Patent Application
20200093086
The present invention relates to the field of watermelon breeding and watermelon improvement. Provided are new diploid watermelon plants (2n=2x=22) and seeds from which such plants can be grown, which produce very small diploid fruits comprising seeds of an average seed length of between 6.0 and 4.0 mm, such as equal to or less than 5.5 mm, due to the combination of the HMBN mutant allele (e.g., in homozygous form), and the ts-gene mutant (deletion of SEQ ID NO: 1 on chromosome 2) (e.g., in homozygous form). The diploid watermelon plants are in one aspect preferably suitable as pollenizers in triploid watermelon (2n=3x=33) production, whereby the pollenizers therefore have a dual purpose: providing sufficient viable pollen to pollinate female flowers of triploid plants (which after pollination produce triploid, seedless watermelon fruits) and/or to provide small (“mini”—or personal size), edible diploid fruits on the pollinizer plants themselves. The fruits are so small, that they can be easily treated like apples, i.e., they can be eaten fresh after e.g., removing the rind, or they can be eaten with a spoon after cutting in half or removing a slice from fruit.
Seedless watermelon (Citrullus lanatus (Thunb.) Matsum. And Nak.) production involves using pollen from diploid male parent plants to fertilize flowers of tetraploid (2n=4x=44) maternal parent plants. Pollination of the tetraploid flowers with diploid pollen leads to hybrid F 1 seeds which are triploid (Kihara, 1951, Proceedings of American Society for Horticultural Science 58: 217-230; Eigsti 1971, Hort Science 6: 1-2). The triploid hybrid plants, grown from these F 1 seeds, are self-infertile as they produce sterile pollen due to chromosome imbalance (Fehr, 1987). The triploid hybrids, therefore, need to be pollinated by a diploid pollenizer to produce watermelon fruit. Triploid plants are, therefore, interplanted with pollenizer plants for fruit production. The “seedless” fruit produced after pollination on the triploid hybrid plant are often not truly seedless, but may contain some undeveloped, small, pale seeds, which are edible.
For optimal seedless watermelon fruit set, sufficient viable pollen is required. Plants are generally planted at a ratio of 1 pollenizer per every 2-4 triploid plants. Triploid plants and pollenizers are either planted in separate rows (e.g., 1 row of pollenizer and 2-4 rows of triploids), or interplanted within rows (e.g., planting 1 pollenizer plant in between 2 to 3 triploid plants in the same row), or interplanted in narrow rows between rows of triploids (see US 2006/0168701 Table 2). The fruit produced on the pollenizer plants preferably has a different rind pattern from the fruit on the triploid hybrids, so that these can be easily distinguished. Until now generally the fruits produced on dedicated pollenizer plants are not harvested or discarded and only the seedless triploid fruits are sold.
In the last years, several dedicated pollenizer plants have been developed, which provide sufficient staminate flowers and sufficient viable pollen throughout the season to increase triploid fruit yield. These dedicated pollenizers include for example varieties Polimax and Jenny (Nunhems), Sidekick (Harris Morin), Companion (Seminis) and the Super-Pollenizers SP-1 and SP-4 (Syngenta). These dedicated pollenizers can be divided into two categories based on their vegetative growth type, which is either of the standard vine length e.g., Jenny and SP-1 and SP-4, or the ‘compact’ vine length, e.g., Companion or Sidekick.
Some pollenizers produce diploid fruits which could be marketable, while others produce fruits that are unsuitable for consumption and marketing. Dittmar (2006, MSc Thesis North Carolina State University, Horticultural Science, Characterization of diploid watermelon pollenizers and utilization for optimal triploid watermelon production and effects of halosulfuron post and post-dir on watermelon) evaluated different pollenizers for the potential marketability of their fruits and concluded that Mickeylee, SF800, MiniPool, Jenny and Pinnacle have a fruit quality that could potentially be marketed. Average fruit weight of these was 5.1 kg, 10.7 kg, 3.9 kg, 3.3 kg and 2.9 kg respectively (Dittmar 2006, supra). The smallest diploid fruits were produced by Sidekick (1.0 kg, with dimensions of 12.3×11.9 cm length:width Dittmar 2006, supra) and SP-1 (2.0 kg, with dimensions of 17.5×15.4 cm length:width (Dittmar 2006, supra), but neither of these produce marketable fruits. The fruits of Sidekick are very poor quality pink-fleshed with a brittle rind and those of SP-1 are white-fleshed and have a low brix value. Due to the non-marketable fruits, these pollenizers are referred to as being “non-harvestable pollenizers”.
US2009/0288183 (Gold Seed Co. LLC) describe a pollinizer called “Escort-4” which produces small fruits having reduced sugar for type 2 diabetics, referred to as a “dual purpose reduced sugar watermelon”. The fruits are said to have an average weight of 4.0 lbs (1.8 kg) and a size of 5-7 inches long (12.7-17.7 cm)×4-5 inches wide (10.1-12.7 cm). The fruits of Escort-4 are said to have approximately ⅓ less sugar content than commercial diploid varieties, such as Sangria (Syngenta Inc.).
It is an object of the invention to provide watermelon plants, in one aspect dual purpose watermelon pollenizers, producing small, edible (i.e., marketable) diploid fruit with an average fruit weight of equal to or less than 2.0 kg, preferably less than 1.8 kg or 1.7 kg, more preferably equal to or less than 1.6, 1.5, 1.4, 1.3, 1.0, 0.9, 0.8, 0.7 kg, and even more preferably equal to or less than about 0.66 kg or 0.65 kg, such as equal to or less than 0.6 kg, 0.5 kg, 0.4 kg or 0.3 kg due to the presence of the HMBN mutant allele in homozygous form and comprising a deletion of SEQ ID NO: 1 on chromosome 2 (the ts-gene mutant), whereby the fruits produce seeds of an average seed length of 6.0 mm or less, preferably 5.5 mm or less (but at least 4.0 mm), at least when the deletion of SEQ ID NO: 1 (the ts-gene mutant) is in homozygous form. The ts-gene mutant is described in Wehner (2007, Cucurbit Genetic Cooperative Report 30:96-120), as referring to the “tomato seed size mutant”, “ts” or “tss”, from the tomato seed Sugar Baby mutant plant, which is originally described by Zhang in Cucurbit Genetics Cooperative Report 19: 66-67 (1996). These Cucurbit Genetic Cooperative Reports can be found on the world wide web at cuke.hort.ncsu.edu/cgc/cgcreports.html.
The ts-gene mutant of the tomato seed Sugar Baby mutant plant was herein mapped to chromosome 2 and, after fine-mapping, it was found to comprise a large deletion of almost 14 kb, depicted in SEQ ID NO: 1. The ts-gene mutant thus contained a large deletion on chromosome 2, which is not deleted in the wild type Sugar Baby plant (indicated as Ts, the wild type allele of the gene). In other words, the tomato seed Sugar Baby mutant plant has the genotype ts/ts (SEQ ID NO:1 Deletion/Seq ID NO: 1 Deletion), while the wild type Sugar Baby plant has the genotype Ts/Ts (SEQ ID NO:1 present/SEQ ID NO:1 present).
Thus, a plant or plant part, or a chromosome 2, comprising the “ts-gene mutant” is herein used synonymous with a plant or a plant part, or a chromosome 2, comprising the deletion of SEQ ID NO: 1 (or the reverse complement sequence thereof) on chromosome 2. Likewise, a plant, or plant part, or a chromosome 2 lacking the ts-gene mutant is a wild type plant, or plant part or a chromosome 2 (comprising the wild type Ts gene), i.e., a plant, or plant part, or chromosome 2, which comprises SEQ ID NO: 1 (i.e., the chromosome 2 does not comprise the deletion of SEQ ID NO: 1).
The “HMBN mutant allele” or the “HMBN allele” refers herein to the mutant allele of a single recessive gene, which is described in U.S. Pat. No. 7,314,979B2 and U.S. Pat. No. 8,034,999B2, incorporated herein by reference. No markers are available, but the multibranching compact phenotype of the HMBN allele is readily recognized when the allele is in homozygous form and an allelism test is described to distinguish the allele from other genes. See, e.g., U.S. Pat. No. 8,034,999B2 at column 9, lines 26-37 and lines 50-57, incorporated herein by reference. The recessive mutant HMBN allele increases secondary branching of the vines, e.g., at 30 cm and 90 cm, compared to plants having the wild type (non-mutant) HMBN allele, i.e., leads to multibranching. Secondary branches means the branches resulting from the splitting of the primary (crown) branches.
“Multibranching phenotype” is thus the increase in secondary branches when the mutant HMBN allele is in homozygous form, compared to a plant comprising the wild type HMBN allele (not the mutant allele) in homozygous form. The number of secondary branches can be measured at e.g., 30 cm of the vine or at e.g., 90 cm of the vine and is, in case the mutant HMBN allele is in homozygous form, greater than 20.0 at 30 cm (see U.S. Pat. No. 8,034,999 at column 10, lines 36-42, incorporated herein by reference) and greater than 19.0 at 90 cm (see U.S. Pat. No. 8,034,999 at column 10, lines 43-50, incorporated herein by reference).
“Allelism test” is a test whereby two plants having similar phenotypes (e.g., multibranching) are crossed, and the segregation of the phenotype is analyzed in the F1 and optionally the F2 generation, whereby one will know if the similar phenotype is caused by the same allele (e.g., the mutant HMBN allele) or by a different allele.
In one embodiment, the fruits are preferably red fleshed, more preferably dark red fleshed, with a RHS rating of 39 or higher (not pink red or coral red or yellow-red or pink). In another embodiment, the average fruit brix is at least about 7.5% or higher.
In one aspect, without limiting the invention, the small fruit size is due to the homozygous presence of the HMBN mutant allele, which is combined with the ts-gene mutant, i.e., the large deletion of SEQ ID NO: 1, resulting in an average seed length of less than 6.0 mm, preferably equal to or less than 5.5 mm, at least when the deletion of SEQ ID NO: 1 (the ts-gene mutant) is present in homozygous form.
Thus, in one aspect, the diploid watermelon plant and plant part provided herein comprises the HMBN mutant allele in homozygous form and further comprises the deletion of SEQ ID NO: 1 on chromosome 2, whereby the fruits produced by said plant are of an average fruit weight of equal to or less than 0.9 kg, preferably equal to or less than 0.7 kg, more preferably equal to or less than 0.66 or 0.65 kg, and comprise seeds having an average seed length of 6.0 mm or less, preferably of 5.5 mm or less, when the deletion of SEQ ID NO: 1 is in homozygous form. In one aspect, the seeds from which the plant itself is grown have an average seed length of 6.0 mm or less, preferably 5.5 mm or less, as the seeds comprise the HMBN mutant allele in homozygous form and preferably also comprise the deletion of SEQ ID NO: 1 (the ts-gene mutant) in homozygous form.
Without intending to limit the scope of the products and methods described herein, the inventors found that the combination of the HMBN allele in homozygous form with the ts-gene mutant (deletion of SEQ ID NO: 1 on chromosome 2), preferably in homozygous form, results in an average fruit weight being reduced to 0.9 kg or less, preferably to 0.7 kg or less, and an average seed length of less than 6.0 mm, e.g., less than or equal to 5.5 mm, but, in one aspect, at least 4.00 mm in length, at least when the ts-gene mutant is in homozygous form. Both genetic elements, the HMBN mutant allele and the ts-gene mutant (i.e., the deletion of SEQ ID NO: 1 on chromosome 2) are present in homozygous form in diploid watermelon variety WH9716, a representative sample of seeds of this line having been deposited under Accession Number NCIMB 42704, and can therefore be obtained by crossing a plant grown from the deposited seeds with another watermelon. As both the genetic elements are recessive, the phenotypes will not be expressed in the F1 plant obtained and further selfing and selection is needed to generate a plant which is homozygous for both genetic elements.
Without wishing to be bound, it is thought that reducing the seed size in plants homozygous for the HMBN allele, by introducing the ts-gene mutant (i.e., a chromosome 2 comprising the deletion of SEQ ID NO: 1), enables the average fruit weight to be reduced to an average fruit weight of equal to or less than 0.9 kg, especially equal to or less than 0.7 kg.
It is a further object of the invention to provide watermelon plants, in one aspect dual purpose watermelon pollenizers, producing small, edible (i.e., marketable) diploid fruit with an average fruit weight of equal to or less than 0.9 kg, such as equal to or less than 0.8 kg, 0.7 kg, 0.66 kg, 0.65 kg, 0.6 kg, 0.5 kg, 0.4 kg or 0.3 kg, but above 0.25 kg. In one embodiment the fruits are preferably red fleshed, more preferably dark red fleshed, e.g., with a RHS rating of 39 or higher (red fleshed means not pink red or coral red or yellow-red or pink). In another embodiment the average fruit brix is at least about 7.5% or higher.
In another aspect, a diploid watermelon plant and plant part is provided herein comprises the HMBN mutant allele in heterozygous form and further comprises the deletion of SEQ ID NO: 1 on chromosome 2 in heterozygous form or in homozygous form.
The verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”, e.g., “a plant” refers also to several cells plants, etc. Similarly, “a fruit” or “a plant” also refers to a plurality of fruits and plants.
As used herein, the term “plant” includes the whole plant or any parts or derivatives thereof, preferably having the same genetic makeup as the plant from which it is obtained, such as plant organs (e.g., harvested or non-harvested fruits, leaves, etc.), plant cells, plant protoplasts, plant cell- or tissue-cultures from which whole plants can be regenerated, plant calli, plant cell clumps, plant transplants, seeds from which the plant can be grown and seeds produced by the plant, seedlings, plant cells that are intact in plants, plant clones or micropropagations, or parts of plants, such as plant cuttings, embryos, pollen, ovules, fruits (e.g., harvested tissues or organs), flowers, leaves, clonally propagated plants, roots, stems, root tips, grafts (scions and/or root stocks) and the like. Also any developmental stage is included, such as seedlings, cuttings prior or after rooting, etc.
It is, thus, understood that herein a watermelon plant, such as a diploid or triploid plant or pollenizer plant, encompasses not only an ungrafted plant, but also a plant with a rootstock of a different plant, such as a gourd or squash rootstock, another watermelon rootstock, a transgenic rootstock, etc.
As used herein, the term “variety” or “cultivar” means a plant grouping within a single botanical taxon of the lowest known rank, which can be defined by the expression of the characteristics resulting from a given genotype or combination of genotypes.
The term “allele(s)” means any of one or more alternative forms of a gene at a particular locus, all of which alleles relate to one trait or characteristic at a specific locus. In a diploid cell of an organism, alleles of a given gene are located at a specific location, or locus (loci plural) on a chromosome. One allele is present on each chromosome of the pair of homologous chromosomes. A diploid plant species may comprise a large number of different alleles at a particular locus. These may be identical alleles of the gene (homozygous) or two different alleles (heterozygous).
The term “HMBN allele” or “HMBN mutant allele” or “multibranching allele” refers to the mutant allele of a recessive gene described in U.S. Pat. No. 7,314,979B2 and U.S. Pat. No. 8,034,999B2, incorporated herein by reference.
The “ts-gene mutant allele” or the “ts-gene allele” refers herein to the single recessive gene described in Wehner (2007, Cucurbit Genetic Cooperative Report 30:96-120), as referring to the “tomato seed size mutant”, “ts” or “tss”, from the tomato seed Sugar Baby mutant plant, which is originally described by Zhang in Cucurbit Genetics Cooperative Report 19: 66-67 (1996). Herein, the “ts-gene mutant allele” also refers to the deletion of SEQ ID NO: 1, or the reverse complement sequence thereof, or a sequence comprising at least 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 1 or to the reverse complement of SEQ ID NO: 1 on chromosome 2 of the watermelon genome.
Similarly, the “wild type Ts-gene allele” or the “Ts-gene wild type allele” refers to the presence of SEQ ID NO: 1, or the reverse complement thereof, or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 or to the reverse complement of SEQ ID NO: 1 on chromosome 2 of the watermelon genome.
The watermelon genome refers to the physical watermelon genome, as e.g., found on the world web at cucurbitgenomics.org/. A Blast of the sequence of SEQ ID NO: 1, or the reverse complement thereof, or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 or to the reverse complement of SEQ ID NO: 1 against one of the watermelon genomes in the database will identify the sequence in the genome on chromosome 2. There are currently three sequenced watermelon genomes in the database, watermelon (97103) genome v1, watermelon (9307) genome v2 and watermelon (Charleston Grey) genome.
The term “locus” (loci plural) means a specific place or places or a site on a chromosome where for example a gene or genetic marker is found.
“Diploid plant” refers to a plant, vegetative plant part(s), or seed from which a diploid plant can be grown, having two sets of chromosome, designated herein as 2n. “Triploid plant” refers to a plant, vegetative plant part(s), or seed from which a triploid plant can be grown, having three sets of chromosomes, designated herein as 3n.
“Tetraploid plant” refers to a plant, vegetative plant part(s), or seed from which a tetraploid plant can be grown, having four sets of chromosomes, designated herein as 4n.
“Pollenizer plant” or “pollenizer” refers to the (inbred or hybrid) diploid plant, or parts thereof (e.g., its pollen or scion), suitable as pollenizer for inducing fruit set on triploid plants. A pollenizer plant is, thus, able to lead to good fruit set (and good triploid fruit yield) of triploid plants, by producing an appropriate amount of pollen at the appropriate day-time and for an appropriate period of time, e.g., at least during peak flowering time of the triploid female plants. A good triploid fruit yield is, for example, a yield comparable to the yield obtainable when using Polimax (produced by Nunhems) as pollenizer (see e.g., Example 3).
“Dual purpose pollenizer” refers to a pollenizer plant which also produces edible diploid fruits on the pollenizer plant itself (through self-pollination) and also is suitable to be used as a pollenizer in triploid (seedless) watermelon production. This definition is independent of whether or not the plant is actually being used as a pollenizer in triploid fruit production, i.e., it can also be used for diploid fruit production on its own.
The term “edible” or “marketable” is used herein to refer to fruits marketable for human consumption, especially fresh consumption of the fruit flesh. The fruits have at harvest at least good, preferably very good flavor properties (i.e., taste and odor). To have good flavor properties the fruits preferably have an average level of Total Soluble Solids of at least about 7.5% or more. Good fruit flesh color is also an important criterion for marketability for human consumption. For red-fleshed fruits it is an embodiment that the flesh color has an average RHS rating of at least 39 or above. If red-fleshed fruits are measured on a scale of 1 (white) to 10 (dark red), the fruits have an average rating of at least 7 or more.
“Hybrid triploid plant” is a triploid plant grown from hybrid, triploid seed obtained from cross fertilizing a male diploid parent with a female tetraploid parent.
“Seedless fruit” are triploid fruit which contain no or few mature seeds. The fruit may contain one or more small, edible, white ovules. Plants which produce seedless fruit may herein be referred to as “seedless”.
“Interplanting” refers to the combination of two or more types of seeds and/or transplants sown or transplanted on the same field, especially the sowing and/or transplanting of pollenizers in the same field as triploid hybrid plants (for seedless fruit production on the triploid plants and diploid fruit production on the pollenizer plants). For example, the pollenizer may either be planted in separate rows or interplanted with the triploid plants in the same row (e.g., in hills within each row). Pollenizers may also be planted in between rows of triploids. Also seeds of pollenizers and triploid hybrids may be mixed prior to seeding, resulting in random seeding. The transplants of the triploid hybrid plants and/or pollenizer plants may also comprise a rootstock of a different plant. Suitable rootstocks are known in the art. Also encompassed are methods where the triploid hybrid plant and the pollenizer plant are grafted together onto one rootstock.
“Planting” or “planted” refers to seeding (direct sowing) or transplanting seedlings (plantlets) into a field by machine or hand.
“Vegetative propagation” refers to propagation of plants from vegetative tissue, e.g., by in vitro propagation or grafting methods (using scions).
“Vegetative type” or “growth type” or “vine type” refers to the combination of growth characteristics of the vegetative parts of a plant line or variety, such as (average) internode length, (average) length of the main vine, (average) length of the shortest and longest branch, average number of primary branches, etc. Three vegetative types can be distinguished: The “normal/standard vine type”, the “compact vine type” and an “intermediate vine type” between these two.
“Compact vine type” refers to the vegetative type of a plant or plant line or variety as e.g., described in U.S. Pat. No. 7,314,979 and U.S. Pat. No. 8,034,999B2 for the HMBN mutant allele, resulting in the plant having an average internode length of about 4.0-6.5 cm, especially an average internode length of equal to or below 6.5 or 6.0 cm, e.g., equal to or below 5.0, 4.5 or 4.0 cm and/or an average longest branch of equal to or less than about 170 cm, preferably 160 cm, preferably equal to or less than 150 cm or 145 cm, preferably equal to or less than 140 or 130 cm. Examples of compact vine types are Companion and Sidekick (U.S. Pat. No. 7,314,979).
A “standard vine type” refers to the vegetative type of a plant or plant line or variety having an average internode length of more than 6.5 cm, preferably equal to or more than about 7, 8, 9, 10, 11 or 12 cm and/or an average longest branch of equal to or more than 225 cm, e.g., equal to or more than 230 cm, 250 cm, 300 cm, 350 cm or more. Examples are varieties Ace, Jenny, SP-1, SP-4.
An “intermediate vine type” refers to the vegetative type of a plant or plant line or variety which falls between the standard and compact vine type as defined above. It has an average internode length of less than 10.5 cm, e.g., equal to or less than 10, 9, 8, 7, 6 or 5 cm, preferably about 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 or 9.0 cm and/or an average longest branch of equal to or less than about 220 cm. The longest branch is on average preferably about 175-220 cm, e.g., preferably about 175, 180, 185, 190, 195, 200, 205, 210, 215 or 220 cm.
Throughout this document “average” and “mean” are used interchangeably and refer to the arithmetic mean.
A plant having “(essentially) all the physiological and morphological characteristics” means a plant having essentially all or all the physiological and morphological characteristics when grown under the same environmental conditions of a plant described herein, e.g., the plant of WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, from which it was derived, e.g., the progenitor plant, the parent, the recurrent parent, the plant used for tissue- or cell culture, etc. For example, the plant may have all fruit and/or all flowering characteristics described. In certain embodiments, the plant having “essentially all the physiological and morphological characteristics” are plants having all the physiological and morphological characteristics, except for certain characteristics, such as one, two or three, mentioned, e.g., the characteristic(s) derived from a converted or introduced gene or trait and/or except for the characteristics which differ in an EDV. So, the plant may have all fruit and/or flowering characteristics described, except for one, two or three characteristics described, in which the plant may thus differ. For example, the fruits may have a higher average brix than in the Examples.
The physiological and/or morphological characteristics mentioned above are commonly evaluated at significance levels of 1%, 5%, 8% or 10% significance level, when measured under the same environmental conditions. For example, a progeny plant of described herein, e.g., of WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721 may have one or more (or all, or all except one, two or three) of the essential physiological and/or morphological characteristics of the plants described herein, e.g., of WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, respectively, or one or more or all (or all except one, two or three) of the fruit and/or flowering characteristics, as determined at the 1% or 5% significance level when grown under the same environmental conditions.
The term “traditional breeding techniques” encompasses herein crossing, selfing, selection, double haploid production, embryo rescue, protoplast fusion, marker assisted selection, mutation breeding etc. as known to the breeder (i.e., methods other than genetic modification/transformation/transgenic methods), by which, for example, a genetically heritable trait can be transferred from one melon line or variety to another.
“Backcrossing” is a traditional breeding technique used to introduce a trait into a plant line or variety. The plant containing the trait is called the donor plant and the plant into which the trait is transferred is called the recurrent parent. An initial cross is made between the donor parent and the recurrent parent to produce progeny plants. Progeny plants which have the trait are then crossed to the recurrent parent. After several generations of backcrossing and/or selfing the recurrent parent comprises the trait of the donor. The plant generated in this way may be referred to as a “single trait converted plant”.
“Progeny” as used herein refers to plants derived from a plant described herein designated WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721. Progeny retain both the HMBN mutant allele and the ts-gene mutant (deletion of SEQ ID NO: 1 on chromosome 2), at least in heterozygous form, but preferably in homozygous form, in its genome. In one aspect, at least the HMBN mutant allele is in homozygous form, while the ts-gene mutant may be either in heterozygous form or in homozygous form. Progeny may be derived by regeneration of cell culture or tissue culture or parts of a plant described herein, e.g., of a plant designated WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, or selfing of a plant described herein, e.g., of a plant designated WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, or by producing seeds of a plant described herein, e.g., of a plant designated WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721. In further embodiments, progeny may also encompass plants derived from crossing of at least one plant described herein, e.g., a plant designated WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, with another watermelon plant of the same or another variety or (breeding) line, backcrossing, inserting of a locus into a plant or selecting a plant comprising a mutation or selecting a variant. A progeny is, e.g., a first generation progeny, i.e., the progeny is directly derived from, obtained from, obtainable from or derivable from the parent plant by, e.g., traditional breeding methods (selfing and/or crossing) or regeneration. However, the term “progeny” generally encompasses further generations such as second, third, fourth, fifth, sixth, seventh or more generations, i.e., generations of plants which are derived from, obtained from, obtainable from or derivable from the former generation by, e.g., traditional breeding methods, regeneration or genetic transformation techniques. For example, a second generation progeny can be produced from a first generation progeny by any of the methods mentioned above. Especially progeny of a plant described herein, e.g., of a plant WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721 which are EDVs or which retain all (or all except 1, 2 or 3) physiological and/or morphological characteristics of the plant described herein, e.g., of a plant WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, or which retain all (or all except 1, 2, or 3) of the fruit characteristics and/or flowering characteristics of the plant described herein, e.g., of a plant WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721 are encompassed herein.
The terms “gene converted” or “conversion plant” in this context refer to watermelon plants which are developed by backcrossing wherein essentially all of the desired morphological and physiological characteristics of parent are recovered in addition to the one or more genes transferred into the parent via the backcrossing technique or via genetic engineering. Likewise a “Single Locus Converted (Conversion) Plant” refers to plants which are developed by plant breeding techniques comprising or consisting of backcrossing, wherein essentially all of the desired morphological and physiological characteristics of a watermelon variety are recovered in addition to the characteristics of the single locus having been transferred into the variety via the backcrossing technique and/or by genetic transformation.
The invention provides plants (and seeds from which such plants are grown) of the species Citrullus lanatus, wherein said plant is optionally, in one aspect, suitable as a pollenizer in triploid watermelon production, is diploid and produces marketable diploid fruits having an average weight of less than 2.0 kg, preferably less than 1.8 kg, more preferably equal to or less than 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.0 kg, more preferably equal to or less than 0.9, 0.8, 0.7, 0.66, 0.65 kg, such as equal to or less than 0.6, 0.5, 0.4, 0.35, 0.3 or 0.25 kg at harvest (i.e., at maturity). In a further embodiment average fruit weight is equal to or less than about 0.9 kg (such as 0.8, 0.7, 0.66, 0.65, 0.6, 0.5, 0.4 kg) but above 0.25 kg. To determine average fruit characteristics, such as average fruit weight, of a plant line or hybrid according to the invention several plants of a line or hybrid are grown in one location and 3, 4, 5 or more fruits are harvested from 2, 3, or more plants of the same line and e.g., weighed. Reference plant lines (such as known pollenizers) can be grown at the same location (e.g., in the same trial or under the same environmental conditions) as a comparison. The average fruit weight according to the invention is in one embodiment preferably less than 0.66 or 0.65 kg, more preferably equal to or less than about 0.6, 0.5, 0.4, 0.3 or 0.25 kg. In another embodiment, the average fruit weight is less than 0.66 or 0.65 kg but above 0.25 kg. Preferably, the plants producing the above fruits comprise the HMBN mutant allele in homozygous form and further comprise the ts-gene mutant (deletion of SEQ ID NO: 1 on chromosome 2) in heterozygous or, preferably, in homozygous form.
When the plants comprise the ts-gene mutant allele in homozygous form, the fruits produced upon self-pollination comprise seeds having an average seed length of equal to or less than 6.0 mm, preferably equal to or less than 5.5. mm, such as equal to or less than 5.52 mm, 5.2 mm, 5.1 mm, or 5.0 mm. The average seed length is preferably at least 4.0 mm. As seed length and width correlate with each other, the seed size may also be measured as the weight of 10 seeds (gram/10 seeds). In one aspect, the fruits produce seeds whereby the weight per 10 seeds is equal to or less than 0.2 g, preferably equal to or less than 0.16 g or 0.15 g, or equal to or less than 0.13 g, at least in fruits of plants comprising the ts-gene mutant allele (i.e., the deletion of SEQ ID NO: 1) in homozygous form. When the deletion of SEQ ID NO: 1 is in homozygous form, this means that both chromosomes 2 of the diploid watermelon lack SEQ ID NO: 1 (and the reverse complement thereof). Each chromosome 2 has a plus and a minus strand, so that each chromosome 2 which lacks SEQ ID NO: 1 (or the reverse complement thereof), lacks both the plus and the minus strand, i.e., lacks SEQ ID NO: 1 and the reverse complement thereof. The presence or absence of SEQ ID NO: 1 (or the reverse complement thereof) in a watermelon or watermelon part can be easily tested by known molecular methods, such as PCR amplification, DNA hybridization, sequencing etc.
Fruit diameters can be variable, but preferably average fruit length is equal to or less than 12 cm, more preferably equal to or less than about 11.5 or 11 cm, such as equal to or less than 10.5, 10.0, 9.5, 9.0, 8.5 or 8.0 cm, while the average fruit width is preferably equal to or less than 11, 10.5 or 10 cm, such as equal to or less than 9.5, 9.0, 8.5, 8.0 cm. Thus, average fruit dimensions range have ranges of 8.0-12.0 cm average length×8.0-11 cm average width. Preferably at least fruit length is on average below 12 cm, preferably below 11.5 cm, more preferably about 11 cm or less. Preferably, both length and width of the fruits is on average about 11 cm or less. In one embodiment the average length by width is 10×11 cm, or less. In one embodiment average fruit dimensions for both lengths and width are equal to or below 10.5 cm. These dimensions are particularly encompassed for the diploid fruit weights of less than 0.66 or 0.65 kg on average. For the heavier diploid fruits (e.g., less than 1.8 kg but above 0.65 kg) also larger dimensions are encompassed herein, such as an average length of equal to or less than 17 cm (such as 16, 15, 14, 13, 12 cm) and an average width of equal to or less than 15 cm, such as 14, 13, 12 cm).
The fruits, i.e., the fruit flesh, produced on the plants are edible at maturity. The flesh color of the mature fruits of the diploid plants according to the invention is in one embodiment red, e.g., having an RHS mini color chart value (Royal Horticultural Society mini color chart) of 39 or higher, especially selected from 39 to 44. Especially fruits with flesh colors RHS 39 A or B; 40 A, B or C; 41A or B, 42A, B, or C; 43A or B; 44A, B or C are encompassed. Also, the average percent Total Soluble Solids (% TTS; herein also referred to as degrees Brix, or ° Brix) of the fruits is at maturity at least about 7.5%, preferably at least about 8% or 8.5%, more preferably at least about 9% or 9.5%, and even more preferably at least about 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, or more. Average TSS can, for example, be determined using a refractometer as described in the Examples. The average percentage TSS of the diploid fruits produced by the watermelon plants according to the invention can be increased by traditional breeding techniques, e.g., by crossing the plants provided herein with watermelon plants comprising high TSS and selection of progeny (e.g., obtainable by one or more selfings and/or backcross populations) producing fruits with higher TSS values while maintaining small fruit size, and retaining the HMBN mutant allele and the ts-gene mutant allele.
The HMBN mutant allele can, for example, be maintained in progeny by selecting for the small fruit size and/or for compact vine type (optionally intermediate vine type) and/or for multibranching in progeny plants homozygous (e.g., after selfing one or more times) for the HMBN mutant allele. Also an allelism test can be done to verify the presence of the HMBN mutant allele, as described in U.S. Pat. No. 7,314,979 and U.S. Pat. No. 8,034,999, incorporated by reference herein.
The ts-gene mutant allele can be maintained in progeny by selecting for the absence of SEQ ID NO: 1 or the heterozygous presence of SEQ ID NO: 1 in progeny plants. This can be done using known molecular biology techniques, such as DNA hybridization, PCR (Polymerase Chain Reaction), sequencing, etc. to detect the presence or absence of SEQ ID NO: 1 in the genomic DNA. For example, a PCR primer pair can be designed which amplifies all or part of SEQ ID NO: 1 (or the reverse complement thereof), and can then be used to determine the presence of SEQ ID NO: 1 in the genomic DNA of the plant or plant part.
In another aspect, the disclosure provides a method for detecting the presence or absence of SEQ ID NO: 1 (or the reverse complement, or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, in the genomic DNA of a watermelon plant or plant part. In another aspect, the disclosure provides a method for selecting a watermelon plant or plant part or seed, comprising determining the presence or absence of SEQ ID NO: 1 (or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1) in the genome of the plant, plant part or seed, and optionally selecting a plant, plant part or seed in which SEQ ID NO: 1 (or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1) is absent in the genome or where only one copy is present in the genome (heterozygous).
In one aspect, a method for screening a watermelon plant or plant part is provided, comprising detecting the presence or absence of a deletion of SEQ ID NO: 1 on chromosome 2 in the genome of said watermelon plant, and optionally detecting the presence or absence of the HMBN mutant allele.
In a further aspect, a method for selecting or breeding a watermelon plant is provided, comprising selecting a plant comprising a deletion of SEQ ID NO: 1 on chromosome 2 in the genome of said watermelon plant (and optionally comprising the HMBN mutant allele in its genome), and optionally crossing said plant, or a progeny of said plant which also comprises the deletion of SEQ ID NO: 1 on chromosome 2 (and optionally comprising the HMBN mutant allele in its genome), with another watermelon plant. In one aspect, the other watermelon plant comprises the HMBN mutant allele in its genome.
A plant produced by the method, comprising a deletion of SEQ ID NO: 1 on chromosome 2 and optionally the HMBN mutant allele, is also encompassed herein. In one aspect, the plant produced is an F1 hybrid plant, produced by crossing a first inbred parent line, comprising a deletion of SEQ ID NO: 1 on chromosome 2 and optionally the HMBN mutant allele, with a second inbred parent line. The second inbred parent line may also comprise a deletion of SEQ ID NO: 1 on chromosome 2 and optionally the HMBN mutant allele. The F1 hybrid may thus comprise the deletion of SEQ ID NO: 1 on chromosome 2 in e.g., homozygous form and optionally the HMBN mutant allele in homozygous form. In another aspect, the method further comprises detecting the presence or absence of a deletion of SEQ ID NO: 1 on chromosome 2 in the genome of the first and/or second inbred parent line, and optionally detecting the presence or absence of the HMBN mutant allele first and/or second inbred parent line.
As discussed herein, the diploid fruits according to the invention are edible, i.e., they have fruit quality characteristics which make them marketable for human consumption. This means that the fruits have good flavor properties (no off-flavors etc.). For fruits to have at least good flavor properties a minimum average brix of at least about 7.5 degrees is desired. Flavor properties can be determined and scored (e.g., as bad, good, very good) by trained test-panels using known methods for evaluating sensory properties of fruits (Karen L. Bett, Ch 13, Fresh-Cut Fruits and Vegetables, Science, Technology, and Market; Edited by Olusola Lamikanra, CRC Press 2002, Print ISBN: 978-1-58716-030-1). Selection for good flavor includes test panels to select against bitterness and other unpleasant flavors, such as caramel flavor. Watermelon checks of varieties which have good flavor properties (e.g., Allsweet, Crimson Sweet) are preferably included in the test.
The fruits should preferably also not be susceptible to what is known as “fruit cracking” and/or should preferably not contain the brittle gene as present in SP-1 and in variety Sidekick. The Super Pollenizers such as SP-1 (as described in WO03/075641) bear brittle fruits, which makes the fruits (in particular the fruit flesh) unmarketable as fresh produce (although the seeds contained within the fruits can be harvested and marketed). Crack-resistance is generally selected for during breeding (e.g., in field observations and/or using for example pressure tests or other tests), as cracking is an undesired fruit quality characteristic. See also Haikun et al. 2010, Acta Hort. (ISHS) 871:223-230).
Other fruit characteristics can be introduced by traditional breeding methods (see further below) and thereby combined with pollenizers producing small, edible diploid fruits according to the invention. For example plants can be selected which produce small, edible fruits, as described above, with increased or reduced rind thickness, increased or reduced rind brittleness, various skin/rind colors (e.g., light green; dark green; green-striped with narrow, medium or wide stripes; grey types; with or without spotting; Golden yellow; black or black green) and rind surfaces (e.g., furrowed or smooth surface), flesh structure/flesh firmness, different fruit shapes (elongate, oval, blocky, spherical or round), higher brix content, higher lycopene and/or vitamin content, different sugar : acid ratios, very good fruit flavour, etc. Also the combination of small edible fruits with another flesh color than red is possible, for example genetic determinants for yellow flesh or orange flesh or white flesh may be introduced, e.g., by backcross breeding with another color-type. See Guner and Wehner 2004, Hort Science 39(6): 1175-1182, in particular pages 1180-1181 describing genes for fruit characteristics. Generally important breeding objectives are early maturity, high fruit yield, high internal fruit quality (good uniform color, high sugar, proper sugar:acid ratio, good flavor, high vitamin and lycopene content, firm flesh texture, non-fibrous flesh texture, freedom from defects such as hollow heart, rind necrosis, blossom-end rot or cross stitch and good rind characteristics and cracking-resistance).
Rind thickness is a characteristic which influences damage during handling and transporting (too thin or too brittle rind), but thin rinds may also be desirable for consumers. In one embodiment the fruits have a thin rind, such as an average rind thickness (measured on the side) of at least about 0.2 cm, 0.3 cm, 0.4 cm, but less than 0.5 cm, more preferably less than or equal to 0.4 cm. Thus in one embodiment the rind is thicker than the rind of SP-4 fruit, but thinner than the rind of Polimax, and optionally thinner than the rind of Sidekick fruit. For certain embodiments thicker rinds may be desired and small fruits having a rind of 0.5 or more cm, such as 0.6 and 0.7 cm are also encompassed herein. The rind of the fruits preferably does not crack easily (i.e., is cracking-resistant), both in fruits with thin rinds and thick rinds.
In one embodiment, the fruits preferably also do not have a brittle rind and/or an explosive rind as described in WO03/075641 on page 13 and 14, i.e., the fruits do not break under pressure in the range of 90 to 140 g/mm2.
Flesh firmness of the diploid fruits is preferably at least about 0.8 (average firmness in kg as in Example 1), more preferably at least about 0.9, 1.0, or more. Flesh firmness can be increased by e.g., crossing with watermelons having firmer fruit flesh and selection for firmer flesh without increasing fruit size. Plants producing fruits with ultra-firm flesh are, for example, described in US2006/0005284.
The diploid plants provided herein are, in one aspect, suitable as pollenizers, which means that they produce sufficient pollen at the right time of the day and for an appropriate period of time to induce fruit set in triploid hybrids, leading to a (average) triploid fruit yield at least comparable to that obtained when using e.g., Polimax as pollinator. However, the plants according to the invention need not be sold or marketed as pollenizer for triploid fruit production and need not be used as pollenizer in triploid fruit production. They may also be marketed and/or used solely for diploid fruit production on their own.
In one embodiment, the plants are pollenizer plants which preferably produce a large number of male flowers at the appropriate time during flowering of the triploid hybrids, preferably at least about 35 open male flowers at day 15 and/or at least about 30 open male flowers at day 22 from the first day of flowering, although the number of male flowers is not critical in determining triploid fruit set, as long as sufficient pollen is produced by the available male flowers to lead to good triploid fruit set. As the pollenizer plants are also used for diploid fruit production, also sufficient female flowers must be produced by the pollenizers to ensure their dual purpose.
As can be seen in the Examples (Table 4), many open male and female flowers were present over the 3 week period counted. At the flowering date (the date when more than 50% of the plant in the plot has male/female flowers) the 11 pollenizer lines had on average 12.6 open male and 3.9 open female flowers. Each line generally had significantly more open male flowers than SP-4 and Polimax. At day 15 and 22 from 1st day of flowering all lines produce significantly more open male flowers than Sidekick, SP-4 and Polimax, all having at least 35 open male flowers at day 15 and at least 30 open male flowers at day 22. However, Polimax is a very good pollenizer for triploid hybrids, despite the fact that the number of male flowers is significantly lower than in the hybrids provided in the Examples. Polimax had only about 18 open male flowers at day 15 and day 20. The pollenizers according to the invention, therefore, may have a lower number of male flowers than in the Examples in Table 3, e.g., plants having about the same number of open male flowers as Polimax are encompassed herein, i.e., pollenizers having at least about 15, 18, 20, 25, 30, or more open male flowers at day 15 and/or at day 22 from the 1st day of flowering are also an embodiment of the invention.
Also the number of open female flowers was good, indicating that the pollinizers are indeed suitable for dual-purposes, i.e., pollination and fruit production of triploid hybrids and/or pollination and fruit production on the diploid plants themselves (fruits produced by self-pollination of the pollenizer plant).
In one embodiment, the pollenizer plants described herein are dual purpose pollenizer plants. In particular, the average number of open male flowers at day 22 from flowering is at least 15, 18, 20, 25 or 30 or more.
In one embodiment, the watermelon plants, e.g., the pollenizer plants described herein, are hybrid diploids (F1 diploids) and not open pollinated (OP). In another aspect, the watermelon plants are open pollinated.
In yet a further embodiment, the watermelon plants, e.g., the dual purpose pollenizer plants described herein, are not of the “edible seed watermelon” or “confectionary” type, as for example produced in variety San Juan (Native Seeds). These types of watermelon are produced to harvest the seeds for consumption or seed oil production and the fruits produce large, black or red edible seeds with soft seed coats, see e.g., Zhang 1996, Cucurbit Genetics Cooperative Report 19:66-67 (article 24) and Zhang and Wang, 1990, Cucurbit Genetics Cooperative Report 13:43-44 (article 16). In contrast, the seeds produced in the diploid fruits according to the invention are small (preferably tomato seed size to medium seed size, but not large) and are not suitable for seed harvest and seed consumption.
In one embodiment, the seeds of the diploid fruits (produced by plants described herein, which comprise the HMBN mutant allele in homozygous form and comprise the deletion of SEQ ID NO: 1 on chromosome 2 in homozygous form) are on average shorter than or equal to 6 mm in length, for example, shorter than or equal to 5.5 mm, 5.25 mm, or 5.0 mm, but are preferably at least 4 mm in length.
In one embodiment, plants described herein comprise the HMBN mutant allele in homozygous form and comprise the ts-gene mutant allele in homozygous form. As mentioned, these plants will produce fruits of equal to or less than 0.9, 0.8, 0.7, 0.66, 0.65 kg and the average seed size of the seeds produced in the fruits (upon self-pollination of the plants) is shorter than or equal to 6 mm in length, for example, shorter than or equal to 5.5 mm, 5.25 mm, or 5.0 mm, but are preferably at least 4 mm length. The seeds comprise the HMBN mutant allele and the ts-gene mutant allele in homozygous form.
In another embodiment, plants described herein comprise the HMBN mutant allele in homozygous form but comprise the ts-gene mutant allele in heterozygous form. These plants will produce fruits of equal to or less than 0.9, 0.8, 0.7, 0.66, 0.65 kg, but the seeds produced in these fruits will segregate for the genotypes Ts/Ts:Ts/ts:ts/ts in a ratio of about 1:2:1.
The watermelon (e.g., dual purpose pollenizer) plants not only produce very small marketable fruits, but also high numbers of marketable fruits. In one embodiment, a plant described herein produces, at maturity, on average at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more fruits per plant, more preferably at least 25 fruits, more preferably at least about 27, 28, 29, 30, 35, 40, 50, 60, 65 or more fruits.
The watermelon plants described herein have, in one aspect, a compact vine type due to the homozygous presence of the HMBN mutant allele, but they can equally have an “intermediate” vine type, as it was found that the phenotype of the vine type caused by the HMBN mutant allele can be also an “intermediate vine type”. Without intending to limit the scope of the products and methods described herein, it is thought that whether a compact vine type or an intermediate vine type is expressed in a plant homozygous for the HMBN mutant allele depends on the genetic background of the plant. For example, the watermelon plants described herein may be crossed with a standard vine type and progeny may be selected which have an intermediate vine type, produce small fruits and comprise at least one copy of the ts-gene mutant. Such plants may thus still comprise the HMBN mutant allele in homozygous form. For example, WH9311 has an intermediate vine type with an average longest branch of 180 cm and an average internode length of 5.3 cm, and it comprises the HMBN mutant allele in homozygous form and the ts-gene mutant in homozygous form. The HMBN mutant allele (in homozygous form) thus can result in a compact vine type or an intermediate compact vine type, depending on the genetic background it is in. The watermelon plants described herein, which are homozygous for the HMBN mutant allele, may thus either have a compact vine type or an intermediate vine type.
It is understood that it is also an object of the invention to provide seeds from which the watermelon plants described herein can be grown. Also seedlings, scions and rootstocks, as well as cells and tissues of the watermelon plants are encompassed herein. Such plant parts comprise the genetic determinants for producing small fruits (with at least one copy of the HMBN mutant allele) and for producing small seeds (with at least one copy of the ts-gene mutant allele) according to the invention. Thus, whole plants obtained from seedlings, scions and rootstocks, as well as cells and tissues of the watermelon plants retain the genetic determinants described, and produce small fruits having small seeds when the genetic determinants are in homozygous form.
It is a further object of the invention to provide a plurality of diploid watermelon fruits obtainable on a plant according to the invention as described above, and/or seeds present in those fruits. Thus, in one embodiment, harvested diploid fruits are provided, such as packaged whole fruits or fruit parts and/or processed fruits or fruit parts. The fruits, i.e., the fruit flesh tissue, comprise the HMBN mutant allele in homozygous form and the ts-gene mutant allele in heterozygous or preferably in homozygous form. Preferably, the fruits comprise the HMBN mutant allele in homozygous form and the ts-gene mutant allele in in homozygous form and the average seed size of the seeds produced in these fruits is equal to or less than 6.0 mm, 5.5 mm, etc. as described herein.
Also progeny of the plants according to the invention are provided herein, such as seeds obtainable by crossing a watermelon plant described herein with another watermelon plant and/or by selfing a plant according to the invention to produce F 1 seeds (and F 1 plants grown from these seeds, as well as fruit produced by self-pollinating the F1 plants).
Further provided are plant cells, cell cultures or tissue cultures of plants according to the invention, as well as root stocks, scions, transplants and vegetative propagations of plants according to the invention or of progeny thereof.
In one aspect, a representative sample of seeds of the plants described herein are deposited under accession number NCIMB 41773 (WH9307), or accession number NCIMB 42704 (WH9716). Both comprise the HMBN mutant allele in homozygous form and the ts-gene mutant allele (deletion of SEQ ID NO: 1) in homozygous form. The HMBN mutant allele and the ts-gene mutant allele can thus be obtained from these deposited seeds and can be crossed into any other watermelon plant using traditional breeding techniques. As mentioned, the transfer of the HMBN mutant allele into another watermelon plant can be easily determined by the phenotype, especially multibranching, conferred by the HMBN mutant allele and the transfer of the ts-gene mutant allele into another watermelon plant can be easily determined by molecular analysis and/or the phenotype conferred by the ts-gene mutant allele.
In one aspect, a watermelon plant (or a seed from which the plant can be grown), or fruit and/or seed thereof, or part of the plant, having essentially all morphological and physiological fruit characteristics and/or flowering characteristics of the plants of which representative samples of seeds have been deposited are provided.
In another aspect a watermelon plant (or a seed from which the plant can be grown), or fruit and/or seed thereof, or part of the plant, having essentially all morphological and physiological characteristics of the plants of which representative samples of seeds have been deposited are provided.
Plants having the genetic determinants for producing small fruits comprising small seeds are, therefore, obtainable from the deposited seeds NCIMB 42704 or NCIMB 41773 or progeny thereof, as both the HMBN mutant allele and the ts-gene mutant allele can be obtained from the seeds. These genetic determinants (i.e., the combination of genes) for producing small fruits comprising small seeds can be transferred to other watermelon plants, for example to create other diploid watermelon plants, e.g., pollenizers, with this phenotype or other diploids (open pollinated or inbred lines, or hybrid diploids). This can be done by using the plants described herein as a parental line in breeding methods, i.e., as male or female parent in a cross with another watermelon plant. Known breeding methods can be used alone or in combination, such as (but not limited to) recurrent selection, pedigree breeding, backcross breeding, inbred development, hybrid testing, marker assisted breeding, etc. Diploids may also be used for tetraploid development, using e.g., colchicine treatment. Progeny are then selected which retain the small fruit dimensions and fruit weight, conferred by the HMBN mutant allele, and which retain at least one copy of the ts-gene mutant allele and optionally a brix of at least 7.5% and optionally dual purpose pollenizer characteristics, all as described herein.
Other watermelon plants may be used as a starting point to develop watermelon plants, e.g., dual purpose pollenizer plants, producing small, marketable fruits according to the invention. For example, small fruited cultivars or lines may be used as starting material, such as for example Sidekick (U.S. Pat. No. 7,314,979), which comprises the HMBN mutant allele and watermelon plants carrying the “tomato seed” mutant (ts-gene mutant, or Sugar baby ts-gene mutant plant) described in Guner and Wehner (2004, HortScience 39(6):1175-1182) and obtainable from gene curator of the Cucurbit Genetics Cooperative), and selecting for e.g., fruit quality characteristics (e.g., high brix, good flavor), small fruit size (e.g., the presence of the HMBN mutant allele conferring small fruit dimensions of e.g., less than 0.9 kg weight) and small seed size in the diploid fruits (especially the presence of the ts-gene mutant allele. i.e., absence of SEQ ID NO: 1), as well as optionally pollenizer characteristics (e.g., many male flowers) as described herein.
Selection for small seed size encompasses selecting for the presence of the ts-gene mutant allele and/or an average seed length of equal to or less than 6 mm or more preferably equal to or less than 5.0 mm, preferably equal to or less than 4.5 mm seed length. Phenotypic selection for an average seed length of equal to or less than 6.0 mm should be done in lines homozygous for the ts-gene mutant allele (i.e., homozygous for the deletion of SEQ ID NO: 1). Selection for the presence of the deletion of SEQ ID NO: 1 is therefore much easier, as this can be done by e.g., PCR analysis, sequencing or DNA hybridization or other methods. For example, PCR primer pairs can be designed to test whether the genomic DNA comprises SEQ ID NO: 1 in one or two copies or lacks SEQ ID NO: 1. Appropriate controls should be included in any analysis. For example, when doing PCR amplifications of SEQ ID NO: 1 or a part of SEQ ID NO: 1, control DNA should be included in the PCR analysis, e.g., DNA of a watermelon plant or plant part comprising SEQ ID NO: 1.
In one embodiment, a breeding method for producing diploid watermelon plants according to the invention is provided, comprising:
a) providing a breeding population of diploid watermelon plants comprising the HMBN mutant allele and the ts-gene mutant allele (deletion of SEQ ID NO: 1), obtainable from e.g., seeds deposited under NCIMB 42704 (WH9716) or progeny thereof, and
b) selecting progeny plants for producing fruits comprising an average fruit weight of equal to or less than 0.9 kg, or 0.7 kg, or 0.65 kg and the presence of the ts-gene mutant allele and/or producing seeds in the fruits comprising an average seed length of equal to or less than 6.0 mm, and optionally high brix, good flesh color and pollenizer characteristics (all as described throughout this application).
The breeding population under a) can, for example, be generated by crossing a watermelon plant comprising the HMBN mutant allele and the ts-gene mutant with a watermelon plant lacking the HMBN allele and lacking the ts-gene mutant allele, and then optionally selfing progeny one or more times. Alternatively, a watermelon plant comprising the HMBN mutant allele can be crossed with a watermelon plant comprising the ts-gene mutant allele to generate progeny comprising both mutant alleles, optionally followed by selfing of the progeny plants.
Preferably, the progeny are selected to be homozygous for the HMBN mutant allele and homozygous for the deletion of SEQ ID NO: 1 on chromosome 2 of their genome. As mentioned previously, no markers for the HMBN allele are available, but the retention of the allele can be seen phenotypically in progeny through the presence of the small fruit weight and the compact vine type or intermediate compact vine type when the allele is in homozygous form.
The presence of the ts-gene mutant allele can be easily determined using DNA based detection methods and/or the average seed length of equal to or less than 6.0 mm when the ts-gene mutant allele is in homozygous form.
Plants obtainable by the above methods are encompassed herein. As discussed herein, the breeding population can be provided by using at least one, preferably two small-fruited parents and crossing these, to generate an F1 and further progeny generations (F2, etc.). For example, one of the plants described herein comprising the HMBN mutant allele and the ts-gene mutant allele (e.g., WH9307 or WH9716 or progeny thereof) is used in step a), and is crossed to another watermelon plant. The progeny generations are then selected for at least the characteristics described in b). Alternatively, the HMBN mutant allele can be obtained from e.g., variety Sidekick and the ts-gene mutant allele can be obtained from the original Sugar Baby ts-gene mutant plant, i.e., these can be used to generate a breeding population comprising both mutant alleles.
Thus, in one aspect, a method for producing a diploid Citrullus lanatus plant is provided, comprising crossing a first Citrullus lanatus plant comprising a deletion of SEQ ID NO: 1 and the HMBN mutant allele, both as present in seeds deposited under NCIMB 42704, with a second Citrullus lanatus plant to obtain progeny plants, and selecting progeny plants that produce fruits having an average fruit weight of equal to or less than 0.9 kg at maturity and and comprise the deletion of SEQ ID NO: 1. Preferably the fruits have red fruit flesh.
In one aspect, the first Citrullus lanatus plant is homozygous for the HMBN mutant allele and for the deletion of SEQ ID NO: 1. In a further aspect, the second Citrullus lanatus plant is also homozygous for the HMBN mutant allele and for the deletion of SEQ ID NO: 1, so that the progeny plants are also homozygous for the HMBN mutant allele and for the deletion of SEQ ID NO: 1.
In one aspect, the first and/or second Citrullus lanatus plant produce fruits with red fruit flesh.
Preferably, the progeny plant has the following characteristics: it comprises the HMBN mutant allele and the ts-gene mutant allele (deletion of SEQ ID NO: 1) in homozygous form and produces fruits of equal to or less than 0.9 kg, preferably equal to or less than 0.7 kg, 0.65 kg or less, at maturity, wherein said fruits are red fleshed and comprise seeds having an average seed length of equal to or less than 6.0 mm, preferably equal to or less than 5.5 mm.
In one aspect, the fruits have a black or black green rind. In another aspect, the rind of the fruits is thin rind thickness, e.g., an average rind thickness on the side of 0.2 to 0.5 cm, preferably 0.2 to 0.4 cm, preferably 0.2 to 0.3 cm.
The plants and plant parts produced by the methods described herein are encompassed, as are seeds from which the plants can be grown. Also the diploid fruits produced by the plants are encompassed herein.
In one embodiment, one or more hybrid plants is provided, obtained from crossing a diploid watermelon plant according to the invention with another watermelon plant and harvesting the F1 seeds of said cross. The F1 seeds may then be grown into F1 plants and self-pollinated or sib-pollinated to produce F2 seeds. If the parents used in the initial cross differ by one or more characteristics (e.g., disease resistance and fruit size), the F2 population will segregate for these trait(s) and the breeder can select plants in this and/or further progeny generations (F3, F4, etc.) which combine the desired traits (e.g., small fruit size and disease resistance). Alternatively, the F1 may be backcrossed to the recurrent parent (e.g., the diploid watermelon plant according to the invention into which a trait is to be introduced) or the F1 may be selfed to produce an F2 population segregating for the trait of interest and selected F2 plants having the trait of interest may be backcrossed to the recurrent parent.
In one aspect, a method for generating F 1 hybrid seeds or F 1 hybrid plants is provided, comprising a) crossing a watermelon inbred line comprising HMBN mutant allele and the ts-gene mutant allele (deletion of SEQ ID NO: 1) both in homozygous form with another watermelon inbred line comprising both the HMBN mutant allele and the ts-gene mutant allele (deletion of SEQ ID NO: 1) in homozygous form and b) harvesting the F1 hybrid seeds produced in said fruits, and c) optionally packaging the seeds. The seeds can be sown and will grow into F1 hybrid plants producing fruits comprising an average fruit weight of equal to or less than 0.9 kg, or 0.7 kg, or 0.65 kg and producing seeds in the fruits comprising an average seed length of equal to or less than 6.0 mm, and optionally high brix (e.g., at least 7.5%, 8%, 9%, 10% or more), good flesh color, e.g., red flesh.
One or more traits not present in the watermelon plants according to the invention can be introduced into a plant according to the invention, while maintaining the genetic determinants for small diploid fruits comprising small seeds, i.e., the HMBN mutant allele and the ts-gene mutant allele. For example other fruit characteristics as described above can be introduced (e.g., darker red flesh color, higher brix, firmer flesh, a different flesh color, etc.), or any other traits can be introduced, such as one or more QTLs for high yield, disease resistance genes, stress tolerance genes (e.g., water stress tolerance), etc. resistance to fungal-, bacterial-, viral-diseases, root-knot nematodes and/or insect pests may be introduced. For example, resistance to Fusarium wilt (Fusarium oxysporum fsp. niveum race 0, race 1 and/or race 2, and/or race 3, and/or other new races which may develop), Anthracnose (Colletotrichum lagenarium races 1-7, or other new races), Gummy stem blight, powdery mildew, Verticillium wilt, bacterial fruit blotch, papaya ringspot virus (PRSV), watermelon mosaic virus (WMV) or zucchini yellow mosaic virus (ZYMV).
Resistance to Fusarium wilt races 0 and 1 is present in many commercial varieties, and also resistance to race 2 has been identified in PI296341 and PI271769 and is also present in SP-4 (U.S. Pat. No. 7,550,652). Anthracnose resistance to race 1 is for examples present in SP-4 and resistance to race 2 in AU-Sweet Scarlet (AW-82-50CS) (Breeder: Alabama Agric. Expt. Station, Auburn University). Crimson Sweet has the Ar-1 gene, which provides resistance to anthracnose races 1 and 3. Gummy stem blight resistance is also found in Plant Introduction lines.
Also provided is a method for producing diploid and triploid watermelon fruits in one field, said method comprising:
a) interplanting diploid pollenizer plants described herein and triploid hybrid plants in one field,
b) allowing pollination of flowers of the triploid hybrid plants with pollen of the diploid pollenizer plants and allowing pollination of flowers of the diploid pollenizer plants with pollen of the diploid pollenizer plants,
c) harvesting fruits produced on the triploid hybrid plants and, optionally, harvesting fruits produced on the diploid pollenizer plants.
Also provided is a method for producing diploid watermelon fruits of an average fruit weight of equal to or less than 0.9 kg, said fruits comprise seeds having an average seed length of equal to or less than 6.0 mm, said method comprising:
a) planting diploid watermelon plants described herein in a field, i.e., comprising the HMBN mutant allele and the ts-gene mutant allele preferably both in homozygous form, and
b) allowing pollination of flowers of the diploid watermelon plants with pollen of the diploid watermelon plants, and optionally
c) harvesting fruits produced on the diploid watermelon plants.
The harvested fruits are also encompassed herein. As mentioned, the fruits are small and comprise small seeds. If the HMBN and ts-mutant allele are both homozygous in the diploid plants, the fruit flesh and the seeds will also be homozygous for both mutant alleles. The fruits can thus be distinguished from other fruits phenotypically and/or genetically, by the absence of SEQ ID NO: 1 in the fruit flesh DNA and in the seed DNA. The presence of the HMBN allele in the seeds of the fruits can also be determined by growing plants from the seeds to determine the growth phenotype of the plant, e.g., the multibranching phenotype and/or the vine type being compact or intermediate vine type. Optionally, an allelism test can be done to verify the presence of the HMBN allele, e.g., as described herein.
In one embodiment, the diploid fruits and/or the triploid fruits are marketable and red-fleshed, white-fleshed, orange-fleshed or yellow-fleshed.
In a further embodiment, the rind of the diploid fruits is not yellow or Golden (as controlled by the recessive gene go) (Barham 1965, Proc Ameri Soc Hort Sci 67: 487-489).
Interplanting in one field may be either done by seeding or transplants of the pollenizer and triploids. Various interplanting methods can be used, as known in the art and various ratios of pollenizer : triploid hybrid may be used. One row of pollenizer plants may for example be present at least every 2, at least every 3 or at least every 4 rows of triploids, but other methods of interplanting may also be used.
Any triploid hybrid may be used, such as known triploid hybrid varieties.
Pollination is usually done by bees, and bee hives can be provided to the fields unless sufficient wild bees are naturally present. Pollination can also be performed by manual or mechanical means. Harvest at maturity may be done by hand or mechanized.
The diploid fruit may be distinguished from the triploid fruit based on the smaller fruit size of the diploid fruit, and/or alternatively by a different rind pattern. In one embodiment the rind of the diploid fruits according to the invention is not yellow or golden. Preferably harvested diploid and triploid fruit are placed into different containers. Thus, in one embodiment a container comprising solely small diploid fruits according to the invention is provided. Any type of container may be used, e.g., cartons, boxes, etc.
Also a method for producing small diploid watermelon fruits having an average weight of less than 1.8 kg, 1.7 kg, 1.0 kg, 0.9 kg, 0.8 kg, 0.7 kg, preferably equal to or less than 0.65 kg (but in one embodiment larger than 0.25 kg), is provided comprising:
a) growing a diploid watermelon plant comprising the HMBN mutant allele and the ts-gene mutant allele, preferably both in homozygous form,
b) pollinating the female flowers of said plant with pollen of said plant, and
c) harvesting the fruits produced on said plant.
Thus, by self-pollination of a diploid plant described herein, small diploid fruits are produced comprising seeds in the fruits having an average seed length of equal to or less than 6.0 mm, preferably equal to or less than 5.5 mm or less. The plants described herein may be grown in a field without other watermelon plants being present, and the small diploid fruits may be harvested and placed in containers for transport. Step (b) may be performed by allowing insect pollination or any other means of pollinating.
In one embodiment, a diploid plant (or a seed from which the plant can be grown) capable of producing small diploid fruits having an average weight of less than 0.9 kg, 0.8 kg, 0.7 kg or equal to or less than 0.65 kg (but in one embodiment larger than 0.25 kg) is provided, wherein a representative sample of seed containing the genetic elements for producing said small fruits comprising seeds having an average seed length of equal to or less than 6.0 mm, i.e., comprising the HMBN mutant allele and the ts-gene mutant allele in homozygous form has been deposited under accession number NCIMB 41773 (WH9307), or accession number NCIMB 42704 (WH9716).
Pollenizer hybrids WH9307 and WH9716 comprise the genetic elements for producing said small fruits with an average seed length of equal to or less than 6.0 mm (i.e., comprising the HMBN mutant allele and the ts-gene mutant allele) and a representative sample of seeds have been deposited under accession number NCIMB 41773 and NCIMB 42704, respectively. Thus, when referring herein to seed deposits of watermelon plants, e.g., pollenizers, described herein, the plants are referred to as a representative plants according to the invention, but seeds of the other hybrids or lines are also suitable and when reference to WH9307 or WH9716 or progeny thereof (or parts of any of these) is made, the other hybrids or lines mentioned herein are equally implied, especially lines and hybrids retaining the combination of HMBN mutant allele and the ts-gene mutant allele (i.e., absence of SEQ ID NO: 1), preferably both in homozygous form.
In one embodiment, a hybrid watermelon seed is provided, having as a male or female parent (preferably as a male parent) a diploid plant comprising the HMBN mutant allele and the ts-gene mutant allele and capable of producing small diploid fruits having an average weight of less than 0.9 kg, 0.8 kg, 0.7 kg or equal to or less than 0.65 kg (but in one embodiment larger than 0.25 kg) said fruits comprising seeds with an average seed length of equal to or less than 6.0 mm is provided, wherein a representative sample of seed containing the genetic elements for producing said small diploid fruits with small seeds inside has been deposited under accession number NCIMB 41773 (WH9307) or accession number NCIMB 42704 (WH9716).
In one embodiment, the fruit of a cross-pollination between a diploid plant as pollen donor capable of producing small diploid fruits having an average weight of less than 0.9 kg, 0.8 kg, 0.7 kg or equal to or less than 0.65 kg (but in one embodiment larger than 0.25 kg) is provided, wherein a representative sample of seed containing the genetic elements for producing said small fruits with small seeds inside has been deposited under accession number NCIMB 41773 (WH9307) or accession number NCIMB 42704 (WH9716), and the pistillate flowers of another watermelon plant is provided. The other watermelon plant is preferably a triploid hybrid and the fruit is triploid and preferably seedless.
In one embodiment, a diploid fruit and/or an inbred diploid seed is provided produced by self-pollinating a diploid watermelon plant comprising the HMBN mutant allele and the ts-gene mutant allele and capable of producing small edible diploid fruits having an average weight of less than 1.8 kg, 1.7 kg, 1.0 kg, 0.9 kg, 0.8 kg, 0.7 kg or equal to or less than 0.65 kg (but in one embodiment larger than 0.25 kg) is provided, wherein a representative sample of seed containing the genetic elements for producing said small diploid fruits with small seeds inside has been deposited under accession number NCIMB 41773 (WH9307) or accession number NCIMB 42704 (WH9716).
In one aspect, plants and fruits described herein and/or the genetic elements described herein (i.e., the combination of the HMBN mutant allele and the ts-gene mutant allele) are obtainable from WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, WH9721 or of progeny of any of these.
Plants or genetic elements obtained (derived), or obtainable (derivable), from plants according to the invention (e.g., from deposited seeds) include, therefore, plants obtained by breeding methods, such as selfing, crossing, backcrossing, recurrent selection, double haploid production, marker assisted selection, clonal propagations, transformants, etc., whereby the derived plants produce small fruits (comprising small seeds inside) as described herein, and comprise the HMBN mutant allele and the ts-gene mutant allele, preferably both in homozygous form.
In another aspect, plants having essentially all the morphological and/or physiological characteristics of WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, WH9721 or of progeny of any of these, are provided. Representative examples of physiological and morphological characteristics are provided in Tables 1-8.
Also provided is a progeny plant of WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320 or WH9321, WH9716, WH9717, WH9721 obtained by further breeding with said plant, wherein said progeny plant has essentially all physiological and morphological characteristics of said plant and/or wherein said progeny plant retains the HMBN mutant allele and the ts-gene mutant allele.
Essentially all physiological and morphological characteristics include herein at least small, edible fruits and dual purpose pollenizer characteristics as described throughout the description and examples. Thus in one aspect essentially all physiological and morphological characteristics refer herein to a plant having the fruit characteristics and/or the flowering (pollenizer) characteristics as described in specification and the Examples.
Also a plant part (e.g., a fruit, tissue, cell, cell culture, vegetative propagation, pollen, etc.) is provided which, when regenerated or grown into a whole plant, has essentially all the morphological and/or physiological characteristics of WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320 or WH9321, WH9716, WH9717, WH9721.
Also provided is a plant derived from (or obtained from) or derivable from (or obtainable from) any of WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320 or WH9321, WH9716, WH9717, WH9721 having one or two physiological and/or morphological characteristics which are different from the WH plant listed and which otherwise has essentially all physiological and morphological characteristics of a plant designated WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320 or WH9321, WH9716, WH9717, WH9721 obtainable by further breeding with the WH plant and/or by selecting a natural or induced mutant, or a somaclonal variant from a population of plants designated WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320 or WH9321 WH9716, WH9717, WH9721.
Thus, the invention provides a seed of watermelon variety WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, wherein a representative sample of said seed has been deposited under Accession Number NCIMB ______.
The invention provides a plant of watermelon variety WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, or a part thereof, wherein a representative sample of seed of said variety has been deposited under Accession Number NCIMB ______.
The invention provides a fruit of watermelon variety WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, or a plant part produced from the plant above.
The invention provides a method of producing a watermelon plant, comprising crossing any one of the plants of WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721 with a second watermelon plant one or more times, and selecting progeny from said crossing.
The invention provides a method of producing a watermelon plant, comprising selfing any one of the plants of WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721 one or more times, and selecting progeny from said selfing.
The invention provides progeny of watermelon variety WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721 obtained by further breeding with said variety.
The invention provides the progeny of watermelon variety WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, wherein said progeny have all the physiological and morphological fruit and/or flowering characteristics of watermelon variety WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721 respectively when grown under the same environmental conditions.
The invention provides the progeny of watermelon variety WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, wherein said progeny have all the physiological and morphological characteristics of watermelon variety WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, respectively, when grown under the same environmental conditions.
The invention provides an Essentially Derived Variety of any one of WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721 having one, two or three physiological and/or morphological characteristics which are different from those of WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721 and which otherwise has all the physiological and morphological characteristics of WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, wherein a representative sample of seed of variety WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721 has been deposited under Accession Number NCIMB ______.
The invention provides a method of producing plants, or a part thereof, of variety WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721 comprising vegetative propagation of variety WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, respectively
In one aspect said vegetative propagation comprises regenerating a whole plant from a part of variety WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721.
In one aspect said part is a cutting, a cell culture or a tissue culture.
The invention provides a vegetative propagated plant of variety WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, or a part thereof, having all the morphological and physiological characteristics of WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, respectively, when grown under the same environmental conditions.
The invention provides a plant part derived from variety WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, or from the vegetatively propagated plant above, wherein said plant part are harvested fruit or parts thereof, pollen, cells, leaves or parts thereof, petioles, shoots or parts thereof, stems or parts thereof, roots or parts thereof, cuttings, or flowers or parts thereof.
The invention provides a food or feed product comprising such a plant part. The plant part is fresh or processed.
The invention provides a watermelon plant produced by growing the seed of WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721.
The invention provides a method of producing a watermelon plant having a desired trait, wherein the method comprises transforming the watermelon plant WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721 with a transgene that confers the desired trait, wherein the transformed plant retains all the phenotypic and morphological characteristics of variety WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, respectively, and contains the desired trait, a representative sample of seed of said variety WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721 having been deposited under Accession Number NCIMB ______.
The invention provides a watermelon plant produced by the method above, wherein the plant comprises the desired trait and all of the physiological and morphological characteristics of WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721.The invention provides a watermelon plant comprising at least a first set of the chromosomes of watermelon line WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, a sample of seed of said line having been deposited under Accession Number NCIMB ______ and further comprising a single locus conversion, wherein said plant has essentially all of the morphological and physiological characteristics of the plant comprising at least a first set of the chromosomes of watermelon line WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721.
The single locus conversion in one aspect confers a trait selected from the group consisting of male sterility, herbicide tolerance, insect resistance, pest resistance, disease resistance, environmental stress tolerance, modified carbohydrate metabolism and modified protein metabolism.
The invention also provides use of any one of WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, or progeny of any one of these, for providing pollen to produce triploid, seedless watermelon fruits.
The invention thus also provides triploid fruits produced from one of WH9306, WH9307, WH9308, WH9309, WH9322, WH9313, WH9317, WH9318, WH9319, WH9320, WH9321, WH9716, WH9717, or WH9721, or progeny of any one of these, as male parent (pollenizer) and a triploid watermelon plant as female parent.
Applicant(s) maintain a deposit of at least 2500 seeds of hybrid pollenizers mentioned in the Examples, and parent inbred lines, at Nunhems B.V. Applicant has deposited hybrid WH9307 at the NCIMB on 12 Nov. 2010 under Accession number NCIMB 41773, and hybrid WH9716 on 12 Dec. 2016 under Accession number NCIMB 42704. Access to this deposit will be available during the pendency of this application to persons determined by the Commissioner of Patent and Trademarks to be entitled thereto upon request.
Subject to 37 C.F.R. § 1.808(b), all restrictions imposed by the depositor on the availability to the public of one or more deposits will be irrevocably removed upon the granting of the patent by affording access to a deposit of at least 2500 seeds with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110 or National Collections of Industrial, Food and Marine Bacteria (NCIMB), 23 St. Machar Drive, Aberdeen, Scotland, AB24 3RY, United Kingdom. The deposit will be maintained for a period of 30 years, or 5 years after the most recent request, or for the enforceable life of the patent whichever is longer, and will be replaced if it ever becomes nonviable during that period. Applicant does not waive any rights granted under this patent on this application or under the Plant Variety Protection Act (7 U.S.C. § 2321 et seq.).
The following non-limiting Examples describe the production of diploid pollenizers and small, edible diploid fruits according to the invention. Unless stated otherwise in the Examples, methods for conventional watermelon breeding are used, such as e.g., described in Maynard 2001, Watermelons—Characteristics, Production and Marketing, ASHS Press; Mohr H.C. Watermelon Breeding in Mark J. Bassett (editor) 1986 Breeding Vegetable Crops, AVI Publishing Company.
Breeding for the hybrids goes back to 1985, to in-house breeding lines and crosses of these with the variety Allsweet. Selected self-pollinations were backcrossed with Tomato Seed OP (Sugar Baby mutant; ts gene mutant plant) and selections were made for red flesh and small seed. Further self-pollinations were selected for red flesh, small seeds and agronomic traits in general. Selections were crossed with a line with many male flowers (variety Sidekick), followed by several selfings and selection for the production of many male flowers and small red fruits. All inbreds and hybrids were selected to be homozygous for the mutant HMBN allele present in Sidekick. This mutant HMBN allele confers smaller fruit size, but it was found that the combination with the is gene mutant in homozygous form enabled a further decrease in average fruit weight and a concomitant decrease in seed size. It is concluded that the combination of the mutant HMBN allele with the ts-gene mutant, whereby both are in homozygous form, enables the generation of watermelon plants producing small fruits (average weight of equal to or less than 0.7 kg) with small seeds (average seed length of less than 6.0 mm, but larger than 4.0 mm).
A field trial was conducted in Italy (Sant'Agata Bolognese-BO). Seeds were sown on 7 Apr. 2010 and transplanted into the field on 21 May 2010 (100 cm within the row, 250 cm between the rows). The plot contained 10 plants per line. Fruits were harvested on 26/27 Jul. 2010 for evaluation.
The number of open male and female flowers were counted for three plants per line on the flowering date (day 1=24 Jun. 2010), and 8, 15 and 22 days after the flowering date (day 8=2 Jul. 2010; day 15=9 Jul. 2010; day 22=16 Jul. 2010). The mean number of three plants per line was calculated.
1) Average fruit number: at maturity (26/27 July) the total number of fruits harvested from two plants was counted and the average fruit number determined.
2) Average fruit weight: mean of the weight of three fruits per line randomly harvested from two plants at maturity
3) Brix: Value is the mean of three reading for three fruit, collected between the centre and the rind of the fruit; expressed in Degrees Brix (°) using the K71901 portable refractometer Mod. RLC ATC 0-18% (OPTECH).
4) Flesh structure (flesh firmness): Value is the mean of the reading of three fruits; expressed in kg using the fruit pressure tester FT 011 (Cientec Instrumentos)
5) Flesh colour: evaluated using the Royal Horticultural Society mini colour chart (world wide web at rhs.org.uk/Plants/RHS-Publications/RHS-colour-charts)
Measurements were done for three fruits:
1) Fruit length (cm), indicated as FRT-cm_L
2) Fruit diameter at midsection (cm), indicated as FRT-cm_D
3) Fruit rind thickness (cm), blossom end, indicated as RND-cm_BE
4) Fruit rind thickness (cm), side of fruit, indicated as RND-cm_S.
Rind thickness is measured from the outer edge of the fruit to the boundary between white mesocarp and colored endocarp.
Table 1 shows that the pollenizers according to the invention produced very small, red, edible fruits.
As can be seen from Table 1, the diploid pollenizers according to the invention produced very small, red-fleshed edible fruits. Fruit weight in Table 1 is significantly smaller than that of commercial pollenizers such as Sidekick, SP-4 and Polimax.
Table 2 shows that the pollenizers according to the invention have on average smaller fruit dimensions than the commercial pollenizers. The fruit length is on average 11.33 cm or smaller, while in Sidekick the average fruit length is 12 cm. The diameter is also smaller, only 10.17 cm or less, compared to 11.33 cm in Sidekick. Thus fruit dimensions of equal to or below 11.33×10.17 cm (e.g., even as small as 9.67×8.67 cm) are significantly smaller than 12.00×11.33 cm in Sidekick.
Table 3 shows mean rind thicknesses per line, measured at two points of the fruit. Thin rinds are an advantage for consumption.
The pollenizers according to the invention have good, although thin, rind thickness of between 0.30 and 0.47 cm. The rind is also not susceptible to cracking, giving the fruits good handling properties. Fruits do also not have a brittle or explosive rind.
The rind pattern of the fruits of all pollenizers according to the invention is a Crimson Sweet type rind pattern (medium-striped or netted), but the small fruit size can also be combined with other rind colors using standard breeding methods.
The hybrids have a relatively compact growth type, with the (average/pollenizer line of the) longest branch falling between 113.5 and 180.0 cm long and the shortest branch being between 57.0 and 80.0 cm long. The average number of primary branches per pollenizer line was between 3 and 3.5. The average number of secondary branches per pollenizer line at 30 cm and at 90 cm was between 56.0 and 88.5 (at 30 cm) and between 77.5 and 144.0 cm. Internode length ranged between 4.3 and 5.3 on average, depending on the line. Leaf length and width is also relatively compact, with leaf widths between 7.1 and 9.4 cm and lengths between 7.8 and 9.1 cm.
Three trials were carried out in Spain using pollenizers WH9317x, WH9318x, WH9320x and WH9321x for triploid fruit production on the triploid hybrid variety ‘Fashion’.
Location: green house
Trial dimensions: 3600 m2
Transplanting date: 19-Mar.-2010
Harvest date: 16-Jun.-2010
Scheme: pollenizers and triploids were in separate rows, alternating one row of triploid and one row of pollenizer. Distance between rows was 3 meter, distance between plants in a row was 1 meter.
Location: open field
Trial dimensions: 2500 m2
Transplanting date: 24-Mar.-2010
Harvest date: 6-Jul.-2010
Scheme: pollenizers and triploids were interplanted in the same rows, with pollenizers making up 25% of the total plants. Distance between rows was 3 meters and distance between plants in a row was 1 meter.
Location: open field
Trial dimensions: 1500 m2
Transplanting date: 20-Mar.-2010
Harvest date: 2-Jul.-2010
Scheme: pollenizers and triploids were interplanted in the same rows, with pollenizers making up 25% of the total plants. Distance between rows was 2 meters and distance between plants in a row was 1.8 meter.
Three further diploid hybrids were developed having dual purpose pollenizer characteristics as described for the above hybrids but having a higher brix content and being thus particularly suitable for fresh consumption similar to apples.
Fruit characteristics—harvested at maturity (method for measurements as in examples 1 and 2)
The watermelon lines described herein were analyzed for their pedigree and all contained the mutant HMBN allele in homozygous form. Furthermore, the seed characteristics were analyzed for three diploid hybrid, which contained the ts-gene mutant in homozygous form (as both parents were homozygous for the ts-gene mutant according to the pedigree).
Seed characteristics of WH9716, WH9717 and WH9721 are shown in Table 9
The ts-gene mutant was mapped and found to be located on chromosome 2 of the watermelon genome. It was found that the ts-gene mutant plant contained a large deletion in chromosome 2 in homozygous form and that this deletion caused the reduction in seed length when the deletion was present in homozygous form (i.e., deletion on both chromosomes). The sequence deleted is shown in SEQ ID NO: 1. The presence of the deletion in homozygous form therefore causes a reduction in average seed size, measurable as a reduction in average seed length to less than 6.0 mm, especially equal to or less than 5.5 mm length (but above 4 mm) or measurable as weight of ten seeds being less than 0.2 grams, especially equal to or less than 0.16 g or 0.15 g, more preferably equal to or less than 0.13 g. This was seen in at least lines and hybrids which comprise the HMBN mutant allele in homozygous form, and produce small fruit sizes of equal to or less than 0.7 kg average fruit weight, especially equal to or less than 0.66 or 0.65 kg.
As shown in Table 10, the small fruited hybrids comprising the HMBN mutant allele in homozygous form and were homozygous for the ts-gene mutant (deletion of SEQ ID NO: 1) showed a reduced seed size. The combination of the HMBN allele and the ts-gene mutant (deletion of SEQ ID NO: 1), both in homozygous form, results in fruits of less than 0.7 kg average weight with seeds of less than 0.6 mm average seed length being produced. Both genetic elements are recessive and thus need to be present in homozygous form. They are both obtainable from watermelon WH9716, a representative sample of seeds having deposited under Accession number NCIMB 42704.
To measure gram per 10 seeds (g/10 seeds), three measurements of a seedlot per genotype were done and the average of the three measurements is provided. Average seed length and average fruit weight was measured according to Exhibit C of the US Department of Agriculture, Objective Description of a variety—Watermelon, see world wide web at ams.usda.gov/services/plant-variety-protection/pvpo-c-forms. Measurements were done on at least 15 fruits per genotype.
Diploid hybrids WH9716, WH9717, WH9721, WH9306, WH9307, WH9308, WH9309 and WH9311 were all homozygous for the deletion of SEQ ID NO: 1, and all produce seeds having an average seed length of less than 5.5 mm.
WH9313, WH9317, WH9318, WH9319, WH9320, WH9321 were heterozygous for the deletion of SEQ ID NO: 1.
BLAST was carried out on the website cucurbitgenomics.org using SEQ ID NO:1.
SEQ ID NO: 1 is present on the watermelon 97103v1 genome on chromosome 2 starting at nucleotide 29916076 and ending at nucleotide 29902113. Likewise, the reverse complement of SEQ ID NO: 1 is present on the watermelon 97103v1 genome starting at nucleotide 29902113 and ending at nucleotide 29916076.
SEQ ID NO: 1 is presented in the Sequence Listing (CRF and paper copy) filed with this application. The Sequence Listing is hereby incorporated by reference.
This application is a continuation-in-part of U.S. patent application Ser. 13/681,388 filed Nov. 19, 2012, which is a continuation-in-part of U.S. patent application Ser. No. 13/518,789 filed Jun. 22, 2012, which is the U.S. national stage application of PCT/EP2011/070817, filed Nov. 23, 2011, which claims the benefit of U.S. Provisional Patent Application 61/416,908, filed Nov. 24, 2010, the contents of each of which are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61416908 | Nov 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13681388 | Nov 2012 | US |
| Child | 16705760 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13518789 | Jul 2012 | US |
| Child | 13681388 | US |
