# Patent

## Title
Lettuce line P2231-3013596

## Abstract

The invention provides seed and plants of the lettuce line designated P2231-3013596. The invention thus relates to the plants, seeds and tissue cultures of lettuce line P2231-3013596, and to methods for producing a lettuce plant produced by crossing a plant of lettuce line P2231-3013596 with itself or with another lettuce 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 lettuce line P2231-3013596, including the gametes of such plants.

## Background
FIELD OF THE INVENTION
The present invention relates to the field of plant breeding and, more specifically, to the development of lettuce line P2231-3013596.
BACKGROUND OF THE INVENTION
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 of different varieties 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 lettuce. Lettuce, Lactuca sativa, is a temperate annual or biennial plant most often grown as a leaf vegetable. Lettuce belongs to the family Asteraceae (or Compositae). Other members of this family include endive, chicory, artichoke, sunflower and safflower. It is closely related to common wild lettuce or prickly lettuce (L. serriola) and less closely related to two other wild lettuces (L. saligna and L. virosa). Lettuce and sunflower are the best genetically characterized members of this family. Four principal types of lettuce include crisphead (mostly iceberg), romaine (cos), leaf and butterhead. Each of these basic groups is comprised of numerous cultivars, each characterized by its own particular morphology, disease resistance and cultural adaptations. These types vary in size, shape, texture, color, nutritional value and taste. The principal nutrients are vitamins A and C and calcium, and the percentage water content in lettuce is high (e.g., 93-96%).
A lettuce plant has a short stem initially, but when it blooms, the stem lengthens and branches, producing many flower heads that look like those of dandelions, but smaller. This is called bolting. When grown to eat, lettuce is harvested before it bolts. In the United States, more than 90% of the nation's lettuce is grown in California and Arizona. In California, variations in temperature in the regions where lettuce is grown allows for a continuous supply of lettuce year-round, although the greatest supply is from May through October.
Lettuce is a diploid species with 2N=18 chromosomes and naturally self-pollinates. The self-pollination feature leads to inbred lines that are uniform and vigorous, and therefore exhibit extensive genetic homozygosity. Virtually all commercial cultivars in use today are inbred lines. Hybrid development has not been pursued with much interest due to the tedious nature of manual crossing and low seed production per cross. Furthermore, there is no consistent large-scale pollen movement by insects or wind.
The principal breeding mechanisms are pedigree breeding and backcrossing, but straight selection of desirable plants within a cultivar is also practiced to slightly modify the cultivar. Choice of breeding or selection methods depends on, for example, the mode of plant reproduction, the heritability of the trait(s) being improved and the type of cultivar used commercially. Since the flowers of lettuce are delicate and emasculation is tedious and difficult, lettuce breeders typically wait until the flowers open and then wash away the self-pollen prior to crossing.
While breeding efforts to date have provided a number of useful lettuce 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 lettuce plant of the line designated P2231-3013596. Also provided are lettuce plants having all the physiological and morphological characteristics of the lettuce line designated P2231-3013596. Parts of the lettuce plant of the present invention are also provided, for example, including pollen, an ovule and a cell of the plant.
The invention also concerns seed of lettuce line P2231-3013596. The lettuce seed of the invention may be provided as an essentially homogeneous population of lettuce seed of the line designated P2231-3013596. Essentially homogeneous populations of seed are generally free from substantial numbers of other seed. Therefore, seed of line P2231-3013596 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 lettuce 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 lettuce plants designated P2231-3013596.
In another aspect of the invention, a plant of lettuce line P2231-3013596 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 lettuce line P2231-3013596 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.
In another aspect of the invention, a tissue culture of regenerable cells of a plant of line P2231-3013596 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 P2231-3013596 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 lettuce plants regenerated from a tissue culture of the invention, the plants having all the physiological and morphological characteristics of line P2231-3013596.
In yet another aspect of the invention, processes are provided for producing lettuce seeds and plants, which processes generally comprise crossing a first parent lettuce plant with a second parent lettuce plant, wherein at least one of the first or second parent lettuce plants is a plant of the line designated P2231-3013596. These processes may be further exemplified as processes for preparing hybrid lettuce seed or plants, wherein a first lettuce plant is crossed with a second lettuce plant of a different, distinct line to provide a hybrid that has, as one of its parents, the lettuce plant line P2231-3013596. 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 lettuce 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 lettuce plants into plants that bear flowers. A third step may comprise preventing self-pollination of the plants, such as by emasculating the male portions of flowers, (i.e., treating or manipulating the flowers to produce an emasculated parent lettuce 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 lettuce plants. Yet another step comprises harvesting the seeds from at least one of the parent lettuce plants. The harvested seed can be grown to produce a lettuce plant or hybrid lettuce plant.
The present invention also provides the lettuce seeds and plants produced by a process that comprises crossing a first parent lettuce plant with a second parent lettuce plant, wherein at least one of the first or second parent lettuce plants is a plant of the line designated P2231-3013596. In one embodiment of the invention, lettuce seed and plants produced by the process are first generation (F1) hybrid lettuce 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 lettuce plant, and methods of use thereof. Therefore, certain exemplary embodiments of the invention provide an F1 hybrid lettuce plant and seed thereof.
In still yet another aspect of the invention, the genetic complement of the lettuce plant line designated P2231-3013596 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 lettuce 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 lettuce plant cells that have a genetic complement in accordance with the lettuce 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 P2231-3013596 or a first generation progeny thereof 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 lettuce plant cells, tissues, plants, and seeds, formed by the combination of a haploid genetic complement of a lettuce plant of the invention with a haploid genetic complement of a second lettuce plant, preferably, another, distinct lettuce plant. In another aspect, the present invention provides a lettuce plant regenerated from a tissue culture that comprises a hybrid genetic complement of this invention.
In still yet another aspect, the invention provides a plant of an inbred lettuce line that exhibits a combination of traits comprising resistance to corky root rot and a tall, wide and heavy head. In certain embodiments, the combination of traits may be defined as controlled by genetic means for the expression of the combination of traits found in lettuce line P2231-3013596.
In still yet another aspect, the invention provides a method of determining the genotype of a plant of lettuce line P2231-3013596 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 P2231-3013596, the method comprising the steps of: (a) preparing a progeny plant derived from line P2231-3013596, wherein said preparing comprises crossing a plant of the line P2231-3013596 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 P2231-3013596. The plant derived from line P2231-3013596 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 P2231-3013596 is obtained which possesses some of the desirable traits of the line as well as potentially other selected traits.
In certain embodiments, the present invention provides a method of producing lettuce comprising: (a) obtaining a plant of lettuce line P2231-3013596, wherein the plant has been cultivated to maturity, and (b) collecting lettuce from the plant.
Any embodiment discussed herein with respect to one aspect of the invention applies to other aspects of the invention as well, unless specifically noted.
The term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and to “and/or.” When used in conjunction with the word “comprising” or other open language in the claims, the words “a” and “an” denote “one or more,” unless specifically noted. The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps. Similarly, any plant that “comprises,” “has” or “includes” one or more traits is not limited to possessing only those one or more traits and covers other unlisted traits.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and any specific examples provided, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.


DETAILED DESCRIPTION OF THE INVENTION
The invention provides methods and compositions relating to plants, seeds and derivatives of lettuce line P2231-3013596. This line shows uniformity and stability within the limits of environmental influence for the traits described hereinafter. Lettuce line P2231-3013596 provides sufficient seed yield. By crossing with a distinct second plant, uniform F1 hybrid progeny can be obtained.
A. Origin and Breeding History of Lettuce Line P2231-3013596
Line P2231-3013596 was derived from the initial cross between cv. Cosmic and PSR 4569 (now PS 6545691). Single plant selections were made in subsequent years in the area of intended commercialization. By F5 a group of 18 families was selected and bulked for trialing and seed increase. An F6 mass was trialed in Year 7 and an increase of F7 seed was produced in Year 8 for commercial trials the same year. The breeding method employed was pedigree selection, using both single plant selection and mass selection practices.
In replicated field trials of P2231-3013596 during the last two years covering generations F6 to F7, neither genetic variants nor off-types were observed in more than 8,000 plants, indicating that this variety is genetically uniform and stable. The origin and breeding history can be summarized as follows.




B. Physiological and Morphological Characteristics of Lettuce Line P2231-3013596
In accordance with one aspect of the present invention, there is provided a plant having the physiological and morphological characteristics of lettuce line P2231-3013596. A description of the physiological and morphological characteristics of lettuce line P2231-3013596 is presented in Table 1.







TABLE 1







Physiological and Morphological Characteristics of Line


P2231-3013596










CHARACTERISTIC
P2231-3013596













1.
Plant Type
Iceberg-Romaine Cross


2.
Seed



Color
Black (Grey Brown)



Light Dormancy
Light Not Required



Heat Dormancy
Susceptible


3.
Cotyledon To Fourth Leaf Stage



Shape of Cotyledons
Intermediate



Shape of Fourth Leaf
Elongated



Length/Width Index of Fourth
17



Leaf: L/W × 10



Apical Margin
Finely Dentate



Basal Margin
Coarsely Dentate



Undulation
Slight



Green Color
Dark Green



Anthocyanin
Absent



Concentration
N/A



Rolling
Absent



Cupping
Uncupped



Reflexing
None


4.
Mature Leaves



Margin



Incision Depth
Absent/Shallow




(Dark Green Boston)



Indentation
Shallowly Dentate




(Great Lakes 65)



Undulations of the Apical
Absent/Slight



Margin
(Dark Green Boston)



Green Color
Dark Green (Vanguard)



Anthocyanin
Absent



Concentration
N/A



Size
Large



Glossiness
Moderate (Salinas)



Blistering
Absent/Slight (Salinas)



Leaf Thickness
Thick



Trichomes
Absent (Smooth)


5.
Plant



Spread of frame Leaves
35 cm



Head Diameter
25 cm



Head Shape
Elongate



Head Size Class
Large



Head Per Carton
24



Head Weight
1431 g



Head Firmness
Very Firm


6.
Butt



Shape
Rounded



Midrib
Prominently Raised




(Great Lakes 659)


7.
Core



Diameter at Base of Head
40 mm



Ratio of Head Diameter/Core
5.5



Diameter



Core Height From Base of Head to
59 mm



Apex


8.
Bolting



Number of Days from First Water
66



Date to Seed Stalk Emergence



Bolting Class
Medium



Height of Mature Seed Stalk
108 cm



Spread of Bolter Plant
48 cm



Bolter Leaves
Curved



Margin
Entire



Color
Medium Green



Bolter Habit



Terminal Inflorescence
Present



Lateral Shoots
Present



Basal Side Shoots
Absent


9.
Maturity



Season and Location
First Water Date to Harvest:



Spring - Huron County, CA
126 days



November 2005



Summer - Salinas County, CA
72 days



April 2005



Fall - Huron County, CA -
85 days



August 2005



Winter - Yuma County, AZ
114 days



October 2005


10.
Adaptation



Regions Tested



Southwest (CA and/or AZ desert)
Adapted



Seasons Tested



Spring - Fresno, CA
Adapted



Summer - Monterey, CA
Adapted



Fall - Fresno, CA
Adapted



Winter, Yuma Co., AZ
Adapted



Greenhouse
Not Tested



Soil Type
Mineral


11.
Viral Diseases



Big Vein
Susceptible



Lettuce Mosaic
Susceptible


12.
Fungal/Bacterial Diseases



Corky Root Rot
Resistant



Downy Mildew
Resistant



Powdery Mildew
Susceptible



Sclerotinia Drop
Susceptible


13.
Insects



Lettuce Aphid
Susceptible


14.
Physiological Stresses



Tipburn
Moderately Resistant/




Moderately Susceptible



Heat
Moderately Resistant/




Moderately Susceptible





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






Line P2231-3013596 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. Lettuce line P2231-3013596, being substantially homozygous, can be reproduced by planting seeds of the line, growing the resulting lettuce 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 Lettuce Line P2231-3013596
One aspect of the current invention concerns methods for crossing the lettuce line P2231-3013596 with itself or a second plant and the seeds and plants produced by such methods. These methods can be used for propagation of line P2231-3013596, or can be used to produce hybrid lettuce seeds and the plants grown therefrom. Hybrid seeds are produced by crossing line P2231-3013596 with second lettuce 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 P2231-3013596 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) when in hybrid combination. 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.
Uniform lines of new varieties may also be developed by way of double-haploids. This technique allows the creation of true breeding lines without the need for multiple generations of selfing and selection. In this manner true breeding lines can be produced in as little as one generation. Haploid embryos may be produced from microspores, pollen, anther cultures, or ovary cultures. The haploid embryos may then be doubled autonomously, or by chemical treatments (e.g. colchicine treatment). Alternatively, haploid embryos may be grown into haploid plants and treated to induce chromosome doubling. In either case, fertile homozygous plants are obtained. In accordance with the invention, any of such techniques may be used in connection with line P2231-3013596 and progeny thereof to achieve a homozygous line.
New varieties may be created, for example, by crossing line P2231-3013596 with any second plant and selection of progeny in various generations and/or by doubled haploid technology. In choosing 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. After one or more lines are crossed, true-breeding lines may be developed.
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 P2231-3013596 for the purpose of developing novel lettuce 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.
D. Performance Characteristics
As described above, line P2231-3013596 exhibits a number of desirable agronomic traits, including resistance to corky root rot, and tall, wide and heavy heads. The performance characteristics of the line were the subject of an objective analysis of the performance traits of the line relative to other lines. The results of the analysis are presented below.







TABLE 2







Performance Characteristics For Line P2231-3013596 and the most similar cultivar, P2428-5006090, for three


important characters.
















Plant
Head
Head
Sum of the three


Trial No
Cultivar
Rep No.
Heighta
Diameterb
Weightc
Standard Deviationsd





Trial 1:
P2231-3013596:
Rep. 1
27.5 ± 2.0
24.1 ± 1.5
1377 ± 78.3
81.8


Evaluated

Rep. 2
26.1 ± 1.8
25.8 ± 1.7
1421 ± 89.6
93.1


Yuma, AZ

Average:
26.8 ± 1.9
25.0 ± 1.6
1399 ± 84.1
87.5



P2428-5006090:
Rep. 1
24.2 ± 0.7
21.7 ± 0.5
1255 ± 23.4
24.6




Rep. 2
25.1 ± 0.8
21.1 ± 0.4
1271 ± 22.7
23.9




Average:
24.7 ± 0.8
21.4 ± 0.5
1263 ± 23.1
24.3


Trial 2:
P2231-3013596:
Rep. 1
26.7 ± 1.6
24.6 ± 1.6
1439 ± 72.5
75.7


Evaluated:

Rep. 2
26.1 ± 1.6
25.2 ± 1.8
1485 ± 84.5
87.9


Salinas, CA

Average:
26.4 ± 1.6
24.9 ± 1.7
1462 ± 78.0
81.9



P2428-5006090:
Rep. 1
23.8 ± 0.7
22.6 ± 0.7
1248 ± 22.1
23.5




Rep. 2
23.9 ± 0.6
23.7 ± 0.6
1253 ± 22.2
23.4




Average:
23.9 ± 0.7
23.2 ± 0.7
1251 ± 22.2
23.5


Range of variation among
P2231-3013596

22.68 to 30.52
21.00 to 28.40
1219.3 to 1583.8


means of statistically
P2428-5006090

22.68 to 25.82
21.04 to 23.51
1267.1 to 1368.4


significant differences


at the 95% level using


the confidence interval [CI =


mean ± (SD × SE)]:





aMean plant height using two sowing dates of 20 plants per replication in cm ± standard deviation.


bMean head diameter using two sowing dates of 20 plants per replication in cm ± standard deviation.


cMean head weight using two sowing dates of 20 plants per replication in grams ± standard deviation.


dSum of the three standard deviations for plant height, head diameter and head weight.






E. Further Embodiments of the Invention
In specific embodiments, the invention provides 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 lettuce 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 lettuce 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 lettuce 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 lettuce 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 lettuce plants of a backcross in which P2231-3013596 is the recurrent parent comprise (i) the desired trait from the non-recurrent parent and (ii) all of the physiological and morphological characteristics of lettuce line P2231-3013596 as determined at the 5% significance level when grown in the same environmental conditions.
Lettuce 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 lettuce 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 lettuce 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 Lettuce Line P2231-3013596 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 lettuce 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 lettuce, are well known to those of skill in the art.
Vectors used for the transformation of lettuce cells are not limited so long as the vector can express an inserted DNA in the cells. For example, vectors comprising promoters for constitutive gene expression in lettuce cells (e.g., cauliflower mosaic virus 35S promoter) and promoters inducible by exogenous stimuli can be used. Examples of suitable vectors include pBI binary vector. The “lettuce cell” into which the vector is to be introduced includes various forms of lettuce cells, such as cultured cell suspensions, protoplasts, leaf sections, and callus.
A vector can be introduced into lettuce cells by known methods, such as the polyethylene glycol method, polycation method, electroporation, Agrobacterium-mediated transfer, particle bombardment and direct DNA uptake by protoplasts. See, e.g., Pang et al. (1996).
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. An example of electroporation of lettuce protoplasts is presented in Chupeau et al. (1989).
A particularly efficient method for delivering transforming DNA segments to plant cells is microprojectile bombardment. 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 macroprojectile 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 lettuce 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.
Microprojectile bombardment techniques are widely applicable, and may be used to transform virtually any plant species. Examples involving microprojectile bombardment transformation with lettuce can be found in, for example, Elliott et al. (2004) and Molinier et al. (2002).
Agrobacterium-mediated transfer is another widely applicable system for introducing gene loci into plant cells. 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). For example, U.S. Pat. No. 5,349,124 describes a method of transforming lettuce plant cells using Agrobacterium-mediated transformation. By inserting a chimeric gene having a DNA coding sequence encoding for the full-length B.t. toxin protein that expresses a protein toxic toward Lepidopteran larvae, this methodology resulted in lettuce having resistance to such insects.
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 resistance, nutritional enhancements and any other gene of agronomic interest. Examples of constitutive promoters useful for lettuce 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 lettuce 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 lettuce plant according to the invention. Non-limiting examples of particular genes and corresponding phenotypes one may choose to introduce into a lettuce 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:
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 lettuce 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 lettuce plant by transformation.
Triploid: A cell or organism having three sets of chromosomes.
H. Deposit Information
A deposit of lettuce line P2231-3013596, 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 Jun. 4, 2008. 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 lettuce line P2231-3013596 is ATCC Accession No. PTA-9244. 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:

    U.S. Pat. No. 5,349,124
    U.S. Pat. No. 5,378,619
    U.S. Pat. No. 5,463,175
    U.S. Pat. No. 5,500,365
    U.S. Pat. No. 5,563,055
    U.S. Pat. No. 5,633,435
    U.S. Pat. No. 5,689,052
    U.S. Pat. No. 5,880,275
    An et al., Plant Physiol., 88:547, 1988.
    Bird et al., Biotech. Gen. Engin. Rev., 9:207, 1991.
    Bustos et al., Plant Cell, 1:839, 1989.
    Callis et al., Plant Physiol., 88:965, 1988.
    Choi et al., Plant Cell Rep., 13: 344-348, 1994.
    Chupeau et al., Bio/Tech., 7:503-508, 1989.
    Dekeyser et al., Plant Cell, 2:591, 1990.
    Elliott et al., Plant Cell Rep., 18:707-714, 2004.
    Ellul et al., Theor. Appl. Genet., 107:462-469, 2003.
    EP 534 858
    Fraley et al., Bio/Technology, 3:629-635, 1985.
    Fromm et al., Nature, 312:791-793, 1986.
    Fromm et al., Plant Cell, 1:977, 1989.
    Gibson and Shillito, Mol. Biotech., 7:125, 1997
    Klee et al., Bio-Technology, 3(7):637-642, 1985.
    Kuhlemeier et al., Plant Cell, 1:471, 1989.
    Marcotte et al., Nature, 335:454, 1988.
    Marcotte et al., Plant Cell, 1:969, 1989.
    Molinier et al., Plant Cell Rep., 21:251-256, 2002.
    Odel et al., Nature, 313:810, 1985.
    Omirulleh et al., Plant Mol. Biol., 21(3):415-428, 1993.
    Pang et al., The Plant J., 9, 899-909, 1996.
    Potrykus et al., Mol. Gen. Genet., 199:183-188, 1985.
    Roshal et al., EMBO J., 6:1155, 1987.
    Ryder, In: Breeding Vegetable Crops, AVI Pub., Westport, Conn., 433-474, 1986.
    Schaffner and Sheen, Plant Cell, 3:997, 1991.
    Schernthaner et al., EMBO J., 7:1249, 1988.
    Siebertz et al., Plant Cell, 1:961, 1989.
    Simpson et al., EMBO J., 4:2723, 1985.
    Terada and Shimamoto, Mol. Gen. Genet., 220:389, 1990.
    Uchimiya et al., Mol. Gen. Genet., 204:204, 1986.
    Wang et al., Science, 280:1077-1082, 1998.
    Williams et al., Nucleic Acids Res., 1 8:6531-6535, 1990.
    WO 99/31248

## Claims

1. A seed of lettuce line P2231-3013596, a sample of seed of said line having been deposited under ATCC Accession Number PTA-9244.

2. A plant grown from the seed of claim 1.

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, pollen, an ovule and a cell.

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

6. A tissue culture of regenerable cells of lettuce line P2231-3013596, a sample of seed of said line having been deposited under ATCC Accession Number PTA-9244.

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 lettuce plant regenerated from the tissue culture of claim 6, wherein the regenerated plant expresses all of the physiological and morphological characteristics of lettuce line P2231-3013596, a sample of seed of said line having been deposited under ATCC Accession Number PTA-9244.

9. A method of producing lettuce seed, comprising crossing the plant of claim 2 with a second lettuce plant.

10. The method of claim 9, wherein the plant of lettuce line P2231-3013596 is the female parent.

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

12. An F1 hybrid plant produced by growing the seed of claim 11.

13. A method for producing a seed of a line P2231-3013596-derived lettuce plant comprising the steps of:
(a) crossing a lettuce plant of line P2231-3013596, a sample of seed of said line having been deposited under ATCC Accession Number PTA-9244, with a second lettuce plant; and
(b) allowing seed of a P2231-3013596-derived lettuce plant to form.

14. The method of claim 13, further comprising the steps of:
(c) crossing a plant grown from said P2231-3013596-derived lettuce seed with itself or a second lettuce plant to yield additional P2231-3013596-derived lettuce seed;
(d) growing said additional P2231-3013596-derived lettuce seed of step (c) to yield additional P2231-3013596-derived lettuce plants; and
(e) repeating the crossing and growing steps of (c) and (d) to generate further P2231-3013596-derived lettuce plants.

15. A method of vegetatively propagating a plant of lettuce line P2231-3013596 comprising the steps of:
(a) collecting tissue capable of being propagated from a plant of lettuce line P2231-3013596, a sample of seed of said line having been deposited under ATCC Accession Number PTA-9244;
(b) cultivating said tissue to obtain proliferated shoots; and
(c) rooting said proliferated shoots to obtain rooted plantlets.

16. The method of claim 15, further comprising growing plants from said rooted plantlets.

17. A method of introducing a desired trait into lettuce line P2231-3013596 comprising:
(a) crossing a plant of line P2231-3013596, a sample of seed of said line having been deposited under ATCC Accession Number PTA-9244, with a second lettuce 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 P2231-3013596, to produce backcross progeny;
(d) selecting backcross progeny comprising the desired trait and the physiological and morphological characteristic of lettuce line P2231-3013596; 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 lettuce line P2231-3013596 when grown in the same environmental conditions.

18. A lettuce plant produced by the method of claim 17.

19. A method of producing a plant of lettuce line P2231-3013596, a sample of seed of said line having been deposited under ATCC Accession Number PTA-9244, comprising an added desired trait, the method comprising introducing a transgene conferring the desired trait into a plant of lettuce line P2231-3013596.

20. A progeny plant of the plant of claim 1 that comprises all of the physiological and morphological characteristics of lettuce line P2231-3013596, a sample of seed of said line having been deposited under ATCC Accession Number PTA-9244.

21. A seed that produces the plant of claim 20.

22. A method of determining the genotype of the plant of claim 2 or a first generation progeny thereof, comprising obtaining a sample of nucleic acids from said plant and detecting in said nucleic acids a plurality of polymorphisms.

23. The method of claim 22, further comprising the step of storing the results of detecting the plurality of polymorphisms on a computer readable medium.

24. A method of producing lettuce comprising:
(a) obtaining the plant of claim 2, wherein the plant has been cultivated to maturity, and
(b) collecting lettuce from the plant.