AU4244699A - Disease resistant cotton - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant: D&PL TECHNOLOGY HOLDING CORP.

Invention Title: DISEASE RESISTANT COTTON.

The following statement is a full description of this invention, including the best method of performing it known to me/us:

I/A

"DISEASE RESISTANT COTTON" BACKGROUND OF THE INVENTION The present invention relates to a cotton (Gossypium) seed, a cotton plant, a cotton variety and a cotton hybrid which contains a mutant disease resistance gene. This invention further relates to a method for producing disease resistant cotton seed and plants.

At least 16 viral diseases have been reported on cotton (Tarr, S.A.J., 1964, Commonw. Mycol. Inst., Kew Surrey, England; Watkins, G.M. 1981, Amer. Phyto. Soc., St. Paul, MN). However, all the causal agents have S 10 not been isolated and characterized; thus, many of these diseases may be caused by the same or similar infectious agents. These diseases are of significant economic importance where cotton is grown in tropical or :i subtropical climates in Africa, Asia, Central America, and South America.

Viral diseases may be divided into groups based on symptoms and 15 vectors. The first, leaf cur, causes thickening and enations of leaf veins, leaf curling, and twisting and curving of stems and petioles. The affected veins appear abnormally dark green and opaque. The virus is transmitted by whitefly, Bemisia tabaci Genn., and is of most importance in Punjab areas of Pakistan and India and Africa. Diseases referred to as mosaics, leaf crumple, and leaf mottle have similar symptoms and all are considered to be geminivirus transmitted by B. tabaci, (Brown, 1998, Proceedings Beltwide Cotton Conferences, Memphis, TN). Plants are often stunted, sterile, and 2 have crinkled, mottled, and blistered leaves. The green blisters or chlorotic patches occur in a mosaic pattern over the leaves. Diseases in this group have been reported in Asia, Africa, South America, and areas of the western United States where cotton is ratooned.

Blue disease is a major viral cause of disease losses in several African and South American countries. This virus is transmitted by Aphis gossypii Glover. Affected plants are dwarfed and have thick, dark blue-green leaves with light areas next to veins and edges that curl down; stems often grow in a zigzag pattern. Diseases referred to as leafroll, vein clearing disease, and veinal mosaic in the USSR, Thailand, Philippines, and South America have symptoms similar to blue disease and also are transmitted by A. gossypii, which indicates similar causal viruses. Vein clearing reported in Texas, USA, apparently is caused by a fourth type of virus, because it is transmitted by the leafhopper, Scaphytopius albifrons Hepner. Virus diseases are controlled by eliminating reservoir hosts and insect vectors.

Cotton blue disease was first documented in 1949 in the Central African Republic (Tarr, 1964). the disease incidence remained relatively moderate until 1966-68 when severe outbreaks were experienced as a result of heavy infestations of the insect vector, Aphis gossypii. During the epidemic years of the mid-to late 1960s, disease incidence was routinely 25-30% in some fields and overall production was reduced by The cotton blue disease is now known to occur in Benin, Chad, Cameroon, Ivory Coast and Zaire.

.:.Cotton expressing blue disease symptoms has been reported to occur in Brazil (Costa, 1996 Annual Review of Phytopathology), Argentina, Paraguay and Bolivia. The cotton aphid, Aphis gossypii, populations infesting the fields were presumed to serve as vectors for this purported virus. Experimental transmissions under glasshouse conditions of blue disease using Aphis gossypii have been successful (Bonacic, et al., 1997, Report on Cotton Blue Disease in Argentina), since all the symptoms were reproduced similarly to those observed in the field dwarf plants, stems in zigzag, hard leaves of dark green-bluish color, with edges curved downward). Positive ELISA reactions between the causal agent of blue disease in South America with antiserum to several luteoviruses were reported by Bonacic, et al., 1997.

The severity and thus the impact of the disease depend on plant age at the time of infection. An early infection (<50 days post-emergence) results in no yield in susceptible cotton varieties whereas, for infections occurring about 100 days post-emergence, 15-20% losses are sustained. Late infections result in minimal to no loss probably because there is a minimum flush growth to support virus replication, and because the metabolism of more mature cotton plants renders them less susceptible to damage. Losses due to infection by the blue disease agent are manifest as a reduction in quality of harvested seed cotton with a low seed index, reduced fiber length and a decrease in fiber resistance. In addition, fewer and smaller flowers are produced and boll shedding occurs.

The so-called blue disease is named for the dramatic dark green to bluish color that the leaves take on when plants are affected. In addition, leaves roll under and become leathery in texture. Epinasty may be very severe, with reddened petioles and veins, and with early-season infection stunting of plants is pronounced. Approximately 3-4% of the affected plants die, whereas recovery from the symptoms is observed in about 6% of the plants.

S•There has been limited attempt to isolate or characterize the causal agent, **although an aphid-transmitted leuteovirus is suspected. Despite efforts to identify alternate host species by transmission studies using A. gossypi, cotton remains the only known host of the disease. The only known natural vector is A. gossypii, as was demonstrated in transmission studies (Cauquil, J. et al., 1971, La maladie bleue du cotonnier en Afrique: transmission de cotonnier a cotonnier par Aphis gossypii Glover, Coton et Fibres Tropicales 26,463-6).

IJ 4 Vermelhao disease or cotton anthocyanosis, was first observed in G.

barbadense plantings in Brazil (Costa, A. et al., 1954, Vermelhao do algodoeiro, Bragantia 13, 237-46). It now occurs throughout the country in both G. barbadense and G. hirsutum cotton varieties. The disease affects 100% of cotton plantings in all 25 counties surveyed in the states of Sao Paulo, Rio Grande Norte and Paraiba. Symptoms are consistently associated with aphid infestations.

Vermelhao disease (cotton anthocyanosis) is characterized by foliar chlorosis followed by patchy purpling or reddening, and eventually leaves become entirely purple/red except for the veins. Symptoms are noticeable first on older leaves and progress to affect the developing leaves. Older affected leaves may be shed while newest leaves are asymptomatic; then newer leaves begin to discolor as described above. If plants are cut back (ratooned), regrowth of asymptomatic leaves occurs, and the progression of symptoms occurs again. Early in the season, affected plants may be observed in scattered areas in the field, and eventually symptoms become widespread as secondary infections occur. Although a plant virus is suspected, there is no direct evidence concerning the aetiology of the disease.

Naturally infected species which serve as sources of inoculum are G.

-oo barbadense, G. hirsutum, Hibiscus cannabinus and H. esculentus.

Aphis gossypii Glover has been shown to vector the disease agent. The disease agent appears to be transmitted in a persistent manner by A. gossypii, with an undetermined latent period. The agent may be transmitted for the life of the vector. Primary spread of the suspect virus occurs when migrating, inoculative A. gossypii colonize cotton seedlings early in the season. Because the agent is not seed-borne, migratory aphid vectors must acquire the agent from alternate host reservoirs. Suspected reservoirs are ratooned cotton, kenaf, okra and several malvaceous weeds, including Sida micrantha and S.

rhombifolia. Inoculum overwinters in these and possibly other reservoirs, and i).

II y is transported by the vector into cotton fields early in the growing season (November January). Secondary spread within cotton occurs following population increases, which stem from founder aphid colonies. A second migration may occur in mid summer (February March) which harbors additional inoculum and adds to the vector population levels in cotton fields.

Thus, several repeated cycles of vector infestations occur, followed by an increase in disease incidence.

Bonsai Bunchy Top (BBT) of cotton is purported to be caused by a virus or virus-like organism and has been characterized and named because of small leaves, small bolls and shortened internodes, particularly in the top terminal area, giving the plant a bunchy top appearance. Some leaves on the BBT infected plant may show a mottled chlorosis or light green patches around the edge with the normal dark green color inside or occasionally a red border around the margin of the leaf. The symptoms of the disorder can be quite variable depending on the severity of attack and the age of the plant when first affected. The cotton plants can be affected early, developing into short stunted plants, with overlapping small leaves giving it a 'climbing ivy' type o of appearance. The attack may also occur later in the plant growth, with the lower part of the plant growing normally with normal sized bolls and internmodes, and the upper portion of the plant having the typical symptoms of small leaves, a prolific number of nodes and small bolls and bunched internodes. If the disease attack occurs late, then the plant grows and looks normal, except for small late season bolls and a compacted terminal area.

S"However, the earlier the attack, the greater the damage with lower yields, poor defoliation and poor pickability. Symptoms of BBT have also been observed S"on Hibiscus trionum, a common summer weed in Australia that was infested with Aphis gossypii and growing adjacent to cotton with BBT symptoms in Australia.

1I 6 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 an improved combination of desirable traits from the parental germplasm. In cotton, the important traits include higher fiber (lint) yield, earlier maturity, improved fiber quality, resistance to diseases and insects, resistance to drought and heat, and improved agronomic traits.

Pureline cultivars of cotton are commonly bred by hybridization of two or more parents followed by selection. The complexity of inheritance, the breeding objectives and the available resources influence the breeding method. Pedigree breeding, recurrent selection breeding and backcross breeding are breeding methods commonly used in self pollinated crops such as cotton. These methods refer to the manner in which breeding pools or populations are made in order to combine desirable traits from two or more *oll cultivars or various broad-based sources. The procedures commonly used for S.i selection of desirable individuals or populations of individuals are called mass 0*o* selection, plant-to-row selection and single seed descent or modified single '0:60 seed descent. One, or a combination of these selection methods, can be used in the development of a cultivar from a breeding population.

Pedigree breeding is primarily used to combine favorable genes into a totally new cultivar that is different in many traits than either parent used in the 25 original cross. It is commonly used for the improvement of self-pollinating V0006 4 crops. Two parents which possess favorable, complementary traits are crossed to produce an F 1 (filial generation An F 2 population is produced by selfing F 1 plants. Selection of desirable individual plants may begin as early as the F 2 generation wherein maximum gene segregation occurs.

f g 7 Individual plant selection can occur for one or more generations.

Successively, seed from each selected plant can be planted in individual, identified rows or hills, known as progeny rows or progeny hills, to evaluate the line and to increase the seed quantity, or, to further select individual plants. Once a progeny row or progeny hill is selected as having desirable traits it becomes what is known as a breeding line that is specifically identifiable from other breeding lines that were derived from the same original population. At an advanced generation F 5 or higher) seed of individual lines are evaluated in replicated testing. At an advanced stage the best lines or a mixture of phenotypically similar lines from the same original cross are tested for potential release as new cultivars.

Descriptions of other breeding methods that are commonly used for different traits and crops can be found in one of several reference books Allard, 1960; Simmonds, 1979; Sneep, et al. 1979; Fehr, 1987).

The single seed descent procedure in the strict sense refers to planting a segregating population, harvesting one seed from every plant, and combining these seeds into a bulk which is planted the next generation. When the population has been advanced to the desired level of inbreeding, the plants from which lines are derived will each trace to different F 2 individuals.

Primary advantages of the seed descent procedures are to delay selection until a high level of homozygosity lack of gene segregation) is achieved in individual plants, and to move through these early generations quickly, usually through using winter nurseries.

The modified single seed descent procedures involve harvesting multiple seed a single lock or a simple boll) from each plant in a population and combining them to form a bulk. Part of the bulk is used to plant the next generation and part is put in reserve. This procedure has been used to save l labor at harvest and to maintain adequate seed quantities of the population.

8 Selection for desirable traits can occur at any segregating generation (F 2 and above). Selection pressure is exerted on a population by growing the population in an environment where the desired trait is maximally expressed and the individuals or lines possessing the trait can be identified. For instance, selection can occur for disease resistance when the plants or lines are grown in natural or artificially-induced disease environments, and the breeder selects only those individuals having little or no disease and are thus assumed to be resistant.

Promising advanced breeding lines are thoroughly tested and compared to popular cultivars in environments representative of the commercial target area(s) for three or more years. The best lines having superiority over the popular cultivars are candidates to become new commercial cultivars. Those lines still deficient in a few traits are discarded or utilized as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing and distribution, usually take from seven to twelve years from the time the first cross is made. Therefor, 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.

go 20 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 lines and widely grown standard cultivars. For many traits a single observation is inconclusive, and replicated observations over time and space **are required to provide a good estimate of a line's genetic worth.

The goal of a commercial cotton breeding program is to develop new, unique and superior cotton cultivars. The breeder initially selects and crosses two or more parental lines, followed by generation advancement and 9 selection, thus producing many new genetic combinations. The breeder can theoretically generate billions of different genetic combinations via this procedure. The breeder has no direct control over which genetic combinations will arise in the limited population size which is grown.

Therefore, two breeders will never develop the same line having the same 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 lines which are developed are unpredictable. This unpredictability is 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 lines he develops, except possibly in a very gross and general fashion. The same breeder cannot produce, with any reasonable likelihood, the same cultivar twice by using the exact same original parents and the same selection techniques. This unpredictability results in the expenditure of large amounts of research moneys to develop superior new cotton cultivars.

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, and the grower, processor and consumer; for special advertising and marketing and'commercial production practices, and new product utilization.

The testing preceding the 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.

Cotton, Gossypium hirsutum, is an important and valuable field crop.

Thus, a continuing goal of plant breeders is to develop stable, high yielding cotton cultivars that are agronomically sound. The reasons for this goal are obviously to maximize the amount and quality of the fiber produced on the land used and to supply fiber, oil and food for animals and humans. To accomplish this goal, the cotton breeder must select and develop plants that have the traits that result in superior cultivars.

The development of new cotton cultivars requires the evaluation and selection of parents and the crossing of these parents. The lack of predictable success of a given cross requires that a breeder, in any given year, make several crosses with the same or different breeding objectives.

The cotton flower is monecious in that the male and female structures are in the same flower. The crossed or hybrid seed is produced by manual crosses between selected parents. Floral buds of the parent that is to be the female are emasculated prior to the opening of the flower by manual removal of the male anthers. At flowering, the pollen from flowers of the parent plants S:i• designated as male, are manually placed on the stigma of the previous 20 emasculated flower. Seed developed from the cross is known as first .i generation (F 1 hybrid seed. Planting of this seed produces F 1 hybrid plants of which half their genetic component is from the female parent and half from the male parent. Segregation of genes begins at meiosis thus producing second generation (F 2 seed. Assuming multiple genetic differences between the original parents, each F 2 seed has a unique combination of genes.

Bonzai Bunchy Top, Blue Disease and Vermelhao diseases are important diseases which can cause severe damage to the cotton crop. Use of a reliable disease resistance gene, if available, would result in improved cotton yield and overall quality.

11 SUMMARY OF THE INVENTION The present invention relates to a cotton seed, a cotton plant, a cotton variety and a method for producing a cotton plant.

More specifically, the invention relates to a cotton plant having the mutant disease resistance gene of the present invention.

The present invention further relates to a method of producing disease resistant cotton seeds and plants by crossing a disease resistant plant of the instant invention with another cotton plant. The invention also relates to the transfer of the genetic disease resistance gene into other genetic backgrounds.

This invention further relates to the seeds of cotton variety DeltaOPAL, to the plants of cotton variety DeltaOPAL and to methods for producing a cotton plant produced by crossing the cotton DeltaOPAL with itself or another cotton line. Thus, any such methods using the cotton variety DeltaOPAL are part of this invention including: selfing, backcrosses, hybrid production, crosses to populations, and the like.

In another aspect, the present invention provides for single trait converted plants of DeltaOPAL. The single transferred trait may preferably be a dominant or recessive allele. Preferably, the single transferred trait will confer such traits as herbicide resistance, insect resistance, resistance for bacterial, S:fungal, or viral disease, male fertility, male sterility, enhanced fiber quality, and industrial usage. The single trait may be a naturally occurring cotton gene or a transgene introduced through genetic engineering techniques.

In another aspect, the present invention provides regenerable cells for use in tissue culture of cotton plant DeltaOPAL. The tissue culture will preferably be capable of regenerating plants having the physiological and morphological characteristics of the foregoing cotton plant, and of regenerating plants having substantially the same genotype as the foregoing cotton plant. Preferably, the regenerable cells in such tissue cultures will be embryos, protoplasts, 12 meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, or stems. Still further, the present invention provides cotton plants regenerated from the tissue cultures of the invention.

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: Lint Yield. As used herein, the term "lint yield" is defined as the measure of the quantity of fiber produced on a given unit of land. Presented below in pounds per acre.

Lint Percent. As used herein, the term "lint percent" is defined as the lint (fiber) fraction of seed cotton (lint and seed).

Gin Tumrnout. As used herein, the term "gin turnout" is defined as a fraction of lint in a machine harvested sample of seed cotton (lint, seed, and trash).

Fiber Length. As used herein, the term "fiber length" is defined as span length in inches of fiber as measured by High Volume Instrumentation

(HVI).

Uniformity Ratio. As used herein, the term "uniformity ratio" is defined as •i 20 a measure of the relative length uniformity of a bundle of fibers as measured by HVI.

Micronaire. As used herein, the term "micronaire" is defined as a measure of the fineness of the fiber. Within a cotton cultivar, micronaire is also a measure of maturity. Micronaire differences are governed by changes in perimeter or in cell wall thickness, or by changes in both. Within a variety, cotton perimeter is fairly constant and maturity will cause a change in micronaire. Consequently, micronaire has a high correlation with maturity e. within a variety of cotton. Maturity is the degree of development of cell wall thickness. Micronaire may not have a good correlation with maturity between 13 varieties of cotton having different fiber perimeter. Micronaire values range from about 2.0 to Below 2.9 Very fine 2.9 to 3.7 3.8 to 4.6 4.7 to 5.5 5.6 Fine Average Coarse Very coarse Possible small perimeter but mature (good fiber), or large perimeter but immature (bad fiber).

Various degrees of maturity and/or perimeter.

Average degree of maturity and/or perimeter.

Usually fully developed (mature), but larger perimeter.

Fully developed, large-perimeter fiber.

Fiber Strength As used herein, the term "fiber strength" is defined as the force required to break a bundle of fibers as measured in grams per millitex on the HVI.

Fiber Elongation As used herein, the term "fiber elongation" is defined as the measure of elasticity of a bundle of fibers as measured by HVI.

Plant Height. As used herein, the term "plant height" is defined as the average height in inches or centimeters of a group of plants.

Stringout Rating (storm resistance). As used herein, the term "stringout rating" is defined as a visual rating prior to harvest of the relative looseness of the seed cotton held in the boll structure on the plant.

Maturity Rating. As used herein, the term "maturity rating" is defined as a visual rating near harvest on the amount of opened bolls on the plant.

Vegetative Nodes. As used herein, the term "vegetative nodes" is defined as the number of nodes from the cotyledonary node to the first fruiting branch on the main stem of the plant.

14 Fruiting Nodes. As used herein, the term "fruiting nodes" is defined as the number of nodes on the main stem from which arise branches which bear fruit or bolls.

Essentially all the physiological and morphological characteristics. A plant having essentially all the physiological and morphological characteristics means a plant having the physiological and morphological characteristics, except for the characteristics derived from the converted trait.

Single trait Converted (Conversion). Single trait converted (conversion) plant refers to plants which are developed by a plant breeding technique called backcrossing or via genetic engineering wherein essentially all of the desired morphological and physiological characteristics of a variety are recovered in addition to the single trait transferred into the variety via the backcrossing technique or via genetic engineering.

Disease Resistance. As used herein the term "disease resistance" is defined as the ability of plants to restrict the activities of a specified pest, such as an insect, fungus, virus, or bacterial.

Disease Tolerance. As used herein the term "disease tolerance" is ::**defined as the ability of plants to endure a specified pest (such as an insect, fungas, virus or bacteria) or an adverse environmental condition and still 20 performing and producing in spite of this disorder.

VRDP. As used herein the term 'V/RDP" is defined as the allele designation for the single dominant allele of the present invention which confers virus resistance. VRDP designates 'V/irus Resistance Deltapine".

DETAILED DESCRIPTION OF THE INVENTION To date, there is no known disease resistant gene in cotton which results in a high level of resistance to BBT, Blue Disease and Vermelhao diseases.

The mutant allele of the present invention allows a high level of resistance to these diseases. This high resistance level is critical for the economical production of cotton seeds in certain geographical regions.

The genetic data indicate that the disease resistance cotton gene of the present invention is controlled monogenically by a single dominant allele designated 'VRDP".

While not wishing to be bound by theory, there are at least three possibilities for the underlying cause of the disease resistance in the present invention: 1) one allele confers resistance to BBT, Blue Disease and Vermelhao; 2) one allele, interacting with other DeltaOPAL traits, confers resistance to BBT, Blue Disease and Vermelhao; 3) one to three alleles confer resistance to BBT, Blue Disease and Vermelhao; 4) or any combination of the above.

EXAMPLES

The following examples are provided to further illustrate the present invention and are not intended to limit the invention beyond the limitations set forth in the appended claims.

Example 1 Cotton cultivar DeltaOPAL has superior characteristics and viral, fungal and bacterial disease resistance which was developed from crossing DP5816 with Sicala 33. The resulting cross was selfed and the F 2 seed harvested as bulk. F 3 seed was grown and selections were made in F 4 single plants. The 20 F 4 plants were grown in a winter nursery as individual progeny rows and S. harvested. Fs bulk selections were yield and fiber tested and seed increased.

F

6 selections were then evaluated for disease resistance yield, plant maturity and fiber quality. Unexpectedly DeltaOPAL variety showed resistance to several virus diseases.

The criteria used to select in various generations include: lint yield, lint turnout, fiber characteristics, maturity, storm resistance, disease resistance, early season vigor.

DeltaOPAL and its parents, DP5816 and Sicala 33, have been screened in 1999 under field conditions for resistance to Blue Disease in Mato Grosso 16 State of Brazil. While DeltaOPAL demonstrated resistance to Blue Disease, both of its parents contained plants with typical Blue Disease symptoms.

During the mid-bloom stage, the percent of plants expressing typical Blue Disease symptoms were counted in an observation trial. The following data was recorded: Sicala 33 had 35% Blue Disease symptoms, DP5816 had 16.3% Blue Disease symptoms, and DeltaOPAL had 0% Blue Disease symptoms.

The cultivar has shown uniformity and stability to the traits, as described in the following variety description information. It has been advanced a sufficient number of generations with careful attention to uniformity of plant type. The line has been increased with continued observation for uniformity.

The following morphological and other characteristics have been measured in the cotton cultivar DeltaOPAL.

*o 17 VARIETY DESCRIPTION INFORMATION Species: Gossypium hirsutum L.

Areas of Adaptation: Australia, Africa, Europe, North and South America General: Plant Habit Intermediate Foliage Dense Stem Lodging Intermediate Fruiting Branch Normal Growth Indeterminate Leaf Color Medium green Boll Shape Length more than width Boll Breadth Broadest at middle Maturity: Days from planting to harvest: 170 days Plant: 1st Fruiting Branch (from cotyledonary node) 13. cm No. of Nodes to 1st Fruiting Branch (Excluding cotyledonary node) 6.3 Mature Plant Height (from cotyledonary node to terminal) 110.6 cm Leaf (Upper most, fully expanded leaf): Type Normal Pubescence Sparse Nectaries Present Stem Pubescence: Intermediate *r Glands: Leaf Normal Stem Normal Calyx Lobe Normal Flower: Petals Cream Pollen Cream Petal Spot Absent Seed: Seed Index (g/100, fuzzy basis) 10.6 Lint Index (g lint/100 seeds) 5.6 Boll: Lint Percent Picked 39.2 Number of Seeds per Boll 29.0 Grams Seed Cotton per Boll 4.6 Number of Locules per Boll Boll Type Open Fiber Properties: Method HVI Length 1.17 inches Uniformity 84% Strength (T1) 31.6 g/tex Elongation (El) 6.2% Micronaire 4.4 Diseases: Alternaria macrospora Moderately resistant Bacterial Blight (Race 18) Resistant Fusarium Wilt Moderately resistant Verticillium Wilt Moderately resistant 19 Blue Disease Resistant Vermelhao Resistant Nematodes, Insects and Pests: Boll Weevil Susceptible Bollworm Susceptible Cotton Aphid Susceptible Cotton Fleahopper Susceptible Cotton Leafworm Susceptible Cutworm Susceptible Fall Armyworm Susceptible Grasshopper Susceptible Lygus Susceptible Pink Bollworm Susceptible Spider Mite Susceptible Stink Bug Susceptible Thrips Susceptible Tobacco Bud Worm Susceptible This invention is also directed to methods for producing a cotton plant by crossing a first parent cotton plant with a second parent cotton plant, wherein 20 the first or second cotton plant is the cotton plant from the line DeltaOPAL.

Further, both the first and second parent cotton plants may be the cultivar DeltaOPAL self-pollination). Therefore, any methods using the cultivar DeltaOPAL are part of this invention: selfing, backcrosses, hybrid breeding, and crosses to populations. Any plants produced using cultivar DeltaOPAL as a parent are within the scope of this invention. As used herein, the term "plant" includes plant cells, plant protoplasts, plant cells of tissue culture from which cotton plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants, such as pollen, flowers, embryos, ovules, seeds, pods, leaves, stems, roots, anthers and the like.

Thus, another aspect of this invention is to provide for cells which upon growth and differentiation produce a cultivar having essentially all of the physiological and morphological characteristics of DeltaOPAL.

Culture for expressing desired structural genes and cultured cells are known in the art. Also as known in the art, cotton is transformable and regenerable such that whole plants containing and expressing desired genes under regulatory control may be obtained. General descriptions of plant expression vectors and reporter genes and transformation protocols can be found in Gruber, et al., 'Vectors for Plant Transformation, in Methods in Plant Molecular Biology Biotechnology" in Glich, et al., (Eds. pp. 89-119, CRC Press, 1993). Moreover GUS expression vectors and GUS gene cassettes are available from Clone Tech Laboratories, Inc., Palo Alto, California while luciferase expression vectors and luciferase gene cassettes are available from Pro Mega Corp. (Madison, Wisconsin). General methods of culturing plant tissues are provided for example by Maki, et al., "Procedures for Introducing Foreign DNA into Plants" in Methods in Plant Molecular Biology Biotechnology, Glich, et al., (Eds. pp. 67-88 CRC Press, 1993); and by Phillips, et al., "Cell-Tissue Culture and In-Vitro Manipulation" in Corn Corn :I Improvement, 3rd Edition; Sprague, et al., (Eds. pp. 345-387) American 20 Society of Agronomy Inc., 1988. Methods of introducing expression vectors Se

C.

into plant tissue include the direct infection or co-cultivation of plant cells with Agrobacterium tumefaciens, Horsch et al., Science, 227:1229 (1985).

Descriptions of Agrobacterium vectors systems and methods for Agrobacterium-mediated gene transfer provided by Gruber, et al., supra.

25 Useful methods include but are not limited to expression vectors introduced into plant tissues using a direct gene transfer method such as microprojectile-mediated delivery, DNA injection, electroporation and the like.

;More preferably expression vectors are introduced into plant tissues using the microprojectile media delivery with the biolistic device Agrobacteriummedicated transformation. Transformant plants obtained with the protoplasm of the invention are intended to be within the scope of this invention.

The present invention contemplates a cotton plant regenerated from a tissue culture of a variety DeltaOPAL) or hybrid plant of the present invention. As is well known in the art, tissue culture of cotton can be used for the in vitro regeneration of a cotton plant. Tissue culture of various tissues of cotton and regeneration of plants therefrom is well known and widely published.

When the term cotton plant is used in the context of the present invention, this also includes any single trait conversions of that variety. The term single trait converted plant as used herein refers to those cotton plants which are developed by a plant breeding technique called backcrossing or via genetic engineering wherein essentially all of the desired morphological and physiological characteristics of a variety are recovered in addition to the single trait transferred into the variety. Backcrossing methods can be used with the present invention to improve or introduce a characteristic into the variety. The term backcrossing as used herein refers to the repeated crossing of a hybrid progeny back to the recurrent parent. The parental cotton plant which contributes the trait for the desired characteristic is termed the nonrecurrent 20 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 cotton plant to which the trait or traits 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 variety of interest (recurrent parent) is crossed to a second variety (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 cotton 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 variety. To accomplish this, a gene or genes of the recurrent variety are modified or substituted with the desired gene(s) 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, 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.

20 Many traits have been identified that are not regularly selected for in the development of a new variety but that can be improved by backcrossing see* S...i techniques. Traits may or may not be transgenic, examples of these traits include but are not limited to, male sterility, herbicide resistance, resistance for bacterial, fungal, or viral disease, insect resistance, male fertility, enhanced fiber quality, industrial usage, yield stability and yield enhancement. These traits are generally inherited through the nucleus.

The cultivar DeltaOPAL is similar to DP5816. While similar, there are numerous differences including: DeltaOPAL is approximately 10.7 cm taller than DP5816. Additionally, DeltaOPAL has a stronger T1 strength in the fiber 23 than DP5816. Unexpectedly, DeltaOPAL has resistance to Bonzai Bunchy Top, Blue disease and Vermelhao diseases.

ooeo o oo ooo o o ••go e go o o oo o oO g o o* 24 Example 2 Studies were conducted with the new virus resistance cotton mutant of the present invention. This mutant was inherited as a single dominant gene designed "VRDP" as shown in this Example 2.

Data and analysis of NuOPAL BC 2

F

2 lines for resistance against Bonzai Bunchy Top in Australia, show that one single dominant gene was involved in this source of resistance. NuOPAL BC 2

F

2 :3 lines (third filial generation) were produced by crossing the non-transgenic variety DeltaOPAL with NuCOTN 37B, containing the Monsanto gene "Bollgard®" line 531, and then backcrossing two times to OPAL. The resulting NuOPAL BC 2

F

1 were selfed to produce BC 2

F

2 seed. These crosses were conducted in a glasshouse in Australia to prevent outcrossing. No selection for resistance to viral diseases or other non-transgenic traits were imposed in the glasshouse.

BC

2

F

2 3 plants, seed harvested off of individual BC 2 F, plants in the glasshouse, were planted in the field at Goondiwindi Australia in December, 1998. During the seedling period, these 37 lines were tested for transgene purity, and found to be 100% pure, then scored for the presence of Bonzai Bunchy Top in April.

:i In the F 1 DeltaOPAL by NuCOTN 37, none of the genes that are unique 20 to DeltaOPAL were fixed in the homozygous state. In the BCF, NuOPAL population approximately half of the unique DeltaOPAL genes were fixed in the homozygous state, and in the BC 2 F, population approximately 75% of the unique genes were fixed with the other 25% heterozygous. When the BC 2

F,

Splants were selfed the 25% heterozygous plants segregated to a 1:2:1 genotypic ratio (homozygous viral resistance, heterozygous viral resistance, and homozygous non-viral resistance). For single gene dominant resistance, the BC 2

F

2 population should have the following phenotypic frequencies, 81.25% viral resistant, 12.5% segregating and 6.25% viral susceptible. When I I these frequencies are applied to a population of 37 lines the expected number of lines in each category is 30:4.6:2.3.

The following Table 1 lists the observed frequency in each category for all 37 NuOPAL lines and the expected frequency when the viral resistance is a single dominant gene.

Table I Bonzai Bunchy Top (BBT) Rating Observed Expected Nil to low BBT symptoms 25 lines 30 lines Segregating for BBT resistance 8 lines 4.6 lines Severe BBT damage 4 lines 2.3 lines The calculated ChiSquare for this population was 4.603 indicating that the probability is approximately 0.1 of getting a deviation at least as large as we observed by chance (ChiSquare is 4.605 for df=2 and Example 3 Cotton variety trial results from Brazil where the 3 cotton diseases were present: Blue Disease, an aphid vectored purported viral disease; Vermelhao, an aphid vectored viral disease; and Red Wilt, a purported bacterial disease, are shown in Tables 2, 3 and 4. DeltaOPAL has a high level of resistance to viral diseases in Brazil as shown in Tables 2, 3, and 4. During the 1997/1998 growing season variety trials were conducted. These trials were rated for the presence of various diseases. Tables 2, 3 and 4 list the average yields and ratings for the 4 replications at each location. No differences in aphid populations between DeltaOPAL and virus susceptible varieties were observed in these trials.

Table 2 4 Replication Variety Strains Trial Located at Rosa Dos Ventos Farm, Goias State, Brazil 1997/1998 Agropem (Maeda) Variety Viral Diseases Bacterial Dis. Seed Cotton* BLUE VERM. RED Kg/ha DeltaEMERALD 2.8 2.3 1.3 3228 DeltaOPAL 1.0 1.0 1.3 3186 PM1266 1.0 1.0 2.0 3161 Redencao 2.0 2.0 2.8 3139 DP4025 1.8 2.0 2.0 2944 SG180 3.5 2.8 1.0 2760 DP4049 2.3 1.8 2.3 2704 2.3 2.0 2.3 2673 DP5111 3.5 3.0 1.3 2643 DP5557 3.5 3.0 1.0 2245 DP5409 3.8 3.5 1.0 2115 SG821 3.0 2.8 1.0 2106 4.0 3.3 1.3 1890 PM1277 3.3 3.0 1.0 1857 3.8 3.5 1.0 1784 DP5690 4.0 3.8 1.3 1693 SG248 4.0 3.3 1.0 1664 DP5305 4.0 3.5 1.3 1449 4.0 3.5 1.3 958 MEAN 3.0 2.7 1.5 2367 r SSorted by Seed Cotton Refers to Ramulose Resistant Ratings numbers are 1=immune 5=highly susceptible BLUE Blue Disease, an aphid vectored purported viral disease RED Red Wilt, a purported bacterial disease VERM Vermelhao, a viral disease Table 3 4 Replication Variety Strains Trial Located at Canada Farm, Goias State, Brazil 1997/1998 Agropem (Maeda) Variety Viral Diseases Bacterial Dis. Seed Cotton* BLUE VERM. RED Kg/ha DeltaOPAL 1.0 1.0 2.5 3438 SG180 3.5 1.3 1.0 2388 DP4025 1.5 1.0 3.3 2375 PM1266 1.3 1.0 2.8 2288 DP4049 2.5 1.3 2.5 2275 DeltaEMERALD 3.0 1.8 1.8 2238 SG821 2.8 1.5 1.0 2013 DeltaPEARL 3.0 1.8 1.5 1913 IAC 20 RR** 2.0 1.5 3.3 1838 DP5557 4.0 1.5 1.0 1800 DP5111 2.8 1.8 1.3 1738 DP5690 3.7 1.5 1.3 1675 Redencao 1.5 1.8 3.3 1613 DP90 3.8 1.5 1.3 1575 DP20 2.8 1.8 1.3 1550 DP5305 3.0 2.0 1.0 1450 SG248 3.0 2.0 1.0 1388 DP5409 3.3 1.5 1.3 1300 PM1277 2.5 1.3 1.3 1150 3.0 1.3 1.3 1075 MEAN 2.7 1.5 .1.8 1854 Sorted by Seed Cotton Refers to Ramulose Resistant Ratings numbers are 1=immune 5=highly susceptible BLUE Blue Disease, an aphid vectored purported viral disease RED Red Wilt, a purported bacterial disease VERM Vermelhao, a viral disease Table 4 4 Replication Variety Strains Trial Located at Rondonopolis Farm, Mato Grosso, Brazil 1997/1998 Agropem (Maeda) Variety Viral Diseases Bacterial Dis. Seed Cotton* BLUE VERM. RED Kglha DP5111 1.8 2.8 1.0 5350 4.3 2.5 1.0 5150 SG248 3.3 2.0 1.3 5113 DeltaEMERALD 2.5 2.0 2.0 5075 3.5 2.3 1.3 4875 DP5690 3.5 2.3 1.3 4825 SG180 2.3 2.0 1.0 4775 DeltaPEARL 2.8 2.3 1.8 4575 PM1266 1.0 1.5 2.8 4538 1.8 1.5 2.8 4513 DeltaOPAL 1.0 1.0 2.3 4350 Redencao 1.0 1.0 4.0 4325 DP5409 3.3 2.3 1.3 4238 DP4025 1.3 1.3 2.5 4213 DP5557 3.0 2.5 1.0 4138 DP5305 2.8 2.3 1.0 4088 DP4049 1.5 1.3 2.8 4025 SG821 3.0 2.3 1.0 3938 PM1277 2.5 2.3 1.3 3825 3.0 2.0 1.0 3413 MEAN 2.5 2.0 1.7 4467 Sorted by Seed Cotton Ratings numbers are 1=immune 5=highly susceptible BLUE Blue Disease, an aphid vectored purported viral disease RED Red Wilt, a purported bacterial disease VERM Vermelhao, a viral disease r r r Example 4 The recently named purported viral disease, Bonzai Bunchy Top (BBT), appeared in many cotton variety comparison sites throughout Australia during the 1998-1999 growing season. This facilitated the quantification of resistance to BBT within several varieties. Two parameters were chosen to quantify the varietal response to BBT percent of plants with BBT symptoms and (b) percent small bolls. Plants deemed infected typically had 6 to 8 compressed internodes with an average internode length of 1.5 cm or less and small leaves. All data was collected from a minimum distance of 5 meters of row consisting of 7 to 11 plants per meter-row. A one-way analysis of variance and Student's t-test was used to statistically analyze the 27 separate observations.

The following Table 5 shows varietal differences in BBT resistance.

Table Bunchy Large bolls Small Small LocationlGrower Variety Top plants (harvestable) bolls bolls Field Data (data averages of at least 5 one meter row sections at full stand) McVeigh Sicot 189 13 68 18 21 Pearl 6 58 28 33 Topaz 21 46 43 48 Emerald 45 60 52 46 Sapphire 9 43 47 52 OPAL 0 71 4 Bailey Sicot 189 28 73 25 34 Pearl 3 90 13 14 Topaz 8 76 25 32 Emerald 17 66 16 Sapphire 22 70 22 31 OPAL 0 78 4 r Table 5 Bunchy Large boils Ismall 1% Small Location/Grower Variety Top plants j(harvestable) Iboils Ibolls Field Data (data averages of at least 5 one meter row sections at full stand) Kent Sicot 189 26 109 36 33 OPAL 0 123 9 7 Bartley Sicot 189 33 64 35 36 Pearl 9 55 24 31 58 20 26 Emerald 53 54 53 49 63 53 31 37 OPAL 0 72 8 9 S. Seis Sicot 189 31 58 27 Pearl 30 145 36 Sicot 189 62 127 66 34 OPAL 4 135 8 Crothers Pearl 100 61 35 36 Emerald 100 32 19 36 OPAL 0 74 25 AVERAGES Sicot 189 32 83 35 31 Pearl 30 82 27 27 15 45 22 27 Emerald 54 45 30 24 42 25 Avg (excluding 31 59 28 29 OPAL averaged IOPAL 1 92 10 9 S S

S

31 The occurrence of BBT symptoms in DeltaOPAL was found to be 0.67%, and the mean occurrence of symptoms in the other 5 varieties was 33.81%.

The probability of this occurring by chance alone is 0.0085% which is highly significant. The percent of small bolls in DeltaOPAL was 9.33% while the percent of small bolls in the other 5 varieties was 26.60% at the lowest and 40.00% at the highest. The probability of this occurring by chance alone is 0.0002%, also highly significant. Since plant ratings of BBT symptoms is a subjective parameter, some of the DeltaOPAL plants were scored as susceptible (less than In addition, not all small bolls are due to BBT infection, thus DeltaOPAL had less than 10% small bolls. However, under BBT infested field conditions in Australia the occurrence of BBT symptoms and percent small bolls is significantly less than other commercial varieties at the 0.01 probability level, demonstrating the high level of resistance in DeltaOPAL to BBT.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

V go 1 11 32 DEPOSIT INFORMATION A deposit of the cotton seed of this invention has been placed on deposit with the American Type Culture Collection, Manassas, Virginia. The date of deposit is16 June 1999 1999 and has ATCC Accession No. PTA- 16 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.

i o o o

Claims (27)

1. A cotton seed containing an allelic DNA genetic factor for disease resistance designed VRDP.

2. The cotton seed of claim 1, wherein said seed contains a dominant allele for disease resistance.

3. A cotton plant, or its parts, produced by growing the seed of claim 1.

4. Pollen of the plant of claim 3. An ovule of the plant of claim 3.

6. A tissue culture of regenerable cells of the plant, or its parts, of claim 3.

7. A cotton plant regenerated from said tissue culture of claim 6.

8. A method for producing a cotton seed comprising crossing a first parent cotton plant with a second parent cotton plant and harvesting the resultant hybrid cotton seed, wherein said first or second parent cotton plant is the cotton plant of claim 3.

9. A hybrid cotton seed produced by the method of claim 8. A hybrid cotton plant, or its parts, produced by growing said hybrid cotton seed of claim 9. S: 20 11. Viable cotton seeds and plants and succeeding generations thereof grown from seeds deposited under ATCC Accession No. PTA-164 on 1 6 /6/99and cotton seeds and plants to which the •disease resistance allele is transferred from said deposited seeds in succeeding generations thereof. 25 12. A cotton seed designated DeltaOPAL, wherein a sample of said seed has been deposited under ATCC Accession No. PTA-164

13. A plant, or its parts, produced by growing the seed of claim 12.

14. Pollen of the plant of claim 13. I 34 An ovule of the plant of claim 13.

16. A cotton plant having essentially all of the physiological and morphological characteristics of the cotton plant of claim 13, or its parts.

17. Tissue culture of the seed of claim 12.

18. A cotton plant regenerated from the tissue culture of claim 17.

19. Tissue culture of regenerable cells of the plant, or its parts, of claim 13. The tissue culture of claim 19 wherein the regenerable cells are embryos, meristematic cells, pollen, leaves, anthers, roots, root tips, flowers, seeds, stems, or protoplasts or calli derived therefrom.

21. A cotton plant regenerated from the tissue culture of claim

22. A method for producing a cotton seed comprising crossing a first parent cotton plant with a second parent cotton plant and harvesting the resultant hybrid cotton seed, wherein said first or second parent cotton plant is the cotton plant of claim 13.

23. A hybrid cotton seed produced by the method of claim 22.

24. A hybrid cotton plant, or its parts, produced by growing said hybrid cotton seed of claim 23. *i

25. Cotton seed produced from said hybrid cotton plant of claim 24.

26. The cotton plant, or its parts, produced from the cotton seed of claim

27. The cotton plant of claim 16, further comprising a single trait conversion.

28. The single trait conversion of the cotton plant of claim 27 where the trait is a transgenic trait.

29. The single trait conversion of the cotton plant of claim 27, where the trait is determined by a dominant allele. I The single trait conversion of the cotton plant of claim 27, where the trait is determined by a recessive allele.

31. The single trait conversion cotton plant of claim 27, where the trait confers herbicide resistance.

32. The single trait conversion of the cotton plant of claim 27, where the trait confers insect resistance.

33. The single trait conversion of the cotton plant of claim 27, where the trait confers resistance to bacterial, fungal or viral disease.

34. The single trait conversion of the cotton plant of claim 27, where the trait confers male sterility. The single trait conversion of the cotton plant of claim 27, where the trait confers improved fiber quality. Dated this 3rd day of August 1999 D&PL TECHNOLOGY HOLDING CORP. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia *S