# Patent

## Title
Squash line ZGN 130-1041T

## Abstract

The invention provides seed and plants of the squash line designated ZGN 130-1041T. The invention thus relates to the plants, seeds and tissue cultures of squash line ZGN 130-1041T, and to methods for producing a squash plant produced by crossing a plant of squash line ZGN 130-1041T with itself or with another squash plant, such as a plant of another line. The invention further relates to seeds and plants produced by such crossing. The invention further relates to parts of a plant of squash line ZGN 130-1041T, including the fruit and gametes of such plants.

## Background
This application claims the priority of U.S. Provisional Appl. Ser. No. 60/968,875, filed Aug. 29, 2007, the entire disclosure of which is incorporated herein by reference.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the field of plant breeding and, more specifically, to the development of squash line ZGN 130-1041T.
2. Description of Related Art
The goal of vegetable breeding is to combine various desirable traits in a single variety/hybrid. Such desirable traits may include greater yield, resistance to insects or pests, tolerance to heat and drought, better agronomic quality, higher nutritional value, growth rate and fruit properties.
Breeding techniques take advantage of a plant's method of pollination. There are two general methods of pollination: a plant self-pollinates if pollen from one flower is transferred to the same or another flower of the same plant or plant variety. A plant cross-pollinates if pollen comes to it from a flower of a different plant variety.
Plants that have been self-pollinated and selected for type over many generations become homozygous at almost all gene loci and produce a uniform population of true breeding progeny, a homozygous plant. A cross between two such homozygous plants produces a uniform population of hybrid plants that are heterozygous for many gene loci. Conversely, a cross of two plants each heterozygous at a number of loci produces a population of hybrid plants that differ genetically and are not uniform. The resulting non-uniformity makes performance unpredictable.
The development of uniform varieties requires the development of homozygous inbred plants, the crossing of these inbred plants, and the evaluation of the crosses. Pedigree breeding and recurrent selection are examples of breeding methods that have been used to develop inbred plants from breeding populations. Those breeding methods combine the genetic backgrounds from two or more plants or various other broad-based sources into breeding pools from which new lines are developed by selfing and selection of desired phenotypes. The new lines are evaluated to determine which of those have commercial potential.
One crop species which has been subject to such breeding programs and is of particular value is squash. The term squash is used to refer to four species of the genus Cucurbita of the family Cucurbitaceae: (1) C. maxima, which includes the Hubbard, buttercup, and some large pumpkins, (2) C. mixta, including cushaw squash, (3) C. moschata, which includes the butternut squash, and (4) C. pepo. Acorn squash, zucchini, yellow crookneck and straightneck, and most pumpkins belong to this last species.
The term squash encompasses pumpkins, marrows, and zucchinis. Exclusively ornamental and functional varieties are included among gourds. There is considerable variation in size, shape and color. A typical categorization is to distinguish between summer and winter varieties. Summer squashes include young vegetable marrows, such as zucchini, and are harvested during the summer months. At this stage, the skin of the fruit is tender and the fruit relatively small. Common fruit forms include straight neck, crooked neck, saucer shaped, and oblong.
Winter squashes, including Hubbard, butternut and pumpkin, are harvested in late summer/early Fall once the rind has hardened. In contrast to summer squash, winter squash is eaten in the mature fruit stage. In some regions the term ‘winter squash’ is used to refer only to mature fruits of the maxima species.
Many different squash cultivars have been produced, and squash breeding efforts have been underway in many parts of the world (see e.g. U.S. Pat. No. 6,916,974, U.S. Pat. No. 6,414,224, U.S. Pat. No. 5,811,642, U.S. Pat. No. 5,457,278, U.S. Pat. No. 4,713,491). Some breeding objectives include varying the color, texture and flavor of the fruit. Other objectives include optimizing flesh thickness, solid content (% dry matter), and sugar content. Breeding programs have also focused on developing plants with earlier fruit maturity, more restricted vine growth, improved disease resistance or tolerance, and improved adaptability to environmental conditions. In the case of pumpkins, one goal is to produce hull-less seeds for the snack food industry. In the case of summer squash, one of the goals is to develop plants with reduced prickly spines. A parthenocarpic hybrid summer squash has been reported (U.S. Pat. No. 6,031,158).
Advances in biotechnology have resulted in genetically engineered squash with resistance or immunity to some viruses. One of the earliest examples is a squash line having resistance to zucchini yellow mosaic virus and watermelon mosaic virus 2 (Fuchs et al., 1995). Development of transgenic squash that is resistant to SqMV and other viruses has also been reported (Pang et al., 2000; U.S. Pat. No. 6,337,431).
While breeding efforts to date have provided a number of useful squash lines with beneficial traits, there remains a great need in the art for new lines with further improved traits. Such plants would benefit farmers and consumers alike by improving crop yields and/or quality.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a squash plant of the line designated ZGN 130-1041T. Also provided are squash plants having all the physiological and morphological characteristics of the squash line designated ZGN 130-1041T. Parts of the squash plant of the present invention are also provided. For example, these include a pollen grain, an ovule, a fruit, and a cell of the plant.
The invention also concerns seed of squash line ZGN 130-1041T. The squash seed of the invention may be provided as an essentially homogeneous population of squash seed of the line designated ZGN 130-1041T. Essentially homogeneous populations of seed are generally free from substantial numbers of other seed. Therefore, seed of line ZGN 130-1041T may be defined as forming at least about 97% of the total seed, including at least about 98%, 99%, or more of the seed. The population of squash seed may be particularly defined as being essentially free from hybrid seed. The seed population may be separately grown to provide an essentially homogeneous population of squash plants designated ZGN 130-1041T.
In another aspect of the invention, a plant of squash line ZGN 130-1041T comprising an added heritable trait is provided. The heritable trait may comprise a genetic locus that is a dominant or recessive allele. In one embodiment of the invention, a plant of squash line ZGN 130-1041T is defined as comprising a single locus conversion. In specific embodiments of the invention, an added genetic locus confers one or more traits such as, for example, herbicide tolerance, insect resistance, disease resistance, and modified carbohydrate metabolism. The trait may be, for example, conferred by a naturally occurring gene introduced into the genome of the line by backcrossing, a natural or induced mutation, or a transgene introduced through genetic transformation techniques into the plant or a progenitor of any previous generation thereof. When introduced through transformation, a genetic locus may comprise one or more transgenes integrated at a single chromosomal location or multiple chromosomal locations.
In another aspect of the invention, a tissue culture of regenerable cells of a plant of line ZGN 130-1041T is provided. The tissue culture will preferably be capable of regenerating plants capable of expressing all of the physiological and morphological characteristics of the line, and of regenerating plants having substantially the same genotype as other plants of the line. Examples of some of the physiological and morphological characteristics of the line ZGN 130-1041T include those traits set forth in the tables herein. The regenerable cells in such tissue cultures may be derived, for example, from embryos, meristems, cotyledons, pollen, leaves, anthers, roots, root tips, pistil, flower, seed and stalks. Still further, the present invention provides squash plants regenerated from a tissue culture of the invention, the plants having all the physiological and morphological characteristics of line ZGN 130-1041T.
In yet another aspect of the invention, processes are provided for producing squash seeds, plants and fruit, which processes generally comprise crossing a first parent squash plant with a second parent squash plant, wherein at least one of the first or second parent squash plants is a plant of the line designated ZGN 130-1041T. These processes may be further exemplified as processes for preparing hybrid squash seed or plants, wherein a first squash plant is crossed with a second squash plant of a different, distinct line to provide a hybrid that has, as one of its parents, the squash plant line ZGN 130-1041T. In these processes, crossing will result in the production of seed. The seed production occurs regardless of whether the seed is collected or not.
In one embodiment of the invention, the first step in “crossing” comprises planting seeds of a first and second parent squash plant, often in proximity so that pollination will occur for example, mediated by insect vectors. Alternatively, pollen can be transferred manually. Where the plant is self-pollinated, pollination may occur without the need for direct human intervention other than plant cultivation.
A second step may comprise cultivating or growing the seeds of first and second parent squash plants into plants that bear flowers. A third step may comprise preventing self-pollination of the plants, such as by emasculating the male flowers, (i.e., treating or manipulating the flowers to produce an emasculated parent squash plant). Self-incompatibility systems may also be used in some hybrid crops for the same purpose. Self-incompatible plants still shed viable pollen and can pollinate plants of other varieties but are incapable of pollinating themselves or other plants of the same line.
A fourth step for a hybrid cross may comprise cross-pollination between the first and second parent squash plants. Yet another step comprises harvesting the seeds from at least one of the parent squash plants. The harvested seed can be grown to produce a squash plant or hybrid squash plant.
The present invention also provides the squash seeds and plants produced by a process that comprises crossing a first parent squash plant with a second parent squash plant, wherein at least one of the first or second parent squash plants is a plant of the line designated ZGN 130-1041T. In one embodiment of the invention, squash seed and plants produced by the process are first generation (F1) hybrid squash seed and plants produced by crossing a plant in accordance with the invention with another, distinct plant. The present invention further contemplates plant parts of such an F1 hybrid squash plant, and methods of use thereof. Therefore, certain exemplary embodiments of the invention provide an F1 hybrid squash plant and seed thereof.
In still yet another aspect of the invention, the genetic complement of the squash plant line designated ZGN 130-1041T is provided. The phrase “genetic complement” is used to refer to the aggregate of nucleotide sequences, the expression of which sequences defines the phenotype of, in the present case, a squash plant, or a cell or tissue of that plant. A genetic complement thus represents the genetic makeup of a cell, tissue or plant, and a hybrid genetic complement represents the genetic make up of a hybrid cell, tissue or plant. The invention thus provides squash plant cells that have a genetic complement in accordance with the squash plant cells disclosed herein, and plants, seeds and plants containing such cells.
Plant genetic complements may be assessed by genetic marker profiles, and by the expression of phenotypic traits that are characteristic of the expression of the genetic complement, e.g., isozyme typing profiles. It is understood that line ZGN 130-1041T could be identified by any of the many well known techniques such as, for example, Simple Sequence Length Polymorphisms (SSLPs) (Williams et al., 1990), Randomly Amplified Polymorphic DNAs (RAPDs), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR), Amplified Fragment Length Polymorphisms (AFLPs) (EP 534 858, specifically incorporated herein by reference in its entirety), and Single Nucleotide Polymorphisms (SNPs) (Wang et al., 1998).
In still yet another aspect, the present invention provides hybrid genetic complements, as represented by squash plant cells, tissues, plants, and seeds, formed by the combination of a haploid genetic complement of a squash plant of the invention with a haploid genetic complement of a second squash plant, preferably, another, distinct squash plant. In another aspect, the present invention provides a squash plant regenerated from a tissue culture that comprises a hybrid genetic complement of this invention.
In certain embodiments, the invention provides a plant of an inbred squash line that exhibits viral resistance to at least a first virus selected from the group consisting of Cucumber Mosaic Virus (CMV), zucchini yellow mosaic potyvirus (ZYMV), and watermelon mosaic virus (WMV), wherein the viral resistance is controlled by genetic means for the expression of such trait found in squash line ZGN 130-1041T.
In still yet another aspect, the invention provides a method of determining the genotype of a plant of squash line ZGN 130-1041T comprising detecting in the genome of the plant at least a first polymorphism. The method may, in certain embodiments, comprise detecting a plurality of polymorphisms in the genome of the plant. The method may further comprise storing the results of the step of detecting the plurality of polymorphisms on a computer readable medium. The invention further provides a computer readable medium produced by such a method.
In still yet another aspect, the present invention provides a method of producing a plant derived from line ZGN 130-1041T, the method comprising the steps of: (a) preparing a progeny plant derived from line ZGN 130-1041T, wherein said preparing comprises crossing a plant of the line ZGN 130-1041T with a second plant; and (b) crossing the progeny plant with itself or a second plant to produce a seed of a progeny plant of a subsequent generation. In further embodiments, the method may additionally comprise: (c) growing a progeny plant of a subsequent generation from said seed of a progeny plant of a subsequent generation and crossing the progeny plant of a subsequent generation with itself or a second plant; and repeating the steps for an additional 3-10 generations to produce a plant derived from line ZGN 130-1041T. The plant derived from line ZGN 130-1041T may be an inbred line, and the aforementioned repeated crossing steps may be defined as comprising sufficient inbreeding to produce the inbred line. In the method, it may be desirable to select particular plants resulting from step (c) for continued crossing according to steps (b) and (c). By selecting plants having one or more desirable traits, a plant derived from line ZGN 130-1041T is obtained which possesses some of the desirable traits of the line as well as potentially other selected traits.
These and other features and advantages of this invention are described in, or are apparent from, the following detailed description of various exemplary embodiments of the devices and methods according to this invention.


DETAILED DESCRIPTION OF THE INVENTION
The invention provides methods and compositions relating to plants, seeds and derivatives of squash line ZGN 130-1041T. This line shows uniformity and stability within the limits of environmental influence for the traits described hereinafter and provides sufficient seed yield. By crossing with a distinct and homozygous second plant, uniform F1 hybrid progeny can be obtained.
ZGN 130-1041T is a green zucchini breeding line with exceptional levels of virus resistance. This genotype in particular shows high level resistance to Cucumber Mosaic Virus (CMV), zucchini yellow mosaic potyvirus (ZYMV), and watermelon mosaic virus (WMV). In addition to the disease resistance, this genotype has a medium green fruit color preferred by growers in many regions, as well as acceptable horticultural characteristics.
The development of the line can be summarized as follows.
A. Origin and Breeding History of Scluash Line ZGN 130-1041T
Squash line ZGN 130-1041T was developed by a selection strategy involving two components: (1) “CAS 46-2002T,” a breeding line having the characteristics of strong female flower production potential, the ZWC multi-virus resistance transgene, and field tolerance to powdery mildew caused by Sphaerotheca fuliginella (Schlechtend.:Fr.) Pollacci, and (2) “ZGN 47-314,” a proprietary breeding line contributing vigor, dark green fruit color, and a long cylindrical fruit shape. The initial cross was made in Florida in April of 2001.
The F1 generation was self pollinated in a California greenhouse in the fall of 2001, to maximize production of F2 seed. The F2 generation was greenhouse grown in 2002, where approximately 500 seedlings were mechanically inoculated with CMV and ZYMV. After development of symptoms, resistant plants were selected and transplanted to a field, where 42 of the survivors were selected for self pollination based upon fruit type and horticultural characteristics. The F3 generation was grown in Florida during the fall of 2002, where 32 F4 selections were made from 14 of the F3 families. Following self pollination, selections were evaluated for resistance to powdery mildew. For the F4 generation, 12 seeds per selection were grown in the spring of 2003. These seedlings were mechanically inoculated with CMV and ZYMV, and survivors were selected for self pollination based on fruit type and horticultural characteristics. The F5 generation was selected in Florida without inoculation during the fall of 2003, and 4 families were retained for further evaluation.
Beginning with the F6 generation, evaluation of these breeding lines was based on performance of both the breeding lines and hybrids created using these breeding lines. For the line designated ZGN 130-1041T, the F6 generation was self pollinated in a California greenhouse during the winter, concurrently with the production of test hybrids. During the subsequent 4 years, more than 100 genetically unique hybrids have been created and evaluated using this lineage. Evaluations of these hybrids have been performed in at least 8 states in the U.S. Hybrids using ZGN 130-1041T show good horticultural characteristics, field tolerance to powdery mildew caused by Sphaerotheca fuliginella (Schlechtend.:Fr.) Pollacci, and resistance to many strains of ZYMV, WMV, and CMV.
Two additional generations of advancement were completed in 2004 through self pollination of greenhouse grown plants. These additional generations further establish uniformity of the parental line, and the founding stock for ZGN 130-1041T was based on a bulk of seed from three F9 generation plants with the same F8 generation source. The pedigree and lineage of this F9 generation is (CAS 46-2002T X ZGN 47-314)-M-5-4-1-2-1-1-M.
ZGN 130-1041T has a complement of resistance traits and horticultural characteristics that make it ideal for many purposes, including for growing green zucchini hybrids adapted to the United States. ZGN 130-1041T may also be useful in the development of hybrids for other markets.
B. Physiological and Morphological Characteristics of Squash Line ZGN 130-1041T
In accordance with one aspect of the present invention, there is provided a plant having the physiological and morphological characteristics of squash line ZGN 130-1041T. A description of the physiological and morphological characteristics of squash line ZGN 130-1041T is presented in Table 1.







TABLE 1







Physiological and Morphological Characteristics of Line ZGN 130-1041T*










CHARACTERISTIC
ZGN 130-1041T













1.
General Descriptors




Fruit Shape/Variety Group
Zucchini



Expected Primary Usage
Culinary



Parts of Plant Providing Expected
Immature Fruit



Primary Usage



Cotyledons Measured Between Full
Length to Width ratio - 1.75



Expansion of 1st and 2nd True Leaves
Apex - Not Notched




Veining - Obvious


2.
Main Stem



Main Color
Dark Green (Nearly Entire Length - Fordhook




Zucchini, Jack O'Lantern, Howden)



White Marks at Nodes
Present



Yellow Marks (Associated with
Absent



Precocious Yellow Gene Complex)



Growth Habit (20 True Leaves)



Bush
True-bush (Fordhook Zucchini, Cocozelle, Ronde




de Nice, benning's Green Tint)



Tendrils (20 True Leaves)
Absent or Rudimentary



Main Stem Internode Dimensions



Observed after 20th Internode



Developed



Length
Constant from 5th to 15th Internode



Width
Decreases from 5th to 15th Internode


3.
Petioles



Derived from Main Stem Observed



after 20th Node Developed



Length to Width Medial Ratio of
49.70



10th



Length to Width Medial Ratio of
45.19



15th Petiole



Spininess (Prickles) Observed after
Moderately Spiny (Cocozelle)



20th Internode



Angle of 6th through 15th on Main
Horizontal (Caserta, less than 10°)



Stem


4.
Laminae



Lobing of 10th and 15th on Main
Medium Lobed



Stem



Dimensions of Leaf after 20th
0.83 Length to Maximal Width Ratio of 10th True



Internode
Leaf



Dimensions of Leaf after 20th
0.85 Length to Maximal Width ratio of 15th True



Internode
Leaf



Silver Blotching or Mottling
Silver Blotching over a Moderate Amount of the




Surface


5.
Flowers



Number per Node
One (almost always) (Fordhook Zucchini,




Cocozelle)



Staminate on day of Anthesis on



Main Stem (Between Nodes 11/20)



Length from Base of Calyx to
71.6 mm



Tip of Corolla



Exterior Width at Top of Calyx
17.3 mm



Cup



Pedicel Length
179.5 mm



Length of Anther Column
11.1 mm



Dominant Color of Corolla of
Orange-yellow (Day of Anthesis)



Staminate Flower



Ring at Base of Interior of Staminate
Absent



Corolla



Ring at Base of Interior of Pistillate
Yellow



Corolla



Pistillate Flower on Day of Anthesis



Length from Base of Calyx to
67.5 mm



Tip of Corolla



Pedicel Length
16.4 mm



Ovary Color (Day Prior to Anthesis)
Green (Black Beauty, Fordhook Zucchini,




Cocozelle, Clarita)


6.
Immature Fruit



Size (3-5 Days Past Anthesis)



Length (through Axis) to Medial
 4.48



Width Ratio



Length (through Axis) to
 4.01



Maximal Width Ratio



Color (3-5 Days Past Anthesis)
Intense Green (Fordhook Zucchini, Black Beauty,




Jack O'Lantern, Senator, Spineless Beauty, Raven)



Fruit Flecks
Small (Nero di Milano, Raven, Magic Lantern)



Fruit Warting
Absent (Cocozelle, Fordhook Zucchini, Ronde de




Nice, Gentry)


6.
Mature Fruit



Dimensions (40 Days Past Anthesis)
3.0 Length (through Axis) to Medial Width Ratio



Warting
Absent (Cocozelle, Fordhook Zucchini, Ronde de




Nice)



Stylar Scar
Flat



Stylar End
Nearly Flat (Fordhook Zucchini, True French)



Peduncle End
Convex



Surface
Neither Netted or Cracked



Exterior Color
Black Green (Fordhook Zucchini, Taybelle)


7.
Seed



Hull (Mature)
Present with Normal Appearance





*These are typical observations. Observations may vary due to environment. Other observations that are substantially equivalent are also within the scope of the invention.






Line ZGN 130-1041T has been self-pollinated and planted for a number of generations to produce the homozygosity and phenotypic stability to make this line useful in commercial seed production. No variant traits have been observed or are expected for this line. Squash line ZGN 130-1041T, being substantially homozygous, can be reproduced by planting seeds of the line, growing the resulting squash plant under self-pollinating or sib-pollinating conditions and harvesting the resulting seeds using techniques familiar to one of skill in the art.
C. Breeding Scluash Line ZGN 130-1041T
One aspect of the current invention concerns methods for crossing the squash line ZGN 130-1041T with itself or a second plant and the seeds and plants produced by such methods. These methods can be used for propagation of line ZGN 130-1041T, or can be used to produce hybrid squash seeds and the plants grown therefrom. Hybrid seeds are produced by crossing line ZGN 130-1041T with second squash parent line.
The development of new varieties using one or more starting varieties is well known in the art. In accordance with the invention, novel varieties may be created by crossing line ZGN 130-1041T followed by multiple generations of breeding according to such well known methods. New varieties may be created by crossing with any second plant. In selecting such a second plant to cross for the purpose of developing novel lines, it may be desired to choose those plants which either themselves exhibit one or more selected desirable characteristics or which exhibit the desired characteristic(s) in progeny. Once initial crosses have been made, inbreeding and selection take place to produce new varieties. For development of a uniform line, often five or more generations of selfing and selection are involved.
Backcrossing can also be used to improve an inbred plant. Backcrossing transfers a specific desirable trait from one inbred or non-inbred source to an inbred that lacks that trait. This can be accomplished, for example, by first crossing a superior inbred (A) (recurrent parent) to a donor inbred (non-recurrent parent), which carries the appropriate locus or loci for the trait in question. The progeny of this cross are then mated back to the superior recurrent parent (A) followed by selection in the resultant progeny for the desired trait to be transferred from the non-recurrent parent. After five or more backcross generations with selection for the desired trait, the progeny are heterozygous for loci controlling the characteristic being transferred, but are like the superior parent for most or almost all other loci. The last backcross generation would be selfed to give pure breeding progeny for the trait being transferred.
The line of the present invention is particularly well suited for the development of new lines based on the elite nature of the genetic background of the line. In selecting a second plant to cross with ZGN 130-1041T for the purpose of developing novel squash lines, it will typically be preferred to choose those plants which either themselves exhibit one or more selected desirable characteristics, or which exhibit the desired characteristic(s) when in hybrid combination. Examples of desirable characteristics related to the rind of the fruit include: a certain coloring, striping, firmness and texture. Desirable characteristics related to the flesh of the fruit include: a certain flavor, taste, texture, thickness, color, sugar content, and solids content (% dry matter). Another desirable set of traits related to the fruit include: a certain fruit size and shape. Other desirable characteristics include: high fruit yield, early maturity, restricted vine growth, improved seed germination, and high seed yield. Disease resistance or tolerance, especially to the diseases mentioned above and throughout this application, is another desirable characteristic. Desirable characteristics related to the environment include: tolerance to environmental stress (e.g. frost, drought, etc.) and adaptability to a wide variety of environmental conditions (e.g. soil, temperature, moisture, etc.).
D. Performance Characteristics
As described above, line ZGN 130-1041T exhibits superior desirable horticultural traits when compared to competing lines, including high level resistance to ZYMV, WMV, and CMV as well as field tolerance to powdery mildew caused by Sphaerotheca fuliginella (Schlechtend.:Fr.) Pollacci. One important aspect of the invention thus provides seed of the variety for commercial use. Such seed can be reproduced by crossing the line under self- or sib-pollinating conditions.
Hybrids made with the line were the subject of objective analyses of performance traits of hybrids made using ZGN 130-1041T compared to other hybrids. The results of the analyses are presented below in Table 2.







TABLE 2







Horticultural Scoring Of Zucchini Hybrids With Or Without ZGN


130-1041T Used As A Parent













Fruit
Fruit
Blossom





Color
Shape
Scar
Spines



Rating
Rating
Rating
Rating
Hybrids
















Average
4.1 ± 0.3
4.4 ± 1.2
4.2 ± 1.0
4.5 ± 0.8
63


without ZGN


130-1041T


as a parent


Average
2.0 ± 1.2
3.0 ± 1.2
4.5 ± 1.4
6.1 ± 1.2
51


with ZGN


130-1041T


as a parent









Fruit Color Rating describes the average and standard deviation of ratings for green color intensity and uniformity on the fruits in the plot on a 1 to 9 scale, where 1 represents uniformly and evenly colored fruit and 9 represents non-uniformly colored, poorly colored, and/or blotchy colored fruit. Fruit Shape Rating describes the average and standard deviation of ratings for how well the fruit produced in the plot match consumer preferences for uniformly cylindrical fruit with an approximate 4:1 length:diameter ratio on a 1 to 9 scale, where 1 represents uniformly cylindrical and ideally proportioned fruit and 9 represents badly misshapen fruit. Blossom Scar Rating represents the average and standard deviation of ratings for the approximate average size of the blossom scar at the distal end of the fruit at harvest maturity on a 1 to 9 scale, where each unit is approximately 1 millimeter. Spines rating represents the average and standard deviation of ratings for the relative size and density of the epidermal spines on the plant on a 1 to 9 scale, where 1 represents plants with a very smooth epidermis and a 9 represents plants with large and dense epidermal spines. Hybrids displays the number of different hybrids rated in each category (with or without ZGN 130-1041T as one parent).
In a separate objective analysis of performance traits of hybrids from a field environment with heavy pressure from Powdery Mildew caused by Sphaerotheca fuliginella (Schlechtend.:Fr.) Pollacci, transgenic multivirus resistant green zucchini type hybrids with and without resistance to this pathogen were compared. The results from this analysis are presented in Table 3.







TABLE 3







Horticultural Scoring Of Transgenic Multivirus Resistant Green


Zucchini Hybrids With Or Without ZGN 130-1041T Used As A Parent










Powdery
Number



Mildew Rating
of hybrids













Average without ZGN 130-1041T as a parent
7.0 ± 0.5
40


Average with ZGN 130-1041T as a parent
5.1 ± 1.3
39









Powdery Mildew Rating describes the average and standard deviation of ratings for intensity of infestation by the fungus Sphaerotheca fuliginella (Schlechtend.:Fr.) Pollacci, where 1 represents complete absence of infestation and 9 represents complete coverage of all aboveground tissues by fungal infestation. A rating of 3 represents a very high level of resistance (minimal evidence of easily visible fungal growth), 5 represents an intermediate level of resistance (field tolerance, moderate fungal growth mostly limited to older leaves), and 7 represents a very low level of resistance (only the youngest leaves show temporary absence of fungal growth). Hybrids displays the number of different hybrids rated in each category (with or without ZGN 130-1041T as one parent).
E. Further Embodiments of the Invention
When the term squash line ZGN 130-1041T is used in the context of the present invention, this also includes plants modified to include at least a first desired heritable trait. Such plants may, in one embodiment, be 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 a genetic locus transferred into the plant via the backcrossing technique. The term single locus converted plant as used herein refers to those squash 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 single locus transferred into the variety via the backcrossing technique.
Backcrossing methods can be used with the present invention to improve or introduce a characteristic into the present variety. The parental squash plant which contributes the locus for the desired characteristic 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 squash plant to which the locus or loci 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 single locus 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 squash 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 single transferred locus from the nonrecurrent parent.
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 a single trait or characteristic in the original variety. To accomplish this, a single locus of the recurrent variety is modified or substituted with the desired locus from the nonrecurrent parent, while retaining essentially all of the rest of the desired genetic, 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 trait to the plant. The exact backcrossing protocol will depend on the characteristic or trait being altered 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.
In one embodiment, progeny squash plants of a backcross in which ZGN 130-1041T is the recurrent parent comprise (i) the desired trait from the non-recurrent parent and (ii) all of the physiological and morphological characteristics of squash line ZGN 130-1041T as determined at the 5% significance level when grown in the same environmental conditions. Squash varieties can also be developed from more than two parents. The technique, known as modified backcrossing, uses different recurrent parents during the backcrossing. Modified backcrossing may be used to replace the original recurrent parent with a variety having certain more desirable characteristics or multiple parents may be used to obtain different desirable characteristics from each.
Many single locus traits have been identified that are not regularly selected for in the development of a new inbred but that can be improved by backcrossing techniques. Single locus traits may or may not be transgenic; examples of these traits include, but are not limited to, male sterility, herbicide resistance, resistance to bacterial, fungal, or viral disease, insect resistance, restoration of male fertility, modified fatty acid or carbohydrate metabolism, and enhanced nutritional quality. These comprise genes generally inherited through the nucleus.
Direct selection may be applied where the single locus acts as a dominant trait. An example of a dominant trait is the anthracnose resistance trait. For this selection process, the progeny of the initial cross are sprayed with anthracnose spores prior to the backcrossing. The spraying eliminates any plants which do not have the desired anthracnose resistance characteristic, and only those plants which have the anthracnose resistance gene are used in the subsequent backcross. This process is then repeated for all additional backcross generations.
Selection of squash plants for breeding is not necessarily dependent on the phenotype of a plant and instead can be based on genetic investigations. For example, one can utilize a suitable genetic marker which is closely genetically linked to a trait of interest. One of these markers can be used to identify the presence or absence of a trait in the offspring of a particular cross, and can be used in selection of progeny for continued breeding. This technique is commonly referred to as marker assisted selection. Any other type of genetic marker or other assay which is able to identify the relative presence or absence of a trait of interest in a plant can also be useful for breeding purposes. Procedures for marker assisted selection applicable to the breeding of squash are well known in the art. Such methods will be of particular utility in the case of recessive traits and variable phenotypes, or where conventional assays may be more expensive, time consuming or otherwise disadvantageous. Types of genetic markers which could be used in accordance with the invention include, but are not necessarily limited to, Simple Sequence Length Polymorphisms (SSLPs) (Williams et al., 1990), Randomly Amplified Polymorphic DNAs (RAPDs), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR), Amplified Fragment Length Polymorphisms (AFLPs) (EP 534 858, specifically incorporated herein by reference in its entirety), and Single Nucleotide Polymorphisms (SNPs) (Wang et al., 1998).
F. Plants Derived from Scluash Line ZGN 130-1041T by Genetic Engineering
Many useful traits that can be introduced by backcrossing, as well as directly into a plant, are those which are introduced by genetic transformation techniques. Genetic transformation may therefore be used to insert a selected transgene into the squash line of the invention or may, alternatively, be used for the preparation of transgenes which can be introduced by backcrossing. Methods for the transformation of plants, including squash, are well known to those of skill in the art. Techniques which may be employed for the genetic transformation of squash include, but are not limited to, electroporation, microprojectile bombardment, Agrobacterium-mediated transformation and direct DNA uptake by protoplasts.
To effect transformation by electroporation, one may employ either friable tissues, such as a suspension culture of cells or embryogenic callus or alternatively one may transform immature embryos or other organized tissue directly. In this technique, one would partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wound tissues in a controlled manner.
A particularly efficient method for delivering transforming DNA segments to plant cells is microprojectile bombardment. Microprojectile bombardment techniques are widely applicable, and may be used to transform virtually any plant species. In this method, particles are coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, platinum, and preferably, gold. For the bombardment, cells in suspension are concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the microprojectile stopping plate.
An illustrative embodiment of a method for delivering DNA into plant cells by acceleration is the Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a surface covered with target squash cells. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectiles aggregate and may contribute to a higher frequency of transformation by reducing the damage inflicted on the recipient cells by projectiles that are too large.
Agrobacterium-mediated transformation of squash explant material and regeneration of whole transformed squash plants has been shown to be an efficient transformation method (U.S. Pat. No. 6,337,431). An advantage of the technique is that DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. Modern Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations (Klee et al., 1985). Moreover, recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate the construction of vectors capable of expressing various polypeptide coding genes. The vectors described have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes. Additionally, Agrobacterium containing both armed and disarmed Ti genes can be used for transformation.
In those plant strains where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene locus transfer. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art (Fraley et al., 1985; U.S. Pat. No. 5,563,055).
Transformation of plant protoplasts also can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, e.g., Potrykus et al., 1985; Omirulleh et al., 1993; Fromm et al., 1986; Uchimiya et al., 1986; Marcotte et al., 1988). Transformation of plants and expression of foreign genetic elements is exemplified in Choi et al. (1994), and Ellul et al. (2003).
A number of promoters have utility for plant gene expression for any gene of interest including but not limited to selectable markers, scoreable markers, genes for pest tolerance, disease tolerance, nutritional enhancements and any other gene of agronomic interest. Examples of constitutive promoters useful for squash plant gene expression include, but are not limited to, the cauliflower mosaic virus (CaMV) P-35S promoter, which confers constitutive, high-level expression in most plant tissues (see, e.g., Odel et al., 1985), including monocots (see, e.g., Dekeyser et al., 1990; Terada and Shimamoto, 1990); a tandemly duplicated version of the CaMV 35S promoter, the enhanced 35S promoter (P-e35S) the nopaline synthase promoter (An et al., 1988), the octopine synthase promoter (Fromm et al., 1989); and the figwort mosaic virus (P-FMV) promoter as described in U.S. Pat. No. 5,378,619 and an enhanced version of the FMV promoter (P-eFMV) where the promoter sequence of P-FMV is duplicated in tandem, the cauliflower mosaic virus 19S promoter, a sugarcane bacilliform virus promoter, a commelina yellow mottle virus promoter, and other plant DNA virus promoters known to express in plant cells.
A variety of plant gene promoters that are regulated in response to environmental, hormonal, chemical, and/or developmental signals can be used for expression of an operably linked gene in plant cells, including promoters regulated by (1) heat (Callis et al., 1988), (2) light (e.g., pea rbcS-3A promoter, Kuhlemeier et al., 1989; maize rbcS promoter, Schaffner and Sheen, 1991; or chlorophyll a/b-binding protein promoter, Simpson et al., 1985), (3) hormones, such as abscisic acid (Marcotte et al., 1989), (4) wounding (e.g., wunl, Siebertz et al., 1989); or (5) chemicals such as methyl jasmonate, salicylic acid, or Safener. It may also be advantageous to employ organ-specific promoters (e.g., Roshal et al., 1987; Schernthaner et al., 1988; Bustos et al., 1989).
Exemplary nucleic acids which may be introduced to the squash lines of this invention include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods rather than classical reproduction or breeding techniques. However, the term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA which is already present in the plant cell, DNA from another plant, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.
Many hundreds if not thousands of different genes are known and could potentially be introduced into a squash plant according to the invention. Non-limiting examples of particular genes and corresponding phenotypes one may choose to introduce into a squash plant include one or more genes for insect tolerance, such as a Bacillus thuringiensis (B.t.) gene, pest tolerance such as genes for fungal disease control, herbicide tolerance such as genes conferring glyphosate tolerance, and genes for quality improvements such as yield, nutritional enhancements, environmental or stress tolerances, or any desirable changes in plant physiology, growth, development, morphology or plant product(s). For example, structural genes would include any gene that confers insect tolerance including but not limited to a Bacillus insect control protein gene as described in WO 99/31248, herein incorporated by reference in its entirety, U.S. Pat. No. 5,689,052, herein incorporated by reference in its entirety, U.S. Pat. Nos. 5,500,365 and 5,880,275, herein incorporated by reference it their entirety. In another embodiment, the structural gene can confer tolerance to the herbicide glyphosate as conferred by genes including, but not limited to Agrobacterium strain CP4 glyphosate resistant EPSPS gene (aroA:CP4) as described in U.S. Pat. No. 5,633,435, herein incorporated by reference in its entirety, or glyphosate oxidoreductase gene (GOX) as described in U.S. Pat. No. 5,463,175, herein incorporated by reference in its entirety.
Alternatively, the DNA coding sequences can affect these phenotypes by encoding a non-translatable RNA molecule that causes the targeted inhibition of expression of an endogenous gene, for example via antisense- or cosuppression-mediated mechanisms (see, for example, Bird et al., 1991). The RNA could also be a catalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desired endogenous mRNA product (see for example, Gibson and Shillito, 1997). Thus, any gene which produces a protein or mRNA which expresses a phenotype or morphology change of interest is useful for the practice of the present invention.
G. Definitions
In the description and tables herein, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, the following definitions are provided:
A: When used in conjunction with the word “comprising” or other open language in the claims, the words “a” and “an” denote “one or more.”
Allele: Any of one or more alternative forms of a gene locus, all of which alleles 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.
Backcrossing: A process in which a breeder repeatedly crosses hybrid progeny, for example a first generation hybrid (F1), back to one of the parents of the hybrid progeny. Backcrossing can be used to introduce one or more single locus conversions from one genetic background into another.
Crossing: The mating of two parent plants.
Cross-pollination: Fertilization by the union of two gametes from different plants.
Diploid: A cell or organism having two sets of chromosomes.
Emasculate: The removal of plant male sex organs or the inactivation of the organs with a cytoplasmic or nuclear genetic factor conferring male sterility or a chemical agent.
Enzymes: Molecules which can act as catalysts in biological reactions.
F1 Hybrid: The first generation progeny of the cross of two nonisogenic plants.
Genotype: The genetic constitution of a cell or organism.
Haploid: A cell or organism having one set of the two sets of chromosomes in a diploid.
Linkage: A phenomenon wherein alleles on the same chromosome tend to segregate together more often than expected by chance if their transmission was independent.
Marker: A readily detectable phenotype, preferably inherited in codominant fashion (both alleles at a locus in a diploid heterozygote are readily detectable), with no environmental variance component, i.e., heritability of 1.
Phenotype: The detectable characteristics of a cell or organism, which characteristics are the manifestation of gene expression.
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.
Regeneration: The development of a plant from tissue culture.
Self-pollination: The transfer of pollen from the anther to the stigma of the same plant.
Single Locus Converted (Conversion) Plant: Plants which are developed by a plant breeding technique called backcrossing, wherein essentially all of the desired morphological and physiological characteristics of a squash variety are recovered in addition to the characteristics of the single locus transferred into the variety via the backcrossing technique and/or by genetic transformation.
Substantially Equivalent: A characteristic that, when compared, does not show a statistically significant difference (e.g., p=0.05) from the mean.
Tetraploid: A cell or organism having four sets of chromosomes.
Tissue Culture: A composition comprising isolated cells of the same or a different type or a collection of such cells organized into parts of a plant.
Transgene: A genetic locus comprising a sequence which has been introduced into the genome of a squash plant by transformation.
Triploid: A cell or organism having three sets of chromosomes.
H. Deposit Information
A deposit of squash line ZGN 130-1041T, disclosed above and recited in the claims, has been made with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209. The date of deposit was 30 Apr. 2007. Upon issuance of a patent, all restrictions upon the deposit will be removed, and the deposit is intended to meet all of the requirements of 37 C.F.R. §1.801-1.809. The accession number for those deposited seeds of squash line ZGN 130-1041T is ATCC Accession No. PTA-8397. The deposit will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced if necessary during that period.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the invention, as limited only by the scope of the appended claims.
All references cited herein are hereby expressly incorporated herein by reference.
REFERENCES
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference:

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## Claims

1. A seed of squash line ZGN 130-1041T, a sample of seed of said line having been deposited under ATCC Accession Number PTA-8397.

2. A plant of squash line ZGN 130-1041T, a sample of seed of said line having been deposited under ATCC Accession Number PTA-8397.

3. A plant part of the plant of claim 2.

4. The plant part of claim 3, wherein said part is selected from the group consisting of a leaf, fruit, pollen, an ovule and a cell.

5. A squash plant, or a part thereof, having all the physiological and morphological characteristics of the squash plant of claim 2.

6. A tissue culture of regenerable cells of squash line ZGN 130-1041T, a sample of seed of said line having been deposited under ATCC Accession Number PTA-8397.

7. The tissue culture according to claim 6, comprising cells or protoplasts from a plant part selected from the group consisting of embryos, meristems, cotyledons, pollen, leaves, anthers, roots, root tips, pistil, flower, seed and stalks.

8. A squash plant regenerated from the tissue culture of claim 6, wherein the regenerated plant expresses all of the physiological and morphological characteristics of squash line ZGN 130-1041T, a sample of seed of said line having been deposited under ATCC Accession Number PTA-8397.

9. A method of producing squash seed, said method comprising crossing a plant of squash line ZGN 130-1041T, a sample of seed of said line having been deposited under ATCC Accession Number PTA-8397, with a second squash plant.

10. The method of claim 9, wherein the plant of squash line ZGN 130-1041T is the female parent.

11. The method of claim 9, wherein the plant of squash line ZGN 130-1041T is the male parent.

12. An F1 hybrid seed produced by the method of claim 9.

13. An F1 hybrid plant produced by growing the seed of claim 12.

14. A method for producing a seed of a line ZGN 130-1041T-derived squash plant, said method comprising the steps of:
(a) crossing a squash plant of line ZGN 130-1041T, a sample of seed of said line having been deposited under ATCC Accession Number PTA-8397, with a second squash plant; and
(b) allowing seed of a ZGN 130-1041T-derived squash plant to form.

15. The method of claim 14, further comprising the steps of:
(c) crossing a plant grown from said ZGN 130-1041T-derived squash seed with itself or a second squash plant to yield additional ZGN 130-1041T-derived squash seed;
(d) growing said additional ZGN 130-1041T-derived squash seed of step (c) to yield additional ZGN 130-1041T-derived squash plants; and
(e) repeating the crossing and growing steps of (c) and (d) to generate further ZGN 130-1041T-derived squash plants.

16. A method of vegetatively propagating a plant of squash line ZGN 130-1041T, said method comprising the steps of:
(a) collecting tissue capable of being propagated from a squash plant of line ZGN 130-1041T, a sample of seed of said line having been deposited under ATCC Accession Number PTA-8397;
(b) cultivating said tissue to obtain proliferated shoots; and
(c) rooting said proliferated shoots to obtain rooted plantlets.

17. The method of claim 16, further comprising growing plants from said rooted plantlets.

18. A method of introducing a desired trait into squash line ZGN 130-1041T, said method comprising:
(a) crossing a plant of line ZGN 130-1041T, a sample of seed of said line having been deposited under ATCC Accession Number PTA-8397, with a second squash plant that comprises a desired trait to produce F1 progeny;
(b) selecting an F1 progeny that comprises the desired trait;
(c) crossing the selected F1 progeny with a plant of line ZGN 130-1041T (ATCC Accession Number PTA-8397) to produce backcross progeny;
(d) selecting backcross progeny comprising the desired trait and the physiological and morphological characteristic of squash line ZGN 130-1041T; and
(e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait and all of the physiological and morphological characteristics of squash line ZGN 130-1041T when grown in the same environmental conditions.

19. A squash plant produced by the method of claim 18.

20. A method of producing a plant of squash line ZGN 130-1041T, a sample of seed of said line having been deposited under ATCC Accession Number PTA-8397, comprising an added desired trait, the method comprising introducing a transgene conferring the desired trait into a plant of squash line ZGN 130-1041T.

21. A plant that comprises all of the physiological and morphological characteristics of squash line ZGN 130-1041T, a sample of seed of said line having been deposited under ATCC Accession Number PTA-8397.

22. A seed that produces the plant of claim 21.

23. A method of determining the genotype of a plant of squash line ZGN 130-1041T, a sample of seed of said line having been deposited under ATCC Accession Number PTA-8397, said method comprising obtaining a sample of nucleic acids from said plant and detecting in said nucleic acids a plurality of polymorphisms.

24. The method of claim 23, further comprising storing the results of detecting the plurality of polymorphisms on a computer readable medium.

25. A squash plant produced by the method of claim 20.