# Patent

## Title
Lettuce cultivar green thunder

## Abstract

A novel romaine lettuce cultivar, designated Green Thunder, is disclosed. The invention relates to the seeds of lettuce cultivar Green Thunder, to the plants of lettuce cultivar Green Thunder and to methods for producing a lettuce plant by crossing the cultivar Green Thunder with itself or another lettuce cultivar. The invention further relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other lettuce cultivars derived from the cultivar Green Thunder.

## Background
BACKGROUND OF THE INVENTION
The present invention relates to a new and distinctive Romaine lettuce (Lactuca sativa) cultivar, designated Green Thunder. All publications cited in this application are herein incorporated by reference.
There are numerous steps in the development of any novel, desirable plant germplasm. Plant breeding begins with the analysis and definition of problems and weaknesses of the current germplasm, the establishment of program goals, and the definition of specific breeding objectives. The next step is selection of germplasm that possess the traits to meet the program goals. The goal is to combine in a single variety or hybrid an improved combination of desirable traits from the parental germplasm. These important traits may include increased head size and weight, higher seed yield, improved color, resistance to diseases and insects, tolerance to drought and heat, and better agronomic quality.
Practically speaking, all cultivated forms of lettuce belong to the highly polymorphic species Lactuca sativa that is grown for its edible head and leaves. As a crop, lettuces are grown commercially wherever environmental conditions permit the production of an economically viable yield. Lettuce is the world's most popular salad. In the United States, the principal growing regions are California and Arizona which produce approximately 287,000 acres out of a total annual acreage of more than 300,000 acres (USDA, 2001). Fresh lettuces are available in the United States year-round although the greatest supply is from May through October. For planting purposes, the lettuce season is typically divided into three categories, early, mid and late, with the coastal areas planting from January to August, and the desert regions from August to December. Fresh lettuces are consumed nearly exclusively as fresh, raw product, occasionally as a cooked vegetable.
Lactuca sativa is in the Cichoreae tribe of the Asteraceae (Compositae family). Lettuce is related to chicory, sunflower, aster, dandelion, artichoke and chrysanthemum. Sativa is one of about 300 species in the genus Lactuca. There are seven different morphological types of lettuces. The Crisphead group includes the iceberg and batavian types. Iceberg lettuce has a large, firm head with a crisp texture and a white or creamy yellow interior. Batavian lettuce predates the iceberg type and has a smaller and less firm head. The Butterhead group has a small, soft head with an almost oily texture. Romaine lettuce, also known as cos lettuce, has elongated upright leaves forming a loose, loaf shaped head. The outer leaves are usually dark green. The Leaf lettuces come in many varieties, none of which form a head. The next three types are seldom seen in the United States: Latin lettuce looks like a cross between romaine and butterhead; stem lettuce has long, narrow leaves and thick, edible stems, and Oilseed lettuce is a type grown for its large seeds that are pressed to obtain oil.
Lactuca sativa is a simple diploid species with nine pairs of chromosomes. Lettuce is an obligate self-pollinating species. This means that the pollen is shed before stigma emergence, assuring 100% self-fertilization. Since each lettuce flower is an aggregate of about 10-20 individual florets (typical of the Compositae family), manual removal of the anther tubes containing the pollen is tedious. As such, a modified method of misting to wash the pollen off prior to fertilization is needed to assure crossing or hybridization. About 60-90 min past sunrise, flowers to be used for crossings are selected. The basis for selection are open flowers, with the stigma emerged and the pollen visibly attached to the single stigma (about 10-20 stigma). Using 3-4 pumps of water from a regular spray bottle, the pollen grains are washed off with enough pressure to dislodge the pollen grains, but not enough to damage the style. Excess water is dried off with clean paper towels. About 30 min later the styles should spring back up and the two lobes of the stigma are visibly open in a “V” shape. Pollen from another variety or donor parent is then introduced by gently rubbing the stigma and style of the donor parent to the maternal parent. Tags with the pertinent information on date and pedigree are then secured to the flowers
Choice of breeding or selection methods depends on the mode of plant reproduction, the heritability of the trait(s) being improved, and the type of cultivar used commercially (e.g., F1 hybrid cultivar, pureline cultivar, etc.). For highly heritable traits, a choice of superior individual plants evaluated at a single location will be effective, whereas for traits with low heritability, selection should be based on mean values obtained from replicated evaluations of families of related plants. Popular selection methods commonly include pedigree selection, modified pedigree selection, mass selection, and recurrent selection.
The complexity of inheritance influences choice of breeding method. Backcross breeding is used to transfer one or a few favorable genes for a highly heritable trait into a desirable cultivar. This approach has been used extensively for breeding disease-resistant cultivars. Various recurrent selection techniques are used to improve quantitatively inherited traits controlled by numerous genes. The use of recurrent selection in self-pollinating crops depends on the ease of pollination, the frequency of successful hybrids from each pollination, and the number of hybrid offspring from each successful cross
Each breeding program should include a periodic, objective evaluation of the efficiency of the breeding procedure. Evaluation criteria vary depending on the goal and objectives, but should include gain from selection per year based on comparisons to an appropriate standard, overall value of the advanced breeding lines, and number of successful cultivars produced per unit of input (e.g., per year, per dollar expended, etc.). Promising advanced breeding lines are thoroughly tested and compared to appropriate standards in environments representative of the commercial target area(s) for three years at least. The best lines are candidates for new commercial cultivars; those still deficient in a few traits are used as parents to produce new populations for further selection. These processes, which lead to the final step of marketing and distribution, usually take from eight to 12 years from the time the first cross is made. Therefore, development of new cultivars is a time-consuming process that requires precise forward planning, efficient use of resources, and a minimum of changes in direction.
A most difficult task is the identification of individuals that are genetically superior, because for most traits the true genotypic value is masked by other confounding plant traits or environmental factors. One method of identifying a superior plant is to observe its performance relative to other experimental plants and to a widely grown standard cultivar. If a single observation is inconclusive, replicated observations provide a better estimate of its genetic worth.
The goal of lettuce plant breeding is to develop new, unique and superior lettuce cultivars. The breeder initially selects and crosses two or more parental cultivars, followed by repeated selfing and selection, producing many new genetic combinations. The breeder can theoretically generate billions of different genetic combinations via crossing, selfing and mutations. The breeder has no direct control at the cellular level. Therefore, two breeders will never develop the same cultivar, or even very similar cultivars, having the same lettuce traits.
Each year, the plant breeder selects the germplasm to advance to the next generation. This germplasm is grown under unique and different geographical, climatic and soil conditions and further selections are then made, during and at the end of the growing season. The cultivars that are developed are unpredictable because the breeder's selection occurs in unique environments, with no control at the DNA level (using conventional breeding procedures), and with millions of different possible genetic combinations being generated. A breeder of ordinary skill in the art cannot predict the final resulting cultivars he develops, except possibly in a very gross and general fashion. The same breeder cannot produce the same cultivar twice by using the exact same original parents and the same selection techniques. This unpredictability results in the expenditure of large research monies to develop superior lettuce cultivars.
The development of commercial lettuce cultivars requires the development of lettuce varieties, the crossing of these varieties, and the evaluation of the crosses. Pedigree breeding and recurrent selection breeding methods are used to develop cultivars from breeding populations. Breeding programs combine desirable traits from two or more varieties or various broad-based sources into breeding pools from which cultivars are developed by selfing and selection of desired phenotypes. The new cultivars are crossed with other varieties and the hybrids from these crosses are evaluated to determine which have commercial potential.
Pedigree breeding is used commonly for the improvement of self-pollinating crops or inbred cultivars of cross-pollinating crops. Two parents which possess favorable, complementary traits are crossed to produce an F1. An F2 population is produced by selfing one or several F1s or by intercrossing two F1s (sib mating). Selection of the best individuals is usually begun in the F2 population; then, beginning in the F3, the best individuals in the best families are selected. Replicated testing of families, or hybrid combinations involving individuals of these families, often follows in the F4 generation to improve the effectiveness of selection for traits with low heritability. At an advanced stage of inbreeding (i.e., F6 and F7), the best cultivars or mixtures of phenotypically similar cultivars are tested for potential release as new cultivars.
Mass and recurrent selections can be used to improve populations of either self- or cross-pollinating crops. A genetically variable population of heterozygous individuals is either identified or created by intercrossing several different parents. The best plants are selected based on individual superiority, outstanding progeny, or excellent combining ability. The selected plants are intercrossed to produce a new population in which further cycles of selection are continued.
Backcross breeding has been used to transfer genes for a simply inherited, highly heritable trait into a desirable homozygous cultivar or line that is the recurrent parent. The source of the trait to be transferred is called the donor parent. After the initial cross, individuals possessing the phenotype of the donor parent are selected and repeatedly crossed (backcrossed) to the recurrent parent. The resulting plant is expected to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait transferred from the donor parent.
The single-seed descent procedure in the strict sense refers to planting a segregating population, harvesting a sample of one seed per plant, and using the one-seed sample to plant the next generation. When the population has been advanced from the F2 to the desired level of inbreeding, the plants from which cultivars are derived will each trace to different F2 individuals. The number of plants in a population declines each generation due to failure of some seeds to germinate or some plants to produce at least one seed. As a result, not all of the F2 plants originally sampled in the population will be represented by a progeny when generation advance is completed.
Descriptions of other breeding methods that are commonly used for different traits and crops can be found in one of several reference books (e.g., “Principles of Plant Breeding” John Wiley and Son, pp. 115-161, 1960; Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr, 1987).
Proper testing should detect any major faults and establish the level of superiority or improvement over current cultivars. In addition to showing superior performance, there must be a demand for a new cultivar that is compatible with industry standards or which creates a new market. The introduction of a new cultivar will incur additional costs to the seed producer, the grower, processor and consumer for special advertising and marketing, altered seed and commercial production practices, and new product utilization. The testing preceding release of a new cultivar should take into consideration research and development costs as well as technical superiority of the final cultivar. For seed-propagated cultivars, it must be feasible to produce seed easily and economically.
Lettuce in general and romaine lettuce in particular is an important and valuable vegetable crop. Thus, a continuing goal of lettuce plant breeders is to develop stable, high yielding lettuce cultivars that are agronomically sound. To accomplish this goal, the lettuce breeder must select and develop lettuce plants with traits that result in superior cultivars.
The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification.
SUMMARY OF THE INVENTION
The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements.
According to the invention, there is provided a novel romaine lettuce cultivar designated Green Thunder. This invention thus relates to the seeds of lettuce cultivar Green Thunder, to the plants of lettuce cultivar Green Thunder and to methods for producing a lettuce plant produced by crossing the lettuce Green Thunder with itself or another lettuce cultivar, and to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic lettuce plants produced by that method. This invention also relates to methods for producing other lettuce cultivars derived from lettuce cultivar Green Thunder and to the lettuce cultivar derived by the use of those methods. This invention further relates to hybrid lettuce seeds and plants produced by crossing the cultivar Green Thunder with another lettuce cultivar.
In another aspect, the present invention provides regenerable cells for use in tissue culture of lettuce cultivar Green Thunder. The tissue culture will preferably be capable of regenerating plants having the physiological and morphological characteristics of the foregoing lettuce plant, and of regenerating plants having substantially the same genotype as the foregoing lettuce plant. Preferably, the regenerable cells in such tissue cultures will be embryos, protoplasts, seeds, callus, pollen, leaves, anthers, pistils, roots, root tips and meristematic cells. Still further, the present invention provides lettuce plants regenerated from the tissue cultures of the invention.
Another aspect of the invention is to provide methods for producing other lettuce plants derived from lettuce cultivar Green Thunder. Lettuce cultivars derived by the use of those methods are also part of the invention.
The invention also relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic lettuce plant produced by that method.
In another aspect, the present invention provides for single gene converted plants of Green Thunder. The single transferred gene may preferably be a dominant or recessive allele. Preferably, the single transferred gene will confer such traits as male sterility, herbicide resistance, insect resistance, resistance for bacterial, fungal, or viral disease, male fertility, enhanced nutritional quality and industrial usage. The single gene may be a naturally occurring lettuce gene or a transgene introduced through genetic engineering techniques.
The invention further provides methods for developing lettuce plants in a lettuce plant breeding program using plant breeding techniques including recurrent selection, backcrossing, pedigree breeding, restriction fragment length polymorphism enhanced selection, genetic marker enhanced selection and transformation. Seeds, lettuce plants, and parts thereof produced by such breeding methods are also part of the invention.
In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by study of the following descriptions.
DEFINITIONS
In the description and tables which follow, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided:
Allele. The allele is any of one or more alternative form of a gene, 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. Backcrossing is a process in which a breeder repeatedly crosses hybrid progeny back to one of the parents, for example, a first generation hybrid F1 with one of the parental genotype of the F1 hybrid.
Essentially all the physiological and morphological characteristics. A plant having essentially all the physiological and morphological characteristics means a plant having essentially all of the physiological and morphological characteristics of the recurrent parent, except for the characteristics derived from the converted gene.
Regeneration. Regeneration refers to the development of a plant from tissue culture.
Single gene converted. Single gene converted or conversion plant refers to plants which are developed by a plant breeding technique called backcrossing wherein essentially all of the desired morphological and physiological characteristics of a cultivar are recovered in addition to the single gene transferred into the cultivar via the backcrossing technique or via genetic engineering.
Maturity Date. Maturity refers to the stage when the plants are of full size or optimum weight, in marketable form or shape to be of commercial or economic value. In romaine types they range from 50-75 days from time of seeding, depending upon the season of the year.
RHS. RHS refers to the Royal Horticultural Society of England which publishes an official botanical color chart quantitatively identifying colors according to a defined numbering system, The chart may be purchased from Royal Horticulture Society Enterprise Ltd RHS Garden; Wisley, Woking; Surrey GU236QB, UK.
Yield (Tons/Acre). The yield in tons/acre is the actual yield of the lettuce at harvest.


DETAILED DESCRIPTION OF THE INVENTION
Lettuce cultivar Green Thunder has superior characteristics and was developed from the cross 00BS-0719, a proprietary romaine breeding line, and Green Forest, which was made in the summer of 2000 in the greenhouse at Synergene Seed in California. The F1 hybrids were grown in a greenhouse during the fall and winter of 2000 and 2001. F2 selection was made at Synergene Seed Spring Nursery in the spring of 2001. The F3 selections were made in the spring of 2002 at Synergene Seed Spring Nursery. F4 plants were selected in 2003 in the Synergene Seed Spring Nursery. F4 plants were selected and bulked in field plots in San Joaquin Valley, Calif. during the summers of 2003 and 2004.
Green Thunder is a romaine lettuce with a very dark green leaf color, thick and slightly blistered leaf texture, highly dense and V-shaped head; it has a very vigorous growth habit and is widely adoptable in a variety of environments. Green Thunder is resistant to tipburn and Sclerotinia. It is also highly tolerant to twisting and mid-rib deformity. Green Thunder has shown a very good adaptability in the coastal California and desert Arizona regions of the United States.
Some of the criteria used for selection in various generations include: color, disease resistance, head weight, number of leaves, appearance and length, yield, emergence, maturity, plant architecture, seed yield and quality.
The cultivar has shown uniformity and stability for the traits, within the limits of environmental influence for the traits. It has been self-pollinated a sufficient number of generations with careful attention to uniformity of plant type. The cultivar has been increased with continued observation for uniformity. No variant traits have been observed or are expected in Green Thunder.
Lettuce cultivar Green Thunder has the following morphologic and other characteristics (based primarily on data collected at Salinas, Calif.).







TABLE 1





VARIETY DESCRIPTION INFORMATION

















Plant Type



Romaine



Seed



Color: Black



Light dormancy: Light not required



Heat dormancy: Susceptible



Cotyledon to Fourth Leaf Stage



Shape of cotyledons: Very Broad



Undulation: Flat



Anthocyanin distribution: Absent



Rolling: Absent



Cupping: Uncupped



Reflexing: None



Mature Leaves



Margin - Incision depth: Absent/Shallow



Margin - Indentation: Entire



Margin - Undulation of the apical margin: Absent/Slightly



Green color: Very dark green



Anthocyanin - Distribution: Absent



Size: Large



Glossiness: Glossy



Blistering: Moderate



Trichomes: Absent



Leaf thickness: Thick



Plant at Market Stage



Head shape: Non-heading, V-shaped



Head size class: Large



Head weight: 940 g



Head firmness: Loose



Core



Diameter at base of head: 4.3 cm



Core height from base of head to apex: 6.7 cm



Maturity



Summer: 52 days



Winter: 92 days



Adaptation



Primary Regions of Adaptation (tested and proven adapted)



Southwest (California, Arizona desert): Adapted



West Coast: Adapted



Southeast: N/A



Northeast: Adapted



Spring area: San Joaquin, Imperial, CA; Yuma, AZ



Summer area: Salinas, Santa Maria, San Juan Bautista, CA



Fall area: Salinas, Santa Maria, Oxnard, CA



Winter area: Yuma, AZ; Imperial, Coachella, CA



Greenhouse: N/A



Soil Type: Both Mineral and Organic



Disease and Stress Reactions



Virus



Big Vein: Intermediate



Lettuce Mosaic: Susceptible



Cucumber Mosaic: Not tested



Broad Bean Wilt: Not tested



Turnip Mosaic: Not tested



Best Western Yellows: Not tested



Lettuce Infectious Yellows: Not tested



Fungal/Bacterial



Corky Root Rot (Pythium Root Rot): Intermediate



Downy Mildew: Susceptible



Powdery Mildew: Not tested



Sclerotinia Rot: Highly resistant



Bacterial Soft Rot (Pseudomonas sp. & others): Not tested



Botrytis (Gray Mold): Susceptible



Insects



Cabbage Loopers: Susceptible



Root Aphids: Susceptible



Green Peach Aphid: Susceptible



Physiological/Stress



Tipbum: Highly resistant



Heat: Intermediate



Drought: Not tested



Cold: Resistant



Salt: Not tested



Brown Rib: Resistant



Post Harvest



Pink Rib: Resistant



Russet Spotting: Not tested



Rusty Brown Discoloration: Not tested



Internal Rib Necrosis (Blackheart, Gray Rib, Gray Streak): Resistant



Brown Stain: Not tested










FURTHER EMBODIMENTS OF THE INVENTION
This invention also is directed to methods for producing a lettuce cultivar plant by crossing a first parent lettuce plant with a second parent lettuce plant wherein either the first or second parent lettuce plant is a lettuce plant of the cultivar Green Thunder. Further, both first and second parent lettuce plants can come from the cultivar Green Thunder. Still further, this invention also is directed to methods for producing a cultivar Green Thunder-derived lettuce plant by crossing cultivar Green Thunder with a second lettuce plant and growing the progeny seed, and repeating the crossing and growing steps with the cultivar Green Thunder-derived plant from 0 to 7 times. Thus, any such methods using the cultivar Green Thunder are part of this invention: selfing, backcrosses, hybrid production, crosses to populations, and the like. All plants produced using cultivar Green Thunder as a parent are within the scope of this invention, including plants derived from cultivar Green Thunder. Advantageously, the cultivar is used in crosses with other, different, cultivars to produce first generation (F1) lettuce seeds and plants with superior characteristics.
As used herein, the term plant includes plant cells, plant protoplasts, plant cell tissue cultures from which lettuce plants can be regenerated, plant calli, plant clumps and plant cells that are intact in plants or parts of plants, such as embryos, pollen, ovules, flowers, seeds, roots, anthers, pistils and the like.
As is well known in the art, tissue culture of lettuce can be used for the in vitro regeneration of a lettuce plant. Tissue culture of various tissues of lettuces and regeneration of plants therefrom is well known and widely published. For example, reference may be had to Teng et al., HortScience. 1992, 27: 9, 1030-1032, Teng et al., HortScience. 1993, 28: 6, 669-1671, Zhang et al., Journal of Genetics and Breeding. 1992, 46: 3, 287-290, Webb et al., Plant Cell Tissue and Organ Culture. 1994, 38: 1, 77-79, Curtis et al., Journal of Experimental Botany. 1994, 45: 279, 1441-1449, Nagata et al., Journal for the American Society for Horticultural Science. 2000, 125: 6, 669-672. It is clear from the literature that the state of the art is such that these methods of obtaining plants are “conventional” in the sense that they are routinely used and have a very high rate of success. Thus, another aspect of this invention is to provide cells which upon growth and differentiation produce lettuce plants having the physiological and morphological characteristics of variety Green Thunder.
With the advent of molecular biological techniques that have allowed the isolation and characterization of genes that encode specific protein products, scientists in the field of plant biology developed a strong interest in engineering the genomes of plants to contain and express foreign genes, or additional, or modified versions of native, or endogenous, genes (perhaps driven by different promoters) in order to alter the traits of a plant in a specific manner. Such foreign additional and/or modified genes are referred to herein collectively as “transgenes”. Over the last fifteen to twenty years several methods for producing transgenic plants have been developed, and the present invention in particular embodiments also relates to transformed versions of the claimed cultivar.
Plant transformation involves the construction of an expression vector that will function in plant cells. Such a vector comprises DNA comprising a gene under control of, or operatively linked to, a regulatory element (for example, a promoter). The expression vector may contain one or more such operably linked gene/regulatory element combinations. The vector(s) may be in the form of a plasmid, and can be used alone or in combination with other plasmids, to provide transformed lettuce plants, using transformation methods as described below to incorporate transgenes into the genetic material of the lettuce plant(s).
Expression Vectors for Lettuce Transformation-Markers
Expression vectors include at least one genetic marker, operably linked to a regulatory element (a promoter, for example) that allows transformed cells containing the marker to be either recovered by negative selection, i.e., inhibiting growth of cells that do not contain the selectable marker gene, or by positive selection, i.e., screening for the product encoded by the genetic marker. Many commonly used selectable marker genes for plant transformation are well known in the transformation arts, and include, for example, genes that code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or an herbicide, or genes that encode an altered target which is insensitive to the inhibitor. A few positive selection methods are also known in the art.
One commonly used selectable marker gene for plant transformation is the neomycin phosphotransferase II (nptII) gene, isolated from transposon Tn5, which when placed under the control of plant regulatory signals confers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci. U.S.A., 80:4803 (1983). Another commonly used selectable marker gene is the hygromycin phosphotransferase gene which confers resistance to the antibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299 (1985).
Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamycin acetyl transferase, streptomycin phosphotransferase and aminoglycoside-3′-adenyl transferase, the bleomycin resistance determinant. Hayford et al., Plant Physiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab et al., Plant Mol. Biol. 14:197 (1990) Hille et al., Plant Mol. Biol. 7:171 (1986). Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate or broxynil. Comai et al., Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) and Stalker et al., Science 242:419-423 (1988).
Selectable marker genes for plant transformation that are not of bacterial origin include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shah et al., Science 233:478 (1986), Charest et al., Plant Cell Rep. 8:643 (1990).
Another class of marker genes for plant transformation requires screening of presumptively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic. These genes are particularly useful to quantify or visualize the spatial pattern of expression of a gene in specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression. Commonly used genes for screening presumptively transformed cells include β-glucuronidase (GUS), α-galactosidase, luciferase and chloramphenicol acetyltransferase. Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131 (1987), DeBlock et al., EMBO J. 3:1681 (1984).
In vivo methods for visualizing GUS activity that do not require destruction of plant tissue are available. Molecular Probes publication 2908, Imagene Green, p. 1-4 (1993) and Naleway et al., J. Cell Biol. 115:151a (1991). However, these in vivo methods for visualizing GUS activity have not proven useful for recovery of transformed cells because of low sensitivity, high fluorescent backgrounds and limitations associated with the use of luciferase genes as selectable markers.
More recently, a gene encoding Green Fluorescent Protein (GFP) has been utilized as a marker for gene expression in prokaryotic and eukaryotic cells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFP may be used as screenable markers.
Expression Vectors for Lettuce Transformation-Promoters
Genes included in expression vectors must be driven by a nucleotide sequence comprising a regulatory element, for example, a promoter. Several types of promoters are well known in the transformation arts, as are other regulatory elements that can be used alone or in combination with promoters.
As used herein, “promoter” includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as “tissue-preferred”. Promoters which initiate transcription only in certain tissue are referred to as “tissue-specific”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue-specific, tissue-preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active under most environmental conditions.
A. Inducible Promoters
An inducible promoter is operably linked to a gene for expression in lettuce. Optionally, the inducible promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in lettuce. With an inducible promoter the rate of transcription increases in response to an inducing agent.
Any inducible promoter can be used in the instant invention. See Ward et al., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promoters include, but are not limited to, that from the ACEI system which responds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2 gene from maize which responds to benzenesulfonamide herbicide safeners (Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al., Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz et al., Mol. Gen. Genetics 227:229-237 (1991)). A particularly preferred inducible promoter is a promoter that responds to an inducing agent to which plants do not normally respond. An exemplary inducible promoter is the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone. Schena et al., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).
B. Constitutive Promoters
A constitutive promoter is operably linked to a gene for expression in lettuce or the constitutive promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in lettuce.
Many different constitutive promoters can be utilized in the instant invention. Exemplary constitutive promoters include, but are not limited to, the promoters from plant viruses such as the 35S promoter from CaMV (Odell et al., Nature 313:810-812 (1985) and the promoters from such genes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last et al., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen. Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3): 291-300 (1992)).
The ALS promoter, Xba1/NcoI fragment 5′ to the Brassica napus ALS3 structural gene (or a nucleotide sequence similarity to said Xba1/NcoI fragment), represents a particularly useful constitutive promoter. See PCT application WO96/30530.
C. Tissue-Specific or Tissue-Preferred Promoters
A tissue-specific promoter is operably linked to a gene for expression in lettuce. Optionally, the tissue-specific promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in lettuce. Plants transformed with a gene of interest operably linked to a tissue-specific promoter produce the protein product of the transgene exclusively, or preferentially, in a specific tissue.
Any tissue-specific or tissue-preferred promoter can be utilized in the instant invention. Exemplary tissue-specific or tissue-preferred promoters include, but are not limited to, a root-preferred promoter, such as that from the phaseolin gene (Murai et al., Science 23:476-482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A. 82:3320-3324 (1985)); a leaf-specific and light-induced promoter such as that from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985) and Timko et al., Nature 318:579-582 (1985)); an anther-specific promoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics 217:240-245 (1989)); a pollen-specific promoter such as that from Zm13 (Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or a microspore-preferred promoter such as that from apg (Twell et al., Sex. Plant Reprod. 6:217-224 (1993).
D. Signal Sequences for Targeting Proteins to Subcellular Compartments
Transport of protein produced by transgenes to a subcellular compartment such as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall or mitochondrion or for secretion into the apoplast, is accomplished by means of operably linking the nucleotide sequence encoding a signal sequence to the 5′ and/or 3′ region of a gene encoding the protein of interest. Targeting sequences at the 5′ and/or 3′ end of the structural gene may determine, during protein synthesis and processing, where the encoded protein is ultimately compartmentalized.
The presence of a signal sequence directs a polypeptide to either an intracellular organelle or subcellular compartment or for secretion to the apoplast. Many signal sequences are known in the art. See, for example Becker et al., Plant Mol. Biol. 20:49 (1992), Knox, C., et al., Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol. 91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuoka et al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell. Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon, et al., Cell 39:499-509 (1984), and Steifel, et al., Plant Cell 2:785-793 (1990).
E. Foreign Protein Genes and Agronomic Genes
With transgenic plants according to the present invention, a foreign protein can be produced in commercial quantities. Thus, techniques for the selection and propagation of transformed plants, which are well understood in the art, yield a plurality of transgenic plants which are harvested in a conventional manner, and a foreign protein then can be extracted from a tissue of interest or from total biomass. Protein extraction from plant biomass can be accomplished by known methods which are discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6 (1981).
According to a preferred embodiment, the transgenic plant provided for commercial production of foreign protein is lettuce. In another preferred embodiment, the biomass of interest is seed. For the relatively small number of transgenic plants that show higher levels of expression, a genetic map can be generated, primarily via conventional RFLP, PCR and SSR analysis, which identifies the approximate chromosomal location of the integrated DNA molecule. For exemplary methodologies in this regard, see Glick and Thompson, Methods in Plant Molecular Biology and Biotechnology CRC Press, Boca Raton 269:284 (1993). Map information concerning chromosomal location is useful for proprietary protection of a subject transgenic plant. If unauthorized propagation is undertaken and crosses made with other germplasm, the map of the integration region can be compared to similar maps for suspect plants, to determine if the latter have a common parentage with the subject plant. Map comparisons would involve hybridizations, RFLP, PCR, SSR and sequencing, all of which are conventional techniques.
Likewise, by means of the present invention, agronomic genes can be expressed in transformed plants. More particularly, plants can be genetically engineered to express various phenotypes of agronomic interest. Exemplary genes implicated in this regard include, but are not limited to, those categorized below:
1. Genes that Confer Resistance to Pests or Disease and that Encode
A. Plant disease resistance genes. Plant defenses are often activated by specific interaction between the product of a disease resistance gene (R) in the plant and the product of a corresponding avirulence (Avr) gene in the pathogen. A plant cultivar can be transformed with a cloned resistance gene(s) to engineer plants that are resistant to specific pathogen strains. See, for example Jones et al., Science 266:789 (1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum); Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistance to Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance to Pseudomonas syringae).
B. A Bacillus thuringiensis protein, a derivative thereof or a synthetic polypeptide modeled thereon. See, for example, Geiser et al., Gene 48:109 (1986), who disclose the cloning and nucleotide sequence of a Bt α-endotoxin gene. Moreover, DNA molecules encoding α-endotoxin genes can be purchased from American Type Culture Collection, Manassas, Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.
C. A lectin. See, for example, the disclosure by Van Damme et al., Plant Molec. Biol. 24:25 (1994), who disclose the nucleotide sequences of several Clivia miniata mannose-binding lectin genes.
D. A vitamin-binding protein such as avidin. See PCT application US93/06487. The application teaches the use of avidin and avidin homologues as larvicides against insect pests.
E. An enzyme inhibitor, for example, a protease or proteinase inhibitor or an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem. 262:16793 (1987) (nucleotide sequence of rice cysteine proteinase inhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotide sequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani et al., Biosci. Biotech. Biochem. 57: 1243 (1993) (nucleotide sequence of Streptomyces nitrosporeus α-amylase inhibitor).
F. An insect-specific hormone or pheromone such as an ecdysteroid or juvenile hormone, a variant thereof, a mimetic based thereon, or an antagonist or agonist thereof. See, for example, the disclosure by Hammock et al., Nature 344:458 (1990), of baculovirus expression of cloned juvenile hormone esterase, an inactivator of juvenile hormone.
G. An insect-specific peptide or neuropeptide which, upon expression, disrupts the physiology of the affected pest. For example, see the disclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloning yields DNA coding for insect diuretic hormone receptor), and Pratt et al., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin is identified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 to Tomalski et al., who disclose genes encoding insect-specific, paralytic neurotoxins.
H. An insect-specific venom produced in nature by a snake, a wasp, etc. For example, see Pang et al., Gene 116:165 (1992), for disclosure of heterologous expression in plants of a gene coding for a scorpion insectotoxic peptide.
I. An enzyme responsible for a hyper-accumulation of a monoterpene, a sesquiterpene, a steroid, a hydroxamic acid, a phenylpropanoid derivative or another non-protein molecule with insecticidal activity.
J. An enzyme involved in the modification, including the post-translational modification, of a biologically active molecule; for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a glucanase, whether natural or synthetic. See PCT application WO 93/02197 in the name of Scott et al., which discloses the nucleotide sequence of a callase gene. DNA molecules which contain chitinase-encoding sequences can be obtained, for example, from the ATCC under Accession Nos. 39637 and 67152. See also Kramer et al., Insect Biochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequence of a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al., Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence of the parsley ubi4-2 polyubiquitin gene.
K. A molecule that stimulates signal transduction. For example, see the disclosure by Botella et al., Plant Molec. Biol 24:757 (1994), of nucleotide sequences for mung lettuce calmodulin cDNA clones, and Griess et al., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequence of a maize calmodulin cDNA clone.
L. A hydrophobic moment peptide. See PCT application WO 95/16776 (disclosure of peptide derivatives of Tachyplesin which inhibit fungal plant pathogens) and PCT application WO 95/18855 (teaches synthetic antimicrobial peptides that confer disease resistance).
M. A membrane permease, a channel former or a channel blocker. For example, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), of heterologous expression of a cecropin-α, lytic peptide analog to render transgenic tobacco plants resistant to Pseudomonas solanacearum. 
N. A viral-invasive protein or a complex toxin derived therefrom. For example, the accumulation of viral coat proteins in transformed plant cells imparts resistance to viral infection and/or disease development effected by the virus from which the coat protein gene is derived, as well as by related viruses. See Beachy et al., Ann. Rev. Phytopathol. 28: 451 (1990). Coat protein-mediated resistance has been conferred upon transformed plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.
O. An insect-specific antibody or an immunotoxin derived therefrom. Thus, an antibody targeted to a critical metabolic function in the insect gut would inactivate an affected enzyme, killing the insect. Cf. Taylor et al., Abstract #497, Seventh Int'l Symposium on Molecular Plant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymatic inactivation in transgenic tobacco via production of single-chain antibody fragments).
P. A virus-specific antibody. See, for example, Tavladoraki et al., Nature 366:469 (1993), who show that transgenic plants expressing recombinant antibody genes are protected from virus attack.
Q. A developmental-arrestive protein produced in nature by a pathogen or a parasite. Thus, fungal endo α-1,4-D-polygalacturonases facilitate fungal colonization and plant nutrient release by solubilizing plant cell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology 10:1436 (1992). The cloning and characterization of a gene which encodes a lettuce endopolygalacturonase-inhibiting protein is described by Toubart et al., Plant J. 2:367 (1992).
R. A developmental-arrestive protein produced in nature by a plant. For example, Logemann et al., Bio/Technology 10:305 (1992), have shown that transgenic plants expressing the barley ribosome-inactivating gene have an increased resistance to fungal disease.
S. A lettuce mosaic potyvirus (LMV) coat protein gene introduced into Lactuca sativa in order to increase its resistance to LMV infection. See Dinant et al., Molecular Breeding. 1997, 3:175-86.
2. Genes that Confer Resistance to an Herbicide, for Example:
A. An herbicide that inhibits the growing point or meristem, such as an imidazalinone or a sulfonylurea. Exemplary genes in this category code for mutant ALS and AHAS enzymes as described, for example, by Lee et al., EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449 (1990), respectively.
B. Glyphosate (resistance impaired by mutant 5-enolpyruvl-3-phosphoshikimate synthase (EPSP) and aroA genes, respectively) and other phosphono compounds such as glufosinate (phosphinothricin acetyl transferase (PAT) and Streptomyces hygroscopicus PAT, bar, genes), and pyridinoxy or phenoxy propionic acids and cyclohexones (ACCase inhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., which discloses the nucleotide sequence of a form of EPSP which can confer glyphosate resistance. A DNA molecule encoding a mutant aroA gene can be obtained under ATCC accession number 39256, and the nucleotide sequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. See also Umaballava-Mobapathie in Transgenic Research. 1999, 8:1, 33-44, that discloses Lactuca sativa resistant to glufosinate. European patent application No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374 to Goodman et al., disclose nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L-phosphinothricin. The nucleotide sequence of a phosphinothricin-acetyl-transferase gene is provided in European application No. 0 242 246 to Leemans et al.; DeGreef et al., Bio/Technology 7:61 (1989), describe the production of transgenic plants that express chimeric bar genes coding for phosphinothricin acetyl transferase activity. Examples of genes conferring resistance to phenoxy propionic acids and cyclohexones, such as sethoxydim and haloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes are described by Marshall et al., Theor. Appl. Genet. 83:435 (1992).
C. An herbicide that inhibits photosynthesis, such as a triazine (psbA and gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al., Plant Cell 3:169 (1991), describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, and DNA molecules containing these genes are available under ATCC Accession Nos. 53435, 67441, and 67442. Cloning and expression of DNA coding for a glutathione S-transferase is described by Hayes et al., Biochem. J. 285:173 (1992).
3. Genes that Confer or Contribute to a Value-Added Trait, Such as:
A. Increased iron content of the lettuce, for example by transforming a plant with a soybean ferritin gene as described in Goto et al., Acta Horticulturae. 2000, 521, 101-109. Parallel to the improved iron content, enhanced growth of transgenic lettuces was also observed in early development stages.
B. Decreased nitrate content of leaves, for example by transforming a lettuce with a gene coding for a nitrate reductase. See for example Curtis et al., Plant Cell Report. 1999, 18: 11, 889-896.
C. Increased sweetness of the lettuce by transferring a gene coding for monellin that elicits a flavor 100,000 times sweeter than sugar on a molar basis. See Penarrubia et al., Biotechnology. 1992, 10: 5, 561-564.
Numerous methods for plant transformation have been developed, including biological and physical plant transformation protocols. See, for example, Miki et al., “Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick B. R. and Thompson, J. E. Eds., CRC Press, Inc., Boca Raton, (1993) pages 67-88. In addition, expression vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are available. See, for example, Gruber et al., “Vectors for Plant Transformation” in Methods in Plant Molecular Biology and Biotechnology, Glick B. R. and Thompson, J. E. Eds., CRC Press, Inc., Boca Raton 1993 pages 89-119.
A. Agrobacterium-Mediated Transformation
One method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. See, for example, Horsch et al., Science 227:1229 (1985). Curtis et al., Journal of Experimental Botany. 1994, 45: 279, 1441-1449, Torres et al., Plant cell Tissue and Organic Culture. 1993, 34: 3, 279-285, Dinant et al., Molecular Breeding. 1997, 3: 1, 75-86. A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided by Gruber et al., supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.
B. Direct Gene Transfer
Despite the fact the host range for Agrobacterium-mediated transformation is broad, some major cereal or vegetable crop species and gymnosperms have generally been recalcitrant to this mode of gene transfer, even though some success has recently been achieved in rice and corn. Hiei et al., The Plant Journal 6:271-282 (1994) and U.S. Pat. No. 5,591,616 issued Jan. 7, 1997. Several methods of plant transformation, collectively referred to as direct gene transfer, have been developed as an alternative to Agrobacterium-mediated transformation.
A generally applicable method of plant transformation is microprojectile-mediated transformation wherein DNA is carried on the surface of microprojectiles measuring 1 to 4 μm. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate plant cell walls and membranes. Russell, D. R., et al. Pl. Cell. Rep. 12 (3 January), 165-169 (1993); Aragao, F. J. L., et al. Plant Mol. Biol. 20 (2 October); 357-359 (1992); Aragao, F. J. L., et al. Pl. Cell. Rep. 12 (9 July) 483-490 (1993); Aragao Theor. Appl. Genet. 93:142-150 (1996); Kim, J.; Minamikawa, T. Plant Science 117: 131-138 (1996); Sanford et al., Part. Sci. Technol. 5:27 (1987); Sanford, J. C., Trends Biotech. 6:299 (1988); Klein et al., Bio/Technology 6:559-563 (1988); Sanford, J. C., Physiol Plant 7:206 (1990); Klein et al., Biotechnology 10:268 (1992).
Another method for physical delivery of DNA to plants is sonication of target cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively, liposome and spheroplast fusion have been used to introduce expression vectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christou et al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake of DNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol or poly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet. 199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982). Electroporation of protoplasts and whole cells and tissues has also been described. Saker, M.; Kuhne, T. Biologia Plantarum 40(4): 507-514 (1997/98), Donn et al., In Abstracts of VIIth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al., Plant Mol. Biol. 24:51-61 (1994). See also Chupean et al., Biotechnology. 1989, 7: 5, 503-508.
Following transformation of lettuce target tissues, expression of the above-described selectable marker genes allows for preferential selection of transformed cells, tissues and/or plants, using regeneration and selection methods now well known in the art.
The foregoing methods for transformation would typically be used for producing a transgenic cultivar. The transgenic cultivar could then be crossed with another (non-transformed or transformed) cultivar in order to produce a new transgenic lettuce cultivar. Alternatively, a genetic trait which has been engineered into a particular lettuce cultivar using the foregoing transformation techniques could be moved into another cultivar using traditional backcrossing techniques that are well known in the plant breeding arts. For example, a backcrossing approach could be used to move an engineered trait from a public, non-elite inbred cultivar into an elite inbred cultivar, or from an inbred cultivar containing a foreign gene in its genome into an inbred cultivar or cultivars which do not contain that gene. As used herein, “crossing” can refer to a simple X by Y cross, or the process of backcrossing, depending on the context.
When the terms lettuce plant, cultivar or lettuce cultivar are used in the context of the present invention, this also includes any single gene conversions of that culitvar. The term single gene 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 cultivar are recovered in addition to the single gene transferred into the cultivar via the backcrossing technique. Backcrossing methods can be used with the present invention to improve or introduce a characteristic into the cultivar. The term backcrossing as used herein refers to the repeated crossing of a hybrid progeny back to one of the parental lettuce plants for that cultivar. The parental lettuce plant which contributes the gene 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 gene or genes from the nonrecurrent parent are transferred is known as the recurrent parent as it is used for several rounds in the backcrossing protocol (Poehlman & Sleper, 1994; Fehr, 1987). In a typical backcross protocol, the original cultivar of interest (recurrent parent) is crossed to a second cultivar (nonrecurrent parent) that carries the single gene 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 gene 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 cultivar. To accomplish this, a single gene of the recurrent cultivar is modified or substituted with the desired gene 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 cultivar. 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 and/or agronomically important 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.
Many single gene traits have been identified that are not regularly selected for in the development of a new cultivar but that can be improved by backcrossing techniques. Single gene traits may or may not be transgenic, examples of these traits include but are not limited to, herbicide resistance, resistance for bacterial, fungal, or viral disease, insect resistance, enhanced nutritional quality, industrial usage, yield stability and yield enhancement. These genes are generally inherited through the nucleus. Several of these single gene traits are described in U.S. Pat. Nos. 5,777,196, 5,948,957 and 5,969,212, the disclosures of which are specifically hereby incorporated by reference.
TABLES
In the tables that follow, the traits and characteristics of lettuce cultivar Green Thunder are given compared to two commercial romaine lettuce cultivars, Green Forest and PIC Cos.
Table 2 below shows the mature seed stalk height for Green Thunder as compared to the seed stalk height for Green Forest and PIC Cos. Seed stalk height is measured in centimeters. An analysis of variance was performed on the data and is shown below the data. As can be seen from the data in Table 2, Green Thunder has a significantly taller mature seed stalk height than either Green Forest or PIC Cos.







TABLE 2







Seed Stalk Height (cm)









Green Thunder
Green Forest
PIC Cos





105
101
100


107
105
102


109
102
100


108
100
 95


100
 98
 96


102
105
105


103
103
 97


111
 99
 98


110
 96
 99


101
 97
 98


102
100
 99


100
106
100


110
 95
 96










Anova: Single Factor


SUMMARY













Groups
Count
Sum
Average
Variance







Green Thunder
13
1368
105.2307692
16.85897



Green Forest
13
1307
100.5384615
12.60256



PIC Cos
13
1285
98.84615385
7.307692











ANOVA













Source of Variation
SS
df
MS
F
P-value
F crit





Between Groups
284.4615385
2
142.2307692
11.6046
0.000129
3.259444


Within Groups
441.2307692
36
12.25641026


Total
725.6923077
38









Table 3 below shows the seed stalk spread for Green Thunder as compared to the seed stalk spread for Green Forest and PIC Cos. Seed stalk spread is measured in centimeters. An analysis of variance was performed on the data and is shown below the data. As can be seen from the data in Table 3, Green Thunder has a significantly wider mature seed stalk spread than Green Forest but a narrower mature seed stalk spread than PIC Cos.







TABLE 3







Seed Stalk Spread (cm)









Green Thunder
Green Forest
PIC Cos





37
44
52


50
42
45


44
42
47


45
40
41


45
40
48


46
45
41


38
44
42


42
42
47


43
35
43


46
44
48


40
36
44


45
44
45


44
40
40










Anova: Single Factor


SUMMARY













Groups
Count
Sum
Average
Variance







Green Thunder
13
565
43.46153846
12.4359



Green Forest
13
538
41.38461538
9.75641



PIC Cos
13
583
44.84615385
12.14103











ANOVA













Source of Variation
SS
df
MS
F
P-value
F crit





Between Groups
78.92307692
2
39.46153846
3.448096
0.042649
3.259444


Within Groups
412
36
11.44444444


Total
490.9230769
38









Tables 4 through 8 show data collected in eight different locations. Location 1 was near Bard, Calif., location 2 was near San Juan Bautista, Calif., locations 3, 5, 6, 7, and 8 were near Salinas, Calif. and location 4 was near Yuma, Ariz.
Table 4 below shows the plant weight in grams at harvest maturity of Green Thunder as compared to Green Forest and PIC Cos. An analysis of variance was performed on the data and is shown below the data. As can be seen from the data in Table 4, Green Thunder is significantly heavier in plant weight at harvest maturity than either Green Forest or PIC Cos.








TABLE 4







Trial
Plant Weight (g) at Harvest Maturity
















Location
Loc1
Loc2
Loc3
Loc4
Loc5
Loc6
Loc7
Loc8






Green
1020
908
1021
1201
1021
1201
540
805


Thunder
1160
1160
984
1136
984
1136
790
780



936
1160
908
1165
908
1165
810
810



908
1020
1025
1146
1025
1146
790
650



908
681
984
990
984
990
810
820



846
1076
1026
1025
1026
1025
570
870



846
790
988
914
988
914
85
650



1020
965
956
986
956
986
860
750



1244
681
1024
1034
1024
1034
820
750



1104
795
1136
1130
1136
1130
790
650


Green
820
622
756
759
756
759
670
680


Forest
795
1076
687
774
687
774
710
690



1076
681
909
804
909
804
610
720



874
795
675
907
675
907
580
820



908
795
756
682
756
682
690
750



820
1076
764
673
764
673
620
800



908
622
904
698
904
698
710
720



1244
795
689
768
6889
768
550
780



681
681
677
759
677
759
690
820



846
846
757
683
757
683
710
810


PIC Cos
846
760
688
766
688
766
610
650



965
820
901
771
901
771
710
720



875
795
679
699
679
699
540
590



705
622
766
706
766
706
600
680



846
740
752
755
752
755
580
710



965
622
742
899
742
899
570
800



875
908
911
878
911
878
810
800



622
908
903
756
903
756
580
720



622
681
699
766
699
766
720
740



705
622
698
751
698
751
710
580










Anova: Two-Factor With Replication
















SUM-











MARY
Loc1
Loc2
Loc3
Loc4
Loc5
Loc6
Loc7
Loc8
Total





Green


Thunder


Count
10
10
10
10
10
10
10
10
80


Sum
9992
9236
10052
10727
10052
10727
6865
7535
75186


Average
999.2
923.6
1005.2
1072.7
1005.2
1072.7
686.5
753.5
939.825


Variance
18355.73
33149.16
3511.067
8995.344444
3511.067
8995.344
56378.06
6300.278
34406.96


Green


Forest


Count
10
10
10
10
10
10
10
10
80


Sum
8972
7989
7574
7507
13774
7507
6540
7590
67453


Average
897.2
798.9
757.4
750.7
1377.4
750.7
654
759
843.1625


Variance
24971.07
27357.88
7490.044
5152.011111
3757250
5152.011
3515.556
2921.111
482030


PIC Cos


Count
10
10
10
10
10
10
10
10
80


Sum
8026
7478
7739
7747
7739
7747
6430
6990
59896


Average
802.6
747.8
773.9
774.7
773.9
774.7
643
699
748.7


Variance
16809.16
12324.18
8992.544
4226.233333
8992.544
4226.233
7734.444
5765.556
10295.5


Total


Count
30
30
30
30
30
30
30
30


Sum
26990
24703
25365
25981
31565
25981
19835
22115


Average
899.6667
823.4333
845.5
866.0333333
1052.167
866.0333
661.1667
737.1667


Variance
25330.09
28242.67
19443.64
27893.41264
1233860
27893.41
21340.83
5409.799










ANOVA















Source of Variation
SS
df
MS
F
P-value
F crit







Sample
1461215
2
730607.6
4.338013203
0.014219
3.03767



Columns
2780088
7
397155.5
2.358127411
0.024316
2.052154



Interaction
2453089
14
175220.6
1.040379891
0.414431
1.737799



Within
36378690
216
168419.9



Total
43073082
239










Table 5 below shows the leaf width of Green Thunder at harvest maturity as compared to Green Forest and PIC Cos. Leaf width is measured in centimeters. An analysis of variance was performed on the data and is shown below the data. As can be seen from the data in Table 5, Green Thunder is significantly wider in leaf width at harvest maturity than Green Forest but not significantly wider than PIC Cos.









TABLE 5









Leaf Width (cm) at Harvest Maturity
















Location:
Loc1
Loc2
Loc3
Loc4
Loc5
Loc6
Loc7
Loc8






Green Thunder
20
21.5
21
18
21
18
22
20



24
21
19
21
19
21
20
22



23
21
21
18
18
18
20
20



21
21
18
18
18
18
20
19



19
16
21
20
21
20
22
19



21.5
20
18
19
18
19
20
20



16
19
18
18
18
18
22
22



21
20
20
21
20
21
20
22



23
20
18
19
17.5
19
22
22



21
19
17
20
17
18
20
20


Green Forest
20.0
16.0
15.0
16.0
15.0
16.0
19.5
22.0



20
20
14
17
14
17
19.5
19



20
16
13
13
17
13
20
20



18
19
17
16
17
16
20
21



19
15
16
13
16
13
19
21



18
21.5
13
14
13
14
19
19



19
14
16
12
16
12
20
22



20.5
16
12
14
12
14
19
22



16
17
14
14
13.5
14
19
19



21
18
13.5
15
14
15
19.5
20


PIC Cos
17
20
20
20
20
20
21
22



19
19
20
16
19.5
16
20
22



17
18
19
17
17
17
21
21



15
17
17
19
17
19
22
22



19
19
20
15
20
15
20
21



17
16
16
17
16
17
22
19



16
18
15
17
15
20
20
19



14
21
17
20
17
20
20
19



17
17
17
17
17
17
21
20



17
15
17
17
17
17
21
21










Anova: Two-Factor With Replication
















SUMMARY
Loc1
Loc2
Loc3
loc4
Loc5
Loc6
Loc7
Loc8
Total





Green Thunder


Count
10
10
10
10
10
10
10
10
80


Sum
209.5
198.5
191
192
187.5
190
208
206
1582.5


Average
20.95
19.85
19.1
19.2
18.75
19
20.8
20.6
19.78125


Variance
5.247222
2.558333
2.322222
1.511111111
2.069444
1.555556
1.066667
1.6
2.745847


Green Forest


Count
10
10
10
10
10
10
10
10
80


Sum
191.5
172.5
143.5
144
147.5
144
194.5
205
1342.5


Average
19.15
17.25
14.35
14.4
14.75
14.4
19.45
20.5
16.78125


Variance
2.225
5.513889
2.558333
2.488888889
2.958333
2.488889
0.191667
1.611111
8.378758


PIC Cos


Count
10
10
10
10
10
10
10
10
80


Sum
168
180
178
175
175.5
178
208
206
1468.5


Average
16.8
18
17.8
17.5
17.55
17.8
20.8
20.6
18.35625


Variance
2.4
3.333333
3.288889
2.722222222
2.913889
3.288889
0.622222
1.6
4.267049


Total


Count
30
30
30
30
30
30
30
30


Sum
569
551
512.5
511
510.5
512
610.5
617


Average
18.96667
18.36667
17.08333
17.03333333
17.01667
17.06667
20.35
20.56667


Variance
6.050575
4.774713
6.691092
6.171264368
5.370402
6.202299
1.002586
1.495402










ANOVA















Source of Variation
SS
df
MS
F
P-value
F crit







Sample
360.3
2
180.15
74.37029958
2.67E−25
3.03767



Columns
481.249
7
68.74985
28.38160993
1.59E−27
2.052154



Interaction
211.4667
14
15.10476
6.235612923
 2.1E−10
1.737799



Within
523.225
216
2.422338



Total
1576.241
239










Table 6 below shows the leaf index of Green Thunder at harvest maturity as compared to Green Forest and PIC Cos. Leaf index is calculated by dividing the leaf length by the leaf width. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 6, Green Thunder has a significantly different leaf index than Green Forest indicating that Green Thunder has a different leaf shape than Green Forest at harvest maturity. However, Green Thunder has a similar leaf index to PIC Cos indicating that Green Thunder has a similar leaf shape at harvest maturity to that of PIC Cos.









TABLE 6









Leaf Index at Harvest Maturity
















Location:
Loc1
Loc2
Loc3
Loc4
Loc5
Loc6
Loc7
Loc8






Green Thunder
1.35
1.33
1.33
1.39
1.33
1.4
1.32
1.5



1.29
1.33
1.37
1.33
1.37
1.3
1.5
1.45



1.3
1.33
1.33
1.39
1.39
1.4
1.45
1.55



1.33
1.33
1.39
1.39
1.39
1.4
1.5
1.58



1.37
1.44
1.33
1.35
1.33
1.4
1.36
1.58



1.33
1.35
1.39
1.37
1.39
1.4
1.55
1.55



1.44
1.37
1.39
1.39
1.39
1.4
1.36
1.36



1.33
1.35
1.35
1.33
1.35
1.3
1.55
1.36



1.3
1.35
1.39
1.37
1.43
1.4
1.45
1.41



1.33
1.37
1.41
1.35
1.42
1.5
1.6
1.6


Green Forest
1.35
1.44
1.47
1.44
1.47
1.4
1.49
1.45



1.35
1.35
1.5
1.41
1.5
1.4
1.54
1.58



1.35
1.44
1.54
1.54
1.42
1.5
1.5
1.55



1.39
1.37
1.53
1.44
1.41
1.4
1.5
1.43



1.21
1.47
1.44
1.54
1.44
1.5
1.53
1.48



1.39
1.33
1.54
1.5
1.54
1.5
1.58
1.58



1.21
1.5
1.44
1.58
1.44
1.6
1.5
1.45



1.34
1.44
1.58
1.5
1.58
1.5
1.58
1.36



1.44
1.41
1.5
1.5
1.56
1.5
1.58
1.58



1.33
1.39
1.56
1.47
1.5
1.5
1.59
1.5


PIC Cos
1.41
1.35
1.35
1.35
1.35
1.4
1.38
1.41



1.37
1.37
1.35
1.44
1.36
1.4
1.4
1.36



1.41
1.39
1.37
1.41
1.41
1.4
1.38
1.43



1.53
1.41
1.41
1.37
1.41
1.4
1.36
1.45



1.37
1.37
1.35
1.47
1.35
1.5
1.55
1.43



1.41
1.44
1.44
1.41
1.44
1.4
1.36
1.58



1.44
1.39
1.47
1.41
1.47
1.4
1.45
1.58



1.5
1.33
1.41
1.35
1.41
1.4
1.5
1.58



1.41
1.41
1.41
1.41
1.41
1.4
1.43
1.5



1.41
1.47
1.41
1.41
1.41
1.4
1.48
1.48










Anova: Two-Factor With Replication
















SUMMARY
Loc1
Loc2
Loc3
loc4
Loc5
Loc6
Loc7
Loc8
Total





Green Thunder


Count
10
10
10
10
10
10
10
10
80


Sum
13.37
13.55
13.68
13.66
13.79
13.9
14.64
14.94
111.53


Average
1.337
1.355
1.368
1.366
1.379
1.39
1.464
1.494
1.394125


Variance
0.00189
0.001139
0.000929
0.000604444
0.001166
0.003222
0.008738
0.008582
0.005698


Green Forest


Count
10
10
10
10
10
10
10
10
80


Sum
13.36
14.14
15.1
14.92
14.86
14.8
15.39
14.96
117.53


Average
1.336
1.414
1.51
1.492
1.486
1.48
1.539
1.496
1.469125


Variance
0.005449
0.002916
0.002356
0.002795556
0.003582
0.004
0.001632
0.005716
0.006907


PIC Cos


Count
10
10
10
10
10
10
10
10
80


Sum
14.26
13.93
13.97
14.03
14.02
14.1
14.29
14.8
113.4


Average
1.426
1.393
1.397
1.403
1.402
1.41
1.429
1.48
1.4175


Variance
0.002671
0.001734
0.00169
0.001423333
0.001507
0.001
0.00421
0.006178
0.003039


Total


Count
30
30
30
30
30
30
30
30


Sum
40.99
41.62
42.75
42.61
42.67
42.8
44.32
44.7


Average
1.366333
1.387333
1.425
1.420333333
1.422333
1.426667
1.477333
1.49


Variance
0.004948
0.002413
0.005426
0.00438954
0.004129
0.004092
0.006703
0.006407










ANOVA















Source of Variation
SS
df
MS
F
P-value
F crit







Sample
0.235641
 2
0.11782
37.63785199
9.47E−15
3.03767



Columns
0.354798
 7
0.050685
16.19152694
4.53E−17
2.052154



Interaction
0.204919
 14
0.014637
4.675831164
1.96E−07
1.737799



Within
0.67616
216
0.00313



Total
1.471518
239










Table 7 below shows the leaf area of Green Thunder at harvest maturity as compared to Green Forest and PIC Cos. Leaf area is calculated by multiplying the leaf length by the leaf width. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 7, Green Thunder has a significantly larger leaf area than both Green Forest and PIC Cos at harvest maturity.









TABLE 7









Leaf Area (cm2) at Harvest Maturity
















Location:
Loc1
Loc2
Loc3
loc4
Loc5
Loc6
Loc7
Loc8






Green Thunder
540
613
588
450
588
450
638
600



744
588
484
588
494
588
600
704



690
588
588
450
450
450
580
620



588
588
450
450
450
450
600
570



494
368
588
540
588
540
660
570



613
540
450
494
450
494
620
620



368
494
450
450
450
450
660
660



588
540
540
588
540
588
620
660



690
540
450
494
438
494
704
682



588
494
408
540
408
486
640
640


Green Forest
540
368
330
368
330
368
566
704



540
540
294
408
294
408
585
570



540
368
260
260
408
260
600
620



450
494
442
368
408
368
600
630



437
330
368
260
368
260
551
651



450
613
260
294
260
294
570
570



437
294
368
228
368
228
600
704



564
368
228
294
228
294
570
660



368
408
294
294
284
294
570
570



588
450
284
330
294
330
605
600


PIC Cos
408
540
540
540
540
540
609
682



494
494
540
368
517
368
560
660



408
450
494
408
408
408
609
630



345
408
408
494
408
494
660
704



494
494
540
330
330
330
620
630



408
368
368
408
368
408
660
570



368
450
330
408
330
540
580
570



294
588
408
540
408
540
600
570



408
408
108
108
108
108
630
600



408
330
408
108
108
108
651
651










Anova: Two-Factor With Replication
















SUMMARY
Loc1
Loc2
Loc3
loc4
Loc5
Loc6
Loc7
Loc8
Total





Green Thunder


Count
10
10
10
10
10
10
10
10
80


Sum
5903
5353
4996
5044
4856
4990
6322
6326
43790


Average
590.3
535.3
499.6
504.4
485.6
499
632.2
632.6
547.375


Variance
11695.12
5092.9
4838.933
3174.044444
4150.933
3038.444
1321.289
2034.711
7410.668


Green Forest


Count
10
10
10
10
10
10
10
10
80


Sum
4914
4233
3128
3104
3242
3104
5817
6279
33821


Average
491.4
423.3
312.8
310.4
324.2
310.4
581.7
627.9
422.7625


Variance
5140.267
10016.46
4153.956
3246.933333
3859.067
3246.933
350.9
2672.1
18801.12


PIC Cos


Count
10
10
10
10
10
10
10
10
80


Sum
4035
4530
4144
3712
3525
3844
6179
6267
36236


Average
403.5
453
414.4
371.2
352.5
384.4
617.9
626.7
452.95


Variance
3683.833
6175.333
17438.04
24042.84444
21363.39
26864.71
1117.656
2345.789
22218.96


Total


Count
30
30
30
30
30
30
30
30


Sum
14852
14116
12268
11860
11623
11938
18318
18872


Average
495.0667
470.5333
408.9333
395.3333333
387.4333
397.9333
610.6
629.0667


Variance
12391.24
8927.361
14234.41
16244.50575
14238.46
16515.44
1333.076
2195.444










ANOVA















Source of Variation
SS
df
MS
F
P-value
F crit







Sample
676150.4
2
338075.2
47.43123725
8.39E−18
3.03767



Columns
2005861
7
286551.6
40.20258531
6.98E−36
2.052154



Interaction
280586.4
14
20041.89
2.811834592
0.000701
1.737799



Within
1539581
216
7127.691



Total
4502179
239










Table 8 below shows the core length of Green Thunder at harvest maturity as compared to Green Forest and PIC Cos. Core length is measured in centimeters. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 8, Green Thunder has a significantly longer core than both Green Forest and PIC Cos at harvest maturity.









TABLE 8









Core Length (cm) at Harvest Maturity
















Location:
Loc1
Loc2
Loc3
Loc4
Loc5
Loc6
Loc7
Loc8






Green Thunder
7.7
9.2
5
6.5
5
6.5
5.5
7



8.6
9
7
7
7
7
6
6.5



8.4
10
7
6
7
6
6.5
6.5



9
9.5
7
6
7
6
6.5
4



8.2
5.5
6.5
7
6.5
7
7
4



8
7.5
6
7
6
7
6
6.5



7
7
6
5.5
6
5.5
5.8
6.8



7.6
8
5.5
5
5.5
5
6.2
7



9
6
6
6
6
6
7
7



8
6.5
6.5
6.5
6.5
6.5
6.5
6.5


Green Forest
7
5.5
5
5
5
5
6.5
6.2



7
9
5
5
5
5
7
7



8.2
7.8
5
5
5
5
6
6.5



7.7
10
5
6
5
6
6.5
6.5



9
6.5
5
5
5
5
6
6.2



7
8
5
5
5
5
6.5
6



9.5
6
5
5
5
5
6.5
6.5



10
7
5
5
5
5
6.5
7



5
6
5
5
5
5
7
7



8.5
9
6
5
6
5
7
6.5


PIC Cos
7
6
5.5
6
5.5
6
5
6.5



7.5
6
6
5
6
5
5
4.5



6.5
6
5.5
5
5.5
5
4
4.2



5
5
5
6
5
6
5
5.6



7
6
6
5
6
5
4
5



6
7
5
6
5
6
4
5



6
7
5
5
5
5
5
5



4
8
4
6
4
5
5
4



5.5
5
5
5
5
5
4
4



5.5
5
5
5
4
4
5
4










Anova: Two-Factor With Replication
















SUMMARY
Loc1
Loc2
Loc3
loc4
Loc5
Loc6
Loc7
Loc8
Total





Green Thunder


Count
10
10
10
10
10
10
10
10
80


Sum
81.5
78.2
62.5
62.5
62.5
62.5
63
61.8
534.5


Average
8.15
7.82
6.25
6.25
6.25
6.25
6.3
6.18
6.68125


Variance
0.398333
2.457333
0.458333
0.458333333
0.458333
0.458333
0.242222
1.368444
1.299264


Green Forest


Count
10
10
10
10
10
10
10
10
80


Sum
78.9
74.8
51
51
51
51
65.5
65.4
488.6


Average
7.89
7.48
5.1
5.1
5.1
5.1
6.55
6.54
6.1075


Variance
2.167667
2.315111
0.1
0.1
0.1
0.1
0.136111
0.129333
1.789563


PIC Cos


Count
10
10
10
10
10
10
10
10
80


Sum
60
61
52
54
51
52
46
47.8
423.8


Average
6
6.1
5.2
5.4
5.1
5.2
4.6
4.78
5.2975


Variance
1.111111
0.988889
0.344444
0.266666667
0.488889
0.4
0.266667
0.668444
0.764804


Total


Count
30
30
30
30
30
30
30
30


Sum
220.4
214
165.5
167.5
164.5
165.5
174.5
175


Average
7.346667
7.133333
5.516667
5.583333333
5.483333
5.516667
5.816667
5.8333333


Variance
2.090851
2.36023
0.560057
0.501436782
0.629023
0.577299
0.976609
1.2685057










ANOVA















Source of Variation
SS
df
MS
F
P-value
F crit







Sample
77.33475
2
38.66737
58.06275418
6.61E−21
3.03767



Columns
121.8153
7
17.40218
26.13104102
9.34E−26
2.052154



Interaction
38.77458
14
2.769613
4.158838409
1.94E−06
1.737799



Within
143.847
216
0.665958



Total
381.7716
239










Table 9 below shows the length of the fourth true leaf of Green Thunder as compared to Green Forest and PIC Cos. Leaf length is measured in centimeters of a 20-day old seedling. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 9, Green Thunder has a significantly shorter fourth leaf length than Green Forest but about the same fourth leaf length as PIC Cos.







TABLE 9







4th Leaf Length (cm)









Green Thunder
Green Forest
PIC Cos





3.8
5.6
3.8


4.3
5.5
3.9


3.9
4.8
3.6


4.1
5.8
4.2


3.9
4  
3.7


4.4
4.8
3.8


4.6
5.5
3.9


3.5
5.5
4  


4.1
5.6
4.1


4.3
5.8
4  


3.8
5.9
3.9


4  
4.5
3.9


4.1
4.6
4  


4.2
5.4
4.1


4  
5.5
4.5










Anova: Single Factor


SUMMARY













Groups
Count
Sum
Average
Variance







Green Thunder
15
61
4.066666667
0.075238



Green Forest
15
78.8
5.253333333
0.321238



PIC Cos
15
59.4
3.96
0.046857











ANOVA













Source of Variation
SS
df
MS
F
P-value
F crit





Between Groups
15.46133333
 2
7.730666667
52.31278
3.96E−12
3.219938


Within Groups
6.206666667
42
0.147777778


Total
21.668
44









Table 10 below shows the width of the fourth true leaf of Green Thunder as compared to Green Forest and PIC Cos. Leaf width is measured in centimeters of a 20-day old seedling. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 10, Green Thunder has a significantly narrower fourth leaf width than Green Forest but about the same fourth leaf width as PIC Cos.







TABLE 10







4th Leaf Width (cm)









Green Thunder
Green Forest
PIC Cos





2.6
2.8
2.3


2.8
3.2
2.5


2.5
2.7
2.2


2.6
3  
2.5


2.4
2.1
2.1


2.5
2.6
2.4


2.6
2.8
2.3


2.2
2.9
2.5


2.6
2.9
2.5


2.8
3  
2.4


2.6
3  
2.4


2.5
2.4
2.2


2.5
2.5
2.4


2.7
3  
2.5


2.8
3  
2.6










Anova: Single Factor


SUMMARY













Groups
Count
Sum
Average
Variance







Green Thunder
15
38.7
2.58
0.026



Green Forest
15
41.9
2.793333333
0.083524



PIC Cos
15
35.8
2.386666667
0.01981











ANOVA













Source of Variation
SS
df
MS
F
P-value
F crit





Between Groups
1.241333333
 2
0.620666667
14.39691
1.73E−05
3.219938


Within Groups
1.810666667
42
0.043111111


Total
3.052
44









Table 11 below shows the leaf index of the fourth true leaf of Green Thunder as compared to Green Forest and PIC Cos. Leaf index is calculated by dividing the leaf length by the leaf width of the fourth true leaf of a 20-day old seedling. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 11, Green Thunder has a significantly different leaf index than both Green Forest and PIC Cos indicating that Green Thunder has a different fourth true leaf shape than either Green Forest or PIC Cos.







TABLE 11







4th Leaf Index









Green Thunder
Green Forest
PIC Cos





1.46
2.07
1.65


1.54
1.72
1.56


1.56
1.78
1.64


1.58
1.93
1.68


1.63
1.9 
1.76


1.76
1.85
1.58


1.77
1.96
1.7 


1.59
1.9 
1.6 


1.58
1.93
1.64


1.54
1.93
1.67


1.46
1.97
1.63


1.6 
1.88
1.73


1.71
1.84
1.67


1.56
1.8 
1.64


1.43
1.83
1.73










Anova: Single Factor


SUMMARY













Groups
Count
Sum
Average
Variance







Green Thunder
15
23.77
1.584666667
0.010241



Green Forest
15
28.29
1.886
0.007526



PIC Cos
15
24.88
1.658666667
0.003155











ANOVA













Source of Variation
SS
df
MS
F
P-value
F crit





Between Groups
0.739791111
2
0.369895556
53.03947
3.22E−12
3.219938


Within Groups
0.292906667
42
0.006973968


Total
1.032697778
44









Table 12 below shows the cotyledon length of Green Thunder as compared to Green Forest and PIC Cos. Cotyledon length was measured in millimeters. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 12, Green Thunder has a significantly shorter cotyledon length than Green Forest but similar cotyledon length to PIC Cos.







TABLE 12







Cotyledon length (mm)









Green Thunder
Green Forest
PIC Cos





15
19
14


15
20
14


15
19
14


16
19
12


16
20
14


15
18
14


13
18
14


13
19
16


15
18
16


15
19
16


15
19
14


15
19
14










Anova: Single Factor


SUMMARY













Groups
Count
Sum
Average
Variance







Green Thunder
12
178
14.83333
0.878788



Green Forest
12
227
18.91667
0.44697



PIC Cos
12
172
14.33333
1.333333











ANOVA













Source of Variation
SS
df
MS
F
P-value
F crit





Between Groups
151.7222222
 2
75.86111
85.58689
8.72E−14
3.284924


Within Groups
29.25
33
0.886364


Total
180.9722222
35









Table 13 below shows the cotyledon width of Green Thunder as compared to Green Forest and PIC Cos. Cotyledon width was measured in millimeters. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 13, Green Thunder has a significantly wider cotyledon width than both Green Forest and PIC Cos.







TABLE 13







Cotyledon Width (mm)









Green Thunder
Green Forest
PIC Cos





10
9
9


10
9
8


10
9
9


10
9
8


10
9
8


11
9
8


 9
9
8


 9
9
8


10
9
9


10
9
9


10
9
8


10
9
8










Anova: Single Factor


SUMMARY













Groups
Count
Sum
Average
Variance







Green Thunder
12
119
9.916667
0.265152



Green Forest
12
108
9
0



PIC Cos
12
100
8.333333
0.242424











ANOVA













Source of Variation
SS
df
MS
F
P-value
F crit





Between Groups
15.16666667
2
7.583333
44.8209
3.92E−10
3.284924


Within Groups
5.583333333
33
0.169192


Total
20.75
35









Table 14 below shows the cotyledon index of Green Thunder as compared to Green Forest and PIC Cos. Cotyledon index is calculated by dividing the cotyledon leaf length by the cotyledon leaf width. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 14, Green Thunder has a significantly different cotyledon index than both Green Forest and PIC Cos indicating that Green Thunder has a different cotyledon leaf shape than both Green Forest and PIC Cos.







TABLE 14







Cotyledon Width (mm)









Green Thunder
Green Forest
PIC Cos





1.5
2.11
1.56


1.5
2.22
1.75


1.5
2.11
1.56


1.6
2.11
1.5 


1.6
2.22
1.75


 1.36
2  
1.75


 1.44
2  
1.75


 1.44
2.11
1.75


1.5
2  
1.78


1.5
2.11
1.78


1.5
2.11
1.75


1.5
2.11
1.75










Anova: Single Factor


SUMMARY













Groups
Count
Sum
Average
Variance







Green Thunder
12
17.94
1.495
0.004227



Green Forest
12
25.21
2.100833
0.005408



PIC Cos
12
20.43
1.7025
0.009948











ANOVA













Source of Variation
SS
df
MS
F
P-value
F crit





Between Groups
2.275038889
2
1.137519
174.2583
2.89E−18
3.284924


Within Groups
0.215416667
33
0.006528


Total
2.490455556
35









DEPOSIT INFORMATION
A deposit of the Synergene Seed & Technology, Inc. Proprietary lettuce cultivar designated Green Thunder disclosed above and recited in the appended claims has been made with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas. Va. 20110. The date of deposit was Aug. 14, 2007. The deposit of 2,500 seeds was taken from the same deposit maintained by Synergene Seed & Technology, Inc. since prior to the filing date of this application. All restrictions upon the deposit have been removed, and the deposit is intended to meet all of the requirements of 37 C.F.R. 1.801-1.809. The ATCC accession number is PTA-8605. 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 as necessary during that period.
While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.

## Claims

1. A seed of lettuce cultivar Green Thunder, wherein a representative sample of seed of said cultivar was deposited under ATCC Accession No. PTA-8605.

2. A lettuce plant, or a part thereof, produced by growing the seed of claim 1.

3. A tissue culture of cells produced from the plant of claim 2, wherein said cells of the tissue culture are produced from a plant part selected from the group consisting of leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, anther, pistil, flower, and stem.

4. A protoplast produced from the plant of claim 2.

5. A protoplast produced from the tissue culture of claim 3.

6. A lettuce plant regenerated from the tissue culture of claim 3, wherein the plant has all the morphological and physiological characteristics of cultivar Green Thunder.

7. A method for producing an F1 hybrid lettuce seed, wherein the method comprises crossing the plant of claim 2 with a different lettuce plant and harvesting the resultant F1 hybrid lettuce seed.

8. A hybrid lettuce seed produced by the method of claim 7.

9. A hybrid lettuce plant, or a part thereof, produced by growing said hybrid seed of claim 8.

10. A method for producing a male sterile lettuce plant wherein the method comprises transforming the lettuce plant of claim 2 with a nucleic acid molecule.

11. A male sterile lettuce plant produced by the method of claim 10.

12. A method for producing an herbicide resistant lettuce plant wherein the method comprises transforming the lettuce plant of claim 2 with a transgene wherein the transgene confers resistance to an herbicide selected from the group consisting of imidazolinone, sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine and benzonitrile.

13. An herbicide resistant lettuce plant produced by the method of claim 12.

14. A method of producing an insect resistant lettuce plant wherein the method comprises transforming the lettuce plant of claim 2 with a transgene that confers insect resistance.

15. An insect resistance lettuce plant produced by the method of claim 14.

16. The lettuce plant of claim 15 wherein the transgene encodes a Bacillus thuringiensis endotoxin.

17. A method of producing a disease resistant lettuce plant wherein the method comprises transforming the lettuce plant of claim 2 with a transgene that confers disease resistance.

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

19. A method of producing a lettuce plant with a value-added trait, wherein the method comprises transforming the lettuce plant of claim 2 with a transgene encoding a protein selected from the group consisting of a ferritin, a nitrate reductase and a monellin.

20. A lettuce plant with a value-added trait produced by the method of claim 19.

21. A lettuce plant, or a part thereof, having all the physiological and morphological characteristics of the cultivar Green Thunder, wherein a representative sample of seed of said cultivar was deposited under ATCC Accession No. PTA-8605.

22. A method of introducing a desired trait into lettuce cultivar Green Thunder wherein the method comprises:
a. crossing a Green Thunder plant, wherein a representative sample of seed was deposited under ATCC Accession No. PTA-8605, with a plant of another lettuce cultivar that comprises a desired trait to produce progeny plants, wherein the desired trait is selected from the group consisting of male sterility, herbicide resistance, insect resistance, and resistance to bacterial disease, fungal disease, or viral disease;
b. selecting progeny plants that have the desired trait to produce selected progeny plants;
c. crossing the selected progeny plants with the Green Thunder plants to produce backcross progeny plants;
d. selecting for backcross progeny plants that have the desired trait and physiological and morphological characteristics of lettuce cultivar Green Thunder listed in Table 1; and
e. repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny plants that comprise the desired trait and all of the physiological and morphological characteristics of lettuce cultivar Green Thunder listed in Table 1.

23. A plant produced by the method of claim 22, wherein the plant has the desired trait and all of the physiological and morphological characteristics of lettuce cultivar Green Thunder listed in Table 1.

24. The plant of claim 23, wherein the desired trait is herbicide resistance and the resistance is conferred to an herbicide selected from the group consisting of imidazolinone, sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine and benzonitrile.

25. The plant of claim 23, wherein the desired trait is insect resistance and the insect resistance is conferred by a transgene encoding a Bacillus thuringiensis endotoxin.

26. The plant of claim 23, wherein the desired trait is male sterility and the trait is conferred by a nucleic acid molecule.