U.S. patent application number 14/264265 was filed with the patent office on 2014-11-06 for potato cultivar j55.
This patent application is currently assigned to J.R. SIMPLOT COMPANY. The applicant listed for this patent is J.R. SIMPLOT COMPANY. Invention is credited to Pete Clark, Susan Fortier Collinge.
Application Number | 20140328995 14/264265 |
Document ID | / |
Family ID | 50514202 |
Filed Date | 2014-11-06 |
United States Patent
Application |
20140328995 |
Kind Code |
A1 |
Clark; Pete ; et
al. |
November 6, 2014 |
POTATO CULTIVAR J55
Abstract
A potato cultivar designated J55 is disclosed. The invention
relates to tubers of potato cultivar J55, to seeds of potato
cultivar J55, to plants and plant parts of potato cultivar J55, to
food products produced from potato cultivar J55, and to methods for
producing a potato plant by crossing potato cultivar J55 with
itself or with another potato variety. The invention also relates
to methods for producing a transgenic potato plant and to the
transgenic potato plants and parts produced by those methods. This
invention also relates to potato plants and plant parts derived
from potato cultivar J55, to methods for producing other potato
plants or plant parts derived from potato cultivar J55 and to the
potato plants and their parts derived from use of those methods.
The invention further relates to hybrid potato tubers, seeds,
plants and plant parts produced by crossing potato cultivar J55
with another potato cultivar.
Inventors: |
Clark; Pete; (Eagle, ID)
; Collinge; Susan Fortier; (Eagle, ID) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
J.R. SIMPLOT COMPANY |
Boise |
ID |
US |
|
|
Assignee: |
J.R. SIMPLOT COMPANY
Boise
ID
|
Family ID: |
50514202 |
Appl. No.: |
14/264265 |
Filed: |
April 29, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61818752 |
May 2, 2013 |
|
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Current U.S.
Class: |
426/637 ;
435/417; 800/260; 800/278; 800/279; 800/281; 800/284; 800/300;
800/301; 800/302; 800/303; 800/317.2 |
Current CPC
Class: |
C12N 15/8243 20130101;
A23L 19/18 20160801; C12N 15/8277 20130101; A01H 5/06 20130101;
A23L 19/15 20160801; C12N 15/8245 20130101; C12N 15/8251 20130101;
A23V 2002/00 20130101; C12N 15/8279 20130101; C12N 15/8275
20130101; C12N 15/8278 20130101; C12N 15/8289 20130101; C12N
15/8274 20130101; C12N 15/8281 20130101; C12N 15/8283 20130101;
A01H 1/02 20130101; C12N 15/8286 20130101; C12N 15/8282 20130101;
C12N 15/8247 20130101 |
Class at
Publication: |
426/637 ;
800/317.2; 435/417; 800/260; 800/278; 800/279; 800/281; 800/284;
800/300; 800/301; 800/302; 800/303 |
International
Class: |
A01H 5/06 20060101
A01H005/06; A23L 1/217 20060101 A23L001/217; A23L 1/2165 20060101
A23L001/2165; C12N 15/82 20060101 C12N015/82 |
Claims
1. A potato tuber, or a part of a tuber, of potato cultivar J55,
wherein a representative sample of said tuber was deposited under
ATCC Accession No. PTA -120601.
2. A potato plant, or a part thereof, produced by growing the
tuber, or a part of the tuber, of claim 1.
3. A potato plant having all of the physiological and morphological
characteristics of the plant of claim 2, and comprising the insert
region of pSIM1278 that is present in cultivar J55 which contains
inverted repeats of potato DNA effective for inhibition of
expression of the endogenous asparagine synthetase-1 gene and the
endogenous polyphenol oxidase-5 gene in addition to inverted
repeats of the endogenous potato promoters for the phosphorylase-L
and dikinase R1 genes.
4. A tissue culture of cells produced from the plant of claim 2,
wherein said cells of the tissue culture are produced from a plant
part selected from the group consisting of leaf, pollen, embryo,
cotyledon, hypocotyl, meristematic cell, root, root tip, pistil,
anther, flowers, stem and tuber, and wherein said tissue cultured
cells comprise the insert region of pSIM1278 that is present in
cultivar J55 which contains inverted repeats of potato DNA
effective for inhibition of expression of the endogenous asparagine
synthetase-1 gene and the endogenous polyphenol oxidase-5 gene in
addition to inverted repeats of the endogenous potato promoters for
the phosphorylase-L and dikinase R1 genes.
5. A potato plant regenerated from the tissue culture of claim 4,
wherein said plant has all of the physiological and morphological
characteristics of potato cultivar J55.
6. A potato seed produced by growing the potato tuber, or a part of
the tuber, of claim 1, wherein said seed comprises the insert
region of pSIM1278 that is present in cultivar J55 which contains
inverted repeats of potato DNA effective for inhibition of
expression of the endogenous asparagine synthetase-1 gene and the
endogenous polyphenol oxidase-5 gene in addition to inverted
repeats of the endogenous potato promoters for the phosphorylase-L
and dikinase R1 genes.
7. A potato plant, or a part thereof, produced by growing the seed
of claim 6.
8. A potato plant regenerated from tissue culture of the potato
plant of claim 7, wherein said regenerated plant comprises the
insert region of pSIM1278 that is present in cultivar J55 which
contains inverted repeats of potato DNA effective for inhibition of
expression of the endogenous asparagine synthetase-1 gene and the
endogenous polyphenol oxidase-5 gene in addition to inverted
repeats of the endogenous potato promoters for the phosphorylase-L
and dikinase R1 genes.
9. A method for producing a potato seed, said method comprising
crossing two potato plants and harvesting the resultant potato
seed, wherein at least one potato plant is the potato plant of
claim 2.
10. A method for producing a potato seed, said method comprising
crossing two potato plants and harvesting the resultant potato
seed, wherein at least one potato plant is the potato plant of
claim 7.
11. A potato seed produced by the method of claim 10, wherein said
seed comprises the insert region of pSIM1278 that is present in
cultivar J55 which contains inverted repeats of potato DNA
effective for inhibition of expression of the endogenous asparagine
synthetase-1 gene and the endogenous polyphenol oxidase-5 gene in
addition to inverted repeats of the endogenous potato promoters for
the phosphorylase-L and dikinase R1 genes.
12. A potato plant, or a part thereof, produced by growing said
potato seed of claim 11.
13. A potato seed produced from the plant of claim 12, wherein said
seed comprises the insert region of pSIM1278 that is present in
cultivar J55 which contains inverted repeats of potato DNA
effective for inhibition of expression of the endogenous asparagine
synthetase-1 gene and the endogenous polyphenol oxidase-5 gene in
addition to inverted repeats of the endogenous potato promoters for
the phosphorylase-L and dikinase R1 genes.
14. The method of claim 9, wherein one of said potato plants is
potato cultivar J55 and the second potato plant is transgenic.
15. A method of producing a potato seed, said method comprising
crossing two potato plants and harvesting the resultant potato
seed, wherein one of said potato plants is the potato plant of
claim 7 and the second potato plant is transgenic.
16. A potato plant, or a part thereof, produced by growing the seed
produced by the method of claim 14, wherein said plant comprises
the insert region of pSIM1278 that is present in cultivar J55 which
contains inverted repeats of potato DNA effective for inhibition of
expression of the endogenous asparagine synthetase-1 gene and the
endogenous polyphenol oxidase-5 gene in addition to inverted
repeats of the endogenous potato promoters for the phosphorylase-L
and dikinase R1 genes.
17. A method of introducing a desired trait into potato cultivar
J55, wherein the method comprises: (a) crossing a J55 plant,
wherein a representative sample of tubers was deposited under ATCC
Accession No. PTA-120601, with a plant of another potato cultivar
that comprises a desired trait to produce progeny plants, wherein
the desired trait is selected from the group consisting of male
sterility, herbicide resistance, insect resistance, modified fatty
acid metabolism, modified carbohydrate metabolism and resistance to
bacterial disease, fungal disease or viral disease; (b) selecting
one or more progeny plants that have the desired trait; (c)
backcrossing the selected progeny plants with J55 plants to produce
backcross progeny plants; (d) selecting for backcross progeny
plants that have the desired trait; and (e) repeating steps (c) and
(d) two or more times in succession to produce selected third or
higher backcross progeny plants that comprise the desired
trait.
18. A potato plant produced by the method of claim 17, wherein the
plant has the desired trait and comprises the insert region of
pSIM1278 that is present in cultivar J55 which contains inverted
repeats of potato DNA effective for inhibition of expression of the
endogenous asparagine synthetase-1 gene and the endogenous
polyphenol oxidase-5 gene in addition to inverted repeats of the
endogenous potato promoters for the phosphorylase-L and dikinase R1
genes.
19. The potato plant of claim 18, wherein the desired trait is
herbicide resistance and the resistance is conferred to an
herbicide selected from the group consisting of imidazolinone,
sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine
and benzonitrile.
20. The potato plant of claim 18, wherein the desired trait is
insect resistance and the insect resistance is conferred by a
transgene encoding a Bacillus thuringiensis endotoxin.
21. The potato plant of claim 18, wherein the desired trait is
modified fatty acid metabolism or modified carbohydrate metabolism
and said desired trait is conferred by a nucleic acid encoding a
protein selected from the group consisting of fructosyltransferase,
levansucrase, .alpha.-amylase, invertase and starch branching
enzyme or DNA encoding an antisense of stearyl-ACP desaturase.
22. A method of producing a commodity plant product, comprising
obtaining the plant of claim 2, or a part thereof, and producing
the commodity plant product from said plant or plant part thereof,
wherein said commodity plant product is selected from the group
consisting of French fries, potato chips, dehydrated potato
material, potato flakes and potato granules.
23. The commodity plant product produced by the method of claim 22,
wherein said product comprises the insert region of pSIM1278 that
is present in cultivar J55 which contains inverted repeats of
potato DNA effective for inhibition of expression of the endogenous
asparagine synthetase-1 gene and the endogenous polyphenol
oxidase-5 gene in addition to inverted repeats of the endogenous
potato promoters for the phosphorylase-L and dikinase R1 genes.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority from U.S.
provisional patent application Ser. No. 61/818,752, filed on May 2,
2013, which is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to a novel potato cultivar
designated J55 and to the tubers, plants, plant parts, tissue
culture and seeds produced by that potato variety. The invention
further relates to food products produced from potato cultivar J55,
such as French fries, potato chips, dehydrated potato material,
potato flakes and potato granules. All publications cited in this
application are herein incorporated by reference.
[0003] The potato is the world's fourth most important food crop
and by far the most important vegetable. Potatoes are currently
grown commercially in nearly every state of the United States.
Annual potato production exceeds 18 million tons in the United
States and 300 million tons worldwide. The popularity of the potato
derives mainly from its versatility and nutritional value. Potatoes
can be used fresh, frozen or dried, or can be processed into flour,
starch or alcohol. They contain complex carbohydrates and are rich
in calcium, niacin and vitamin C.
[0004] The quality of potatoes in the food industry is adversely
affected by two critical factors: (1) potatoes contain large
amounts of asparagine, a non-essential free amino acid that is
rapidly oxidized to form acrylamide, a carcinogenic product, upon
frying or baking; and (2) potatoes are highly susceptible to
enzymatic browning and discoloration, an undesirable event which
happens when polyphenol oxidase leaks out from the damaged plastids
of bruised potatoes. In the cytoplasm, the enzyme oxidizes phenols,
which then rapidly polymerize to produce dark pigments. Tubers
contain large amounts of phosphorylated starch, some of which is
degraded during storage to produce glucose and fructose. These
reducing sugars react with amino acids to form Maillard products
including acrylamide when heated at temperatures above 120.degree.
C. Two enzymes involved in starch phosphorylation are water
dikinase R1 and phosphorylase-L (R1 and PhL). Browning is also
triggered non-enzymatically as a consequence of the partial
degradation of starch into glucose and fructose.
[0005] To date, there are no potato plant varieties that produce
tubers with low acrylamide content, increased black spot bruise
tolerance and reduced senescence sweetening. Thus, there is a need
to develop potato varieties with reduced levels of toxic compounds,
but without the use of unknown or foreign nucleic acids. The
present invention satisfies this need.
[0006] The foregoing examples of the related art and limitations
related therewith are intended to be illustrative and not
exclusive. Other limitations of the related art will become
apparent to those of skill in the art upon a reading of the
specification.
SUMMARY OF THE INVENTION
[0007] The following embodiments and aspects thereof are described
in conjunction with systems, tools and methods which are meant to
be exemplary, not limiting in scope. In various embodiments, one or
more of the above-described problems have been reduced or
eliminated, while other embodiments are directed to other
improvements.
[0008] To this end, the present invention provides novel potato
variety J55 transformed with nucleic acid sequences that are native
to the potato plant genome and does not contain foreign DNA,
Agrobacterium DNA, viral markers or vector backbone sequences.
Rather, the DNA inserted into the genome of the potato variety J55
is a non-coding polynucleotide native to potato or native to wild
potato, a potato sexually-compatible plant, that silences genes
involved in the expression of black spot bruises, asparagine
accumulation and senescence sweetening.
[0009] Thus, in one embodiment, the present invention provides a
plant vector, referred to as pSIM278, that comprises a first
silencing cassette containing two copies of a DNA segment
comprising, in anti-sense orientation, a fragment of the asparagine
synthetase-1 gene (fAsn1) and the 3'-untranslated sequence of the
polyphenol oxidase-5 gene; and a second silencing cassette
containing two copies of a DNA segment comprising, in anti-sense
orientation, a fragment of the potato phosphorylase-L (pPhL) gene
and a fragment of the potato R1 gene. The pSIM1278 vector comprises
a 9,511 bp backbone region that supports maintenance of the plant
DNA prior to plant transformation and is not transferred into plant
cells upon transformation of the plant cells, and a 10,147 bp DNA
insert region comprising native DNA that is stably integrated into
the genome of the plant cells upon transformation.
[0010] In a different embodiment, the invention provides a plant
cell transformed with the plant vector of the invention. In a
further embodiment, the invention provides a potato plant variety
comprising one or more cells transformed with the vector of the
invention. In one aspect of the invention, the potato plant variety
expresses at least one of the two silencing cassettes of the
vector, and expression of the silencing cassette results in the
down-regulation of the asparagine synthetase-1 gene and the
polyphenol oxidase-5 gene in the tubers of the intragenic plant. In
a preferred aspect of the invention, the tubers of the potato plant
variety expressing at least one silencing cassette display two or
more desirable traits that are not present in the tubers of
untransformed plants of the same variety. In the most preferred
aspect of the invention, the two or more desirable traits are
selected from the group consisting of low asparagine accumulation,
reduced black-spot bruising and reduced heat-induced acrylamide
formation.
[0011] In a different aspect of the invention, the potato plant
variety expresses both silencing cassettes of the plant DNA vector,
and expression of the silencing cassettes results in the
down-regulation of the asparagine synthetase-1 gene, the polyphenol
oxidase-5 gene, the phosphorylase-L gene and the dikinase R1 gene
in the tubers of the potato plant variety. In a preferred aspect of
the invention, the tubers of the potato plant variety expressing
two silencing cassettes of the plant DNA vector display two or more
desirable traits that are not present in the tubers of
untransformed plants of the same variety. In a preferred
embodiment, the two or more desirable traits are selected from the
group consisting of low asparagine accumulation, reduced black-spot
bruising, reduced accumulation of reducing sugars during storage
and reduced heat-induced acrylamide formation. In one aspect of the
invention, the potato plant variety expressing the two silencing
cassettes of the plant DNA vector is the Atlantic J55 variety.
[0012] Thus, according to the invention, there is provided a new
potato cultivar of the genus and species Solanum tuberosum L.
designated J55. This invention thus relates to potato cultivar J55,
to the tubers of potato cultivar J55, to the plants of potato
cultivar J55, to the seeds of potato cultivar J55, to the food
products produced from potato cultivar J55, and to methods for
producing a potato plant produced by selfing potato cultivar J55 or
by crossing potato cultivar J55 with another potato cultivar, and
the creation of variants by mutagenesis or transformation of potato
cultivar J55.
[0013] Thus, any such methods using the cultivar J55 are
embodiments of this invention: selfing, backcrosses, hybrid
production, crosses to populations, and the like. All plants
produced using potato cultivar J55 as at least one parent are
within the scope of this invention. Advantageously, the potato
cultivar could be used in crosses with other, different, potato
plants to produce first generation (F.sub.1) potato hybrid tubers,
seeds and plants with superior characteristics.
[0014] In another embodiment, the present invention provides for
single or multiple gene converted plants of potato cultivar J55. In
one embodiment, the transferred gene(s) may be a dominant or
recessive allele(s). In some embodiments, the transferred gene(s)
will confer such traits as herbicide resistance, insect resistance,
resistance for bacterial, fungal, or viral disease, male fertility,
male sterility, enhanced nutritional quality, uniformity, and
increase in concentration of starch and other carbohydrates,
decrease in tendency to bruise and decrease in the rate of
conversion of starch to sugars. The gene(s) may be a naturally
occurring potato gene or a transgene introduced through genetic
engineering techniques, backcrossing or mutation.
[0015] In another embodiment, the present invention provides
regenerable cells for use in tissue culture of potato cultivar J55.
In one embodiment, the tissue culture will be capable of
regenerating plants having all the physiological and morphological
characteristics of the foregoing potato plant, and of regenerating
plants having substantially the same genotype as the foregoing
potato plant. In some embodiments, the regenerable cells in such
tissue cultures will be embryos, protoplasts, meristematic cells,
callus, pollen, leaves, anthers, pistils, cotyledons, hypocotyl,
roots, root tips, flowers, seeds, petioles, tubers, eyes or stems.
Still further, the present invention provides potato plants
regenerated from tissue cultures of the invention.
[0016] In a further embodiment, the invention provides a food
product made from a tuber of potato plant variety Atlantic J55.
Preferably, the food product is a heat-treated product. Even more
preferably, the food product is a French fry, potato chip,
dehydrated potato material, potato flakes, or potato granules.
[0017] In addition to the exemplary aspects and embodiments
described above, further aspects and embodiments will become
apparent by study of the following descriptions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 depicts the pSIM1278 transformation vector. The
vector backbone region, on the left, is 9,511 bp long, as it starts
at position 9,957 bp and ends at position 19,468 bp. The backbone
DNA consists mainly of bacterial DNA which provides support
maintenance of the DNA insert prior to plant transformation. The
DNA insert region (right side), including flanking Border
sequences, is 10,147 bp long (from 19,469 bp to 19,660 bp and from
1 bp to 9,956 bp). The DNA insert consists of native DNA only and
was stably integrated into the potato genome upon
transformation.
[0019] FIG. 2 provides a schematic representation of the silencing
cassettes in the DNA insert inserted in the pSIM1278 transformation
vector. Each silencing cassette contains two copies of two gene
fragments separated by a spacer. Two copies of a DNA segment
comprising fragments of four targeted genes, namely Asn-1, Ppo-5,
Ph1 and R1, were inserted as inverted repeats between two
convergent promoters, indicated as Pro, that are predominantly
active in tubers. Plants containing the resulting silencing
cassette produce a diverse and unpolyadenylated array of RNA
molecules in tubers that dynamically and vigorously silence the
intended target genes. The size of the RNA molecules was generally
smaller than the distance between the two promoters employed
because convergent transcription results in collisional
transcription.
DETAILED DESCRIPTION OF THE INVENTION
[0020] In the description and tables which follow, a number of
terms are used. In order to provide a clear and consistent
understanding of the specification and claims, including the scope
to be given such terms, the following definitions are provided:
[0021] Allele. An allele is any of one or more alternative forms of
a gene which relate to one trait or characteristic. In a diploid
cell or organism, the two alleles of a given gene occupy
corresponding loci on a pair of homologous chromosomes.
[0022] Amino acid sequence. As used herein, includes an
oligopeptide, peptide, polypeptide, or protein and fragments
thereof that are isolated from, native to, or naturally occurring
in a plant, or are synthetically made but comprise the nucleic acid
sequence of the endogenous counterpart.
[0023] Artificially manipulated. as used herein, "artificially
manipulated" means to move, arrange, operate or control by the
hands or by mechanical means or recombinant means, such as by
genetic engineering techniques, a plant or plant cell, so as to
produce a plant or plant cell that has a different biological,
biochemical, morphological, or physiological phenotype and/or
genotype in comparison to unmanipulated, naturally-occurring
counterpart.
[0024] Asexual propagation. Producing progeny by generating an
entire plant from leaf cuttings, stem cuttings, root cuttings,
tuber eyes, stolons, single plant cells protoplasts, callus and the
like, that does not involve fusion of gametes.
[0025] Backbone. Nucleic acid sequence of a binary vector that
excludes the DNA insert sequence intended for transfer.
[0026] Backcrossing. Backcrossing is a process in which a breeder
repeatedly crosses hybrid progeny back to one of the parents, for
example, a first generation hybrid F.sub.1 with one of the parental
genotypes of the F.sub.1 hybrid.
[0027] Bacterial Ring Rot. Bacterial ring rot is a disease caused
by the bacterium Clavibacter michiganense ssp. Bacterial ring rot
derives its name from a characteristic breakdown of the vascular
ring within the tuber. This ring often appears as a creamy-yellow
to light-brown, cheesy rot. On the outer surface of the potato,
severely diseased tubers may show slightly sunken, dry and cracked
areas. Symptoms of bacterial ring rot in the vascular tissue of
infected tubers can be less obvious than described above, appearing
as only a broken, sporadically appearing dark line or as a
continuous, yellowish discoloration.
[0028] Black spot bruise. Black spots found in bruised tuber tissue
are a result of a pigment called melanin that is produced following
the injury of cells and gives tissue a brown, gray or black
appearance. Melanin is formed when phenol substrates and an
appropriate enzyme come in contact with each other as a result of
cellular damage. The damage does not require broken cells. However,
mixing of the substrate and enzyme must occur, usually when the
tissue is impacted. Black spots occur primarily in the
perimedullary tissue just beneath the vascular ring, but may be
large enough to include a portion of the cortical tissue.
[0029] Border-like sequences. A "border-like" sequence is isolated
from the selected plant species that is to be modified, or from a
plant that is sexually-compatible with the plant species to be
modified, and functions like the border sequences of Agrobacterium.
That is, a border-like sequence of the present invention promotes
and facilitates the integration of a polynucleotide to which it is
linked. A DNA insert of the present invention preferably contains
border-like sequences. A border-like sequence of a DNA insert is
between 5-100 bp in length, 10-80 bp in length, 15-75 bp in length,
15-60 bp in length, 15-50 bp in length, 15-40 bp in length, 15-30
bp in length, 16-30 bp in length, 20-30 bp in length, 21-30 bp in
length, 22-30 bp in length, 23-30 bp in length, 24-30 bp in length,
25-30 bp in length, or 26-30 bp in length. A DNA insert left and
right border sequence are isolated from and/or native to the genome
of a plant that is to be modified. A DNA insert border-like
sequence is not identical in nucleotide sequence to any known
Agrobacterium-derived T-DNA border sequence. Thus, a DNA insert
border-like sequence may possess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides that are
different from a T-DNA border sequence from an Agrobacterium
species, such as Agrobacterium tumefaciens or Agrobacterium
rhizogenes. That is, a DNA insert border, or a border-like sequence
of the present invention has at least 95%, at least 90%, at least
80%, at least 75%, at least 70%, at least 60% or at least 50%
sequence identity with a T-DNA border sequence from an
Agrobacterium species, such as Agrobacterium tumefaciens or
Agrobacterium rhizogenes, but not 100% sequence identity. As used
herein, the descriptive terms "DNA insert border" and "DNA insert
border-like" are exchangeable. A border-like sequence can be
isolated from a plant genome and be modified or mutated to change
the efficiency by which it is capable of integrating a nucleotide
sequence into another nucleotide sequence. Other polynucleotide
sequences may be added to or incorporated within a border-like
sequence of the present invention. Thus, a DNA insert left border
or a DNA insert right border may be modified so as to possess 5'-
and 3'-multiple cloning sites, or additional restriction sites. A
DNA insert border sequence may be modified to increase the
likelihood that backbone DNA from the accompanying vector is not
integrated into the plant genome.
[0030] Consisting essentially of. A composition "consisting
essentially of" certain elements is limited to the inclusion of
those elements, as well as to those elements that do not materially
affect the basic and novel characteristics of the inventive
composition. Thus, so long as the composition does not affect the
basic and novel characteristics of the instant invention, that is,
does not contain foreign DNA that is not from the selected plant
species or a plant that is sexually compatible with the selected
plant species, then that composition may be considered a component
of an inventive composition that is characterized by "consisting
essentially of" language.
[0031] Cotyledon. A cotyledon is a type of seed leaf. The cotyledon
contains the food storage tissues of the seed.
[0032] Degenerate primer. A "degenerate primer" is an
oligonucleotide that contains sufficient nucleotide variations that
it can accommodate base mismatches when hybridized to sequences of
similar, but not exact, homology.
[0033] Dicotyledon (dicot). A flowering plant whose embryos have
two seed leaves or cotyledons. Examples of dicots include, but are
not limited to, tobacco, tomato, potato, sweet potato, cassava,
legumes including alfalfa and soybean, carrot, strawberry, lettuce,
oak, maple, walnut, rose, mint, squash, daisy, and cactus.
[0034] DNA insert. According to the present invention, the DNA
insert to be inserted into the genome of a plant comprises
polynucleotide sequences native to that plant or has native genetic
elements to that plant. In one example, for instance, the DNA
insert of the potato variety J55 of the present invention is a
10,147 bp non-coding polynucleotide that is native to potato or
wild potato, a potato sexually-compatible plant, that is stably
integrated into the genome of the plant cells upon transformation
and silences genes involved in the expression of black spot
bruises, asparagine accumulation and senescence sweetening. The DNA
insert preferably comprises two expression cassettes and is
inserted into a transformation vector referred to as the pSIM1278
transformation vector. The first cassette comprises fragments of
both the asparagine synthetase-1 gene (Asn1) and the polyphenol
oxidase-5 gene (Ppo5), arranged as inverted repeats between the Agp
promoter of the ADP glucose pyrophosphorylase gene (Agp) and the
Gbss promoter of the granule-bound synthase gene (Gbss). These
promoters are predominantly active in tubers. The function of the
second cassette is to silence the promoters of the starch
associated gene dikinase-R1 (R1) and the phosphorylase-L gene
(PhL). This cassette is comprised of fragments of the promoters of
the starch associated gene dikinase-R1 (R1) and the phosphorylase-L
gene (PhL), operably linked to the same Agp and Gbss promoters as
the first cassette. These expression cassettes contain no foreign
DNA, and consist of DNA only from either the selected plant species
or from a plant that is sexually compatible with the selected plant
species.
[0035] Embryo. The embryo is the immature plant contained within a
mature seed.
[0036] Foreign. "Foreign," with respect to a nucleic acid, means
that that nucleic acid is derived from non-plant organisms, or
derived from a plant that is not the same species as the plant to
be transformed or is not derived from a plant that is not
interfertile with the plant to be transformed, does not belong to
the species of the target plant. According to the present
invention, foreign DNA or RNA represents nucleic acids that are
naturally occurring in the genetic makeup of fungi, bacteria,
viruses, mammals, fish or birds, but are not naturally occurring in
the plant that is to be transformed. Thus, a foreign nucleic acid
is one that encodes, for instance, a polypeptide that is not
naturally produced by the transformed plant. A foreign nucleic acid
does not have to encode a protein product. According to the present
invention, a desired intragenic plant is one that does not contain
any foreign nucleic acids integrated into its genome.
[0037] Gene. As used herein, "gene" refers to the coding region and
does not include nucleotide sequences that are 5'- or 3'- to that
region. A functional gene is the coding region operably linked to a
promoter or terminator. A gene can be introduced into a genome of a
species, whether from a different species or from the same species,
using transformation or various breeding methods.
[0038] Gene Converted (Conversion). Gene converted (conversion)
plant refers to plants which are developed by a plant breeding
technique called backcrossing wherein essentially all of the
desired morphological and physiological characteristics of a
variety are recovered in addition to the one or more genes
transferred into the variety via the backcrossing technique, via
genetic engineering or via mutation. One or more loci may also be
transferred.
[0039] Genetic rearrangement. Refers to the re-association of
genetic elements that can occur spontaneously in vivo as well as in
vitro which introduce a new organization of genetic material. For
instance, the splicing together of polynucleotides at different
chromosomal loci, can occur spontaneously in vivo during both plant
development and sexual recombination. Accordingly, recombination of
genetic elements by non-natural genetic modification techniques in
vitro is akin to recombination events that also can occur through
sexual recombination in vivo.
[0040] Golden nematode. Globodera rostochiensis, commonly known as
golden nematode, is a plant parasitic nematode affecting the roots
and tubers of potato plants. Symptoms include poor plant growth,
wilting, water stress and nutrient deficiencies.
[0041] Hypocotyl. A hypocotyl is the portion of an embryo or
seedling between the cotyledons and the root. Therefore, it can be
considered a transition zone between shoot and root.
[0042] In frame. Nucleotide triplets (codons) are translated into a
nascent amino acid sequence of the desired recombinant protein in a
plant cell. Specifically, the present invention contemplates a
first nucleic acid linked in reading frame to a second nucleic
acid, wherein the first nucleotide sequence is a gene and the
second nucleotide is a promoter or similar regulatory element.
[0043] Integrate. Refers to the insertion of a nucleic acid
sequence from a selected plant species, or from a plant that is
from the same species as the selected plant, or from a plant that
is sexually compatible with the selected plant species, into the
genome of a cell of a selected plant species. "Integration" refers
to the incorporation of only native genetic elements into a plant
cell genome. In order to integrate a native genetic element, such
as by homologous recombination, the present invention may "use"
non-native DNA as a step in such a process. Thus, the present
invention distinguishes between the "use of" a particular DNA
molecule and the "integration" of a particular DNA molecule into a
plant cell genome.
[0044] Introduction. As used herein, refers to the insertion of a
nucleic acid sequence into a cell, by methods including infection,
transfection, transformation or transduction.
[0045] Isolated. "Isolated" refers to any nucleic acid or compound
that is physically separated from its normal, native environment.
The isolated material may be maintained in a suitable solution
containing, for instance, a solvent, a buffer, an ion, or other
component, and may be in purified, or unpurified, form.
[0046] Leader. Transcribed but not translated sequence preceding
(or 5' to) a gene.
[0047] Locus. A locus confers one or more traits such as, for
example, male sterility, herbicide tolerance, insect resistance,
disease resistance, waxy starch, modified fatty acid metabolism,
modified phytic acid metabolism, modified carbohydrate metabolism,
and modified protein metabolism. The trait may be, for example,
conferred by a naturally occurring gene introduced into the genome
of the variety by backcrossing, a natural or induced mutation, or a
transgene introduced through genetic transformation techniques. A
locus may comprise one or more alleles integrated at a single
chromosomal location.
[0048] Marketable Yield. Marketable yield is the weight of all
tubers harvested that are between 2 and 4 inches in diameter.
Marketable yield is measured in cwt (hundred weight) where cwt=100
pounds.
[0049] Monocotyledon (monocot). A flowering plant whose embryos
have one cotyledon or seed leaf Examples of monocots include, but
are not limited to turf grass, maize, rice, oat, wheat, barley,
sorghum, orchid, iris, lily, onion, and palm.
[0050] Native. A "native" genetic element refers to a nucleic acid
that naturally exists in, originates from, or belongs to the genome
of a plant that is to be transformed. Thus, any nucleic acid, gene,
polynucleotide, DNA, RNA, mRNA, or cDNA molecule that is isolated
either from the genome of a plant or plant species that is to be
transformed or is isolated from a plant or species that is sexually
compatible or interfertile with the plant species that is to be
transformed, is "native" to, i.e., indigenous to, the plant
species. In other words, a native genetic element represents all
genetic material that is accessible to plant breeders for the
improvement of plants through classical plant breeding. Any
variants of a native nucleic acid also are considered "native" in
accordance with the present invention. In this respect, a "native"
nucleic acid may also be isolated from a plant or sexually
compatible species thereof and modified or mutated so that the
resultant variant is greater than or equal to 99%, 98%, 97%, 96%,
95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%,
82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%,
69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, or 60% similar in
nucleotide sequence to the unmodified, native nucleic acid isolated
from a plant. A native nucleic acid variant may also be less than
about 60%, less than about 55%, or less than about 50% similar in
nucleotide sequence. A "native" nucleic acid isolated from a plant
may also encode a variant of the naturally occurring protein
product transcribed and translated from that nucleic acid. Thus, a
native nucleic acid may encode a protein that is greater than or
equal to 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%,
88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%,
75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%,
62%, 61%, or 60% similar in amino acid sequence to the unmodified,
native protein expressed in the plant from which the nucleic acid
was isolated.
[0051] Native genetic elements. "Native genetic elements" can be
incorporated and integrated into a selected plant species genome
according to the present invention. Native genetic elements are
isolated from plants that belong to the selected plant species or
from plants that are sexually compatible with the selected plant
species. For instance, native DNA incorporated into cultivated
potato (Solanum tuberosum) can be derived from any genotype of S.
tuberosum or any genotype of a wild potato species that is sexually
compatible with S. tuberosum (e.g., S. demissum).
[0052] Naturally occurring nucleic acid. Naturally occurring
nucleic acid are found within the genome of a selected plant
species and may be a DNA molecule or an RNA molecule. The sequence
of a restriction site that is normally present in the genome of a
plant species can be engineered into an exogenous DNA molecule,
such as a vector or oligonucleotide, even though that restriction
site was not physically isolated from that genome. Thus, the
present invention permits the synthetic creation of a nucleotide
sequence, such as a restriction enzyme recognition sequence, so
long as that sequence is naturally occurring in the genome of the
selected plant species or in a plant that is sexually compatible
with the selected plant species that is to be transformed.
[0053] Operably linked. Combining two or more molecules in such a
fashion that in combination they function properly in a plant cell.
For instance, a promoter is operably linked to a structural gene
when the promoter controls transcription of the structural
gene.
[0054] Plant. As used herein, the term "plant" includes but is not
limited to angiosperms and gymnosperms such as potato, tomato,
tobacco, alfalfa, lettuce, carrot, strawberry, sugarbeet, cassava,
sweet potato, soybean, maize, turf grass, wheat, rice, barley,
sorghum, oat, oak, eucalyptus, walnut, and palm. Thus, a plant may
be a monocot or a dicot. The word "plant," as used herein, also
encompasses plant cells, seed, plant progeny, propagule whether
generated sexually or asexually, and descendents of any of these,
such as cuttings or seed. Plant cells include suspension cultures,
callus, embryos, meristematic regions, callus tissue, leaves,
roots, shoots, gametophytes, sporophytes, pollen, seeds and
microspores. Plants may be at various stages of maturity and may be
grown in liquid or solid culture, or in soil or suitable media in
pots, greenhouses or fields. Expression of an introduced leader,
trailer or gene sequences in plants may be transient or permanent.
A "selected plant species" may be, but is not limited to, a species
of any one of these "plants."
[0055] Plant Parts. As used herein, the term "plant parts" (or a
potato plant, or a part thereof) includes but is not limited to
protoplast, leaf, stem, root, root tip, anther, pistil, seed,
embryo, pollen, ovule, cotyledon, hypocotyl, flower, tuber, eye,
tissue, petiole, cell, meristematic cell, and the like.
[0056] Plant species. The group of plants belonging to various
officially named plant species that display at least some sexual
compatibility.
[0057] Plant transformation and cell culture. Broadly refers to the
process by which plant cells are genetically modified and
transferred to an appropriate plant culture medium for maintenance,
further growth, and/or further development.
[0058] Precise breeding. Refers to the improvement of plants by
stable introduction of nucleic acids, such as native genes and
regulatory elements isolated from the selected plant species, or
from another plant in the same species as the selected plant, or
from species that are sexually compatible with the selected plant
species, into individual plant cells, and subsequent regeneration
of these genetically modified plant cells into whole plants. Since
no unknown or foreign nucleic acid is permanently incorporated into
the plant genome, the inventive technology makes use of the same
genetic material that is also accessible through conventional plant
breeding.
[0059] Progeny. As used herein, includes an F.sub.1 potato plant
produced from the cross of two potato plants where at least one
plant includes potato cultivar J55 and progeny further includes,
but is not limited to, subsequent F.sub.2, F.sub.3, F.sub.4,
F.sub.5, F.sub.6, F.sub.7, F.sub.8, F.sub.9, and F.sub.10
generational crosses with the recurrent parental line.
[0060] Quantitative Trait Loci (QTL). Quantitative trait loci (QTL)
refer to genetic loci that control to some degree numerically
representable traits that are usually continuously distributed.
[0061] Recombinant. As used herein, broadly describes various
technologies whereby genes can be cloned, DNA can be sequenced, and
protein products can be produced. As used herein, the term also
describes proteins that have been produced following the transfer
of genes into the cells of plant host systems.
[0062] Regeneration. Regeneration refers to the development of a
plant from tissue culture.
[0063] Regulatory sequences. Refers to those sequences which are
standard and known to those in the art, that may be included in the
expression vectors to increase and/or maximize transcription of a
gene of interest or translation of the resulting RNA in a plant
system. These include, but are not limited to, promoters, peptide
export signal sequences, introns, polyadenylation, and
transcription termination sites. Methods of modifying nucleic acid
constructs to increase expression levels in plants are also
generally known in the art (see, e.g. Rogers et al., 260 J. Biol.
Chem. 3731-38, 1985; Cornejo et al., 23 Plant Mol. Biol. 567:
81,1993). In engineering a plant system to affect the rate of
transcription of a protein, various factors known in the art,
including regulatory sequences such as positively or negatively
acting sequences, enhancers and silencers, as well as chromatin
structure may have an impact. The present invention provides that
at least one of these factors may be utilized in engineering plants
to express a protein of interest. The regulatory sequences of the
present invention are native genetic elements, i.e., are isolated
from the selected plant species to be modified.
[0064] Selectable marker. A "selectable marker" is typically a gene
that codes for a protein that confers some kind of resistance to an
antibiotic, herbicide or toxic compound, and is used to identify
transformation events. Examples of selectable markers include the
streptomycin phosphotransferase (spt) gene encoding streptomycin
resistance, the phosphomannose isomerase (pmi) gene that converts
mannose-6-phosphate into fructose-6 phosphate; the neomycin
phosphotransferase (nptII) gene encoding kanamycin and geneticin
resistance, the hygromycin phosphotransferase (hpt or aphiv) gene
encoding resistance to hygromycin, acetolactate synthase (als)
genes encoding resistance to sulfonylurea-type herbicides, genes
coding for resistance to herbicides which act to inhibit the action
of glutamine synthase such as phosphinothricin or basta (e.g., the
bar gene), or other similar genes known in the art.
[0065] Sense suppression. Reduction in expression of an endogenous
gene by expression of one or more an additional copies of all or
part of that gene in transgenic plants.
[0066] Specific gravity. As used herein, "specific gravity" is an
expression of density and is a measurement of potato quality. There
is a high correlation between the specific gravity of the tuber and
the starch content and percentage of dry matter or total solids. A
higher specific gravity contributes to higher recovery rate and
better quality of the processed product.
[0067] T-DNA-Like. A "T-DNA-like" sequence is a nucleic acid that
is isolated from a selected plant species, or from a plant that is
sexually compatible with the selected plant species, and which
shares at least 75%, 80%, 85%, 90%, or 95%, but not 100%, sequence
identity with Agrobacterium species T-DNA. The T-DNA-like sequence
may contain one or more border or border-like sequences that are
each capable of integrating a nucleotide sequence into another
polynucleotide.
[0068] Total Yield. Total yield refers to the total weight of all
harvested tubers.
[0069] Trailer. Transcribed but not translated sequence following
(or 3' to) a gene.
[0070] Transcribed DNA. DNA comprising both a gene and the
untranslated leader and trailer sequence that are associated with
that gene, which is transcribed as a single mRNA by the action of
the preceding promoter.
[0071] Transformation of plant cells. A process by which DNA is
stably integrated into the genome of a plant cell. "Stably" refers
to the permanent, or non-transient retention and/or expression of a
polynucleotide in and by a cell genome. Thus, a stably integrated
polynucleotide is one that is a fixture within a transformed cell
genome and can be replicated and propagated through successive
progeny of the cell or resultant transformed plant. Transformation
may occur under natural or artificial conditions using various
methods well known in the art. Transformation may rely on any known
method for the insertion of nucleic acid sequences into a
prokaryotic or eukaryotic host cell, including
Agrobacterium-mediated transformation protocols, viral infection,
whiskers, electroporation, heat shock, lipofection, polyethylene
glycol treatment, micro-injection, and particle bombardment.
[0072] Transgene. A gene that will be inserted into a host genome,
comprising a protein coding region. In the context of the instant
invention, the elements comprising the transgene are isolated from
the host genome.
[0073] Transgenic plant. A genetically modified plant which
contains at least one transgene.
[0074] Variant. A "variant," as used herein, is understood to mean
a nucleotide or amino acid sequence that deviates from the
standard, or given, nucleotide or amino acid sequence of a
particular gene or protein. The terms, "isoform," "isotype," and
"analog" also refer to "variant" forms of a nucleotide or an amino
acid sequence. An amino acid sequence that is altered by the
addition, removal or substitution of one or more amino acids, or a
change in nucleotide sequence, may be considered a "variant"
sequence. The variant may have "conservative" changes, wherein a
substituted amino acid has similar structural or chemical
properties, e.g., replacement of leucine with isoleucine. A variant
may have "nonconservative" changes, e.g., replacement of a glycine
with a tryptophan. Analogous minor variations may also include
amino acid deletions or insertions, or both. Guidance in
determining which amino acid residues may be substituted, inserted,
or deleted may be found using computer programs well known in the
art such as Vector NTI Suite (InforMax, MD) software.
[0075] Vine Maturity. Vine maturity refers to a plant's ability to
continue to utilize carbohydrates and photosynthesize. Vine
maturity is scored on a scale of 1 to 5 where 1=dead vines and
5=vines green, still flowering.
[0076] The insertion of desirable traits into the genome of potato
plants presents particular difficulties because potato is
tetraploid, highly heterozygous and sensitive to in-breeding
depression. It is therefore very difficult to efficiently develop
transgenic potato plants that produce less acrylamide and less
harmful Maillard-reaction products, including
N-Nitroso-N-(3-keto-1,2-butanediol)-3'-nitrotyramine (Wang et al.,
Arch Toxicol 70: 10-5, 1995), 5-hydroxymethyl-2-furfural (Janzowski
et al., Food Chem Toxicol 38: 801-9, 2000), and other Maillard
reaction products with mutagenic properties (Shibamoto, Prog Clin
Biol Res 304: 359-76, 1989), during processing using conventional
breeding.
[0077] Several methods have been tested and research is ongoing to
reduce acrylamide through process changes, reduction in dextrose,
and additives such as asparaginase, citrate, and competing amino
acids. The required capital expense to implement process changes
throughout the potato industry would cost millions of dollars. In
addition to the expense, these process changes have significant
drawbacks including potentially negative flavors associated with
additives such as asparaginase or citrate. Typically, fry
manufacturers add dextrose during processing of french fries to
develop the desired golden brown color, but dextrose also increases
the formation of acrylamide through the Maillard reaction.
Significant reductions in acrylamide occur by merely omitting
dextrose from the process; however, the signature golden brown
colors must then be developed some other way (such as though the
addition of colors like annatto) The use of alternate colors,
results in an absence of the typical flavors that develop through
those browning reactions. Another challenge with the use of
additives to reduce reactants like asparagine is moisture migration
that occurs during frozen storage with the resulting return of
asparagine to the surface and increased acrylamide. Finally, the
blackening that occurs after potatoes are bruised affects quality
and recovery in processing French fries and chips. Damaged and
bruised potatoes must be trimmed or are rejected before processing,
resulting in quality challenges or economic loss.
[0078] The "native technology" strategy of the present invention
addresses the need of the potato industry to improve the agronomic
characteristics and nutritional value of potatoes by reducing the
expression of polyphenol oxidase-5 (PPO-5), which is responsible
for black spot bruise, the expression of asparagine synthetase-1
(Asn-1), which is responsible for the accumulation of asparagine, a
precursor in acrylamide formation, and/or the expression of
phosphorylase-L and kinase-R1, which are enzymes associated with
the accumulation of reducing sugars that normally react with amino
acids, such as asparagine, and form toxic Maillard products,
including acrylamide. The partial or complete silencing of these
genes in tubers decreases the potential to produce acrylamide. Use
of the native technology of the invention allows for the
incorporation of desirable traits into the genome of commercially
valuable potato plant varieties by transforming the potatoes only
with "native" genetic material, that is genetic material obtained
from potato plants or plants that are sexually-compatible with
potato plants, that contains only non-coding regulatory regions,
without the integration of any foreign genetic material into the
plant's genome. Desirable traits include high tolerance to
impact-induced black spot bruise, reduced formation of the
acrylamide precursor asparagine and reduced accumulation of
reducing sugars, with consequent decrease in accumulation of toxic
Maillard products, including acrylamide, improved quality and food
color control. The incorporation of these desirable traits into
existing potato varieties is impossible to achieve through
traditional breeding because potato is tetraploid, highly
heterozygous and sensitive to inbreeding depression.
[0079] The non-coding potato plant DNA insert sequences used in the
present invention are native to the potato plant genome and do not
contain any Agrobacterium DNA. The DNA insert preferably comprises
two expression cassettes and is inserted into a transformation
vector referred to as the pSIM1278 transformation vector. The first
cassette comprises fragments of both the asparagine synthetase-1
gene (Asn1) and the polyphenol oxidase-5 gene (Ppo5), arranged as
inverted repeats between the Agp promoter of the ADP glucose
pyrophosphorylase gene (Agp) and the Gbss promoter of the
granule-bound synthase gene (Gbss). These promoters are
predominantly active in tubers. The function of the second cassette
is to silence the promoters of the starch associated gene
dikinase-R1 (R1) and the phosphorylase-L gene (PhL). This cassette
is comprised of fragments of the promoters of the starch associated
gene dikinase-R1 (R1) and the phosphorylase-L gene (PhL), operably
linked to the same Agp and Gbss promoters as the first cassette.
These expression cassettes contain no foreign DNA, and consist of
DNA only from either the selected plant species or from a plant
that is sexually compatible with the selected plant species.
[0080] The commercially valuable potato plant variety used in the
present invention is Atlantic. Atlantic plants are moderately
large, with thick, upright stems, and slightly swollen, sparsely
pubescent nodes. Leaves are bright, medium green, smooth, and
moderately pubescent with prominent wings, large asymmetrical
primary leaflets and numerous secondary and tertiary leaflets.
Flowers are profuse with green, awl-shaped, pubescent calyx lobes,
pale lavender corolla, orange anthers and abundant, viable pollen.
The Atlantic cultivar is tolerant to scab and Verticillium wilt,
resistant to pinkeye, and highly resistant to Race A of golden
nematode, viruses, tuber net necrosis, and black spot bruise.
Tubers of Atlantic are susceptible to internal heat necrosis,
particularly in sandy soils in warm, dry seasons. Hollow heart in
the larger diameter tubers (>0.83 mm) can be serious in some
growing areas. Tubers are oval to round with light to heavy scaly
netted skin, moderately shallow eyes and white flesh, and tuber
dormancy is medium-long. With high yield potential, high specific
gravity and uniform tuber size and shape, Atlantic is the standard
variety for chipping from the field or from very short-term storage
(Webb et al., 1978). The variety is fertile and mainly grown in the
Northeast and Southeast, especially for the production of
chips.
[0081] The present invention provides a potato variety of
significant market value--namely Atlantic--transformed with the
transformation vector pSIM1278, identified using the polymerase
chain reaction rather than markers, and successfully propagated.
Also provided are food products made from the tubers of the potato
plant variety J55 of the present invention. Potato cultivar J55 has
the following unique plant variety identifier with the Organization
for Economic Cooperation and Development (OECD): SPS-OOJ55-2.
[0082] Targeted gene silencing with native DNA reduces the level of
the RNA transcripts of the targeted genes in the tubers of the
potato plant variety J55. Asn1 and Ppo5 gene silencing is
sufficient to significantly reduce acrylamide formation by two to
four fold without further inhibiting the starch associated genes
kinase-R1 (R1) and phosphorylase-L (PhL). Thus, the tubers of the
intragenic potato plant variety J55 of the invention incorporate
highly desirable traits, including a reduced ratio in free amide
amino acids asparagine and glutamine, which is associated with
reduced acrylamide formation upon frying or baking Specifically,
the potato variety J55 of the present invention is characterized by
two- to more than four-fold reduction in free-asparagine content.
Furthermore, the potato variety J55 of the invention displays a
delay in the degradation of starch into the reducing sugars glucose
and fructose during storage. Impairment of starch-to-sugar
conversion further reduces senescence sweetening and acrylamide
formation and limits heat-induced browning.
[0083] Potato variety J55 of the present invention is therefore
extremely valuable in the potato industry and food market, as its
tubers produce significantly less acrylamide upon heat processing
and do not carry any potentially harmful foreign genes.
EXAMPLES
[0084] The present invention uses native technology to integrate
native non-coding DNA into the genome of selected potato plant
varieties to develop new intragenic potato plant varieties. The
method includes trait identification, design of vectors,
incorporation of vectors into Agrobacterium, selection of the
recipient potato variety, plant transformation, evidence of absence
of open reading frames, and confirmation that the new potato plant
varieties contain only the native DNA. The potato cultivar J55 of
the present invention has a lowered potential to form acrylamide
and lower amounts of sucrose than its untransformed
counterpart.
Example 1
The pSIM1278 Transformation Vector
[0085] The transformation vector pSIM1278 used in the invention was
derived from pSIM106, which was created by ligating a 0.4-kb potato
plant DNA fragment (deposited as GenBank accession no. AY566555)
with a 5.9-kb SacII-SphI fragment of pCAMBIA1301 (CAMBIA, Canberra,
Australia), carrying bacterial origins of replication from plasmids
pVS1 and pBR322, and the nptIII gene for bacterial resistance to
kanamycin. An expression cassette comprising the Agrobacterium ipt
gene preceded by the Ubi-3 promoter (Garbarino and Belknap, 1994)
and followed by the Ubi-3 terminator was introduced as a 2.6-kb
SacII fragment into the vector backbone (Rommens et al., 2004).
Insertion of the native 10-kb DNA segment carrying two silencing
cassettes into the DNA insert of pSIM106 yielded pSIM1278. This
vector was used for all transformations. The pSIM1278 vector map is
shown in FIG. 1. The vector backbone region is 9,511 bp, as it
starts at position 9,957 bp and ends at position 19,468 bp. The
backbone DNA consists mainly of bacterial DNA and provides support
maintenance of the DNA insert prior to plant transformation. The
backbone portion is not transferred into the plant cells. The
various elements of the backbone are described in Table 1.
TABLE-US-00001 TABLE 1 Other Genbank Start-End effects on Accession
Point in Genetic Element Origin Intended Function plant Number
pSIM1278 Reference Pat promoter (pPat) Solanum Drives expression of
the ipt None HM439286 17,479- Unpublished including the coding
tuberosum var. backbone marker gene 19,2178 sequence for a 76-
Ranger Russet amino-acid potato ubiquitin monomer Isopentenyl
transferase Agrobacterium condensation of AMP and Cytokinin
NC_002377.1 16,744- Smigocki (ipt) gene tumefaciens
isopentenylpyrophosphate formation 17,466 and Owens, to form
isopentenyl-AMP, 1988 a cytokinin Terminator of the Solanum
Terminate ipt gene None GP755544.1 16,038- Garbarino ubiquitin-3
gene tuberosum transcription 16,392 and Belknap, (tUbi3) 1994
Neomycin E. coli Aminoglycoside None FJ362602.1 15,048- Courvalin
et phosphotransferase III phosphotransferase 15,842 al., 1977
(nptIII) gene Origin of replication for E. coli Start position for
plasmid None J01784.1 14,477- pBR322 (pBR322 ori) replication in
bacterial cells 14,757 (pBR322 bom) E. coli pBR322 region for None
J01749.1 14,077- replication in E. coli 14,337 pVS1 replicon
Pseudomonas pVS1 region for replication None AJ537514.1 12,667-
(pVS1Rep) fluorescens in Agrobacterium (4,501-5,501) 13,667 plasmid
pVS1 pVS1 partitioning Pseudomonas pVS1 stability None AJ537514.1
11,074- protein StaA (PVS1 fluorescens (6,095-7,095) 12,074 Stat)
plasmid pVS1 overdrive Agrobacterium Enhances cleavage at the None
K00549.1 9,963- tumefaciens Right Border site (103-132) 9,992
Example 2
The Plant DNA Insert and its Open Reading Frames (ORFs)
[0086] The DNA insert region, including the flanking border
sequences, used in the pSIM1278 is 10,147 bp long, from 19,469 bp
to 19,660 bp and from 1 bp to 9,956 bp. The DNA insert consists of
native DNA only and is stably integrated into the potato genome.
The DNA insert or a functional part thereof, is the only genetic
material of vector pSIM1278 that is integrated in the potato plant
varieties of the invention. The DNA insert is described in FIG. 2
and Table 2 below.
TABLE-US-00002 TABLE 2 Genbank Start-End Accession Point in Genetic
Element Origin Intended Function Number pSIM1278 Reference Left
Border (LB) site Synthetic Site for secondary cleavage to AY566555
19,469-19,493 van Haaren release single-stranded DNA (bases 1-25)
et al. 1989 insert from pSIM1278 DNA flanking the LB sequence S.
tuberosum Supports secondary cleavage AY566555 19,494-19,655 var.
Ranger at LB (bases 26-187) Russet KpnI restriction site S.
tuberosum Site for connection of AF393847.1 19,656-1 DNA insert
with LB flanking sequence. Promoter for the ADP glucose S.
tuberosum One of the two convergent HM363752 2-2,261
pyrophosphorylase gene var. Ranger promoters that drives (pAgp),
1st copy Russet expression of an inverted repeat containing
fragments of Asn1 and Ppo5, especially in tubers Fragment of the
asparagine S. tuberosum Generates with (9) double HM363759
2,262-2,666 Chawla et synthetase-1 (Asn1) gene (1st var. Ranger
stranded RNA that triggers al. 2012 copy antisense orientation)
Russet the degradation of Asn1 transcripts to impair asparagine
formation 3'-untranslated sequence of the S. verrucosum Generates
with (8) double HM363754 2,667-2,810 polyphenol oxidase-5 gene
stranded RNA that triggers (Ppo5) (1st copy, in antisense the
degradation of Ppo5 orientation) transcripts to block black spot
bruise development Xba1 restriction site S. tuberosum Cloning site.
U26831.1 2,811-2,816 Spacer-1 S. tuberosum Sequence between the 1st
HM363753 2,817-2,973 var. Ranger inverted repeats Russet
3'-untranslated sequence of the S. verrucosum Generates (6) double
stranded HM363754 2,974-3,117 polyphenol oxidase-5 gene RNA that
triggers the (Ppo5) (2nd copy, in sense degradation of Ppo5
orientation) transcripts to block black spot bruise development
Fragment of the asparagine S. tuberosum Generates with (5) double
HM363759 3,118-3,523 Chawla et synthetase-1 (Asn1) gene (2nd var.
Ranger stranded RNA that triggers al. 2012 copy, in sense
orientation) Russet the degradation of Asn1 transcripts to impair
asparagine formation EcoR1 restriction site S. tuberosum Cloning
site AY027522 3,524-3,529 Promoter for the granule-bound S.
tuberosum One of the two convergent HM363755 3,530-4,215 starch
synthase (pGbss) gene var. Ranger promoters that drives (1st copy,
convergent Russet expression of an inverted orientation relative to
the 1st repeat containing fragments copy of pAgp) of Asn1 and Ppo5,
especially in tubers Spe1 restriction site S. tuberosum Cloning
site AY341425 4,216-4,231 pAgp, 2nd copy S. tuberosum One of the
two convergent HM363752 4,232-6,491 var. Ranger promoters that
drives Russet expression of an inverted repeat containing fragments
of the promoters of PhL and R1, especially in tubers Fragment of
promoter for the S. tuberosum Generates with (16) double HM363758
6,492-7,000 potato phosphorylase-L (pPhL) var. Ranger stranded RNA
that triggers gene (1st copy, in antisense Russet the degradation
of PhL orientation) transcripts to limit the formation of reducing
sugars through starch degradation Fragment of promoter for the S.
tuberosum Generates with (15) double HM363757 7,001-7,532 potato R1
gene (pR1) (1st var. Ranger stranded RNA that triggers copy, in
antisense orientation) Russet the degradation of R1 transcripts to
limit the formation of reducing sugars through starch degradation
Pst1 restriction site S. tuberosum Cloning site AY341425
7,533-7,538 Spacer-2 S. tuberosum Sequence between the 2nd HM363756
7,539-7,796 var. Ranger inverted repeat Russet
[0087] The DNA insert described in Table 2 that was used to create
potato line J55 of the present invention does not activate adjacent
genes and does not adversely affect the phenotype of potato plant
variety J55. In addition, the potato plant variety J55 of the
invention does not produce novel proteins associated with open
reading frames encoded by the DNA insert.
Example 3
The Agrobacterium Strain and Transfection
[0088] The C58-derived Agrobacterium strain AGL1 was developed by
precisely deleting the transfer DNA of the hyper-virulent plasmid
pTiBo542 (Lazo et al., 1991). A transposon insertion in the general
recombination gene (recA) stabilizes recombinant plasmid vectors
such as pSIM1278 (FIG. 1). AGL1 displays resistance against
carbenicillin and rifampicin, and is eliminated from transformed
potato tissue using timentin.
[0089] Stock plants of the Atlantic variety were maintained in
magenta boxes with 40 ml half-strength M516 medium containing 3%
sucrose and 2 g/l gelrite (propagation medium). Potato internode
segments of four to six mm were cut from four-week old plants,
infected with the Agrobacterium AGL1 strain carrying pSIM1278, and
transferred to tissue culture media containing 3% sucrose and 6 g/l
agar (co-cultivation medium). Infected explants were transferred,
after two days, to M404 medium containing 3% sucrose, 6 g/l agar
and 150 mg/l timentin to eliminate Agrobacterium (hormone-free
medium). Details of the methods are described in Richael et al.
(2008).
[0090] After one month, the infected explants were transferred to
fresh medium lacking any synthetic hormones and incubated in a
Percival growth chamber under a 16 hr photoperiod at 24.degree. C.
where they started to form shoots. Many shoots expressed the ipt
gene and displayed a cytokinin overproduction phenotype; these
shoots were not considered for further analyses. PCR genotyping
demonstrated that about 0.3 to 1.5% of the remaining shoots
contained at least part of the P-DNA while lacking the ipt gene.
Thus, no markers were used to select for the transformed plants.
Details on ipt-based marker-free plant transformation were
published by Richael et al. (2008).
[0091] The process of eliminating Agrobacterium started two days
after explant infection. For this purpose, tissues were subjected
to the antibiotic timentin (150 mg/L) until proven to be free of
live Agrobacterium. Proof was obtained by incubating stem fragments
of transformed events on nutrient broth-yeast extract (NBY medium)
for 2 weeks at 28.degree. C. (repeated twice). In accordance with
97 CFR Part 340, transformed plants were transported and planted in
the field only when free of live Agrobacterium.
[0092] Potato plant variety J55 was analyzed by DNA gel blot
analyses to determine the structure and copy number of integrated
DNA insert sequences and to confirm the absence of vector backbone
sequences. In addition, molecular characterization was used to
determine the sequence of the junctions flanking the DNA insert and
show stability of the inserted DNA. Sequencing information of the
junctions provided the basis for developing specific PCR tests for
the intragenic potato plant variety J55. Potato cultivar J55 was
found to contain one full copy of the DNA insert and an additional
truncated and linked copy of the DNA insert that lacks the R1/PHL
promoter silencing cassette, as deduced from hybridization results
when various DNA digests were hybridized with AGP, ASN, PHL and GBS
molecular probes.
Example 4
Evidence for the Absence of the Vector Backbone DNA
[0093] Unlike many commercial transgenic crops, potato cultivar J55
of the invention was confirmed to be free of Agrobacterium-derived
DNA sequences that are used for transformation, such as vector
backbone DNA, by three different methods: 1) First, the presence or
absence of the negative selectable isopentenyl isomerase (ipt)
marker gene in the vector backbone was determined, as inadvertent
transfer of backbone DNA comprising the ipt gene expression
cassette from Agrobacterium to plant cells would trigger ipt gene
expression and, consequently, the formation of the cytokinin-type
hormone isopentenyladenosine, 2) Southern blot hybridization was
then used on the transformed potato plants that had passed the
first screening method to confirm the absence of backbone DNA, and
3) PCR was then designed to amplify fragments indicative of
junctions between DNA insert border regions and flanking backbone
DNA or regions within the backbone DNA that flank the DNA insert.
The efficacy of the method was confirmed by using pSIM1278 DNA as a
positive control. Potato cultivar J55 of the present invention did
not produce PCR bands indicative of the presence of vector backbone
DNA.
Example 5
Stability of the Inserted DNA
[0094] The stability of DNA inserts was evaluated in the original
transformants and again in propagated plant material using both DNA
gel blot hybridization and trait evaluation. These studies were
carried out to ensure that intragenic events expressed the
incorporated traits in a consistent and reliable manner.
Instability might be triggered by rare recombination events or
could also be caused by methylation. Because potatoes are normally
propagated clonally, standard assessments for sexually propagated
crops were not directly applicable, and tubers rather than seeds
were used to define subsequent generations. Results of DNA blot
hybridization demonstrate consistent bands were present in multiple
generations, thus indicating stability. Further evidence for
stability was obtained by confirming trait efficacy in generations
one and two tuber seed.
[0095] DNA insert stability was demonstrated in the
originally-transformed material (G0) by extracting and evaluating
DNA from leaves of plants that had been propagated in vitro and
never planted in soil. For generation-1 (G1) analyses, two
propagated plants from each intragenic variety and one plant from
each control were planted in the greenhouse; one of the tubers
harvested from each plant was planted to obtain leaves from G1
plants that were used to isolate DNA and evaluate the G1
generation. Tubers from this generation were planted again, and
leaves of the resulting G2 plants allowed a characterization of
that generation.
[0096] Hybridization of DNA isolated from the Atlantic potato
cultivar J55 of the present invention with the GBS probe revealed
three common bands (8.6, 7.8 and 7.1-kb) and hybridization with the
AGP probe revealed four bands (7.2, 5.0, 2.0 and 1.4-kb) in all
lanes. These bands are indicative of DNA fragments of the
unmodified genome. The presence of additional bands in the DNA
isolated from the J55 variety, one 2.2-kb band indicative of an
internal DNA insert fragment and others representing a DNA insert
junction fragment (2.3 and 4.1-kb with GBS and 1.95, 2.3 and 4.8-kb
with AGP), indicated that the inserts of the original transformant
(G0) remained stable into the first and second vegetative
generations G1 and G2.
[0097] The Atlantic G2-tubers could not be assayed for Ppo activity
using the catechol assay because cut tuber surfaces of the
untransformed Atlantic variety do not develop a brown precipitant
when exposed to catechol. It is possible that untransformed
Atlantic potatoes do not produce the enzyme that catalyzes the
oxidation of polyphenols. Instead, the stability of the inserted
DNA was confirmed by PCR analysis, which demonstrated that J55
tubers contained an amplified 0.8-kb product, which is part of the
Asn1/Ppo5 gene, and did not display instability in G2 tubers.
Example 6
Junction Analysis and Variety-Specific Detection
[0098] At least one DNA insert/flanking plant DNA junction was
sequenced using either Adapter Ligation-Mediated PCR or Thermal
Asymmetric Interlaced PCR. The junction sequence was used to design
primers for potato cultivar J55, and these primers were applied for
variety-specific PCR-based detection methods. Primers can be used
to amplify a J55-specific DNA fragment of 401-kb, resulting in a
line specific test method for variety J55. The methods developed
were used to monitor plants and tubers in field and storage to
confirm the absence of intragenic material in tubers or processed
food, and to ensure the purity of organic seed.
Example 7
Efficacy and Tissue-Specificity of Gene Silencing
[0099] Gene silencing methods were employed to lower the activity
of the Asn1, Ppo5, PhL, and R1 native proteins, and transcript
levels rather than protein amounts were evaluated to link new
phenotypic traits to changes at the molecular level
[0100] Since strong silencing of the Asn1 gene involved in ASN
(asparagine) formation in leaves and stems might adversely affect
growth, the Agp promoter and the Gbss promoter, which are tuber-
and stolon-specific promoters and are much less active in
photosynthetically-active tissues and roots, were used to drive
gene silencing in tubers and stolons. The transcript levels of the
four targeted genes in various tissues of plant variety J55 and its
untransformed counterpart were determined by Northern blot
analysis.
[0101] In tubers of untransformed controls, transcript levels were
"high" (easily detectable by northern blot hybridization) for the
Asn1, PhL, and R1 genes and "low" for the Ppo5 gene. A comparison
of northern blots indicated that the Asn1, Ppo5, and PhL were
expressed at similar levels in tubers from greenhouse and field. In
contrast, the R1 gene was silenced slightly more effectively in
greenhouse-grown control tubers than in tubers from the field.
[0102] Strongly reduced transcript levels for the Asn1 and Ppo5
genes in tubers of variety J55 were associated with low-acrylamide
potential.
[0103] Transcript levels for the PhL gene were partially reduced in
the tubers of line J55. This change was linked to reduced amounts
of glucose and fructose. R1 transcripts were partially reduced
("whispered") in tubers of J55 to help limit the degradation of
starch into sugars.
[0104] The Asn1, Ppo5 and R1 genes were expressed at low levels in
stolons of untransformed plants, with undetectable levels for Ppo5
in Atlantic varieties and controls. In contrast, high transcript
levels in the controls were associated with the PhL gene.
[0105] The amounts of transcript for the R1 gene were slightly
reduced ("whispered") in stolons of variety J55. This molecular
change contributed to the limited degradation of starch into
sugars.
[0106] In leaf tissues, the transcript levels for the Asn1 gene
were similar for line J55 and its untransformed counterpart. The
transcript levels for the Ppo5 gene expression were in all cases
undetectable, whereas the levels for the PhL gene were consistently
high among the original variety Atlantic and the transformed
derivative J55. The transcript levels for the R1 gene were
unaltered in variety J55 when compared to its control.
[0107] In stem tissues, Asn1 gene transcript levels were similar
for event J55 and its control. The transcript levels for the Ppo5
gene were reduced in the variety J55 of the invention. PhL gene
expression was very similar in line J55 and its control and R1 gene
expression was not reduced.
[0108] In root tissue, transcript levels for both the Asn1 and Ppo5
genes were reduced in variety J55. These results indicated that the
promoters used to drive silencing are partially functional in
underground tissues.
[0109] In floral tissue, the Asn1 gene transcript levels were lower
in the variety J55 of the invention than in the original variety
Atlantic. Transcripts were not detectable for the Ppo5 gene and
expression levels of the PhL and R1 genes were similar to
controls.
[0110] These results demonstrated that the expression levels of the
Asn1 and Ppo5 genes were down-regulated in tubers and stolons in
potato variety J55, and that the R1 and PhL genes were at least
partially silenced in tubers and stolons in variety J55. Silencing
was most effective in tubers and stolons.
[0111] The selected potato variety J55 was more strongly affected
in the expression levels of the Asn1 and Ppo5 genes than in those
of the RI and PhL genes. These results coincided with the
inventor's intent of (1) preventing the formation of Ppo protein
and free ASN to the greatest extent possible, and (2) only
partially blocking the conversion of starch into glucose and
fructose.
[0112] The occasional changes in transcript levels in tissues other
than tubers and stolons demonstrated some leakiness of the
tuber/stolon-specific promoter. It is also possible that small RNAs
produced in tubers through expression of the silencing cassettes
migrate into other tissues, especially roots and stems (Molnar et
al., 2010). In most cases of altered expression levels in tissues
other than tubers and stolons, the differences were minor. A
summary of the down-regulated transcript levels in specific tissues
of intragenic potato cultivar J55 is shown in Table 3. In Table 3,
A=Asn1, P=Ppo5, L=PhL, and R=R1. Underlined letters in Table 3
indicate down-regulated gene expression levels.
TABLE-US-00003 TABLE 3 Variety Tubers Stolons Roots Stems Leaves
Flowers J55 A P L R A P R A R P L R A
Example 8
Field Performance and Tuber Evaluation
[0113] The 2009, 2010, and 2011 trials were planted mechanically to
facilitate harvests and ensure that intragenic potatoes were kept
separate from unmodified material. For the 2009 evaluations,
"nuclear seed" minitubers from each event and the control varieties
were used to plant four or five single-row plots (20
minitubers/plot) whereby the plots were randomly distributed within
blocks across the field. This randomized complete block design
(RCB) is typical for the evaluation of new potato varieties and
events. The approach taken in 2010 and 2011 was to use three random
plots per event and control per site, also using the RCB design
with the number or replications (plots per event) equal to the
number of blocks. Each plot in 2010 consisted of three rows of 20
seed pieces each from "nuclear seed" minitubers produced in Cherry
County, NE, in 2009 for Atlantic. The Atlantic seed for the 2011
trials was first generation (G1) produced in Cherry County, NE in
2010. In all trials, the seed of the intragenic line was handled
similar to its unmodified control. Field grown tubers are desirable
over minitubers as seed because they generate more vigorous and
uniform plants that produce higher tuber yield and quality.
[0114] Each plot was evaluated qualitatively, in some cases using
standardized monitoring scales, for differential responses to
insect, disease, and environmental stresses that were not induced
artificially but might occur spontaneously during the growing
season. Mid-season monitoring was conducted 2009, 2010, and 2011
just prior to or during early row closure and flowering (June-July
for most trails except for the Florida trials, which were evaluated
in April), to assess plant vigor, leaf color, leaf size, leaf curl,
disease symptoms (presence/absence), and insect-associated plant
damage. In 2011, specific insects, diseases, and abiotic stressors
common to the growing region were evaluated. Disease and insect
pressure are generally highest during the mid and late season, but
the plants were monitored for symptoms caused by pathogens and
insects from July to September (March through May for Florida
trials). Late season monitoring of vine maturity and diseases was
performed once prior to vine killing, a process intended to ensure
tuber maturation and late-season skin set. Vine killing is induced
either by mowing or flailing the vines or by using approved
herbicides such as Reglone according to the manufacturer's
recommendations (JR Johnson, Roseville, Minn.). At this time,
plants were also assessed for disease symptoms and insect damage.
In some cases when disease symptoms were identified, a sprout test
was also conducted to confirm the findings.
[0115] Means, standard deviations, and 90% confidence intervals
were calculated using JMP 9.0.2. A conventional varieties range was
generated by obtaining the minimum and maximum mean values
(year*location*entry) of all conventional varieties included in the
experiments. All characteristics for the Atlantic varieties were
analyzed in JMP 9.0.2 by combining data from multiple years and
locations.
Example 9
Potato Cultivar J55 Characterization Summary
[0116] Potato variety J55 addresses the need of the potato industry
to improve quality by reducing acrylamide through lowering the
concentration of the reactants, namely asparagine and reducing
sugars. Potato variety J55 was transformed with nucleic acid
sequences that are native to the potato plant genome and does not
contain foreign DNA, Agrobacterium DNA, viral markers or vector
backbone sequences In addition, agronomic studies were conducted to
ensure that the events grew the same as conventional controls, with
the exception of the characteristics associated with the trait
Agronomic Characteristics
[0117] Evaluations of agronomic characteristics of potato variety
J55 event and control grown in 2009, 2010, and 2011 are shown in
Tables 4-7. Results were analyzed by statistical methods where
possible. Overall the data suggest that there are no major
differences between the Atlantic control and the J55 Atlantic
event.
[0118] Table 4 shows the number of site years for each
characteristic tested for variety J55. The agronomic
characteristics for J55 and the Atlantic control are shown in Table
5. No statistically significant differences were detected between
J55 and the control for five of the agronomic characteristics.
Leaflet curl and vine maturity rating data were not able to be
statistically compared, as the mean value of J55 was the same as
the control for leaflet curl, and the observed value of J55 was
outside the combined conventional varieties range (2.98 vs.
3.00-3.50, respectively). Statistically significant differences
were detected between J55 and the control for early emergence (68.6
vs. 74.3, respectively) and senescence (52.37 vs. 47.37); however,
the value of J55 was within the conventional varieties range for
both cases. In Table 5, the stems per plant and final emergence
data is from 2011 only; conventional varieties (ConV) range equals
a range of mean values of conventional Ranger, Burbank and Atlantic
varieties; NA means that a statistical comparison was not
possible.
[0119] The yield and grading characteristics of J55 and the
Atlantic control are shown in Table 6. There were no statistically
significant differences detected for any of the seven analyzed
agronomic characteristics. Statistical analysis was not possible
for % grade B but the mean of J55 was within the conventional
varieties range. In Table 6, conventional varieties (ConV) range
equals a range of mean values of conventional Ranger, Burbank and
Atlantic varieties; NA means that a statistical comparison was not
possible.
[0120] The flower colors of J55 and the Atlantic control are shown
in Table 7. Purple and mixed flower colors were observed in
different plots for each entry.
TABLE-US-00004 TABLE 4 Number of Site Years Characteristic J55
Early Emergence 13 Final Emergence 4 Stems Per Plant 7 Plant Vigor
7-8 Foliage Color 14-15 Leaflet Size 14-15 Leaflet Curl 14-15
Senescence 6 Vine Size NA Vine Maturity Rating 9-10 Flower Color 7
Total Yield 14 %4-6 oz. NA %6-10 oz. NA %10-14 oz. NA % > 14 oz.
NA Specific Gravity 14 High Sugar NA Sugar Ends NA Total Internal
Defects 14 % U.S. #1 14 % Grade B 14 % Grade A 14 % Oversize 14 %
Pick-outs 14
TABLE-US-00005 TABLE 5 Commercial Atlantic Reference Characteristic
Statistic J55 Ctrl Range Early Emergence Mean 68.6 74.3 7.0-100.0
SD 26.2 30.4 90% CI 62.4-74.7% 67.2-81.4% p-Value 0.0308 Final
Emergence Mean 99.3 99.0 79.0-100.0 SD 2.2 2.5 90% CI 98.6-100.0%
98.2-99.8% p-Value 0.6324 Stems Per Plant Mean 3.3 3.1 1.6-5.0 SD
1.22 1.14 90% CI 2.86-3.76 2.72-3.55 p-Value 0.0593 Plant Vigor
Mean 3.2 3.2 2.3-4.3 SD 0.72 0.46 90% CI 2.98-3.30 3.06-3.27
p-Value 0.5226 Foliage Color Mean 3.2 3.1 2.3-4.0 SD 0.51 0.32 90%
CI 3.07-3.30 3.04-3.19 p-Value 0.6899 Leaflet Size Mean 3.0 3.0
2.0-3.0 SD 0.26 0.00 90% CI 2.98-3.09 3.00-3.00 p-Value 0.9144
Leaflet Curl Mean 3.0 3.0 1.0-3.0 SD 0.00 0.00 90% CI 3.00-3.00
3.00-3.00 p-Value NA Senescence Mean 52.4 47.4 8.7-91.7 SD 30.38
32.56 90% CI 40.28-64.46 34.42-60.32 p-Value 0.0044 Vine Maturity
Mean 3.0 3.0 2.3-4.5 Rating SD 0.94 0.11 90% CI 2.73-3.22 3.00-3.06
p-Value NA
TABLE-US-00006 TABLE 6 Commercial Atlantic Reference Characteristic
Statistic J55 Ctrl Range Total Yield Mean 46.2 45.0 17.2-80.1 SD
18.9 18.4 90% CI 40.5-51.9 39.4-50.5 p-Value 0.3913 % US#1 Mean
86.3 87.1 64.0-95.0 SD 8.2 6.7 90% CI 83.9-88.8 85.1-89.1 p-Value
0.4341 % Grade B Mean 11.8 11.9 3.0-36.0 SD 7.2 7.3 90% CI 9.6-14.0
9.7-14.1 p-Value NA % Grade A Mean 78.5 78.8 61.0-92.3 SD 7.6 8.6
90% CI 76.2-80.8 76.2-81.4 p-Value 0.8392 % Oversize Mean 7.8 8.3
0.0-33.0 SD 8.9 9.9 90% CI 5.1-10.4 5.3-11.3 p-Value 0.6937 % Pick
Outs Mean 2.0 1.0 0.0-6.0 SD 4.8 2.1 90% CI 0.5-3.4 0.4-1.6 p-Value
0.1049 Specific Gravity Mean 1.093 1.092 1.074-1.109 SD 0.009 0.009
90% CI 1.090-1.095 1.090-1.095 p-Value 0.4442 Total Internal Mean
36.6 30.6 0.0-120.0 Defects SD 24.8 30.5 90% CI 29.1-44.0 21.5-39.8
p-Value 0.1472
TABLE-US-00007 TABLE 7 Number of Plots Purple or White Entry Mixed
Flowers Flowers J55 22 0 Atlantic Ctrl 22 0
[0121] Based on the data presented in Tables 4-7 for potato
cultivar J55, it can be concluded that there are no major
differences in agronomic characteristics, flower color, yield and
grading, and ecological interactions between the untransformed
Atlantic variety and potato cultivar J55. Therefore, based on the
multi-year data, the Atlantic variety J55 poses no significant risk
of persistence in the environment as a result of weediness or plant
pest potential.
Asparagine and Acrylamide Levels
[0122] Silencing of the asparagine synthetase gene resulted in
average reductions of 78% free asparagine in potato variety J55.
The lower levels of asparagine, which combines with reducing sugars
in the Maillard reaction to form acrylamide, result in average
reductions of 72% acrylamide in potato chips. The differences in
asparagine and acrylamide between the Atlantic control and potato
J55 are shown in Table 8. Before testing for acrylamide, the
control and J55 were processed into potato chips. All results were
from tubers analyzed near the time of harvest.
TABLE-US-00008 TABLE 8 Free Percent Percent Asparagine Reduction
Acrylamide Reduction Variety (ppm) from Control (ppb) from Control
Atlantic 2268 0% 842.7 0% Control J55 502.4 78% 233.4 72%
[0123] Potato cultivar J55 is an Atlantic variety with improved
quality that has reduced acrylamide levels.
Further Embodiments of the Invention
[0124] The research leading to potato varieties which combine the
advantageous characteristics referred to above is largely
empirical. This research requires large investments of time, labor,
and money. The development of a potato cultivar can often take up
to eight years or more from greenhouse to commercial usage.
Breeding begins with careful selection of superior parents to
incorporate the most important characteristics into the progeny.
Since all desired traits usually do not appear with just one cross,
breeding must be cumulative.
[0125] Present breeding techniques continue with the controlled
pollination of parental clones. Typically, pollen is collected in
gelatin capsules for later use in pollinating the female parents.
Hybrid seeds are sown in greenhouses and tubers are harvested and
retained from thousands of individual seedlings. The next year one
to four tubers from each resulting seedling are planted in the
field, where extreme caution is exercised to avoid the spread of
virus and diseases. From this first-year seedling crop, several
"seed" tubers from each hybrid individual which survived the
selection process are retained for the next year's planting. After
the second year, samples are taken for density measurements and fry
tests to determine the suitability of the tubers for commercial
usage. Plants which have survived the selection process to this
point are then planted at an expanded volume the third year for a
more comprehensive series of fry tests and density determinations.
At the fourth-year stage of development, surviving selections are
subjected to field trials in several states to determine their
adaptability to different growing conditions. Eventually, the
varieties having superior qualities are transferred to other farms
and the seed increased to commercial scale. Generally, by this
time, eight or more years of planting, harvesting and testing have
been invested in attempting to develop the new and improved potato
cultivars.
[0126] With the advent of molecular biological techniques that have
allowed the isolation and characterization of genes that encode
specific protein products, scientists in the field of plant biology
developed a strong interest in engineering the genome of plants to
contain and express foreign genes, or additional, or modified
versions of native, or endogenous, genes (perhaps driven by
different promoters) in order to alter the traits of a plant in a
specific manner. Such foreign additional and/or modified genes are
referred to herein collectively as "transgenes". Over the last
fifteen to twenty years several methods for producing transgenic
plants have been developed, and the present invention, in
particular embodiments, also relates to transformed versions of the
claimed variety or line.
[0127] Plant transformation involves the construction of an
expression vector which will function in plant cells. Such a vector
comprises DNA comprising a gene under control of, or operatively
linked to, a regulatory element (for example, a promoter). The
expression vector may contain one or more such operably linked
gene/regulatory element combinations. The vector(s) may be in the
form of a plasmid, and can be used alone or in combination with
other plasmids, to provide transformed potato plants, using
transformation methods as described below to incorporate transgenes
into the genetic material of the potato plant(s).
[0128] Traditional plant breeding typically relies on the random
recombination of plant chromosomes to create varieties that have
new and improved characteristics. According to standard, well-known
techniques, genetic "expression cassettes," comprising genes and
regulatory elements, are inserted within the borders of
Agrobacterium-isolated transfer DNAs ("T-DNAs") and integrated into
plant genomes. Agrobacterium-mediated transfer of T-DNA material
typically comprises the following standard procedures: (1) in vitro
recombination of genetic elements, at least one of which is of
foreign origin, to produce an expression cassette for selection of
transformation, (2) insertion of this expression cassette, often
together with at least one other expression cassette containing
foreign DNA, into a T-DNA region of a binary vector, which usually
consists of several hundreds of basepairs of Agrobacterium DNA
flanked by T-DNA border sequences, (3) transfer of the sequences
located between the T-DNA borders, often accompanied with some or
all of the additional binary vector sequences from Agrobacterium to
the plant cell, and (4) selection of stably transformed plant cells
that display a desired trait, such as an increase in yield,
improved vigor, enhanced resistance to diseases and insects, or
greater ability to survive under stress.
[0129] Thus, genetic engineering methods rely on the introduction
of foreign, not-indigenous nucleic acids, including regulatory
elements such as promoters and terminators, and genes that are
involved in the expression of a new trait or function as markers
for identification and selection of transformants, from viruses,
bacteria and plants. Marker genes are typically derived from
bacterial sources and confer antibiotic or herbicide resistance.
Classical breeding methods are laborious and time-consuming, and
new varieties typically display only relatively modest
improvements.
[0130] In the "anti-sense" technology, the sequence of native genes
is inverted to silence the expression of the gene in transgenic
plants. However, the inverted DNA usually contains new and
uncharacterized open reading frames inserted between the promoter
and the terminator that encode foreign amino acid sequences that
may be undesirable as they interfere with plant development and/or
reduce their nutritional value.
Expression Vectors for Potato Transformation: Marker Genes
[0131] Expression vectors include at least one genetic marker,
operably linked to a regulatory element (a promoter, for example)
that allows transformed cells containing the marker to be either
recovered by negative selection, i.e., inhibiting growth of cells
that do not contain the selectable marker gene, or by positive
selection, i.e., screening for the product encoded by the genetic
marker. Many commonly used selectable marker genes for plant
transformation are well known in the transformation arts, and
include, for example, genes that code for enzymes that
metabolically detoxify a selective chemical agent which may be an
antibiotic or an herbicide, or genes that encode an altered target
which is insensitive to the inhibitor. A few positive selection
methods are also known in the art.
[0132] One commonly used selectable marker gene for plant
transformation is the neomycin phosphotransferase II (nptII) gene
which, when under the control of plant regulatory signals, confers
resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.
U.S.A., 80:4803 (1983). Another commonly used selectable marker
gene is the hygromycin phosphotransferase gene which confers
resistance to the antibiotic hygromycin. Vanden Elzen et al., Plant
Mol. Biol., 5:299 (1985).
[0133] Additional selectable marker genes of bacterial origin that
confer resistance to antibiotics include gentamycin acetyl
transferase, streptomycin phosphotransferase and
aminoglycoside-3'-adenyl transferase, the bleomycin resistance
determinant. Hayford et al., Plant Physiol. 86:1216 (1988), Jones
et al., Mol. Gen. Genet., 210:86 (1987), Svab et al., Plant Mol.
Biol. 14:197 (1990) Hille et al., Plant Mol. Biol. 7:171 (1986).
Other selectable marker genes confer resistance to herbicides such
as glyphosate, glufosinate or bromoxynil. Comai et al., Nature
317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990)
and Stalker et al., Science 242:419-423 (1988).
[0134] Selectable marker genes for plant transformation not of
bacterial origin include, for example, mouse dihydrofolate
reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and
plant acetolactate synthase. Eichholtz et al., Somatic Cell Mol.
Genet. 13:67 (1987), Shah et al., Science 233:478 (1986), Charest
et al., Plant Cell Rep. 8:643 (1990).
[0135] Another class of marker genes for plant transformation
requires screening of presumptively transformed plant cells rather
than direct genetic selection of transformed cells for resistance
to a toxic substance such as an antibiotic. These genes are
particularly useful to quantify or visualize the spatial pattern of
expression of a gene in specific tissues and are frequently
referred to as reporter genes because they can be fused to a gene
or gene regulatory sequence for the investigation of gene
expression. Commonly used genes for screening presumptively
transformed cells include .beta.-glucuronidase (GUS),
.beta.-galactosidase, luciferase and chloramphenicol
acetyltransferase. Jefferson, R. A., Plant Mol. Biol. Rep. 5:387
(1987), Teeri et al., EMBO J. 8:343 (1989), Koncz et al., Proc.
Natl. Acad. Sci. USA 84:131 (1987), DeBlock et al., EMBO J. 3:1681
(1984).
[0136] In vivo methods for visualizing GUS activity that do not
require destruction of plant tissue are available. Molecular Probes
publication 2908, IMAGENE GREEN, p. 1-4 (1993) and Naleway et al.,
J. Cell Biol. 115:151a (1991). However, these in vivo methods for
visualizing GUS activity have not proven useful for recovery of
transformed cells because of low sensitivity, high fluorescent
backgrounds and limitations associated with the use of luciferase
genes as selectable markers.
[0137] More recently, a gene encoding Green Fluorescent Protein
(GFP) has been utilized as a marker for gene expression in
prokaryotic and eukaryotic cells. Chalfie et al., Science 263:802
(1994). GFP and mutants of GFP may be used as screenable
markers.
Expression Vectors for Potato Transformation: Promoters
[0138] Genes included in expression vectors must be driven by a
nucleotide sequence comprising a regulatory element, for example, a
promoter. Several types of promoters are well known in the
transformation arts as are other regulatory elements that can be
used alone or in combination with promoters.
[0139] As used herein, "promoter" includes reference to a region of
DNA upstream from the start of transcription and 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 plant cells. Examples of promoters
under developmental control include promoters that preferentially
initiate transcription in certain tissues, such as leaves, roots,
seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such
promoters are referred to as "tissue-preferred". Promoters that
initiate transcription only in a certain tissue are referred to as
"tissue-specific". A "cell-type" specific promoter primarily drives
expression in certain cell types in one or more organs, for
example, vascular cells in roots or leaves. An "inducible" promoter
is a promoter which is under environmental control. Examples of
environmental conditions that may effect transcription by inducible
promoters include anaerobic conditions or the presence of light.
Tissue-specific, tissue-preferred, cell type specific, and
inducible promoters constitute the class of "non-constitutive"
promoters. A "constitutive" promoter is a promoter that is active
under most environmental conditions.
A. Inducible Promoters
[0140] An inducible promoter is operably linked to a gene for
expression in potato. Optionally, the inducible promoter is
operably linked to a nucleotide sequence encoding a signal sequence
which is operably linked to a gene for expression in potato. With
an inducible promoter the rate of transcription increases in
response to an inducing agent.
[0141] Any inducible promoter can be used in the instant invention.
See Ward et al., Plant Mol. Biol. 22:361-366 (1993). Exemplary
inducible promoters include, but are not limited to, that from the
ACEI system which responds to copper (Mett et al., PNAS
90:4567-4571 (1993)); In2 gene from maize which responds to
benzenesulfonamide herbicide safeners (Hershey et al., Mol. Gen
Genetics 227:229-237 (1991) and Gatz et al., Mol. Gen. Genetics
243:32-38 (1994)) or Tet repressor from Tn10 (Gatz et al., Mol.
Gen. Genetics 227:229-237 (1991)). A particularly preferred
inducible promoter is a promoter that responds to an inducing agent
to which plants do not normally respond. An exemplary inducible
promoter is the inducible promoter from a steroid hormone gene, the
transcriptional activity of which is induced by a
glucocorticosteroid hormone. Schena et al., Proc. Natl. Acad. Sci.
USA 88:0421 (1991).
B. Constitutive Promoters
[0142] A constitutive promoter is operably linked to a gene for
expression in potato or the constitutive promoter is operably
linked to a nucleotide sequence encoding a signal sequence which is
operably linked to a gene for expression in potato.
[0143] Many different constitutive promoters can be utilized in the
instant invention. Exemplary constitutive promoters include, but
are not limited to, the promoters from plant viruses such as the
35S promoter from CaMV (Odell et al., Nature 313:810-812 (1985))
and the promoters from such genes as rice actin (McElroy et al.,
Plant Cell 2: 163-171 (1990)); ubiquitin (Christensen et al., Plant
Mol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol.
Biol. 18:675-689 (1992)); pEMU (Last et al., Theor. Appl. Genet.
81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730 (1984))
and maize H3 histone (Lepetit et al., Mol. Gen. Genetics
231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):
291-300 (1992)).
[0144] The ALS promoter, Xba1/Nco1 fragment 5' to the Brassica
napus ALS3 structural gene (or a nucleotide sequence similarity to
said Xba1/Nco1 fragment), represents a particularly useful
constitutive promoter. See PCT application WO 96/30530.
C. Tissue-Specific or Tissue-Preferred Promoters
[0145] A tissue-specific promoter is operably linked to a gene for
expression in potato. Optionally, the tissue-specific promoter is
operably linked to a nucleotide sequence encoding a signal sequence
which is operably linked to a gene for expression in potato. Plants
transformed with a gene of interest operably linked to a
tissue-specific promoter produce the protein product of the
transgene exclusively, or preferentially, in a specific tissue.
[0146] Any tissue-specific or tissue-preferred promoter can be
utilized in the instant invention. Exemplary tissue-specific or
tissue-preferred promoters include, but are not limited to, a
root-preferred promoter--such as that from the phaseolin gene
(Murai et al., Science 23:476-482 (1983) and Sengupta-Gopalan et
al., Proc. Natl. Acad. Sci. USA 82:3320-3324 (1985)); a
leaf-specific and light-induced promoter such as that from cab or
rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985) and Timko
et al., Nature 318:579-582 (1985)); an anther-specific promoter
such as that from LAT52 (Twell et al., Mol. Gen. Genetics
217:240-245 (1989)); a pollen-specific promoter such as that from
Zm13 (Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or a
microspore-preferred promoter such as that from apg (Twell et al.,
Sex. Plant Reprod. 6:217-224 (1993)).
Signal Sequences for Targeting Proteins to Subcellular
Compartments
[0147] Transport of protein produced by transgenes to a subcellular
compartment such as the chloroplast, vacuole, peroxisome,
glyoxysome, cell wall or mitochondrion or for secretion into the
apoplast, is accomplished by means of operably linking the
nucleotide sequence encoding a signal sequence to the 5' and/or 3'
region of a gene encoding the protein of interest. Targeting
sequences at the 5' and/or 3' end of the structural gene may
determine, during protein synthesis and processing, where the
encoded protein is ultimately compartmentalized.
[0148] The presence of a signal sequence directs a polypeptide to
either an intracellular organelle or subcellular compartment or for
secretion to the apoplast. Many signal sequences are known in the
art. See, for example, Becker et al., Plant Mol. Biol. 20:49
(1992); Close, P. S., Master's Thesis, Iowa State University
(1993); Knox, C., et al., Plant Mol. Biol. 9:3-17 (1987); Lerner et
al., Plant Physiol. 91:124-129 (1989); Frontes et al., Plant Cell
3:483-496 (1991); Matsuoka et al., Proc. Natl. Acad. Sci. 88:834
(1991); Gould et al., J. Cell. Biol. 108:1657 (1989); Creissen et
al., Plant J. 2:129 (1991); Kalderon, et al., Cell 39:499-509
(1984); Steifel, et al., Plant Cell 2:785-793 (1990).
Foreign Protein Genes and Agronomic Genes
[0149] With transgenic plants according to the present invention, a
foreign protein can be produced in commercial quantities. Thus,
techniques for the selection and propagation of transformed plants,
which are well understood in the art, yield a plurality of
transgenic plants which are harvested in a conventional manner, and
a foreign protein then can be extracted from a tissue of interest
or from total biomass. Protein extraction from plant biomass can be
accomplished by known methods which are discussed, for example, by
Heney and Orr, Anal. Biochem. 114:92-6 (1981).
[0150] According to a preferred embodiment, the transgenic plant
provided for commercial production of foreign protein is a potato
plant. In another preferred embodiment, the biomass of interest is
seed or tubers. For the relatively small number of transgenic
plants that show higher levels of expression, a genetic map can be
generated, primarily via conventional RFLP, PCR and SSR analysis,
which identifies the approximate chromosomal location of the
integrated DNA molecule. For exemplary methodologies in this
regard, see Glick and Thompson, Methods in Plant Molecular Biology
and Biotechnology CRC Press, Boca Raton 269:284 (1993). Map
information concerning chromosomal location is useful for
proprietary protection of a subject transgenic plant. If
unauthorized propagation is undertaken and crosses made with other
germplasm, the map of the integration region can be compared to
similar maps for suspect plants, to determine if the latter have a
common parentage with the subject plant. Map comparisons would
involve hybridizations, RFLP, PCR, SSR and sequencing, all of which
are conventional techniques.
[0151] Likewise, by means of the present invention, agronomic genes
can be expressed in transformed plants. More particularly, plants
can be genetically engineered to express various phenotypes of
agronomic interest. Exemplary genes implicated in this regard
include, but are not limited to, those categorized below:
1. Genes That Confer Resistance to Pests or Disease and That
Encode:
[0152] A. Plant disease resistance genes. Plant defenses are often
activated by specific interaction between the product of a disease
resistance gene (R) in the plant and the product of a corresponding
avirulence (Avr) gene in the pathogen. A plant variety can be
transformed with cloned resistance gene(s) to engineer plants that
are resistant to specific pathogen strains. See, for example Jones
et al., Science 266:789 (1994) (cloning of the tomato Cf-9 gene for
resistance to Cladosporium fulvum); Martin et al., Science 262:1432
(1993) (tomato Pto gene for resistance to Pseudomonas syringae pv.
tomato encodes a protein kinase); Mindrinos et al. Cell 78:1089
(1994) (Arabidopsis RSP2 gene for resistance to Pseudomonas
syringae).
[0153] B. A gene conferring resistance to a pest, such as soybean
cyst nematode. See e.g., PCT Application WO 96/30517; PCT
Application WO 93/19181.
[0154] C. A Bacillus thuringiensis protein, a derivative thereof or
a synthetic polypeptide modeled thereon. See, for example, Geiser
et al., Gene 48:109 (1986), who disclose the cloning and nucleotide
sequence of a Bt .delta.-endotoxin gene. Moreover, DNA molecules
encoding .delta.-endotoxin genes can be purchased from American
Type Culture Collection, Manassas, Va., for example, under ATCC
Accession Nos. 40098, 67136, 31995 and 31998.
[0155] D. A lectin. See, for example, Van Damme et al., Plant
Molec. Biol. 24:25 (1994), who disclose the nucleotide sequences of
several Clivia miniata mannose-binding lectin genes.
[0156] E. A vitamin-binding protein such as avidin. See PCT
application US 93/06487 which teaches the use of avidin and avidin
homologs as larvicides against insect pests.
[0157] F. An enzyme inhibitor, for example, a protease or
proteinase inhibitor or an amylase inhibitor. See, for example, Abe
et al., J. Biol. Chem. 262:16793 (1987) (nucleotide sequence of
rice cysteine proteinase inhibitor), Huub et al., Plant Molec.
Biol. 21:985 (1993) (nucleotide sequence of cDNA encoding tobacco
proteinase inhibitor I), Sumitani et al., Biosci. Biotech. Biochem.
57:1243 (1993) (nucleotide sequence of Streptomyces nitrosporeus
.alpha.-amylase inhibitor) and U.S. Pat. No. 5,494,813 (Hepher and
Atkinson, issued Feb. 27, 1996).
[0158] G. An insect-specific hormone or pheromone such as an
ecdysteroid or juvenile hormone, a variant thereof, a mimetic based
thereon, or an antagonist or agonist thereof. See, for example, the
disclosure by Hammock et al., Nature 344:458 (1990), of baculovirus
expression of cloned juvenile hormone esterase, an inactivator of
juvenile hormone.
[0159] H. An insect-specific peptide or neuropeptide which, upon
expression, disrupts the physiology of the affected pest. For
example, see the disclosures of Regan, J. Biol. Chem. 269:9 (1994)
(expression cloning yields DNA coding for insect diuretic hormone
receptor), and Pratt et al., Biochem. Biophys. Res. Comm. 163:1243
(1989) (an allostatin is identified in Diploptera puntata). See
also U.S. Pat. No. 5,266,317 to Tomalski et al., who disclose genes
encoding insect-specific, paralytic neurotoxins.
[0160] I. An insect-specific venom produced in nature by a snake, a
wasp, etc. For example, see Pang et al., Gene 116:165 (1992), for
disclosure of heterologous expression in plants of a gene coding
for a scorpion insectotoxic peptide.
[0161] J. An enzyme responsible for a hyperaccumulation of a
monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a
phenylpropanoid derivative or another non-protein molecule with
insecticidal activity.
[0162] K. An enzyme involved in the modification, including the
post-translational modification, of a biologically active molecule;
for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic
enzyme, a nuclease, a cyclase, a transaminase, an esterase, a
hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase,
an elastase, a chitinase and a glucanase, whether natural or
synthetic. See PCT application WO 93/02197 (Scott et al.), which
discloses the nucleotide sequence of a callase gene. DNA molecules
which contain chitinase-encoding sequences can be obtained, for
example, from the ATCC under Accession Nos. 39637 and 67152. See
also Kramer et al., Insect Biochem. Molec. Biol. 23:691 (1993), who
teach the nucleotide sequence of a cDNA encoding tobacco hornworm
chitinase, and Kawalleck et al., Plant Molec. Biol. 21:673 (1993),
who provide the nucleotide sequence of the parsley ubi4-2
polyubiquitin gene.
[0163] L. A molecule that stimulates signal transduction. For
example, see the disclosure by Botella et al., Plant Molec. Biol.
24:757 (1994), of nucleotide sequences for mung bean calmodulin
cDNA clones, and Griess et al., Plant Physiol. 104:1467 (1994), who
provide the nucleotide sequence of a maize calmodulin cDNA
clone.
[0164] M. A hydrophobic moment peptide. See PCT application WO
95/16776, which discloses peptide derivatives of Tachyplesin which
inhibit fungal plant pathogens, and PCT application WO 95/18855
which teaches synthetic antimicrobial peptides that confer disease
resistance.
[0165] N. A membrane permease, a channel former or a channel
blocker. For example, see the disclosure of Jaynes et al., Plant
Sci 89:43 (1993), of heterologous expression of a cecropin-.beta.
lytic peptide analog to render transgenic tobacco plants resistant
to Pseudomonas solanacearum.
[0166] O. A viral-invasive protein or a complex toxin derived
therefrom. For example, the accumulation of viral coat proteins in
transformed plant cells imparts resistance to viral infection
and/or disease development effected by the virus from which the
coat protein gene is derived, as well as by related viruses. See
Beachy et al., Ann. Rev. Phytopathol. 28:451 (1990). Coat
protein-mediated resistance has been conferred upon transformed
plants against alfalfa mosaic virus, cucumber mosaic virus and
tobacco mosaic virus.
[0167] P. An insect-specific antibody or an immunotoxin derived
therefrom. Thus, an antibody targeted to a critical metabolic
function in the insect gut would inactivate an affected enzyme,
killing the insect. Cf Taylor et al., Abstract #497, Seventh Int'l
Symposium on Molecular Plant-Microbe Interactions (Edinburgh,
Scotland) (1994) (enzymatic inactivation in transgenic tobacco via
production of single-chain antibody fragments).
[0168] Q. A virus-specific antibody. See, for example, Tavladoraki
et al., Nature 366:469 (1993), who show that transgenic plants
expressing recombinant antibody genes are protected from virus
attack.
[0169] R. A developmental-arrestive protein produced in nature by a
pathogen or a parasite. Thus, fungal
endo-.alpha.-1,4-D-polygalacturonases facilitate fungal
colonization and plant nutrient release by solubilizing plant cell
wall homo-.alpha.-1,4-D-galacturonase. See Lamb et al.,
Bio/Technology 10:1436 (1992). The cloning and characterization of
a gene which encodes a bean endopolygalacturonase-inhibiting
protein is described by Toubart et al., Plant J. 2:367 (1992).
[0170] S. A developmental-arrestive protein produced in nature by a
plant. For example, Logemann et al., Bio/Technology 10:305 (1992),
have shown that transgenic plants expressing the barley
ribosome-inactivating gene have an increased resistance to fungal
disease.
[0171] T. Genes involved in the Systemic Acquired Resistance (SAR)
Response and/or the pathogenesis-related genes. Briggs, S. Current
Biology, 5(2) (1995).
[0172] U. Antifungal genes. See Cornelissen and Melchers, Plant
Physiol., 101:709-712 (1993); Parijs et al., Planta 183:258-264
(1991) and Bushnell et al., Can. J. of Plant Path. 20(2):137-149
(1998).
[0173] V. Genes that confer resistance to Phytophthora blight, such
as the R1, R2, R3, R4 and other resistance genes. See, Naess, S.
K., et. al., (2000) Resistance to late blight in Solanum
bulbocastanum is mapped to chromosome 8. Theor. Appl. Genet. 101:
697-704 and Li, X., et. al., (1998) Autotetraploids and genetic
mapping using common AFLP markers: the R2 allele conferring
resistance to Phytophthora infestans mapped on potato chromosome 4.
Theor. Appl. Genet. 96: 1121-1128.
2. Genes That Confer Resistance to an Herbicide, For Example:
[0174] A. An herbicide that inhibits the growing point or meristem,
such as an imidazolinone or a sulfonylurea. Exemplary genes in this
category code for mutant ALS and AHAS enzyme as described, for
example, by Lee et al., EMBO J. 7:1241 (1988), and Miki et al.,
Theor. Appl. Genet. 80:449 (1990), respectively.
[0175] B. Glyphosate (resistance impaired by mutant
5-enolpyruvlshikimate-3-phosphate synthase (EPSP) and aroA genes,
respectively) and other phosphono compounds such as glufosinate
(phosphinothricin acetyl transferase (PAT) and Streptomyces
hygroscopicus PAT bar genes), and pyridinoxy or phenoxy proprionic
acids and cyclohexones (ACCase inhibitor-encoding genes). See, for
example, U.S. Pat. No. 4,940,835 to Shah, et al., which discloses
the nucleotide sequence of a form of EPSP which can confer
glyphosate resistance. A DNA molecule encoding a mutant aroA gene
can be obtained under ATCC accession number 39256, and the
nucleotide sequence of the mutant gene is disclosed in U.S. Pat.
No. 4,769,061 to Comai. European patent application No. 0 333 033
to Kumada et al., and U.S. Pat. No. 4,975,374 to Goodman et al.,
disclose nucleotide sequences of glutamine synthetase genes which
confer resistance to herbicides such as L-phosphinothricin. The
nucleotide sequence of a PAT gene is provided in European
application No. 0 242 246 to Leemans et al. DeGreef et al.,
Bio/Technology 7:61 (1989) describe the production of transgenic
plants that express chimeric bar genes coding for phosphinothricin
acetyl transferase activity. Exemplary of genes conferring
resistance to phenoxy proprionic acids and cyclohexones, such as
sethoxydim and haloxyfop are the Acc1-S1, Acc1-S2, and Acc2-S3
genes described by Marshall et al., Theor. Appl. Genet. 83:435
(1992).
[0176] C. An herbicide that inhibits photosynthesis, such as a
triazine (psbA and gs+ genes) or a benzonitrile (nitrilase gene).
Przibila et al., Plant Cell 3:169 (1991), describe the
transformation of Chlamydomonas with plasmids encoding mutant psbA
genes. Nucleotide sequences for nitrilase genes are disclosed in
U.S. Pat. No. 4,810,648 to Stalker and DNA molecules containing
these genes are available under ATCC Accession Nos. 53435, 67441
and 67442. Cloning and expression of DNA coding for a glutathione
S-transferase is described by Hayes et al., Biochem. J. 285:173
(1992).
[0177] D. Acetohydroxy acid synthase, which has been found to make
plants that express this enzyme resistant to multiple types of
herbicides, has been introduced into a variety of plants. See
Hattori et al., Mol. Gen. Genet. 246:419, 1995. Other genes that
confer tolerance to herbicides include a gene encoding a chimeric
protein of rat cytochrome P4507A1 and yeast NADPH-cytochrome P450
oxidoreductase (Shiota et al., Plant Physiol., 106:17, 1994), genes
for glutathione reductase and superoxide dismutase (Aono et al.,
Plant Cell Physiol. 36:1687, 1995), and genes for various
phosphotransferases (Datta et al., Plant Mol. Biol. 20:619,
1992).
[0178] E. Protoporphyrinogen oxidase (protox) is necessary for the
production of chlorophyll, which is necessary for all plant
survival. The protox enzyme serves as the target for a variety of
herbicidal compounds. These herbicides also inhibit growth of all
the different species of plants present, causing their total
destruction. The development of plants containing altered protox
activity which are resistant to these herbicides are described in
U.S. Pat. Nos. 6,288,306; 6,282,837; 5,767,373; and international
publication WO 01/12825.
3. Genes that Confer or Contribute to a Value-Added Trait, Such
as:
[0179] A. Modified fatty acid metabolism, for example, by
transforming a plant with an antisense gene of stearyl-ACP
desaturase to increase stearic acid content of the plant. See
Knultzon et al., Proc. Natl. Acad. Sci. USA 89:2625 (1992).
[0180] B. Decreased phytate content--1) Introduction of a
phytase-encoding gene would enhance breakdown of phytate, adding
more free phosphate to the transformed plant. For example, see Van
Hartingsveldt et al., Gene 127:87 (1993), for a disclosure of the
nucleotide sequence of an Aspergillus niger phytase gene. 2) A gene
could be introduced that reduced phytate content. In maize, for
example, this could be accomplished by cloning and then
reintroducing DNA associated with the single allele which is
responsible for maize mutants characterized by low levels of phytic
acid. See Raboy et al., Maydica 35:383 (1990).
[0181] C. Modified carbohydrate composition effected, for example,
by transforming plants with a gene coding for an enzyme that alters
the branching pattern of starch. See Shiroza et al., J. Bacteriol.
170:810 (1988) (nucleotide sequence of Streptococcus mutants
fructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet.
20:220 (1985) (nucleotide sequence of Bacillus subtilis
levansucrase gene), Pen et al., Bio/Technology 10:292 (1992)
(production of transgenic plants that express Bacillus
lichenifonnis .alpha.-amylase), Elliot et al., Plant Molec. Biol.
21:515 (1993) (nucleotide sequences of tomato invertase genes),
Sogaard et al., J. Biol. Chem. 268:22480 (1993) (site-directed
mutagenesis of barley .alpha.-amylase gene), and Fisher et al.,
Plant Physiol. 102:1045 (1993) (maize endosperm starch branching
enzyme II).
[0182] D. Elevated oleic acid via FAD-2 gene modification and/or
decreased linolenic acid via FAD-3 gene modification. See U.S. Pat.
Nos. 6,063,947; 6,323,392; and international publication WO
93/11245.
4. Genes that Control Male Sterility
[0183] A. Introduction of a deacetylase gene under the control of a
tapetum-specific promoter and with the application of the chemical
N--Ac--PPT. See international publication WO 01/29237.
[0184] B. Introduction of various stamen-specific promoters. See
international publications WO 92/13956 and WO 92/13957.
[0185] C. Introduction of the barnase and the barstar genes. See
Paul et al., Plant Mol. Biol. 19:611-622, 1992).
Methods for Potato Transformation
[0186] Numerous methods for plant transformation have been
developed including biological and physical plant transformation
protocols. See, for example, Miki et al., "Procedures for
Introducing Foreign DNA into Plants" in Methods in Plant Molecular
Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds.
(CRC Press, Inc. Boca Raton, 1993) pages 67-88. In addition,
expression vectors and in-vitro culture methods for plant cell or
tissue transformation and regeneration of plants are available.
See, for example, Gruber et al., "Vectors for Plant Transformation"
in Methods in Plant Molecular Biology and Biotechnology, Glick, B.
R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)
pages 89-119.
[0187] A. Agrobacterium-mediated Transformation--One method for
introducing an expression vector into plants is based on the
natural transformation system of Agrobacterium. See, for example,
Horsch et al., Science 227:1229 (1985). A. tumefaciens and A.
rhizogenes are plant pathogenic soil bacteria which genetically
transform plant cells. The Ti and Ri plasmids of A. tumefaciens and
A. rhizogenes, respectively, carry genes responsible for genetic
transformation of the plant. See, for example, Kado, C. I., Crit.
Rev. Plant Sci. 10:1 (1991). Descriptions of Agrobacterium vector
systems and methods for Agrobacterium-mediated gene transfer are
provided by Gruber et al., supra, Miki et al., supra and Moloney et
al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.
5,563,055 (Townsend and Thomas), issued Oct. 8, 1996.
[0188] B. Direct Gene Transfer--Several methods of plant
transformation collectively referred to as direct gene transfer
have been developed as an alternative to Agrobacterium-mediated
transformation. A generally applicable method of plant surface of
microprojectiles measuring 1 to 4 .mu.m. The expression vector is
introduced into plant tissues with a biolistic device that
accelerates the microprojectiles to speeds of 300 to 600 m/s which
is sufficient to penetrate plant cell walls and membranes. Sanford
et al., Part. Sci. Technol. 5:27 (1987); Sanford, J. C., Trends
Biotech. 6:299 (1988); Klein et al., Bio/Tech. 6:559-563 (1988);
Sanford, J. C. Physiol Plant 7:206 (1990); Klein et al.,
Biotechnology 10:268 (1992). See also U.S. Pat. No. 5,015,580
(Christou, et al.), issued May 14, 1991 and U.S. Pat. No. 5,322,783
(Tomes, et al.), issued Jun. 21, 1994.
[0189] Another method for physical delivery of DNA to plants is
sonication of target cells. Zhang et al., Bio/Technology 9:996
(1991). Alternatively, liposome and spheroplast fusion have been
used to introduce expression vectors into plants. Deshayes et al.,
EMBO J., 4:2731 (1985); Christou et al., Proc Natl. Acad. Sci. USA
84:3962 (1987). Direct uptake of DNA into protoplasts using
CaCl.sub.2 precipitation, polyvinyl alcohol or poly-L-ornithine has
also been reported. Hain et al., Mol. Gen. Genet. 199:161 (1985)
and Draper et al., Plant Cell Physiol. 23:451 (1982).
Electroporation of protoplasts and whole cells and tissues have
also been described. Donn et al., In Abstracts of VIIth
International Congress on Plant Cell and Tissue Culture IAPTC,
A2-38, p 53 (1990); D'Halluin et al., Plant Cell 4:1495-1505 (1992)
and Spencer et al., Plant Mol. Biol. 24:51-61 (1994).
[0190] Following transformation of potato target tissues,
expression of the above-described selectable marker genes allows
for preferential selection of transformed cells, tissues and/or
plants, using regeneration and selection methods well known in the
art.
[0191] The foregoing methods for transformation would typically be
used for producing a transgenic variety. The transgenic variety
could then be crossed with another (non-transformed or transformed)
variety in order to produce a new transgenic variety.
Alternatively, a genetic trait that has been engineered into a
particular potato line using the foregoing transformation
techniques could be moved into another line using traditional
backcrossing techniques that are well known in the plant breeding
arts. For example, a backcrossing approach could be used to move an
engineered trait from a public, non-elite variety into an elite
variety, or from a variety containing a foreign gene in its genome
into a variety or varieties that do not contain that gene. As used
herein, "crossing" can refer to a simple X by Y cross or the
process of backcrossing depending on the context.
[0192] Persons of ordinary skill in the art will recognize that
when the term potato plant is used in the context of the present
invention, this also includes derivative varieties that retain the
essential distinguishing characteristics of J55, such as a gene
converted plant of that variety or a transgenic derivative having
one or more value-added genes incorporated therein (such as
herbicide or pest resistance). Backcrossing methods can be used
with the present invention to improve or introduce a characteristic
into the variety. The term "backcrossing" as used herein refers to
the repeated crossing 1, 2, 3, 4, 5, 6, 7, 8, 9 or more times of a
hybrid progeny back to the recurrent parents. The parental potato
plant which contributes the gene(s) for the one or more desired
characteristics is termed the nonrecurrent or donor parent. This
terminology refers to the fact that the nonrecurrent parent is used
one time in the backcross protocol and therefore does not recur.
The parental potato plant to which the gene or genes from the
nonrecurrent parent are transferred is known as the recurrent
parent as it is used for several rounds in the backcrossing
protocol. In a typical backcross protocol, the original variety of
interest (recurrent parent) is crossed to a second variety
(nonrecurrent parent) that carries the gene(s) of interest to be
transferred. The resulting progeny from this cross are then crossed
again to the recurrent parent and the process is repeated until a
potato plant is obtained wherein essentially all of the desired
morphological and physiological characteristics of the recurrent
parent are recovered in the converted plant, in addition to the one
or more genes transferred from the nonrecurrent parent.
[0193] The selection of a suitable recurrent parent is an important
step for a successful backcrossing procedure. The goal of a
backcross protocol is to alter or substitute one or more traits or
characteristics in the original variety. To accomplish this, one or
more genes of the recurrent variety are modified, substituted or
supplemented with the desired gene(s) from the nonrecurrent parent,
while retaining essentially all of the rest of the desired genes,
and therefore the desired physiological and morphological
constitution of the original variety. The choice of the particular
nonrecurrent parent will depend on the purpose of the backcross.
One of the major purposes is to add some commercially desirable,
agronomically important trait to the plant. The exact backcrossing
protocol will depend on the characteristic or trait being altered
or added to determine an appropriate testing protocol. Although
backcrossing methods are simplified when the characteristic being
transferred is a dominant allele, a recessive allele may also be
transferred. In this instance, it may be necessary to introduce a
test of the progeny to determine if the desired characteristic has
been successfully transferred.
[0194] Likewise, transgenes can be introduced into the plant using
any of a variety of established recombinant methods well-known to
persons skilled in the art, such as: Gressel, 1985,
Biotechnologically Conferring Herbicide Resistance in Crops: The
Present Realities, In Molecular Form and Function of the Plant
Genome, L. van Vloten-Doting, (ed.), Plenum Press, New York;
Huttner, S. L., et al., 1992, Revising Oversight of Genetically
Modified Plants, Bio/Technology; Klee, H., et al., 1989, Plant Gene
Vectors and Genetic Transformation: Plant Transformation Systems
Based on the use of Agrobacterium tumefaciens, Cell Culture and
Somatic Cell Genetics of Plants; Koncz, C., et al., 1986, The
Promoter of T.sub.L-DNA Gene 5 Controls the Tissue-Specific
Expression of Chimeric Genes Carried by a Novel Type of
Agrobacterium Binary Vector; Molecular and General Genetics;
Lawson, C., et al., 1990, Engineering Resistance to Mixed Virus
Infection in a Commercial Potato Cultivar: Resistance to Potato
Virus X and Potato Virus Y in Transgenic Russet Burbank,
Bio/Technology; Mitsky, T. A., et al., 1996, Plants Resistant to
Infection by PLRV. U.S. Pat. No. 5,510,253; Newell, C. A., et al.,
1991, Agrobacterium-Mediated Transformation of Solanum tuberosum L.
Cv. Russet Burbank, Plant Cell Reports; Perlak, F. J., et al.,
1993, Genetically Improved Potatoes: Protection from Damage by
Colorado Potato Beetles, Plant Molecular Biology; all of which are
incorporated herein by reference for this purpose.
[0195] Many traits have been identified that are not regularly
selected for in the development of a new variety but that can be
improved by backcrossing and genetic engineering techniques. These
traits may or may not be transgenic; examples of these traits
include but are not limited to: herbicide resistance; resistance to
bacterial, fungal or viral disease; insect resistance; uniformity
or increase in concentration of starch and other carbohydrates;
enhanced nutritional quality; decrease in tendency of tuber to
bruise; and decrease in the rate of starch conversion to sugars.
These genes are generally inherited through the nucleus. Several of
these traits are described in U.S. Pat. No. 5,500,365, U.S. Pat.
No. 5,387,756, U.S. Pat. No. 5,789,657, U.S. Pat. No. 5,503,999,
U.S. Pat. No. 5,589,612, U.S. Pat. No. 5,510,253, U.S. Pat. No.
5,304,730, U.S. Pat. No. 5,382,429, U.S. Pat. No. 5,503,999, U.S.
Pat. No. 5,648,249, U.S. Pat. No. 5,312,912, U.S. Pat. No.
5,498,533, U.S. Pat. No. 5,276,268, U.S. Pat. No. 4,900,676, U.S.
Pat. No. 5,633,434 and U.S. Pat. No. 4,970,168.
Deposit Information
[0196] A tuber deposit of the J.R. Simplot Company proprietary
POTATO CULTIVAR J55 disclosed above and recited in the appended
claims has been made with the American Type Culture Collection
(ATCC), 10801 University Boulevard, Manassas, Va. 20110. The date
of deposit was Sep. 25, 2013. The deposit of 25 vials of
microtubers was taken from the same deposit maintained by J.R.
Simplot Company since prior to the filing date of this application.
All restrictions will be irrevocably removed upon granting of a
patent, and the deposit is intended to meet all of the requirements
of 37 C.F.R. .sctn..sctn.1.801-1.809. The ATCC Accession Number is
PTA-120601. The deposit will be maintained in the depository for a
period of thirty years, or five years after the last request, or
for the enforceable life of the patent, whichever is longer, and
will be replaced as necessary during that period.
[0197] While a number of exemplary aspects and embodiments have
been discussed above, those of skill in the art will recognize
certain modifications, permutations, additions and sub-combinations
thereof. It is therefore intended that the following appended
claims and claims hereafter introduced are interpreted to include
all such modifications, permutations, additions and
sub-combinations as are within their true spirit and scope.
* * * * *