Broccoli hybrid SVBL0308

Abstract

To provide new broccoli varieties.SOLUTION: The invention provides seeds and plants of broccoli hybrid SVBL0308. The invention thus relates to the plants, seeds and tissue cultures of broccoli hybrid SVBL0308 and to methods for producing a broccoli plant produced by crossing such plants with themselves or with another plant, such as a broccoli plant of another genotype. The invention further relates to seeds and plants produced by such crossing. The invention further relates to plants, seeds and tissue cultures of broccoli hybrid SVBL0308 comprising introduced beneficial or desirable traits.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 216,110, filed June 29, 2021, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to the field of plant breeding, and more particularly to the development of broccoli hybrid SVBL0308. [Background technology]

[0003] The goal of vegetable breeding is to combine a variety of desirable traits in a single variety. Such desirable traits may include any trait deemed useful or desirable by the breeder or consumer, including higher yield, resistance to insect or disease pests, tolerance to environmental stresses, and nutritional value.

[0004] Breeding techniques take advantage of how plants pollinate. There are two general pollination methods: a plant is self-pollinating when pollen from one flower is transferred to the same or another flower of the same plant or plant variety. A plant is cross-pollinating when pollen comes from a flower of a different plant variety.

[0005] Plants that have been self-pollinated and selected for variety over multiple generations become homozygous at almost all loci and produce a uniform population of homozygous plants that are true breeding progeny. Crossing two such homozygous plants with different genotypes produces a uniform population of hybrid plants that are heterozygous at many loci. Conversely, crossing two plants that are each heterozygous at multiple loci produces a population of genetically distinct, non-uniform hybrid plants. The resulting heterogeneity makes performance unpredictable.

[0006] The development of uniform varieties requires the development of homozygous inbred plants, the crossing of these inbred plants, and the evaluation of the crosses. Pedigree breeding and recurrent selection are examples of breeding methods that have been used to develop inbred plants from breeding populations. These breeding methods combine genetic backgrounds from two or more plants or a variety of other sources into a breeding pool from which new lines and hybrids are developed by selfing and selection for desired phenotypes. The new lines and hybrids are evaluated to determine which of them have commercial potential. Summary of the Invention

[0007] In one aspect, the present invention provides broccoli plants of hybrid SVBL0308. Also provided are broccoli plants having all the physiological and morphological characteristics of such plants. Parts of these broccoli plants are also provided, including, for example, pollen, ovules, embryos, florets, flower heads, seeds, and cells of the plant.

[0008] Another aspect of the present invention provides a plant of broccoli hybrid SVBL0308 that contains an added genetic trait. The genetic trait can include a genetic locus, i.e., for example, a dominant or recessive allele. In one embodiment of the present invention, a plant of broccoli hybrid SVBL0308 is defined as containing a single locus conversion. In a specific embodiment of the present invention, the added genetic locus confers one or more traits, such as herbicide tolerance, insect resistance, disease resistance, and altered carbohydrate metabolism. In a further embodiment, the trait can be conferred by a naturally occurring gene introduced into the genome of the line by backcrossing, by natural or artificial mutation, or by a transgene introduced into the plant or any of its ancestral ancestors using genetic transformation techniques. When introduced by transformation, the genetic locus can include one or more genes integrated at a single chromosomal location.

[0009] In some embodiments, the single locus conversion comprises one or more site-specific alterations to the plant genome, such as, but not limited to, one or more nucleotide modifications, deletions, or insertions. A single locus may comprise one or more genes or nucleotides integrated or mutated at a single chromosomal location. In one embodiment, the single locus conversion can be introduced by genetic engineering techniques, including, for example, engineered nuclease-mediated genome editing (GEEN). Engineered nucleases include, but are not limited to, Cas endonucleases; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); engineered meganucleases known as homing endonucleases; and other endonucleases for DNA- or RNA-dependent genome editing that are well known to those skilled in the art.

[0010] The present invention also relates to seeds of broccoli hybrid SVBL0308. In certain embodiments, the broccoli seeds of the present invention can be provided as an essentially homogeneous population of broccoli seeds of broccoli hybrid SVBL0308. An essentially homogeneous population of seeds generally does not contain a significant number of other seeds. Thus, seeds of broccoli hybrid SVBL0308 can be defined as forming at least about 97% of all seeds, for example, at least about 98%, 99%, or more of the seeds. The seed population can be grown separately to produce an essentially homogeneous population of broccoli plants designated SVBL0308.

[0011] In yet another aspect of the present invention, tissue cultures of regenerable cells of hybrid SVBL0308 broccoli plants are provided. The tissue cultures are preferably capable of regenerating broccoli plants capable of expressing all of the physiological and morphological characteristics of the starting plant, as well as plants having substantially the same genotype as the starting plant. Examples of some physiological and morphological characteristics of hybrid SVBL0308 include those shown in the tables herein. Regenerable cells in such tissue cultures can be obtained, for example, from embryos, meristems, cotyledons, pollen, leaves, anthers, roots, root tips, pistils, flowers, seeds, and stems. Furthermore, the present invention provides broccoli plants regenerated from tissue cultures of the present invention, which have all of the physiological and morphological characteristics of hybrid SVBL0308.

[0012] In yet another aspect of the present invention, processes for producing broccoli seeds, plants, and parts thereof are provided, generally comprising crossing a first parent broccoli plant with a second parent broccoli plant, where at least one of the first or second parent broccoli plants is a plant of broccoli line BRM8Z14-4006 or broccoli line BRM8Z13-3998. These processes may be further exemplified as processes for producing hybrid broccoli seeds or plants, in which a first broccoli plant is crossed with a second broccoli plant of a different and distinct genotype to produce a hybrid having a plant of broccoli line BRM8Z14-4006 or broccoli line BRM8Z13-3998 as one parent. In these processes, crossing results in the production of seed. Seed production occurs regardless of whether the seed is collected.

[0013] In one embodiment of the present invention, the first step of "crossing" involves sowing seeds of the first and second parent broccoli plants in close proximity so that pollination occurs, often via, for example, an insect vector. Alternatively, pollen can be transferred artificially. If the plant is self-pollinating, pollination can occur without the need for direct human intervention other than plant cultivation.

[0014] A second step can include cultivating or growing seeds of the first and second parent broccoli plants into flowering plants. A third step can include preventing self-pollination of the plants, for example, by emasculating the flowers (i.e., killing or removing the pollen).

[0015] A fourth step for hybridization can include cross-pollinating between the first and second parent broccoli plants. A further step includes harvesting seeds from at least one of the parent broccoli plants. The harvested seeds can be grown to produce broccoli plants or hybrid broccoli plants.

[0016] The present invention also provides broccoli seeds and plants produced by a process comprising crossing a first parent broccoli plant with a second parent broccoli plant, wherein at least one of the first or second parent broccoli plants is a plant of broccoli hybrid SVBL0308, broccoli line BRM8Z14-4006, or broccoli line BRM8Z13-3998. In one embodiment of the present invention, the broccoli seeds and plants produced by the process are first-generation (F1) hybrid broccoli seeds and plants produced by crossing a plant with another distinct plant according to the present invention. The present invention further contemplates plant parts of such F1 hybrid broccoli plants and methods of use thereof. Accordingly, certain exemplary embodiments of the present invention provide F1 hybrid broccoli plants and seeds thereof.

[0017] In yet another aspect, the present invention provides a method for producing a plant derived from hybrid SVBL0308, the method comprising: (a) generating a progeny plant derived from hybrid SVBL0308, wherein the generating comprises crossing the hybrid SVBL0308 plant with a second plant; and (b) crossing the progeny plant with itself or a second plant to produce seeds of a next-generation progeny plant. In a further embodiment, the method may further comprise (c) growing a next-generation progeny plant from the seeds of the next-generation progeny plant and crossing the next-generation progeny plant with itself or a second plant; and repeating the steps for 3 to 10 more generations to produce plants derived from hybrid SVBL0308. Plants derived from hybrid SVBL0308 may be inbred, and the repeated crossing steps may be defined as including sufficient inbreeding to produce an inbred line. In the method, it may be desirable to select a particular plant obtained from step (c) for continued crossing via steps (b) and (c). By selecting plants that have one or more desired traits, plants derived from hybrid SVBL0308 are obtained that have some of the desired traits of the line / hybrid and possibly other selected traits.

[0018] In one embodiment, the present invention provides a method for producing a food or feed product, the method comprising: (a) obtaining a plant or part thereof of broccoli hybrid SVBL0308; and (b) producing a food or feed product from the plant or part thereof. In one embodiment, the food or feed product is soup. In another embodiment, the plant has been grown to maturity.

[0019] In one aspect, the present invention provides an edible composition comprising at least one cell of broccoli hybrid SVBL0308. In one embodiment, the edible composition is defined as a food or feed product. In another embodiment, the edible composition is defined as a soup.

[0020] In some embodiments, the present invention provides methods for producing an edible composition, the method comprising: (a) obtaining a plant or part thereof of broccoli hybrid SVBL0308; and (b) producing an edible composition from the plant or part thereof. In one embodiment, the edible composition is defined as a food or feed product. In another embodiment, the edible composition is defined as a soup. In yet another embodiment, the plant is grown to maturity.

[0021] In another aspect of the present invention, genetic complementation of broccoli hybrid SVBL0308 is provided. The term "genetic complementation" is used to refer to a collection of nucleotide sequences whose expression, in the present case, defines the phenotype of a broccoli plant, or a cell or tissue of that plant. Thus, genetic complementation refers to the genetic complementation of a cell, tissue, or plant, and hybrid genetic complementation refers to the genetic complementation of a hybrid cell, tissue, or plant. Thus, the present invention provides broccoli plant cells having a genetic complementation with the broccoli plant cells disclosed herein, as well as seeds and plants containing such cells.

[0022] The genetic complement of a plant can be assessed by genetic marker profiles and the expression of phenotypic traits characteristic of the genetic complement, such as isozyme classification profiles. It is understood that hybrid SVBL0308 can be identified by any of a number of well-known techniques, such as simple sequence length polymorphism (SSLP) (Williams et al., Nucleic Acids Res., 18:6531-6535, 1990), random amplified fragment polymorphism DNA (RAPD), DNA amplification fingerprinting (DAF), sequence-characterized amplified region (SCAR), arbitrarily sequence-primed polymerase chain reaction (AP-PCR), amplified fragment length polymorphism (AFLP) (EP534858, the entire contents of which are specifically incorporated herein by reference), and single nucleotide polymorphism (SNP) (Wang et al., Science, 280:1077-1082, 1998).

[0023] In yet another aspect, the present invention provides a hybrid genetic complement, represented by broccoli plant cells, tissues, plants, and seeds, formed by combining the haploid genetic complement of a broccoli plant of the present invention with the haploid genetic complement of a second broccoli plant, preferably another separate broccoli plant. In another aspect, the present invention provides a broccoli plant regenerated from tissue culture comprising the hybrid genetic complement of the present invention.

[0024] In yet another aspect, the present invention provides a method for determining the genotype of a broccoli hybrid SVBL0308 plant, the method comprising detecting at least a first polymorphism in the plant's genome. In certain embodiments, the method may comprise detecting multiple polymorphisms in the plant's genome. The method may further comprise storing results of detecting the multiple polymorphisms on a computer-readable medium. The present invention further provides a computer-readable medium produced by such a method.

[0025] Any embodiment discussed herein with respect to one aspect of the invention also applies to other aspects of the invention unless specifically stated otherwise.

[0026] The term "about" is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. The use of the word "or" in the claims is used to mean "and / or" unless otherwise clearly indicated to refer to alternatives only or that the alternatives are mutually exclusive. When used in the claims with the word "comprise" or other open-ended language, the words "a" and "an" mean "one or more" unless specifically stated otherwise. The terms "comprise," "have," and "comprise" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprises," "comprising," "have," "having," "includes," and "including," are also open-ended. For example, any method that "comprises," "having," or "includes" one or more steps is not limited to having only those one or more steps, but also includes other unlisted steps. Similarly, any plant that "comprises," "has," or "includes" one or more traits is not limited to having only those one or more traits, but also covers other traits not listed.

[0027] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and any specific examples provided, while indicating specific embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention provides methods and compositions relating to plants, seeds and derivatives of broccoli hybrid SVBL0308, broccoli line BRM8Z14-4006 or broccoli line BRM8Z13-3998.

[0029] Broccoli hybrid SVBL0308, also known as 19-8Z-BRO-0308, contains the Myb28 allele that confers increased glucosinolates as described in US Pat. No. 9,617,554, specifically incorporated herein by reference.

[0030] A. Origin and breeding history of broccoli hybrid SVBL0308 The parents of broccoli hybrid SVBL0308 are broccoli line BRM8Z14-4006 and broccoli line BRM8Z13-3998. The parent lines are uniform and stable, as are the hybrids produced from them. During repeated breeding, a small percentage of variation may occur within commercially acceptable limits for almost any characteristic. However, variation is not expected.

[0031] B. Physiological and morphological characteristics of broccoli hybrid SVBL0308 According to one aspect of the present invention, there is provided a plant having the physiological and morphological characteristics of broccoli hybrid SVBL0308. A description of the physiological and morphological characteristics of such a plant is provided in the table below. [Table 1-1] [Table 1-2] [Table 1-3]

[0032] C. Breeding of Broccoli Plants One aspect of the present invention relates to a method for producing seeds of broccoli hybrid SVBL0308, comprising crossing broccoli lines BRM8Z14-4006 and BRM8Z13-3998. Alternatively, in other embodiments of the present invention, broccoli hybrid SVBL0308 can be crossed with itself or any second plant. Such methods can be used for the propagation of broccoli hybrid SVBL0308 or to produce plants derived from broccoli hybrid SVBL0308. Plants derived from broccoli hybrid SVBL0308 can, in some embodiments, be used for the development of new broccoli varieties.

[0033] The development of new varieties using one or more starting varieties is well known in the art. In accordance with the present invention, new varieties can be created by crossing broccoli hybrid SVBL0308 and subsequent breeding for multiple generations according to such well-known methods. New varieties can be created by crossing with any second plant. In selecting such second plants to cross for the purpose of developing new lines, it may be desirable to select plants that exhibit one or more selected desired characteristics by themselves or that exhibit the desired characteristic(s) when combined in a hybrid. Once the initial cross is made, inbreeding and selection are performed to create new varieties. Five or more generations of selfing and selection are often required to develop a uniform line.

[0034] Uniform lines of new varieties can also be developed by doubled haploids. This technique allows for the creation of true breeding lines without the need for multiple generations of selfing and selection. In this way, true breeding lines can be produced in as little as one generation. Haploid embryos can be produced from pollen grains, pollen, anther cultures, or ovule cultures. The haploid embryos can then be doubled automatically or by chemical treatment (e.g., colchicine treatment). Alternatively, the haploid embryos can be grown into haploid plants and treated to induce chromosome doubling. In either case, fertile homozygous plants are obtained. According to the present invention, any of these techniques can be used in combination with the plants of the present invention and their progeny to obtain homozygous lines.

[0035] Backcrossing may also be used to improve inbred plants. Backcrossing transfers a specific desirable trait from one inbred or off-inbred source to an inbred lacking that trait. This can be accomplished, for example, by first crossing a superior inbred line (A) (the recurrent parent) to a donor inbred line (the non-recurrent parent) that carries the appropriate locus or loci for the trait in question. The progeny of this cross are then bred back with the superior recurrent parent (A), followed by selection of the resulting progeny for the desired trait to be transferred from the non-recurrent parent. After five or more generations of backcrossing with selection for the desired trait, the progeny will possess the transferred characteristic but resemble the superior parent at most or almost all other loci. The final backcross generation will be selfed to obtain pure-breeding progeny for the transferred trait.

[0036] The plants of the present invention are particularly well suited for the development of new lines based on the selective nature of the plant's genetic background. When selecting a second plant to cross with broccoli hybrid SVBL0308 for the purpose of developing a new broccoli line, it is typically preferred to select a plant that exhibits one or more selected desired characteristics by itself or that exhibits the desired characteristic(s) when combined in a hybrid. In specific embodiments, examples of desirable traits may include high seed yield, high seed germination rate, seedling vigor, high yield, disease resistance or tolerance, and adaptability to soil and climatic conditions. Consumer-oriented traits, such as flower head shape, nutritional value, and flavor, are other examples of traits that may be incorporated into new lines of broccoli plants developed according to the present invention.

[0037] D. Further Embodiments of the Invention Certain aspects of the present invention provide plants as described herein that have been modified to contain at least a first desired genetic trait. Such plants, in some embodiments, can be produced by a plant breeding technique known as backcrossing, and essentially all morphological and physiological characteristics of a variety are restored to normal in addition to the locus transferred to the plant via the backcrossing technique. As used herein, the term "single-locus transformed plant" refers to a broccoli plant produced by a plant breeding technique known as backcrossing or genetic engineering, and in addition to the transfer of a single locus to a variety, essentially all morphological and physiological characteristics of the variety are restored to normal in addition to the backcrossing technique or genetic engineering, respectively. By "essentially all morphological and physiological characteristics," we mean that the characteristics of the plant that cause it to exist differently than when compared in the same environment are restored to normal or preserved, other than incidental mutations that may arise during backcrossing, the introduction of a transgene, or the use of genetic engineering techniques.

[0038] Backcrossing techniques can be used with the present invention to improve existing varieties or to introduce traits into existing varieties. The parent broccoli plant that provides the locus for the desired trait is called the nonrecurrent parent or donor parent. This terminology means that the nonrecurrent parent is used only once in the backcrossing protocol, i.e., it is not used repeatedly. The parent broccoli plant that receives the transfer of one or more loci from the nonrecurrent parent is known as the recurrent parent because it is used multiple times in the backcrossing protocol.

[0039] In a typical backcrossing protocol, the original variety of interest (the recurrent parent) is crossed with a second variety (the nonrecurrent parent) that carries the single locus of the introgression of interest. The progeny of such a cross are then crossed back to the recurrent parent, and the process is repeated until the converted plant possesses essentially all of the morphological and physiological characteristics of the recurrent parent in addition to the single locus inherited from the nonrecurrent parent.

[0040] Selection of an appropriate recurrent parent is a critical step in the successful implementation of the backcrossing procedure. The goal of a backcrossing protocol is to alter or replace a single trait or characteristic possessed by the original variety. To accomplish this, a single locus in the recurrent variety is modified or replaced with the desired locus from the nonrecurrent parent, while essentially retaining all but the desired gene, thereby preserving the desired physiological and morphological makeup of the original variety. The choice of a particular nonrecurrent parent will depend on the purpose of the backcross; one of the primary goals is to add some commercially desirable trait to the plant. The exact backcrossing protocol will vary depending on the characteristic or trait to be altered and the genetic distance between the recurrent and nonrecurrent parents. Backcrossing is simplified when the trait being transferred is a dominant allele, but recessive alleles or additional alleles (between recessive and dominant) can also be transferred. In this case, it may be necessary to conduct tests on the progeny to determine whether the desired trait was successfully transferred.

[0041] In one embodiment, progeny broccoli plants from a backcross using a plant described herein as the recurrent parent contain (i) the desired traits from the non-recurrent parent and (ii) all the physiological and morphological characteristics of the recurrent parent, as determined at a 5% significance level observed when grown under the same environmental conditions.

[0042] New varieties can also be produced from more than two parents. A technique known as modified backcrossing uses a different recurrent parent during backcrossing. Modified backcrossing can be used to replace the original recurrent parent with a variety that has a specific, more desirable characteristic, or multiple parents can be used to obtain different desirable characteristics from each.

[0043] By developing molecular markers associated with specific traits, it becomes possible to add additional traits to established germlines, such as those described herein, ultimately resulting in substantially the same basic genetic resource with one or more novel traits added. Molecular breeding, as described, for example, in Moose and Mumm, 2008 (Plant Physiology, 147:969-977), provides a mechanism for incorporating one or more traits or QTLs into elite lines. This molecular breeding-facilitated transfer of one or more traits into elite lines can involve incorporating specific genomic fragments associated with the particular trait of interest into the elite line by using assays of adjacent or associated markers to identify the incorporated genomic fragments. In the embodiments described herein, for example, one, two, three, or four genomic loci can be integrated into the elite line in this manner. When this elite plant containing additional loci is further crossed with another elite parent plant to produce hybrid progeny, at least eight separate additional loci can be incorporated into the hybrid. These additional loci can confer traits such as disease resistance or fruit quality traits, for example. In some embodiments, each locus can confer a separate trait. In other embodiments, the loci must be homozygous and incorporated into each parent plant to confer the trait to the hybrid. In yet other embodiments, multiple loci can be combined to confer a single, consistent phenotype of the desired trait.

[0044] Numerous single-locus traits have been identified that are not typically selected for in the development of new inbred lines, but can be improved with backcrossing techniques. Single-locus traits may or may not be due to a transgene; examples of these traits include, but are not limited to, herbicide resistance, resistance to bacterial, fungal, or viral diseases, insect resistance, modified fatty acid or carbohydrate metabolism, and altered nutritional value. These generally involve genes that are inherited via the nucleus.

[0045] When a single locus functions as a dominant trait, direct selection can be applied. For this selection process, the progeny of the initial cross are assayed for the presence of virus resistance, or the corresponding gene, before backcrossing. Selection eliminates any plants that do not have the desired gene and resistance trait, and only plants that have the trait are used in subsequent backcrosses. This process is then repeated in all further backcross generations.

[0046] Selection of broccoli plants for breeding does not necessarily depend on the plant's phenotype, but can instead be based on genetic studies. For example, appropriate genetic markers closely linked to the trait of interest can be utilized. One of these markers can be used to identify the presence or absence of the trait in the progeny of a particular cross and can be used to select the progeny during continued breeding. This technique is generally referred to as marker-assisted selection. Any other type of genetic marker or other assay capable of identifying the relative presence or absence of a trait of interest in a plant is also useful for breeding purposes. Marker-assisted selection procedures are well known in the art. Such methods are particularly useful in cases of recessive traits and variable phenotypes, or in cases where traditional assays are expensive, time-consuming, or otherwise disadvantageous. Additionally, marker-assisted selection can be used to identify plants containing desired genotypes at the seed, sowing, or plant stage, or to identify or assay the purity of cultivars, to classify the genetic diversity of germplasm collections, and to monitor specific alleles or haplotypes within established cultivars.

[0047] Types of genetic markers that may be used in accordance with the present invention include Simple Sequence Length Polymorphisms (SSLP) (Williams et al., Nucleic Acids Res., 1 8:6531 6535, 1990), Randomly Amplified Polymorphic DNA (RAPD), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCAR), Arbitrary Primed Polymerase Chain Reaction (AP-PCR), Amplified Fragment Length Polymorphisms (AFLP) (EP 534 858, the entire contents of which are expressly incorporated herein by reference), and Single Nucleotide Polymorphisms (SNP) (Wang et al., Science, 280:1077-1082, 1998), but are not necessarily limited to these.

[0048] In certain embodiments of the present invention, marker-assisted selection can be used to increase the efficiency of backcross breeding schemes to produce broccoli lines containing desired traits. This technique is commonly referred to as marker-assisted backcrossing (MABC). This technique is well known in the art and involves the use of three or more levels of selection, such as foreground selection to identify the presence of desired loci, which can complement or replace phenotypic screening protocols, recombination selection to minimize drug binding, and background selection to maximize reversion of the recurrent parental genome to a normal state.

[0049] E. Plants obtained through genetic engineering A variety of genetic engineering techniques have been developed and can be used by those skilled in the art to introduce traits into plants. In certain claimed embodiments, a single locus or transgene is modified or introduced into the genome of the progeny of the varieties described herein to introduce the trait into the broccoli plant. Genetic engineering methods for modifying, deleting, or inserting genes and polynucleotides of interest into the genomic DNA of plants are well known in the art.

[0050] In certain embodiments of the present invention, superior broccoli lines can be produced through site-specific modification of plant genome.Genetic engineering methods include, for example, sequence-specific nucleases, such as zinc finger nucleases (see, for example, US Patent Application Publication No. 2011-0203012); engineered meganucleases or native meganucleases; TALE-endonucleases (see, for example, US Patent Application Publication No. 8,586,363 and US Patent Application Publication No. 9,181,535); and RNA-guided endonucleases, such as RNA-guided endonucleases in CRISPR / Cas systems (see, for example, US Patent Application Publication No. 8,697,359 and US Patent Application Publication No. 8,771,945 and US Patent Application Publication No. 2014-0068797).Therefore, some embodiments of the present invention relate to using nucleases or any related proteins to carry out genome modification. This nuclease can be provided heterologously within the donor template DNA for templated genome editing, or in a separate molecule or vector. The recombinant DNA construct can also include one or more guide RNAs that direct the nuclease to the site in the plant genome that will be modified. In addition, a method for modifying or introducing a single locus has been described, for example, by using a single-stranded oligonucleotide to introduce base pair modifications into the broccoli plant genome (see, for example, Sauer et al., Plant Physiol, 170(4):1917-1928, 2016).

[0051] Methods for site-specific modification or introduction of a single locus are well known in the art and include the use of sequence-specific nucleases, such as those described above, or protein-guide RNA complexes that cleave genomic DNA to create a double-strand break (DSB) or nick at the locus. As is well known in the art, during the process of repairing the DSB or nick caused by the nuclease enzyme, a donor template, transgene, or expression cassette polynucleotide can be integrated into the genomic DSB or nick site. The presence of homologous arms in the integrating DNA can facilitate the acceptance and targeting of the insertion sequence into the plant genome during the repair process via homologous recombination or non-homologous end joining (NHEJ).

[0052] In another embodiment of the invention, transformation may be used to insert a selected transgene into a plant of the invention or to prepare a transgene that can be introduced by backcrossing. Methods of plant transformation that are well known to those skilled in the art and available for many crop species include, but are not limited to, electroporation, microprojectile bombardment, Agrobacterium-mediated transformation, and direct DNA uptake by protoplasts.

[0053] For efficient transformation by electroporation, friable tissues such as cell suspension cultures or embryogenic callus can be used, or immature embryos or other organized tissues can be transformed directly. In this technique, the cell walls of selected cells are partially degraded by exposure to pectin-degrading enzymes (pectolyases) or by mechanically damaging the tissue in a controlled manner.

[0054] An efficient method for delivering transforming DNA segments into plant cells is microprojectile bombardment. In this method, particles are coated with nucleic acids and delivered into the cells by propelling force. Exemplary particles are composed of tungsten, platinum, and, preferably, gold. For use in bombardment, the cell-containing suspension can be concentrated on a filter or solid medium, or immature embryos or other target cells can be placed on solid culture medium. The cells to be bombarded are placed at an appropriate distance below the macroprojectile stopping plate.

[0055] An exemplary embodiment of a method for accelerating and delivering DNA to plant cells is the Biolistics Particle Delivery System, which can be used to propel DNA-coated particles or cells onto a surface coated with target cells using a screen, such as a stainless steel or Nytex screen. The screen disperses the particles, preventing large particle clumps from being delivered to the recipient cells. Microprojectile technology is widely available and can be used to transform virtually any plant species.

[0056] Agrobacterium-mediated transfer is another widely available system for introducing genetic loci into plant cells. The advantage of this technique is that DNA can be introduced into whole plant tissues, thereby eliminating the need to regenerate intact plants from protoplasts. Modern Agrobacterium transformation vectors are replicable in both E. coli and Agrobacterium and are easy to manipulate (Klee et al., Nat. Biotechnol., 3(7):637-642, 1985). Furthermore, recent technological advances in vectors for Agrobacterium-mediated gene transfer have redefined the placement of genes and restriction sites in vectors, facilitating the construction of vectors capable of expressing genes encoding various polypeptides. The vectors described herein contain a convenient multilinker region flanked by promoters and polyadenylation sites for direct expression of inserted polypeptide-encoding genes. In addition, Agrobacterium containing both modified and unmodified Ti genes can be used for transformation.

[0057] In plant strains where Agrobacterium-mediated transformation is efficient, it is the method of choice due to the ease and defined nature of the gene locus transfer. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art (Fraley et al., Nat. Biotechnol., 3:629-635, 1985; U.S. Patent No. 5,563,055).

[0058] Transformation of plant protoplasts can also be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, e.g., Potrykus et al., Mol. Gen. Genet., 199:183-188, 1985; Omirulleh et al., Plant Mol. Biol., 21(3):415-428, 1993; Fromm et al., Nature, 312:791-793, 1986; Uchimiya et al., Mol. Gen. Genet., 204:204, 1986; Marcotte et al., Nature, 335:454, 1988). Plant transformation and expression of foreign genetic elements are exemplified by Choi et al. (Plant Cell Rep., 13: 344-348, 1994) and Ellul et al. (Theor. Appl. Genet., 107:462-469, 2003).

[0059] Numerous promoters are useful for directing plant gene expression of genes of interest, including, but not limited to, selectable markers, scorable markers, genes for pest resistance, disease resistance, nutritional enhancement, and any other gene of agricultural interest. Examples of constitutive promoters useful for plant gene expression include the cauliflower mosaic virus (CaMV) P-35S promoter (see, e.g., Odel et al., Nature, 313:810, 1985), which exhibits constitutive high-level expression in most plant tissues, including monocotyledons (see, e.g., Dekeyser et al., Plant Cell, 2:591, 1990; Terada and Shimamoto, Mol. Gen. Genet., 220:389, 1990); the enhanced 35S promoter (P-e35S), which is a tandemly duplicated form of the CaMV 35S promoter; the nopaline synthase promoter (An et al., Plant Physiol., 88:547, 1988); and the octopine synthase promoter (Fromm et al., Plant Cell, 1:977, 1989); and the Figwort mosaic virus (P-FMV) promoter described in U.S. Pat. No. 5,378,619, an enhanced version of the FMV promoter (P-eFMV) in which the promoter sequence of P-FMV is duplicated in tandem, the cauliflower mosaic virus 19S promoter, the sugarcane bacillus virus promoter, the Commelinus yellow mottle virus promoter, and other plant DNA virus promoters known to express in plant cells, but are not limited to these.

[0060] Various plant gene promoters that are regulated in response to environmental, hormonal, chemical, and / or developmental signals can also be used to express operably linked genes in plant cells, and examples of such promoters include: (1) heat (Callis et al., Plant Physiol., 88:965, 1988); (2) light (e.g., the pea rbcS-3A promoter, Kuhlemeier et al., Plant Cell, 1:471, 1989; the maize rbcS promoter, Schaffner and Sheen, Plant Cell, 3:997, 1991; or the chlorophyll a / b-binding protein promoter, Simpson et al., EMBO J., 4:2723, 1985); and (3) hormones, such as abscisic acid (Marcotte et al., Plant Cell, 1:969, (1989), (4) wound-regulated promoters (e.g., Wunl, Siebertz et al., Plant Cell, 1:961, 1989); or (5) chemically regulated promoters, such as methyl jasmonate, salicylic acid, or safeners. Organ-specific promoters (e.g., Roshal et al., EMBO J., 6:1155, 1987; Schernthaner et al., EMBO J., 7:1249, 1988; Bustos et al., Plant Cell, 1:839, 1989) can also be advantageously used.

[0061] Representative nucleic acids of the invention that can be introduced into plants include, for example, DNA sequences or genes from another species, or genes or sequences derived from or present in the same species but introduced into the recipient cell by genetic engineering rather than traditional breeding or reproduction techniques. However, the term "exogenous" also refers to genes not normally present in the cell being transformed, or perhaps simply not present in the same form or structure as found in the DNA segment or gene being transformed, or genes normally present and desired to be expressed in a manner different from their natural expression pattern, e.g., overexpressed. Thus, the term "exogenous" gene or DNA is intended to refer to any gene or DNA segment introduced into a recipient cell, regardless of whether a similar gene is already present in the recipient cell. Types of DNA included in exogenous DNA include DNA already present in the plant cell, DNA from another plant, DNA from a different organism, or exogenously produced DNA, such as a DNA sequence containing an antisense message for a gene or encoding a synthetic or modified version of a gene.

[0062] Hundreds, if not thousands, of different genes are known that could potentially be introduced into the broccoli plants of the present invention. Examples of particular genes, and corresponding phenotypes, that may be selected for introduction into broccoli plants include, but are not limited to, one or more genes for insect resistance, e.g., Bacillus thuringiensis (Bt) genes, pest resistance, e.g., genes for fungal disease control, herbicide resistance, e.g., genes that confer glyphosate tolerance, and genes for quality improvement, e.g., yield, nutritional enhancement, environmental or stress tolerance, or any desired change in plant physiology, growth, development, morphology, or plant product(s). For example, the structural gene may be any gene that confers insect resistance, such as, but not limited to, the Bacillus insect control protein gene described in WO 99 / 31248, U.S. Patent No. 5,689,052, the entire contents of which are incorporated herein by reference, and U.S. Patent Nos. 5,500,365 and 5,880,275, the entire contents of which are incorporated herein by reference. In another embodiment, the structural gene may confer tolerance to the herbicide glyphosate, conferred by various genes, such as, but not limited to, the Agrobacterium strain CP4 glyphosate resistance EPSPS gene (aroA:CP4) described in U.S. Patent No. 5,633,435, the entire contents of which are incorporated herein by reference, or the glyphosate oxidoreductase gene (GOX) described in U.S. Patent No. 5,463,175, the entire contents of which are incorporated herein by reference.

[0063] Alternatively, the DNA coding sequence can encode a non-translated RNA molecule that targets and inhibits the expression of the endogenous gene, thereby affecting these phenotypes, for example, through antisense or co-suppression-mediated mechanisms (see, e.g., Bird et al., Biotech. Gen. Engin. Rev., 9:207, 1991). The RNA can be a catalytic RNA molecule (i.e., a ribozyme) engineered to cleave the desired endogenous mRNA product (see, e.g., Gibson and Shillito, Mol. Biotech., 7:125, 1997). Thus, any gene that produces a protein or mRNA that results in a phenotype or morphology of interest is useful in practicing the present invention.

[0064] F. Edible composition As used herein, "edible composition" refers to a composition that can be ingested by a mammal, such as a food, feed, or pharmaceutical composition. As used herein, "food" and "feed" refer to a substance that can be used or prepared as a food for an animal or human, and include substances that can be used in the preparation of food or that can be used as a food additive. Typical foods or feeds include, but are not limited to, soups, juices, smoothies, spreads, yogurt, sauces, gravies, quiches, pies, prepared vegetable products, such as vegetable bakes, and mixed vegetable products. Additionally, the edible compositions described herein can be ingested as additives or supplements. They can be formulated with nutritional substances, such as various vitamins and minerals, and can be in a substantially liquid composition, a substantially solid composition, or gelatin. The edible compositions described herein can, in some embodiments, be in powder form.

[0065] As used herein, a "pharmaceutical composition" refers to a composition that includes a pharmaceutically acceptable carrier. In certain embodiments, the term "pharmaceutically acceptable" refers to a carrier that is suitable for administration to an animal or a human. Edible compositions of the present disclosure can include, for example, any pharmaceutically acceptable carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Edible compositions of the present disclosure can be administered, for example, as a tablet, capsule, elixir, solution, or suspension.

[0066] The edible compositions described herein may be produced using any method known in the art, including, but not limited to, mixing, blending, freezing, heating, cooling, freeze-drying, pasteurization, and cooking using any suitable method.

[0067] The broccoli of the present invention can be used in any product containing broccoli, including, but not limited to, broccoli, broccoli parts, broccoli extracts or derivatives, or broccoli cells. Broccoli can be used as an ingredient, component, preparation, extract, or derivative. Broccoli can be present as identifiable or non-identifiable parts. Broccoli can be fresh, frozen, or dried. Broccoli can be used in any food, beverage, extract, supplement, dietary supplement, or the like, for any purpose, including, but not limited to, human or animal food, feed, or supplement. In particular, broccoli can be used in soups, convenience meals, snack bars, beverages, smoothies, milkshakes, tablets, capsules, injectables, vitamins, or other similar products, including fresh or frozen intact broccoli products, or intact or processed broccoli.

[0068] G. Definition In the description and tables herein, a number of terms are used. For a clear and consistent understanding of the specification and claims, the following definitions are provided:

[0069] Allele: Any of one or more alternative forms of a genetic locus, all of whose alleles are associated with a single trait or characteristic. In diploid cells or organisms, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

[0070] Backcrossing: The process by which breeders repeatedly cross hybrid progeny, such as a first generation hybrid (F1), with one of the parents of the hybrid progeny. Backcrossing can be used to introduce one or more single locus conversions or transgenes from one genetic background into another.

[0071] Hybridization: The crossing of two parent plants.

[0072] Cross-pollination: fertilization by the joining of two gametes from different plants.

[0073] Diploid: a cell or organism that has two sets of chromosomes.

[0074] Emasculation: Removal of the male organs of a plant or their inactivation using cytoplasmic or nuclear genetic elements or chemicals that result in male sterility.

[0075] Enzyme: A molecule that acts as a catalyst in a biological reaction.

[0076] F1 hybrid: first generation offspring of a cross between two non-isogenic plants.

[0077] Genotype: the genetic makeup of a cell or organism.

[0078] Haploid: A cell or organism that has one set of two chromosomes in diploid form.

[0079] Linkage: the tendency of alleles on the same chromosome to segregate together more frequently than would be expected by chance if their inheritance were independent.

[0080] Marker: A phenotype that has no component of environmental variance, i.e., a heritability of 1, is easily detectable, and is preferably inherited in a codominant manner (both alleles of a locus in a diploid heterozygote are easily detectable).

[0081] Phenotype: The detectable characteristics of a cell or organism, characterized by the manifestation of gene expression.

[0082] Quantitative trait loci (QTL): Quantitative trait loci (QTL) are genetic loci that control, to some extent, a quantifiable trait that is usually continuously distributed.

[0083] Resistant: As used herein, the terms "resistant" and "tolerant" are used interchangeably to describe plants that do not show symptoms of a particular pest, pathogen, abiotic influence, or environmental condition. These terms are also used to describe plants that show some symptoms but are capable of producing acceptable yields of marketable product. Some plants that are designated as resistant or tolerant are so designated only in the sense that the plant's development is stunted and yield is reduced, but it is still capable of producing a crop.

[0084] Regeneration: The development of plants from tissue culture.

[0085] Royal Horticultural Society (RHS) Colour Chart Values: The RHS colour chart is a standardised standard that allows for the accurate identification of any colour. A colour's name on the chart describes its hue, brightness and saturation. Colours are accurately named on the RHS colour chart by specifying the group name, sheet number and symbol, for example, Yellow-Orange Group 19A, or Red Group 41B.

[0086] Self-pollination: the transfer of pollen from anther to the stigma of the same plant.

[0087] Single-locus transformed plant (single-locus transformed plant): A plant produced by the plant breeding technique called backcrossing or genetic manipulation of a locus in which essentially all of the desired morphological and physiological characteristics of a broccoli variety have been restored to their normal state in addition to the characteristics of the single locus.

[0088] Substantially equivalent: Characteristics that are not statistically significantly different from the mean when compared (e.g., p=0.05).

[0089] Tissue culture: A composition containing isolated cells of the same or different types, or a collection of such cells that make up part of a plant.

[0090] Transgene: A genetic locus containing sequences introduced into the genome of a broccoli plant by transformation or site-specific modification.

[0091] H. Deposit information A deposit of broccoli hybrid SVBL0308, as described above and claimed in the claims, was made with the Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA), 60 Bigelow Drive, East Boothbay, Maine, 04544 USA. The deposit date of the seed deposit of broccoli hybrid SVBL0308 was May 27, 2021. The accession number of the seed deposit of broccoli hybrid SVBL0308 is NCMA Accession No. 202105012. Upon the granting of a patent, all restrictions on the deposit will be removed, and the deposit will comply with all requirements of 37 C.F.R. §§ 1.801-1.809. The deposit has been accepted pursuant to the Budapest Treaty and will be maintained in the depositary institution for a period of 30 years, or 5 years from the last request, or for the life of the patent, whichever is longer, with replacement as required within that period.

[0092] Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be made within the scope of the invention as defined by the appended claims.

[0093] All documents cited herein are expressly incorporated herein by reference in their entirety.

Claims

1. A broccoli plant of broccoli hybrid SVBL0308, wherein a sample of the seeds of said hybrid is deposited under NCMA accession number 202105012, said plant.

2. A broccoli seed that produces the plant according to claim 1.

3. A plant part of the plant according to claim 1, said plant part comprising cells of said plant.

4. A broccoli plant having all of the physiological and morphological characteristics of the plant according to claim 1.

5. A tissue culture of regenerable cells of the plant according to claim 1.

6. A broccoli plant regenerated from the tissue culture according to claim 5, said plant having all of the physiological and morphological characteristics of broccoli hybrid SVBL0308.

7. A method for vegetatively propagating the plant according to claim 1, comprising: (a) taking a tissue capable of propagation from the plant according to claim 1, and (b) propagating a broccoli plant from said tissue The method as described above.

8. A method for producing a broccoli plant containing an added trait, said method comprising introducing a transgene conferring said trait into the plant according to claim 1.

9. A broccoli plant produced by the method according to claim 8.

10. A broccoli plant of broccoli hybrid SVBL0308, wherein a sample of the seeds of said hybrid is deposited under NCMA accession number 202105012, further comprising a transgene, said plant.

11. wherein said transgene confers a trait selected from the group consisting of male sterility, herbicide tolerance, insect pest resistance, disease and pest resistance, disease resistance, modified fatty acid metabolism, environmental stress tolerance, modified carbohydrate metabolism, and modified protein metabolism, the plant according to claim 10.

12. A broccoli plant of broccoli hybrid SVBL0308, wherein a sample of the seeds of said hybrid is deposited under NCMA accession number 202105012, further comprising single locus conversion, said plant.

13. wherein said single locus conversion confers a trait selected from the group consisting of male sterility, herbicide tolerance, insect pest resistance, disease and pest resistance, disease resistance, modified fatty acid metabolism, environmental stress tolerance, modified carbohydrate metabolism, and modified protein metabolism, the plant according to claim 12.

14. A method for producing seeds of a broccoli plant derived from the broccoli hybrid SVBL0308, comprising: (a) crossing the plant according to claim 1 with itself or with a different broccoli plant; and (b) forming seeds of a broccoli plant derived from the broccoli hybrid SVBL0308. The method as described above.

15. A method for producing seeds of a broccoli plant derived from the broccoli hybrid SVBL0308, comprising: (a) producing a broccoli plant derived from the broccoli hybrid SVBL0308 from seeds produced by crossing the plant according to claim 1 with itself or with a different broccoli plant; and (b) crossing the broccoli plant derived from the broccoli hybrid SVBL0308 with itself or with a different broccoli plant to obtain seeds of a further broccoli plant derived from the broccoli hybrid SVBL0308.

16. The method further comprises: using the seeds from step (b) to produce the broccoli plant as described in step (a) and repeating the production and crossing steps of (a) and (b) for at least one generation to produce additional seeds of a broccoli plant derived from the broccoli hybrid SVBL0308. The method according to claim 15.

17. A method for manufacturing a food product or a feed product, comprising: (a) obtaining the plant according to claim 1 or a part thereof; and (b) manufacturing the food product or the feed product from the plant or a part thereof.

18. The method according to claim 17, wherein the food product or the feed product is defined as a soup.

19. An edible composition comprising at least one cell of the broccoli hybrid SVBL0308, wherein a sample of the seeds of the hybrid is deposited under NCMA accession number 202105012.

20. The edible composition according to claim 19, defined as a food product or a feed product.

21. The edible composition according to claim 20, defined as a soup.

22. A dietary supplement comprising at least one cell of the broccoli hybrid SVBL0308, wherein a sample of the seeds of the hybrid is deposited under NCMA accession number 202105012.

23. A method for manufacturing an edible composition, comprising: (a) Obtaining the plant according to claim 1 or a part thereof, and (b) Manufacturing the edible composition from the plant or a part thereof The method comprising the above.