Polyploidization of interspecific hybrids to create rootstocks for cucurbit

Abstract

The present disclosure relates to Cucurbitaceae allotetraploid plants and hybrid allopolyploid plants having desirable traits, such as resistance to an abiotic or biotic stressor, which may be used as rootstock for cultivated varieties. The disclosure further relates to composite Cucurbitaceae plants comprising the allopoly ploid plants described herein as the rootstock. The disclosure further relates to methods of producing Cucurbitaceae allopolyploid plants and hybrid allopolyploid plants.

Description

IN THE UNITED STATES PATENT AND TRADEMARK OFFICEPCT APPLICATIONPOLYPLOIDIZATION OF INTERSPECIFIC HYBRIDS TO CREATE ROOTSTOCKS FOR CUCURBITCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 699,304 filed on September 26, 2024, the contents of which are herein incorporated by reference in their entirety.FIELD

[0002] The present disclosure relates to the fields of agriculture, plant biotechnology, and molecular biology. More specifically, the disclosure relates to allopolyploid plants and methods of producing allopolyploid plants having desirable traits and methods of using them.BACKGROUND

[0003] It is becoming more challenging for farmers to satisfy the increasing worldwide demand for food. Traditional breeding methods and / or transgenic technologies can develop improved plant varieties, however incompatibility between species, complex multifactorial and polygenic traits, and restrictions on genetically modified crops can prevent and / or hinder progress. One way to introduce new traits is to conduct interspecific crosses of cultivated varieties with wild species having a desirable trait, such as increased tolerance against abiotic stresses such as drought tolerance, salinity tolerance, flooding / water tolerance and heat and cold temperature tolerance. These traits may be fixed through chromosome doubling (generating allopolyploids) and further crossed with other allopolyploids to generate F1hybrids. Additionally, as the roots supply vital nutrients to the plant, these F1allopolyploid hybrids may be used as rootstocks improve plant performance and yield of cultivated varieties.SUMMARY OF THE DISCLOSURE

[0004] In some aspects, the techniques described herein relate to a method for producing a composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress, including: (i) selecting a first Cucurbitaceae variety which is stress-tolerant against at least one abiotic or biotic stress; (ii) crossing said first Cucurbitaceae variety with a secondCucurbitaceae variety of a different species sexually compatible with the first Cucurbitaceae variety to produce an interspecific hybrid seed; (iii) growing the interspecific hybrid seed to produce an interspecific hybrid Cucurbitaceae plant; (iv) applying a chromosome doubling treatment to the interspecific hybrid plant, or a part thereof, to generate a chimeric interspecific hybrid Cucurbitaceae plant; (v) collecting seed from an allotetraploid fruit of said chimeric interspecific hybrid plant; (vi) growing the seed to produce an allotetraploid Cucurbitaceae rootstock plant with tolerance against at least one abiotic or biotic stress and, optionally, further propagating said plant; and (vii) grafting a scion of a cultivated Cucurbitaceae variety to the allotetraploid Cucurbitaceae rootstock to produce a composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress.

[0005] In some aspects, the techniques described herein relate to a method for producing a composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress, including: (i) selecting a first Cucurbitaceae variety which is stress-tolerant against at least one abiotic or biotic stress; (ii) fusing a protoplast isolated from said first Cucurbitaceae variety with another protoplast isolated from a Cucurbitaceae variety sexually incompatible with the first Cucurbitaceae variety; (iii) selecting a heterokaryon; (iv) regenerating an allotetraploid Cucurbitaceae rootstock plant with tolerance against at least one abiotic or biotic stress from the heterokaryon; and, optionally, further propagating said plant; and (v) grafting a scion of a cultivated Cucurbitaceae variety to the allotetraploid Cucurbitaceae rootstock to produce a composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress.

[0006] In some aspects, the techniques described herein relate to a method for producing a stress-tolerant hybrid allopolyploid Cucurbitaceae plant or seed, including: (i) selecting a first Cucurbitaceae variety which is stress-tolerant against at least one abiotic or biotic stress; (ii) crossing said first Cucurbitaceae variety with a second Cucurbitaceae variety of a different species sexually compatible with the first Cucurbitaceae variety to produce an interspecific hybrid seed; (iii) growing the interspecific hybrid seed to produce an interspecific hybrid Cucurbitaceae plant; (iv) applying a chromosome doubling treatment to the interspecific hybrid plant, or a part thereof, to generate a chimeric interspecific hybrid Cucurbitaceae plant; (v) collecting seed from an allotetraploid fruit of said chimeric interspecific hybrid plant; (vi) growing the seed to produce a first allotetraploid Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress and, optionally, further propagating said plant; (vii) crossing the first allotetraploid Cucurbitaceae plant with a second allotetraploid Cucurbitaceae plant to produce hybrid allopolyploid seed; (viii) harvesting the hybrid allopolyploid seed; and (ix)optionally growing the hybrid allopolyploid seed to produce a stress-tolerant hybrid allopolyploid Cucurbitaceae plant.

[0007] In some aspects, the techniques described herein relate to a method for producing a stress-tolerant hybrid allopolyploid Cucurbitaceae plant, including: (i) selecting a first Cucurbitaceae variety which is stress-tolerant against at least one abiotic or biotic stress; (ii) fusing a protoplast isolated from said first Cucurbitaceae variety with another protoplast isolated from a second Cucurbitaceae variety sexually incompatible with the first Cucurbitaceae variety to produce a heterokaryon; (iii) regenerating a first allotetraploid Cucurbitaceae plant from the heterokary on; (iv) fusing a protoplast isolated from the first allotetraploid Cucurbitaceae plant with another protoplast isolated from a second allotetraploid Cucurbitaceae plant to a produce hybrid allopoly ploid heterokaryon; and (v) regenerating a hybrid allopolyploid Cucurbitaceae plant from the hybrid allopolyploid heterokaryon to produce a stress-tolerant hybrid allopolyploid Cucurbitaceae plant.

[0008] In some aspects, the techniques described herein relate to a hybrid allopolyploid Cucurbitaceae plant or plant part with tolerance against at least one abiotic or biotic stress, including: (i) at least one chromosome from each of a first and a second Cucurbitaceae variety, wherein the second Cucurbitaceae variety exhibits at least one tolerance against at least one abiotic or biotic stress which is not present in said first Cucurbitaceae variety; and (ii) at least one chromosome for a cultivated Cucurbitaceae variety of a species different from said first and second Cucurbitaceae varieties.

[0009] In some aspects, the techniques described herein relate to a composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress, said composite Cucurbitaceae plant including: (i) as a rootstock an allotetraploid Cucurbitaceae plant, and (ii) as a scion a cultivated Cucurbitaceae variety.

[0010] In some aspects, the techniques described herein relate to a method for producing a composite Cucurbitaceae plant with an improved agronomic trait, including: selecting first and second Cucurbitaceae plants having one or more desirable traits; generating an interspecific hybrid plant from said first and second Cucurbitaceae plants; applying a chromosome doubling treatment to the interspecific hybrid plant, or a part thereof, to generate a chimeric interspecific hybrid Cucurbitaceae plant; collecting seed from an allotetraploid fruit of said chimeric interspecific hybrid plant; growing the seed to produce an allotetraploid Cucurbitaceae rootstock plant with one or more desirable traits and, optionally, further propagating said plant, and grafting a scion to the allotetraploid Cucurbitaceae rootstock to produce a composite Cucurbitaceae plant, wherein the scion is a commercial Cucurbitaceae variety, and wherein afruit or vegetable harvested from the scion has an improved agronomic trait compared to the same variety grown without the allotetraploid Cucurbitaceae rootstock.

[0011] The foregoing was intended as a summary only and of only some of the aspects of the disclosure. It was not intended to define the limits or requirements of the disclosure. Other aspects of the disclosure will be appreciated by reference to the detailed description of the embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 is a flow diagram showing the steps of generating an allotetraploid plant.

[0013] FIG. 2 is a flow diagram showing the steps of generating an allotetraploid hybrid and using the resulting F1as rootstock for a cultivated variety.

[0014] FIG. 3 shows comparative morphology of diploids and tetrapioid hybrids of Cucumis melo and Cucumis metuliferus . The tetrapioid hybrids exhibit larger leaves with more pronounced serration and larger, thicker flowers compared to their diploid counterparts.

[0015] FIG. 4 compares pollen size between diploid and tetrapioid hybrids of Cucumis melo and Cucumis metuliferus under lOx and lOOx magnification.

[0016] FIG. 5 shows a composite plant having ‘Alcazaba’ melon as the scion and allotetraploid ‘MR1B2’ as the rootstock.

[0017] FIG. 6 shows a composite plant having ‘Kenza’ melon as the scion and allotetraploid MR1B2’ as the rootstock.

[0018] FIG. 7A and FIG. 7B are photographs of a grafted melon trial conducted at CT Tecnova, Almeria, Spain. FIG. 7A shows the melon plants on 14 August 2024, showing early - stage growth and establishment. FIG. 7B shows the same melon plants on 3 September 2024, illustrating advanced growth stages, increased foliage, and fruit development.

[0019] FIG. 8 is a bar graph showing fresh biomass production of grafted melon varieties ‘Alcazaba’ and ‘Kenza’ grafted onto rootstocks ‘MR-1A’ (interspecific hybrid, diploid) and ‘MR-1B2’ (allotetraploid), compared to self-grafted controls.

[0020] FIG. 9 shows comparative morphology of diploid ‘Wei-Zhen 103’ and allotetraploid ‘MC 24-01 ’ (hybrids of Cucurbita maxima and Cucurbita moschata). The allotetraploid ‘MC 24-01’ exhibits larger leaves colored a darker green, with more serration, and larger pollen compared to the diploid counterpart ‘Wei-Zhen 103’.

[0021] FIG. 10 shows comparative morphology of diploid ‘Wei-Zhen 105’ and allotetraploid ‘MC 24-02’ (hybrids of Cucurbita maxima and Cucurbita moschata'). The allotetraploid ‘MC24-02’ exhibits larger leaves colored a darker green, with more serration, and larger fruit compared to the diploid counterpart ‘Wei-Zhen 105’.

[0022] FIG. 11 is a photograph of roots of ‘Wei-Zhen 105’ (left) compared to ‘MC 24-02’ (right).DETAILED DESCRIPTIONDefinitions

[0023] While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0024] Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. For example, the phrase “a cell” refers to one or more cells, and in some embodiments can refer to a tissue and / or an organ. Similarly, the phrase “at least one”, when employed herein to refer to an entity, refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or more of that entity, including but not limited to all whole number values between 1 and 100 as well as whole numbers greater than 100.

[0025] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” The term “about” when immediately preceding a numerical value means a range (e.g.. plus or minus 10% of that value). For example, “about 50” can mean 45 to 55, “about 25,000” can mean 22,500 to 27,500, etc., unless such an interpretation would result in a value above or below range of possible values, such as below 0% or above 100% of a possible value. Furthermore, the phrases “less than about” a value or “greater than about” a value should be understood in view of the definition of the term “about” provided herein, as applied to any recited endpoint. Similarly, the term “about” when preceding a series of numerical values or a range of values (e.g., “about 10, 20, 30” or “about 10-30”) refers, respectively to all values in the series, or the endpoints of the range. Unless otherwise indicated, it is to be understood that all numbers expressing quantities, ratios, and numerical properties of ingredients, reaction conditions, and so forth, used in the specification and claims are contemplated to be able to be modified in all instances by the term “about”.

[0026] The term “approximately” when immediately preceding a numerical value means a range (e.g., plus or minus 5% of that value). For example, “approximately 50” can mean 47.5 to 52.5. “approximately 25,000” can mean 23.750 to 26.250, etc., unless such an interpretation would result in a value above or below range of possible values, such as below 0% or above100% of a possible value. Furthermore, the phrases " less than approximately” a value or “greater than approximately” a value should be understood in view of the definition of the term “approximately” provided herein, as applied to any recited endpoint. Similarly, the term “approximately” when preceding a series of numerical values or a range of values (e.g., “approximately 10, 20, 30” or “approximately 10-30”) refers, respectively to all values in the series, or the endpoints of the range. Unless otherwise indicated, it is to be understood that all numbers expressing quantities, ratios, and numerical properties of ingredients, reaction conditions, and so forth, used in the specification and claims are contemplated to be able to be modified in all instances by the term “approximately.”

[0027] The term “including all ranges and subranges therebetween” or equivalents, are used herein to denote the intention that disclosure of any range or series of possible values, inherently also discloses all ranges and subranges encompassed by the highest and lowest values disclosed. This term includes the entire range from highest to lowest disclosed values, as well as subranges from any two or more disclosed points. This term is also intended to disclose any subranges encompassed anywhere within the highest and lowest disclosed values, including between two points that are explicitly recited in the document, up to one decimal point. Thus, disclosure of values 0, 5, 10, 15, 20, including all ranges and subranges therebetween, should be interpreted as also encompassing a range from 0-20, a range from 0-5 or 5-15, as well as a range from 2-16, or 3. 1 to 19.8, etc. Unless otherwise indicated, it is to be understood that all numbers expressing quantities, ratios, and numerical properties of ingredients, reaction conditions, and so forth, used in the specification are contemplated to be able to be modified in all instances by the term “including all ranges and subranges therebetween.”

[0028] As used herein, the term “and / or” when used in the context of a list of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase “A, B, C, and / or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D (e g., AB, AC, AD, BC, BD, CD, ABC, ABD, and BCD). In some embodiments, one or more of the elements to which the “and / or” refers can also individually be present in single or multiple occurrences in the combinations(s) and / or subcombination( s) .

[0029] The term “allopolyploidy” refers to a cell or plant having two or more complete sets of chromosomes derived from different species.

[0030] The term “allotetraploid” refers to a hybrid cell or plant derived from different species and possessing four times the chromosomes in a haploid organism. For example, aninterspecific hybridization followed by chromosome doubling would generate an allotetraploid. In some cases, an allotetraploid may exhibit a certain degree of aneuploidy and crossover events.

[0031] As used herein, the term “aneuploid” refers to a cell or plant having an incomplete set of chromosomes. An aneuploid may have for example, missing or extra chromosome(s).

[0032] As used herein, an “anti-mitotic” or “anti-mitotic agent” refers to a compound or chemical that is used to block cell growth by stopping mitosis (cell division) used in plant breeding to induce chromosome doubling. Examples of anti-mitotic agents include, but are not limited to, colchicine, trifluralin, oryzalin, and amiprophos-methyl (APM).

[0033] As used herein, the term “at least a portion” or “fragment” of a nucleic acid or polypeptide means a portion having the minimal size characteristics of such sequences, or any larger fragment of the full-length molecule, up to and including the full-length molecule.

[0034] A “chimera,” “chimeric tissue” or “chimeric plant” is a plant or tissue that consists of two or more genetically distinct groups of cells.

[0035] As used herein, the term “cisgenesis” refers to genetic modification of a recipient organism with one or more genes (cisgenes) from a crossable, sexually compatible, organism.

[0036] As used herein, a “composite” or “composite plant” refers to a plant comprising two distinct varieties that have been grafted together (rootstock + scion) to form one plant.

[0037] “Colchicine” is a pale-yellow alkaloid, C22H25NO6, obtained from the autumn crocus and used in plant breeding to induce chromosome doubling.

[0038] As used herein, the term "enhanced abiotic stress tolerance" refers to the ability of a plant or plant part to grow, reproduce and / or survive under abiotic stress conditions, as compared to one or more controls (a plant which is not stress tolerant). "Enhanced abiotic stress tolerance" may refer to any improvement in a plant's or plant part's ability to thrive and / or endure when grown under abiotic stress conditions, or may refer to a plant’s ability to maintain growth and yield under abiotic stress conditions, including, but not limited to, decreased water loss, decreased accumulation of one or more reactive oxygen species, decreased accumulation of one or more salts, increased salt excretion, increased accumulation of one or more dehydrins, improved root architecture, improved osmotic pressure regulation, increased accumulation of one or more late embryogenesis abundant proteins, increased survival rate, increased growth rate, increased height, increased chlorophyll content, improvement of fruit quality, and / or increased yield (e.g., increased biomass, increased seed yield, increased grain yield at standard moisture percentage, increases shoot length, decreased electrolyte leakage, increased grain weight per plot, increased percent yield recovery, decreased yield reduction, and / or decreasedpercent barren) when grown under abiotic stress conditions. A plant or plant part that exhibits enhanced abiotic stress tolerance may be designated as "abiotic stress tolerant."

[0039] As used herein, the term "enhanced drought tolerance" refers to an improvement in one or more water optimization traits as compared to one or more controls (a plant which is not stress tolerant). A plant or plant part that exhibits decreased water loss, decreased accumulation of one or more reactive oxygen species, decreased accumulation of one or more salts, increased salt excretion, increased accumulation of one or more dehydrms, improved root architecture, improved osmotic pressure regulation, increased accumulation of one or more late embryogenesis abundant proteins, increased survival rate, increased growth rate, increased height, increased chlorophyll content and / or increased yield as described above, as compared to a control plant when each is grown under the same drought stress conditions displays enhanced drought tolerance and may be designated as "drought tolerant." In some embodiments, the plant or plant part exhibits an increased survival rate after being subjected to drought stress conditions (e.g., an irrigation withholding experiment).

[0040] As used herein, the term "enhanced osmotic stress tolerance" refers to an improvement in one or more osmotic pressure optimization traits as compared to one or more controls (a plant which is not stress tolerant). A plant or plant part that exhibits decreased water loss, decreased accumulation of one or more reactive oxygen species, decreased accumulation of one or more salts, increased salt excretion, increased accumulation of one or more dehydrins, improved root architecture, improved osmotic pressure regulation, increased accumulation of one or more late embryogenesis abundant proteins, increased survival rate, increased growth rate, increased height, increased chlorophyll content and / or increased yield as described above, when each is grown under the same osmotic stress conditions displays enhanced osmotic stress tolerance and may be designated as "osmotic stress tolerant." In some embodiments, the plant or plant part exhibits an increased survival rate after being subjected to mannitol -induced osmotic stress conditions (e.g., incubation in a 200 mM mannitol solution).

[0041] As used herein, the term "enhanced salt stress tolerance" refers to an improvement in one or more salt optimization traits as compared to one or more controls (a plant which is not stress tolerant). A plant or plant part that exhibits decreased water loss, decreased accumulation of one or more reactive oxygen species, decreased accumulation of one or more salts, increased salt excretion, increased accumulation of one or more dehydrins, improved root architecture, improved osmotic pressure regulation, increased accumulation of one or more late embryogenesis abundant proteins, increased survival rate, increased growth rate, increased height, increased chlorophyll content and / or increased yield as described above, as comparedto a control plant when each is grown under the same salt stress conditions displays enhanced salt stress tolerance and may be designated as "salt stress tolerant." In some instances, “enhanced salt stress tolerance" means that the reduction of total dry mass of the stress tolerant plant under salt stress conditions is not more than 75% of the total dry mass of a plant which is not a salt stress tolerant plant but which under normal condition exhibits the same dry mass as the stress tolerant plant. In some instances, “enhanced salt stress tolerance” means that the reduction of yield of the stress tolerant plant under salt stress conditions is not more than 20% of the total yield of a plant which is not a salt stress tolerant plant but which under normal condition exhibits the same dry mass as the stress tolerant plant. Salt tolerance can be evaluated as described in Negrao et. al. Annals of botany 119. 1, 1-11 (2017) and Morton et. al. The Plant Journal 97.1. 148-163 (2019).

[0042] As used herein, the term "enhanced temperature stress tolerance" refers to an improvement in one or more temperature tolerance traits as compared to one or more controls (a plant which is not stress tolerant). A plant or plant part that exhibits decreased water loss, decreased accumulation of one or more reactive oxygen species, decreased accumulation of one or more salts, increased salt excretion, increased accumulation of one or more dehydrins, improved root architecture, improved osmotic pressure regulation, increased accumulation of one or more late embryogenesis abundant proteins, increased survival rate, increased grow th rate, increased height, increased biomass, increased chlorophyll content, increased grain yield as described above, as compared to a control plant when each is grown under the same temperature stress conditions displays enhanced temperature stress tolerance and may be designated as "temperature stress tolerant."

[0043] It is to be understood that "drought tolerant." "osmotic stress tolerant," "salt stress tolerant," and "temperature stress tolerant" plants and plant parts may also be referred to as "abiotic stress tolerant" because drought stress, osmotic stress, salt stress and temperature stress are all abiotic stresses.

[0044] As used herein, the term "enhanced biotic stress tolerance" refers to an improvement in the ability of a plant or plant part to grow, reproduce and / or survive under biotic stress conditions, as compared to one or more controls (a plant which is not stress tolerant). "Enhanced biotic stress tolerance" may refer to any improvement in a plant's or plant part's ability to thrive and / or endure when grown under biotic stress conditions, including, but not limited to, decreased plant vigor reduction, increased cell lignification, improved root architecture, improved osmotic pressure regulation, increased accumulation of one or more late embryogenesis abundant proteins, increased survival rate, increased growth rate, increasedheight, increased chlorophyll content and / or increased yield (e.g., increased biomass, increased seed yield, increased grain yield at standard moisture percentage, increases shoot length, decreased electrolyte leakage, increased grain weight per plot, increased percent yield recovery, decreased yield reduction, and / or decreased percent barren) when grown under biotic stress conditions. A plant or plant part that exhibits enhanced biotic stress tolerance may be designated as "biotic stress tolerant."

[0045] "Grafting" is the operation by which a scion is grafted onto a rootstock. Grafting a susceptible scion onto a resistant rootstock can provide a resistant cultivar without the need to breed the resistance into the scion cultivar. In addition, grafting may enhance tolerance of a susceptible scion to abiotic stress, increase yield, and result in more efficient water and nutrient uses.

[0046] As used herein, an “intergeneric cross” refers to the hybridization of two individuals, each from different genera of the same family. “Intergeneric hybrid” means a plant, cell, or plant part derived from an intergeneric cross.

[0047] As used herein, an “interspecific cross” refers to the hybridization of two individuals, each from different species of the same genus. “Interspecific hybrid” means a plant, cell, or plant part derived from an interspecific cross.

[0048] The term “engineered” or “genetically engineered” refers to any man-made manipulation of a genome of a cell of interest.

[0049] As used herein, the term “naturally occurring” refers to a gene or plant derived from a naturally occurring source or method. In some aspects, a naturally occurring gene refers to a gene of a wild type (non-transgene) gene, whether located in its endogenous setting w ithin the source organism, or if placed in a “heterologous” setting, when introduced in a different organism. A “non-naturally occurring” plant is a man-made plant created by either manipulating the chromosome number (e.g. an allotetraploid) and / or grafting two distinct species together to form one composite plant.

[0050] A “rootstock” is a plant in w hich the low er part of a plant (including the roots) is capable of receiving a scion in a grafting process.

[0051] RHS refers to the Royal Horticultural Society of England which publishes an official botanical color chart quantitatively identifying colors according to a defined numbering system. The chart may be purchased from Royal Hort. Society Enterprise Ltd. RHS Garden; Wisley, Woking, Surrey GU236QB, UK.

[0052] “Salt stress” is the accumulation of excessive salt contents in the soil or other growing medium which can result in the inhibition of crop growth.

[0053] A “scion” is a plant in which the upper part of the plant is capable of being grafted onto a rootstock in a grafting process.

[0054] “Sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the number of residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have "sequence similarity " or "similarity ." Means for making this adjustment are well-known to those of skill in the art, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988). The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, for example, NCBI Basic Local Alignment Search Tool (BLAST®) (Altschul et al. 1990 J. Mol. Biol. 215: 403-10), which is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp. blastn, blastx, tblastn and tblastx, and the Clustal W and Clustal X (Larkin et al. 2007 Bioinformatics, 23, 2947-294, Clustal W and Clustal X version 2.0) as well as Clustal Omega. Unless otherwise stated, references to sequence identity used herein refer to the Clustal Omega.

[0055] A plant cell is a cell of a plant, taken from a plant, or derived through culture from a cell taken from a plant. Thus, the term “plant cell” includes for example, cells within seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, shoots, gametophytes, sporophytes, pollen, and microspores.

[0056] The phrase “plant part” refers to a part of a plant, including single cells and cell tissues such as plant cells that are intact in plants, cell clumps, and tissue cultures from which plants can be regenerated. Plant part also refers to those cells and tissues that cannot be used to regenerate a plant, but w ere nonetheless derived from a plant. Examples of plant parts include, but are not limited to, single cells, plant tissues, pollen, ovules, leaves, embry os, roots, root tips, anthers, flowers, fruits, stems, shoots, and seeds, as well as scions, rootstocks, protoplasts, calli, and the like.

[0057] As used herein, the term "plant" or “whole plant” refers to a plant at any stage of growth having both arial and root biomass.

[0058] The term “plant variety” is used herein as a group of plants that share a set of characteristics. The 1991 UPOV Convention's Article 1 (vi) states that a plant variety is a grouping within a botanical taxon of the low est known rank. The grouping can be defined by the characteristics that result from a specific genotype or combination of genotypes. This includes synthetic varieties and hybrids.

[0059] As used herein, the term “resistant”, or “resistance”, describes a plant, line or variety that show s fewer or reduced symptoms than a susceptible (or more susceptible) plant, line or variety. This term is also applied to plants that show no symptoms, and may also be referred to as “high / standard resistance”.

[0060] As used herein, the term “tolerant” or “tolerance” describes a plant, line, or variety that show-s some symptoms, but that are still able to produce marketable product with an acceptable yield. These lines may also be referred to as having “moderate / intermediate resistance”.

[0061] As defined by the International Seed Federation (ISF), a non-governmental, non-profit organization representing the seed industry (see “Definition of the Terms Describing the Reaction of Plants to Pests or Pathogens and to Abiotic Stresses for the Vegetable Seed Industry". May 2005), the recognition of whether a plant is affected by or subject to a pest, pathogen or abiotic stress can depend on the analytical method employed. Resistance is defined by the ISF as the ability of plant types to restrict the growth and development of a specified pest or pathogen and / or the damage they cause when compared to susceptible plant varieties under similar environmental conditions and pest or pathogen pressure. Resistant plant types may still exhibit some disease symptoms or damage. Tw o levels of resistance are defined. The term “high / standard resistance” is used for plant varieties that highly restrict the growth and development of the specified pest or pathogen under normal pest or pathogen pressure when compared to susceptible varieties. “Moderate / intermediate resistance” is applied to plant types that restrict the growth and development of the specified pest or pathogen, but exhibit a greater range of symptoms or damage compared to plant types with high resistance. Plant types with intermediate resistance will show less severe symptoms than susceptible plant varieties, when grown under similar field conditions and pathogen pressure. Methods of evaluating resistance are well known to one skilled in the art. Such evaluation may be performed by visual observation of a plant or a plant part (e g., leaves, roots, flowers, fruits et. al) in determining the severity’ of symptoms. For example, when each plant is given a resistance score on a scale of 1 to 5 based on the severity of the reaction or symptoms, with 1 being the resistance scoreapplied to the most resistant plants (e.g.. no symptoms, or with the least symptoms), and 5 the score applied to the plants with the most severe symptoms, then a line is rated as being resistant when at least 75% of the plants have a resistance score at a 1, 2, or 3 level, while susceptible lines are those having more than 25% of the plants scoring at a 4 or 5 level. If a more detailed visual evaluation is possible, then one can use a scale from 1 to 10 so as to broaden out the range of scores and thereby hopefully provide a greater scoring spread among the plants being evaluated.

[0062] In addition to such visual evaluations, disease evaluations can be performed by determining the pathogen bio-density in a plant or plant part using electron microscopy and / or through molecular biological methods, such as protein hybridization (e.g., ELISA, measuring pathogen protein density) and / or nucleic acid hybridization (e.g.. RT-PCR, measuring pathogen RNA density). Depending on the particular pathogen / plant combination, a plant may be determined resistant to the pathogen, for example, if it has a pathogen RNA / DNA and / or protein density that is about 50%, or about 40%, or about 30%, or about 20%, or about 10%, or about 5%, or about 2%. or about 1%, or about 0.1%, or about 0.01%. or about 0.001%, or about 0.0001% of the RNA / DNA and / or protein density in a susceptible plant.

[0063] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al.. John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds.. Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998); and Current Protocols in Molecular Biology (Ausubel et al. eds., John Wiley & Sons 2003), including supplements 1-1 17, the disclosures of which are incorporated herein by reference.

[0064] ‘ ‘Cultivated melon” or “domesticated melon” refers to plants of Cucumis melo L. i.e. varieties, breeding lines or cultivars, cultivated by humans and having good agronomic characteristics, and producing edible and marketable fruits of good size and quality and uniformity. Such plants are not “wild melon” plants, i.e. plants which generally have much poorer yields and poorer agronomic characteristics than cultivated plants and are less uniform genetically and in their physiological and / or morphological characteristics.

[0065] “Cultivated watermelon” or “domesticated watermelon” or “Citrullus lanatus'’’ refers to plants of Citrullus lanatus ssp. vulgaris, i.e. varieties, breeding lines or cultivars, cultivated byhumans and having good agronomic characteristics, and producing edible and marketable fruits of good size and quality and uniformity’. Such plants are not “wild watermelon” plants, i.e. plants which generally have much poorer yields and poorer agronomic characteristics than cultivated plants and are less uniform genetically and in their physiological and / or morphological characteristics.

[0066] " Cucurbita pepo plant” or “cultivated C. pepo" or “domesticated C. pepo" refers to plants of C. pepo, i.e. varieties, breeding lines or cultivars, cultivated by humans and having good agronomic characteristics, and producing edible and marketable fruits of good size and quality and uniformity'. Such plants are not “wiId” plants, i.e. plants which generally have much poorer yields and poorer agronomic characteristics than cultivated plants and are less uniform genetically and in their physiological and / or morphological characteristics.

[0067] “Landrace(s)” refers to cultivars developed in local geographic regions, which often show a high degree of genetic variation in their genome and exhibit a high degree of morphological and / or physiological variation within the landrace (e.g. large variation in fruit size, etc.), and are significantly less uniform than commercialized plants. As used herein, “Landraces” are distinct from “commercial” plants.

[0068] “Wild” varieties are plants found growing naturally in the wild, which generally have much poorer yields and poorer agronomic characteristics than cultivated plants and are less uniform genetically and in their physiological and / or morphological characteristics.Overview

[0069] The present disclosure relates to allotetr apioid Cucurbitaceae plants and hybrid allopolyploid Cucurbitaceae plants having desirable traits, such as resistance to an abiotic or biotic stressor, which may be used as rootstock for commercial varieties. The disclosure further relates to composite plants comprising the allotetraploid and hybrid allopolyploid Cucurbitaceae plants described herein as the rootstock, and commodity plant products produced from these composite plants, for example fruits and vegetables from the scion having an improved output trait (for example increased fruit size, nutrient content, shelf-life, etc.) that is attributable to the rootstock. The disclosure further relates to methods of producing allotetraploid Cucurbitaceae plants and plant parts and hybrid allopolyploid Cucurbitaceae plants and plant parts.Plants for use with the disclosed methods

[0070] Cucurbitaceae, also known as cucurbits, is a gourd family of about 125 genera and about 960 species (Mukheijee P.K., et al. “Therapeutic importance of Cucurbitaceae: A medicinallyimportant family” J. of Ethnopharmacology, 2022, Vol. 282). The family includes both food and ornamental plants, annual and perennials, native to temperate and tropical areas.

[0071] Cucurbitaceae is broken down into two subfamilies, Cucurbitoideae and Zanonioideae. Most of the edible fruits and vegetables come from subfamily Cucurbitoideae, which is further broken down into 15 tribes, shown in Table 1.Table 1: Tribes of the subfamily Cucurbitoideae

[0072] Some of the more well-known cucurbits include bottle gourd (Lagenaria siceraria), bur cucumber (genus Sicyos), bryony (genus Bryonia), colocynth (Citrullus colocynthis), watermelon (Citrullus lanatus), chayote (Sechmm edule), cucumber (Cucumis sativus), gherkin (Cucumis anguria), melon (Cucumis melo), muskmelon, loofah (genus Luffa), musk cucumber (Sicana odorifera). snake gourd (Trichosanthes cucumerind), squash (genus Cucurbita),calabazilla (Cucurbita foetidissima), pumpkin (Cucurbita pepo, sometimes Cucurbita moschata). winter squash (Cucurbita maxima), yellow-flowered gourd (Cucurbita pepo, subspecies ovifera), zucchini (Cucurbita pepo), wax gourd (Benincasa hispida) (Britannica, The Editors of Encyclopaedia. "Cucurbitaceae". Encyclopedia Britannica, 12 Jun. 2024, britannica.com / plant / Cucurbitaceae. Accessed 19 August 2024).Tribe Sicyoeae

[0073] Tribe Sicyoeae is large, comprised of approximately 18 genera and about 266 species. The burr cucumber, also known as star cucumber, and the loofa plants, are within this tribe.

[0074] In some embodiments, the Cucurbitaceae variety used in the methods and composite plants disclosed herein (e.g., the " first” and / or “second” Cucurbitaceae variety) is from the subfamily Cucurbitoideae. Tribe Sicyoeae. In some embodiments, the Cucurbitaceae variety is from the subfamily Cucurbitoideae, Tribe Sicyoeae, genus Sicyos. In some embodiments, the Cucurbitaceae variety is Sicyos angulatus.

[0075] In some embodiments, the Cucurbitaceae variety is from the subfamily Cucurbitoideae, Tribe Sicyoeae, genus Luffa. In some embodiments, the Cucurbitaceae variety is selected from Luffa acutangula, L. aegyptiaca. L. astorii, L. operculata L., L. quinqueflda, L. saccate, and L. sepium. In some embodiments, the Cucurbitaceae variety is selected from Luffa echinata. L. graveolens, L. tuberosa, and L. umbellata. In some embodiments, the first Cucurbitaceae variety is Luffa aegyptiaca and the second Cucurbitaceae variety is Luffa acutangula.

[0076] In some embodiments, the Cucurbitaceae variety is from the subfamily Cucurbitoideae, Tribe Sicyoeae, genus Sechium.

[0077] In some embodiments, the Cucurbitaceae variety is from the subfamily Cucurbitoideae, Tribe Sicyoeae, genus Trichosanthes.Tribe Benincaseae

[0078] Tribe Benincaseae is the second largest tribe, comprised of approximately 26 genera and about 214 species. Melons and cucumbers are within this tribe.

[0079] In some embodiments, the Cucurbitaceae variety is from the subfamily Cucurbitoideae, Tribe Benincaseae. In some embodiments, the Cucurbitaceae variety is from the subfamily Cucurbitoideae, Tribe Benincaseae. genus Benincasa. In some embodiments, the Cucurbitaceae variety is Benincasa hispida, also known as ash gourd, white pumpkin or white gourd (Xie, D., et al. The wax gourd genomes offer insights into the genetic diversity and ancestral cucurbit karyotype. Nat Commun 10, 5158 (2019)). Example cultivated varieties of wax gourd include, but are not limited to ‘Benefit’, ‘Blue Hili’. ‘Calm Heart’. ‘Cheerer',‘Christine’. ‘Concord’, ‘Green Tiger’. ‘KY Trim’, ‘KY Trim 2’. ‘Super Soup’, and ‘WG97- 140’.

[0080] In some embodiments, the Cucurbitaceae variety is from the subfamily Cucurbitoideae, Tribe Benincaseae, genus Citrullus. In some embodiments, the Cucurbitaceae is a variety of Citrullus lanatus. In some embodiments, the Cucurbitaceae variety is selected from Citrullus amarus, Citrullus colocynthis, Citrullus ecirrhosus, Citrullus rehmii. There are over a thousand cultivated watermelon varieties worldwide. Example cultivated watermelon varieties include, but are not limited to ‘Charleston Grey’, ‘Sugar Baby’, 'Jubilee Bush’, ‘Odell’s White’, ‘Moon and Stars’, ‘Bradford Watermelon’, ‘Georgia Rattlesnake’, ‘Ravenscroff , ‘Crimson Sweet', ‘Amarillo’, ‘Orange Crisp', ‘Calhoun Grey’, ‘Sugarlee’, ‘Smokylee', and ‘Au-Producer’.

[0081] In some embodiments, the Cucurbitaceae variety is selected from Citrullus lanatus ssp. vulgris var. cordophanus. Citrullus mucosospermus, Citrullus colocynthis , Citrullus amarus, Citrullus colocynthis, Citrullus ecirrhosus, Citrullus naudinianus, and Citrullus rehmii.

[0082] In some embodiments, the Cucurbitaceae variety is from the subfamily Cucurbitoideae, Tribe Benincaseae, genus Coccinia. Ctenolepis. Diplocyclos, Lagenaria, Melothria, Peponium, Solena, or Zehneria. In some embodiments, the Cucurbitaceae is a variety of Lagenaria siceraria.

[0083] In some embodiments, the Cucurbitaceae variety is from the subfamily Cucurbitoideae, Tribe Benincaseae. genus Cucumis. In some embodiments, the Cucumis species is C. melo L., or a subspecies thereof. In some embodiments, the Cucumis species is C. sativus L., or a subspecies thereof.

[0084] In some embodiments, the disclosure relates to allotetraploids and hybrids thereof comprised of Cucumis species, and methods of producing and using the same. See also Skalova, D. et al., Polyploidization facilitates biotechnological In Vitro techniques in the genus Cucumis. J. of Biomed. and Biotech., V:2010; Cho, W. et al., Induction of polyploidy in Cucumis melo ‘Chammel’ and evaluation of morphological and cytogenetic changes. Hort. Sci. and Tech. 2021; Chen, J. and Adelberg, J. Interspecific hybridization in Cucumis — progress, problems, and perspectives. HortScience. Vol. 35(1). 2000; Chen, J. and Kirkbride Jr.. J.H., A new synthetic species of Cucumis (Cucurbitaceae) from interspecific hybridization and chromosome doubling. Brittonia, 52(4), 2000.

[0085] In some embodiments, the Cucumis variety is selected from C. melo subsp. melo, C. melo subsp. agrestis. C. melo subsp. acidulous, C. melo var. momordica. C. callosus. C.trigonus. and C. picrocarpus. In some embodiments, the first Cucurbitaceae variety is Cucumis melo and the second Cucurbitaceae variety is Cucumis metuliferus.

[0086] In some embodiments, the Cucumis variety is selected from a C. melo L. variety or subspecies. Cucumis melo L. is a diploid species with twelve pairs of highly differentiated chromosomes. The genome includes over 375 Mb of sequence with an estimated 27,427 protein-coding genes (Garcia-Mas et al., (2012. The genome of melon (Cucumis melo L.). PNAS July issue). The more common cultivated melon plants fall into four main groups. First are the true cantaloupes of Europe. These have thick, scaly, rough, often deeply grooved, but not netted rinds. Second are the muskmelons, mostly grown in the United States, where they are incorrectly called cantaloupes. These have finely netted rinds with shallow ribs. Third are the casaba or winter melons with large fruits. These have smooth, often yellow rinds. The honeydew melons are in this third group. Fourth are a group of elongated melons of India, China and Japan which are grow n as vegetables. In some embodiments, the cultivated Cucumis is selected from C. melo var. cantalupensis , C. melo var. inodorous and C. melo var. reticulatus. Examples of cultivated C. melo L. include, but are not limited to ananas melons, Athena cantaloupes, canary melons, muskmelons, casaba melons, charentais melons. Christmas melons, crenshaw melons, galia melons, honeydew melons, oriental melons, Persian melons, and the true cantaloupe.

[0087] In some embodiments, the Cucurbitaceae variety is from the subfamily Cucurbitoideae, Tribe Benincaseae, genus Cucumis. species sativus L. In some embodiments, the Cucurbitaceae variety is selected from Cucumis sativus var. hokutosei. C. sativus var. long green, C. sativus var. sativus, C. sativus cv. winter long. Examples of cultivated C. sativus L. include, but are not limited to, English cucumbers, white cucumbers, Persian cucumbers, garden cucumbers, lemon cucumbers, Kirby cucumbers, gherkins, and Armenian cucumbers.

[0088] In some embodiments, the Cucurbitaceae variety is selected from Cucumis sativus var. hardwickii, C. sativus var. sikkimensis. and C. sativus xishuangbannanensis.Tribe Cucurbiteae

[0089] Tribe Cucurbiteae is comprised of about 12 genera. Cultivated gourds and squash, such as acom squash, butternut squash, calabaza. cushaw squash, delicata squash, honeynut squash, kabocha, red kuri squash, spaghetti squash, gem squash, and zucchini are within this tribe.

[0090] In some embodiments, the Cucurbitaceae variety is from the subfamily Cucurbitoideae, Tribe Cucurbiteae. In some embodiments, the Cucurbitaceae variety is from the subfamily Cucurbitoideae, Tribe Cucurbiteae, genus Cucurbita.

[0091] In some embodiments, the Cucurbitaceae variety is from the subfamily Cucurbitoideae, Tribe Cucurbiteae, genus Cucurbita, and selected from species C. argyrosperma, C. californica, C. cor data. C. cylindrata. C. digitata. C. ecuadorensis. C. flcifolia. C. foetidissima, C. galeottii, C. gracilior, C. lundelliana, C. martinezii, C. maxima, C. moschata, C. okeechobeensis, C. palmata, C. pedatifolia, C. pepo, C. radicans, and C. scabridifolia.

[0092] In some embodiments, the Cucurbitaceae variety is selected from Cucurbita moschata, Cucurbita maxima x Cucurbita moschata. and Cucurbita flcifolia. In some embodiments, the first Cucurbitaceae variety is Cucurbita maxima and the second Cucurbitaceae variety is Cucurbita moschata. See also Liu Z. et al., Characteristics of interspecific hybridization and inbred progeny of pumpkin (Cucurbita moschata Duch.) and winter squash (Cucurbita maxima Duch.). Horticulturae, 2022, 8, 596.

[0093] C. maxima is a diploid species with a chromosome number of 2n = 40. Examples of cultivated varieties include, but are not limited to 'Alibi', 'Citadel', 'Eureka', 'Supremo', 'Vlasstar', 'Corinto', 'Excelsior', 'Katrina', 'Lisboa', 'Noykya', 'Socrates', 'Tyria', 'Unistars', 'Crimson', 'Sugar', 'Sangria', 'Bristol', 'Dasher', 'General', 'Intimidator', 'Marketmore', 'Speedway', 'Diplomat', 'Passport', 'San', 'Sun', 'Athena', 'Divergent', 'Goddess', 'Gold', 'Halona', 'Sarah's', 'Wrangler', 'Gypsy', and 'Sorbet'.

[0094] Examples of wild species of Cucurbita include, but are not limited to C. texana, C. fraterna, C. pepo subsp fraterna, and C. pepo subsp olifera var. texana.

[0095] In some embodiments, the Cucurbitaceae variety is from the subfamily Cucurbitoideae, Tribe Gomphogyneae, Tribe Momordiceae, Tribe Bryonieae, Tribe Triceratieae, Tribe Zanonieae, Tribe Actinostemmateae, Tribe Thladiantheae, Tribe Siraitieae, Tribe Joliffieae, Tribe Schizopeponeae, Tribe Coniandreae, or Tribe Indofevilleae.Generation of allopolyploids

[0096] There are numerous steps in the development of any novel, desirable plant germplasm. Plant breeding begins with the analysis and definition of problems and weaknesses of the current germplasm, the establishment of program goals, and the definition of specific breeding objectives. The next step is selection of germplasm that possesses the traits to meet the program goals. The goal is to combine in a single variety or hybrid an improved combination of desirable traits from the parental germplasm.

[0097] These important traits may include enhanced tolerance to abiotic and / or biotic stressors, increased fruit number, fruit size and fruit weight, higher seed yield, improved color, resistance to diseases and insects, tolerance to drought and heat, better uniformity, higher nutritional value and better agronomic quality, growth rate, high seed germination, seedling vigor, early fruitmaturity, ease of fruit setting, adaptability for soil and climate conditions, firmness, content in soluble solids, acidity and viscosity. With mechanical harvesting of many crops, harvestability and field holding are also very important.

[0098] Polyploidy is the presence of more than two homologous sets of chromosomes in the cell’s nucleus (Soltis et al. 2009). This phenomenon has largely influenced plant evolution and speciation (Van de Peer, 2017). Some advantages of polyploidy are the increase in organ size ("gigas” effect), buffering of deleterious mutations, and increased heterozygosity. (Sattler et. al., 2016).

[0099] While autopolyploids (sets of chromosomes derived from the same species) are often sterile, allopolyploids (sets of chromosomes derived from different species) show restored fertility and heterosis (Comai, 2005).

[0100] Allotetraploids are hybrid cells or plants derived from different species and possessing four times the chromosome number of a haploid organism. As shown in FIG. 1 using a melon plant as an example, an allotetraploid may be generated by crossing (103) a first Cucurbitaceae variety (for example, a cultivated variety), with a second Cucurbitaceae variety (for example, a wild variety having a desired trait such as abiotic stress-tolerance).

[0101] Following the interspecific cross, polyploidization is used to fix F1heterosis by chemically -induced chromosome doubling (105). The resulting chimeric plant (107) has both diploid and tetrapioid cells. Seeds from fruit of the chimeric plant are collected (109) and sowed. Alternatively, allotetraploids may be generated via protoplast fusion.

[0102] Resulting plants are examined for ploidy and an allotetraploid plant is selected (1 11 ). The allotetraploid plant may be used as a plant itself, or as a rootstock for a commercial variety. As shown in FIG. 2, it may be crossed with another allotetraploid to generate a hybrid allopolyploid (203). This hybrid allopolyploid (203) may be used as a plant itself, as a rootstock for commercial varieties (205), or as breeding material. The allotetraploid and / or hybrid allopolyploid may further be subjected to chromosome doubling agents to generate allooctoploids.Interspecific hybrids

[0103] In an embodiment of the present disclosure, a cultivated variety is crossed with a wild variety. In some embodiments, a cultivated variety is crossed with a landrace. In some embodiments, a landrace variety is crossed with a wild variety.

[0104] In some embodiments, the disclosure relates to allotetraploids and hybrid allopolyploids comprised of Cucumis melo and Cucumis metuliferus.

[0105] In some embodiments, the disclosure relates to allotetraploids and hybrid allopolyploids comprised of Cucumis melo and Cucumis pubescens.

[0106] In some embodiments, the disclosure relates to allotetraploids and hybrid allopolyploids comprised of Cucumis melo and Cucumis anguria.

[0107] In some embodiments, the disclosure relates to allotetraploids and hybrid allopolyploids comprised of Cucurbita species, and methods of producing and using the same.

[0108] In some embodiments, the disclosure teaches an allotetraploid or hybrid allopolyploid comprised of Cucurbita maxima and Cucurbita moschata.Chemically induced chromosome doubling

[0109] Following interspecific hybridization, a chromosome doubling agent is applied. The chromosome doubling agent may be an anti-mitotic agent including, but not limited to, colchicine, trifluralin, oryzalin, amiprophos-methyl, and other polyploidy inducing agent(s). Tetrapioids can occur spontaneously in nature or be induced using spindle fiber inhibitors, such as colchicine. The technique of colchicine-induced polyploidization has been used since the 1930's. Colchicine inhibits the assembly of tublin subunits into spindle fibers, such that no chromosome movement can occur and hence, cells at the metaphase stage of mitosis accumulate. When the chromatids separate, but are not divided into separate cells by the spindle, the chromosome number is doubled creating an autopolyploid.

[0110] When creating a polyploid for breeding purposes, the layer of meristematic cells that gives rise to the gametophytic tissue needs to be doubled. To optimize the probability of successful doubling, a high number of small, actively growing meristems are treated. Colchicine concentration may vary depending on the tissue and species, but may be used, for example, at a concentration of 0.1% to 2.0%. Methods for treating seeds with colchicine or other spindle fiber inhibitors are well-known in the art, as discussed in Poehlman. J. M., Breeding Field Crops, University of Missouri, Holt, Rinehart and Winston Inc. (1966); Watts,L., Flower and Vegetable Plant Breeding, Grower Books (1980); Callaway D. J. and CallawayM. B., Breeding Ornamental Plants, Timber Press Inc. (2000).Protoplast fusion

[0111] In another embodiment, protoplast fusion can also be used to generate allotetraploids and / or hybrid allopolyploids. Protoplast fusion is an induced or spontaneous union, such as a somatic hybridization, between two or more protoplasts (cells of which the cell walls are removed by enzymatic treatment) to produce a single bi- or multi-nucleate cell. The fused cell that may even be obtained with plant species that cannot be interbred in nature is tissue cultured into a hybrid plant exhibiting the desirable combination of traits.

[0112] In some embodiments, the disclosure teaches a method for producing an allotetraploid or hybrid allopolyploid plant having a desirable trait, comprising: fusing a protoplast isolated from species with another protoplast isolated from another species having a desirable trait; selecting a heterokaryon; and regenerating an allopolyploid plant from the heterokaryon. In some embodiments, the two species are sexually incompatible. In some embodiments, the protoplast fusion is asymmetric. In some embodiments, the mitochondria and / or chloroplasts are only provided by the cultivated variety. In some embodiments, the mitochondria and / or chloroplasts are only provided by the wild variety. In some embodiments, the nucleus is only provided by the cultivated variety. In some embodiments, the nucleus is only provided by the wild variety. In some embodiments, the disclosure teaches fusing protoplasts from two different allotetraploids to generate a hybrid allopolyploid.

[0113] In some embodiments, the disclosure relates to allotetraploids and hybrid allopolyploids produced by the methods disclosed herein. In some embodiments, the disclosure relates to a seed of allotetraploid designated ‘MR 1B2’, wherein a sample of seed of said allotetraploid has been deposited under NCMA Accession Number >

[0114] In some embodiments, the disclosure relates to allotetraploids and hybrid allopolyploids produced by the methods disclosed herein. In some embodiments, the disclosure relates to a seed of allotetraploid designated ‘MC 24-01’, wherein a sample of seed of said allotetraploid has been deposited under NCMA Accession Number >

[0115] In some embodiments, the disclosure relates to allotetraploids and hybrid allopolyploids produced by the methods disclosed herein. In some embodiments, the disclosure relates to a seed of allotetraploid designated ‘MC 24-02’, wherein a sample of seed of said allotetraploid has been deposited under NCMA Accession Number >Generation of allopolyploid hybrids

[0116] Allotetraploids may be intercrossed and bred to other allopolyploids to create additional hybrid lines. In some embodiments, the disclosure teaches a method for producing seed for the production of a hybrid plant comprising the steps of crossing a first allotetraploid plant with a second allotetraploid plant and harvesting the resulting F i hybrid seed. Different allotetraploids may also be combined via protoplast fusion, as described above. In some embodiments, one or both of the allotetraploids has a desirable trait, such as a stress tolerance. In some embodiments, the first and second allotetraploids have different desirable traits. For example, the first provides at least one tolerance against at least one stress factor which is not provided by the second, such as. one is stress-tolerant against an abiotic stress, while the otheris stress tolerant against a biotic stress. In another embodiment, the disclosure relates to hybrid allopolyploid plants and parts thereof grown from hybrid allopolyploid seed.Embryo Rescue

[0117] Alternatively, embryo rescue may be employed in the generation of interspecific hybrids and / or allopolyploid hybrids. Embryo rescue can be used as a procedure to isolate embryos from crosses to rapidly move to the next generation of backcrossing or selfing or wherein plants fail to produce viable seed. In this process, the fertilized ovary or immature seed of a plant is tissue cultured to create new plants (see Pierik, 1999, In Vitro Culture of Higher Plants, Springer, ISBN 079235267X, 978-0792352679, which is incorporated herein by reference in its entirety ).Tissue Culture

[0118] As is well known in the art, tissue culture can be used for the in vitro regeneration of plants. By way of example, a tissue culture comprising organs has been used to produce regenerated plants as described in Girish- Chandel et al., Advances in Plant Sciences. 2000, 13: 1, 11-17, Costa et al.. Plant Cell Report. 2000, 19: 3327-332, Plastira et al., Acta Horticulturae . 1997, 447. 231-234. Zagorska et al.. Plant Cell Report. 1998, 17: 12 968-973. Asahura et al.. Breeding Science . 1995, 45: 455-459, Chen et al., Breeding Science . 1994, 44: 3, 257-262, Patil et al., Plant and Tissue and Organ Culture. 1994, 36: 2,255-258. It is clear from the literature that the state of the art is such that these methods of obtaining plants are routinely used and have a very high rate of success. Thus, another aspect of this disclosure is to provide cells which upon growth and differentiation produce allopolyploid plants.

[0119] As used herein, the term “tissue culture” indicates a composition comprising isolated cells of the same or a different type or a collection of such cells organized into parts of a plant. Exemplary types of tissue cultures are protoplasts, calli, plant clumps, and plant cells that can generate tissue culture that are intact in plants or parts of plants, such as embryos, pollens, flowers, seeds, leaves, stems, roots, root tips, anthers, pistils, meristematic cells, axillary buds, ovaries, seed coats, endosperms, hypocotyls, cotyledons and the like. Means for preparing and maintaining plant tissue culture are well known in the art. By way of example, a tissue culture comprising organs has been used to produce regenerated plants. U.S. Patent Nos. 5,959,185. 5,973,234, and 5,977,445 describe certain techniques, the disclosures of which are incorporated herein by reference.Asexual propagation

[0120] Sometimes referred to as vegetative propagation, asexual propagation of plants involves taking a plant part, for example a stem or root, and regenerating it into a new plant.Types of asexual propagation include, for example, cuttings, layering, division, separation, grafting, budding, and micropropagation. In some embodiments, following protoplast fusion or chromosome doubling, the allopolyploid is asexually propagated. In some embodiments, following crossing two allopolyploids, the resulting F1hybrid allopolyploid is asexually propagated.Desirable traits

[0121] In some embodiments, the disclosure teaches a method for producing an allotetraploid plant or part thereof having a desirable trait, comprising: crossing a first variety with a second, related variety having a desirable trait to produce interspecific hybrid seed; growing the interspecific hybrid seed to produce an interspecific hybrid plant; applying a chromosome doubling agent to the interspecific hybrid plant, or a vegetative cutting thereof, to generate a chimeric interspecific hybrid; growing the chimeric interspecific hybrid to produce seed; collecting seed; growing the seed; and selecting an allopolyploid plant having a desirable trait. In some embodiments, the first variety is a cultivated variety or commercial variety . In some embodiments, the second, related variety is a wild variety or a landrace variety.

[0122] In some embodiments, the desirable trait may include increased fruit number, fruit size and fruit weight, higher seed yield, improved color, resistance to diseases, pests and insects, tolerance to drought, heat, cold, salinity, better uniformity, higher nutritional value and better agronomic quality, growth rate, high seed germination, seedling vigor, early flowering, early fruit maturity, ease of fruit setting, adaptability for soil and climate conditions, root vigor, plant vigor, fruit firmness, content in soluble solids, acidity and viscosity. With mechanical harvesting of, fruit setting concentration, harvestability and field holding are also very important.

[0123] In some embodiments, plants are selected based on particularly desirable traits that may be incorporated by the methods of this disclosure. In some aspects, the desirable trait is improved resistance to abiotic and biotic stressors. Biotic stressors include different viral, fungal, and bacterial pathogens and improved resistance to insect pests. Improved resistance to insect pests is another desirable trait that may be incorporated into new allopolyploid plants developed by this disclosure.

[0124] In some embodiments, the desirable trait is resistance to a biotic stressor such as disease resistance, a pest resistance, a bacterial resistance, a fungal resistance, an insect resistance, and a nematode resistance. Diseases affecting cucurbits include, but are not limited to, Powdery mildew (Podosphaera xanthii), Fusarium wilt race 0,1,2 (Fusarium oxysporum melonis), Downy mildew (Pseudoperonospora cubensis), Cucumber Mosaic virus,Watermelon Mosaic virus, Zucchini Mosaic virus, Papaya Ringspot virus, Cucurbit Yellow Stunting disorder virus, Tomato Leaf Curl New Delhi virus, Melon Necrotic Spot Virus, melon aphid (Aphis gossyppi). In some embodiments, the allopolyploid Cucumis or Cucurbita plants are resistant to, or can be used as rootstock to confer resistance to, fusarium.

[0125] In some embodiments, the desirable trait is resistance to an abiotic stressor such as drought tolerance, salinity tolerance, flooding / water tolerance, and heat and cold temperature tolerance.

[0126] Salinization is an adverse result of irrigation (Tanji, 1990). Salts come from primary minerals in soil. All surface and ground waters contain dissolved salts picked up from soil and geologic materials that the water has come in contact with. Water used for irrigation leaves salts behind when it evaporates or is transpired by agricultural plants. The accumulating salts can negatively impact all stages of plant growth, from seed germination through seed set. Yet, irrigation is necessary to attain higher agricultural productivities to meet the growing demand for food and feed.

[0127] The need for salt and drought tolerant crops is steadily increasing, as fresh water supplies diminish, irrigation increases, and salinization threatens ever greater acres across the world. The present disclosure provides a method for transferring the salt tolerance of some wild varieties to cultivated varieties.

[0128] In some embodiments, the selected varieties having one or more desirable traits have a homozygosity of at least 80% and / or are varieties which are strict or preferential selfpollinators.

[0129] In some embodiments the desirable trait is a single gene trait. Single gene traits may or may not be transgenic. It should be appreciated that in certain embodiments, plants may be selected based on the absence, suppression or inhibition of a certain feature or trait (such as an undesirable feature or trait) as opposed to the presence of a certain feature or trait (such as a desirable feature or trait).

[0130] Selecting plants based on genotypic information is also envisaged (for example, including the pattern of plant gene expression, genotype, or presence of genetic markers). Where the presence of one or more genetic marker is assessed, the one or more marker may already be known and / or associated with a particular characteristic of a plant; for example, a marker or markers may be associated with an increased grow th rate or metabolite profile. This information could be used in combination with assessment based on other characteristics in a method of the disclosure to select for a combination of different plant characteristics that may be desirable. Such techniques may be used to identify novel quantitative trait loci (QTLs). Byway of example, plants may be selected based on growth rate, size (including but not limited to weight, height, leaf size, stem size, branching pattern, or the size of any part of the plant), general health, survival, tolerance to adverse physical environments and / or any other characteristic, as described herein before.

[0131] Further non-limiting examples include selecting plants based on: speed of seed germination; quantity of biomass produced; increased root, and / or leaf / shoot growth that leads to an increased yield (fruit) or biomass production; effects on plant growth that results in an increased seed yield for a crop; effects on plant growth which result in an increased yield; effects on plant grow th that lead to an increased resistance or tolerance to disease including fungal, viral or bacterial diseases, to mycoplasma, or to pests such as insects, mites or nematodes in which damage is measured by decreased foliar symptoms such as the incidence of bacterial or fungal lesions, or area of damaged foliage or reduction in the numbers of nematode cysts or galls on plant roots, or improvements in plant yield in the presence of such plant pests and diseases; effects on plant growth that lead to increased metabolite yields; effects on plant growth that lead to improved aesthetic appeal which may be particularly important in crops grown for their form, color or taste.

[0132] In some embodiments, the desirable trait is an improved output trait in the fruit or vegetable of the scion variety produced from the composite plant, compared to those of the same variety grown without a rootstock or with a different rootstock. In some embodiments, the output trait is increased sweetness, increased shelf-life, larger fruit or vegetable size, improved flavor, improved texture, improved color, increased nutrient content, altered nutrient content, or a combination thereof. In some embodiments, the increased nutrient is an antioxidant, fiber, potassium, vitamin C, protein, calcium, vitamin K, vitamin A. magnesium, boron, zinc, or combination thereof. In some embodiments, the increased nutrient is a carotenoids, terpenoid, saponin, phytochemical, or combination thereof.Interploidy hybridization

[0133] In addition to crosses with other allopolyploids, the allotetraploids of the present disclosure may also be crossed with plants having different ploidy levels. In some embodiments, the allopolyploid plants and / or hybrids of the present disclosure are crossed with a diploid cultivar. In some embodiments, the allopolyploid plant is an allotetraploid crossed with a diploid cultivar to produce a tripl oid plant.Composite (non-naturally occurring grafted) plants

[0134] Grafting is a method of asexual plant propagation widely used in agriculture and horticulture where the tissues of one plant are encouraged to fuse with those of another.Grafting involves combining two independent plant parts into one plant. Such combination may be performed in various ways, including, but not limited to cleft grafting, side grafting, whip grafting, stub grafting, awl grafting, veneer grafting, bark grafting, tongue grafting, splice grafting, tip-cleft grafting, saddle grafting, approach grafting, and budding grafting (patch budding, chip budding, T-budding) (for further details see Gamer R. J., The Grafter's Handbook, 5th Ed edition (March 1993) Cassell Academic; ISBN: 0304342742). Grafting produces a non-naturally occurring composite plant.

[0135] An embodiment of the present disclosure relates to composite plants comprising the allotetraploids or allopolyploids described herein as rootstock and methods of generating said composite plants. In some embodiments, the scion is a cultivated variety. In some embodiments, the cultivated variety is the same variety used in the initial interspecific cross or protoplast fusion to generate the allotetraploid or hybrid allopolyploid rootstock, and thus rootstock and scion share at least one allele. In some embodiments, the rootstock and scion share at least one chromosome. In some embodiments, the rootstock and scion share a set of chromosomes. In some embodiments, the disclosure relates to a chimeric plant tissue generated by grafting a cultivated variety as the scion to an allotetraploid or hybrid allopolyploid plant described herein as the rootstock. In some embodiments, the chimeric plant tissue comprises a first plant cell and a second plant cell, wherein the first plant cell is an allotetraploid or allopolyploid comprising chromosomes from a cultivated variety and at least one species related to the cultivated variety.

[0136] The allopolyploid plants described herein are graft compatible and may be suitable for use as rootstock for cultivated varieties of a different species or genera, and overcome previous graft incompatibility of some scion+rootstock combinations. Some intrafamilial grafts are compatible within Solanaceae and Cucurbitaceae, for example. The allopolyploid plants and methods of producing disclosed herein may possess any number of desirable traits and confer as much to the scion, and fruits and vegetables produced therefrom, including, but not limited to, resistance and / or tolerance to salinity stress, cold stress, heat stress, drought stress, disease resistance, fungal resistance, pest resistance, bacterial resistance, insect resistance, and nematode resistance. Additionally, the allotetraploid and hybrid allopolyploid plants described herein may further increase yield of the scion plant and / or improve an output trait, such as nutrient content of the fruits or vegetables harvested from the scion. The allotetraploid and hybrid allopolyploid plants disclosed herein are especially suitable for automated grafting by an automated grafting machine due to their high uniformity. Thus, in another embodiment, the disclosure teaches methods of conferring a desirable trait from a wild or landrace species to acultivated variety by grafting. In some embodiments, the allotetraploid or hybrid allopolyploid is used as a rootstock. In some embodiments, the disclosure relates to a method of producing a composite plant wherein the rootstock is an allotetraploid or hybrid allopolyploid as described herein having an abiotic stress tolerance (for example, resistance to salt stress), and the scion has a biotic stress tolerance (for example, resistance to a pest or pathogen).

[0137] Grafting is a process that has been used for many years in crops such as members of the Cucurbitaceae family. The variety used as the scion (usually an elite commercial variety), is grafted onto a rootstock variety, usually comprising a desirable trait such as resistance to abiotic or biotic stress. The resistant rootstock thus remains healthy and provides nutrients from the soil to the scion. In some recent developments, it has also been shown that some rootstocks are also able to improve the agronomic value for the grafted plant and in particular the equilibrium between the vegetative and generative development that are difficult to balance some cultivation.

[0138] The allotetraploids and hybrid allopoly ploids described herein can be bred with other allopolyploids to generate hybrid allopolyploids, and / or used as rootstock for a number of commercial varieties and other species. For example, the Cucurbita maxima x C. moschata described above may be used as rootstock for watermelon, cucumber, and melon (Traka- MavronaE. et al., “Response of squash (Cucurbita spp.) as rootstock for melon (Cucumis melo L.).” Scientia Hort. 83 (2000) pp. 353-362). Examples of cultivated varieties include, but are not limited to 'Alibi', 'Citadel', 'Eureka'. 'Supremo'. 'Vlasstar', 'Corinto', 'Excelsior', 'Katrina', 'Lisboa', 'Noykya', 'Socrates', 'Tyria', 'Unistars', 'Crimson', 'Sugar', 'Sangria', 'Bristol', 'Dasher', 'General', 'Intimidator', 'Marketmore', 'Speedway', 'Diplomat', 'Passport', 'San', 'Sun', 'Athena', 'Divergent', 'Goddess', 'Gold', 'Halona', 'Sarah's', 'Wrangler', 'Gypsy', and 'Sorbet'.

[0139] Additional example commercial varieties that may be used with the disclosed allopolyploids and methods include, but are not limited to, C melo var. cantalupensis . C. melo var. inodorous and C. melo var. reliculatus.

[0140] “Wild plants " or “wild melon" may also be used for various interspecific crosses, including for example, ecotypes, landraces, or wild varieties, such as for example accessions of Cucumis melo ssp. agrestis, C. melo ssp. melo, C. melo ssp. acidulous. C. callosus. C. trigonus, C. picrocarpus, and Cucumis melo ear. momordica. These wild varieties, while perhaps exhibiting poor yield and / or quality, may nonetheless possess valuable desirable trails that can be transferred to commercial vaneties using the disclosed methods of generating allopolyploids and using them as rootstock for commercial varieties.

[0141] Similarly, desirable traits from wild watermelon species, such as Citrullus mucosospermus, Citrullus colocynthis and C. amanis, can be transferred to commercial watermelon species such as Citrullus ianatus using the disclosed methods.

[0142] There are several methods for grafting. Examples of suitable grafting methodologies include, without limitation, cleft grafting, approach grafting, micrografting, tube grafting, side insertion grafting, and top insertion grafting. Cleft grafting involves cutting a V-shape into the rootstock and inserting a complementing wedge-shaped scion. The graft may be then held with a small clip until healing occurs. Approach grafting, also known as tongue approach grafting (TAG), involves notching opposing sides of the stems of the root-stock and scion, and then using a clip to hold the stems together while they fuse. Once the graft has healed, the scion of the desired rootstock plant may be removed above the graft site, and the unused rootstock from scion plant may be detached from the scion below the graft site. Micrografting, also known as splice grafting, is a technique that has been recently integrated into micropropagation production for hybrid tomato. Micrografting involves utilizing micropropagated scion shoots that may be grafted onto approximately three-week-old rootstock seedlings. In some embodiments, micrografting is utilized for commercial scale grafting. Tube grafting involves severing the scion and rootstock as seedlings and attaching the severed rootstock seedling to the severed scion seedling with a small, silicone tube with or without a clip. Tube grafting can be highly effective, as it may be carried out when plants are very small, thereby eliminating the need for large healing chambers while increasing the output. Although less frequently used on a commercial scale, side insertion grafting and top insertion grafting are also contemplated herein. See also (Lee, 1994; Lee and Oda, 2003; Hanna, 2012; Lee and Oda, 2003; Oda, 1995; Rivard and Louws, 2006; Vu et al., 2015; Bausher, 2013; Rivard and Louws, 2006; Kubota et al., 2008; and Lee, 2003).DEPOSIT INFORMATION

[0143] A deposit of allopolyploid seed of this disclosure is maintained by RedSea Science and Technology Inc., 14 Ridge Square NW, Suite 300, Washington, DC 20016.

[0144] In addition, a sample of 625 seeds of the varieties disclosed herein will be deposited with an International Depositary Authority as established under the Budapest Treaty according to 37 CFR 1.803(a)(1).

[0145] Applicant will deposit seeds at the Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA), located at the Bigelow Laboratory for Ocean Science at 60 Bigelow Drive East Boothbay. ME 04544.

[0146] To satisfy the enablement requirements of 35 U.S.C. 112, and to certify that the deposit of the allopolyploids of the present disclosure meets the criteria set forth in 37 CFR 1.801- 1.809 and Manual of Patent Examining Procedure (MPEP) 2402-2411.05, Applicant hereby makes the following statements regarding the deposited seed:1. During the pendency of this application, access to the disclosure will be afforded to the Commissioner upon request;2. All restrictions on availability to the public will be irrevocably removed upon granting of the patent under conditions specified in 37 CFR 1 .808;3. The deposit will be maintained in a public repository for a period of 30 years or 5 years after the last request or for the effective life of the patent, whichever is longer;4. A test of the viability of the biological material at the time of deposit will be conducted by the public depository under 37 CFR 1.807; and5. The deposit will be replaced if it should ever become unavailable.

[0147] Access to this deposit will be available during the pendency of this application to persons determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. § 1.14 and 35 U.S.C. § 122. Upon allowance of any claims in this application, all restrictions on the availability to the public of the variety will be irrevocably removed by affording access to a deposit of at least 625 seeds of the same variety' with the NCMA.

[0148] Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials, similar or equivalent to those described herein, can be used in the practice or testing of the present invention, the non-limiting exemplary methods and materials are described herein.

[0149] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure.

[0150] Many modifications and other embodiments of the disclosures set forth herein will come to mind to one skilled in the art to which these disclosures pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within thescope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0151] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.EXAMPLES

[0152] The following examples are provided to illustrate further the various applications and are not intended to limit the disclosure beyond the limitations set forth in the appended claims.Example 1: Allotetraploid Variety ‘MR 1B2’

[0153] Allotetraploid variety ‘MR 1B2’ was generated by an interspecific cross of a Cucumis melo variety and a Cucumis metidiferus variety.

[0154] The Cucumis melo variety flowers were used as mothers, emasculated and pollinated with pollen extracted from an accession of Cucumis metuliferus, selected based on plant vigor, resistance to number of diseases such as powdery mild and Fusarium wilt.

[0155] F1hybrid seeds were sown in commercial seedling trays with either 50 or 72 cells, following standard seedling production practices. Once the seedlings emerged from the soil, a drop of Oryzalin solution (35-50 pM) was applied between the two cotyledons. Seedlings were then maintained under normal production conditions, ensuring proper watering. Seedlings exhibiting delayed growth, characterized by dark green, more serrated leaves, were selected (FIG. 3). These selected seedlings were transplanted into greenhouse beds after developing 3- 4 true leaves. Plants were monitored for traits indicative of tetraploidy, such as changes in leaf and flower morphology (FIG. 3), and those showing these traits were self-pollinated. Ploidy was confirmed by assessing pollen grain size and morphology (FIG. 4). Seeds were harvested from mature fruit, typically 35-45 days after pollination, depending on temperature conditions. Further ploidy confirmation and seed increase were conducted using the self-pollinated progeny to ensure uniformity and stability of the tetrapioid cucurbit variety. Allotetraploid variety 'MR 1B2’ was selected based on uniformity and seed yield.

[0156] The closest rootstock variety to allotetraploid variety ‘MR 1B2’ is its diploid version ‘MR 1A’. However, as shown in Table 2 below, allotetraploid variety ‘MR 1B2’ has different morphology compared to diploid ‘MR 1 A’ (See also FIG. 3 and FIG. 4).Table 2: Characteristics of ‘MR 1B2’ compared to ‘MR 1A’

[0157] Allotetraploid variety ‘MR 1B2’ shows uniformity' and stability for the traits due to the fixation of heterozygosity achieved by the chromosome doubling agent.

[0158] Allotetraploid variety ‘MR 1B2’ has been tested as a rootstock. Commercial varieties ‘Alcazaba’ (FIG. 5) and ‘Kenza’ (FIG. 6) were grafted onto allotetraploid variety ‘MR 1B2’ rootstock to generate 18 composite plants per scion / rootstock combination. For comparison,‘Alcazaba’ and ‘Kenza’ were also grafted onto diploid rootstock variety ‘MR 1A’, generating 18 composite plants per scion / rootstock combination. For comparison, plants of ‘Alcazaba’ and ‘Kenza’ were self-grafted at the same time. This trial was conducted at the Experimental Center of the National Technological Center of Auxiliary Industry of Agriculture, TECNOVA Foundation, located at Majada Ortigas (36°53’N; 222’W, 184 m above sea level), in the municipality of Viator, in the province of Almeria, in southeast of Spain. The trial was carried out by the technical team of the National Technological Center for the Auxiliary Industry of Agriculture, TECNOVA Foundation (FIG. 7). All grafting was done manually using standard techniques. The Alcazaba-MR 1B2 composite plants produced an average of 1340 g of biomass per plant, whereas the Alcazaba-MR 1A composite plants produced an average of 833 g of biomass per plant. In comparison, the Alcazaba self-grafted controls produced an average of 781 g of biomass per plant. Thus, the Alcazaba-MR 1B2 composite plant had a 61% increase in biomass over the Alcazaba-MR 1 A composite plant and a 72% increase over the Alcazaba self-grafted controls (FIG. 8). Similarly, the Kenza-MR 1B2 composite plants produced an average of 1083 g of biomass per plant, whereas the Kenza-MR 1A composite plants produced an average of 787 g of biomass per plant, and the Kenza self-grafted controls produced an average of 695 g of biomass per plant. Thus, the Kenza-MR 1B2 composite plant had a 38% increase in biomass over the Kenza-MR 1 A composite plant and a 56% increase over the Kenza self-grafted controls (FIG. 8).Example 2 - Allotetraploid Variety ‘MC 24-01’

[0159] Allotetraploid variety ‘MC 24-01’ was generated by an interspecific cross of a Cucurbita maxima variety and a Cucurbita moschata variety.

[0160] The C. maxima variety flowers were used as mothers, emasculated and pollinated with pollen extracted from an accession of C. moschata, selected based on plant vigor and disease resistance such as Fusarium wilt and stress tolerance such as cold and hot temperatures.

[0161] F1hybrid seeds were sown in commercial seedling trays with either 50 or 72 cells, following standard seedling production practices. Once the seedlings emerged from the soil, a drop of Oryzalin solution (35-50 pM) was applied between the two cotyledons. Seedlings were then maintained under normal production conditions, ensuring proper watering. Seedlings exhibiting delayed growth, characterized by dark green, more serrated leaves, were selected (FIG. 9). These selected seedlings were transplanted into greenhouse beds after developing 3- 4 true leaves. Plants were monitored for traits indicative of tetraploidy, such as changes in leaf and flower morphology, and those showing these traits were self-pollinated. Ploidy wasconfirmed by assessing pollen grain size and morphology (FIG. 9). Seeds were harv ested from mature fruit, typically 35-45 days after pollination, depending on temperature conditions. Further ploidy confirmation and seed increase were conducted using the self-pollinated progeny to ensure uniformity and stability of the tetrapioid cucurbit variety. Allotetraploid variety ‘MC 24-01’ was selected based on uniformity and seed yield.

[0162] The closest rootstock variety to allotetraploid variety ‘MC 24-01' is its diploid version ‘Wei-Zhen 103’. However, as shown in Table 3 below, allotetraploid variety ‘MC 24-01’ has different morphology compared to diploid variety ‘Wei-Zhen 103’ (See also FIG. 9).Table 3: Characteristics of ‘MC 24-01’ compared to diploid ‘Wei-Zhen 103’

[0163] Allotetraploid variety MC 24-01 ’ shows uniformity and stability for the traits due to the fixation of heterozygosity achieved by the chromosome doubling agent.

[0164] Allotetraploid variety ‘MC 24-01’ was evaluated as a rootstock in Weifang City , Shandong Province, at the Peking University Institute of Advanced Agricultural Sciences during the 2025 growing season, compared to three commercial rootstocks (Wei-Zhen 201. Wei -Zhen 103 and Wei -Zhen 105). The experiment was conducted using a randomized block design with three replicates (10 x 10 columns per block). For each material, 120 seeds were sown, 60 seedlings were grafted, and 30 plants were transplanted to the field, yielding 30 replicates per material. The scion used was the commercial line G42, with G42-selfgrafted and G42-ungrafted controls.

[0165] Rootstocks were grown ungrafted to observe morphology and vegetative traits. ‘MC 24-01’ seedlings displayed a 94% emergence rate. Root assays in paper bags showed that 10 days post germination, root length averaged 33.5 cm, comparable to the commercial rootstock average (34.0 cm). Biomass measurements showed root dry weight of 15.8 g. representing a 30% increase over the commercial average (12.2 g). Shoot dry weight was stable (22.3 g vs. 22.4 g average), and total biomass was essentially equivalent (38.1 g vs. 38.0 g). In field conditions, the main vine circumference at flowering was consistently thicker in ‘MC 24-01’ compared to other commercial rootstocks, indicating improved stem robustness. ‘MC 24-01 ’ produced an average 100-seed weight of 20.17 g, corresponding to a 39% increase compared to the commercial average (14.50 g).

[0166] In field trials under a plastic tunnel, composite plants were generated with watermelon variety G24 as the scion. Grafted plants were transplanted under a randomized block design with three replicates and using three commercial rootstocks as controls (Wei-Zhen 201, Wei- Zhen 103 and Wei-Zhen 105). For each material, 120 seeds were sown, 60 seedlings grafted, and 30 transplanted (10 per block). G24-MC 24-01 composite plants produced a total of 74 fruits, averaging 2.5 fruits per plant, compared to an average of 2.6 fruits per composite plant comprising G24 and a commercial rootstock. Average fruit weight in G24-MC 24-01 composite plants was 1.62 kg, a 10% increase compared to the commercial composite average of 1.47 kg. Yield per plant reached 3.99 kg, a 10% improvement over the commercialcomposite average of 3.63 kg. These results demonstrate that ‘MC 24-01’ delivers larger fruit compared to standard commercial rootstocks.

[0167] In addition to yield and vigor traits, ‘MC 24-01’ was generated with the objective of enhancing stress tolerance. The observed increases in root biomass and stem thickness are consistent with improved water and nutrient uptake, traits that are expected to enhance performance under heat and limited-water conditions. Ongoing disease evaluations include Fusarium wilt screening, where allotetraploid rootstocks such as ‘MC 24-01 ’ are anticipated to display stronger resistance compared to commercial standards.Example 3 - Allotetraploid Variety ‘MC 24-02’

[0168] Allotetraploid variety ‘MC 24-02’ was generated by an interspecific cross of a Cucurbita maxima variety and a Cucurbita moschata variety.

[0169] The C. maxima variety flowers were used as mothers, emasculated and pollinated with pollen extracted from an accession of C. moschata. selected based on plant vigor and disease resistance such as Fusarium wilt and stress tolerance such as cold and hot temperatures.

[0170] F1hybrid seeds were sown in commercial seedling trays with either 50 or 72 cells, following standard seedling production practices. Once the seedlings emerged from the soil, a drop of Oryzalin solution (35-50 pM) was applied between the two cotyledons. Seedlings were then maintained under normal production conditions, ensuring proper watering. Seedlings exhibiting delayed growth, characterized by dark green, more serrated leaves, were selected. These selected seedlings were transplanted into greenhouse beds after developing 3-4 true leaves. Plants were monitored for traits indicative of tetraploidy. such as changes in leaf and flower morphology, and those showing these traits were self-pollinated. Ploidy was confirmed by assessing pollen grain size and morphology. Seeds were harvested from mature fruit, typically 35-45 days after pollination, depending on temperature conditions. Further ploidy confirmation and seed size increase were conducted using the self-pollinated progeny to ensure uniformity and stability of the tetraploid cucurbit variety. Allotetraploid variety ‘MC 24-01’ was selected based on uniformity and seed yield.

[0171] The closest rootstock variety to allotetraploid variety ‘MC 24-02’ is ‘Wei-Zhen 105’. However, as shown in Table 4 below, allotetraploid variety ‘MC 24-02’ has different morphology compared to diploid variety ‘Wei-Zhen 105’ (FIG. 10).Table 4: Characteristics of ‘MC 24-02’ compared to diploid ‘Wei-Zhen 105’

[0172] Allotetraploid variety ‘MC 24-02' was evaluated as a rootstock in Weifang City, Shandong Province, at the Peking University Institute of Advanced Agricultural Sciences during the 2025 growing season, compared to three commercial rootstocks (Wei-Zhen 201, Wei -Zhen 103 and Wei -Zhen 105). The experiment was conducted using a randomized block design with three replicates (10 x 10 columns per block). For each material, 120 seeds were sown, 60 seedlings were grafted, and 30 plants were transplanted to the field, yielding 30replicates per material. The scion used was the commercial line G42, with G42-selfgrafted and G42-ungrafted controls.

[0173] Rootstocks were grown ungrafted to observe morphology and vegetative traits. ‘MC 24-02’ seedlings exhibited strong vigor with a 100% emergence rate. Root assays in paper bags showed that 10 days post germination, root length averaged 36.5 cm, an approximately 12% increase compared to the commercial rootstock average of 32.5 cm (FIG. 11). Biomass measurements showed "MC 24-02’ had an average shoot dry weight of 29.3 g, an approximately 30% increase compared to the average 22.4 g for the commercial rootstocks. Root dry weight of ‘MC 24-02’ was comparable to that of the commercial rootstocks, 12.0 g vs. average 11.8 g, however total biomass of ‘MC 24-02’ was 41.3 g. an approximately 9% increase compared to an average of 37.9 g for the commercial rootstocks. In field conditions, the main vine circumference at flowering was thicker in ‘MC 24-02’ than in commercial rootstocks, indicating superior stem robustness. ‘MC 24-02’ produced an average 100-seed weight of 28.40 g, nearly double that of the commercial average of 14.50 g.

[0174] In field trials under plastic tunnel with watermelon variety G24 as a scion, composite plants were transplanted under a randomized block design with three replicates and using three commercial rootstocks (Wei-Zhen 201, Wei-Zhen 103 and Wei-Zhen 105) as controls. For each material, 120 seeds were sown, 60 seedlings grafted, and 30 transplanted (10 per block). G24-MC 24-02 composite plants produced a total of 77 fruits, averaging 2.6 fruits per plant, equivalent to the commercial rootstock average of 2.6 fruits per plant. Average fruit weight in G24-MC 24-02 composite plants was 1.50 kg, matching the commercial average of 1.50 kg. Yield per plant was 3.85 kg, essentially equivalent to the commercial rootstock average of 3.96 kg. These results indicate that ‘MC 24-02’ performs at least as well as commercial rootstocks in fruit yield while offering stronger vegetative vigor and seed traits.

[0175] ‘MC 24-02’ was also developed for stress tolerance applications. Its strong seedling vigor, extended root length, and superior shoot biomass indicate a higher capacity to sustain growth under abiotic stresses such as salinity and high temperature. Furthermore, ‘MC 24-02’ is currently under evaluation for tolerance to soilbome pathogens, including Fusarium wilt, where its enhanced vegetative robustness is expected to translate into improved resistance relative to commercial rootstocks.Example 4: Overcoming graft incompatibility

[0176] Many grafting combinations within the Cucurbitaceae family are unsuccessful due to grafting incompatibility (see for example Table 5). For example, melon (Cucumis melo) has been shown to be incompatible when grafted onto luffa, wax gourd, bottle gourd, bitter gourd,and watermelon rootstocks (Mu X. et al., Compatibility Evaluation and Anatomical Observation of Melon Grafted Onto Eight Cucurbitaceae Species, Frontiers in Plant Science, Vol. 12, 2021). Reasons for graft incompatibility include, for example, poor vascular connection, excess callose deposition at the graft union and starch buildup, biochemical incompatibility that interferes with graft healing, lack of aligned cell signaling and hormonal responses due to genetic divergence, necrotic layer formation at the graft interface that prevents tissue fusion, and stem size differences (Kgabo Pofu & Phatu Mashela (2012) Improving survival of inter-generic grafts of nematode-susceptible watermelon cultivars and nematoderesistant Cucumis species, Acta Agriculturae Scandinavica, Section B - Soil & Plant Science, 62:4, 383-386)Table 5: Examples of graft incompatibility within Cucurbitaceae

[0177] The allotetraploid rootstocks and composite plants described herein are well suited to overcome some of the incompatibilities discussed above. For example, because the allotetraploid rootstock may be comprised of a wild species or landrace accessions with a cultivated variety, there is less genetic distance / divergence between scion and rootstock, facilitating improved compatibility. In addition, the greater stem diameter and enlarged root systems observed in allotetraploid lines are expected to promote stronger vascular connections, more efficient water and nutrient transport, and enhanced overall scion vigor.

[0178] Beyond compatibility, allotetraploid rootstocks are hypothesized to provide advantages under abiotic and biotic stress conditions. Larger and deeper root systems may increase water uptake and tolerance to drought or salinity, while thicker stems and greater biomass may enhance resilience under high temperature stress.

[0179] The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification.NUMBERED EMBODIMENTS1. A method for producing a composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress, comprising:(i) selecting a first Cucurbitaceae variety which is stress-tolerant against at least one abiotic or biotic stress;(ii) crossing said first Cucurbitaceae variety with a second Cucurbitaceae variety of a different species sexually compatible with the first Cucurbitaceae variety to produce an interspecific hybrid seed;(iii) growing the interspecific hybrid seed to produce an interspecific hybrid Cucurbitaceae plant;(iv) applying a chromosome doubling treatment to the interspecific hybrid plant, or a part thereof, to generate a chimeric interspecific hybrid Cucurbitaceae plant;(v) collecting seed from an allotetraploid fruit of said chimeric interspecific hybrid plant;(vi) growing the seed to produce an allotetraploid Cucurbitaceae rootstock plant with tolerance against at least one abiotic or biotic stress and, optionally, further propagating said plant; and(vii) grafting a scion of a cultivated Cucurbitaceae variety to the allotetraploid Cucurbitaceae rootstock to produce a composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress.2. A method for producing a composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress, comprising:(i) selecting a first Cucurbitaceae variety which is stress-tolerant against at least one abiotic or biotic stress;(ii) fusing a protoplast isolated from said first Cucurbitaceae variety with another protoplast isolated from a Cucurbitaceae variety sexually incompatible with the first Cucurbitaceae variety;(iii) selecting a heterokaryon;(iv) regenerating an allotetraploid Cucurbitaceae rootstock plant with tolerance against at least one abiotic or biotic stress from the heterokaryon; and, optionally, further propagating said plant: and(v) grafting a scion of a cultivated Cucurbitaceae variety to the allotetraploid Cucurbitaceae rootstock to produce a composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress. 3. A method for producing a stress-tolerant hybrid allopolyploid Cucurbitaceae plant or seed, comprising:(i) selecting a first Cucurbitaceae variety which is stress-tolerant against at least one abiotic or biotic stress;(ii) crossing said first Cucurbitaceae variety with a second Cucurbitaceae variety of a different species sexually compatible with the first Cucurbitaceae variety to produce an interspecific hybrid seed;(iii) growing the interspecific hybrid seed to produce an interspecific hybrid Cucurbitaceae plant;(iv) applying a chromosome doubling treatment to the interspecific hybrid plant, or a part thereof, to generate a chimeric interspecific hybrid Cucurbitaceae plant;(v) collecting seed from an allotetraploid fruit of said chimeric interspecific hybrid plant;(vi) growing the seed to produce a first allotetraploid Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress and, optionally, further propagating said plant;(vii) crossing the first allotetraploid Cucurbitaceae plant with a second allotetraploid Cucurbitaceae plant to produce hybrid allopolyploid seed;(viii) harvesting the hybrid allopolyploid seed; and(ix) optionally growing the hybrid allopolyploid seed to produce a stress-tolerant hy brid allopolyploid Cucurbitaceae plant. 4. A method for producing a stress-tolerant hybrid allopolyploid Cucurbitaceae plant, comprising:(i) selecting a first Cucurbitaceae variety which is stress-tolerant against at least one abiotic or biotic stress;(ii) fusing a protoplast isolated from said first Cucurbitaceae variety with another protoplast isolated from a second Cucurbitaceae variety sexually incompatible with the first Cucurbitaceae variety to produce a heterokaryon;(iii) regenerating a first allotetraploid Cucurbitaceae plant from the heterokaryon;(iv) fusing a protoplast isolated from the first allotetraploid Cucurbitaceae plant with another protoplast isolated from a second allotetraploid Cucurbitaceae plant to a produce hybrid allopolyploid heterokaryon; and(v) regenerating a hybrid allopolyploid Cucurbitaceae plant from the hybrid allopolyploid heterokaryon to produce a stress-tolerant hybrid allopolyploid Cucurbitaceae plant. 5. A method for producing a composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress, comprising:(i) providing a hybrid allopolyploid Cucurbitaceae plant produced by the method of embodiment 3 or 4 as a rootstock; and(ii) grafting a scion of a cultivated Cucurbitaceae variety to the allotetraploid Cucurbitaceae rootstock to produce a composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress. 6. The method of embodiment lor 3, wherein the part thereof is a vegetative cutting. 7. The method of embodiment 3 or 4, wherein the second allotetraploid Cucurbitaceae plant exhibits at least one tolerance against at least one abiotic or biotic stress which is not present in the first allopolyploid Cucurbitaceae plant. 8. The method of embodiment 1, 2, or 5, wherein the cultivated Cucurbitaceae is an inbred or essentially homozygous. 9. The method of embodiment 1, 2, or 5, wherein the cultivated Cucurbitaceae is a hybrid. 10. The method of any one of embodiments 1-5, wherein the first Cucurbitaceae variety is a wild variety. 11. The method of any one of embodiments 1-5, wherein the first or second Cucurbitaceae variety is a landrace variety. 12. The method of any one of embodiments 1-5, wherein the first Cucurbitaceae is a wild variety and the second Cucurbitaceae variety is a cultivated variety. 13. The method of any one of embodiments 1-5, wherein the first Cucurbitaceae is a landrace variety and the second Cucurbitaceae variety is a cultivated variety. 14. The method of any one of embodiments 1-5, wherein the first or second Cucurbitaceae variety is an F1hybrid. 15. The method of embodiment 1, 2 or 5, wherein the scion is a commercial Cucurbitaceae variety.16. The method of any one of embodiments 1-5, wherein the first or second Cucurbitaceae variety has an abiotic stress tolerance selected from the group consisting of cold tolerance, high temperature tolerance, drought tolerance, and salt tolerance. 17. The method of any one of embodiments 1-5, wherein the first or second Cucurbitaceae variety has a biotic stress tolerance selected from the group consisting of a disease resistance, a pest resistance, a bacterial resistance, a fungal resistance, an insect resistance, and a nematode resistance. 18. The method of any one of embodiments 1 -5, wherein the first Cucurbitaceae variety has a different biotic or abiotic stress tolerance than the second Cucurbitaceae variety. 19. The method of any one of embodiments 1, 2 or 5. wherein the cultivated Cucurbitaceae variety used as scion is less tolerant against at least one biotic or abiotic stress in comparison to the allotetraploid Cucurbitaceae rootstock. 20. The method of embodiment 2 or 4, wherein the protoplast fusion is asymmetrical and mitochondria are only provided by a cultivated Cucurbitaceae variety to generate the first allotetraploid plant. 21. A hybrid allopolyploid Cucurbitaceae plant or plant part with tolerance against at least one abiotic or biotic stress, comprising:(i) at least one chromosome from each of a first and a second Cucurbitaceae variety, wherein the second Cucurbitaceae variety exhibits at least one tolerance against at least one abiotic or biotic stress which is not present in said first Cucurbitaceae variety; and(ii) at least one chromosome for a cultivated Cucurbitaceae variety of a species different from said first and second Cucurbitaceae varieties. 22. The hybrid allopolyploid Cucurbitaceae plant of embodiment 21, wherein said plant is produced by the method of embodiment 3 or 4. 23. The hybrid allopolyploid Cucurbitaceae plant part of embodiment 21, wherein said plant part is a seed. 24. A composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress, said composite Cucurbitaceae plant comprising:(i) as a rootstock the hybrid allotetraploid Cucurbitaceae plant of any one of embodiments 21-23; and(ii) as a scion a cultivated Cucurbitaceae variety. 25. A composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress, said composite Cucurbitaceae plant comprising:(i) as a rootstock an allotetraploid Cucurbitaceae plant comprising i. at least one chromosome from a wild or landrace Cucurbitaceae variety which exhibits at least one tolerance against at least one abiotic or biotic stress; and ii. at least one chromosome from a cultivated Cucurbitaceae variety(ii) as a scion a cultivated Cucurbitaceae variety. 26. The composite Cucurbitaceae plant of embodiment 25, wherein said composite Cucurbitaceae plant is produced by the method of embodiment 1 or 2. 27. The composite Cucurbitaceae plant of any of embodiment 24-26, wherein said composite Cucurbitaceae plant has a higher tolerance against at least one biotic or abiotic stress in comparison to the cultivated Cucurbitaceae variety used a scion when grown under the same conditions without the allotetraploid rootstock. 27.1 The composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the allotetraploid rootstock and scion comprise chromosomes from different genera. 27.2 The composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the allotetraploid rootstock comprises chromosomes from at least one of Lagenaria siceraria, Benincasa hispida, and Momordica charantia and the scion is a cultivated Cucumis melo variety. 27.3 The composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the allotetraploid rootstock comprises chromosomes from at least one of Cucumis myriocarpus. and Cucumis africcmus the scion is a cultivated Citrullus lanatus variety’. 27.4 The composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the allotetraploid rootstock comprises chromosomes from Cucumis melo the scion is a cultivated Luffci cylindrica variety. 28. The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from the subfamily Cucurbitoideae. 29. The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Sicyoeae. 29.1. The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceaeplant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Sicyoeae, genus Luffa. 29.2 The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first Cucurbitaceae variety is Luffa aegyptiaca and the second Cucurbitaceae variety is Luffa acutangula. 30. The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Benincaseae. 30.1 The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Benincaseae, genus Benincasa. 30.2 The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Benincaseae, genus Citrullus. 30.3 The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are selected from Citrullus amarus, Citrullus colocynthis, Citrullus ecirrhosus, Citrullus rehmii, and combinations thereof 30.4 The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Benincaseae, genus Cucumis. 30.5 The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first or second Cucurbitaceae variety are subspecies of Cucumis melo L. 30.6 The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceaeplant of any one of embodiments 24-27, wherein the first Cucurbitaceae variety is Cucumis melo and the second Cucurbitaceae variety is Cucumis metuliferus. 30.7 The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first or second Cucurbitaceae variety are subspecies of Cucumis sativus L. 30.8 The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are species from the Lagenaria genus. 31. The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Cucurbiteae. 31.1 The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first or second Cucurbitaceae variety are from Tribe Cucurbiteae, genus Cucurbita. 31.2 The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are subspecies of Cucurbita pepo. 31.3 The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are selected from Cucurbita moschata, Cucurbita maxima x Cucurbita moschata, and Cucurbita ficifolia. 31.4 The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first variety is Cucurbita maxima and the second variety is C. moschata. 32. The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceaeplant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Momordiceae. 33. The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Gomphgyneae. 34. The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Bryonieae. 35. The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Triceratieae. 36. The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Zanonieae. 37. The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Actinostemmateae. 38. The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Thladiantheae. 39. The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Siraitieae. 40. The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Joliffieae.41. The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Schizopeponeae. 42. The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Coniandreae. 43. The method of any one of embodiments 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the first and / or second Cucurbitaceae variety are from Tribe Indofevilleae. 44. The composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the scion is a species of Citrullus or hybrid thereof. 45. The composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the scion is C. lanatus or a hybrid thereof. 46. The composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the scion is a species of Cucumis or hybrid thereof 47. The composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the scion is a species of Cucurbita or hybrid thereof. 48. The composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the scion is a species of Lagenaria or hybrid thereof 49. The composite Cucurbitaceae plant of embodiment 46, wherein the rootstock comprises species of Cucurbita. 50. The composite Cucurbitaceae plant of any one of embodiments 24-27, wherein the scion is a variety selected from the group consisting of: cucumber, zucchini, pumpkin, wax gourd, bottle gourd, bitter gourd, ridge gourd, sponge gourd, chayote, snake gourd, melon, homed cucumber, watermelon, snake melon, and musky gourd. 51. A commodity plant product produced from the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of embodiments 21-23, or the composite Cucurbitaceae plant of any one of embodiments 24-27. 52. The commodity plant product of embodiment 51, wherein the plant product is a fruit or vegetable. 53. The commodity plant product of embodiment 52, wherein the plant product is a seed.54. The commodity plant product of embodiment 52 or 53, wherein the fruit, vegetable, or seed has an improved output trait compared to the same variety grown without grafting to a rootstock or grafting to a different rootstock. 55. The commodity plant product of embodiment 54, wherein the fruit, vegetable, or seed output trait is selected from the group consisting of: increased sweetness, increased shelflife, larger fruit size, improved flavor, improved texture, improved color, increased nutrient content, altered nutrient profile, or a combination thereof 56. The commodity plant product of embodiment 55, wherein the fruit, vegetable, or seed, has an increased nutrient content selected from potassium, zinc, copper, syringic acid, protocatechuic acid, catechin, kaempferol, vitamin C, vitamin A, fiber, lycopene, potassium, calcium, protein, magnesium, boron, and combinations thereof. 57. The composite Cucurbitaceae plant of any one of embodiments 24-27 or 44-50, wherein the rootstock comprises at least one allele from the scion variety. 58. A chimeric plant tissue comprising a first plant cell and a second plant cell, wherein the first plant cell is an allopolyploid comprising chromosomes of a first and a second Cucurbitaceae variety and, and wherein the second plant cell is a cultivated Cucurbitaceae variety. 59. A method for producing a composite Cucurbitaceae plant with an improved agronomic trait, comprising: selecting first and second Cucurbitaceae plants having one or more desirable traits; generating an interspecific hybrid plant from said first and second Cucurbitaceae plants; applying a chromosome doubling treatment to the interspecific hybrid plant, or a part thereof, to generate a chimeric interspecific hybrid Cucurbitaceae plant; collecting seed from an allotetraploid fruit of said chimeric interspecific hybrid plant; growing the seed to produce an allotetraploid Cucurbitaceae rootstock plant with one or more desirable traits and, optionally, further propagating said plant, and grafting a scion to the allotetraploid Cucurbitaceae rootstock to produce a composite Cucurbitaceae plant, wherein the scion is a commercial Cucurbitaceae variety, and wherein a fruit or vegetable harvested from the scion has an improved agronomic trait compared to the same variety grown without the allotetraploid Cucurbitaceae rootstock.60. The method of embodiment 59, wherein the first and second Cucurbitaceae plants are sexually compatible and the generating an interspecific hybrid plant is achieved via crossing. 61. The method of embodiment 59, wherein the first and second Cucurbitaceae plants are sexually incompatible and the generating an interspecific hybrid plant is achieved via protoplast fusion. 62. The method of any one of embodiment 59-61, wherein the improved agronomic trait is increased sweetness, increased shelf-life, larger fruit size, improved flavor, improved texture, improved color, increased nutrient content, altered nutrient profile, or a combination thereof. 63. A plant, plant part, or plant cell of a cucurbit variety designated ‘MR 1B2?, wherein seed of said cucurbit variety has also been deposited under NCMA No. XXXXXXXXX. 64. The cucurbit plant part of embodiment 63, wherein the part is selected from the group consisting of a seed, leaf, a flower, a fruit, a stalk, a root, a rootstock, a scion, a meristem, and a cell. 65. The plant part of embodiment 64, wherein the plant part is a rootstock. 66. A tissue culture of regenerable cells produced from the cucurbit plant, plant part, or plant cell of embodiment 63. 67. A cucurbit plant regenerated from the tissue culture of embodiment 66, said plant having all the physiological and morphological characteristics of cucurbit variety designated ‘MR 1B2’ deposited under NCMA No. XXXXXXXXX, when grown under the same environmental conditions. 68. A method for harvesting a cucurbit fruit, the method comprising: (a) growing the cucurbit plant of embodiment 63 to produce a cucurbit fruit, and (b) harvesting said cucurbit fruit. 69. A method for producing a cucurbit seed, the method comprising: (a) crossing a first cucurbit plant with a second cucurbit plant and (b) harvesting the resultant cucurbit seed, wherein said first cucurbit plant and / or second cucurbit plant is the cucurbit plant of embodiment 63. 70. A method of vegetatively propagating cucurbit variety designated ‘MR 1B2', the method comprising: (a) collecting a part capable of being propagated from the plant of embodiment 63 and (b) regenerating a plant from said part. 71. The method of embodiment 70, further comprising (c) harvesting a fruit from said regenerated plant.72. A plant obtained by the method of embodiment 70, wherein said plant has all of the physiological and morphological characteristics of cucurbit designated ‘MR 1B2’ deposited under NCMA No. XXXXXXXXX. 73. A cucurbit fruit produced by the method of embodiment 71. 74. A method of producing a cucurbit plant obtained from cucurbit variety designated ‘MR 1B2’. the method comprising: (a) growing the seed produced by the method of embodiment 69 to obtain a progeny cucurbit plant. 75. The method of embodiment 74, further comprising the steps of:(b) crossing the progeny cucurbit plant obtained from step (a) with itself or a second cucurbit plant to produce a progeny seed of a subsequent generation;(c) growing the progeny seed of the subsequent generation to produce a progeny plant of a subsequent generation; and(d) crossing the progeny plant of a subsequent generation with itself or a second cucurbit plant to produce a cucurbit seed of a further subsequent generation. 76. The method of embodiment 75, further comprising: (e) repeating steps (c) and (d) at least once to produce a cucurbit plant further derived from cucurbit variety designated ‘MR 1B2’. 77. The plant, plant part, or plant cell of embodiment 63, further comprising a single locus conversion and otherwise all of the essential morphological and physiological characteristics of cucurbit variety designated ‘MR 1B2’ deposited under NCMA No. XXXXXXXXX, when grown under the same environmental conditions. 78. The plant, plant part, or plant cell of embodiment 77, wherein the single locus conversion confers said plant with male sterility, male fertility, herbicide resistance, insect resistance, disease resistance, water stress tolerance, heat tolerance, improved standability, enhanced plant vigor, improved shelf life, delayed senescence or controlled ripening, and / or increased nutritional quality. 79. The plant, plant part, or plant cell of embodiment 77, wherein the single locus conversion is an artificially mutated gene or a nucleotide sequence. 80. The plant, plant part, or plant cell of embodiment 77, wherein the single locus conversion is introduced into the plant by a genetic transformation or a gene editing technique with a nuclease selected from the group consisting of Zinc finger nuclease (ZFN), Transcription Activation-Like Effector Nuclease (TALEN), Clustered Regularly Interspaced Short Palindromic Repeats-associated Cas endonuclease (CRISPR-Cas), meganuclease, homing endonuclease, and RNA-guided nuclease.81. A method of producing a composite cucurbit plant, the method comprising: grafting a rootstock or a scion of the cucurbit plant of embodiment 63 to another cucurbit plant. 82. A method for producing nucleic acids, the method comprising: isolating nucleic acids from the plant, plant part or plant cell of embodiment 63. 83. A method of producing a commodity plant product, the method comprising: obtaining the plant, plant part, or plant cell of embodiment 63 and producing said commodity plant product therefrom. 84. A method for producing a hybrid allotetraploid cucurbit plant, comprising: crossing cucurbit variety designated ‘MR 1B2’ with a second allotetraploid cucurbit plant to produce hybrid allotetraploid cucurbit seed; collecting the hybrid allotetraploid cucurbit seed; and growing the hybrid allotetraploid cucurbit seed to produce a hybrid allotetraploid cucurbit plant. 85. A composite plant, wherein the rootstock of the composite plant is cucurbit variety designated ‘MR 1B2’, wherein seed of said cucurbit variety has also been deposited under NCMA No. XXXXXXXXX 86. A plant, plant part, or plant cell of a cucurbit variety' designated ‘MC 24-01’, wherein seed of said cucurbit variety' has also been deposited under NCMANo. XXXXXXXXX. 87. The cucurbit plant part of embodiment 86, wherein the part is selected from the group consisting of a seed, leaf, a flower, a fruit, a stalk, a root, a rootstock, a scion, a meristem, and a cell. 88. The plant part of embodiment 87, wherein the plant part is a rootstock. 89. A tissue culture of regenerable cells produced from the cucurbit plant, plant part, or plant cell of embodiment 86. 90. A cucurbit plant regenerated from the tissue culture of embodiment 89, said plant having all the physiological and morphological characteristics of cucurbit variety designated ‘MC 24-01’ deposited under NCMA No. XXXXXXXXX, when grown under the same environmental conditions. 91. A method for harvesting a cucurbit fruit, the method comprising: (a) growing the cucurbit plant of embodiment 86 to produce a cucurbit fruit, and (b) harvesting said cucurbit fruit. 92. A method for producing a cucurbit seed, the method comprising: (a) crossing a first cucurbit plant with a second cucurbit plant and (b) harvesting the resultant cucurbit seed, wherein said first cucurbit plant and / or second cucurbit plant is the cucurbit plant of embodiment 86.93. A method of vegetatively propagating cucurbit variety designated 'MC 24-01’, the method comprising: (a) collecting a part capable of being propagated from the plant of embodiment 86 and (b) regenerating a plant from said part. 94. The method of embodiment 93, further comprising (c) harvesting a fruit from said regenerated plant. 95. A plant obtained by the method of embodiment 93, wherein said plant has all of the physiological and morphological characteristics of cucurbit designated ‘MC 24-01’ deposited under NCMA No. XXXXXXXXX 96. A cucurbit fruit produced by the method of embodiment 94. 97. A method of producing a cucurbit plant obtained from cucurbit variety designated ‘MC 24-01’. the method comprising: (a) growing the seed produced by the method of embodiment 92 to obtain a progeny cucurbit plant. 98. The method of embodiment 97, further comprising the steps of:(b) crossing the progeny cucurbit plant obtained from step (a) with itself or a second cucurbit plant to produce a progeny seed of a subsequent generation;(c) growing the progeny seed of the subsequent generation to produce a progeny plant of a subsequent generation; and(d) crossing the progeny plant of a subsequent generation with itself or a second cucurbit plant to produce a cucurbit seed of a further subsequent generation. 99. The method of embodiment 98, further comprising: (e) repeating steps (c) and (d) at least once to produce a cucurbit plant further derived from cucurbit variety designated ‘MC 24-01'. 100. The plant, plant part, or plant cell of embodiment 86. further comprising a single locus conversion and otherwise all of the essential morphological and physiological characteristics of cucurbit variety designated ‘MC 24-01 ’ deposited under NCMA No. XXXXXXXXX, when grown under the same environmental conditions. 101. The plant, plant part, or plant cell of embodiment 100, wherein the single locus conversion confers said plant with male sterility, male fertility, herbicide resistance, insect resistance, disease resistance, water stress tolerance, heat tolerance, improved standability, enhanced plant vigor, improved shelf life, delayed senescence or controlled ripening, and / or increased nutritional quality. 102. The plant, plant part, or plant cell of embodiment 100, wherein the single locus conversion is an artificially mutated gene or a nucleotide sequence.103. The plant, plant part, or plant cell of embodiment 100, wherein the single locus conversion is introduced into the plant by a genetic transformation or a gene editing technique with a nuclease selected from the group consisting of Zinc finger nuclease (ZFN), Transcription Activation-Like Effector Nuclease (TALEN), Clustered Regularly Interspaced Short Palindromic Repeats-associated Cas endonuclease (CRISPR-Cas), meganuclease, homing endonuclease, and RNA-guided nuclease. 104. A method of producing a composite cucurbit plant, the method compnsing: grafting a rootstock or a scion of the cucurbit plant of embodiment 86 to another cucurbit plant. 105. A method for producing nucleic acids, the method comprising: isolating nucleic acids from the plant, plant part or plant cell of embodiment 86. 106. A method of producing a commodity plant product, the method comprising: obtaining the plant, plant part, or plant cell of embodiment 86 and producing said commodity plant product therefrom. 107. A method for producing a hybrid allotetraploid cucurbit plant, comprising: crossing cucurbit variety designated MC 24-01’ with a second allotetraploid cucurbit plant to produce hybrid allotetraploid cucurbit seed; collecting the hybrid allotetraploid cucurbit seed; and growing the hybrid allotetraploid cucurbit seed to produce a hybrid allotetraploid cucurbit plant. 108. A composite plant, wherein the rootstock of the composite plant is cucurbit variety designated ‘MC 24-01 ’, wherein seed of said cucurbit variety has also been deposited under NCMA No. XXXXXXXXX. 109. A plant, plant part, or plant cell of a cucurbit variety designated ‘MC 24-02’, wherein seed of said cucurbit variety has also been deposited under NCMA No. XXXXXXXXX. 110. The cucurbit plant part of embodiment 109, wherein the part is selected from the group consisting of a seed, leaf, a flower, a fruit, a stalk, a root, a rootstock, a scion, a meristem, and a cell. 111. The plant part of embodiment 110. wherein the plant part is a rootstock. 112. A tissue culture of regenerable cells produced from the cucurbit plant, plant part, or plant cell of embodiment 109. 113. A cucurbit plant regenerated from the tissue culture of embodiment 112, said plant having all the physiological and morphological characteristics of cucurbit variety designated ‘MC 24-02’ deposited under NCMA No. XXXXXXXXX, when grown under the same environmental conditions.114. A method for harvesting a cucurbit fruit, the method comprising: (a) growing the cucurbit plant of embodiment 109 to produce a cucurbit fruit, and (b) harvesting said cucurbit fruit. 115. A method for producing a cucurbit seed, the method comprising: (a) crossing a first cucurbit plant with a second cucurbit plant and (b) harvesting the resultant cucurbit seed, wherein said first cucurbit plant and / or second cucurbit plant is the cucurbit plant of embodiment 109. 116. A method of vegetatively propagating cucurbit variety designated MC 24-02’, the method comprising: (a) collecting a part capable of being propagated from the plant of embodiment 109 and (b) regenerating a plant from said part. 117. The method of embodiment 93, further comprising (c) harvesting a fruit from said regenerated plant. 118. A plant obtained by the method of embodiment 116, wherein said plant has all of the physiological and morphological characteristics of cucurbit designated ‘MC 24-02’ deposited under NCMA No. XXXXXXXXX. 119. A cucurbit fruit produced by the method of embodiment 117. 120. A method of producing a cucurbit plant obtained from cucurbit variety' designated ‘MC 24-02’, the method comprising: (a) growing the seed produced by the method of embodiment 115 to obtain a progeny cucurbit plant. 121. The method of embodiment 120. further comprising the steps of:(b) crossing the progeny cucurbit plant obtained from step (a) with itself or a second cucurbit plant to produce a progeny seed of a subsequent generation;(c) growing the progeny seed of the subsequent generation to produce a progeny plant of a subsequent generation; and(d) crossing the progeny plant of a subsequent generation with itself or a second cucurbit plant to produce a cucurbit seed of a further subsequent generation. 122. The method of embodiment 121, further comprising: (e) repeating steps (c) and (d) at least once to produce a cucurbit plant further derived from cucurbit variety designated ‘MC 24-02’. 123. The plant, plant part, or plant cell of embodiment 109, further comprising a single locus conversion and otherwise all of the essential morphological and physiological characteristics of cucurbit variety designated ‘MC 24-02’ deposited under NCMA No. XXXXXXXXX, when grown under the same environmental conditions.124. The plant, plant part, or plant cell of embodiment 123, wherein the single locus conversion confers said plant with male sterility, male fertility, herbicide resistance, insect resistance, disease resistance, water stress tolerance, heat tolerance, improved standability, enhanced plant vigor, improved shelf life, delayed senescence or controlled ripening, and / or increased nutritional quality . 125. The plant, plant part, or plant cell of embodiment 123, wherein the single locus conversion is an artificially mutated gene or a nucleotide sequence. 126. The plant, plant part, or plant cell of embodiment 123, wherein the single locus conversion is introduced into the plant by a genetic transformation or a gene editing technique with a nuclease selected from the group consisting of Zinc finger nuclease (ZFN), Transcription Activation-Like Effector Nuclease (TALEN). Clustered Regularly Interspaced Short Palindromic Repeats-associated Cas endonuclease (CRISPR-Cas), meganuclease, homing endonuclease, and RNA-guided nuclease. 127. A method of producing a composite cucurbit plant, the method comprising: grafting a rootstock or a scion of the cucurbit plant of embodiment 109 to another cucurbit plant. 128. A method for producing nucleic acids, the method comprising: isolating nucleic acids from the plant, plant part or plant cell of embodiment 109. 129. A method of producing a commodity plant product, the method comprising: obtaining the plant, plant part, or plant cell of embodiment 109 and producing said commodity plant product therefrom. 130. A method for producing a hybrid allotetraploid cucurbit plant, comprising: crossing cucurbit variety designated ‘MC 24-02’ with a second allotetraploid cucurbit plant to produce hybrid allotetraploid cucurbit seed; collecting the hybrid allotetraploid cucurbit seed; and growing the hybrid allotetraploid cucurbit seed to produce a hybrid allotetraploid cucurbit plant. 131. A composite plant, wherein the rootstock of the composite plant is cucurbit variety designated ‘MC 24-02’, wherein seed of said cucurbit variety has also been deposited under NCMANo. XXXXXXXXX.

Claims

CLAIMSWhat is claimed is:

1. A method for producing a composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress, comprising:(i) selecting a first Cucurbitaceae variety which is stress-tolerant against at least one abiotic or biotic stress;(ii) crossing said first Cucurbitaceae variety with a second Cucurbitaceae variety of a different species sexually compatible with the first Cucurbitaceae variety to produce an interspecific hybrid seed;(iii) growing the interspecific hybrid seed to produce an interspecific hybrid Cucurbitaceae plant;(iv) applying a chromosome doubling treatment to the interspecific hybrid plant, or a part thereof, to generate a chimeric interspecific hybrid Cucurbitaceae plant;(v) collecting seed from an allotetraploid fruit of said chimeric interspecific hybrid plant;(vi) growing the seed to produce an allotetraploid Cucurbitaceae rootstock plant with tolerance against at least one abiotic or biotic stress and, optionally, further propagating said plant; and(vii) grafting a scion of a cultivated Cucurbitaceae variety to the allotetraploid Cucurbitaceae rootstock to produce a composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress.

2. A method for producing a composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress, comprising:(i) selecting a first Cucurbitaceae variety which is stress-tolerant against at least one abiotic or biotic stress;(ii) fusing a protoplast isolated from said first Cucurbitaceae variety with another protoplast isolated from a Cucurbitaceae variety sexually incompatible with the first Cucurbitaceae variety;(iii) selecting a heterokaryon;(iv) regenerating an allotetraploid Cucurbitaceae rootstock plant with tolerance against at least one abiotic or biotic stress from the heterokaryon; and, optionally, further propagating said plant; and(v) grafting a scion of a cultivated Cucurbitaceae variety to the allotetraploid Cucurbitaceae rootstock to produce a composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress.

3. A method for producing a stress-tolerant hybrid allopolyploid Cucurbitaceae plant or seed, comprising:(i) selecting a first Cucurbitaceae variety which is stress-tolerant against at least one abiotic or biotic stress;(ii) crossing said first Cucurbitaceae variety with a second Cucurbitaceae variety of a different species sexually compatible with the first Cucurbitaceae variety to produce an interspecific hybrid seed;(iii) growing the interspecific hybrid seed to produce an interspecific hybrid Cucurbitaceae plant;(iv) applying a chromosome doubling treatment to the interspecific hybrid plant, or a part thereof, to generate a chimeric interspecific hybrid Cucurbitaceae plant;(v) collecting seed from an allotetraploid fruit of said chimeric interspecific hybrid plant;(vi) growing the seed to produce a first allotetraploid Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress and, optionally, further propagating said plant;(vii) crossing the first allotetraploid Cucurbitaceae plant with a second allotetraploid Cucurbitaceae plant to produce hybrid allopolyploid seed;(viii) harvesting the hybrid allopolyploid seed; and(ix) optionally growing the hybrid allopolyploid seed to produce a stress-tolerant hybrid allopolyploid Cucurbitaceae plant.

4. A method for producing a stress-tolerant hybrid allopolyploid Cucurbitaceae plant, comprising:(i) selecting a first Cucurbitaceae variety which is stress-tolerant against at least one abiotic or biotic stress;(ii) fusing a protoplast isolated from said first Cucurbitaceae variety with another protoplast isolated from a second Cucurbitaceae variety sexually incompatible with the first Cucurbitaceae variety to produce a heterokaryon;(iii) regenerating a first allotetraploid Cucurbitaceae plant from the heterokaryon;(iv) fusing a protoplast isolated from the first allotetraploid Cucurbitaceae plant with another protoplast isolated from a second allotetraploid Cucurbitaceae plant to a produce hybrid allopolyploid heterokaryon; and(v) regenerating a hybrid allopolyploid Cucurbitaceae plant from the hybrid allopolyploid heterokaryon to produce a stress-tolerant hybrid allopolyploid Cucurbitaceae plant.

5. A method for producing a composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress, comprising:(i) providing a hybrid allopolyploid Cucurbitaceae plant produced by the method of claim 3 or 4 as a rootstock; and(ii) grafting a scion of a cultivated Cucurbitaceae variety to the allotetraploid Cucurbitaceae rootstock to produce a composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress.

6. The method of claim 1 or 3, wherein the part thereof is a vegetative cutting.

7. The method of claim 3 or 4, wherein the second allotetraploid Cucurbitaceae plant exhibits at least one tolerance against at least one abiotic or biotic stress which is not present in the first allopolyploid Cucurbitaceae plant.

8. The method of claim 1, 2, or 5, wherein the cultivated Cucurbitaceae is an inbred or essentially homozygous.

9. The method of claim 1, 2, or 5, wherein the cultivated Cucurbitaceae is a hybrid.

10. The method of any one of claims 1 -5, wherein the first Cucurbitaceae variety is a wild variety.

11. The method of any one of claims 1 -5, wherein the first or second Cucurbitaceae variety is a landrace variety.

12. The method of any one of claims 1-5, wherein the first Cucurbitaceae is a wild variety and the second Cucurbitaceae variety is a cultivated variety.

13. The method of any one of claims 1-5, wherein the first Cucurbitaceae is a landrace variety and the second Cucurbitaceae variety is a cultivated variety.

14. The method of any one of claims 1-5, wherein the first or second Cucurbitaceae variety is an F1hybrid.

15. The method of claim 1, 2 or 5, wherein the scion is a commercial Cucurbitaceae variety.

16. The method of any one of claims 1-5, wherein the first or second Cucurbitaceae variety has an abiotic stress tolerance selected from the group consisting of cold tolerance, high temperature tolerance, drought tolerance, and salt tolerance.

17. The method of any one of claims 1-5, wherein the first or second Cucurbitaceae variety has a biotic stress tolerance selected from the group consisting of a disease resistance, a pest resistance, a bacterial resistance, a fungal resistance, an insect resistance, and a nematode resistance.

18. The method of any one of claims 1-5, wherein the first Cucurbitaceae variety has a different biotic or abiotic stress tolerance than the second Cucurbitaceae variety.

19. The method of any one of claims 1. 2 or 5, wherein the cultivated Cucurbitaceae variety used as scion is less tolerant against at least one biotic or abiotic stress in comparison to the allotetraploid Cucurbitaceae rootstock.

20. The method of claim 2 or 4, wherein the protoplast fusion is asymmetrical and mitochondria are only provided by a cultivated Cucurbitaceae variety to generate the first allotetraploid plant.

21. A hybrid allopolyploid Cucurbitaceae plant or plant part with tolerance against at least one abiotic or biotic stress, comprising:(i) at least one chromosome from each of a first and a second Cucurbitaceae variety, wherein the second Cucurbitaceae variety exhibits at least one tolerance against at least one abiotic or biotic stress which is not present in said first Cucurbitaceae variety; and(ii) at least one chromosome for a cultivated Cucurbitaceae variety of a species different from said first and second Cucurbitaceae varieties.

22. The hybrid allopolyploid Cucurbitaceae plant of claim 21 , wherein said plant is produced by the method of claim 3 or 4.

23. The hybrid allopolyploid Cucurbitaceae plant part of claim 21, wherein said plant part is a seed.

24. A composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress, said composite Cucurbitaceae plant comprising:(i) as a rootstock the hybrid allopoly ploid Cucurbitaceae plant of any one of claims 21-23, and(ii) as a scion a cultivated Cucurbitaceae variety.

25. A composite Cucurbitaceae plant with tolerance against at least one abiotic or biotic stress, said composite Cucurbitaceae plant comprising:(i) as a rootstock an allotetraploid Cucurbitaceae plant comprisingi. at least one chromosome from a wild or landrace Cucurbitaceae variety which exhibits at least one tolerance against at least one abiotic or biotic stress; and ii. at least one chromosome from a cultivated Cucurbitaceae variety(ii) as a scion a cultivated Cucurbitaceae variety.

26. The composite Cucurbitaceae plant of claim 25, wherein said composite Cucurbitaceae plant is produced by the method of claim 1 or 2.

27. The composite Cucurbitaceae plant of any of claim 24-26, wherein said composite Cucurbitaceae plant has a higher tolerance against at least one biotic or abiotic stress in comparison to the cultivated Cucurbitaceae variety used a scion when grown under the same conditions without the allotetraploid rootstock.

28. The method of any one of claims 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of claims 21-23, or the composite Cucurbitaceae plant of any one of claims 24-27, wherein the first and / or second Cucurbitaceae variety are species from the Cucumis genus.

29. The method of any one of claims 1-20, the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of claims 21-23, or the composite Cucurbitaceae plant of any one of claims 24-27, wherein the first and / or second Cucurbitaceae variety are species from the Cucurbila genus.

30. The method of any one of claims 1-20. the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of claims 21-23, or the composite Cucurbitaceae plant of any one of claims 24-27, wherein the first and / or second Cucurbitaceae variety' are species from the Citrullus genus.

31. The method of any one of claims 1-20. the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of claims 21-23, or the composite Cucurbitaceae plant of any one of claims 24-27, wherein the first variety is Cucurbita maxima and the second variety is C. moschata.

32. The method of any one of claims 1-20. the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of claims 21-23, or the composite Cucurbitaceae plant of any one of claims 24-27, wherein the first variety is Cucumis melo and the second variety is Cucumis metuliferus .

33. A commodity' plant product produced from the hybrid allopolyploid Cucurbitaceae plant or plant part of any one of claims 21-23, or the composite Cucurbitaceae plant of any one of claims 24-27.

34. The commodity plant product of claim 33, wherein the plant product is a fruit or vegetable.

35. The fruit or vegetable of claim 34, wherein the fruit or vegetable has an improved output trait compared to the same variety grown without grafting to a rootstock.

36. The fruit or vegetable of claim 35, wherein the output trait is selected from the group consisting of: increased sweetness, increased shelf-life, larger fruit size, improved flavor, improved texture, improved color, increased nutrient content, altered nutrient profile, and combinations thereof37. The fruit or vegetable of claim 36, wherein the nutrient selected from the group consisting of potassium, zinc, copper, syringic acid, protocatechuic acid, catechin, kaempferol, vitamin C. vitamin A, fiber, lycopene, potassium, calcium, protein, magnesium, boron, and combinations thereof.

38. A method for producing a composite Cucurbitaceae plant with an improved agronomic trait, comprising: selecting first and second Cucurbitaceae plants having one or more desirable traits; generating an interspecific hybrid plant from said first and second Cucurbitaceae plants; applying a chromosome doubling treatment to the interspecific hybrid plant, or a part thereof, to generate a chimeric interspecific hybrid Cucurbitaceae plant; collecting seed from an allotetraploid fruit of said chimeric interspecific hybrid plant; growing the seed to produce an allotetraploid Cucurbitaceae rootstock plant with one or more desirable traits and, optionally, further propagating said plant, and grafting a scion to the allotetraploid Cucurbitaceae rootstock to produce a composite Cucurbitaceae plant, wherein the scion is a commercial Cucurbitaceae variety , and wherein a fruit or vegetable harvested from the scion has an improved agronomic trait compared to the same variety grown without the allotetraploid Cucurbitaceae rootstock.

39. The method of claim 38, wherein the first and second Cucurbitaceae plants are sexually compatible and the generating an interspecific hybrid plant is achieved via crossing.

40. The method of claim 38, wherein the first and second Cucurbitaceae plants are sexually incompatible and the generating an interspecific hybrid plant is achieved via protoplast fusion.

41. The method of claim 38, wherein the improved agronomic trait is increased sweetness, increased shelf-life, larger fruit size, improved flavor, improved texture, improved color, increased nutrient content, altered nutrient profile, or a combination thereof.

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