limitation,
seeds, suspension cultures, embryos, meristematic regions, callus
tissue, protoplasts, leaves, roots, shoots, somatic and zygotic
embryos, as well as any part of a reproductive or vegetative tissue
or organ.
[0042] By "promoter" is meant a region of nucleic acid, upstream
from a translational start codon, which is involved in recognition
and binding of RNA polymerase and other proteins to initiate
transcription. A "plant promoter" is a promoter capable of
initiating transcription in a plant cell, and may or may not be
derived from a plant cell.
[0043] By "tissue-specific promoter" is meant that the expression
from the promoter is directed to a subset of the tissues of the
plant. It will be understood that not every cell in a given tissue
needs to be expressing from the promoter in order for the promoter
to be considered tissue-specific.
[0044] By "heterologous" is meant that the nucleic acid molecule
originates from a foreign source or, if from the same source, is
modified from its original form. Thus, a "heterologous promoter" is
a promoter not normally associated with the duplicated enhancer
domain of the present invention. Similarly, a heterologous nucleic
acid molecule that is modified from its original form or is from a
source different from the source from which the promoter to which
it is operably linked was derived.
[0045] The term "plant" includes any cell having a chloroplast, and
can include whole plants, plant organs (e.g., stems, leaves, roots,
etc.), seeds, and cells. The class of plants that can be used in
the method of the invention is generally as broad as the class of
higher plants amenable to transformation techniques, including both
monocots and dicots.
[0046] By "plant component" is meant a part, segment, or organ
obtained from an intact plant or plant cell. Exemplary plant
components include, without limitation, somatic embryos, leaves,
fruits, scions, cuttings, and rootstocks.
[0047] By "phytotoxin" is meant a substance that is capable of
killing a plant cell. Phytotoxins are preferably from a pathogen
such as a fungus or a bacterium. For use in the present invention,
they may be purified or unpurified. In one example, a phytotoxin is
present in a filtrate from a culture of a pathogen such as a
bacterium or a fungus. The identity of the phytotoxin (e.g., its
chemical structure) need not be known for use in the methods of the
invention.
[0048] By "pathogen" is meant an organism whose infection of viable
plant tissue elicits a disease response in the plant tissue. Such
pathogens include, without limitation, bacteria and fungi. Plant
diseases caused by these pathogens are described in Chapters 11-16
of Agrios, Plant Pathology, 3rd ed., Academic Press, Inc., New
York, 1988.
[0049] Examples of bacterial pathogens include, without limitation,
Agrobacterium vitis, Agrobacterium tumefaciens, Xylella fastidosa,
and Xanthomonas ampelina. Examples of fungal pathogens include,
without limitation, Uncinula necator, Plasmopara viticola, Botrytis
cinerea, Guignardia bidwellii, Phomophsis viticola, Elsinoe
ampelina, Eutypa lata, Armillaria mellea, and Verticllium
dahliae.
[0050] By "pathogen culture" is meant a culture in which a pathogen
has grown. A filtrate of the culture is preferably substantially
free of the pathogen.
[0051] By "increased level of resistance" is meant a greater level
of resistance or tolerance to a disease-causing pathogen or pest in
a resistant grapevine (or scion, rootstock, cell, or seed thereof)
than the level of resistance or tolerance or both relative to a
control plant (i.e., a grapevine that has not been subjected to in
vitro selection to any plant pathogen or toxin-containing filtrate
thereof). In preferred embodiments, the level of resistance in a
resistant plant of the invention is at least 5-10% (and preferably
at least 30% or 40%) greater than the resistance of a control
plant. In other preferred embodiments, the level of resistance to a
disease-causing pathogen is at least 50% greater, 60% greater, and
more preferably even more than 75% or even 90% greater than the
level of resistance of a control plant; with up to 100% above the
level of resistance as compared to the level of resistance of a
control plant being most preferred. The level of resistance or
tolerance is measured using conventional methods. For example, the
level of resistance to a pathogen may be determined by comparing
physical features and characteristics (for example, plant height
and weight, or by comparing disease symptoms, for example, delayed
lesion development, reduced lesion size, leaf wilting, shriveling,
and curling, decay of fruit clusters, water-soaked spots, leaf
scorching and marginal burning, and discoloration of cells) of
resistant grape plants with control grape plants. Quantitation can
be performed on the level of populations. For example, if 4 out of
40 control plants are resistant to a given pathogen, and 20 out of
40 plants of the invention are resistant to that pathogen, than the
latter plant is 20/4 or 500% more resistant to the pathogen.
[0052] By "transformed" is meant any cell which includes a nucleic
acid molecule (for example, a DNA sequence) which is inserted by
artifice into a cell and becomes part of the genome of the organism
(in either an integrated or extrachromosomal fashion for example, a
viral expression construct which includes a subgenomic promoter)
which develops from that cell. As used herein, the transformed
organisms or cells are generally transformed grapevines or
grapevine components and the nucleic acid molecule (for example, a
transgene) is inserted by artifice into the nuclear or plastidic
compartments of the plant cell.
[0053] By "transgene" is meant any piece of a nucleic acid molecule
(for example, DNA) which is inserted by artifice into a cell, and
becomes part of an organism (or a descendant thereof) by being
integrated into the genome or maintained extrachromosomally which
develops from that cell. Such a transgene may include a gene which
is partly or entirely heterologous (i.e., foreign) to the
transgenic organism, or may represent a gene homologous to an
endogenous gene of the organism.
[0054] By "transgenic plant" is meant a plant containing a
transgene. Those in the art will recognize that, once a transgenic
plant has been produced, it may be propagated sexually or
asexually; if a descendant contains a transgene, it is considered
to be a transgenic plant.
[0055] By "protein" is meant any combination of two or more
covalently-bonded amino acids, regardless of post-translational
modifications.
[0056] By "PR-5" is meant a protein that is substantially identical
to VVTL-1 (SP accession no. 004708) and, when overexpressed in a
grape plant, confers on the plant increased pathogen
resistance.
[0057] Sequence identity is typically measured using sequence
analysis software with the default parameters specified therein
(e.g., Sequence Analysis Software Package of the Genetics Computer
Group, University of Wisconsin Biotechnology Center, 1710
University Avenue, Madison, Wis. 53705). This software program
matches similar sequences by assigning degrees of homology to
various substitutions, deletions, and other modifications.
Conservative substitutions typically include substitutions within
the following groups: glycine, alanine, valine, isoleucine,
leucine; aspartic acid, glutamic acid, asparagine, glutamine;
serine, threonine; lysine, arginine; and phenylalanine,
tyrosine.
[0058] By "high stringency conditions" is meant hybridization in
2.times.SSC at 40.degree. C. with a DNA probe length of at least 40
nucleotides. For other definitions of high stringency conditions,
see F. Ausubel et al., Current Protocols in Molecular Biology, pp.
6.3.1-6.3.6, John Wiley & Sons, New York, N.Y., 1994, hereby
incorporated by reference.
[0059] By "substantially pure polypeptide" is meant a polypeptide
that has been separated from the components that naturally
accompany it. Typically, the polypeptide is substantially pure when
it is at least 60%, by weight, free from the proteins and
naturally-occurring organic molecules with which it is naturally
associated. Preferably, the polypeptide is at least 75%, more
preferably at least 90%, and most preferably at least 99%, by
weight, pure. A substantially pure polypeptide may be obtained, for
example, by extraction from a natural source, by expression of a
recombinant nucleic acid encoding the polypeptide, or by chemically
synthesizing the protein. Purity can be measured and further
enhanced by any appropriate method, e.g., by column chromatography,
polyacrylamide gel electrophoresis, or HPLC analysis.
[0060] Methods of measuring protein amounts are known in the art.
Any of these methods is useful for quantitating the level of total
protein or of a specific protein. For example, proteins can be
separated by polyacrylamide gel electrophoresis and individual
proteins quantitated using densitometry.
[0061] A polypeptide is substantially free of naturally associated
components when it is separated from those contaminants that
accompany it in its natural state. Thus, a polypeptide which is
chemically synthesized or produced in a cellular system different
from the cell from which it naturally originates will be
substantially free from its naturally associated components.
Accordingly, substantially pure polypeptides include those which
naturally occur in eukaryotic organisms but are synthesized in E.
coli or other prokaryotes.
[0062] By "substantially pure nucleic acid" is meant nucleic acid
that is free of the genes which, in the naturally-occurring genome
of the organism from which the nucleic acid of the invention is
derived, flank the nucleic acid. The term therefore includes, for
example, a recombinant nucleic acid that is incorporated into a
vector; into an autonomously replicating plasmid or virus; into the
genomic nucleic acid of a prokaryote or a eukaryote cell; or that
exists as a separate molecule (e.g., a cDNA or a genomic or cDNA
fragment produced by PCR or restriction endonuclease digestion)
independent of other sequences. It also includes a recombinant
nucleic acid that is part of a hybrid gene encoding additional
polypeptide sequence.
[0063] The invention features plants that are resistant to
pathogens and method for their production. Other features and
advantages of the invention will be apparent from the following
detailed description, and from the claims.
DESCRIPTION OF THE DRAWINGS
[0064] FIG. 1A is a photograph of a plant culture plate showing the
embryogenic mass of `Chardonnay` obtained from a liquid culture
medium. This photograph was taken approximately ten weeks after the
initiation of a liquid cell culture.
[0065] FIG. 1B is a photograph of a plant culture plate showing
cotyledonary stage somatic embryos. This photograph was taken
approximately twelve weeks after the initiation of embryo
development.
[0066] FIG. 1C is a photograph of a plant culture plate showing
mature somatic embryos starting to precociously germinate in liquid
culture. Note the elongation of roots in many embryos. This
photograph was taken approximately twenty weeks after the
initiation of embryo development.
[0067] FIG. 1D is a photograph of a plant culture plate showing
initial stages of somatic embryo differentiation in a solid medium
after five weeks of culturing. These somatic embryos were obtained
from embryogenic cell masses that were cultured in a liquid medium
(from FIG. 1A).
[0068] FIG. 1E is a photograph of a plant culture plate showing the
early stages of somatic embryo development in a solid medium. Very
early somatic embryos are hyaline and start turning opaque after a
few days.
[0069] FIG. 1F is a photograph of a plant culture plate showing
mature somatic embryos germinating on a MS basal medium with 3%
sucrose.
[0070] FIG. 2 shows growth of resistant proembryogenic masses in
suspension culture after 4 cycles (10 days each) of in vitro
selection with medium containing 40% Elsinoe ampelina culture
filtrate.
[0071] FIGS. 3A and 3B are photographs showing inhibition of
mycelial growth in dual culture by in vitro selected line. In vitro
selected (left) and non-selected (right) PEMs from suspension were
cultured in semisolid medium for 6 weeks and a small mycelial plug
(5 mm diameter) was placed at the center after 6 weeks. Photograph
taken 10 days after fungal inoculation. (FIG. 3A) Elsinoe ampelina,
(FIG. 3B) Fusarium oxysporium isolated from watermelon.
[0072] FIG. 4 is a photograph showing mycelial growth inhibition of
E. ampelina in conditioned medium assay. Spent liquid medium, after
growing in vitro selected (RC1 and RC2) and non-selected (C) PEMs
in suspension, was solidified on glass cover slips, with potato
dextrose agar to give a final strength of 0.75N. Mycelial plug of
Elsinoe ampelina was inoculated and allowed to grow on the plates.
Photographed two weeks after fungal inoculation.
[0073] FIGS. 5A-5C are photographs of SDS-PAGE of extracellular
proteins precipitated from spent liquid medium after growing
selected and non-selected PEMs (FIG. 5A), somatic embryos (FIG.
5B), and inter-cellular washing fluids (ICWF) (FIG. 5C) in
suspension culture. The gels were silver stained. Lanes (S)
molecular weight markers, (C) non-selected control, (1) resistant
line RC1, (2) resistant line RC2.
[0074] FIGS. 6A-6C show chitinase activity in the extracellular
proteins precipitated from spent liquid medium after growing
selected and non-selected PEMs in suspension culture. Chitinase
activity was detected after native PAGE (FIG. 6A) or SDS-PAGE (FIG.
6B) using a glycol chitin assay. FIG. 6C: Immunodetection of
chitinase activity after SDS-PAGE in the extracellular proteins
using barley chitinase antiserum. Lanes (c) non-selected control,
(s) Chitinase standard from Serratia marcescens (Sigma, St. Louis,
Mo.) (1) resistant line RC1 (2) resistant line RC2.
[0075] FIG. 7 shows mature somatic embryos of selected and
non-selected cultures growing in solid medium containing 40% (v/v)
Elsinoe ampelina culture filtrate. (Left) Somatic embryos of RI in
regular embryogenesis medium, (Center) Somatic embryos of RC1 in
embryogenesis medium containing 40% (v/v) culture filtrate, (Right)
Somatic embryos of non-selected control in embryogenesis medium
containing 40% (v/v) culture filtrate.
[0076] FIG. 8 shows greenhouse grown, somatic embryo derived plants
of selected and non-selected cultures were sprayed with Elsinoe
ampelina spore suspension (1.times.10.sup.6 spores per ml). Plants
from non-selected somatic embryos exhibited anthracnose symptoms 4
days after inoculation (inset), while the in vitro selected plant
did not show any symptom.
[0077] FIG. 9 is a schematic illustration showing the nucleotide
and amino acid sequence of the 33 kDa protein.
[0078] FIGS. 10A-10C are a series of photographs showing
immuno-detection of a 22 kDa protein with PR-5 antiserum.
Extracellular proteins (ECPs) from PEMs and heart stage somatic
embryos were separated by SDS-PAGE on a 12% mini-gel and
transferred to a ImmunBlot.TM. membrane and detected with PR-5
antiserum from `Pinto bean`. Proteins from PEMs are shown in FIG.
10A, heart stage somatic embryos in FIG. 10B, and ICWF of
regenerated plants in FIG. 10C. Lanes: C--non-selected control,
RC1--in vitro selected line 1, and RC2--in vitro selected line
2.
[0079] FIG. 11 is a schematic illustration showing a comparison of
the amino terminal amino acid residues of a 9 kDa protein from ECP
of heart stage somatic embryos. This protein had high homology with
several nsLTP: Vitis nsLTP P4 (SP accession no. P80274), Vitis
nsLTP (Salzman et al., Plant Physiol 117:465-472, 1998), Sorghum
nsLTP (SP accession no. Q43194), rice nsLTP (SP accession no.
P23096). X denotes unidentified amino acid residue.
[0080] FIG. 12 is a schematic illustration showing a comparison of
the amino terminal amino acid residues of a 22 kDa protein from ECP
of heart stage somatic embryos. This protein had high homology with
several TLPs: Vvtl 1 (SP accession no. 004708), Tobacco TLP--E22
(accession no. P13046), Tobacco TLP--E2 (accession no. P07052),
Vvosm (accession no. Y10992), and grape osmotin (GO; Salzman et
al., supra). X denotes unidentified amino acid residue.
[0081] FIG. 13 is a schematic illustration showing a comparison of
the amino terminal amino acid residues of the two .about.22 kDa
proteins from regenerated, in vitro selected plants. X denotes
unidentified amino acid residue.
DETAILED DESCRIPTION OF THE INVENTION
[0082] We have developed a method for growing perennial grape
embryogenic cultures that is useful for the regeneration of grape
plants. The unique germplasm resulting from our culture system has
been observed to produce grape plants with an enhanced ability to
recreate embryogenic cultures. Furthermore, we have developed a
process for growing large quantities of somatic grape embryos from
such perennial embryogenic cultures in a relatively short period
using a liquid suspension culture. The culture method is useful,
for example, for selecting somatic grape embryos capable of
surviving in the presence of a pathogen. We have discovered that
plants derived from these somatic embryos are also more resistant
to pathogens. The plants of the invention are likely to have
resistance to many pathogens. The "Compendium of Grape Diseases"
(APS Press (1988) R. C. Pearson & A. C. Goheen, Eds.; hereby
incorporated by reference) describes a wide variety of grape plant
diseases and the pathogens that cause them. These include, without
limitation, botrytis bunch rot and blight (Botrytis cinerea); black
rot (Guignardia bodwelli); phomopsis cane and leaf spot (Phomopsis
viticola); anthracnose (Elsinoe ampelina); bitter rot (Greeneria
uvicola); white rot (Coniella diplodiella); ripe rot
(Colletotrichum gloeosporioides); macrophoma rot (Botryosphavria
dothidea); angular leaf spot (Mycosphaerella nagulata); diplodia
cane dieback and bunch rot (Diplodia natelensis); rust (Physopella
ampelopsidis); leaf blight (Pseudocerospora vitis); leaf blotch
(Brioisia ampelaphaga); zonate leaf spot (Cristulariella moricola);
septoria leaf spot (Septoria spp.); eutypa dieback (Eutypa lata);
black dead arm (Botryosphaeria steuensil); phymatotrichum root rot
(Phymatotrichum omnivorum); verticillium wilt (Verticillium
dahliae); dematophora root rot; (Dematophora necatrix);
phytophthora crown and root rot (Phytophthora spp.); crown gall
(Agrobacterium spp.); bacteria blight (Xanthomas ampelina);
Pierce's disease (Xylella fastidiosa); flavescence doree; and bois
noir and vergilbungskrankheit, and other grapevine yellows.
[0083] The regeneration methods described herein have been used for
the successful regeneration by somatic embryogenesis of a variety
of grapevine rootstock and scion cultivars, including Autumn
Seedless, Blanc du Bois, Cabernet Franc, Cabernet Sauvignon,
Chardonnay (e.g., CH 01 and CH 02), Dolcetto, Merlot, Pinot Noir
(e.g., PN and PN Dijon), Semillon, White Riesling, Lambrusco,
Stover, Thompson Seedless, Niagrara Seedless, Seval Blanc,
Zinfindel, Vitis rupestris St. George, Vitis rotundifolia Carlos,
Vitis rotundifolia Dixie, Vitis rotundifolia Fry, and Vitis
rotundifolia Welder. The methods of the invention are generally
applicable for a variety of grape plants (for example, Vitis spp.,
Vitis spp. hybrids, and all members of the subgenera Euvitis and
Muscadinia), including scion or rootstock cultivars. Exemplary
scion cultivars include, without limitation, those which are
referred to as table or raisin grapes Alden, Almeria, Anab-E-Shahi,
Autumn Black, Beauty Seedless, Black Corinth, Black Damascus, Black
Malvoisie, Black Prince, Blackrose, Bronx Seedless, Burgrave,
Calmeria, Campbell Early, Canner, Cardinal, Catawba, Christmas,
Concord, Dattier, Delight, Diamond, Dizmar, Duchess, Early Muscat,
Emerald Seedless, Emperor, Exotic, Ferdinand de Lesseps, Fiesta,
Flame seedless, Flame Tokay, Gasconade, Gold, Himrod, Hunisa,
Hussiene, Isabella, Italia, July Muscat, Khandahar, Katta,
Kourgane, Kishmishi, Loose Perlette, Malaga, Monukka, Muscat of
Alexandria, Muscat Flame, Muscat Hamburg, New York Muscat, Niabell,
Niagara, Olivette blanche, Ontario, Pierce, Queen, Red Malaga,
Ribier, Rish Baba, Romulus, Ruby Seedless, Schuyler, Seneca, Suavis
(IP 365), Thompson seedless, and Thomuscat. They also include those
used in wine production, such as Aleatico, Alicante Bouschet,
Aligote, Alvarelhao, Aramon, Baco blanc (22A), Burger, Cabernet
franc, Cabernet, Sauvignon, Calzin, Carignane, Charbono, Chardonnay
(e.g., CH 01, CH 02, CH Dijon), Chasselas dore, Chenin blanc,
Clairette blanche, Early Burgundy, Emerald Riesling, Feher Szagos,
Fernao Pires, Flora, French Colombard, Fresia, Furmint, Gamay,
Gewurztraminer, Grand noir, Gray Riesling, Green Hungarian, Green
Veltliner, Grenache, Grillo, Helena, Inzolia, Lagrein, Lambrusco de
Salamino, Malbec, Malvasia bianca, Mataro, Melon, Merlot, Meunier,
Mission, Montua de Pilas, Muscadelle du Bordelais, Muscat blanc,
Muscat Ottonel, Muscat Saint-Vallier, Nebbiolo, Nebbiolo fino,
Nebbiolo Lampia, Orange Muscat, Palomino, Pedro Ximenes, Petit
Bouschet, Petite Sirah, Peverella, Pinot noir, Pinot Saint-George,
Primitivo di Gioa, Red Veltliner, Refosco, Rkatsiteli, Royalty,
Rubired, Ruby Cabernet, Saint-Emilion, Saint Macaire, Salvador,
Sangiovese, Sauvignon blanc, Sauvignon gris, Sauvignon vert,
Scarlet, Seibel 5279, Seibel 9110, Seibel 13053, Semillon, Servant,
Shiraz, Souzao, Sultana Crimson, Sylvaner, Tannat, Teroldico, Tinta
Madeira, Tinto cao, Touriga, Traminer, Trebbiano Toscano,
Trousseau, Valdepenas, Viognier, Walschriesling, White Riesling,
and Zinfandel. Rootstock cultivars include Couderc 1202, Couderc
1613, Couderc 1616, Couderc 3309 (Vitis riparia X rupestris), Dog
Ridge, Foex 33 EM, Freedom, Ganzin 1 (A.times.R #1), Harmony, Kober
5BB, LN33, Millardet & de Grasset 41B (Vitis vinifera X
berlandieri), Millardet & de Grasset 420A, Millardet & de
Grasset 101-14 (Vitis riparia X rupestris), Oppenheim 4 (SO4),
Paulsen 775, Paulsen 1045, Paulsen 1103, Richter 99, Richter 110,
Riparia Gloire, Ruggeri 225, Saint-George, Salt Creek, Teleki 5A,
Vitis rupestris Constantia, Vitis california, and Vitis girdiana,
Vitis rotundifolia, Vitis rotundifolia Carlos, Teleki 5C (Vitis
berlandieri X riparia), 5BB Teleki (selection Kober, Vitis
berlandieri X riparia), SO.sub.4 (Vitis berlandieri X rupestris),
and 039-16 (Vitis vinifera X Muscadinia).
[0084] Using plant tissue culture methods described herein, we have
also developed in vitro selection methods which enable those
skilled in the art to develop pathogen-resistant grapevines. One
such application is the selection of mutations in grape cell
cultures. In this application, cells that are resistant or
susceptible to a particular condition are selected based on
increased or selective growth. The cells can further be exposed to
a mutagen that results in changes in the DNA of the exposed cells.
The mutagenized DNA can then be identified using standard
techniques.
[0085] A second, related application is the selection of
pathogen-resistant cells. Cells are cultured in the presence of a
phytotoxin from a plant pathogen. Cells that show resistance can
then be used to regenerate a pathogen-resistant plant.
[0086] A third application is the transfer of genetic information
into grape cells. The genetic information can include nucleic acid
sequence encoding a selectable marker. Culturing cells in the
presence of the selective pressure (e.g., in the presence of
filtrate from a culture of E. ampelina at a concentration that
kills cells not expressing a nucleic acid of the invention, such as
SEQ ID NO: 6, but does not kill cells that are expressing the
nucleic acid) results in the proliferation or survival only of the
cells that have the desired genetic information. Those in the art
will recognize that determination of the concentration of filtrate
or related compounds may be determined by performing a standard
dose-response assay.
[0087] There now follows a description for each of the
aforementioned methods. These examples are provided for the purpose
of illustrating the invention, and should not be construed as
limiting.
EXAMPLE 1
Perennial Grape Embryonic Culture System
[0088] The following method has proven effective for the production
of perennial embryogenic grape cultures, and for the regeneration
of grapevine by somatic embryogenesis.
[0089] Explant Tissue and Culture Initiation
[0090] In the culture initiation step, explant material was
collected from the field, greenhouse, or in vitro shoot
micropropagation cultures of grapevine and placed into in vitro
culture. This explant material was typically collected from leaves,
anthers, or tendrils, but is also obtained from other vegetative or
reproductive tissues of grapevine. Once collected, the explant
tissue, if desired, was surfaced sterilized according to standard
methods, and then placed on a suitable solid culture initiation
medium in a petri plate.
[0091] Any of a number of well known media, e.g., Murashige and
Skoog (MS) and Nitsch's medium, may be used. Such media typically
include inorganic salts, vitamins, micronutrients, a nitrogen
source, and a carbon source such as sucrose, maltose, glucose,
glycerol, inositol, and the like. For example, sucrose may be added
at a concentration of between about 1 g/L to about 200 g/L; and
preferably at a concentration of between about 30 g/L to about 90
g/L. Moreover, the composition of such plant tissue culture media
may be modified to optimize the growth of the particular plant cell
employed. For example, the culture initiation medium may be
prepared from any of the basal media found Table 1. TABLE-US-00001
TABLE 1 COMPOSITION OF MEDIA COMMONLY USED IN THE EXAMPLES
Component (mg/L unless otherwise specified) MS Modified MS Nitsch
KNO.sub.3 1900.0 3033.3 950.0 NH.sub.4NO.sub.3 1650.0 -- 720.0
NH.sub.4Cl -- 363.7 -- MgSO.sub.4.7H.sub.2O 370.0 370.0 185.0
CaCl.sub.2 440.0 440.0 166.0 KH.sub.2PO.sub.4 170.0 170.0 68.0
Na.sub.2EDTA 37.23 37.23 37.3 FeSO.sub.4.7H.sub.2O 27.95 27.95
27.95 MnSO.sub.4.H.sub.2O 16.9 16.9 18.9 KI 0.83 0.83 --
H.sub.3BO.sub.3 6.2 6.2 10.0 ZnSO.sub.4.7H.sub.2O 8.6 8.6 10.0
Na.sub.2MoO.sub.4.2H.sub.2O 0.25 0.25 0.25 CuSO.sub.4.5H.sub.2O
0.025 0.025 0.025 CoCl.sub.2.6H.sub.2O 0.025 0.025 0.025 Glycine
2.0 2.0 -- Nicotinic acid 0.5 0.5 1.0 Pyridoxin HCl 0.5 0.5 1.0
Thiamine HCl 0.1 0.1 1.0 Inositol 0.1 g/L 1.0 g/L 0.1 g/L Sucrose
30.0 g/L; 30.0 g/L; 60.0 g/L; 20.0 g/L 60.0 g/L 90.0 g/L Activated
Charcoal -- 0.5 g/L; 1.0 g/L; -- 2.0 g/L Agar 7.0 g/L 7.0 gm 8.0
g/L pH 5.5 5.5 5.5
If desired, the initiation medium may contain an auxin or a mixture
of auxins at a concentration of about 0.01 mg/L to about 100 mg/L,
depending on the cultivar of interest, which is effective for
inducing the production of embryogenic cells or embryogenic cell
masses on the explant tissue. For example, explant tissue can be
maintained on an agar-solidified Nitsch's-type medium supplemented
with, for example, between about 0.01 mg/L and about 10 mg/L of
2,4-D, and preferably between about 0.5 mg/L and about 3.0 mg/L of
2,4-D. 2,4-D is just one example of an auxin which is useful in the
methods of the invention. Other auxins include, for example, NAA,
NOA, IAA, dicamba, and picloram. Additionally, if desired, other
plant growth regulators may be included in the medium at standard
concentrations. For example, cytokinins (e.g., a
naturally-occurring or synthetic cytokinin, such as BA or zeatin),
if present, may be used at a concentration of from about 0.01 mg/L
to about 10 mg/L, and preferably about 0.3 mg/L, depending on the
cultivar of interest. In some instances, other classes of growth
regulators, such as ABA or GA, may be included at appropriate
standard concentrations. For example, ABA may be added at a
concentration of about 0.5 mg/L to about 20 mg/L, and preferably at
a concentration of about 5 mg/L; and GA may be added at a
concentration of about 0.1 mg/L to about 30 mg/L, and preferably at
a concentration of about 5 mg/L. The addition of plant growth
regulators at this stage is not necessary for the induction of
embryogenesis. Additionally, the initiation medium may also include
activated charcoal (0.1-2.0 g/L) or a similar adsorbent known to
those in the art.
[0092] Culturing of explant tissue during this stage is preferably
carried out in the dark at 22-30.degree. C., although it may also
be carried out under very low light conditions, or in full light.
After approximately one to four weeks in culture, explant tissue
cultures are then placed in full light with a 16 hr photoperiod.
Cultures are scanned weekly for the presence of emerging
embryogenic cells or embryogenic cell masses. Embryogenic cells or
cell masses are identified based on morphology. Embryogenic cell
masses, in general, tend to be white to pale yellow in color, and
are often hyaline. They may be recognized from a very early, small
stage (10-20 cell aggregates), based upon their color and friable,
granular appearance. Embryogenic cultures are also identified by
their compact nature with cells that are rich in cytoplasm (as seen
under the microscope). The embryogenic cultures appear at varying
frequencies depending on a multitude of factors including, but not
limited to, genotype, nature and type of explant, medium
composition, and season of harvest. Careful visual selection to
ensure transfer of appropriate embryo-like structures is required
for culture maintenance. Once identified, embryogenic cells or cell
masses are then transferred to culture maintenance medium, as
described herein.
[0093] Culture Maintenance
[0094] Embryogenic cells or embryogenic cell masses are carefully
removed and transferred to a culture maintenance medium. Again, any
of a number of well known media, e.g., MS and Nitsch's medium, may
be used. Although not generally required, plant growth regulators
may be added as described above.
[0095] In general, embryogenic tissue can be maintained by
subculturing at regular intervals (e.g., every one to four weeks,
or every four to eight weeks) to new maintenance medium, as
described herein. Alternatively, embryogenic tissue can be placed
in a liquid culture medium (e.g., MS, B-5, or Nitsch) and grown as
a liquid embryogenic suspension as described herein. Embryogenic
cell masses are grown to increase embryogenic cell biomass as
required by division of expanding cultures during transfer. The
cultures can be prompted to develop toward increasing embryogenesis
or toward less embryogenesis and more unorganized embryogenic cell
growth by repeated manipulation of the culture, which includes
careful selection of embryogenic cells and cell masses during
transfer. Repeated transfer of embyogenic cells or cell masses has
not only been found to enrich the growth of embryogenic tissue, but
also to facilitate the process of somatic embryogensis. The
cultures are perennial in that they typically persist for over two
years.
[0096] A key component of the present approach involves the careful
selection of embryogenic cells from explanted tissue, followed by
recurrent selection and subculturing of the selected embryogenic
tissue. This material has not only been found to be useful in the
regeneration of whole grape plants from somatic embryos, but has
also been found to have a significantly increased capacity for
embryogenesis, including the production of somatic embryos. By
carrying out this procedure, the growth of embryogenic cells is
enriched, speeding the process of somatic embryo formation and
subsequent plant regeneration.
[0097] The explant material taken from plants that were grown from
somatic embryos was observed to exhibit an enhanced embryogenic
potential, when compared to explant material taken from clonal
explant tissue which had not been cultured for the production of
embryogenic cells. This increase in embryogenic potential was
observed to increase after two or more successive initiation,
culture and plant regeneration cycles (e.g., clonal
plant-->explant-->embryogenic culture initiation-->somatic
embryo-->somatic embryo-derived plant-->explant). It is not
necessary to use a somatic embryo-derived plant as the source of
the explant; somatic embryos or even embryogenic cultures that have
been transferred to new medium will also produce new somatic
embryos with increased embryogenic potential. Such explant material
is conveniently maintained as in vitro axillary shoot cultures,
which serves as the source for vegetative explants; however, other
methods of plant maintenance are also acceptable.
[0098] Germination and Plantlet Growth
[0099] Somatic embryos obtained from the above-described cultures
are subsequently germinated into grape plantlets according to
standard methods. For example, somatic embryos are placed on the
surface of a germination medium (e.g., MS medium) in sterile petri
plates. The cultures containing the embryos are incubated in a
growth chamber under lighted conditions (16 hr photoperiod). During
germination the root emerges and the epicotyl begins to grow. When
grape plantlets that are grown on germination medium reach
sufficient size (1 cm, with at least two leaves), they can be
removed from the culture dishes and planted in a sterilized potting
mixture. Plantlets are typically transferred into nursery
containers in a soiless potting mix (e.g., Vermiculite, Perlite, or
ProMix.TM., V.J. Growers, Apopka, Fla.). If desired, plantlets can
be placed in a growth chamber or in a greenhouse moisture chamber
and incubated under high humidity conditions (90% humidity) for
plantlet growth and acclimatization. Subsequently, acclimatized
plantlets can be transferred outdoors to a vineyard or to a
greenhouse.
[0100] In one example, we describe the production of an embryogenic
perennial culture of Vitis vinifera cv. `Thompson Seedless.` Mother
plant-derived cultures were obtained from a leaf that was
surface-disinfected and inoculated onto culture initiation medium
described by Nitsch (1968) and modified by Gray D. J. ("Somatic
Embryogenesis in Grape." In: Somatic embryogenesis in woody
perennials, Vol. 2, Gupta P. K., Jain S. M., and Newton R. J.
(Eds.), Kluwer Academic, Dordrecht, The Netherlands, pp. 191-217,
1995). This medium contained about 1.1 mg/L of 2,4-D and about 0.05
mg/L of BA. After explanting the tissue, the culture vessels were
incubated in complete darkness for six weeks. Most of the explanted
tissue was observed to form a mass of undifferentiated, highly
vacuolated cells within this six week period. Embryogenic cultures,
identified by their compact nature and the presence of cells that
were rich in cytoplasm, were repeatedly subcultured. The resulting
culture was obtained by the selection method described above,
followed by subculturing (for about 6 weeks) until enough
embryogenic culture was available. A somatic embryo originally
obtained from the mother plant (i.e., 1.sup.st generation embryo)
was germinated, and its shoot tip used to create an in vitro
micropropagation culture. Leaves from the plant derived from that
culture were then used to produce a new embryogenic culture
(2.sup.nd generation). A somatic embryo obtained from that culture
was similarly used to create the third generation. The embryogenic
response of Vitis vinifera cv. `Thompson Seedless` from in vitro
micropropagation culture-derived leaves is presented in Table 2.
These results show the comparison of leaves from potted mother
plant-derived cultures with leaves from plants derived from
cultures obtained from third-generation germinated somatic embryos.
TABLE-US-00002 TABLE 2 Culture No. leaves No. embryogenic %
response derivation cultured cultures per leaf Mother plant 195
0.sup.* 0 3.sup.rd generation somatic 200 14 7.sup.# embryo
.sup.*Other experiments have yielded one embryogenic culture.
.sup.#The percent response per leaf has been found to be has high
as 30%.
[0101] In addition, perennial embryogenic cultures from other
grapevines have also been produced using the methods described
herein, including Vitis longii, Vitis rotundifolia (cv. Carlo and
Dixie), Vitis rupestris, Vitis vinifera (cv. Autumn Seedless,
Cabernet Sauvignon, Cabernet Franc, Chardonnay, Dolcetto, Gamay
Beaujolais, Lambrusco, Pinot Noir, Semillon, Tokay, White Riesling,
Zinfindel, and the like), and several Vitis hybrids (cv. Blanc du
Bois, Niagara Seedless, Seyval Blanc, Stover, Southern Home and the
like).
EXAMPLE 2
Production of Highly Embryogenic Grape Cells Using Liquid
Suspension or Solid Cultures
[0102] A method has also been developed for the production of large
quantities of grapevine somatic embryos using either a liquid cell
suspension culture or a solid culture system. These methods are
particulary useful for producing highly embryogenic cells that are
capable of regenerating into whole plants. Below, a simple protocol
for efficient somatic embryogenesis of grapevine using either a
liquid cell suspension culture or a solid culture system is
presented.
[0103] In general, the method includes a multistage culturing
process typically involving (i) culture initiation; (ii)
identification and isolation of embryogenic cells or embryogenic
cell masses; (iii) production of perennial embryogenic cultures;
and (iv) concentration of highly embryogenic cell clusters. The
method involves the following steps.
[0104] Explant tissue is placed on a suitable culture initiation
medium, as is described herein. After approximately six weeks on
culture initiation medium, embryogenic cells and embryogenic cell
masses are identified. Once identified, embryogenic cultures, which
may be less than 1 mm in diameter, are isolated and cultured on
fresh initiation medium to encourage growth, as described herein.
Subculturing of the embryogenic cultures typically results in the
formation of somatic embryos. Embryogenic cultures and early stage
somatic embryos obtained from these cultures are then further
cultured in a suitable liquid plant growth medium. For example, the
plant tissue culture nutrient media, consisting B-5 medium (Gamborg
et al., Exp. Cell. Res. 50:151-158, 1968; Sigma Chemicals, St.
Louis, Mo.), that has been modified as described by DeWald et al.
(J. Amer. Soc. Hort. Sci. 114:712-716, 1989) and Litz et al.
("Somatic embryogenesis in mango," 1995, supra). This modified
medium consists of B-5 major salts, MS minor salts and vitamins,
glutamine (about 400 mg/L), and sucrose or commercial table sugar
(about 60 g/L). Before autoclaving, the pH of the medium is
adjusted to about 5.8. Although 2,4-D (about 0.5-2.0 mg/L) is the
preferred growth regulator used in this medium, other growth
regulators, such as, for example, dicamba, picloram, NOA, or
2,4,5-trichlorophenoxy acetic acid, may be also used at appropriate
concentrations, for example, those described above. Flasks
containing the embryogenic cell cultures, somatic embryos, or both
are subsequently incubated at about 26.degree. C. on a rotary
shaker at 125 rpm in darkness or diffuse light. The cultures are
then subcultured as described herein, typically once every ten to
fourteen days, but subculturing regimens may vary depending on the
growth and proliferation of embryogenic cell clusters.
[0105] In about six to eight weeks, a fine cell suspension culture
is produced, which consists of highly-vacuolated elongated cells
(non-embryogenic cells), and also a lesser number of small,
cytoplasm-rich, isodiametric cells (embryogenic cells). Once
sufficient culture is produced, the differentiated embryos can be
removed from the culture by sieving, and the differentiated embryos
are discarded. Continued maintenance of the sieved embryogenic cell
suspension culture in modified B-5 liquid medium, with periodic
subcultures, has been found to increase the biomass of embryogenic
cell clusters.
[0106] After approximately twelve to sixteen weeks, a large mass of
highly concentrated embryogenic grape cells is typically observed.
The time taken for the concentration of embryogenic cells or
embryogenic cell masses may vary depending on several factors,
including the cultivar, genotype, and culture conditions.
Embryogenic cells at this stage are especially useful in virtually
any type of genetic transformation method. These embryogenic cells
can also be induced to differentiate into somatic embryos according
to any standard method, e.g., by culturing the cells in modified
B-5 liquid medium devoid of growth regulators for a period of about
four to six weeks. Alternatively, the early stage somatic embryos
may be plated in medium solidified by the addition of a suitable
gelling agent such as gellan gum, agarose, agar, or any other
similar agent, for further differentiation of somatic embryos in
complete darkness. If desired, torpedo/cotyledonary-stage embryos
can be individually subcultured on a standard maturation medium,
e.g., a maturation medium consisting of MS nutrient formulations.
Mature somatic embryos are then transferred to a growth chamber for
germination, and regeneration to plants in an appropriate
container. The frequency of somatic embryo formation using this
procedure is typically high.
[0107] There now follows a description of the results for the
production of embryogenic cells and cell masses obtained from a
liquid suspension and solid cultures of `Thompson Seedless` and two
different clones of `Chardonnay,` CH 01 and CH 02.
[0108] Asynchronous somatic embryos of Vitis vinifera cv. `Thompson
Seedless` and `Chardonnay` CH 01 and CH 02 obtained from perennial
embryogenic tissue were further cultured in a liquid medium to
produce a callus tissue suspension culture. After about fourteen
days and following about two to three subcultures (a subculture was
performed about every fourteen days), an amorphous, yellowish to
creamy white colored callus was produced. As a result of the
production of callus tissue, the liquid culture media in the tissue
culture flask appeared as a dense suspension. Microscopic
examination revealed that the callus cells were elongated and
highly vacuolated, and exhibited no signs of embryogenic capacity.
Amorphous callus continued to proliferate, even when the somatic
embryos used to initiate the culture were removed from the
culture.
[0109] After approximately six weeks in modified B-5 liquid medium,
we observed the production of small clusters of cytoplasm-rich
cells as white clumps (FIG. 1A). These embryogenic masses were
observed to proliferate exponentially, and grew to the capacity of
the flask in about ten to twelve weeks.
[0110] Continued maintenance of these embryogenic masses as a
single unit (i.e., in one flask) is often detrimental, as the
cultures have been found to deteriorate in quality, and eventually
turn brown. Dividing these embryogenic cultures into smaller units
during subculturing assists to proliferate and increase the biomass
of the divided cultures. Among the two cultivars tested, both
clones of `Chardonnay` were found to be equally fast growing and
outgrew `Thompson Seedless.` While the embryogenic masses of
`Chardonnay` were creamy white to yellowish in color, those of
`Thompson Seedless` were dull white or brownish. In addition,
`Thompson Seedless` appeared to be more sensitive to culture
density, as the cells were observed to turn dark if the culture
density was not corrected. The preferred culture density was
approximately 400 mg of embryogenic cells per 40 mL of liquid
modified B5 medium in a 125 mL flask.
[0111] Somatic Embryo Production in Liquid Culture
[0112] Embryogenic masses were passed through a 960 micron nylon
sieve and collected in a sterile beaker. Sieving of the embryogenic
masses to initiate embryogenesis in liquid culture was found to
serve two purposes. First, a fair degree of synchronization of
embryo differentiation was obtained. Second, the formation of
somatic embryo abnormalities during differentiation, such as
fasciation or fusion, was reduced. After four to six weeks in
liquid medium, small, white somatic embryos in the globular or
early heart stage were observed. Sieving the cultures at this stage
did not facilitate an increase in embryo differentiation.
[0113] After approximately eight weeks, somatic embryos were
clearly visible, and a few embryos were found to have reached the
cotyledonary stage of embryo development. Sieving the
differentiated embryos and culturing them in a separate flask,
however, facilitated faster differentiation, as well as
synchronization of embryo devlopment.
[0114] Both `Chardonnay` clones--CH 01 and CH 02--were found to
readily differentiate into somatic embryos. Appropriate sieving and
density adjustment (performed by culturing about 1000 mg of somatic
embryos per 40 mL medium) ensured greater synchronization and
singulation, as well as embryo differentiation (FIG. 1B). In
approximately twelve to fourteen weeks after subculture in liquid
embryogenesis medium, singulated somatic embryos started to turn
green and radicles elongated, showing the onset of precocious
germination (FIG. 1C).
[0115] Cultures of `Thompson Seedless` initially were found not to
advance beyond the heart stage in liquid culture. In addition, the
embryos were found to be more clustered, often resulting in the
formation of fused somatic embryos. Removal of the abnormal embryos
and lowering the culture density by half resulted in normal somatic
embryogenesis in liquid culture. These somatic embryos reached
maturity in about fourteen to eighteen weeks.
[0116] Somatic Embryo Production in Solid Medium
[0117] Embryogenic cells or embryogenic cell masses obtained from
liquid cultures were observed to differentiate into somatic embryos
as early as three weeks after culture initiation. After four weeks
of culture, microscopic examination also revealed the formation of
globular and heart shaped somatic embryos on the callus tissue
(FIG. 1D). The somatic embryos were hyaline, and resembled that of
a hyperhydric state (FIG. 1E); however, the embryos continued to
differentiate, and were found to develop into mature somatic
embryos in another three to four weeks. These somatic embryos were
observed to develop a suspensor (FIG. 1E). In addition, embryogenic
cells were observed to develop into a mass of asynchronous somatic
embryos.
[0118] One of the interesting observations from these experiments
was that the majority of somatic embryos arose as individual units,
and not as small clumps, although there were a few clumps of
somatic embryos. In such cases, the number of somatic embryos
ranged from six to ten in each clump, and these embryos were easily
separated from the callus tissue. Embryos found in the cotyledonary
stage were isolated on a weekly basis, and subcultured for
maturation. Each clump of embryogenic mass continued producing
somatic embryos for at least twelve weeks. Embryogenic cell masses
tended to turn brown in solid medium, containing Gel-Gro (ICN
Biochemicals), but this discoloration did not adversely affect
culture viability. About four or five weeks later, clusters of
somatic embryos started to appear on the surface of the brown
embryogenic cells or embryogenic cell masses.
[0119] Somatic Embryo Maturation, Germination and Plant
Regeneration
[0120] Three maturation media--mango maturation medium (Litz et
al., 1995, supra); mango maturation medium solidified with agar (7
g/L); and MS basal medium with 3% sucrose--were studied to evaluate
the ability to promote somatic embryo germination and plant
regeneration. Our results indicated that a MS basal medium
containing 3% sucrose was the most effective at promoting embryo
maturation, germination, and plant regeneration, for both embryos
derived from solid medium and for the precociously germinated
embryos that were obtained from liquid medium cultures (FIG. 1D).
Although there was good germination on mango maturation medium with
agar, the quality of the regenerants was not as good as with MS
salts with 3% sucrose. Embryos from the two systems studied (i.e.,
liquid and solid media) showed variation between themselves in
germination and regeneration. Although embryos have precociously
germinated in liquid cultures, continued germination in these
cultures was not observed. On transfer to solid medium, however,
the embryos were found to continue the germination process, and
resulted in the formation of grape plants with a dense root system.
Continued maintenance in liquid medium after radicle emergence lead
to hyperhydricity and eventually plant regeneration was reduced
from these embryos. Accordingly, it is preferred that the somatic
embryos should be removed from the liquid as soon as they
precociously germinate and transferred to solid medium.
[0121] Long-term Preservation of Suspension-Derived Grapevine
Somatic Embryos and Regeneration of Plants
[0122] We have established a method for the long-term storage of
somatic embryos. Mature somatic embryos from suspension cultures of
`Chardonnay` were blot-dried on sterile filter paper in a
laminar-flow hood and then stored in sterile petri plates at
6.degree. C. Samples were periodically drawn from these plates and
germinated on MS medium with 3% sucrose. Germination (i.e., the
emergence of roots from the somatic embryo) and plant regeneration
were recorded. Table 3 shows the data from clone CH 02 after 22
months in storage, and Table 4 shows the data from clone CH 01
after 5 months in storage. TABLE-US-00003 TABLE 3 Trial Number
Number Germinated Number Percent Number of Embryos (Percent
Germinated) of Plants Yield 1 87 81 (93.1) 69 79.3 2 42 40(95.2) 35
83.3 3 41 41 (100.0) 30 73.2 Total 170 162 (95.3) 134 78.8
[0123] TABLE-US-00004 TABLE 4 Trial Number Number Germinated Number
Percent Number of Embryos (Percent Germinated) of Plants Yield 1 15
15 (100.0) 13 86.7 2 15 15 (100.0) 13 86.7 3 15 13 (86.7) 9 60.0 4
15 12 (80.0) 7 46.7 5 15 13 (86.7) 9 60.0 6 15 11 (73.3) 9 60.0 7
15 15 (100.0) 14 93.3 8 15 13 (86.7) 9 60.0 Total 120 107 (89.2) 83
69.2
[0124] Direct Seeding of Suspension Culture-Derived Grapevine
Somatic Embryos
[0125] `Chardonnay` and `Thompson Seedless` grapevine somatic
embryos were produced from liquid cultures as described herein.
Suspension-derived, mature somatic embryos were blot dried briefly
in the laminar flow hood and germinated directly in Magenta vessels
containing one of the following potting media: i) sand; ii)
ProMix.TM. commercial potting mixture (CPM); or CPM overlaid with
sand. Each vessel containing 20 mL of distilled water and the
potting medium was sterilized by autoclaving for 30 min and cooled
overnight prior to inoculating the somatic embryos. Three somatic
embryos were placed in each vessel. Seeding was carried out under
aseptic conditions and the containers were closed and incubated at
26.degree. C. with a 16 hr photoperiod at 75 .mu.mol s.sup.-1
m.sup.-2 light intensity. Results revealed that CPM overlaid with
sand was ideal for plant development. Although sand promoted more
germination, the resulting plants were chlorotic and their survival
rate was poor. There was more contamination of somatic embryos on
pure CPM. The present study offers scope for large-scale
multiplication of grapes using suspension cultures and sets the
platform for growing grape somatic embryos in bioreactors.
[0126] The experimental results described above were carried out
using the following techniques.
[0127] Culture Initiation
[0128] Embryogenic cultures were initiated from anthers and ovaries
of the cultivar "Chardonnay" (Clones CH 01 and CH 02), and from the
leaves of the cultivar "Thompson Seedless" according to standard
methods, e.g., those described herein. Somatic embryos of these
cultures, initiated and maintained in modified MS medium, were used
to initiate liquid cell suspension cultures. Typically these
cultures are highly asynchronous in embryonic development and
differentiation and, therefore, each inoculum consisted of somatic
embryos at various stages of development.
[0129] Establishment of Liquid Cultures from Differentiated Somatic
Embryos
[0130] The composition of the liquid medium was adapted from the
medium described by Litz et al., supra as follows. Callus induction
was achieved by the addition of 1 mg/L of 2,4-D in the medium. The
pH of the medium was adjusted to about 5.8, and dispensed as 40 mL
aliquots in 125 mL Erlenmeyer flasks. The flasks were tightly
covered with heavy duty aluminum foil before autoclaving. After
cooling, approximately one gram of the somatic embryos was
transferred to the liquid medium using a sterilized spatula under
aseptic conditions. The neck of the flask was sealed with Parafilm,
and the cultures were then incubated in semi-darkness (diffused
light) on a rotary shaker at about 120 rpm. The cultures were
subcultured at least one time every two weeks.
[0131] Flasks containing the suspension cultures were removed from
the orbital shaker and the cultures were allowed to settle for
about 15 minutes. The supernatant was gently decanted into a
sterile flask, leaving the embryogenic cells in a minimal volume
(approximately 5 mL). Approximately 35 mL of fresh liquid medium
was added to the embryogenic cells and swirled quickly. The entire
contents of the flask were then transferred to a sterile 125 .mu.L
flask. This second flask, containing the embryogenic cells, was
then sealed with Parafilm and returned to the orbital shaker.
[0132] The amorphous callus generated from the somatic embryos was
collected as follows. The embryogenic suspension, including
differentiated somatic embryos and callus, was allowed to settle in
the flasks. About half of the supernatant medium was decanted, and
the remainder was swirled and quickly filtered through a
presterilized, nylon mesh (960 microns), placed over a 150 mL
beaker. While the differentiated somatic embryos were retained in
the mesh, the fine callus that passed through along with the liquid
medium was collected in the beaker. The callus that was collected
in the beaker was next filtered through a sterile, double-folded,
Kimwipe placed over a sterile funnel. The amorphous callus that
adhered to the Kimwipe was subsequently removed from the Kimwipe
using a sterilized spatula, and resuspended in fresh liquid culture
medium. Approximately 100 mg of the callus was suspended in each
flask. These liquid cultures were subcultured as described herein
approximately once every fourteen days in modified B-5 liquid
medium containing 2,4-D.
[0133] Somatic Embryo Production in Suspension Culture
[0134] Embryogenic cells or cell masses that were initiated in
liquid suspension cultures were sieved using a 960 micron sieve,
and the finer fraction was harvested in liquid embryogenesis
medium, under aseptic conditions. The medium composition was the
same as that of the initiation medium; however, 2,4-D was omitted
from the medium and about 0.05 mg/L of BA was added. After
adjusting the pH to 5.8, the medium was dispensed as 40 mL aliquots
in 125 mL Erlenmeyer flasks, covered with aluminum foil and
autoclaved. Approximately 100 mg of callus was cultured in each
flask. The cultures were maintained in semidarkness at 25.degree.
C. on a rotary shaker at 120 rpm, and subcultured once every 14
days. Sieving of cultures was done as necessary, in order to
synchronize differentiated somatic embryos. Finer mesh sieves
(e.g., 520 micron sieves), if necessary, may also be employed.
[0135] Germination of Somatic Embryos from Suspension Cultures and
Regeneration
[0136] Greening somatic embryos having elongated radicles were
sieved from the suspension cultures. Somatic embryos were
individually picked and cultured. Three different media-mango
maturation medium (Litz et al., supra), mango maturation medium
solidified with agar (7 g/L) instead of Gel-Gro, and MS basal
medium with 3% sucrose-were tested for germination and plant
regeneration. Plant growth regulators were omitted from these media
preparations. Twenty-five embryos were cultured in each standard
petri plate, and eight plates of each medium was tested. After
sealing with Parafilm, the cultures were incubated in a growth
chamber under a 16 hour photoperiod. Plantlets with four true
leaves were subsequently transferred to soil.
[0137] Somatic Embryo Production in Solid Medium
[0138] Embryogenic cells and embryogenic cell masses produced in
suspension cultures were harvested as described above and then
transferred to solid embryogenesis medium. The medium consisted of
the same compounds as the liquid embryogenesis medium, and
solidified with 2.0 g/L Gel-Gro or 7 g/L agar. Approximately 50 mg
of callus was placed as a clump onto a medium-containing petri
plate and each plate had two such clumps. After inoculating, the
petri plates were sealed with Parafilm and incubated in complete
darkness. Subculturing was performed after somatic embryo
differentiation was observed. Somatic embryos produced from the
embryogenic cells or embryogenic cell masses were counted on a
weekly basis, starting from six weeks after culture. Embryos of
cotyledonary stage were counted and subcultured for maturation.
[0139] Maturation and Germination of Somatic Embryos from Solid
Medium
[0140] Mature somatic embryos that were approximately 5 mm in
length were isolated from the asynchronous mass and cultured on
maturation medium. Twenty-five mature somatic embryos were cultured
in each standard petri plate on MS medium with 3% sucrose. The
cultures were kept in the dark until they germinated. After
elongation of radicle, they were transferred to light under a 16
hour photoperiod. Plantlets with at least four true leaves were
subsequently transferred to soil.
EXAMPLE 3
Selection of Disease Resistant Embryogenic Cells and Plants of
Grapevine
[0141] The perennial grape embryogenic cultures of the invention
can be used for the selection or screening for grape cells having
resistance to toxic substances, such as those present in a filtrate
produced by a fungal culture. Such pathogens include, without
limitation, bacteria and fungi. Plant diseases generally caused by
these pathogens are described in Chapters 11-16 of Agrios, Plant
Pathology, 3rd ed., Academic Press, Inc., New York, 1988, hereby
incorporated by reference. The "Compendium of Grape Diseases" (APS
Press (1988) R. C. Pearson & A. C. Goheen, Eds.) describes
diseases that affect grape plants. Examples of bacterial pathogens
include, without limitation, Agrobacterium vitis, Agrobacterium
tumefaciens, Xylella fastidosa, and Xanthomonas ampelina. Examples
of fungal pathogens include, without limitation, Plasmopara
viticola, Botrytis cinerea, Guignardia bidwellii, Phomophsis
viticola, Elsinoe ampelina, Eutypa lata, Armillaria mellea, and
Verticllium dahliae. Others are described herein.
[0142] By exposing embryogenic cultures to a phytotoxin (e.g.,
crude culture filtrate or a purified phytotoxin obtained from a
plant pathogen), resistant grape cells can be selected and
propagated. Grape cells that survive the selection pressure are
expected to resist not only the selecting toxin, but also the
original microbe that produces the toxin. Moreover, due to the
dynamics of the selection process, induced resistance may also
function against an array of disease-causing organisms beyond the
original microbe used for selection. Because the selection is
carried out at the cellular level, it is likely that grape plants
regenerated from the cells will show the selected characteristic.
In particular, this system allows one skilled in the art to select
or screen for the desired characteristic from among thousands of
cells in a single culture flask or petri plate.
EXAMPLE 4
Methods for Selecting Pathogen-Resistant Somatic Embryos and
Producing Plants
[0143] Various microbes attack grapevine and cause a number of
diseases. These diseases include fungal diseases of leaves and
fruits (such as black rot and anthracnose), fungal diseases of the
vascular system and roots (such as Esca, Black Measles, Black Dead
Arm, and Eutypa dieback) and bacterial diseases (such as crown gall
and Pierce's disease).
[0144] One disease affecting grapevine is anthracnose, also known
as bird's eye spot disease, which is caused by the fungus, E.
ampelina. Under favorable conditions, this fungus attacks almost
all the aerial parts of the grapevine, including fruits, causing
extensive damage to the crop. Anthracnose causes the appearance of
circular lesions with brown or black margins and round or angular
edges on the grapevine plant. The center of the lesions becomes
grayish white and eventually dries up and falls off, leaving a
`shot-hole` appearance. The disease especially affects young
leaves, preventing normal development. New shoots are also affected
and acquire an obvious, burnt appearance. Fruit clusters are also
susceptible to fungal infection throughout their development;
lesions on the berries extend into the pulp, often inducing
cracking.
[0145] Preparation of Phytotoxin
[0146] An E. ampelina culture filtrate having toxic activity was
prepared as follows. Full-strength Czapekk-Dox broth medium (Fisher
Scientific, Springfield, N.J.) was prepared by dissolving the
required amount of the broth mixture in deionized (DI) water. The
medium was dispensed as 50 mL aliquots in 125 mL Erlenmeyer flasks.
After autoclaving and cooling, 100 .mu.L of a E. ampelina spore
suspension was added to each flask (Day 1); the flask was then
incubated in a rotary shaker at 25.degree. C. at 120 rpm for one
week in the dark. After one week, the contents in each flask were
transferred to 100 mL of full strength Czapek-Dox in a 250 mL
Erlenmeyer flask and the incubation was continued for two more
weeks. At the end of this period (i.e., three weeks from Day 1),
the fungal culture filtrate was collected by filtering the contents
of each flask through a sterile, multi-layer cheese cloth. The
crude culture filtrate was stored at -4.degree. C. until further
use.
[0147] Prior to addition to the culture media for in vitro
selection, the frozen culture filtrate was thawed (without heating)
at room temperature, pH adjusted to 5.8, and filter-sterilized
through a 0.2 micron filter (Nalgene, Rochester, N.Y.). This
filter-sterilized pathogen filtrate was found to retain its toxic
activity, as determined by its ability to cause grape plant cell
death.
[0148] Selection
[0149] The E. ampelina culture filtrate was next added to liquid
suspension cultures of V. vinifera cv. `Chardonnay` embryogenic
cells and embryogenic cell masses in modified B-5 medium to select
cells having resistance to the toxic fungal culture filtrate. The
grape embryogenic cells and embryogenic cell masses were grown as
described above; however, in this in vitro selection, the medium in
which the cells were grown was supplemented with known volumes of
E. ampelina culture filtrate. Appropriate dilutions of pathogen
filtrate were determined by examining the toxicity of the filtrate
using serial dilution analysis. These experiments demonstrated that
a 40% (v/v) culture filtrate was useful for in vitro selection.
[0150] Cultures of `Chardonnay` embryogenic cells and embryogenic
cell masses were maintained in liquid medium containing 40% (v/v)
fungal culture filtrate at about 26.degree. C. on a rotary shaker
(125 rpm) in diffuse light. Subculturing was done once every ten
days; during each subculture, filter-sterilized culture filtrate
was used to dilute the medium. Selection with culture filtrate was
continued for four or five cycles (each cycle=ten days) of
subculture. While most of the embryogenic cells died, a very few
cells, often less than 1%, survived the selection pressure.
Resistant culture lines were established by withdrawing the
selection pressure after four or five cycles and letting the
surviving cells grow in modified B-5 medium devoid of culture
filtrate. These resistant lines were proliferated, and somatic
embryo were produced using the methods described herein.
[0151] Embryogenic cell cultures obtained from the selection
process were subsequently tested for resistance to E. ampelina
using a number of in vitro bioassays. We first analyzed whether the
resistant grapevine lines were producing an activity that could
inhibit the growth of the fungus. To this end, the culture medium
(i.e., conditioned culture medium) from a resistant culture line
was tested for an inhibitory activity against the fungus.
Conditioned media was collected from different cell cultures having
resistance to E. ampelina, and used in several concentrations to
prepare fungal growth media. An actively growing mycelial colony
was placed in the center of a petri plate containing a fungal
growth medium prepared with or without (control) conditioned medium
from a resistant grapevine culture line and incubated under
standard conditions. The results of these experiments showed that
the growth of the fungus was inhibited by a fungal growth medium
containing 25% or more of the conditioned medium.
[0152] To further demonstrate the presence of anti-fungal activity
in resistant grapevine cultures, resistant lines, as well as
control lines, were placed in solid plant growth medium in six and
twelve o'clock positions in petri plates, and incubated in darkness
for four weeks. After this period, a plug of mycelium from an
actively growing E. ampelina colony was placed in the center of the
petri plates and incubated under standard conditions. The fungus
was observed to grow rapidly and infect the control cultures.
Conversely, fungal growth was inhibited on the plates containing
grape cell cultures having resistance to the E. ampelina culture
filtrate. Hyphae did not grow freely through the medium in plates
containing these resistant cultures, as compared to fungal hyphae
growth through the medium in plates containing control (i.e.,
non-resistant) cultures. A thick mat of mycelium, as seen in the
plates containing control cultures, was never formed in the plates
containing the E. ampelina resistant cultures. This capacity of the
resistant cultures to inhibit the growth of E. ampelina was
retained nine months after selection, demonstrating that the
genetic changes in the resistant cultures were stable.
[0153] In addition, the grapevine cultures that were resistant to
E. ampelina were tested for resistance to a second fungal pathogen,
Fusarium (F.) oxysporum. Resistant lines, as well as control lines,
were placed in solid plant growth medium in six and twelve o'clock
positions in petri plates, and incubated in darkness for four
weeks. After this period, a plug of mycelium from an actively
growing F. oxysporum colony was placed in the center of the petri
plates and incubated at room temperature under a 16 hour
photoperiod. The fungus was observed to grow rapidly and infect the
control cultures. Conversely, growth of F. oxysporum was inhibited
on the plates containing grape cell cultures having resistance to
the E. ampelina culture filtrate. Hyphae did not grow freely
through the medium in plates containing these resistant cultures,
as compared to fungal hyphae growth through the medium in plates
containing control (i.e., non-resistant) cultures. A thick mat of
Fusarium mycelia, as seen in the plates containing control
cultures, was never formed in the plates containing the E. ampelina
resistant cultures. This experiment demonstrated that the resistant
grapevine cultures were not only resistant to E. ampelina, but were
also resistant to F. oxysporum.
[0154] Further analysis was made to determine if the
fungal-resistant grapevine cultures could give rise to somatic
embryos that were also resistant to E. ampelina. Somatic embryos
derived from resistant cultures and control cultures were grown
either in medium containing 40% (v/v) of fungal culture filtrate or
in control medium containing no fungal culture filtrate. While
somatic embryos derived from the resistant cultures formed and
germinated normally in both the fungal culture filtrate-containing
medium and control medium, somatic embryos derived from control
cultures turned necrotic and eventually died in the fungal culture
filtrate-containing medium, but did not die in the control medium.
The necrosis of the controls in the fungal culture
filtrate-containing medium was rapid enough to turn the control
somatic embryos dark within seventy-two hours of culture
initiation. The results from these experiments demonstrated that
the somatic embryos obtained from resistant cell cultures were also
resistant to the fungal filtrate. Furthermore, these resistant
somatic embryos were observed to withstand a concentration of E.
ampelina culture filtrate that was equal to that withstood by their
progenitor resistant embryogenic cells and embryogenic cell
masses.
[0155] Pathogen-Resistant Plants
[0156] Embryogenic cultures were selected in vitro against fungal
culture filtrate produced by E. ampelina. Plants were regenerated
from the selected cultures and acclimatized in the greenhouse.
Plants from selected lines and unselected controls were sprayed
with a spore suspension (1.times.10.sup.6 spores/mL) until runoff.
The plants were individually bagged to maintain humidity (a
condition is optimum for the pathogen to cause anthracnose disease)
for 3 days. The bags were then removed and the plants were scored
for anthracnose symptoms. All of the unselected controls exhibited
a very high degree of susceptibility, and in most cases there was
defoliation due to the disease within three days. Among the 40
different plants from the two selected lines, only one plant showed
mild anthracnose symptoms. These data show that the resistance
acquired by the embryogenic cells during in vitro selection can be
translated into whole plant resistance against the pathogen.
[0157] In Vitro Selection and Establishment of Resistant Lines
[0158] PEMs became brown and necrotic within a few days of culture
in culture filtrate-containing medium. The medium also turned dark
brown in these flasks. As selection progressed, browning of the
medium was gradually reduced, which was accompanied by necrosis of
most of the PEMs. Only a few PEMs (or cells within a few PEMs)
survived selection pressure through four or five cycles of
selection (FIG. 2). Cultures that survived four and five cycles of
selection were designated as `resistant culture 1` (RC1) and
`resistant culture 2` (RC2), respectively. By continuous
subculturing of these resistant cultures in suspension, we
increased the tissue mass in approximately 5 months after
withdrawing selection pressure. These cultures were used in
subsequent studies and for plant regeneration. There was no
browning in cultures that were grown in medium containing 40% (v/v)
of Czapek-Dox broth. PEMs in these flasks grew normally as in the
non-selected controls. This indicates that the necrosis was caused
by compounds, that were produced by the fungus and released into
the culture filtrate.
[0159] Dual Culture
[0160] Mycelium of E. ampelina grew uninhibited on plates
containing PEMs and somatic embryos from non-selected control.
Within a week after fungal inoculation, mycelium covered the entire
plate, growing on the embryogenic tissue as well. However, both
selected lines (RC1 and RC2) inhibited the growth of mycelium
significantly (FIG. 3A). Even after 10 days, the mycelial growth
did not reach the PEMs. A clear zone of inhibition could be
observed for several days. A similar trend was observed with F.
oxysporium, which is not a pathogen of grapevine (FIG. 3B).
Mycelial growth was white, fluffy and rapid on the non-selected
selected controls. On the selected lines, however, the fluffy
growth was restricted to the central region of the plate. There was
more vertical mycelial growth compared to the concentric pattern
seen with non-selected control.
[0161] Conditioned Medium Test
[0162] The fungus grew well on coverslips bearing PDA or
conditioned medium from non-selected controls. There was no
difference in growth between the two. On the other hand, mycelial
growth was inhibited on coverslips with conditioned medium from
both resistant lines (FIG. 4). Microscopical examination revealed
that the hyphal tips were smaller and many had burst, probably soon
after they started growing onto these coverslips. Additionally,
mycelial growth was uninhibited around these coverslips. This
suggests that the coated coverslips contained anti-fungal compounds
that had been secreted into the culture medium by selected
cultures.
[0163] Electrophoresis of Extracellular Proteins
[0164] Significant differences in extracellular protein profile
between the in vitro selected lines and non-selected controls could
be seen in the SDS-PAGE, both in PEMs and differentiated somatic
embryos. PEMs of both selected lines secreted additional proteins
of 8, 22 and 33 kDa (FIG. 5A). Heart stage somatic embryos of
non-selected controls exhibited two proteins of 35 and 36 kDa,
while there was only one protein of 36 kDa in the selected lines
(FIG. 5B). In addition, the 22 and 33 kDa proteins secreted by PEMs
of selected lines were also present during this stage of
embryogenesis, but the 8 kDa protein was absent. It is possible
that this protein was present, but ran out of the gel, since
shorter electrophoretic runs could not resolve this region
adequately.
[0165] Chitinase Activity in Extracellular Proteins
[0166] Native PAGE, which can resolve even isozymes of the same
size, indicates that selected lines have multiple chitinases. Two
of these are induced by selection. One isozyme, with the least
mobility, was greatly elevated in the selected lines in comparison
with the control. After SDS-PAGE analysis, a 36 kDa protein
exhibited chitinase activity in both selected lines and the
non-selected controls as revealed by glycol chitin gel assay. At
least a twenty-fold increase in chitinase activity of the 36 kDa
isozyme was seen in the resistant lines as revealed by
densitometric analysis. A 28 kDa protein also showed chitinase
activity in the resistant lines. The results indicate that new
isozymes of chitinases are expressed after selection and that the
secretion of chitinase increases after in vitro selection in
grapevine embryogenic cultures.
[0167] Immunological Detection of Chitinase
[0168] The 36 kDa protein in the ECP of both resistant lines
strongly reacted with chitinase antiserum. There was no reaction in
the ECP of non-selected controls, though chitinase activity was
detected in the glycol chitin assay. This indicates that the 36 kDa
protein observed in the non-selected control and in the selected
lines may not be the same protein. The 28 kDa peptide that was
present in the selected lines as detected in the glycol chitin
assay, did not react with this antiserum.
[0169] Retesting of Somatic Embryos after In Vitro Selection
[0170] Mature somatic embryos of non-selected controls grew
normally on germination medium, but they did not germinate on
medium containing 40% (v/v) fungal culture filtrate. Most of them
turned necrotic within 4 days after culture. Somatic embryos from
both resistant lines germinated and grew into plants on both media
(FIG. 7), indicating that the acquired resistance is stable and not
epigenetic. More than 50 plants were regenerated from somatic
embryos from each of the resistant lines and established in the
greenhouse. Plant establishment was accomplished at 8 months after
selection and testing of plants occurred when they were 18 months
old.
[0171] In Vitro Leaf Bioassay
[0172] Leaves from non-selected controls developed black lesions at
the infected sites within three days of spore inoculation. The
lesions spread rapidly and the entire leaf became necrotic within a
week. Leaves from both in vitro selected lines were very slow in
exhibiting the lesions. It took more than 10 days for the lesions
to appear. The lesions did not spread as in the controls, even
after two weeks of incubation, indicating that the resistance
acquired by PEMs during in vitro selection persisted in the
plants.
[0173] Testing the Regenerated Plants for Resistance
[0174] After removing bags, the leaves of inoculated plants were
examined for disease symptoms. Most of the young leaves from
non-selected control plants were crinkled with spreading lesions.
Some leaves exhibited `shothole symptoms`, characteristic of
anthracnose disease (FIG. 8). Few leaves turned necrotic within
this three day period. There was extensive defoliation among
non-selected controls. Thirty nine out of forty in vitro selected
plants from both resistant lines remained healthy even after
several days. Only one plant tested this way showed mild symptoms
of leaf curl; no lesions were observed, however. Defoliation was
very minimal and often only the older leaves were lost.
[0175] Re-isolation of Fungus from the Infected Plants
[0176] Fungal mycelium grew rapidly from symptomatic leaves of
control plants. Mycelial growth was identical to that of the
original control culture. Microscopic observations of conidia
confirmed them to be E. ampelina. Koch's postulate was accomplished
using these conidia to infect grapevine leaves.
[0177] Identification of Differentially Expressed Proteins
[0178] Extracellular proteins from resistant and control
embryogenic cultures were analyzed to determine if any activation
of defense genes was apparent in the embryogenic cells or somatic
embryos resistant to E. ampelina. Analysis of extracellular
proteins (i.e., proteins secreted in the liquid culture medium)
revealed changes in protein profiles between the control and
resistant embryogenic cultures. In addition, chitinase was observed
to be secreted in abundance by the resistant embryogenic cultures
in comparison with control cultures. This secretion of chitinase
was observed even eight months after selection. These results
demonstrated that the resistant cultures retained an activity many
generations (in terms of cell divisions) after the selection
pressure had been removed; hence, the E. ampelina resistance was a
stable genetic mutation.
[0179] Extraction of proteins in the intercellular fluids was more
difficult than described for other species. Extracted proteins are
preferably separated by electrophoresis within a few hours, since
storing them even at -50.degree. C. leads to loss of proteins. ICWF
extractions were analysed several times in order to confirm the
separation of proteins. Two prominent, differentially expressed,
proteins of 8 and 22 kDa could be identified consistently in the
ICWF of selected lines. While there were two proteins of 1.6 and 22
kDa in the ICWF of RC1 and RC2, a weak 21.6 kDa protein was present
in the ICWF of non-selected control plants (FIG. 5C).
[0180] Immunodetection
[0181] The 22 kDa protein from both resistant lines reacted with
pinto bean PR 5 antiserum. There was no reaction in the control.
This protein could be detected both at the PEM stage (FIG. 10A) and
also at the heart stage somatic embryo, using the same antiserum,
indicating persistent expression of this protein. At the somatic
embryo stage, however, an additional band of approximately 26 kD
also cross reacted with this antiserum (FIG. 10B) in the resistant
line RC2. There were two bands of 22 and 23 kDa (referred to herein
as the 22 kDa doublet) in the ICWF from plants of both resistant
lines that reacted with the PR-5 antiserum. There was also faint
reaction in the ICWF from non-selected controls (FIG. 10C). Thus
there is a doublet between 22 kDa and 23 kDa that includes two
PR-5-related proteins.
[0182] Identification of Differentially Expressed Proteins Using
N-terminal Amino Acid Sequencing
[0183] The sequence of the N-terminal 21 amino acids of the 8 kDa
protein was determined by Edman degradation method to be
TVTXGQVASAVGPXISYLQ (SEQ ID NO: 1). Sequence similarity searches
revealed that this protein exhibits a high similarity with
non-specific lipid transfer proteins (nsLTP). Among the nsLTPs that
showed high similarity was a 9 kDa protein identified from
grapevine somatic embryos and identified as LTP P4 (Coutos-Thevenot
et al., Eur. J. Biochem. 217:885-889, 1993). In additon, it also
exhibited 75% similarity with another 9 kDa protein from grapevine
berries (Salzman et al., supra). Thus the 8 kDa protein was
identified as a nsLTP (FIG. 11). Amino acid sequence information
could not be obtained for the 14 kDa protein that was
differentially expressed by heart stage somatic embryos presumably
because the N-terminus of this protein was blocked.
[0184] One of the N-terminal amino acid sequences (ATFDILNKXTYTVXA;
SEQ ID NO: 2) of the 22 kDa protein doublet secreted by heart stage
somatic embryos of in vitro selected lines matched that of a
thaumatin/osmotin-like protein (VVTL-1) isolated from grapevine
berries (Tattersal et al., Plant Physiol. 114:759-769, 1997). In
addition, it also exhibited very high sequence similarity with the
N-terminal sequences of several other TLPs. Among these, two
tobacco thaumatin-like proteins, E22 and E2, exhibited 92% sequence
similarity (FIG. 12). The amino fragment from the second protein
(ATFNIQNKGGYTVXA; SEQ ID NO: 3) had homology to grapevine osmotin.
Both proteins from ICWF exhibited high homology with the
corresponding 22 kDa protein doublet secreted by heart stage
somatic embryos. It is evident that the 22 kDa protein doublet is
differentially and constitutively expressed by the selected lines,
predominantly as a secreted protein and could be traced from the
early PEM stage to all the way in regenerated plants.
[0185] N-terminal sequence of the 33 kDa protein from heart stage
embryos (ASLADQQANEFTKV; SEQ ID NO: 4) did not reveal any
significant sequence similarity in the database search. A cDNA
encoding the 33 kDa protein was cloned as follows. Primers were
designed based on amino terminal and carboxy terminal amino acid
sequence information generated from the 33 kDa protein. Using
these, we amplified the fragment from the genomic DNA and then
cloned and sequenced the fragment. The primer designed based on the
carboxy terminal fragment did not help in amplifying, but a
palindromic sequence to the primer designed based on the N terminal
fragment existed at the 3' end of the DNA sequence. The sequences
for both the DNA (SEQ ID NO: 6) and the putative protein (SEQ ID
NO: 5) are depicted in FIG. 9.
[0186] Pathogen Resistance
[0187] The methods of the invention are useful for providing
resistance to other grapevine diseases. Grape plants exhibiting
resistance to a number of different diseases may be generated from
embryogenic cells and embryogenic cell masses that are selected for
resistance to the etiologic agent of a particular disease, a toxin
produced by the agent, or the etiologic agent (or toxin) of another
grapevine disease. For example, embryogenic cells and embryogenic
cell masses may be grown in a liquid suspension culture in the
presence of a filter-sterilized culture filtrate prepared from a
pathogen, at a concentration of culture filtrate that is ideal for
in vitro selection. After four or five cycles of recurrent
selection in such a liquid medium containing culture filtrate, with
subculturing performed every ten days as described above, the
surviving cells are allowed to expand in a liquid medium lacking
the culture filtrate. From these cells, somatic embryogenesis may
be performed to produce cells and plants showing increased
resistance to the powdery mildew disease, as well as to diseases
caused by other fungi and/or bacteria. The filtrate may be the cell
supernatant from the culture. In some cases, it may be preferable
to culture the pathogen in the presence of plant cells, harvest and
lyse and/or homogenize the cells, and then collect the supernatant
following centrifugation. Such a filtrate is particularly useful
when the pathogen is a virus or a bacterium.
[0188] The method described herein can be modified to select for
cells that have been transformed with a nucleic acid sequence. Cell
transformation, while a standard technique, does not result in
every cell containing the nucleic acid of interest. It is standard
laboratory practice to include in the transformation nucleic acid
sequence that confers a growth advantage in a specific selection
medium. Thus, only the cells of interest (i.e., the ones that are
transformed) are able to grow or survive in the selection medium.
The proteins described herein (and the nucleic acids encoding them)
can be used as selectable markers in such methods. In this example,
the selection medium includes a pathogen, or a pathogen filtrate or
conditioned medium. Cells that have been transformed with the
nucleic acid sequence encoding the protein that confers pathogen
resistance will survive, while cells that have not been transformed
will die.
[0189] It will be understood that a protein that confers resistance
to one pathogen may also confer resistance to additional pathogens.
Plants resistant to anthracnose may be additionally resistant to
additional pathogens. For example, a plant that is resistant to
both anthracnose and black rot (caused by the fungus, Guignardia
bidwellii) may be additionally resistant to Botrytis bunch rot and
blight (caused by the fungus, Botrytis cinerea). The rapid
generation of these resistant grape plants using the methods of the
invention allows for such combination of resistance not just for
fungi, but for other grapevine pathogens (e.g., bacteria and
viruses).
[0190] Evaluation of the level of pathogen protection conferred to
a plant by the selection methods described herein is determined
according to conventional methods.
EXAMPLE 5
Grapevine Transformation
[0191] The method described herein can be used to produce
transformed plants. Cells can be transformed at any step in the
process of making a somatic embryo-derived. Thus, tissue or cells
suitable for transformation include explanted tissue, embryogenic
cells, embryogenic cell masses, and somatic embryos (including
mature somatic embryos).
[0192] Cell cultures produced according to the methods of the
invention may be transformed with DNA comprising a desired
transgene, such as the DNA of SEQ ID NO: 6). Such cells, for
example, may be transformed with genes which confer resistance to
pathogens, diseases, or pests, or any combination thereof. For
example, a number of Bacillus thurigiensis genes which encode
proteins that are toxic to a number of pests are well known and
useful in the methods of the invention. Several standard methods
are available for introduction of a transgene into a plant host,
thereby generating a transgenic plant.
[0193] Upon construction of the plant expression vector, several
standard methods are available for introduction of the vector into
a plant host, thereby generating a transgenic plant. These methods
include (1) Agrobacterium-mediated transformation (A. tumefaciens
or A. rhizogenes) (see, e.g., Lichtenstein and Fuller In: Genetic
Engineering, vol 6, P W J Rigby, ed, London, Academic Press, 1987;
and Lichtenstein, C. P., and Draper, J,. In: DNA Cloning, Vol II,
D. M. Glover, ed, Oxford, IRI Press, 1985)); (2) the particle
delivery system (see, e.g., Gordon-Kamm et al., Plant Cell 2:603
(1990); or BioRad Technical Bulletin 1687, supra); (3)
microinjection protocols (see, e.g., Green et al., supra); (4)
polyethylene glycol (PEG) procedures (see, e.g., Draper et al.,
Plant Cell Physiol. 23:451, 1982; or e.g., Zhang and Wu, Theor.
Appl. Genet. 76:835, 1988); (5) liposome-mediated DNA uptake (see,
e.g., Freeman et al., Plant Cell Physiol. 25:1353, 1984); (6)
electroporation protocols (see, e.g., Gelvin et al., supra;
Dekeyser et al., supra; Fromm et al., Nature 319:791, 1986; Sheen
Plant Cell 2:1027, 1990; or Jang and Sheen Plant Cell 6:1665,
1994); and (7) the vortexing method (see, e.g., Kindle supra). The
method of transformation is not critical to the invention. Any
method which provides for efficient transformation may be employed.
As newer methods are available to transform crops or other host
cells, they may be directly applied.
[0194] The following is an example outlining one particular
technique, an Agrobacterium-mediated plant transformation. By this
technique, the general process for manipulating genes to be
transferred into the genome of plant cells is carried out in two
phases. First, cloning and DNA modification steps are carried out
in E. coli, and the plasmid containing the gene construct of
interest is transferred by conjugation or electroporation into
Agrobacterium. Second, the resulting Agrobacterium strain is used
to transform plant cells. Thus, for the generalized plant
expression vector, the plasmid contains an origin of replication
that allows it to replicate in Agrobacterium and a high copy number
origin of replication functional in E. coli. This permits facile
production and testing of transgenes in E. coli prior to transfer
to Agrobacterium for subsequent introduction into plants.
Resistance genes can be carried on the vector, one for selection in
bacteria, for example, streptomycin, and another that will function
in plants, for example, a gene encoding kanamycin resistance or
herbicide resistance. Also present on the vector are restriction
endonuclease sites for the addition of one or more transgenes and
directional T-DNA border sequences which, when recognized by the
transfer functions of Agrobacterium, delimit the DNA region that
will be transferred to the plant.
[0195] In another example, plant cells may be transformed by
shooting into the cell tungsten microprojectiles on which cloned
DNA is precipitated. In the Biolistic Apparatus (Bio-Rad) used for
the shooting, a gunpowder charge (22 caliber Power Piston Tool
Charge) or an air-driven blast drives a plastic macroprojectile
through a gun barrel. An aliquot of a suspension of tungsten
particles on which DNA has been precipitated is placed on the front
of the plastic macroprojectile. The latter is fired at an acrylic
stopping plate that has a hole through it that is too small for the
macroprojectile to pass through. As a result, the plastic
macroprojectile smashes against the stopping plate, and the
tungsten microprojectiles continue toward their target through the
hole in the plate. For the instant invention the target can be any
plant cell, tissue, seed, or embryo. The DNA introduced into the
cell on the microprojectiles becomes integrated into either the
nucleus or the chloroplast.
[0196] In general, transfer and expression of transgenes in plant
cells are now routine practices to those skilled in the art, and
have become major tools to carry out gene expression studies in
plants and to produce improved plant varieties of agricultural or
commercial interest.
[0197] While the expression of one of the proteins of the invention
is likely to confer on a plant increased disease resistance, it may
be preferable to express two, three, or even all four proteins in a
plant to achieve maximal pathogen resistance. This can be achieved
either by the selection method described herein, or by producing a
plant having transgenes encoding the four sequences.
EXAMPLE 6
Generation of Antibodies, Nucleic Acids, and Proteins
[0198] Using standard techniques, such as those described above,
one in the art can identify full-length proteins and nucleic acids
from any variety of grape plant. For example, an amino terminal
peptide fragment can be used to generate a degenerate nucleic acid
probe for PCR, Southern blotting, or colony hybridization. Using a
nucleic acid sequence, one can identify orthologues in other
variety of plants or in plants other than grape plants. The
proteins or polypeptides of the invention can be used to raise
antibodies or binding portions thereof or probes. The antibodies
can be monoclonal or polyclonal. A description of the theoretical
basis and practical methodology of fusing such cells is set forth
in Kohler and Milstein, Nature, 256:495, 1975), and Milstein and
Kohler, Eur. J. Immunol., 6:511, 1976), hereby incorporated by
reference. Procedures for raising polyclonal antibodies are also
well known to the skilled artisan. This and other procedures for
raising polyclonal antibodies are disclosed in Harlow et. al.,
editors, Antibodies: A Laboratory Manual (1988), which is hereby
incorporated by reference.
[0199] In addition to utilizing whole antibodies, binding portions
of such antibodies can be used. Such binding portions include Fab
fragments, F(ab').sub.2 fragments, and Fv fragments. These antibody
fragments can be made by conventional procedures, such as
proteolytic fragmentation procedures, as described in Goding,
Monoclonal Antibodies: Principles and Practice, New York: Academic
Press, pp. 98-118 (1983), hereby incorporated by reference.
[0200] The present invention also relates to probes found either in
nature or prepared synthetically by recombinant DNA procedures or
other biological procedures. Suitable probes are molecules which
bind to the proteins of the present invention. Such probes can be,
for example, proteins, peptides, lectins, or nucleic acid
probes.
[0201] Antibodies raised against the proteins or polypeptides of
the present invention or binding portions of these antibodies can
be utilized in a method for selection of plants having increased
resistance to a plant pathogen. A variety of assay systems can be
employed, such as enzyme-linked immunosorbent assays,
radioimmunoassays, gel diffusion precipitin reaction assays,
immunodiffusion assays, agglutination assays, fluorescent
immunoassays, protein A immunoassays, or immunoelectrophoresis
assays.
[0202] The sequences of the present invention can also be used to
identify proteins that are substantially identical to those
described herein. By "substantially identical" is meant a protein
or nucleic acid exhibits at least 70%, preferably 80%, and most
preferably 90%, 95%, or even 98% identity to a reference amino acid
sequence or nucleic acid sequence. For proteins, the length of
comparison sequences will generally be at least 15 amino acids,
preferably at least 20 amino acids, more preferably at least 25
amino acids, and most preferably 35 amino acids or greater. For
nucleic acids, the length of comparison sequences will generally be
at least 50 nucleotides, preferably at least 60 nucleotides, more
preferably at least 75 nucleotides, and most preferably 110
nucleotides or greater.
[0203] Sequence identity, at the amino acid levels, is typically
measured using sequence analysis software (for example, Sequence
Analyis Software Package of the Genetics Computer Group, Univerity
of Wisconsin Biotechnology Center, 1710 University Avenue, Madison,
Wis. 53705, BLAST, or PILEUP/PRETTYBOX prgrams). Such software
matches identical or similar sequences by assigning degrees of
homology to various substitutions, deletions, and/or other
modifications.
[0204] The present invention also includes nucleic acids that
selectively hybridize to the DNA sequence of the present invention.
Hybridization may involve Southern analysis (Southern Blotting), a
method by which the presence of DNA sequences in a target nucleic
acid mixture are identified by hybridization to a labeled
oligonucleotide or DNA fragment probe. Southern analysis typically
involves electrophoretic separation of DNA digests on agarose gels,
denaturation of the DNA after electrophoretic separation, and
transfer of the DNA to nitrocellulose, nylon, or another suitable
membrane support for analysis with a radiolabeled, biotinylated, or
enzyme-labeled probe as described in Sambrook et al., (1989)
Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.
[0205] Hybridization often includes the use of a probe. It is
generally preferred that a probe of at least 20 nucleotides in
length be used, preferably at least 50 nucleotides, more preferably
at least about 100 nucleotides.
[0206] A nucleic acid can hybridize under moderate stringency
conditions or high stringency conditions to a nucleic acid
disclosed herein. High stringency conditions are used to identify
nucleic acids that have a high degree of homology or sequence
identity to the probe. High stringency conditions can include the
use of a denaturing agent such as formamide during hybridization,
e.g., 50% formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM NaCl, and 75 mM sodium citrate at 42.degree. C. Another
example is the use of 50% formamide, 5.times.SSC (0.75 M NaCl,
0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%
sodium pyrophosphate, 5.times. Denhart's solution, sonicated salmon
sperm DNA (50 ug/mL) 0.1% SDS, and 10% dextran sulfate at
42.degree. C., with washes at 42.degree. C. in 0.2.times.SSC and
0.1% SDS. Alternatively, low ionic strength washes and high
temperature can be employed for washing.
[0207] Moderate stringency conditions are hybridization conditions
used to identify nucleic acids that have less homology or identity
to the probe than do nucleic acids under high stringency. All of
these techniques are well known to the artisan skilled in molecular
biology.
Materials and Methods
[0208] In vitro Selection, Culture Establishment and Plant
Regeneration
[0209] Suspension cultures, somatic embryogenesis and plant
regeneration of `Chardonnay` (Clone 02Ch; Stimson Lane Wineries,
Prosser, Wash.) consisting of actively growing PEMs were
established as follows. Log phase cultures were sieved using a 960
.mu.M sieve to generate a synchronized culture. Approximately 1.0 g
of PEMs were subjected to recurrent selection in suspension culture
with a modified culture medium containing 40% (v/v) fungal culture
filtrate. The liquid medium was prepared and cooled to room
temperature and the culture filtrate was added after filter
sterilization to eliminate any loss of filtrate activity due to
autoclaving. The culture filtrate was obtained by growing a
virulent strain of E. ampelina spores for 3 weeks in Czapek-Dox
broth. Cell free extract was collected and stored at -20.degree. C.
until further use. The pH of the culture was adjusted to 5.8 before
adding to the medium. Selection was carried out for four or five
cycles, each cycle lasting for 10 days. At the end of 4.sup.th and
5.sup.th cycles, putative resistant cultures were proliferated in
regular suspension culture medium and established as `resistant
culture 1` (RC1) and `resistant culture 2` (RC2), respectively.
Somatic embryogenesis was achieved by culturing the selected PEMs
in auxin-free suspension culture medium and the resulting somatic
embryos were germinated in solid medium. Regenerated plants were
acclimatized in potting mixture and established in a greenhouse. A
set of control, non-selected PEMs were cultured in a similar way
and plants regenerated from these non-selected cultures served as
control for rest of the experiments.
[0210] Dual Cultures
[0211] PEMs of resistant and unchallenged controls, both of which
were maintained in suspension culture for more than 20 weeks after
selection, were used for dual culture. PEMs were collected on a
sterile filter paper and approximately 1.0 g of PEMs were cultured
on semisolid medium at opposite sides of a 100.times.15 mm petri
dish. The medium had the same components as liquid medium, but was
solidified with TC agar at 7.0 g/l. The cultures were sealed with
Parafilm.TM. and incubated in darkness at 25.+-.2.degree. C. After
5 weeks, a mycelial plug (5 mm in diameter) from an actively
growing fungal culture was placed at the center of the plates.
Cultures were tested against two different fungi, E. ampelina
(against which the PEMs were selected) and Fusarium oxysporium (a
root pathogen isolated from watermelon). After inoculating mycelial
plugs the cultures were sealed and incubated at 25.+-.2.degree. C.
at 16 h photoperiod. There were 5 petri plates for each fungus and
the experiment was repeated twice. Mycelial growth on the plates
was measured daily and photographed after 10 days of culture.
[0212] Conditioned Medium Assay
[0213] Spent liquid medium was collected from a resistant line and
unchallenged control and centrifuged at 2500 rpm for 10 min to
remove cellular debris. After filter-sterilization, the supernatant
was diluted with an equal volume of warm, 1.5 N (58.5 gl.sup.-1)
potato dextose agar (PDA) medium to give a final concentration of
0.75 N (29.25 gl.sup.-1). Sterile glass slide coverslips were
soaked in the molten medium rapidly (before the medium solidified)
and placed on 0.75 N PDA plates. Three coverslips were placed on
each plate. Coverslips soaked in 0.75 N PDA and plated as before
served as an additional control. After cooling the plates
overnight, a mycelial plug from E. ampelina was placed at the
center of the plate and incubated at 25.+-.2.degree. C. Mycelial
growth on the coverslips containing the conditioned medium was
evaluated daily and photographed after seven days of culture.
[0214] Extraction of ECPs
[0215] Spent medium was collected in sterile flasks during
subculture and filtered through a double layer Kimwipe.TM. to
eliminate any cellular debris. ECP was precipitated from the
filtered medium by adding three volumes of ice cold, 95% ethanol
and kept overnight at 0.degree. C. Proteins were pelleted by
centrifugation, concentrated in a vacuum concentrator and
resuspended in sterile distilled water. Protein quantitation was
done by the Bradford protein assay, using bovine serum albumin as a
standard. Protein samples were stored at -20.degree. C. until
further use.
[0216] Extraction of ICWF and Protein Concentration
[0217] Fully expanded, flaccid leaves were collected from
greenhouse-grown plants early in the morning. The leaves were
washed thoroughly with distilled water and blot-dried. Lamina were
cut into 2 cm wide strips and vacuum infiltrated for 15 min in a
buffer containing 100 mM Tris-HCl, 2.0 mM CaCl.sub.2, 10 mM EDTA,
50 mM P-mercaptoethanol and 0.5 M sucrose, at the rate of 10 ml/g
of leaf tissue. After infiltration, the leaf strips were gently
blotted and rolled into 0.5 ml microfuge tubes (without caps) with
a 0.2 mm dia hole at the bottom. Only 2-3 strips were loaded in
each microfuge tube. These tubes were then loaded onto a 1.5 ml
centrifuge tubes. The set-up was spun at 7500 rpm for 15 min at
room temperature. ICWF collects as a dense drop in the 1.5 ml
centrifuge tubes. To concentrate the proteins, ICWF was diluted
with 4 volumes of distilled water and the proteins were
precipitated with 3 volumes of ice-cold, 95% ethanol overnight at
0.degree. C. Proteins were pelleted by centrifugation, concentrated
in a vacuum concentrater and re-suspended in sterile distilled
water.
[0218] Electrophoresis of Proteins
[0219] SDS-PAGE was carried out using 1 mm thick mini gels. Protein
samples were diluted with equal volume of SDS-PAGE buffer (Sigma,
St. Louis, Mo.) and the diluted samples were heated in a boiling
water bath for 5 min and cooled. Samples were spun at 10,000 rpm
for 5 min at room temperature to remove any insoluble particles.
Total protein of 10 .mu.g was loaded onto each lane and
electrophoresed for approximately 80 min at 200 V. The gels were
then either silver-stained using SilverSnap.TM. (Pierce, Rockford.
IL) or stained with colloidal Coomassie Blue (Sigma, St. Louis Mo.)
and photographed using a Kodak DC 120 digital camera.
[0220] Chitinase activity was analyzed as follows. After native
PAGE, the gels were rinsed in 150 .mu.M sodium acetate (pH 5.0) for
15 min. The gels were placed on a clean glass plate and overlaid
with a 7.5% gel containing 0.01% (v/v) glycol chitin. After
removing air bubbles, the gel sandwich was incubated at 37.degree.
C. under moist conditions. The overlay gels were removed and
stained with 0.01% fluorescent brightener (Calcoflour white M2R) in
Tris-HCl buffer (pH 8.9) for 10 min and rinsed thoroughly in
distilled water overnight. Chitinase activity derived from various
chitinase isozymes was visible as dark (lytic) bands in the overlay
gels.
[0221] Running gels in SDS-PAGE were incorporated with 0.02% glycol
chitin while casting the gels. After electrophoresis, the gels were
incubated in 200 mM sodium acetate solution at pH 5.0 containing 1%
of Triton-X 100 for 4 h at 37.degree. C.
[0222] After incubation, the gels were washed 3 times with
distilled water, stained with 0.01% (v/v) fluorescent brightener in
500 mM Tris-HCl (pH 8.9) for 10 min and destained overnight in
distilled water. Chitinase isozymes were identified as lytic bands
on a UV-transilluminator and photographed using a Kodak DC 120
digital camera with orange filter.
[0223] Immunodetection of Chitinase
[0224] SDS-PAGE was carried out as described above and the proteins
were transferred to a PVDF membrane (Bio-Rad, Almeda, Calif.) in a
mini transblot gel transfer cell. Following transfer of proteins,
the membrane was probed with an antiserum raised against a barley
seed chitinase at 1:1000 dilution. The antigen-antibody complex was
detected by a goat-anti rabbit horseradish peroxidase
(Bio-Rad).
[0225] Re-testing the In Vitro Selected Cultures for Resistance to
Culture Filtrate
[0226] Mature somatic embryos from both selected cultures and
non-selected control were germinated on a solid germination medium
containing 40% (v/v) fungal culture filtrate. There were five
plates per treatment, each containing 15 embryos. After culturing,
the plates were incubated in the dark. Three weeks after
incubation, embryos that germinated were counted as being
resistant. A similar set of embryos were germinated in a medium
without culture filtrate as an additional control.
[0227] Plant Regeneration and Establishment in Greenhouse
[0228] Plants regenerated from somatic embryos were transferred to
starter plugs containing sterile commercial potting mixture and
kept under 16 h photoperiod for in vivo acclimitization. After
approximately one month, soil-acclimatized plants were transferred
to the greenhouse. Well-established and vigorously growing plants,
approximately 18 months after regeneration, were used for further
studies.
[0229] In Vitro Leaf Bioassay for Anthracnose Resistance
[0230] Fully expanded green, young leaves, approximately 6 cm wide
were collected from 10 different plants in each of two selected
lines and the non-selected control. These leaves were inoculated
with 100 .mu.l of a spore suspension containing 1.times.10.sup.6
spores per ml. There were three inoculations on each leaf in the
inter-venal region. Immediately after inoculation, the leaves were
incubated under humid conditions in moist chambers at 25+2.degree.
C. and 16 h photoperiod. After one week, the leaves were evaluated
for anthracnose symptoms. The assay was repeated twice.
[0231] Test for Anthracnose Resistance in Selected Plants
[0232] Eighteen month old, greenhouse grown in vitro selected
plants and non-selected controls regenerated from somatic embryos
were used in this study. Clones from the original `Chardonnay`
(`02Ch`), from which the cultures were initiated, were also used as
an additional control. Plants that were actively growing with young
leaves were chosen for this test. The plants were sprayed with a
spore suspension containing 1.times.10.sup.6 spores per ml on both
sides of the leaves until runoff. They were then individually
covered with a polythene bag carefully so that leaves did not touch
the bag which was sealed around the pot. These plants were
incubated in the growth room, at 25+/-2.degree. C. and 16 h
photoperiod. After 72 h of incubation, the bags were carefully
removed and the plants were observed for disease symptoms. Plants
exhibiting crinkling of leaf lamina or typical shot hole symptoms
were scored as susceptible. The experiment was repeated twice using
different sets of plants from each selected line and control. For
each test, there were at least 20 plants from each selected line
and 6 plants from the control.
[0233] Recovery of Pathogen after Infection
[0234] Leaves that showed anthracnose symptoms were removed and
washed well with distilled water. They were air dried under the
laminar flow hood for two days. Pieces of lamina and midrib from
these air dried leaves were then cultured in PDA. A small plug of
mycelium from the original culture that was used to infect the
leaves was also cultured, for comparison. The cultures were
incubated at 16 h photoperiod and 25.+-.2.degree. C.
[0235] Immunodetection of Proteins
[0236] After SDS-PAGE, proteins were transferred to ImmunoBlot.TM.
PVDF membrane (Bio-Rad, Almeda, Calif.) in a mini trans-blotter
according to manufacturer's instructions. Transfer was carried out
under high intensity electric field (100 V) for 2 hr. The membrane
was washed thoroughly in washing buffer and rinsed thrice in
distilled, deionized water for 10 min each, then blocked overnight
in a 3% bovine serum albumin solution at room temperature. After
another cycle of washing and rinsing, proteins were probed with PR
5 antiserum raised against pinto bean thaumatin-like protein
(provided by Dr. O. P. Sehgal, University of Missouri, Columbia,
Mo.) at a dilution of 1:500 for 2 hr at room temperature with
gentle shaking. Color development was carried out using Opti-4Cn
kit (Bio-rad, Almeda, Calif.), according to manufacturer's
instructions.
[0237] N-terminal Amino Acid Sequencing
[0238] For N-terminal amino acid sequencing, proteins were
transferred to an ImmunoBlot PVDF membrane using a buffer lacking
glycine. After transfer, proteins were stained with Coomassie blue
and appropriate bands were identified based on their molecular
weight and cut out using a sterile scalpel. Amino-terminal amino
acid sequence determination was accomplished by the automated Edman
degradation method, in the Protein Chemistry Core Laboratory,
University of Florida, Gainesville, using a protein sequencer,
Model 494HT (Applied Biosystems, Foster City, Calif.).
Phenylthiohydantoin amino acid derivatives were automatically
detected by a 120A analyzer used in conjunction with the
sequencer.
Other Embodiments
[0239] All publications mentioned in this specification are herein
incorporated by reference to the same extent as if each independent
publication was specifically and individually indicated to be
incorporated by reference.
[0240] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses, or adaptations following, in general,
the principles of the invention and including such departures from
the present disclosure within known or customary practice within
the art to which the invention pertains and may be applied to the
essential features hereinbefore set forth.
Sequence CWU 1
1
6 1 19 PRT Vitis vinifera VARIANT (1)...(19) Xaa = Any Amino Acid 1
Thr Val Thr Xaa Gly Gln Val Ala Ser Ala Val Gly Pro Xaa Ile Ser 1 5
10 15 Tyr Leu Gln 2 15 PRT Vitis vinifera VARIANT (1)...(15) Xaa =
Any Amino Acid 2 Ala Thr Phe Asp Ile Leu Asn Lys Xaa Thr Tyr Thr
Val Xaa Ala 1 5 10 15 3 15 PRT Vitis vinifera VARIANT (1)...(15)
Xaa = Any Amino Acid 3 Ala Thr Phe Asn Ile Gln Asn Lys Gly Gly Tyr
Thr Val Xaa Ala 1 5 10 15 4 14 PRT Vitis vinifera 4 Ala Ser Leu Ala
Asp Gln Gln Ala Asn Glu Phe Thr Lys Val 1 5 10 5 251 PRT Vitis
vinifera 5 Ala Asn Glu Phe Thr Asn Leu Leu Tyr Cys Ile Gln Lys Arg
Lys Lys 1 5 10 15 Lys Tyr Val Ile Phe Gly Val Cys Asp Val Tyr Gly
Ile His Gln Gly 20 25 30 Gly Ile Ile Leu Gly Pro Ser Gly Leu Gly
Lys Ser Pro Ala Phe Ser 35 40 45 Lys Trp Val Phe Pro Glu Ser Ser
Ile Tyr Phe Ser Gln Thr Val Ala 50 55 60 Leu Phe Gly Cys Met Ile
Phe Met Phe Leu Val Gly Val Lys Met Asp 65 70 75 80 Thr His Leu Met
Arg Lys Ser Gly Arg Arg Gly Val Val Ile Gly Phe 85 90 95 Cys Asn
Phe Phe Leu Pro Leu Ile Ile Val Val Gly Leu Ala His Asn 100 105 110
Leu Arg Lys Thr Lys Thr Leu Gly His Asn Ile Ser Asn Ser Ile Tyr 115
120 125 Cys Val Ala Thr Leu Met Ser Met Ser Ser Ser His Val Ile Thr
Cys 130 135 140 Leu Leu Thr Asp Ile Lys Ile Leu Asn Ser Glu Leu Gly
Arg Leu Ala 145 150 155 160 Leu Ser Ser Ser Met Ile Ser Gly Leu Cys
Ser Trp Thr Leu Ala Leu 165 170 175 Gly Ser Tyr Val Ile Phe Gln Gly
Ser Thr Gly Gln Tyr Glu Ser Met 180 185 190 Leu Ala Leu Ser Leu Ser
Phe Ile Ile Leu Val Leu Ile Ile Val Tyr 195 200 205 Ile Leu Arg Pro
Ile Met Asp Trp Met Val Glu Gln Thr Ala Glu Gly 210 215 220 Lys Pro
Ile Lys Glu Ser Tyr Val Phe Ser Ile Phe Val Met Ile Leu 225 230 235
240 Gly Ser Ala Phe Leu Gly Glu Leu Ile Gly Leu 245 250 6 777 DNA
Vitis vinifera 6 agaattccaa caggccaatg agttcaccaa tttactgtac
tgcatccaaa agaggaaaaa 60 gaagtatgta atatttggtg tgtgtgatgt
ttatggtatt catcagggag gtattatcct 120 gggaccgtcg ggtttaggaa
aatctccagc attctccaaa tgggttttcc cagagagcag 180 catttatttc
agccaaaccg tcgccttatt tgggtgcatg atctttatgt tcctagttgg 240
agtgaaaatg gatacacatc tgatgaggaa gtcaggaagg agaggagtag tcataggctt
300 ctgcaacttc ttcttgccat tgataattgt ggttggcttg gctcacaatc
tcagaaaaac 360 taagaccttg ggccacaata taagcaattc tatttactgt
gtagcaacac tgatgagcat 420 gagttcctcc catgtcatta cttgccttct
aactgatatc aagatcctca actccgagct 480 gggaaggtta gccctatcct
catctatgat aagtggcctg tgcagttgga ccctggcatt 540 gggctcatat
gtaatatttc aaggctcaac tggtcagtat gaaagcatgc tagcattatc 600
cttgtcattt atcatcttgg tgcttatcat tgtatacatt ctgcggccta ttatggattg
660 gatggttgaa cagactgctg aaggaaaacc aatcaaggag agctatgtct
ttagcatctt 720 tgtgatgatc ttagggagtg ccttccttgg tgaactcatt
ggcctgttgg aattctt 777
* * * * *