阅读说明：本技术 具有改进的营养价值的甘蓝植物 (Brassica oleracea plants with improved nutritional value ) 是由 F·G·范登博施 G·N·科里瓦尔 R·F·米森 于 2013-09-13 设计创作，主要内容包括：本发明提供涉及与标准甘蓝品种相比萝卜硫苷增加的组合物和方法。本发明也涉及具有所需硫代葡萄糖苷含量的杂交品种的生产。本发明进一步提供包含这种性状并包含来自甘蓝麦的不与来自甘蓝麦的ELONG等位基因遗传连锁的Myb28等位基因的植物、植物部分和种子。(The present invention provides compositions and methods relating to increased glucoraphanin compared to standard cabbage varieties. The invention also relates to the production of hybrid varieties having a desired glucosinolate content. The invention further provides plants, plant parts and seeds comprising such a trait and comprising a Myb28 allele from Brassica oleracea that is not genetically linked to an ELONG allele from Brassica oleracea.)

1. A cabbage plant comprising a Myb28 allele from glycine max and lacking an ELONG allele from glycine max that is genetically linked to the Myb28 allele, wherein the Myb28 allele confers increased glucosinolates when compared to a plant lacking the Myb28 allele.

2. The plant of claim 1, wherein the plant is a cauliflower plant.

3. The plant of claim 1, wherein said plant is inbred.

4. The plant of claim 1, wherein said plant is hybrid.

5. The plant of claim 1, wherein the plant is homozygous for the Myb28 allele from Brassica oleracea.

6. The plant of claim 1, wherein the plant is heterozygous for the Myb28 allele from Brassica oleracea.

7. The plant of claim 1, wherein said ELONG allele is from Brassica oleracea.

8. A plant part of the plant of claim 1.

9. The plant part of claim 8, wherein said part is a leaf, ovule, floret, pollen, head, or cell.

10. Producing a seed of the plant of claim 1.

11. A brassica oleracea plant comprising a chromosome fragment comprising the Myb28 allele from glycine-bluegrass and lacking an ELONG allele from glycine-bluegrass that is genetically linked to the Myb28 allele, wherein the fragment confers increased glucosinolates relative to a plant lacking the Myb28 allele, and wherein a seed sample comprising the chromosome fragment is deposited under ATCC accession No. PTA-13165.

12. Producing a seed of the plant of claim 11.

13. A plant part of the plant of claim 11.

14. The plant part of claim 13, wherein said part is a leaf, ovule, floret, pollen, head, or cell.

15. A recombinant DNA fragment comprising the Myb28 allele from glycine-cyanea and the ELONG allele from brassica oleracea.

16. The DNA segment of claim 15, further defined as being comprised within a cell.

17. The DNA segment of claim 15, further defined as being comprised within a seed.

18. The DNA segment of claim 15, further defined as being comprised in a plant.

19. A method for obtaining a brassica plant comprising a desired glucosinolate composition, comprising:

(a) obtaining a brassica plant that is heterozygous for a Myb28 allele from glycine max, which Myb28 allele confers increased glucosinolates and is genetically linked in the plant to an ELONG allele from glycine max;

(b) obtaining progeny of the plant; and

(c) selecting at least a first progeny plant that undergoes recombination such that said progeny comprise the Myb28 allele but do not comprise an ELONG allele from Glycyrrhiza maxima, wherein said progeny plant has the desired thioglucoside composition due to the presence of the Myb28 allele and thus the absence of the ELONG allele from Glycyrrhiza maxima.

20. The method of claim 19, wherein selection of a progeny plant comprises identifying a progeny plant that (1) comprises a genetic marker that is genetically linked to the Myb28 allele in glycine-bluegrass and/or lacks a genetic marker present at a corresponding locus in the brassica plant, and (2) lacks a genetic marker that is genetically linked to the ELONG allele from glycine-bluegrass and/or comprises a genetic marker present at a corresponding locus from the brassica plant.

21. The method of claim 20, wherein selection of the progeny plant comprises detecting polymorphisms found in the plant genome flanking the complement of SEQ ID No. 1 and SEQ ID No. 2.

22. The method of claim 21, wherein the allele of (a) is detected by a PCR-based method using an oligonucleotide primer pair.

23. The method of claim 20, wherein selecting the progeny plant comprises detecting a polymorphism in the progeny plant shown in figure 5.

24. The method of claim 19, wherein the brassica plant is a brassica oleracea plant.

25. A plant or progeny thereof produced by the method of claim 19, comprising the Myb28 allele but not the ELONG allele from glycine-bluegrass.

26. A part of the plant of claim 25, said part selected from the group consisting of a cell, a seed, a root, a stem, a leaf, a head, a flower, and pollen.

27. A method for producing a hybrid cabbage plant with increased glucosinolate content comprising crossing a first cabbage parent plant with a second cabbage plant of a different genotype, wherein the first parent plant comprises the Myb28 allele from glycine-bluegrass and lacks an ELONG allele from glycine-bluegrass that is genetically linked to the Myb28 allele, wherein the Myb28 allele confers increased glucosinolate relative to a plant lacking the Myb28 allele.

28. The method of claim 27, further comprising producing a plurality of hybrid brassica oleracea plants, comprising crossing a first brassica oleracea parent plant with a plurality of second brassica oleracea plants of different genotypes.

29. A method of producing a brassica oleracea plant having a desired increased glucosinolate content, comprising introgressing into the plant a chromosome fragment comprising the Myb28 allele from brassica oleracea and lacking the ELONG allele from brassica oleracea that is genetically linked to the Myb28 allele, wherein the fragment confers the desired glucosinolate content relative to the plant lacking the fragment, wherein a seed sample comprising the chromosome fragment is deposited under ATCC accession No. PTA-13165.

Technical Field

The present invention relates to the development and use of Brassica oleracea (Brassica oleracea) plants having recombinant chromosome fragments.

Background

Thioglucosides are allelochemicals present in plant species of 16 families, particularly the Brassicaceae family (Brassicaceae), of which broccoli is a well-known example. Although there are more than 120 identified glucosinolates in nature, the closely related taxonomic groups typically contain only a few of these compounds. The predominant glucosinolates in cauliflower, such as glucoraphanin and curvularin, are biochemically derived from the amino acid methionine. In this thioglucoside pathway, the activity of methionine through the ELONG (elongation) locus is converted to homomethionine and dihomomethionine by the addition of a single carbon unit to the tail at a time. Homomethionine is finally converted into 3-methylthiopropylthioglucoside (chikusetoside; "MSP") and dihomomethionine is converted into 4-methylthiobutylthioglucoside (sulforaphide; "MSB"). These glucosinolates (curvularin and glucoraphanin) are potent inducers of phase II detoxification enzymes such as glutathione-S-transferase and quinone reductase, which promote the metabolism and excretion of potential carcinogens.

Brief description of the invention

In one aspect, the invention provides a Brassica oleracea plant comprising a Myb28 allele from Brassica villosa (Brassica villosa) and lacking an ELONG allele from Brassica villosa that is genetically linked to the Myb28 allele, wherein the Myb28 allele confers increased thioglucoside when compared to a plant lacking the Myb28 allele.

In one embodiment, the plant is a cauliflower plant. In other embodiments, the plant is inbred or hybrid. In another embodiment, the plant is homozygous for the Myb28 allele from glycine max. In yet another embodiment, the plant is heterozygous for the Myb28 allele from glycine max. In yet another embodiment, the ELONG allele is from brassica oleracea.

In another aspect, the invention provides a plant part of the invention. In some embodiments, the plant part may be further defined as a leaf, ovule, floret, pollen, head (head), or cell.

In yet another aspect, the present invention provides seeds for producing the plants of the invention.

In yet another aspect, the present invention provides a brassica oleracea plant comprising a chromosome fragment comprising the Myb28 allele from glycine-bluegrass and lacking the ELONG allele from glycine-bluegrass that is genetically linked to the Myb28 allele, wherein the fragment confers increased glucosinolates relative to a plant lacking the Myb28 allele, and wherein a seed sample comprising the chromosome fragment is deposited under ATCC accession No. PTA-13165. In one embodiment, the present invention provides seeds for producing such plants. In another embodiment, the invention provides a plant part, wherein said part is a leaf, ovule, floret, pollen, head or cell. In another embodiment, the plant is a brassica oleracea plant.

In another aspect of the invention, there is provided a recombinant DNA fragment comprising the Myb28 allele from Brassica oleracea and the ELONG allele from Brassica oleracea. In one embodiment, the DNA fragment is further defined as being comprised within a cell. In another embodiment, the DNA fragment is further defined as being comprised within a seed. In yet another embodiment, the DNA fragment is further defined as being comprised within a plant.

In another aspect, the present invention provides a method for obtaining a brassica plant comprising a desired glucosinolate composition, comprising: a) obtaining a brassica plant that is heterozygous for a Myb28 allele from glycine max, which Myb28 allele confers increased glucosinolates and is genetically linked in the plant to an ELONG allele from glycine max; (b) obtaining progeny of the plant; and (c) selecting at least a first progeny plant in which recombination occurs such that the progeny comprises the Myb28 allele but not the ELONG allele from glycine-bluegrass, wherein the progeny plant has the desired thioglucoside composition due to the presence of the Myb28 allele but not the ELONG allele from glycine-bluegrass.

In one embodiment, selection of a progeny plant comprises identifying a progeny plant that (1) comprises a genetic marker that is genetically linked to the Myb28 allele in glycine-bluegrass and/or lacks a genetic marker present at a corresponding locus in the brassica plant, and (2) lacks a genetic marker that is genetically linked to the ELONG allele from glycine-bluegrass and/or comprises a genetic marker present at a corresponding locus from the brassica plant.

In another embodiment, selection of progeny plants comprises detecting complement of SEQ ID NO:1 and SEQ ID NO:2 in the genome of said plant flanked by complementary sequences of seq id no 2. In a further embodiment, such alleles are detected by a PCR-based method using oligonucleotide primer pairs. In another embodiment, the selection of the progeny plant comprises detecting a polymorphism in the progeny plant shown in figure 5. In a further embodiment, the brassica plant may be a brassica oleracea plant.

In a still further aspect, the invention provides a plant produced by the method of the invention, or progeny thereof, comprising the Myb28 allele but not the ELONG allele from glycine-cyanea. In one embodiment, the invention provides a part of such a plant. In another embodiment, the plant part is selected from the group consisting of a cell, a seed, a root, a stem, a leaf, a head, a flower, and pollen.

In another aspect, the present invention provides a method for producing a hybrid cabbage plant with increased glucosinolate content comprising crossing a first cabbage parent plant with a second cabbage plant of a different genotype, wherein the first parent plant comprises the Myb28 allele from glycine-bluegrass and lacks an ELONG allele from glycine-bluegrass that is genetically linked to the Myb28 allele, wherein the Myb28 allele confers increased glucosinolate relative to a plant lacking the Myb28 allele. In one embodiment, the method further comprises producing a plurality of hybrid brassica oleracea plants, comprising crossing a first brassica oleracea parent plant with a plurality of second brassica oleracea plants of different genotypes.

In yet another aspect, the present invention provides a method of producing a brassica oleracea plant having a desired increased glucosinolate content, comprising introgressing into the plant a chromosomal fragment comprising the Myb28 allele from glycine-bluegrass and lacking the ELONG allele from glycine-bluegrass that is genetically linked to the Myb28 allele, wherein the fragment confers the desired glucosinolate content relative to the plant lacking the fragment, wherein a seed sample comprising the chromosomal fragment is deposited under ATCC accession No. PTA-13165.

The term "about" is used to indicate that a value includes the standard deviation of error for the device or method used to determine the value. The term "or" as used in the claims is intended to mean "and/or" unless explicitly indicated to refer to the alternative alone or the alternatives are mutually exclusive, although the present disclosure supports the definition of alternative and "and/or" alone. In the claims, the words "a" and "an" when used in conjunction with the word "comprising" or other open-ended terms mean "one or more" unless specifically stated otherwise. The terms "comprising," "having," and "including" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprising," "having," "containing," and "including," is also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to having only those one or more steps, and also covers other unlisted steps. Similarly, any plant that "comprises," "has," or "includes" one or more traits is not limited to having only such one or more traits, and also covers other unlisted traits.

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

Drawings

FIG. 1 depicts the heads from hybrid broccoli varieties Ironman (left) and RX05991199 (right). The cultivars were grown at 50x50cm intervals in summer.

FIG. 2 shows a horizontal cross section of stems from hybrid broccoli varieties Ironman (left) and RX05991199 (right). The cultivars were grown in autumn at 50x50cm intervals.

FIG. 3 shows the contours of the head and stem from hybrid broccoli varieties Ironman (left) and RX05991199 (right). The cultivars were grown in autumn at 50x50cm intervals.

FIG. 4 shows the results of analysis of different ELONG alleles with QTL1-BoGLS-ELONG markers. The V allele is a Brassica oleracea allele associated with Myb28, which results in a high 3-MSP/4-MSB ratio and a high total glucosinolates. A. The B and C alleles are examples of alleles found in broccoli that do not contain the brassica oleng allele.

FIG. 5 shows an alignment (SEQ ID NOs: 8-9) between the consensus sequence of the Myb28 locus from Brassica Oleracea and the consensus sequence of the corresponding locus from a cauliflower, such as cabbage (Oleracea), without increased levels of glucosinolates contained in cauliflower variety FT 69.

Detailed Description

The present invention provides methods and compositions related to plants, seeds and derivatives of Brassica oleracea plants comprising a novel recombinant introgression from Brassica oleracea capable of conferring increased 4-Methylsulfinylbutylthioglucoside (MSB), also known as glucoraphanin. It has also been surprisingly found that plants comprising the introgression are able to stably produce hybrids with increased glucoraphanin content relative to chikungunya, whereas the results are significantly more variable when using parental lines comprising non-recombinant introgression, for example in the case of the Myb28 donor parent (us patent application publication No. 2011/0055945) of hybrid PS05151639 comprising Myb28 and the ELONG alleles from glycine lucium. The ability to produce a variety of superior hybrid progeny with increased glucosinolate content and/or glucoraphanin to garbanin ratio from a single inbred parent is important in that the number of superior cabbage parent lines available to produce hybrid varieties is limited. Thus reduced introgression actually increases the utility of a given inbred and allows, for example, the production of a variety of hybrids, each with a desired glucoraphanin content, that are likely to be adapted to different growth environments, end uses, or other criteria.

A new "reduced introgression" comprising the Myb28 locus from glycine max and lacking the ELONG locus from glycine max to date genetically linked thereto is able to stably confer an increased glucoraphanin relative to chikungunya on hybrids derived from plants comprising the introgression. One aspect of the present invention therefore relates to a method for obtaining a brassica oleracea plant comprising at least one such reduced introgression, wherein the brassica oleracea plant formed and/or progeny derived therefrom have a desired glucoraphanin content relative to a control plant lacking said introgression. The present invention thus provides plants having the desired glucoraphanin content conferred by the reduced introgression of the present invention. In certain embodiments, a method for obtaining such a plant comprises obtaining a brassica oleracea plant that is heterozygous for the Myb28 allele from glycine max, which Myb28 allele confers increased glucosinolates and is genetically linked in the plant to the ELONG allele from glycine max; obtaining progeny of such a plant; and selecting one or more such progeny plants, wherein genetic recombination occurs such that the progeny comprises the Myb28 allele from glycine-bluegrass but does not comprise the ELONG allele from glycine-bluegrass. Such progeny or further progeny thereof may also have the desired glucoraphanin content due to the presence of the Myb28 allele and thus the absence of the ELONG allele from Glycyrrhiza glabra. In particular embodiments, the method may comprise obtaining a progeny plant comprising the Myb28 and/or the ELONG allele by identifying one or more genetic markers that are genetically linked to such allele. Identifying a genetic marker may comprise phenotypic, genetic or biochemical tests, and may comprise screening for the presence of, for example, one or more of the alleles described herein, including for example, the Myb28 allele from glycine-bluegrass, the ELONG allele from glycine-bluegrass and the ELONG allele from brassica oleracea, in the parent and/or progeny plants.

It was found that certain traits such as glucoraphanin content relative to chikungunyin content or unpredictability of glucosinolate species in hybrid progeny heterozygous for the cabbage wheat Myb28 allele co-localized (co-locus) with the Myb28 allele from kale and the glucosinolate trait in the ELONG allele from cabbage gene introgression. Thus, the formation of a "reduced" introgression is understood to be caused by recombination events in the vicinity of Myb28 and the ELONG QTL. Lines that contain reduced introgression, i.e., have undergone a recombination event with increased glucosinolates close to the QTL, can be effectively screened by using molecular and/or phenotypic markers. Thus, a population of plants that segregates (i.e. is heterozygous) relative to the QTL specified by Myb28 and ELONG introgression, or progeny of such a population, may be screened for plants having a recombinant phenotype, e.g. increased glucoraphanin levels relative to chikungunya levels.

In other embodiments, the methods of the invention may comprise identifying a brassica oleracea plant comprising a reduced introgression from brassica oleracea and comprising meiotic recombination between Myb28 and the ELONG alleles described herein. In particular embodiments, identifying the introgression can include measuring glucoraphanin and/or garcinin using standard protocols. In certain embodiments, a plant of the invention comprising a reduced introgression as disclosed herein comprises an increased average glucoraphanin ratio relative to chikusetoside as compared to a plant comprising the Myb28 and ELONG alleles from glycine max or a plant lacking the Myb28 allele from glycine max. In one embodiment, such a plant comprising an increased average sulforaphane ratio relative to chikungunya is an inbred line, and in another embodiment is defined as hybrid F1 comprising as one or more parents a plant of the present invention having reduced introgression. In a particular embodiment, plants of the invention are provided that comprise glucoraphanin in a ratio of about 10: 1, 12: 1, 15: 1, 18: 1, 20: 1, 23: 1, 25: 1, 28: 1, 30: 1, 35: 1, and about 40: 1. In one aspect, the increase in sulforaphane content can be calculated with reference to a standard cabbage variety, such as the broccoli variety Ironman.

A. Breeding of cabbage lines with increased sulforaphane

One aspect of the present invention relates to methods of crossing a plant comprising a reduced Myb28/ELONG introgression as provided herein with itself or a second plant, and seeds and plants produced by such methods. These methods can be used to produce and propagate cultivated cabbage plants having a desired glucosinolate composition, including MSB and/or MSP content. Yet further, the plants of the invention with increased thioglucoside content comprise improved plant nutritional value relative to plants that do not contain the increased thioglucoside. The method may also be used to produce hybrid cabbage seeds and plants grown therefrom. Hybrid seed is produced by crossing this line with a second cabbage parent line. The hybrid may be heterozygous or homozygous for the reduced introgression.

Cabbage wheat is an endemic wild variety in the north and middle of west, and thus the Myb28 allele may be obtained by one skilled in the art from a plant selected from the wild. Alternatively, the Myb28 allele is known in the art and can be obtained from other sources for use in the present invention, including SNR 347(FT 69; in Mithen et al, the or Appl Genet, 106: 727-734; 2003, 428-11-69), BR384-014, SNP13(580333), SNP88 (BRM51-1210), BR384-020, B1639(ATCC accession No. PTA-9676, Italian cabbage, Brassica oleracea var. italica, Collection date 2008, 12.23), BRM51-1162(ATCC accession No. PTA-9675, Italian cabbage, Brassica oleracea. italica, Collection date 2008, 12.23) and RX05991199 (ATCC accession No. PTA-13165, cauliflower, cabbage, Broccoli, Brassica oleracea, Collection date, 12.20). According to the present invention, the plants provided herein will typically lack an ELONG allele from glycine max which is genetically linked to the Myb28 allele. This may be achieved according to the present invention by crossing a plant comprising Myb28 and an ELONG allele from glycine-bluegrass with a brassica plant, including a standard brassica variety, which does not comprise Myb28 and an ELONG allele from glycine-bluegrass. This includes, inter alia, many broccoli varieties well known in the art.

The goal of vegetable breeding is to combine various desirable traits in a single variety/hybrid. Such desirable traits may include any trait deemed beneficial by growers and/or consumers, including high yield, insect or disease resistance, tolerance to environmental stress, and nutritional value. Breeding techniques for attempting to obtain desired traits utilize pollination methods of plants. There are two conventional pollination methods: plants are self-pollinated if pollen from one flower passes to the same or another flower of the same plant, and cross-pollinated if pollen reaches it from a flower of a different plant.

Development of uniform varieties requires the generation of homozygous inbred plants, crossing of these inbred plants, and evaluation of the crosses. Pedigree breeding and recurrent selection are examples of breeding methods that have been used to generate inbred plants from breeding populations. Those breeding methods incorporate genetic background from two or more plants or a variety of other broad sources into a breeding pond, and new lines and hybrids derived from the new lines are produced from the breeding pond by inbreeding and selection of the desired phenotype.

According to the present invention, new varieties can be created by crossing the plants of the present invention and then selecting generations and inbreeding as desired to produce uniform lines. A new variety can also be created by crossing with any second plant. In selecting such second plants for the purpose of crossing to develop a new line, it may be desirable to select those plants which themselves exhibit one or more selected desired characteristics or which exhibit desired characteristics in hybrid combination. Once the primary cross is performed, inbreeding and selection are performed to produce new varieties. In order to develop uniform lines, selfing and selection for five or more generations is typically included.

Homogeneous lines of new varieties can also be developed by means of haploid doubling. This technique allows the establishment of true breeding lines (true breeding lines) without the need for multiple generations of selfing and selection. In this way, true breeding lines can be produced in as few as one generation. Haploid embryos can be produced from microspores, pollen, anther culture, or ovary culture. The haploid embryos can then be multiplied either spontaneously or by chemical treatment (e.g., colchicine treatment). Alternatively, haploid embryos can be grown into haploid plants and processed to induce chromosome doubling. In either case, fertile homozygous plants are obtained. According to the present invention, any of such techniques may be used in conjunction with the plants of the present invention and their progeny to obtain homozygous lines.

Backcrossing can also be used to improve inbred plants. Backcrossing transfers a particular desired trait, such as increased glucoraphanin, from an inbred or non-inbred source to a variety lacking that trait. This can be done, for example, by first crossing the parent (a) (recurrent parent) with a donor inbred line (non-recurrent parent) carrying one or more appropriate loci for the trait in question. The progeny of this cross are then matched back to the recurrent parent (a) and the desired trait to be transferred from the non-recurrent parent is selected in the resulting progeny. After five or more backcross generations of the desired trait are selected, the progeny are heterozygous for the locus controlling the transferred trait, but similar to the first parent for most or nearly all of the other loci. The final backcross generation will be selfed to provide true breeding progeny of the transferred trait.

Selection of the appropriate recurrent parent is an important step for a successful backcrossing procedure. The goal of the backcrossing scheme is to alter or replace a single trait or characteristic of the original variety. To achieve this goal, individual loci of a recurrent variety are modified or replaced with desired loci from non-recurrent parents, while substantially maintaining all of the remaining desired genetic makeup of the original variety, and thus maintaining the desired physiological and morphological constitution. The choice of a particular non-recurrent parent will depend on the purpose of the backcross; one of the main objectives is to add some commercially desirable traits to the plant. The exact backcrossing protocol will depend on the trait or trait to be altered to determine the appropriate test protocol. Although the backcrossing process is simplified when the transferred trait is a dominant allele, the recessive allele may also be transferred. It may be necessary to introduce a progeny test to determine if the desired trait has been successfully transferred.

Cabbage varieties may also be developed from more than two parents. This technique, called modified backcrossing, uses different recurrent parents during backcrossing. Improved backcrossing can be used to replace the original recurrent parent or multiple parents with a variety having certain more desirable characteristics can be used to obtain different desired characteristics from each parent.

A number of individual locus traits have been identified which are not regularly selected in the development of new inbred systems, but which can be improved by backcrossing techniques. A single locus trait may or may not be transgenic; examples of such traits include, but are not limited to, male sterility, herbicide resistance, resistance to bacterial, fungal or viral diseases, insect resistance, restoration of male fertility, improved fatty acid or carbohydrate metabolism, and increased nutritional quality. These contain genes that are usually inherited through the nucleus.

Direct selection can be applied, where a single locus acts as a dominant trait. The choice of cabbage plants for breeding does not necessarily depend on the phenotype of the plant, but may instead be based on genetic studies. For example, suitable genetic markers that are closely genetically linked to the trait of interest may be utilized. One of these markers can be used to identify the presence or absence of a trait in the progeny of a particular cross, and can be used to select progeny for continued breeding. This technique is commonly referred to as marker assisted breeding. Any other kind of genetic marker or other test that is capable of identifying the relative presence or absence of a trait of interest in a plant may also be used for breeding purposes.

The procedure of marker assisted selection is particularly useful for introgression of a given trait. Types of well-known genetic markers that may be used in accordance with the present invention include, but are not necessarily limited to, Simple Sequence Length Polymorphism (SSLP), enzymatically amplified polymorphic sequence (CAP), randomly amplified polymorphic DNA (rapd), DNA Amplification Fingerprint (DAF), sequence characterized amplified region (scarr), arbitrary primer polymerase chain reaction (AP-PCR), Amplified Fragment Length Polymorphism (AFLP), and Single Nucleotide Polymorphism (SNP).

B. Plants derived by genetic engineering from the plants of the invention

Many useful traits that can be introduced by backcrossing as well as directly into plants are those that can be introduced by genetic transformation techniques. Genetic transformation can therefore be used to insert a selected transgene into a brassica plant of the invention, or alternatively, to prepare a transgene which can be introduced by backcrossing. Methods for transforming plants, including brassica, are well known to those skilled in the art.

The vector used for transforming the plant cell is not limited as long as the vector can express the inserted DNA in the cell. For example, a vector comprising a promoter for constitutive gene expression in brassica cells (e.g., cauliflower mosaic virus 35S promoter) and a promoter inducible by external stimuli can be used. Examples of suitable vectors include the pBI binary vector. The "brassica cell" into which the vector is to be introduced includes various forms of brassica cells, such as cultured cell suspensions, protoplasts, leaf sections, and callus.

The vector can be introduced into the Brassica cell by known methods, such as polyethylene glycol, polycation, electroporation, Agrobacterium-mediated transfer, particle bombardment, and direct uptake of protoplast DNA.

To effect transformation by electroporation, friable tissue such as suspension cultures of cells or embryogenic callus may be used, or immature embryos or other organized tissues may be directly transformed. In this technique, the cell wall of selected cells can be partially degraded by exposing the cells to pectin degrading enzymes (pectin lyases) or mechanically damaging the tissue in a controlled manner.

One effective method of delivering transforming DNA fragments to plant cells is microprojectile bombardment. In this method, particles are coated with nucleic acids and delivered into cells by propulsive force. Exemplary particles include those composed of tungsten, platinum, and preferably gold. For bombardment, the cells in suspension are concentrated on a filter or solid medium. Alternatively, immature embryos or other target cells may be placed on solid media. The cells to be bombarded can be positioned at a suitable distance below a microparticle stopping plate (microprojectile stopping plate). Microprojectile bombardment techniques are widely applicable and can be used to efficiently transform any plant species.

Agrobacterium-mediated transfer is another widely applicable system for introducing loci into plant cells. The advantage of this technique is that DNA can be introduced into whole plant tissue, thereby eliminating the need to regenerate whole plants from protoplasts. Modern Agrobacterium transformation vectors are capable of replication in E.coli as well as in Agrobacterium (and other Rhizobium species), allowing for convenient manipulation. In addition, recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the placement of genes and restriction sites in the vectors to facilitate the construction of vectors capable of expressing various polypeptide-encoding genes. The described vectors have convenient polylinker regions flanking the promoter and polyadenylation site for direct expression of the inserted polypeptide-encoding gene. In addition, Agrobacterium containing armed and disarmed Ti genes can be used for transformation.

In those plant lines where Agrobacterium-mediated transformation is effective, it is a method of particular choice due to the easy-to-implement and defined nature of locus transfer. The use of agrobacterium-mediated plant integration vectors to introduce DNA into plant cells is well known in the art (U.S. Pat. No. 5,563,055). For example, U.S. Pat. No. 5,349,124 describes methods for transforming plant cells using Agrobacterium-mediated transformation. The method confers resistance to such insects to plants by inserting a chimeric gene having a DNA coding sequence encoding a full length b.t. toxin protein which expresses a protein toxic to lepidopteran larvae.

Many promoters can be used for plant gene expression of any gene of interest, including, but not limited to, selectable markers, scorable markers, genes for pest tolerance, disease resistance, nutrient enhancement, and any other agronomically significant gene. Examples of constitutive promoters for brassica gene expression include, but are not limited to, the cauliflower mosaic virus (CaMV) P-35S promoter, which confers constitutive high-level expression in most plant tissues, including monocots, continuous repeats of the CaMV 35S promoter, enhanced 35S promoter (P-e35S), nopaline synthase promoter, octopine synthase promoter; and the figwort mosaic virus (P-FMV) promoter and enhanced FMV promoter (P-eFMV) as described in U.S. patent No. 5,378,619 (where the promoter sequence of the P-FMV repeats in tandem), the cauliflower mosaic virus 19S promoter, the sugarcane bacilliform virus promoter, the dayflower yellow mottle virus promoter, and other plant DNA virus promoters known to be expressed in plant cells.

Exemplary nucleic acids that can be introduced into a plant of the invention include, for example, a DNA sequence or gene from another species, or even a gene or sequence derived from or present in the same species but introduced into a recipient cell by genetic engineering methods rather than traditional breeding or breeding techniques. However, the term "exogenous" is also used to refer to a gene that is not normally present in the cell being transformed or may be present only in a form, structure, etc., as found in a transformed DNA fragment or gene, or that normally exists and is desired to be expressed (e.g., overexpressed) in a pattern other than the native expression pattern. Thus, the term "exogenous" gene or DNA is used to refer to any gene or DNA fragment introduced into a recipient cell, regardless of whether a similar gene may already be present in such cell. The kind of DNA contained in the foreign DNA may include DNA already present in a plant cell, DNA from another plant, DNA from a different organism or externally generated DNA, for example a DNA sequence containing an antisense messenger of a gene, or a DNA sequence encoding a synthetic or modified form of a gene.

Hundreds or thousands of different genes are known and can potentially be introduced into brassica plants according to the invention. Non-limiting examples of specific genes and corresponding phenotypes that may be selected for introduction into brassica plants include one or more genes for insect tolerance (such as Bacillus thuringiensis (B.t.)) genes), pest tolerance (such as genes for fungal disease control), herbicide tolerance (such as genes that confer glyphosate tolerance), and quality improvement such as yield, nutrient enhancement, environmental or stress tolerance, or any desired change in plant physiology, growth, development, morphology or plant product. For example, a structural gene would include any gene that confers insect tolerance, including but not limited to a bacillus insect control protein gene, as described in WO99/31248, which is incorporated herein by reference in its entirety; as described in U.S. patent No. 5,689,052, which is incorporated herein by reference in its entirety; as described in U.S. patent nos. 5,500,365 and 5,880,275, which are incorporated herein by reference in their entirety. In another embodiment, the structural gene may confer herbicide glyphosate tolerance as conferred by genes including, but not limited to, the agrobacterium strain CP4 glyphosate resistant EPSPS gene (aroA: CP4) as described in U.S. patent No. 5,633,435 (which is incorporated herein by reference in its entirety) or the glyphosate oxidoreductase Gene (GOX) as described in U.S. patent No. 5,463,175 (which is incorporated herein by reference in its entirety).

Alternatively, the DNA coding sequence may affect these phenotypes by encoding an untranslated RNA molecule that causes targeted suppression of endogenous gene expression, e.g., via antisense or co-suppression mediated mechanisms. RNA can also be a catalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desired endogenous mRNA product. Thus, any gene that produces a protein or mRNA that expresses a phenotypic or morphologic change of interest can be used in the practice of the present invention.

D. Definition of

A number of terms are used in the specification and tables herein. In order to provide a clear and consistent understanding of the specification and claims, the following definitions are provided:

allele: any one or more alternative forms of a locus, all of which alleles are associated with a trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

Backcrossing: the process of crossing a hybrid progeny, such as a first generation hybrid (F1), with one of the parents of the hybrid progeny is repeated by the breeder. Backcrossing can be used to introduce one or more single locus transformations from one genetic background into another.

The cultivated species: is suitable for consumption and meets the requirements of commercial cultivation. One example is a broccoli variety. In addition to the plant itself and parts thereof suitable for consumption, such as the head or leaves, the present invention includes plant parts or derivatives suitable for propagation. Examples of parts suitable for propagation are organ tissues, such as leaves, stems, roots, shoots, etc., protoplasts, somatic embryos, pollen sacs, petioles, cells in culture, etc. Derivatives suitable for propagation are, for example, seeds. Plants according to the invention may be cultivated or propagated in a conventional manner, but may also be cultivated or propagated from plant parts by tissue culture techniques.

And (3) hybridization: mating of the two parental plants.

Cross pollination: fertilization is by the combination of two gametes from different plants.

Diploid: a cell or organism having two sets of chromosomes.

Enzyme: molecules that can be used as catalysts in biological reactions.

F1 hybrid: first generation progeny of a cross of two non-syngeneic plants.

Genotype: genetic composition of a cell or organism.

Haploid: a cell or organism having one of two sets of chromosomes that is diploid.

Linkage: a phenomenon in which alleles on the same chromosome tend to segregate together more frequently than would be expected if their transfer were independent.

A marker: an easily detectable phenotype, preferably inherited in a co-dominant form (two alleles at a locus in a diploid heterozygote are easily detectable), with no environmental variation component, i.e., heritability of 1.

Phenotype: a detectable characteristic of a cell or organism, which characteristic is an expression of gene expression.

Quantitative Trait Locus (QTL): quantitative Trait Loci (QTLs) refer to loci that control, to some extent, a generally continuous distribution of numerically representable traits.

Recombination events are understood to mean meiotic crossovers.

Regeneration: plants develop from tissue culture.

Self-pollination: pollen is transferred from the anther to the stigma of the same plant.

Single locus transformed (transformed) plants: plants developed by a plant breeding technique known as backcrossing in which substantially all of the desired morphological and physiological characteristics of a broccoli variety are restored, except for the characteristics of a single locus transferred into the variety via backcrossing techniques and/or by genetic transformation.

Substantially equivalent to: when compared, no feature of statistically significant difference (e.g., p 0.05) from the mean was shown.

Tissue culture: a composition comprising isolated cells of the same or different species or a collection of such cells organized into plant parts.

And (3) transgenosis: a genetic locus comprising a sequence that has been introduced into the genome of a cauliflower plant by transformation.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the invention, which is limited only by the scope of the appended claims.

All references cited herein are expressly incorporated herein by reference.

E. Preservation information

The deposit consists of at least 2500 seeds of cauliflower hybrid RX05991199, which includes a reduced introgression of the Myb28 allele from brassica oleracea and the ELONG allele from brassica oleracea as described herein. This deposit was made at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va.20110-2209 USA. This deposit is designated ATCC accession number PTA-13165. The date of deposit is 8 months and 20 days 2012. The right to obtain the deposit is provided to the qualified person as required during the pendency of this application. The deposit will be deposited at the ATCC depository (which is a public depository) for a period of 30 years or 5 years following the latest requirements, or for the applicable term of this patent, whichever is longer, and will be replaced if non-viable during that period. The applicant does not exempt any infringement of its rights granted to this patent or any other form of variety protection, including plant variety protection regulation (7 u.s.c.2321etseq.).

Examples