WO1999004619A1 - Novel brassica variety - Google Patents

Abstract

The present invention provides methods of producing new Brassica cultivars that retain a high degree of both inherent and acclimation-specific freezing tolerance, but lack a vernalization requirement. The present invention further provides novel Brassica cultivars, including VERN-, a cultivar that lacks a vernalization requirement and is tolerant to freezing.

Description

NOVEL BRASSICA VARIETY

FIELD OF THE INVENTION

The present invention is related to the production and cultivation of novel Brassica cultivars.

BACKGROUND OF THE INVENTION

Brassica

The Brassicaceae (or Cruciferae) is a family of approximately 350 genera and 3,200 species of pungent or acrid plants commonly referred to as the "mustard family." Included within this family are many edible species, including cabbage, broccoli, cauliflower, kale, kohlrabi, turnips, radish, and rutabaga. The edible genera include

Brassica (rape, cabbage, and mustard), Lepidium (cress), Nasturtium (watercress), Raphanus (radish), Romarmoracia (horseradish), and Wasabia (Japanese horseradish or wasabe). The ornamental genera include Aethionema (stonecress), Alyssoides (bladderpod), Alyssum (madwort), Arabis (rock cress), Aubrieta, Aurinia, Brassica (ornamental kale), Cardamine, Cheiranthes (wallflower), Crambe (colewort),

Diplotaxis (rocket), Draba, Erysimum (wallflower or blister cress), Hesperis (rocket), Iberis (candytuft), Lobularia, Lunaria (honesty), and Matthiola (stock).

Brassica sp. include plants commonly referred to as "rape" "rapeseed," or "canola." These plants are annual or biennial, and resemble mustard in appearance. Rape is primarily grown as an oil source, as well as for stock feed and green manure.

Brassica napus and Brassica rapa, as well as other species, are important worldwide as oilseed crops, with the oil found in mature seeds known as canola oil. Canola must meet standards based on seed, meal and oil characteristics. For example, the seed must be of the genus Brassica and contain <1.0% of all fatty acids as erucic acid (22:1) and <18 μmoles of glucosinolates per gram of whole seed at a moisture content of 8.5%; and the meal must contain no more than 30 μmoles of glucosinolates per gram of oil free meal, at a moisture content of 8.5% (Canadian Canola Council 1997; and Canola Growers Manual, 1991). Thus, there is great interest in producing Brassica plants that produce large quantities of high quality canola oil.

The rape Brassica can be grouped into one of two different growth habits, referred to as annual spring (or summer) cultivars and biennial winter cultivars. The winter types are planted in the fall, overwinter as seedlings, and then complete their development the following spring. The winter type cultivars require vernalization at low temperatures to induce flowering and seed development. Concurrent with vernalization in winter cultivars is the acquisition of a degree of freezing tolerance. The spring types do not have a vernalization requirement. The vernalization requirement reduces the chance of premature flower induction that may occur following brief periods of warm weather during the winter. While this is advantageous to producers, it is detrimental to breeders, who must add six weeks to every generation time for winter cultivars. However, compared to spring types, winter types have numerous attributes such as superior lodging resistance, increased yield, better disease resistance, and higher inherent and acclimation-specific freezing tolerance.

Low Temperature Effects and Freezing Tolerance

As biological stress includes any change in environmental conditions that might reduce or adversely affect growth or development, exposure of plants to low temperatures places stress on plants. This stress triggers a series of responses apparent in freezing tolerant species, including low temperature stress, low temperature adjustment, the acquisition of acclimation-specific freezing tolerance (acclimation), and vernalization. Adjustment to low temperature stress and acclimation occur simultaneously, making them difficult to differentiate. A number of mechanisms have evolved that enable plants to cope with a variety of adverse environmental conditions. In order to survive exposure to freezing temperatures, plants must possess high levels of both inherent and acclimation- specific tolerance. Inherent tolerance is genetically determined and allows plants to cope with short periods of high, sub-zero temperatures. Acclimation, developed in response to prolonged exposure to low temperatures, increases the degree of freezing tolerance in plants capable of acclimating. This process involves complex physiological, molecular, and biochemical processes (See e.g., Singh and Laroche, Biochem. Cell. Biol., 66:650-657 [1988]). Low temperature acclimation has been shown to cause alterations at all gene expression levels (See, Thomashow, Adv. Genet., 28:99-130 [1990]). The genes encoding a number of low temperature-induced proteins have been isolated from several plants including B. napus (Orr et al., Plant Physiol., 98:1532-1534 [1992]; and Weretilynk et al., Plant Physiol., 101:171-177 [1993]), Arabidopsis thaliana (Kurkela and Frank, Plant Mol. Biol., 15:137-144 [1990]; Gilmour et al, Plant Mol. Biol.,

18:13-21 [1992]; Lin and Thomashow Plant Physiol., 99:519-525 [1992]; and Palva, Gene expression under low temperature stress, in Basra (ed.), Stress-Induced Gene Expression in Plants, Harwood Academic, New York, pp. 103-130 [1994]), Spinacia oleracea (Guy, Ann. Rev. Plant Physiol., Plant Mol. Biol., 47:187-223 [1990]), Hordeum vulgare (Cativelli and Bartels, Plant Physiol., 93:1504-1510 [1990]), and

Medicago sativa (Monroy et al, Plant Physiol., 102:873-879 [1993]). Gene subsets or families appear to be regulated at the level of RNA transcription or protein translation during exposure to low temperature (Guy, supra; Johnson-Flanagan et al, Plant Physiol., 81:301-308 [1991]; and Hawkins et al, Genome 39:704-710 [1996]). However, no function has been assigned to any of the low temperature-induced genes described.

Selection for freezing tolerant lines or cultivars has been difficult because of the numerous responses associated with exposure to low temperature, including low temperature adjustment and vernalization. Further, many breeders working to improve freezing tolerance have relied upon field survival as the major selection criterion

(Marshall, in Christiansen and Lewis (eds)., Breeding For Tolerance to Heat and Cold: Breeding Plant for Favorable Environments, John Wiley and Sons, New York, pp. 47-69 [1982]). This is difficult to assess, as there are many other factors that control field survival, including ice encasement and the consequent anoxia (Andrews and Morrison, Agronomy J., 84:960-962 [1992]). Selection is further complicated by the fact that the acclimation process is cumulative and can be stopped, reversed or restarted (Gusta et al, Can. J. Bot., 60:301-305 [1982]; and Roberts, Can. J. Bot., 57:413-419 [1979]). The fact that plant material must be acclimated for up to six weeks for maximum attainment of freezing tolerance has also complicated the breeding and selection process for hardier canola cultivars.

Furthermore, data collected to date indicate that freezing tolerance is complex and follows a polygenic pattern (See, Stushnoff et al, Breeding and selection of resistance to low temperature, in Voss (ed.), Plant Breeding— A Contemporary Basis, pp. 115-136 [1984]). For example, Sutka (Sutka, Theor. Appl. Genet, 59:145-152

[1981]) showed that 15 of 21 chromosomes can be implicated in freezing tolerance in Triticum aestivum (winter wheat). In B. napus, there are a minimum of four linkage groups that appear to segregate with freezing tolerance (See, Teutonico et al, Crop Sci., 33:103-107 [1995]). It is difficult to ascertain the highest level of inherent and acclimation-specific freezing tolerance achieved to date in winter canola, as many breeding programs do not assess freezing tolerance. Nonetheless, it is possible that the highest tolerances are in the range of -5°C and -15°C, based on the fact that most tolerant canola quality rape was produced in the Pacific Northwest, a region that can be very cold. In the early years of the development of tolerant canola, Cascade was used as a foundation stock, as it was the most freezing tolerant material available at the time. Since then, freezing tolerance in canola has not been significantly improved. This appears to be the result of a lack of suitable germplasm, an inability to rapidly and accurately assess tolerance, and the complexity of plant responses to low temperature. Thus, due in part to the increased demand for quality canola oil, there remains a need in the art to increase the freezing tolerance of winter canola cultivars, including an increased need for an understanding of the genetic control of agronomic traits (Ferreira et al , Theor. Appl. Genet., 89:615-621 [1994). Development of cultivars better suited to the often harsh conditions in the temperate growing regions (e.g., drought, heat, disease and in particular, low temperature), has become an important consideration for canola breeders.

SUMMARY OF THE INVENTION

The present invention is related to novel Brassica cultivars, and methods for developing novel Brassica plants.

The present invention provides methods for producing improved cultivars. In preferred embodiments, the improved cultivars are members of the genus Brassica. In particularly preferred embodiments, the cultivars are canola. In one embodiment, the present invention provides a method for producing an improved Brassica cultivar, comprising the steps of: providing a first parent Brassica line and a second parent

Brassica line; crossing the first and second parent Brassica lines to produce a reciprocal cross; culturing the reciprocal cross to produce a microspore culture; and treating the microspore culture to produce a doubled haploid cultivar.

In another embodiment, the method further comprises the step of vernalizing the reciprocal cross. In yet another embodiment, the method further comprises the step of vernalizing the doubled haploid. Thus, it is contemplated that either or both the reciprocal cross and doubled haploid undergo vernalization.

In an alternative embodiment, the first and second parent Brassica lines are winter type. In one preferred embodiment, the first parent Brassica line is selected from the group consisting of Cascade and Rebel. In yet another alternate embodiment, the present invention further comprises the step of growing the doubled haploid cultivar to produce seeds. In another embodiment, the present invention provides the step of harvesting the seeds.

The present invention also provides the doubled haploid cultivars. In preferred embodiments, the doubled haploid cultivar is vernalization minus and/or freezing tolerant.

The present invention further provides a method for producing Brassica cultivars, comprising the steps of crossing first and second Brassica plants, where at least one of the Brassica plants is characterized by at least one genetic factor which confers freezing tolerance, the genetic factor being capable of transmission to the Brassica cultivar substantially as a recessive gene.

In addition, the present invention provides the cultivars produced according to the method. In one preferred embodiment, the genetic factor confers vernalization independence (i.e., the plant does not require vernalization). In an alternative preferred embodiment of the method, the Brassica plant is selected from the group consisting of Cascade and Rebel. In a most preferred embodiment, the Brassica plant is selected from the group consisting of Rebel, VERN-, and any line that does not require vernalization (e.g., F₂ progeny containing the approximately 2.8 kb band, but not the approximately 3.0 kb band described in Example 9, and shown in Figures 6A-C). The present invention also provides a winter Brassica cultivar expressing characteristics of spring Brassica cultivars. In preferred embodiments, the cultivar is vernalization independent, and/or freezing tolerant. In a particularly preferred embodiment, the cultivar is VERN- or Rebel.

The present invention also provides methods and models to elucidate the genetic components of Brassica associated with various traits, including, but not limited to freezing tolerance and vernalization. It is contemplated that the present invention will find use in establishing the genetic elements associated with these and other economically important traits and characteristics. In particular, it is contemplated that the vernalization independent cultivars (i.e., spring-like characteristics) that retain the desirable qualities of winter types will be used to develop additional cultivars. The present invention will also find use in the development of improved cultivars of other members of the Brassicaceae. It is further contemplated that the cultivars of the present invention will find widespread use in agricultural areas in which freezing tolerance and/or vernalization are considerations in making choices as to the type of canola to cultivate. DESCRIPTION OF THE FIGURES

Figures 1A-C are graphs of freezing tolerance for three DH, lines, including VERN-. Figures ID and IE are graphs showing the inherent and acclimation-specific freezing tolerance of Cascade, Rebel, F„ and the DH, line, VERN-. Figure 2 shows a graph of an LOD plot of flowering time.

Figure 3 shows the segregation of acclimation-specific freezing tolerance.

Figures 4A and 4B provide representative blots probed with the cDNA clone BN28 from B. napus.

Figure 5 is a representative blot probed with genomic clones A) EC2E5, and B) WG1F6.

Figure 6 is a table showing the recombination analysis of DH, lines.

Figure 7A is a blot showing RFLP complementation analysis of parents and F,. Figures 7B and 7C are a drawing and blot (respectively), showing the complementation analysis of the parent lines, VERN-, an F, plant, and multiple F₂s. Figure 8 is a blot showing RFLP analysis of BC,F₂ segregants.

Figure 9A shows the RFLP results obtained with the EcoRI-digested samples, and Figure 9B shows the results obtained with the PvwII-digested samples.

Figure 10 shows a Northern blot demonstrating expression of BN28.

Figure 11 shows an immunoblot analysis of BN28 protein accumulation. Figure 12 provides a graph showing the marker loci of Cascade, Rebel, and

VERN-.

GENERAL DESCRIPTION OF THE INVENTION

The present invention provides methods of producing new Brassica cultivars that retain a high degree of both inherent and acclimation-specific freezing tolerance, but lack a vernalization requirement.

The present invention provides several distinct advantages over currently used winter cultivars, as it provides plant lines with acclimation-specific freezing tolerance in excess of -18°C, representing an improvement of at least 3 °C over previously reported tolerance levels. However, the phenotypic differences associated with inherent and acclimation-specific freezing tolerance in Brassica napus represent but one of the improvements provided by the lines of the present invention.

As the improved lines are derived from canola quality material, the present invention also provides methods to increase freezing tolerance in winter canola in demand for high quality oil production. The methods of the present invention are also useful for improving the freezing tolerance of spring canola. Spring-type canola cultivars often suffer from late spring frosts, while the plants are at the seedling stage, and then in the fall during maturation. For example, the inherent tolerance of -8.5°C observed during the development of the present invention could be transferred to spring canola, representing significant progress in developing tolerance to these frosts.

Definitions

Prior to providing a further description of the invention, and to facilitate understanding the invention, a number of terms are defined below.

As used herein, the term "Brassica" refers to any member of the genus Brassica. The term "Brassicaceae" (or "Cruciferae") refers to the family of plants commonly referred to as the "mustard family."

As used herein, the term "winter type" refers to plants which typically have a vernalization requirement. These plants are "vernalization dependent."

As used herein, the term "spring type" (or "summer type") refers to plants that do not have a vernalization requirement. These plants are "vernalization independent."

As used herein, the term "cultivar" refers to any cultivated variety produced by horticultural techniques. The term encompasses any horticultural variety, strain, or race that has originated and persists under cultivation.

As used herein, the term "strain" refers to an intraspecific group of organisms that possess only one or a few distinctive traits, are usually genetically homozygous

(i.e., pure breeding) for those traits, and are often maintained as an artificial breeding group for domestication (e.g., agricultural applications), or for genetic experimentation. The term also encompasses "variety," although this term is applied when the differences between the intraspecific groups is substantial.

As used herein, the term "inflorescence" refers to the flowering part of a plant, the arrangement of flowers on a plant, as well as the process of coming into bloom (i.e., blossoming).

As used herein, the term "anthesis" refers to the period during which a flower opens (or the act of a flower opening), or coming into full bloom.

As used herein, the term "stigma" refers to the receptive surface usually located at the apex of the style of a flower, on which compatible pollen grains germinate. The term "pollen grain" refers to microspores in flowering plants that germinate to form male gametophytes (pollen grain and pollen tube).

As used herein, the term "graft" refers to the union of a piece of one plant to another, established plant.

As used herein, the term "meristem" refers to regions where cells are actively dividing.

As used herein, the term "bolt" refers to the rapid growth of a stem prior to flowering.

As used herein, the term "annual" refers to plants that complete their life cycle within a single growing season. The term encompasses plants that grow from seed, bloom, fruit, and die in the course of the same year. However, it is also intended to encompass plants that may be carried over two or more years by preventing them from setting seed.

As used herein, the term "biennial" refers to plants that require two seasons of growth. As used herein, the term "vernalization" refers to plants that require exposure to low temperature in order to complete their life cycle.

As used herein, the term "freezing tolerance" refers to the ability of a plant to withstand the effects of subzero temperatures (i.e., temperatures below freezing [0°C]).

As used herein, the term "double haploid" refers to plants produced by manipulation of the developing pollen grains to induce development of a sporophyte plant (i.e., a haploid plant), that is further treated so that its single haploid set of chromosomes is doubled. These plants are 100% homozygous. In the case where the "parent" plant used as a source of developing pollen is heterozygous, each doubled haploid plant produced is genetically different, since these plants represent the haploid products of different meioses where recombination of alleles of different genes may occur. Inheritance analysis of the alleles of genes using doubled haploid plants is equivalent to the direct analysis of the gametes produced by a heterozygote.

As used herein, the term "haploid" refers to a plant having one set of unpaired chromosomes. As used herein, term "diploid" refers to plants having two sets of chromosomes.

As used herein, the term "allopolyploidy" refers to hybrids arising from the combination of chromosomes from two different species or strains.

As used herein, the term "aneuploidy" refers to the condition in which the chromosome numbers are not exact multiples of the haploid set (i.e., there are missing or extra chromosomes present within the nucleus).

As used herein, the term "hybrid" refers to the offspring of two plants of the same, different, or closely related species that differ in one or more genes. The term "heterosis" refers to hybrid vigor" (i.e., increased vigor, size, and/or fertility of a hybrid, as compared with its parents). "Hybridogenesis" refers to a form of clonal reproduction in species hybrids, whose gametes carry only the nuclear genome derived from one of the parental species. A "hybrid swarm" is a continuous series of morphologically distinct hybrids resulting from hybridization of two species followed by crossing and backcrossing of subsequent generations. "Hybrid sterility" refers to the failure of hybrids between different species to produce viable offspring. "Hybrid breakdown" refers to the reduction in fitness of F₂ and/or backcross populations from fertile hybrids produced by intercrossing genetically disparate populations or species. The term is also used in reference to a postzygotic reproductive isolating mechanism. When used in relationship to the creation of hybrids, the term "hybridization" refers to the mating of individuals belonging to genetically disparate populations or to different species, or in Mendelian terms, hybridization is the mating of any two unlike genotypes or phenotypes.

As used herein, the term "reciprocal cross" refers to the crossing of two plants of different varieties or cultivars. The term encompasses crosses of the forms A (female) x B (male) and B (female) x A male), where the "A" and "B" individuals differ in genotype and/or phenotype. For example, the term encompasses the crossing of Cascade and Rebel, as described in Example 2. "Reciprocal genes" refer to complementary genes, while "reciprocal hybrids," are hybrid offspring derived from reciprocal crosses of parents from different species. "Reciprocal recombination" refers to the production of new linkage arrangements that are different from those of the maternal and paternal homologues, that occur in the gametes of dihybrids.

As used herein, the term "self refers to an individual plant produced by self- fertilization, as opposed to cross-breeding.

As used herein, the term "cross-fertilization" or "cross-breeding" refers to the fertilization of the ovules of one flower by the pollen of another, between individuals of the same or different species, resulting in the production of hybrid plants.

As used herein, "F," refers to offspring resulting from the first experimental crossing of plants (i.e., the first filial generation). The parental generation with which the genetic experiment starts is referred to as "P," or "P." As used herein, the term "backcross" refers to a cross between an offspring and one of its parents or an individual genetically identical to one of its parents. A "backcross parent" is that parent of a hybrid with which it is again crossed, or with which is is repeatedly crossed. A backcross may involve individuals identical to the parent, rather than the parent itself. "B,," "B₂," "B₃," etc., refer to the first, second, third, etc. backcross generations. The first backcross is created by mating an individual with one of its parents or with an individual of that identical genotype. The offspring resulting from this cross are the "B, generation." The second backcross is created by crossing B, individuals again with individuals of a genotype identical to the parent referred to in the first backcross, etc. As used herein, the term "back mutation" refers to reverse mutations. As used herein, the term "polymorphism" refers to the occurrence within and among populations of several phenotypic forms associated with the alleles of one gene or homologues of one chromosome. The term is also used in reference to the large number of variants seen in different individuals. As used herein, the terms "pleotropism" and "pleotropy" refer to the production of multiple phenotypic effects by a single gene. The term encompasses multiple phenotypic effects that are distinct and seemingly unrelated. "Pleomorphism" refers to the occurrence of variable phenotypes in a genetically uniform group of organisms.

As used herein, the term "homologous chromosomes" refers to plants that have matching chromosome pairs. The term encompasses paired chromosomes, one of paternal and one of maternal origin. Homologous chromosomes are morphologically alike and contain loci for the same genes. It also refers to chromosomes that pair during the process of meiosis. Each homologue is a duplicate of one of the maternally or paternally contributed chromosomes. Homologous chromosomes contain the same sequence of genes. Therefore, each gene is present in duplicate.

As used herein, the term "homozygous" refers to plants that have identical genes on homologous chromosomes. The term is also used in reference to the situation in which, at a particular locus, the alleles on a chromosome pair are identical.

As used herein, the term "heterozygous" refers to plants that have both dominant and recessive genes for a particular characteristic on homologous chromosomes. The term is also used in reference to the situation in which, at a particular locus, the alleles on a chromosome pair are different.

As used herein, the term "heredity," refers to the transmission of traits to successive generations. As used herein, the term "alternation of generations" refers to the reproductive cycles in which a haploid phase and a diploid phase alternate, during the course of a plant's life cycle. In mosses and vascular plants, the haploid phase is the gametophyte, while the diploid phase is the sporophyte. As used herein, the term "independent segregation," refers to the independent, random inheritance of genes on different chromosomes. It is also sometimes referred to as "independent assortment."

As used herein, the term "karyotype" refers to the chromosomal constituents of a cell or individual. The term is often used in reference to the arrangement of chromosomes during a particular stage in meiosis, in which the chromosomes may be observed, counted, and potential heredity problems identified.

As used herein, the term "meiosis" refers to the process during which one round of chromosomal replication (duplication) is followed by two rounds of division, to produce four haploid cells. Nuclear division of sex cells results in the formation of cells with half the normal amount of genetic information. Thus, each of these "haploid" cells contain one of each pair of the individual's chromosomes. The term "mitosis" refers to the process of cell division in which two cells ("daughter cells") are produced from one cell ("parent" or "mother" cell) in which each of the daughter cells contains the same genetic complement as the parent cell.

As used herein, the term "phenotype" refers to the observable characteristics of an individual, which results from the expression of the individual's genotype. The term "genotype" refers to the genetic makeup of the individual. Often, it is used in reference to the alleles of particular gene or limited number of genes under investigation.

As used herein, the term "clones" refers to genetically identical organisms produced vegetatively from a single parent.

As used herein, the term "spore" refers to a reproductive cell that grows directly into a new plant. The term "microspore" refers to a spore that develops into a male gametophyte. The term "gametophyte" refers to the haploid phase, in which mitosis results in the production of gametes in plants undergoing an alternation of generation. The term "sporophyte" refers to a diploid, spore-producing plant in the alternation of generations.

As used herein, the term "germination" refers to the beginning of growth of a seed, spore, or pollen grain. As used herein, the term "stably maintained" refers to organisms that maintain at least one of their unique properties or elements (i.e., the element that is desired) through multiple generations. For example, it is intended that the term encompass recombinant winter Brassica plants that have lost the vernalization requirement typical of winter canola. It is not intended that the term be limited to any particular species, cultivar or strain.

The term "gene" refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or precursor. The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired enzymatic activity is retained.

The term "allele" refers to alternate forms of a gene that can exist at a particular locus.

The term "gene frequency" refers to the percentage of a given type of allele in a population. The term "gene pool" refers to the total complement of all genes present in members of the population. The term "genetic equilibrium" refers to the situation in which allele frequencies are maintained at the same rate in successive generations (i.e., the allele frequencies are neither increasing nor decreasing in successive generations within the population).

As used herein, the term "genome" refers to the array of genes carried by an individual organism. In plants, the genome is comprised of multiple chromosomes.

As used herein, the term "intron," refers to non-coding segments of DNA located between coding regions in a gene which is transcribed, but does not appear in the mRNA, nor final gene product. The term "exon" refers to coding regions of DNA.

As used herein, the term "inverted repeat refers to two copies of the same DNA sequence which are in oriented in opposite directions on the same molecule.

As used herein, the term "linkage" refers to the situation in which two or more non-allelic genes tend to be inherited together. Linked genes are are located on the same chromosome, although they can be separated by crossing over. The term "linkage disequilibrium" refers to the finding that some gene pairs are found together more frequently than would be expected by chance. Thus, they are present more often that the product of their individual gene frequencies. The term "linkage group" refers to a group of genes with loci on the same chromosome. The term "locus" refers to the site on a chromosome at which a certain gene is located (plural: loci).

The term "wild-type" refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene. In contrast, the term "modified" or "mutant" refers to a gene or gene product which displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product. The term "oligonucleotide" as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof.

Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends.

When two different, non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence, and the 3' end of one oligonucleotide points towards the 5' end of the other, the former may be called the "upstream" oligonucleotide and the latter the "downstream" oligonucleotide.

The term "primer" refers to an oligonucleotide which is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated. An oligonucleotide "primer" may occur naturally, as in a purified restriction digest or may be produced synthetically.

A primer is selected to be "substantially" complementary to a strand of specific sequence of the template. A primer must be sufficiently complementary to hybridize with a template strand for primer elongation to occur. A primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being substantially complementary to the strand. Non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize and thereby form a template primer complex for synthesis of the extension product of the primer.

"Hybridization" methods involve the annealing of a complementary sequence to the target nucleic acid (the sequence to be detected). The ability of two polymers of nucleic acid containing complementary sequences to find each other and anneal through base pairing interaction is a well-recognized phenomenon. The initial observations of the "hybridization" process by Marmur and Lane, Proc. Natl. Acad. Sci. USA 46:453 (1960) and Doty et al, Proc. Natl. Acad. Sci. USA 46:461 (1960) have been followed by the refinement of this process into an essential tool of modern biology. Nonetheless, a number of problems have prevented the wide scale use of hybridization as a tool in diagnostics. Among the more formidable problems are: 1) the inefficiency of hybridization; 2) the low concentration of specific target sequences in a mixture of genomic DNA; and 3) the hybridization of only partially complementary probes and targets.

With regard to efficiency, it is experimentally observed that only a fraction of the possible number of probe-target complexes are formed in a hybridization reaction. This is particularly true with short oligonucleotide probes (e.g., less than 100 bases in length). There are three fundamental causes: a) hybridization cannot occur because of secondary and tertiary structure interactions; b) strands of DNA containing the target sequence have rehybridized (reannealed) to their complementary strand; and c) some target molecules are prevented from hybridization when they are used in hybridization formats that immobilize the target nucleic acids to a solid surface.

Even where the sequence of a probe is completely complementary to the sequence of the target (i.e., the target's primary structure), the target sequence must be made accessible to the probe via rearrangements of higher-order structure. These higher-order structural rearrangements may concern either the secondary structure or tertiary structure of the molecule. Secondary structure is determined by intramolecular bonding. In the case of DNA or RNA targets this consists of hybridization within a single, continuous strand of bases (as opposed to hybridization between two different strands). Depending on the extent and position of intramolecular bonding, the probe can be displaced from the target sequence preventing hybridization.

Solution hybridization of oligonucleotide probes to denatured double-stranded DNA is further complicated by the fact that the longer complementary target strands can renature or reanneal. Again, hybridized probe is displaced by this process. This results in a low yield of hybridization (low "coverage") relative to the starting concentrations of probe and target.

With regard to low target sequence concentration, the DNA fragment containing the target sequence is usually in relatively low abundance in genomic DNA. This presents great technical difficulties; most conventional methods that use oligonucleotide probes lack the sensitivity necessary to detect hybridization at such low levels.

One attempt at a solution to the target sequence concentration problem is the amplification of the detection signal. Most often this entails placing one or more labels on an oligonucleotide probe. In the case of non-radioactive labels, even the highest affinity reagents have been found to be unsuitable for the detection of single copy genes in genomic DNA with oligonucleotide probes. (See Wallace et al, Biochimie 67:755 [1985]). In the case of radioactive oligonucleotide probes, only extremely high specific activities are found to show satisfactory results. (See Studencki and Wallace, DNA 3:1 [1984] and Studencki et al, Human Genetics 37:42 [1985]). With regard to complementarity, it is important for some diagnostic applications to determine whether the hybridization represents complete or partial complementarity. For example, where it is desired to detect simply the presence or absence of DNA encoding a particular protein, it is only important that the hybridization method ensures hybridization when the relevant sequence is present; conditions can be selected where both partially complementary probes and completely complementary probes will hybridize. Other diagnostic applications, however, may require that the hybridization method distinguish between partial and complete complementarity. It may be of interest to detect genetic polymorphisms.

Unless combined with other techniques (such as restriction enzyme analysis), methods that allow for the same level of hybridization in the case of both partial as well as complete complementarity are typically unsuited for such applications; the probe will hybridize to both the normal and variant target sequence. Hybridization, regardless of the method used, requires some degree of complementarity between the sequence being assayed (the target sequence) and the fragment of DNA used to perform the test (the probe). (Of course, one can obtain binding without any complementarity but this binding is nonspecific and to be avoided.)

The "complement" of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other, is in "antiparallel association." Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention and include, for example, inosine and 7-deazaguanine. Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs.

As used herein the terms "protein" and "polypeptide" refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably. The terms "native gene" or "native gene sequences" are used to indicate DNA sequences encoding a particular gene which contain the same DNA sequences as found in the gene as isolated from nature. In contrast, "synthetic gene sequences" are DNA sequences which are used to replace the naturally occurring DNA sequences when the naturally occurring sequences cause expression problems in a given host cell. For example, naturally-occurring DNA sequences encoding codons which are rarely used in a host cell may be replaced (e.g., by site-directed mutagenesis) such that the synthetic DNA sequence represents a more frequently used codon. The native DNA sequence and the synthetic DNA sequence will preferably encode the same amino acid sequence.

"Nucleic acid sequence" as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand. Similarly, "amino acid sequence" as used herein refers to peptide or protein sequence. "Peptide nucleic acid" as used herein refers to an oligomeric molecule in which nucleosides are joined by peptide, rather than phosphodiester, linkages.

A "deletion" is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.

An "insertion" or "addition" is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to, naturally occurring sequences.

As used herein, the term "non-polar" ("nonpolar") insertion refers to an insertion of a DNA fragment that does not negatively affect the expression of genes located downstream of the insertion. As used herein, the term "insertional inactivation" refers to the abolition of the functional properties of a gene product by insertion of a foreign DNA sequence into the coding or regulatory portion of the gene.

A "substitution" results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.

As used herein, the term "substantially purified" refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated. An "isolated polynucleotide" is therefore a substantially purified polynucleotide.

As used herein, the term "probe" refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of hybridizing to another oligonucleotide or polynucleotide of interest. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labelled with any "reporter molecule," so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is further contemplated that the oligonucleotide of interest (i.e., to be detected) will be labelled with a reporter molecule. It is also contemplated that both the probe and oligonucleotide of interest will be labelled. It is not intended that the present invention be limited to any particular detection system or label.

As used herein, the term "target" refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction. Thus, the "target" is sought to be sorted out from other nucleic acid sequences. A "segment" is defined as a region of nucleic acid within the target sequence.

"Amplification" is defined as the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction (PCR) or other technologies well known in the art (e.g., Dieffenbach and Dveksler, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview NY [1995]). As used herein, the term "polymerase chain reaction" ("PCR") refers to the method of K.B. Mullis (U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188, hereby incorporated by reference), which provides methods for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle"; there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the "polymerase chain reaction" (hereinafter "PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified".

As used herein, the term "polymerase" refers to any polymerase suitable for use in the amplification of nucleic acids of interest. It is intended that the term encompass such DNA polymerases as Taq DNA polymerase obtained from Thermus aquaticus, although other polymerases, both thermostable and thermolabile are also encompassed by this definition.

With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications. Amplified target sequences may be used to obtain segments of DNA (e.g., genes) for insertion into recombinant vectors.

As used herein, the terms "PCR product" and "amplification product" refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.

As used herein, the term "nested primers" refers to primers that anneal to the target sequence in an area that is inside the annealing boundaries used to start PCR.

(See, K.B. Mullis, et al, Cold Spring Harbor Symposia, Vol. LI, pp. 263-273 [1986]). Because the nested primers anneal to the target inside the annealing boundaries of the starting primers, the predominant PCR-amplified product of the starting primers is necessarily a longer sequence, than that defined by the annealing boundaries of the nested primers. The PCR-amplified product of the nested primers is an amplified segment of the target sequence that cannot, therefore, anneal with the starting primers. As used herein, the term "amplification reagents" refers to those reagents (deoxyribonucleoside triphosphates, buffer, etc.), needed for amplification except for primers, nucleic acid template and the amplification enzyme. As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.

As used herein, the terms "vector" and "vehicle" are used interchangeably in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another. The terms "shuttle vector" or "bifunctional vector" refer to a cloning vector (i.e., vector) that is capable of replication in two different organisms. These vectors can "shuttle" between the two hosts. For example, the present invention encompasses shuttle vectors that are capable of replicating in various cultivars of Brassica.

The terms "expression vector" or "expression cassette" as used herein, refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), a ribosome binding site, and an initiation codon, often along with other sequences. The term "expression" may refer to "gene expression" and/or "protein expression."

As used herein, the term "multiple cloning site module" or refers to nucleic acid that contains multiple cloning sites (i.e., "restriction sites," "MCS," or "polylinker"). It is intended that the term encompass DNA that contain unique, as well as non-unique restriction sites. It also is intended to encompass multiple cloning site modules that contain foreign (i.e., exogenous) DNA inserted within the DNA containing the MCS. This foreign DNA may be inserted within the MCS by recombinant techniques. The DNA may also contain foreign DNA that is inserted in locations other than the MCS.

The terms "in operable combination," "in operable order," and "operably linked" as used herein, refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced. As used herein, the term "replicon" refers to a genetic element that behaves as an autonomous unit during DNA replication. The term also encompasses nucleic acid regions or units that have a single site for origin of replication.

As used herein the term "portion" when in reference to a gene refers to fragments of that gene. The fragments may range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, "a nucleotide comprising at least a portion of a gene" may comprise fragments of the gene or the entire gene.

As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base- pairing rules. For example, for the sequence "A-G-T," is complementary to the sequence "T-C-A." Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.

The term "homology" refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid is referred to using the functional term "substantially homologous." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target. The art knows well that numerous equivalent conditions may be employed to comprise either low or high stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency hybridization different from, but equivalent to, the above listed conditions. The term "hybridization" as used herein includes "any process by which a strand of nucleic acid joins with a complementary strand through base pairing" (Coombs, Dictionary of Biotechnology,

Stockton Press, New York NY [1994].

"Stringency" typically occurs in a range from about T_m-5°C (5°C below the T_m of the probe) to about 20°C to 25°C below T_m. As will be understood by those of skill in the art, a stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences.

As used herein, the term "T_m" is used in reference to the "melting temperature." The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the T_m of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of the T_m value may be calculated by the equation: T_m = 81.5 + 0.41(% G + C), when a nucleic acid is in aqueous solution at 1 M NaCl (See e.g., Anderson and Young, Quantitative Filter Hybridisation, in Nucleic Acid Hybridisation [1985]). Other references include more sophisticated computations which take structural as well as sequence characteristics into account for the calculation of T_m.

As used herein the term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bounds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The twσ complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution (e.g., C₀t or Rgt analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized to a solid support (e.g., a nylon membrane or a nitrocellulose filter as employed in Southern and Northern blotting, dot blotting or a glass slide as employed in in situ hybridization, including FISH [fluorescent in situ hybridization]).

As used herein, the term "antisense" is used in reference to RNA sequences which are complementary to a specific RNA sequence (e.g., mRNA). Antisense RNA may be produced by any method, including synthesis by splicing the gene(s) of interest in a reverse orientation to a viral promoter which permits the synthesis of a coding strand. Once introduced into a cell, this transcribed strand combines with natural mRNA produced by the cell to form duplexes. These duplexes then block either the further transcription of the mRNA or its translation. In this manner, mutant phenotypes may be generated. The term "antisense strand" is used in reference to a nucleic acid strand that is complementary to the "sense" strand. The designation (-) ( . e. , "negative") is sometimes used in reference to the antisense strand, with the designation (+) sometimes used in reference to the sense (i.e., "positive") strand.

The term "antigenic determinant" as used herein refers to that portion of an antigen that makes contact with a particular antibody (i.e., an "epitope"). When a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants. An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.

The terms "specific binding" or specifically binding" when used in reference to the interaction of an antibody and a protein or peptide means that the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the protein; in other words the antibody is recognizing and binding to a specific protein structure rather than to proteins in general. For example, if an antibody is specific for epitope "A," the presence of a protein containing epitope "A" (or free, unlabelled "A") in a reaction containing labelled "A" and the antibody will reduce the amount of labelled "A" bound to the antibody.

As used herein, the term "immunogen" refers to a substance, compound, molecule, or other moiety which stimulates the production of an immune response. The term "antigen" refers to a substance, compound, molecule, or other moiety that is capable of reacting with products of the immune response. For example, BN28 or other proteins associated with freezing tolerance may be used as immunogens to elicit an immune response in an animal to produce antibodies directed against the protein used as an immunogen. The protein may then be used as an antigen in an assay to detect the presence of antibodies directed against the protein in the serum of the immunized animal. "Alternations in the polynucleotide" as used herein comprise any alteration in the sequence of polynucleotides encoding any protein, in, including deletions, insertions, and point mutations that may be detected using hybridization assays. Included within this definition is the detection of alterations to the genomic DNA sequence which encodes (e.g., by alterations in pattern of restriction enzyme fragments capable of hybridizing to any sequence (e.g., by RFLP analysis), the inability of a selected fragment of any sequence to hybridize to a sample of genomic DNA (e.g., using allele-specific nucleotide probes), improper or unexpected hybridization, such as hybridization to a locus other than the normal chromosomal locus for genes of interest (e.g., using FISH to metaphase chromosomes spreads, etc.). A "variant" in regard to amino acid sequences is used to indicate an amino acid sequence that differs by one or more amino acids from another, usually related amino acid. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "non-conservative" changes, e.g., replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions (i.e., additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNAStar software. Thus, it is contemplated that this definition will encompass variants of any gene of interest. Such variants can be tested in functional assays or by other means.

The term "sample" as used herein is used in its broadest sense. For example, it refers to any type of material obtained from plants, plant cells or tissue cultures, cell lines.

Brassica Types

Winter types of Brassica cultivars are planted in the fall, overwinter, and complete their development the following spring. During the overwintering period, the plants must meet a vernalization requirement in order to flower. This requirement is met by extended exposure to low, non-freezing temperatures. For example, B. napus cv Cascade requires at least 6 weeks of exposure to 4°C, in order to meet its vernalization requirement. Prior to vernalization, plant development is arrested at the rosette stage Once the requirement is met, the plants bolt, and become reproductive, first forming inflorescence meristems, followed shortly thereafter by flower meristems. Subsequent steps in floral development are analogous in winter and spring types.

The novel spring type B. napus with a high degree of freezing tolerance provided by the present invention (e.g., VERN-) resulted from significant genetic changes that led to the loss of the vernalization requirement of its parent plants. Freezing tolerance was assessed in a segregating double haploid population (DH,) produced using microspore culture. Individual DH, lines provided a homozygous non- segregating source of plant material. The relationship between inherent and acclimation-specific freezing tolerance was assessed using phenotypic characterization. Genotypic characterization was used to examine the segregation of acclimation-specific freezing tolerance.

The observed polymorphism between the parents and the novel cultivar of the present invention can be detected with a number of restriction enzymes in VERN-, but never in the parent plants, suggesting that there has been a rearrangement or deletion of more than a few base pairs. The 600:0 ratio for vernalization in the F,s, followed by the 85:1 ratio in the DH,s indicated that the change arose between these generation. The VERN- line of the present invention differs significantly from other described vernalization mutants (See, Koorneef et al, Mol. Gen. Genet., 229:57-66 [1991]; Chandler et al, Plant J., 10:637-644 [1996]; and Martinez-Zapatre and

Somerville, Plant Physiol., 92:770-776 [1995]). First, it has a high degree of freezing tolerance. Second, it appears to be on the vernalization-dependent pathway, rather the vernalization-independent pathway. Finally, it is recessive for the spring growth habit.

The latter two properties are characteristic of the late-flowering ecotypes of Arabidopsis (Dennis et al, Cell. Develop. Biol., 7:441-448 [1996]; Lee et al, Mol.

Gen. Genet., 237:171-176 [1993]; Clarke and Dean, Mol. Gen. Genet., 242:81-89

[1994]; and Lee and Amasino, Plant Physiol., 108:157-162 [1995]).

In addition, comparisons between VERN-, vernalized VERN+, and spring cultivars demonstrated that the vernalization response was ameliorated in VERN-, as it flowered in the same time-frame as the spring types. All of the other known vernalization mutants simply delay or accelerate flowering.

Earliness is a function of the length of both the vegetative phase and the reproductive phase. In VERN-, it was noticed that both phases were generally shorter, relative to the spring cultivars. The fact that VERN- reached the 4-leaf stage in advance of the winter parents suggested that rapid vegetative growth is a function of genetic alteration(s). In addition, once flowering commenced, it was more prolific and proceeded more quickly in VERN-.

The freezing tolerance of VERN- was significantly greater than that of the tolerant parent, Cascade. This result is significant, as a lack of adequate freezing tolerance in winter canola is consistently and repeatedly cited as a major constraint to expansion of the area for production of canola. Although an understanding of the mechanism is not necessary to use the present invention, the improvement was noted in the F, generation, indicating that it may have occurred because of heterosis or the genetic similarity between the parents. Teutonico et al. (Teutonico et al, Mol. Breed., 1 :329-339 [1995]) reported transgressive segregants in their population of canola.

However, the highest tolerance obtained by these authors was only -13°C, well below the -18°C suggested with the present invention. Thus, the VERN- line of the present invention will find use in areas where it is desirable to have a canola line with increased freezing tolerance. Freezing also severely limits the acreage available to grow spring canola.

Spring frosts can kill or set back rosette-stage plants, resulting in yield reductions at the end of the growing season. Fall frosts adversely affect quality (Johnson-Flanagan et al, Plant Physiol., 81 :301-308 [1990]). These problems associated with freezing are overcome by the VERN- plant line of the present invention, as it is a spring type with an inherent tolerance of -8.5°C expressed at both the vegetative and reproductive stages. These plants will not be adversely affected by frosts that rarely exceed -5°C.

Breeding Strategies in Brassica Development

Production of new Brassica cultivars has generally made use of traditional methods of plant breeding, coupled with phenotypic assessment. The phenotype is measured using visible parameters such as plant vigor and general appearance, or quantifiable traits, such as yield, oil and protein composition and production (See e.g., Rafalski et al, RAPD Markers— A New Technology for Genetic Mapping and Plant Breeding, CAB Int'l, pp. 645-648 [1991]). Although traditional methods have been commonly used to date, in complex systems, such as low temperature responses, this selection process is difficult.

The pedigree method of breeding is used in populations of self and cross- pollinated species for the development of desirable homogenous lines (See e.g., Fehr, Principles of Cultivar Development, vol. 1, Macmillan Publishing, Ames, Iowa [1993]). Pedigree breeding produces F,s by hybridization of two parental lines, which are then grown to maturity for F₂ seed (Snape, Doubled Haploid Breeding: Theoretical Basis and Practical Applications, Second Symposium on Genetic Manipulations in Crops, CIMMYT and IRRI, [1982]). Thus, F₂ individuals are recombinant products of the original parent, and are maintained in a heterozygous state. In order to stabilize the genotype, successive rounds of selfing and selection are required. Generally, sufficient homozygosity can be attained after five or six generations (Fehr, supra). One advantage of the pedigree method is that recombination occurs during each meiotic generation allowing for expression and selection of favorable traits. However, the time needed to complete the selfing process is a major limitation to this approach, especially where environmental interactions require seasonal field assessment and selection of individual generations.

The double haploid breeding method makes use of the ability to develop individuals from gametes without fertilization. In order to be successful, tissue culture technology must be able to produce large numbers of embryos representing the genetic diversity of the parents. An advantage of this system is that whole plants are regenerated from individual cells, with each cell having an unique genotype.

Haploid embryo production in the Brassicaceae is achieved by the isolation of microspores from the pollen sac. At this stage, the microspore is uninucleate, and has not yet undergone the first mitotic division (Fan et al, Protoplasma 147:191-199

[1988]). In vitro culture of microspores under sterile conditions leads to the formation of haploid embryos. These embryos undergo cell divisions and grow in a manner similar to zygotic embryos. When placed in appropriate media, the embryos form roots and shoots, similar to germinating seeds. Unless spontaneous doubling occurs, the resulting DH, plants remain sterile, and must have their chromosome number doubled with colchicine. Colchicine is an alkaloid drug that inhibits spindle fiber formation during metaphase and anaphase of mitosis, preventing separation of the paired chromatids after splitting of the centromere. The result is a single cell that is homozygous at all loci. One advantage of double haploid technology is that it fixes recombinant gametes directly as homozygous lines in a single generation, whereas five or six generations of selfing and selection are needed in conventional pedigree programs. However, a disadvantage of double haploid breeding observed in previous work, is the inability to combine favorable loci through generations of recombination. The DH, lines produced during the development of the present invention are stable and no longer segregate for freezing tolerance. In order to complete the required requisites, several plants had to be grown, spanning four generations of self- pollination. Statistical assessment of inherent freezing tolerance and acclimation- specific freezing tolerance of DH, lines demonstrated a lack of segregation within individual lines. The stability of the population was important in order to allow characterization of the lines.

Vernalization and Freezing Tolerance

As indicated above, exposure to low temperatures not only induces freezing tolerance and adjustment to low temperature, it is also required for vernalization in winter species. Fulfillment of vernalization requirements results in the transition from vegetative to reproductive growth (Dennis et al, Cell Develop. Biol., 7:441-448

[1996]).

There is a broad range of vernalization responses, ranging from an absolute requirement to a very weak requirement. Those plants with a weak requirement eventually flower under favorable conditions in the absence of vernalization. Further, exposure to extended photoperiods and/or high light intensity overcomes the low temperature requirement. This weak requirement is referred to as a quantitative response. For example, this is the response demonstrated by most of the Arabidopsis ecotypes studied (See, Lee et al, Mol. Gen. Genet, 237:171-176 [1993]; and Napp- Zinn, On the genetic basis of vernalization requirement in Arabidopsis thaliana, in Champagnat and Jacques (eds.), La Phsiologie de lafloraison, Coll. Int. CNRS, Paris, pp. 217-220 [1985]). In these plants, exposure to vernalization conditions serves to hasten floral induction. On the other hand, those plants with a strong vernalization requirement must be exposed to low temperature in order to flower. This is referred to as a qualitative response, and is the response observed in winter Brassica sp. (See.Hodgson, Aus. J. Agric. Sci. Cambridge 84:693-710 [1978]; and Tommey and Evans, Ann. Appl. Biol., 118:201-208 [1991]).

However, little is known about the genetics of vernalization in Brassica. Early work on B. napus suggested that vernalization is controlled by two recessive genes

(Thurling and Das, Aust J. Agr. Res., 30:261-269 [1979]). Similarly, studies on B. oleracea indicate polygenic inheritance (Pelofske and Baggett, [1979]; Teutonico and Osborn, Theor. Appl. Genet., 90:727-732 [1994]), with the annual growth habit being dominant (Baggett and Kean, Hort Sci., 24:262-264 [1989]; Teutonico and Osborn, Theor. Appl. Genet., 90:727-732 [1994]). More recently, linkage maps have shown one region that is strongly linked to vernalization and days to flowering and two to other regions with smaller effects on days to flowering (Ferreira et al. Theor. Appl. Genet., 89:885-894 [1995]).

In winter cereals, vernalization has been reported to be linked, either pleotropically or genetically, to freezing tolerance (Brule-Babel and Fowler, Crop Sci.,

28:879-884 [1988]). Recent reports demonstrate an obligate relationship between vernalization and freezing tolerance, with tolerance decreasing significantly once the vernalization requirement is met (Fowler et al, Theor. Appl. Genet., 93:554-559 [1996]; and Fowler et al, Can. J. Plant Sci., 76:37-42 [1995]). There is limited evidence which suggests that freezing tolerance and vernalization are not linked in Brassica. For example, Markowski and Rapacz (Markowski and Rapacz, J. Agron. Crop Sci., 173:184-192 [1994]) indicated that there was no relationship between the degree of freezing tolerance and the degree of vernalization required in B. napus lines. In addition, genetic analysis showed that separate linkage groups exist in both B. napus and B. rapa for the capacity to attain freezing tolerance and vernalization (Ferreira et al, Theor. Appl. Genet, 90:727-732 [1995]; and Teutonico et al, Mol. Breed., 1:329-339 [1995]). However, although the lines developed in these studies expressed considerable differences in their ability to acclimate and in their vernalization requirements, no lines were found that failed to express either trait. Consequently, it has been generally assumed by those in the art, that freezing tolerance and vernalization cannot be inherited separately, despite the lack of statistical correlation between the traits.

The complexity of the genetics of freezing tolerance has further hindered the determination of the relationship between freezing tolerance and vernalization. For example, Teutonico et al. (Teutonico et al.,Mo\. Breed., 1:329-339 [1995]) showed that freezing tolerance in the Brassicas may be controlled by a number of linkage groups spread throughout the genome. They also observed that regions linked to freezing tolerance in B. rapa were not linked with tolerance in B. napus. The complexity of the trait is compounded by the fact that plants have both inherent tolerance and acclimation-specific tolerance (Stone et al. Proc. Natl. Acad. Sci., 90:7869-7873 [1993]; and Teutonico et al, Mol. Breed., 1 :329-339 [1995]). These two traits are under separate genetic control (Stone et al, supra).

The present invention also provides means for determining the relationship between freezing tolerance and vernalization. Vernalization has been reported to be linked, either pleotropically or genetically to freezing tolerance (Brule-Babel and Fowler, Crop Sci., 28:879-884 [1988]). Early work suggested that vernalization is controlled by two recessive genes (Thurling and Das, Aust. J. Agr. Res., 30:261-269 [1979]), although more recent reports indicate that there is a genetic region that is strongly associated with vernalization requirements and days required to flowering

(Ferreira et al, Theor. Appl. Genet., 90:727-732 [1995]). In addition, other reports indicate that separate linkage groups exist for the capacity to attain a level of freezing tolerance.

To date, the only loci that appear to be true vernalization loci are FRI (Lee et al, [1993], supra; Clarke and Dean, [1994], supra; and Lee and Amasino [1995], supra), and FLC (Lee et al, Plant J., 6:903-909 [1994]). These are found on the vernalization-dependent pathway (Dennis et al, [1996], supra). Other loci, such as vrnl and 2 (Chandler et al, [1996], supra), and fca, fy, and fve (Burns et al, Proc. Natl. Acad. Sci., 90:287-291 [1993]), have arisen as recessive mutations of the spring ecotypes. These mutations would be expected to confer a vernalization requirement by blocking the vernalization-independent pathway (Martinez-Zapater and Somerville, [1990], supra; and Dennis et al, [1996], supra).

However, prior to the development of the present invention, the relationship between vernalization and freezing tolerance remained unclear. Recent work on

Arabidopsis suggests that freezing tolerance and vernalization are controlled by completely separate pathways (Chandler et al, [1996], supra). However, it is difficult to draw a conclusion from this report, as the mutants were generated from the annual ecotype Landsberg erecta, in which flowering is largely dictated by light conditions, with vernalization playing a secondary role. In addition, this ecotype only attains a moderate degree of freezing tolerance following exposure to low, non-freezing temperatures (Chandler et al, [1996], supra). Furthermore, analysis of the inheritance of BN28 throughout the Brassicaceae revealed that species-specific loci exist.

The results obtained during the development of the present invention clearly demonstrate that freezing tolerance and vernalization can be inherited separately. For example, VERN- has lost the vernalization requirement, while expressing a higher degree of freezing tolerance than that expressed by either parent, both in the absence of acclimation and following acclimation. As such, the linkage between vernalization and freezing tolerance has been broken in VERN-. Thus, the methods used to produce VERN- will find use in producing superior plants with advantages such as high freezing tolerance and earlier maturation. Unlike previously used methods to breed for improved tolerance to low temperature, the methods of the present invention provide the means to successfully accomplish the production of plants with high freezing tolerance levels and other highly desirable characteristics.

DETAILED DESCRIPTION OF THE INVENTION

In order to identify the difference between the requirements for vernalization and freezing tolerance, an isogenic line was developed from doubled haploid progeny of reciprocal crosses between B. napus cv Rebel and B. napus cv Cascade. Both of these parent cultivars are winter types, with strong vernalization responses. Rebel was found to have little capacity for acquisition of freezing tolerance, while Cascade is highly freezing tolerant. Analysis of approximately 100 isogenic lines revealed random segregation of freezing tolerance. A single DH, line ("6-200" or "VERN-") was scored as "vern-," indicating that it lacked a vernalization requirement, yet retained a high degree of freezing tolerance.

As both parents have similar lineages, the Cascade x Rebel cross provided maximal selection pressure for freezing tolerance, and limited other genetic differences. Microspore culture methods used in conjunction with colchicine treatment yielded approximately 100 isogenic doubled lines (DH,). Spontaneous doubling accounted for 3% of the doubling that occurred. The remainder of the lines were doubled with colchicine. Aside from fertility, no other phenotypic differences were apparent between any of the colchicine-treated material and the spontaneous diploids. Derived lines were tested for acquisition of freezing tolerance and vernalization requirements. Freezing tolerance measurements were completed on original materials, as well as on selfed (DH₂) progeny, in order to ensure that no further segregation had occurred. Selfed progeny performed similarly to the original plant line. Based on the data obtained during the development of the present invention, the segregation of freezing tolerance was determined. DH, plants were grouped into 2°C intervals and the distribution was determined. Three major groups were apparent, at a ratio of approximately 1 :1 :1. The lack of a genetic trend further indicates that freezing tolerance is a multigenetic trait. Although an understanding of the degree of transgressive segregation appearing in isogenic plants is not necessary to use the present invention, isogenic plants may contain QTLs with more tolerant phenotypes than Cascade.

During the development of the present invention, phenotypic and genotypic analyses of isogenic DH, lines indicated that freezing tolerance and vernalization are not linked, although both occur under similar environmental conditions. Furthermore, on the basis of the segregation ratio of BC,F, and BC,F₂, it was concluded that vern- is a homozygous recessive locus. Reciprocal backcrosses to Rebel and Cascade resulted in the complementation of the vern- phenotype, and reinstatement of the vernalization requirement in 100% of 600 BC,F, plants. In the BC,F₂, the phenotypic segregation ratio for 80 plants was 61:19 (i.e., almost exactly a ratio of 3:1). These results confirmed the homozygous recessive nature of VERN-. Molecular analysis of VERN- revealed a distinct polymorphism at a vernalization locus. Complementation analysis and F₂ segregation using parental backcrosses suggested that a single recessive gene controls vernalization requirements at this locus, and has no influence over freezing tolerance. These results indicated that the linkage between freezing tolerance and vernalization can be broken in winter types of B. napus, as well as other cultivars with the same phenotypes.

By comparing spring and winter cultivars, it was possible to identify the spring and winter type alleles at each marker or gene locus. The data indicate that VERN- carries spring type alleles at all the loci examined. So, too, does Rebel, with the exception of one marker, wg7b3 in LG12. At this locus, Rebel carries a winter type allele and Cascade has the spring type allele. Thus, VERN- appears to have inherited spring type alleles from both Cascade and Rebel.

In additional experiments, B. napus homologous clones of two flowering time genes of Arabidopsis (Co and FCA) were used as probes to determine the genotypes of VERN- and its parents. Clone FCA#17 covered the B. napus FCA gene from within intron 3 into the RRM2 (RNA Recognition Motif 2). One polymorphism between

Cascade and Rebel was detected within the clone. VERN- and Rebel shared the same allele at this locus.

Two genomic Co clones from linkage groups N10 and N12 in the map of Parkin et al. (Parkin et al, Genome 38:1122-1131 [1995]; corresponding to LGsl2 and 11 in the map of Osborn et al. [Osborn et al, Genetics 146:1123-1129 [1997], respectively) were also used. These clones detected polymorphisms between Cascade and Rebel. Again, VERN- had Rebel's alleles at the two Co loci. EXPERIMENTAL

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof. In the experimental disclosure which follows, the following abbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N (Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); μg (micrograms); ng (nanograms); 1 or L (liters); ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); μE (microeinstein);°C (degrees Centigrade); rpm (revolutions per minute); Kd or KDa (kilodalton); Kb (kilobase); MW (molecular weight); PCR (polymerase chain reaction); RAPD (randomly amplified polymorphic DNA); RFLP (restriction fragment length polymorphism); R₀t or Rgt (the product of RNA concentration and the time of incubation in an RNA-driven hybridization; the analog of C₀t values used to describe DNA-driven hybridization reactions); BSA (bovine serum albumin); PBS (phosphate buffered saline); Tris (Tris(hydroxymethyl) methylamine); TBS (Tris buffered saline); TAE (10 mM Tris, 1% acetic acid, 1 mM EDTA); TE (10 mM Tris, 1 mM EDTA); SSC (salt, sodium citrate buffer); DEPC (diethy pyrocarbonate); EDTA (ethylenediamine tetraacetic acid); ddH₂0 (double distilled deionized water); SDS (sodium dodecyl sulfate); SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis); QTL (quantitative trait loci); Fl or F, (first generation offspring); F2 or F₂ (second generation offspring); DH1 or DH, (double haploid, first generation); BC, (backcross first generation); cv (cultivar); FT or ft. (freezing tolerance); LT50 or LT₅₀ (temperature at which 50% death occurs (i.e., 50% of the population dies); vern+ (vernalization required); vern- (no vernalization requirement; VERN-); reb (Rebel); cas (Cascade); SAS (statistical analysis software); GLM (general linear model); S.E. (standard error); BBL (Becton Dickinson Microbiology Systems, Cockeysville, MD); DIFCO or Difco (Difco Laboratories, Detroit, MI); Remel (Remel, Lenexa, KS); Scientific Products (McGaw Park, IL); Fisher (Fisher Scientific, New York, NY)U.S. Biochemical (U.S. Biochemical Corp., Cleveland, OH); Scientific Products (McGraw Park, IL); Sigma (Sigma Chemical Co., St. Louis, MO.); Biorad (BioRad Laboratories, Richmond, CA); Complete Plant Products (Complete Plant Products, Brampton, Ontario); Conviron (Conviron, Winnipeg, Manitoba); Bach-Simpson (Bach-Simpson Ltd., London, Ontario, Canada); Schleicher and Schuell (Schleicher and Schuell, Keene, NH); Calbiochem

(Calbiochem, San Diego, CA); Pharmacia (Pharmacia Biotech, Piscataway, NJ); Boehringer-Mannheim (Boehringer-Mannheim Corp., Concord, CA); Amersham (Amersham, Inc., Arlington Heights, IL); NEB (New England Biolabs, Beverly, MA); Pierce (Pierce Chemical Co., Rockford, IL); Eppendorf (Eppendorf Scientific, Madison, WI); and Molecular Dynamics (Molecular Dynamics, Sunnyvale, CA);

ATCC (American Type Culture Collection, Rockville, MD); U.S. Biochemical (U.S. Biochemical Corp., Cleveland, OH); Scientific Products (McGraw Park, IL); Oxoid (Oxoid, Basingstoke, England); BBL (Becton Dickinson Microbiology Systems, Cockeysville, MD); DIFCO (Difco Laboratories, Detroit, MI); Life Technologies, BRL, and Gibco-BRL (Life Technologies, Gaithersburg, MD); New England Nuclear

(New England Nuclear Research Products, Boston, MA); and SAS (SAS Institute, Inc., Cary, North Carolina).

EXAMPLE 1 Plant Material and Growth Conditions Seeds of B. napus cv Cascade, cv Rebel, F,s, DH,s, BC,F,, and BC,F₂, as well as other cultivars used in the following experiments (e.g., the spring cultivars tested in these experiments included Westar, Legend, Excel and Quantum, as well as winter- type sister line to VERN-, "VERN+") were planted in 15 cm pots containing a soil mixture of sand, peat and vermiculite (1:1:1), and grown under greenhouse conditions, with a 16 hour photoperiod (16 hours of "day" and 8 hours of "night"), augmented with high intensity discharge (HID, Sylvania, Canada) sodium vapor lights, to maintain a minimum light intensity of 275-300 μE s^'1. The temperature regime was 20°C day, and 16°C night. Unless otherwise indicated, the light and temperature regimes were maintained for the entire experiment. For example, VERN- was also grown under greenhouse conditions with a non-inductive photoperiod of 10 hours, using the same temperature and light intensity as described above. The plants were watered daily to field capacity, and fertilized every 14 days using 20:20:20 (N-P-K) (Complete Plant Products). The main reason for choosing the parent cultivars (Cascade and Rebel) was their difference in freezing tolerance (the TL₅₀ for Cascade is -15.5°C, while the TL₅₀ for Rebel is -7.5°C. Although some common parentage is shared between the cultivars, they are considered to be genetically dissimilar. Both cultivars are canola quality and were developed at the Agricultural Experimental Station in Moscow, Idaho.

The Cascade used in the development of the present invention was selected in the F₆ generation from crosses between Indore and three edible oil lines, Sipal, WW 827, and Liraglu. Segregating generations were advanced by single seed descent and F₃ to F₆ generations were screened for low levels of glucosinolate in mature seed (<8% seed moisture; Auld et al, Crop Sci., 27:1309-1310 [1987]).

Rebel is an F₆ line derived by a cross between OAC Triton and WRE 17. OAC Triton is a triazine-tolerant spring type from the University of Guelph (Beversdorf et al, Sci., 64:1007-1009 [1984]). WRE 17 is a line derived by single seed descent from a cross between Sipal and Indore. Rebel was selected using the same methods that were used with Cascade.

EXAMPLE 2 Development of Homozygous Lines Reciprocal Crosses

At the 4-leaf stage, the parent plants were transferred to a controlled environment chamber at 4°C for six weeks to complete vernalization with a 16 hour photoperiod of 400-450 μEm^'V, and then transferred to a programmable growth cabinet (Conviron PGW 36), under 10°C (day) and 6°C (night) conditions to synchronize bud development. Buds that started to mature and change color from dark green to yellow (indicating impending anthesis) were chosen for crossing. All crosses were completed in reciprocal fashion between vernalized winter cultivars Cascade (freezing tolerant; TL₅₀ = -15°C) and Rebel (freezing sensitive; TL₅₀ = -6.5°C). A total of nine (9) reciprocal crosses were completed, to produce 18 plants. Individual buds were opened using ethanol-sterilized forceps. Sepals, petals, and anthers were removed from the female flower prior to dehiscence of pollen. Stigmas were pollinated with mature pollen from the appropriate donor parent, and enclosed in pollination bags until silique (pod) elongation was apparent (indicating embryo fertilization). This helped to ensure that contamination from any airborne pollen did not occur. Once all reciprocal crosses had been completed, the plants were returned to the greenhouse to allow the F, seeds to mature and be harvested.

Microspore Culture

F, seed was planted and grown as described above. Plants used for microspore culture were maintained in the growth cabinet after vernalization at 10°C (day) and 6°C (night) conditions, with a 16 hour photoperiod of 275-300 μE m'V¹, to synchronize bud development. Buds to be used for microspore embryo production were 3-5 mm in length and were collected from healthy plants just prior to anthesis.

Buds were placed into a wire strainer and surface sterilized in 7% sodium hypochlorite (100 ml) for 15 minutes, then rinsed three times in 150 ml of sterile water for 5 minutes each rinse. All subsequent work was conducted in a laminar flow hood, using autoclaved solutions and instruments, to help ensure that sterile conditions were maintained.

Buds were transferred to a mortar and gently crushed in 1-3 ml of B5 medium containing 13% sucrose, as described by Coventry et al. (Coventry et al, Manual for Microspore Culture Techniques in Brassica napus,OAC Publ. 0489, University of

Guelph, Guelph, Ont, [1988]), to release the microspore cells. The suspension was filtered through a 0.44 μm nylon filter into a 15 ml Falcon tube (Fisher), and the nylon filter was rinsed twice with 1 ml aliquots of B5 medium. The volume in all of the tubes was then adjusted to 10 ml B5 medium prior to centrifugation in a tabletop centrifuge (Model IEC-HN-SII, Needam, Mass.), at 1000 rpm for 3 minutes. The supernatant was removed and the pellet was resuspended in 5 ml of B5 medium containing 13% sucrose. Three more washes were completed with centrifugation at 800 rpm, 500 rpm, and 500 rpm, respectively. The final pellet was suspended in NLN medium containing 13% sucrose (Lichter, Z. Pflanzephysiol., 105:285-291 [1982]). The volume of the suspension was adjusted to 1 ml for every original bud used

(approximately 20 per isolation). The suspension was then poured into sterile petri plates (60 x 15 mm; Fisher), and sealed with Parafilm®. The samples in the plates were heat shocked for 3 days at 37°C, in the dark, with no agitation. The plates were then transferred to, and kept on a shaker (55-60 rpm), at room temperature in the dark, for 4-6 weeks. Embryos were selected and plated onto solid B5 medium containing

3%) agar (Difco-Bacto agar) supplemented with 3% sucrose, and placed on a light bench with a light intensity of 150 μmole m^'V, at room temperature, until roots and true leaves were apparent. DH, plantlets were transferred to sterile soil, and placed in a mist chamber under high humidity for 14 days to reduce desiccation and excessive stress, and were then transferred to the greenhouse, and allowed to grow until 3-5 mature leaves were apparent, at which time they were transferred to 4°C for a 6 week vernalization period. Fertility Assessment and Colchicine Treatment

Vernalized DH, plants were moved to the greenhouse and assessed for fertility. Bolting plants were analyzed for the presence of pollen as an indicator of spontaneous doubling (i.e., spontaneous diploidization). Where chromosome doubling had not occurred, up to 5 cuttings were taken from a single DH, plant, dipped in powdered rooting compound (0.2% indole-3-butyric acid)(Plant Products Co.), and rooted in moist soil in a misting chamber. Rooted cuttings were trimmed to 6 cm, and foliage was trimmed from the shoots to approximately 20 cm. Plants were then placed in an aqueous colchicine solution (3.14 g/1), as described by Coventry et al. (Coventry et al, supra), and placed under high intensity light (300 μmole m^'V), for 2 hours to maximize transpiration and colchicine uptake. The plants were thoroughly rinsed in water, potted in 15 cm pots, and placed in the greenhouse. A single doubled shoot from each DH, line was maintained and bagged to obtain selfed seed.

Approximately 100 DH, lines were derived from tissue culture. Spontaneous doubling accounted for 3% of the doubling that occurred. The remainder of the lines were doubled with colchicine. Aside from fertility, no other phenotypic differences were apparent between any of the colchicine-treated material and the spontaneous diploids.

EXAMPLE 3 Vernalization and Acclimation

In order that the flowering response of VERN+ could be compared to that of Westar and VERN-, it was necessary to determine suitable vernalization conditions. In these experiments, two parameters were checked: a) the stage at which the seedlings were vernalized (i.e., seed, cotyledons, 2-leaf and 4-leaf) and; b) length of cold treatment (i.e., 3, 4, 5, and 6 weeks). Seed was allowed to imbibe for 24 hours prior to vernalization. Seedlings were grown in the greenhouse as described above, transferred to the growth chamber for vernalization, and then returned to the greenhouse. The final leaf number of 10 plants was determined and the results were analyzed using one-way ANOVA (p<0.05) using MSExcel. Based on these data, it was determined that 4 leaf staged plants should be vernalized for 6 weeks. The relationship between leaf number and flowering time was also determined.

In experiments to determine the flowering responses of the cultivars under different growth conditions, VERN-, Westar and VERN+ plants were grown in growth chambers with 22°C (day) and 17°C (night) temperatures, under 1 of 2 photoperiods of 400-450 μE M^"2 s^"1; a 16 hour inductive photoperiod and an 8 hour non-inductive photoperiod, with and without vernalization. The same photoperiod was maintained throughout vernalization. It was determined that VERN- and Westar seedlings had to be vernalized at 17 DAP (Days After Planting) when grown under a 16 hour photoperiod, as this was the latest time at which the seedlings were in the vegetative stage of development. The maximum time to assessment was 150 DAP or 150 DAV (Days After Vernalization).

For vernalization experiments, with the exception of the experiment designed to determine the minimum leaf number needed on VERN+ in order to flower, plants were moved, at the 4-leaf stage, to 4°C for six weeks. Minimum leaf number was determined by moving plants at the cotyledonary stage, and first, second, third, and fourth leaf stage to 4°C for 3, 4, 5, and 6 weeks. The light intensity and photoperiod were maintained as described in Example 1. Following six weeks of acclimation, VERN- expressed a high degree of freezing tolerance, with a TL₅₀ of >-18°C, as compared to a TL₅₀ of -15°C for Cascade, and only -7.5°C for Rebel. In addition, the inherent tolerance of VERN- was high, compared with the tolerant parent (TL₅₀ of -8.5°C for VERN). This tolerance was expressed during both vegetative and reproductive development. Figure 1 provides a graph showing the frequency distribution of ion leakage (TL₅₀), for eight lines.

The loss of the vernalization requirement was confirmed in both greenhouse and field trials of original and selfed seed. As shown in Table 1 below, VERN- flowered 3-5 days earlier than the 4 spring cultivars in the growth chamber. In the greenhouse, flowering was comparable to that of the spring cultivars, while in the field, VERN- again flowered earlier. In Table 1, the asterisk (*) indicates that the values were significant at the 5% level.

Table 1. Flowering Times of VERN- and Registered Spring Cultivars

In comparison with three commonly used spring types (B. napus cv Altex, B. napus cv Legend, and B. napus cv. Alto), VERN- reached the 4-leaf stage and then bolted significantly faster. In addition, VERN- reached the 4-leaf stage at a more rapid rate than either of the parents. VERN- then continued to rapidly produce leaves, resulting in more leaves at flowering than were present on the spring cultivars. Flowering in both VERN- and Legend began at 35 days, and ceased at 50 days, a full

5 days earlier than either Altex or Alto. Both the number of siliques and seed yield were high in VERN- and Alto. These results indicated that more flowers were produced in a given time frame (i.e., 15 days) in these plants, as compared to Legend. Further phenotypic characterization involved field assessments that compared VERN- with Excel and Quantum at two locations (one in Alberta and one in

Saskatchewan). These cultivars were studied as blackleg tolerance is required in the field. VERN- was found to be developmentally faster and provided a higher yield than either cultivar. Excel (the early cultivar), took 5 days longer than VERN- to reach the 4-leaf stage; 2 days later, initiation of flowering and maturation occurred. Quantum was approximately 5 days slower in comparison with VERN-. Once again, the yields of VERN- were superior to the spring cultivars.

The following tables summarize the results obtained in the greenhouse and field assessments of VERN-. Table 2 provides a comparison of the gross morphological characters associated with growth and development from germination through maturation. The data are expressed as the mean +/- S.E. (n=12), divided into three replicates, with each having 4 randomized pots per line; each pot contained 1 plant. Tables 3 and 4 provide comparison of gross field and greenhouse characters of growth and development of VERN-, Excel, and Quantum. In Table 4, the values represent the mean ± the standard error with n=10; these values are significant at the 5% level. The field data were collected from 3 locations (1 in Alberta, and 2 in Saskatchewan), from 10 plants in each plot.

Table 2. Greenhouse Assessment of VERN-

Table 3. Field Assessment of VERN-

Table 4. Greenhouse Assessment of VERN-

The results from greenhouse, field, and gross morphological assessments suggested that VERN- produced more vegetative growth, despite an earlier transition to reproductive development. In order to determine whether this was a function of the mutation or was a trait of winter types, additional information was required. To make this determination, the effect of vernalization time and seedling stage on leaf number at flowering and time to flowering was determined for the full sib, VERN+. It was determined that four-leaf staged seedlings vernalized for 6 weeks had the least leaves and flowered the fastest. Data from VERN+ vernalized for 6 weeks at the 4-leaf stage, and from VERN-, indicated that rapid vegetative growth is a winter trait. Furthermore, the results showed that time to flowering is not compromised in VERN-.

EXAMPLE 4 Assessment of Freezing Tolerance

Freezing tolerance was determined on non-acclimated plants, and plants that had been acclimated for 6 weeks at 4°C, using an electrolyte leakage assay. Inherent freezing tolerance was assessed on non-acclimated material that was maintained in the greenhouse. Twenty-four randomly selected DH, lines were assayed. Acclimation- specific freezing tolerance was determined on the two parent lines (Cascade and Rebel), six F, plants, and 70 DH, lines that had been exposed to 4°C for six weeks, as described for vernalization. Assessments were completed on all 78 lines. The experiments were replicated three times, with five plants per replicate.

The second and third leaves of 4-leaf stage plants and apical leaves of bolting plants were harvested and washed in distilled water. Discs of leaf tissue (1 cm) were removed with a cork borer, being careful to avoid any veins, and placed on moistened filter paper (Whatman) in small petri dishes (2 discs per plate). The plates were placed in a programmable freezer, and the temperature was lowered to 0°C over a 1 hour period. A set of plates was removed at this temperature to act as a non- frozen control. The temperature was then lowered to -2.5°C, and nucleation was initiated by touching the filter paper with a metal probe cooled in liquid nitrogen. Samples were maintained overnight at -2.5°C. The temperature was then lowered at a constant rate of -2.5°C per hour, and samples were removed at 2.5°C intervals between -2.5°C and -

18.5°C. Samples removed from the freezer were allowed to thaw at 4°C for at least 12 hours.

Electrolyte leakage was determined by placing the thawed discs (including the filter paper) in tubes containing 10 ml of double distilled water, and shaking (45 rpm) overnight at room temperature. Freeze-induced leakage was determined by using a radiometer (Model CAM 83, Bach-Simpson) to measure the conductivity of the samples. Total leakage was determined after boiling the samples for 3 minutes, cooling them to room temperature, and shaking for at least 1 hour at 45 rpm. Ion leakage was measured in each case and injury was expressed as a percentage of total (boiled). The temperature at which 50%) leakage occurred, termed "TL₅₀," was used as a measure of plant viability at that particular temperature, given that 50%) leakage is equivalent to 99% cell death and consequent plant mortality (Sukumaran and Weiser, Hort. Sci., 7:467-468 [1972]; and Boothe et al, Plant Physiol., 108:795-803 [1995]). The developmental age of plant material selected for freeze testing has been determined to be important in order to attain accurate ion leakage results, with older leaves showing reduced levels of freezing tolerance relative to younger leaves. Thus, second and third leaves from a four-leaf stage plant were used. These leaves were initiated at the same time, and were of similar physiological and developmental age. These leaves also provided sufficient plant material for an accurate assessment of freezing tolerance from individual plants, as well as plant lines.

Acclimation capacity was determined as the difference in freezing tolerance between non-acclimated and acclimated material (i.e., the difference between the inherent and acclimation-specific freezing tolerance represents the acclimation capacity of the line). The acclimation capacity of these 24 randomly selected lines ranged from a low of 1.0°C to a high of 14°C. The results also indicated that there are three distinct aspect of freezing tolerance present in these DH, lines—inherent freezing tolerance, acclimation-specific tolerance, and acclimation capacity.

Figures 1A-C show the inherent and acclimation-specific freezing tolerance in representative three DH, lines. The values represent the mean +/- standard error, with n=10 and n=15 for inherent and acclimation-specific tolerance, respectively. Figure

1A shows that inherent freezing tolerance and acclimation-specific freezing tolerance are not correlated. Likewise, inherent freezing tolerance is not correlated with acclimation capacity, as shown in Figure IB. Figures ID and E are graphs showing the inherent and acclimation specific-freezing tolerance in Cascade, Rebel, F„ and the DH, line, VERN-.

Thus, as indicated above and in Figure 1 , VERN- expressed a high degree of freezing tolerance, with a TL₅₀ of > -18°C, as compared to a TL₅₀ of -15°C for Cascade and only -7.5°C for Rebel. In addition, inherent tolerance was high compared with the tolerant parent (i.e., TL₅₀ of -8.5°C for VERN- and TL₅₀ of -5.5°C for Cascade). This tolerance was expressed during both vegetative and reproductive development.

In addition, thirty-two DH lines were assessed for acclimation-specific freezing tolerance as above. RFLP data were collected and used to determine possible associations using clones ec3e5, wglgό, wglg3, wglfό, wg4h3, ec4h3, tg5b2, and tglc8, which map to the freezing tolerance associated (FTA) linkage groups in Brassica rapa, as outlined by Teutonico et al. (Teutonico et al, Theor. Appl. Genet., 89:885-894 [1995]). As indicated below in Example 8, low-temperature induced cDNA clones BN28, BN115, cor 6.6 and cor 15 were also tested. Statistical analysis for association with acclimation-specific freezing tolerance was completed based on X² values derived using SAS ver 6.03 (SAS Institute Inc. 1988), as described in Example 5, below. As indicated in the following Table (Table 5), strong associations between acclimation-specific freezing tolerance and loci wglgό of LG4 and ec2e5 of LG7 were identified. At these loci, VERN- inherited freezing tolerance alleles from Cascade. At other loci, there was either no association or no polymorphism detected between the parents.

Table 5. Association of RFLP Marker Loci for Freezing Tolerance Linkage Groups

Analysis of a backcross between VERN- to Cascade (vern+), showed total complementation by re-establishment of the vernalization requirement in all of the F, progeny tested. Assessment of F₂ segregation showed that the vernalization loci mapping with WG6B10 segregated as a homozygous recessive, as shown by the 3:1 vern+:VERN- ratio observed. Preliminary genotype analysis of the F₂ population confirmed the observed phenotype.

EXAMPLE 5 Statistical Analysis of Freezing Tolerance Data

SAS version 6.03 was used to assess the significance of segregation of freezing tolerance in the DH, lines at the level of inherent freezing tolerance and acclimation- specific freezing tolerance. From the DH, population, 24 lines were randomly selected for determination of inherent freezing tolerance. Analysis of inherent tolerance was completed using two replicates. Each replicate contained 5 plants per line (n= 10). For each plant, leakage measurements were averaged from 3 discs at each of 5 different temperatures (0, -2.5, -5, -7.5, and -10°C). Analysis of acclimation-specific freezing tolerance was completed using three replicates. Each replicate contained 5 plants per line (n=15). For each plant, leakage measurements were averaged from 5 discs, taken at each of 5 different temperatures (0, -7.5, -12.5, -15, and -17.5°C). The resulting means were used to generate a graph, from which the TL₅₀s were determined. The difference between inherent (TL₅₀) and acclimation-specific freezing tolerance (TL₅₀) relationship was the acclimation capacity. The design used was ANOVA univariate analysis using the general linear models (GLM) procedure on replicate by line to examine consistency within each replicate. The line by line analysis was completed in the same manner and used to compare differences between individual DH, lines as seen by differences in freezing tolerance. Coefficient of variance was calculated using (root mean squared/mean) as a second measure of significance in the test.

Cascade was found to be freezing tolerant (-15.5°C), while Rebel was freezing sensitive (-7.5°C). Segregation of acclimation-specific freezing tolerance in the DH, lines showed a trimodal distribution that ranged from -3.0°C to greater than -18°C, as shown in Figure 3. There appeared to be a large proportion of transgressive segregation, which favored a level of freezing tolerance greater than that of the tolerant parent, Cascade. The distribution curve did not conform to any Mendelian ratio. Figure 3 shows the segregation of freezing tolerance based on the TL₅₀s of 78 individual lines of B. napus, including the 2 parent lines, six F„ and 70 DH, lines. In comparing reciprocal crosses, differences in the level of freezing tolerance could not be attributed to maternal effects. Freezing tolerance appeared to segregate randomly throughout the DH, lines, regardless of the direction of the cross.

Comparison of REPLICATE X LINE using ANOVA univariate analysis revealed no significant difference between the replicates. However, when comparing LINE X LINE, highly significant differences were apparent. A correlation of variance value of 5.8% (LINE X LINE) confirmed that significant levels of variation existed between segregating lines.

Correlation analysis was completed between non-acclimated and acclimated plant tissue, to examine the possibility that inherent freezing tolerance could be used as a tool in predicting a line's acclimation capacity. The results showed that neither acclimation-specific freezing tolerance nor acclimation capacity can be predicted using inherent freezing tolerance as an indicator.

EXAMPLE 6 Phenotypic Characterization

Both vernalized and non-vernalized plants were grown under greenhouse conditions and assessed on the basis of the following parameters: (1) days to fourth leaf, bolting, transition from vegetative to reproductive meristem, first flower, completion of flowering, and maturity; (2) number of expanded leaves; and (3) yield. Field testing of the non- vernalized plants was conducted at two locations.

In experiments to analyze plant development, Westar and VERN- were grown in a growth chamber under a 16 hour photoperiod of 400-450 μE m^"2 s^"1, at 22°C (day) and 17°C (night). VERN+ was vernalized as above, then moved to the growth chamber. Sampling of Westar and VERN- began at 14 days after planting and continued up to the time that the first floral buds in the primary inflorescence opened. VERN+ sample collection began immediately after vernalization. Shoot tips were collected and leaves carefully removed to expose the apical meristem; meristem tissue was fixed and processed for scanning electron microscopy (SEM). Floral development of VERN- and the spring cultivar, Westar, was then followed by SEM, according to the morphological landmarks of Smyth et al. (Smyth et al, Plant Cell., 2:755-767 [990]). The sample size was at least 10 meristems for each collection time. Comparisons were made between VERN- and VERN+, in order to determine whether vegetative and reproductive development in VERN- was more "winter-like" or "springlike." The results indicated that unlike Arabidopsis (Bagnall, [1993]), there is not a linear relationship between flowering time and leaf number, (as indicated in Table 6, below). These observations necessitated recording both chronological time and leaf number (developmental time).

Table 6. Correlation Analysis Between Leaf Number and Flowering Time in VERN+

In addition, non-vernalized material was grown in the greenhouse for a minimum of 180 days, in order to determine the absolute vernalization requirements of the parents, F,s, and DH, lines. Phenotypic analysis of vernalized and non-vernalized

DH, lines revealed that all but one line (VERN-) required six weeks of vernalization, in order to complete development and set viable seed.

As the vernalization requirement of some plants can be replaced by extended photoperiods, the possibility that the photoperiod in the greenhouse could promote flowering was addressed. Two approaches were taken to make this determination.

First, VERN- was grown under a short (10 hour) photoperiod, and the winter parents (Cascade and Rebel) were allowed to grow for over 180 days. These results indicated that the winter types failed to flower, even after an extended growth period, and confirmed that VERN- had lost the vernalization requirement present in both parents. Thus, since VERN- flowered, the photoperiod in the greenhouse did not replace the vernalization requirement.

To confirm the phenotype of the parents, Cascade and Rebel were allowed to grow for up to 300 days under greenhouse and growth chamber conditions, with and without vernalization. Cascade failed to flower unless vernalized, indicating that it has an absolute vernalization requirement. On the other hand, Rebel flowered, albeit abnormally and very late, in the absence of vernalization. The flowers were pale, malformed and had very little pollen. Following vernalization, flowering time was significantly reduced and the transition to reproductive development was normal (See, Tables 7 and 8, below). In Table 7, the values represent the mean + standard error, with n = 10). In Table 8, all of the values given (i.e., other than "Did Not Flower") were significant at the 5% level. As such, Rebel would be considered to have a vernalization requirement. These results confirmed that VERN- had lost the vernalization requirement that was present in the parents.

Table 7. Vernalization Response of Cascade and Rebel

Table 8. Mean Final Leaf Number Under Inductive and Non-inductive Growth Conditions

Under a 16 hour photoperiod, both Westar and VERN- underwent the transition from vegetative to reproductive growth at 20 days after planting, as indicated by the formation of the inflorescence meristem. Subsequent floral meristem development was slightly slower in VERN-. However, this did not result in a significant difference in time to anthesis. Comparisons between VERN- and VERN+ showed that floral development was faster by as much as 4 days in VERN+. Again, however, this did not translate into a significant difference in time to anthesis. Assessment of reproductive development in terms of leaf number showed that

VERN- was not developmentally compromised by a lack of vernalization (See e.g., Table 8). Under a 16 hour photoperiod, VERN- had less leaves than did the vernalized winter types, Rebel and VERN+. Admittedly, vernalization reduced the leaf number in VERN-, but the same response was seen in the spring type, Westar. This reduction in leaf number in response to vernalization in spring types is generally accepted (Dennis et al., supra).

VERN-'s response to photoperiod and vernalization was intermediate between the winter and spring types (See, Table 8). The winter type VERN+, did not flower without vernalization and was insensitive to photoperiod. In the absence of vernalization, Rebel was very slow to flower under a long photoperiod and simply did not flower under a short photoperiod. The spring type, Westar, was photoperiod sensitive; under an 8 hour photoperiod, leaf number was nearly double that produced under a 16 hour photoperiod. Westar was also responsive to vernalization, and this was most apparent under the short photoperiod. In comparison to Westar, VERN- had a weaker response to photoperiod, and a weaker response to vernalization under the short photoperiod, but a stronger response under the long photoperiod.

EXAMPLE 7 Segregation Analysis

In these experiments, the genetic behavior of the lack of a vernalization requirement in VERN- was investigated. VERN- was backcrossed with Cascade, to produce the BC,F, generation. This was then self-pollinated to produce the BC,F₂ generation. The vernalization requirement was determined as described above (Example 3). Vernalization requirement assessments for BC,F, and BC,F₂ individuals were made at 100 and 300 days after planting. One hundred days generally represents the complete life cycle of a spring type B. napus cultivar. If a line did not flower within the 100 day time frame, it was scored as a winter type (i.e., requiring vernalization); the 300 day period represented an extremity.

BC,F₂ were assessed for the appearance of bolting, and first anthesis, as evidence for the lack of the vernalization requirement. Absolute vernalization was gauged as the presence versus the absence of bolting after 95 days of growth.

Analysis of association was completed based on X² values derived from orthogonal functions according to Mather (1951) using 61 BC,F₂ plants. Linkage group analysis was completed using MAPMAKER/QTL vl.l program (Lincoln et al., Whitehead Institute Technical Report, Cambridge, MA [1992]). A LOD score of 2.0 was chosen as the threshold for declaring linkage. As shown in Table 9, four of the five markers showed significant association with the vernalization requirement. Marker tg6al2a had the strongest association (X²=18.25, pO.OOl). Interval mapping analysis was also performed to examine the association of these markers with the flowering time variation in the BC,F₂ population. As shown in Figure 2, the LOD plot produced by MAPMAKER/QTL displayed 1 score peak, which was located in the interval between tg6al2a and wg5a5, close to marker tg6a!2a. In this Figure, the marker loci are indicated on the horizontal axis and the LOD score is indicated on the vertical axis. In this Figure, "TTF" refers to the time to flower (in days).

Table 9. Association of RFLP Marker Loci for Vernalization Linkage Groups in BC^ Progeny

When assessed at 100 days after planting, all BC,F, flowered, while in the BC,F₂, 192 individuals flowered and 84 did not. This segregation pattern indicates that the lack of vernalization requirement in VERN- is a dominant characteristic. When evaluated for 300 days, 1 out of 69 and 3 out of 135 BC,F₂ individuals had not bolted or shown any sign of flowering. The ratios were close to the expected, if 3 or 4 loci with additive effects are assumed (X²=0.003-0.05, 0.8 < p < 0.99).

As LG9 has been reported as having strong association with the vernalization requirement and major effects on flowering time in the F, -derived DH population from 'Major' x 'Stellar' (Ferreira et al, Theor. Appl. Genet., 90:727-732 [1995]), a subset of the above BC,F₂ population was analyzed with the 9 RFLP markers in LG9 of the map described by Ferreira et al. (Ferreira et al, supra). Polymorphisms were detected between VERN- and Cascade at 6 of the 9 marker loci. Segregation data for these 6 markers were collected from 62 individuals of the population and analyzed for linkage relationship. Five marker loci were grouped together; 3 loci retained their order as in the original map of Ferreira et al. (Ferreira et al., supra); and the other 2 loci, tg6al2a and wg5a5, inverted their positions on the partial linkage map. Marker locus wg2dllb was originally mapped to the bottom of LG9 (Ferreira et al, supra), but was placed into another group by MAPMAKER using the segregation data in the BC,F₂. Although an understanding of the mechanism is not necessary in order to use the present invention, it is not known whether this is a result of a structural rearrangement or scarcity of markers in the neighboring region.

EXAMPLE 8 DNA Extraction At the 4-leaf stage, leaf tissue was taken, flash frozen in liquid nitrogen and stored at -80°C for genotypic assessment. DNA was extracted from approximately 5 g of young leaf tissue using the method of Dellaporta (Dellaporta, Plant Mol. Biol. Reptr., 1 :19-21 [1983]), with modifications according to Hawkins et al. (Hawkins et al, Genome 39:704-710 [1996]). Briefly, the plant tissue was ground to a fine powder with a mortar and pestle under liquid nitrogen, and then ground in 10 ml of extraction buffer (100 mM Tris- HC1, pH 8.0, 500 mM NaCl, 50 mM EDTA, pH 8.0, to which 7 μl of 144 mM of 2- mercaptoethanol was added fresh). To the slurry, 1 ml of 20% SDS was added and the sample was mixed thoroughly, then 70 μl of 50 mg/ml proteinase K was added and mixed, and the sample was incubated at 65 °C for 1 hour. To this, 2 ml of 5 M potassium acetate was added and sample mixed gently, but thoroughly, then placed on ice for 15 minutes. Samples were then centrifuged for 10 minutes at 10,000xg, and the supernatant filtered through a layer of Miracloth (CalBiochem). Samples were extracted with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), and centrifuged for 10 minutes at 10,000xg. The aqueous phase was removed, placed in a clean tube, and precipitated with 0.6 volumes of isopropanol. DNA was then spooled out of solution, rinsed three times in 70% ethanol, air dried, and dissolved in TE8 (10 mM Tris-HCl, pH 8.0, 1 ml EDTA, pH 8.0). Samples were treated with RNAse as known in the art (See e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, [1989]), then 5 M NaCl was added to a final concentration of 2.5 M (Fang et al, BioTech., 13:52-55 [1992]). DNA was precipitated with 2 volumes of 95% ethanol, placed at -20°C for 30 minutes, pelleted at 4,500xg for 10 minutes at 4°C, rinsed three times in 70% ethanol, and dried. Extracted DNA was resuspended in a minimal volume of TE8 (10 mM Tris- HC1, 1 mM EDTA, pH 8), and quantified using ethidium bromide fluorometry (Karsten and Wallenberger, Anal. Biochem., 77:464-470 [1972]). Briefly, 2 μl of DNA were mixed with 2.4 ml PBS (170 mM NaCl, 3.3 mM KC1, 10 mM Na₂HP0₄, 1.8 mM KH₂P0₄, pH 7.2), and 0.1 ml ethidium bromide (100 μg/ml). Samples were read on a

Varian SF-330 (Varian) spectrofluorometer, with excitation at 360 nm, and absorbance at 580 nm, and were compared with a calibration curve constructed using known quantities of lambda DNA.

Use of low temperature-induced clones (BN28, BN115, and corό.ό) revealed no polymorphism within the parents, or any of the DH, progeny. Figures 4A and 4B show the RFLP analysis of parent and DH, lines. These are representative blots probed with the cDNA clone BN28 from B. napus. In this Figure, "(cas)" indicates Cascade, "(reb)" indicates Rebel, "(F,)" indicates F,, and "(DH,)" indicates DH, lines. Acclimation- specific freezing tolerance was denoted as: (+) freezing tolerant; and (-) freezing sensitive. Only one cDNA (corό.ό) was previously shown to be associated with freezing tolerance, and this was in B. rapa (Teutonico et al, [1995], supra). However, it was not found to be linked nor associated with the population identified in this Example.

Genomic screening showed that 2 of 8 genomic clones, identified by Teutonico et al. (Teutonico et al, supra) as being linked to freezing tolerance had polymorphic differences between the parents. The polymorphism was complemented in the F,, and segregated within the DH, lines analyzed, as shown in Figures 5 and 6. Figure 5 is a representative blot probed with genomic clones A) EC2E5 and B) WG1F6. The same designations for the lines as used in Figure 4 are used in Figure 5. Figure 6 is a table showing the recombination analysis of DH, lines. In this Figure, the recombination frequency is scored against the freezing tolerance phenotype. WG1G6 did not appear to be associated with freezing tolerance, whereas EC2E5 appeared to be associated with freezing tolerance.

EXAMPLE 9

Southern Blotting

As described in this Example, the genetic basis for the loss of the vernalization requirement in VERN- was explored by probing Southern blots of VERN-, the parent cultivars, as well as a number of spring cultivars with clones that had been shown to map to vernalization and flowering time loci (designated VRN1 to VRN3) (Ferreira et al. supra; and Osborn et al, Genetics 146:1123-1129 [1997]). Figure 12 provides a graph showing the marker loci of Cascade, Rebel, and VERN-, using the marker order described by Ferreira et al. (Ferreira et al, supra).

Probe Preparation The genomic clones were isolated from a genomic library of B. napus, and were a gift from Dr. T.C. Osborn. BN115 and BN28 are clones from a low temperature cDNA library of B. napus cv Jet Neuf. The BN clones were a gift from Dr. J. Singh. The cold-regulated clone (corό.ό) came from a cDNA library of cold- acclimated Arabidopsis thaliana, and was a gift from Dr. M. Thomashow. All of the clones had T7 and T3 priming sites adjacent to the multiple cloning site of the clone.

This allowed for amplification of the inserts using PCR with Tag DNA polymerase. For each amplification, 0.1 μg of template was used. T7 and T3 primers were purchased from Promega. PCR was completed using a Techne PHC-2 thermocycler, using the fastest possible ramping times. DNA was initially denatured at 95°C for 5 minutes, followed by 35 cycles of 95°C for 1 minute, 39°C for 1 minute, and 72°C for

2 minutes. A final elongation at 72°C for 10 minutes was used to ensure that all products would be of equal length. The purity of the amplification products was assessed by electrophoresis of 2 μl of sample in 0.8% TAE agarose gel, and visualized by ethidium bromide staining as known in the art (See e.g., Sambrook et al, supra). Samples were purified using G-50 Sephadex in a spun column as known in the art (See, Sambrook et al, supra). Samples were quantified by fluorometry as described above, and 75-100 ng were used for each labelling reaction in the Southern blot procedure.

Blotting

Genomic DNA (20 or 30 μg) of VERN-, Cascade, Rebel, other DH, lines, and a spring control (Westar) were restricted according to the manufacturer's instructions using EcoRI or HmdIII (Boeringer Mannheim). Digested samples were separated on an 0.8% agarose gel in lx TAΕ at 80 volts for 3-5 hours (Sambrook et al. supra). Equal loading of restricted samples was assessed using ethidium bromide fluorescence of separated samples. Gels were capillary blotted onto Zeta-probe nylon membranes (Bio-Rad), using the semi-dry method of Rutledge et al. (Rutledge et al, Mol. Gen. Genet, 229:31-40 [1985).

DNA was cross-linked to the membrane by illumination at 254 nm, then baked at 80°C for 30 minutes under vacuum. Blots were pre-hybridized, and hybridized according to the membrane manufacturer's instructions, using 50% formamide at 43°C. Membranes were hybridized with ³²Pα dCTP (Amersham) labeled probe (Random Priming Kit, BRL #55567656), and rinsed at room temperature as follows—once for 2 minutes in 2x SSC and 0.1% SDS; once for 10 minutes in lx SSC and 0.1% SDS, once for 10 minutes in 0.5x SSC and 0.1% SDS; and a final wash at 65°C for 10 minutes in O.lx SSC and 0.1%) SDS. The membranes were then exposed to XAR-5 film (Kodak) with enhancing screens at -80°C, for 2-5 days. Individual lines were scored as being freezing tolerant (+) or scored as being freezing sensitive (-). Freezing tolerant (+) lines had TL₅₀s greater than or equal to -14.5°C. Freezing sensitive (-) lines had TL₅₀s less than or equal to -9°C. All plants falling between "tolerant" and "sensitive" were scored as "intermediates." Figure 4 is a Southern blot of EcoRI digests probed with WG6B 10. In this Figure, "Cas" indicates the lane containing Cascade, "Reb" indicates the lane containing Rebel, and "Wes" indicates the lane containing "Westar." The row indicated as "vern" shows whether the particular samples were vern+ or vern-. Also, "bcF,," indicates two lanes loaded with BCF,.

The EcoRI digests of genomic DNA probed with WG6B10 showed that VΕRN- is polymorphic to Cascade and Rebel. Figure 7A is a representative blot probed with WG6B10. The lanes containing Cascade (cas), Rebel (reb), and F, are indicated. The same polymorphic difference was observed between Westar and the winter cultivars. The spring types both lacked a band at 3.0 kb. A different polymorphism was also noted between Cascade and Rebel at this locus. Although this suggests that there may be more than one gene responsible for the phenotype, the bands always segregated with each other, indicated that they are closely linked. The results clearly indicate that there is no polymorphism in the winter type parents, but a polymorphic difference exists in Westar and the DH, line (6-200; VΕRN-), which also shows a spring type genotype. The polymorphism detected in VERN- is clearly not linked to freezing tolerance, as this line maintains good levels of both inherent and acclimated freezing tolerance, yet it no longer needs vernalization.

It was determined that the band at approximately 2.8 kb was found to be necessary, while the band at approximately 3.0 kb was lacking for the vern- phenotype. As shown in Figures 7B and 7C, VERN- and Rebel both have bands at "x kb," while Cascade has a band at "n kb." In Figure 7B and 7C, the "x kb" indicates the band at approximately 2.8 kb, while the "n kb" indicates the band at approximately 3.0 kb. In Figure 7C, the asterisked band in the VERN- lane (as well as the faint band at the same location in the F, lane) indicates a band that was apparently incompletely digested. In other blots, this band did not appear, and the last band in the lane was much larger (i.e., when the substrate is sufficiently digested, the DNA is present in the last band of the blot).

The approximately 3.0 kb band appears to be dominant, as plants that contain this band required vernalization (indicated by a "+" in Figure 7B). Plants that only have the approximately 2.8 kb band lacked the vernalization requirement (indicated by a "-" in Figure 7B). This makes the VERN- and Rebel cultivars, as well as the other lines containing the 2.8 kb band, but not the 3.0 kb band valuable as parent lines for development of spring-type cultivars (i.e., lacking the vernalization requirement), from winter type lines.

The wildtype genotype was restored when VERN- was backcrossed to either Cascade or Rebel, as predicted based on the phenotype of the BC,F, generation. As phenotypic and genotypic complementation between the winter cultivars and VERN- was demonstrated in the BC,F, lines using the clone WG6B10, the relative linkage was investigated. An example of a Southern blot used in this investigation is shown in

Figure 8. In this Figure, the vernalization requirement was denoted as "(+)" indicating lines with a requirement for vernalization, and "(-)" indicating lines that did not require vernalization.

Of 80 individual BC,F₂ plants, 23 had the VERN- polymorphism. As the phenotypic ratio was 61:19, this indicated a recombination frequency of approximately

8%o. In comparison, flanking clones WG7F3 and WG8G1 had recombination frequencies of 27.9%) and 25%, respectively, indicating that VERN- may more closely associated with WG6B10. The following Table (Table 10) shows the results of recombination analysis of backcross progeny of Cascade x VERN-. Probes from genomic clones were used. Recombination frequency was scored against the vernalization phenotype. Phenotypic assessment of vernalization requirement was based on the presence of reproductive bud formation was done at 35 and 70 days after planting.

Table 10. Recombination Analysis of Backcross Progeny Cascade x VERN- Cross

The marker interval wg7fa-wg6bl0 in LG9 was found to be strongly associated with the major flowering time locus VRN1 (Ferreira et al supra); this interval and its adjacent regions (i.e., from wg7f3a to wg5a5) in VERN- appear to have been inherited from Rebel. The genomic region around marker locus ec3g3c in LG12 contains VRN2

(Osborn et al. supra). It was also determined that VERN- carries Rebel's alleles at this locus as well. Comparison of the genotypes at ec3g3c, and its two adjacent loci (i.e., wg7b3 and wglg4), revealed that 2 crossing over events may have occurred in the F, plant that produced the VERN- line. This was the only locus at which VERN- carries alleles from Cascade. All other loci examined showed polymorphic differences between Cascade and Rebel, with Rebel as the contributor to the VERN- genotype. Thus, with regard to the marker interval wg9c7-wgόb2 in LG16 (associated with VFN3), VERN- and Rebel shared the same alleles, and Cascade had different alleles.

EXAMPLE 10 Occurrence and Inheritance of BN28

While molecular markers have been used in the search for cold-responsive loci (Cai et al, Theor. Appl. Genet., 89:606-614 [1994]), difficulties with this approach have been encountered due to the fact that genes for quantitative traits can act separately or pleotropically. Furthermore, inheritance patterns can be very complex. Searches for low temperature-induced genes resulted in the identification of such a gene in B. napus cv Jet Neuf, termed "BN28." Recently, a homologous gene was isolated from B. rapa (brkinl).

In this Example, the occurrence and inheritance of BN28 within the Brassicaceae family. As B. napus is an allotetraploid, it was thought that the gene encoding BN28 might be present in some form within all of the Brassica species.

This was investigated in this Example.

PCR and Southern Blots

DNA from various lines was prepared as described above and PCR was performed using 1 mg of target DNA and Taq DNA polymerase as describe above. Primers specific to the 5' and 3' ends of the coding region of BN28 were used (BN28 forward primer 5'-ATGTCAGAGACCAACAAGAAT-3' (SEQ ID NO:l), and BN28 reverse primer 5'-GTCTTGTCCTTCACGAAGTT-3' (SEQ ID NO:2). Amplified products were separated on 1.4% agarose gels in lx TAE at 5 volts/min., and visualized by ethidium bromide staining, as described above. The PCR results indicated that a single fragment was amplified in all diploid species tested. The size of the fragments were: B. nigra, 465 bp; B. oleracea, 425 bp; and B. rapa, 450 bp. Electrophoresis of a mixture of amplification products from each of the diploid species into resolvable fragments indicated that each fragment is of unique size. B. napus is the only allotetraploid that contained representative fragments from both of its original parents (B. oleracea and B. rapa). B. carinata contained the same sized fragment as B. rapa, and B. juncea contained the same sized fragment as B. oleracea. S. alba and S. arvensis amplified the same sized fragment as B. nigra.

Southern blots of 20 μg genomic DNA were digested as described in Example 9, above, with the exception that EcoRI and Pvu2 (Boehringer Mannheim) were used. The blot results indicated that each of the diploid parents contained a unique homologue of BN28, as shown in Figure 9. Diploid Brassicas are considered to be secondary aneuploids derived by duplication of chromosomes from an extinct common ancestor having six chromosomes. Duplication and extensive redistribution then gave rise to the three diploids outlined by U (U, Japan J. Bot, 7:389-452 [1935]).

Figure 9A shows the results obtained with the EcoRI-digested samples, and Figure 9B shows the results obtained with the Pvuϊl -digested samples. In Figure 9, "(Bn)" indicates B. nigra, "(Bo)" indicates B. oleracea, "(Br)" indicates B. rapa, while the B. napus cultivars were "(c)" Cascade, "(w)" Westar, "(Be)" indicates B. carinata, "(Bj)" indicates B. juncea, "(ar)" indicates S. arvensis, and "(al)" represents S. alba. The results indicate that specific homologous of BN28 are present in each of the diploids, and these homologues are transferred to individual allotetraploids, with B. napus being the only one to acquire homologous from both original parents.

Again, B. napus was the only allotetraploid to contain restriction fragments from both diploid parents. The other species tested, B. carinata, B. juncea, and S. arvensis, contained a gene from one diploid parent, in the same manner as the PCR analysis. S. alba contained a unique profile from all of the species represented. Coupled to PCR data, the Southern blots provided unambiguous identification of all genomes represented herein.

RNA Extraction

Total RNA was extracted from control and acclimated plant tissue (4°C for 14 days), using the hot phenol method of Verwoerd et al. (Verwoerd et al, Nucl. Acid Res., 17:2362-2366 [1989]). RNA was quantified using ethidium bromide fluorometry as described above in Example 8. Fluorometric values attained for RNA were multiplied by a DNA to RNA conversion factor of 2.17. Then, 30 mg of total RNA were separated on 1.4% agarose gels as known in the art. Gels were rinsed briefly in SSC and transferred to Zeta-Probe membranes as described in Example 9. RNA was cross-linked to the membrane by illumination, and hybridized with a ³²PαdCT-labelled

BN28 cDNA probe as described above, washed at high stringency twice for 10 minutes in 0.1 x SSC and 0.1% SDS at 60 °C, and exposed to XAR-5 film (Kodak) for 6-8 hours BN28 hybridizes to an mRNA transcript of approximately 0.5 kB in cold- acclimated tissues of B. napus. Analysis of acclimated and non-acclimated Brassicas was accomplished by probing equally loaded blots of total RNA for the presence of BN28 transcript using radiolabelled BN28 cDNA. No message was detected in any of the non-acclimated samples. After 10 days of low temperature acclimation, all of the tested species showed high levels of expression of BN28 mRNA with the exception of B. carinata, which showed a slightly lower level of accumulation. No significant difference in the size of the transcripts was detected in the different species examined. These Northern blot results are shown in Figure 10. In this Figure, the non- acclimating conditions are indicated with an "0" (i.e., 0 days), and low temperature conditions are indicated with a "14" (i.e., 14 days). The species abbreviations used in this Figure are the same as those described for Figure 9. These Northern analyses confirmed that each species expresses a BN28-like gene that was induced in response to low temperature. Even though different forms of BN28 were present, the coding regions appear to be sufficiently homologous to hybridize to a common cDNA probe under high stringency conditions. Although such an understanding is not necessary for practicing the present invention, similar patterns of stress induction may suggest that each of the three homologues present in the Brassicaceae contain conserved regulatory regions. In addition, although all of the Brassicaceae examined had low temperature- induced homologues of BN28, differences in protein expression were apparent.(See, Figure 11). Although such an understanding is not necessary to successfully practice the present invention, the lack of protein accumulation in B. nigra and S. arvensis suggests that gene silencing has occurred in these cultivars.

Protein Extraction and Immunoblotting

Total SDS-soluble proteins were extracted from approximately 0.1 g of plant tissue in 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1% SDS, and 10 mM PMSF, as known in the art. Protein concentrations were determined using the BCA method of Smith et al. (Smith et al, 150:76-85 [1985]). Then, 30 mg aliquots were mixed with an equal volume of 2x SDS loading buffer, and boiled for 5 minutes prior to loading Samples were separated on a 15% SDS-PAGE gel using a modified Tris-Tricine running buffer (Shagger and von Jagow, Anal. Biochem., 166:368-379 [1987]), at 30 milliamps (mA), until the tracking dye ran off the end of the gel.

Proteins were transferred to 0.22 μm supported nitrocellulose membranes (Schleicher and Schuell) in carbonate buffer, at 300 mA for 2 hours at 4°C. Blots were then reacted with BN28 specific antibody (Boothe et al, Plant Physiol., 108:795- 803 [1995]). The antibody was detected using alkaline phosphatase conjugated secondary antibody (Sigma) and visualized using the 5-bromo-4-chloro-3-indolyl phosphate-p-toluidine salt (BCIP)/p-nitroblue tetrazolium chloride salt (NBT) reagent system (Sigma).

The calculated molecular mass of BN28 protein is 6.6 kD, although the polypeptide migrates at a slightly lower apparent molecular mass. No accumulation of BN28 protein was detected in the non-acclimated samples. However, after 14 days of low temperature acclimation, all but two of the species tested showed protein accumulation. The results are shown in Figure 11. The same abbreviations as used in Figure 10 are used in the immunoblot in Figure 11. Again, there was no significant difference in apparent size in any of the species showing accumulation of BN28 protein. There was no detectable signal in B. nigra and S. arvensis. Testing several samples at several developmental stages, including very young and mature leaves, or increasing the protein concentration to 200 μg failed to produce any detectable signal.

It is clear from the above that the present invention provides compositions and methods for the production and use of improved Brassica, as well as methods and compositions for the development of additional strains and/or cultivars of agricultural importance.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in plant biology, molecular biology, plant genetics, or related fields are intended to be within the scope of the present invention.

Claims

CLAIMSWhat is claimed is:

1. A method for producing an improved Brassica cultivar, wherein said Brassica cultivar is vernalization negative, comprising the steps of: a) providing: i) a first parent Brassica line; and ii) a second parent Brassica line; b) crossing said first and second parent Brassica lines to produce a reciprocal cross; c) culturing said reciprocal cross to produce a microspore culture; and d) treating said microspore culture to produce a doubled haploid cultivar, wherein said doubled haploid is vernalization independent.

2. The method of Claim 1, further comprising the step of vernalizing said reciprocal cross.

3. The method of Claim 2, further comprising the step of vernalizing said doubled haploid.

4. The method of Claim 1, wherein said first and second parent Brassica lines are winter type.

5. The method of Claim 4, wherein said first parent Brassica line is selected from the group consisting of Cascade and Rebel.

6. The method of Claim 1, further comprising the step of growing said doubled haploid cultivar to produce seeds.

7. The method of Claim 6, further comprising the step of harvesting said seeds.

8. The method of Claim 1, wherein said doubled haploid cultivar is freezing tolerant.

9. A doubled haploid cultivar produced according to the method of Claim 1.

10. The doubled haploid cultivar of Claim 9, wherein said doubled haploid is freezing tolerant.

11. A winter Brassica cultivar expressing characteristics of spring Brassica cultivars.

12. The winter Brassica cultivar of Claim 11, wherein said cultivar is vernalization independent.

13. The winter Brassica cultivar of Claim 11, wherein said cultivar is

VERN-.

14. The winter Brassica cultivar of Claim 11, wherein said cultivar is freezing tolerant.

15. The winter Brassica cultivar of Claim 11, wherein one of the parents of said cultivar is Cascade.

16. The winter Brassica cultivar of Claim 11, wherein one of the parents of said cultivar is Rebel.

17. A method for producing Brassica cultivars, comprising the steps of crossing first and second Brassica plants, where at least one of said Brassica plants is characterized by at least one genetic factor which confers freezing tolerance, said genetic factor being capable of transmission to said Brassica cultivar substantially as a recessive gene.

18. A Brassica cultivar produced according to the method of Claim 17.

19. The method of Claim 17, wherein said genetic factor further confers vernalization independence.

20. The method of Claim 17, wherein said Brassica plant is selected from the group consisting of Cascade and Rebel.