# Patent

## Title
Melon plants

## Abstract

The present invention relates to novel plants, in particular to melon plants capable of producing fruits with a new pleasant taste and to seeds thereof. The present invention further relates to fruits of melon plants of the present invention, wherein such fruits have organic acid content, low pH and high sugar contents. The present invention further relates to methods of making and using plants and fruits disclosed herein.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 11/031,578, filed Jan. 7, 2005, which claims the benefit of U.S. Provisional Application No. 60/535,631, filed Jan. 9, 2004. The above applications are incorporated herein by reference in their entireties.


FIELD OF THE INVENTION
The present invention relates to novel plants, in particular to melon plants capable of producing fruits with a new pleasant taste. In particular, the fruits of the melon plants of the present invention have altered organic acid contents, lower pH when compared to current commercial sweet melon fruits, and high sugar contents.
BACKGROUND OF THE INVENTION
Melon (Cucumis melo L.) is a commercial crop grown worldwide. Cucumis melo is a member of the family Cucurbitaceae. The Cucurbitaceae comprises about 90 genera and 700 to 760 species, mostly of the tropics. The family includes pumpkins, squashes, gourds, watermelon, loofah and several weeds. Cucumis melo L. includes a very wide variety of cultivars producing fruits of different shape, external appearance and flesh color. Commercial melons generally produce sweet fruits known for example as Charentais, cantaloupe, honeydew, amarello, Piel de sapo, Kirkagak, Hamy, Ananas, Galia, Oriental that are usually consumed as dessert fruits. Cucumis melo L. also includes non-sweet, commercial cultivars consumed in the Middle to Far East in salad, cooking or pickling, as for example Alficoz, Faqqous, Chito, Conomon (Pitrat et al (2000) Eucarpia meeting Proceedings: 29-36). The taste and aroma of melon fruits is determined by a number of factors, including sugars, aroma volatiles, free amino acids, organic acids, pH and soluble minerals (Wang et al. (1996) J. Agric. Food Chem. 44: 210-216). Among the four primary tastes (sweet, sour, bitter, salty), sweetness is considered to be a very important component of good tasting melon fruits. In commercial melon fruits the sweet taste mostly results from high levels of sucrose (Burger et al. (2002) J. Amer. Soc. Hort. Sci. 127(6): 938-943). Sucrose is accumulated at the end of fruit development, during ripening process (Shaffer et al (1987) Phytochemistry 26: 1883-1887). Melon fruits also initially accumulate hexoses, mainly fructose and glucose, which are the dominant reducing sugars (Stepanski et al (1999) Genetic Resources and Crop Evolution 46: 53-62). An important taste component in melon fruits is sweetness, which is mainly the result of sugars accumulation. Sweetness correlates not only with the total sugars content, but also with the type of sugars. For example, 1 gram of glucose is the sweet equivalent of 0.7 gram of sucrose; 1 gram of fructose is the sweet equivalent of 1.7 gram of sucrose; 1 gram of inverted sugar, i.e. glucose plus fructose generated from 1 gram of sucrose, is the sweet equivalent of 1.3 gram of sucrose (J. A. BABOR et J. IBARZ (1935) Quimica General Moderna).
The flesh of sweet melon fruits has a pH usually above 6.0, but melon accessions are also known to have a much lower pH, as low as below 5.0. This low pH is widespread over many different melon types as for example Faqqous, Chito, Conomon, Momordica, Agrestis (Stepanski et al). In most of these cases, these melon types combine low pH and low sugar content, e.g. sucrose (Stepanski et al). These fruits are generally not edible in fresh consumption without dressing or cooking and, in some cases, they are even bitter. In most of these melon accessions with low pH, the mesocarp, which is the edible part of the fruit, represents a minor part of the total fruit, while the seed cavity and placenta represent a major part of the total fruit fresh weight. This is in contrast to sweet dessert melons, where the mesocarp represents a major part of the fruit. Also, in many cases, the fruit size or weight of the melon having low pH is below commercially acceptable ranges.
The fruit flesh of some melons has a sour taste (Kubicki (1962) Genetica Polonica 3:265-274). The cause for the sour taste remains unclear, but it has been linked with low pH in the fruit flesh (U.S. Pat. No. 5,476,998 and Danin-Poleg et al. (2002) Euphytica 125: 373-384). Single genes for sour taste (So) and pH have also been reported, although their genetic association is not clear (Danin-Poleg et al.).
Attempts have been made to produce melon fruits combining sour and sweet tastes. For example, Najd melons based on Arabic wild varieties have been reported (Ibrahim and Al-Zeir (1992) HortScience 27: 276-277). U.S. Pat. No. 5,476,998 also described melons with a sour taste, with a mean pH value of 4.8 and a total soluble content of about 11. The melons in U.S. Pat. No. 5,476,998 are derived from MR-1, also known as PI124111 (Thomas, Eucarpia '92 pp 142), which is a C. melo Var. Momordica (Roxburg). Fruits of this melon showed low pH (Danin-Poleg et al.). This melon variety also involves poor fruit traits such as very mealy, non-aromatic and non-sweet flesh, very climacteric behavior and thin skin bursting at ripening. These undesired traits are difficult to remove by breeding and it is therefore expected that the development of commercial products from such a variety would be lengthy and difficult. Accordingly, there is an unmet need for melons producing fruits with alternative or improved tastes. In particular, there is an unmet need for melon fruits having new combinations of organoleptic characteristics and aroma.
SUMMARY OF THE INVENTION
The instant invention addresses the need for melon fruits with alternative or improved tastes. Accordingly, the instant invention discloses melon plants capable of producing fruits with novel combinations of organic acid contents and compositions, pH, and sugars contents and compositions. The invention also discloses methods of making and methods of using plants of the present invention and their fruits.
The inventors of the instant application have identified that there is a wide variability in the contents and composition of organic acids in melon fruits. In particular, the inventors of the present invention have identified that melon plants produce fruits with varying contents of citric acid and varying ratios of citric acid to malic acid. Accordingly, the inventors of the present invention have combined appropriate content of organic acids, low pH and desired sugar contents in a melon fruit to obtain novel and extremely pleasant tastes.
Accordingly, in one embodiment, the instant invention discloses melon plants capable of producing fruit with low pH and desired combinations of citric and malic acid contents, while maintaining or increasing levels of sugars currently observed in sweet melons. In one embodiment, the instant invention discloses melon plants capable of producing fruit with low pH and desired combinations of citric and malic acid contents, combined with desired combinations of sucrose and hexoses contents. In one embodiment, the present invention provides melon plants capable of producing fruits with elevated contents of citric acid and lower pH, when compared to current commercial melons. In one embodiment, melon plants of the present invention are obtained by introducing a low pH trait in a melon plant not comprising said trait. In one embodiment, melon plants of the present invention are obtained by introducing a low pH gene in a melon plant not comprising said gene. In one embodiment, the low pH gene is obtainable from line IND-35, deposited with NCIMB under Accession number NCIMB 41202. Accordingly, the present invention discloses melon plants comprising a low pH trait, and producing fruit comprising desired contents and compositions of organic acids, pH and contents and compositions of sugars. In one embodiment, increased citric acid contents are achieved while maintaining low levels of malic acid in the fruit. In one embodiment, fruits of melon plant of the instant invention have high ratios of citric acid to malic acid.
In one embodiment, the melon plants of the instant invention are capable of producing fruit with citric acid content equal or higher than about 400 mg per 100 g fresh weight (fwt). In one embodiment, melon plants of the instant invention are capable of producing fruit with a pH of about 4.2 to about 5.6. In one embodiment, melon plants of the instant invention are capable of producing fruit with a sugar content equal or higher than about 5.0 g per 100 g fwt. In one embodiment, melon plants of the instant invention are capable of producing fruit with a sugar content equal or higher than about 7.0 g per 100 g fwt.
In one embodiment, the present invention discloses melon plants capable of producing fruit with citric acid content equal or higher than about 400 mg per 100 g fwt, pH of about 4.2 to about 5.6, and sugar content equal or higher than about 5.0 g per 100 g fwt.
In one embodiment, the present invention discloses a C. melo plant capable of producing a fruit comprising at maturity:

    
    
        a) about 400 mg to about 1,200 mg citric acid per 100 g fwt;
        b) pH of about 4.2 to about 5.6; and
        c) about 5.0 g to about 15.0 g sugar per 100 g fwt.
    
    


In one embodiment, the citric acid content of a fruit of a plant according to the present invention is about 400 mg to about 1,000 mg citric acid per 100 g fwt. In one embodiment, melon plants of the present invention produce edible fruits, preferably with a round or oval shape, and preferably weighting over 450 grams. The flesh of the melons of the present invention is preferably green, yellow, white or orange.
In one embodiment, the instant invention discloses melon plants capable of producing a very sweet juicy fruit with tart-refreshing sour taste, and referred herein to as “Citric+” plants or fruits. Such fruits comprise high contents of citric acid and low pH, increasing the acid taste and providing a tart perception, which covers still too flat and poor flavors of early mature fruits.
In one embodiment, a fruit of a Citric+plant of the present invention at maturity comprises:

    
    
        a) about 600 to about 1,200 mg citric acid per 100 g fwt;
        b) pH of about 4.2 to about 5.1; and
        c) about 5.0 g to about 15.0 g sugar per 100 g fwt.
    
    


In one embodiment, the citric acid content of a fruit of a plant according to the present invention is about 600 mg to about 1,000 mg citric acid per 100 g fwt.
In one embodiment, the present invention discloses a melon plant capable of producing a sweet aromatic fruit with mild-fruity sour taste, referred herein as “Citric-” melon plant or fruit. Such fruits comprise relatively high contents of citric acid and mildly low pH. This mild low pH makes a fruity sour perception, which covers other still too flat and poor flavors of early mature fruits.
In one embodiment, the fruit of a Citric-plant of the present invention at maturity comprises:

    
    
        a) about 400 to about 650 mg citric acid per 100 g fwt;
        b) pH of about 4.6 to about 5.6; and
        c) about 5.0 g to about 15.0 g sugar per 100 g fwt.
    
    


In one embodiment, the present invention discloses a melon plant capable of producing fruit with low pH and a flesh of deep orange color. In one embodiment, the present invention discloses a C. melo plant capable of producing fruit with pH of about 4.5 to about 5.6, wherein said fruit has orange flesh rated 4 or higher.
In one embodiment, characteristics of melon fruits described herein are measured on fruits of melon plants grown in open fields or in plastic houses, and harvested at maturity. In one embodiment fruits are harvested from early maturity to late maturity (stages 2-4, as described herein). In one embodiment, a fruit of the present invention is at maturity when its sucrose contents is at or over 2 g sucrose per 100 g fwt.
In one embodiment, the present invention discloses a C. melo plant capable of producing fruit comprising an acid savor of about 1.6 to about 3.8 and a sugar savor of about 4.3 to about 5.8. In one embodiment, the present invention discloses a C. melo plant capable of producing fruit comprising an acid savor of about 2.5 to about 3.8 and a sugar savor of about 4.3 to about 5.6. In one embodiment, the present invention discloses a C. melo plant capable of producing fruit comprising an acid savor of about 1.6 to about 3.0 and a sugar savor of about 5.2 to about 5.8. In one embodiment, the acid savor and sugar savor are determined by an Expert panel, for example as described in Example 12 herein. In one embodiment, such fruit comprises a pH and citric acid content as described herein. In one embodiment, such fruit comprises a pH, organic acid contents and compositions and sugar contents and compositions as described herein.
In one embodiment, the present invention discloses a C. melo plant comprising a DNA sequence, which co-segregates with a low pH trait. In one embodiment, the DNA sequence is a template for amplification of a DNA fragment described herein using the primers described herein. In one embodiment, the present invention discloses such primers and DNA fragments amplified using these primers. In one embodiment, a DNA fragment described herein is amplified from DNA of said plant using the primers described herein. The DNA fragments are used as molecular markers for a low pH trait. In one embodiment, a DNA fragment of about 168 bp to about 178 bp is amplified from the DNA of said plant when the primers capable of identifying the CMAT141 marker are used. In one embodiment, a DNA fragment of 168 bp, 173 bp, 169 bp, 172 bp or 178 bp is amplified when the primers capable of identifying the CMAT141 marker are used. In one embodiment, a DNA fragment of less than 176 bp is amplified when the primers capable of identifying the CMAT141 marker are used. In one embodiment, a DNA fragment of less than 175 bp is amplified when the primers capable of identifying the CMAT141 marker are used. In one embodiment, a DNA fragment of about 218 bp to about 253 bp is amplified when the primers capable of identifying the NE0585 marker are used. In one embodiment, a DNA fragment of 230 bp, 232 bp, 218 bp, 229 bp, 234 bp or 239 bp is amplified when the primers capable of identifying the NE0585 marker are used. In one embodiment, a DNA fragment of about 121 bp to about 145 bp is amplified when the primers capable of identifying the NE1746 marker are used. In one embodiment, a DNA fragment of 124 bp, 127 bp, 133 bp, 142 bp or 145 bp is amplified when the primers capable of identifying the NE1746 marker are used.
In one embodiment, the present invention discloses a C. melo plant comprising a DNA sequence, which is a template for amplification of a DNA fragment indicative for the presence of a low pH trait in said plant (acid fragment) or for the absence of the low pH trait (basic fragment) in said plant. In one embodiment, the present invention discloses a C. melo plant comprising a DNA sequence, which is a template for amplification of a basic fragment linked to a low pH trait, wherein said DNA sequence is linked to said low pH trait. In one embodiment, the C. melo plant comprises such a DNA sequence on one side of a low pH gene. In one embodiment, the C. melo plant comprises such a DNA sequence on both sides of a low pH gene. In one embodiment, a fruit of such a plant comprise a pH within the ranges disclosed herein. In one embodiment, fruits of such a plant comprise the contents and compositions of sugars as described herein. In one embodiment, fruits of such a plant comprise the contents and compositions of organic acids as described herein. In one embodiment, fruits of such a plant comprise the pH, contents and compositions of sugars and contents and compositions of organic acids as described herein.
In one embodiment, the characteristics of a fruit according to the present invention described herein remain stable after the fruit reaches maturity. In one embodiment, such characteristics remain stable after a fruit reaches maturity when the fruit is kept on the plant. In one embodiment, such characteristics remain stable when the fruit is harvested and kept in storage after harvest. This allows for a reduced harvest frequency, and to store or ship a fruit of the present invention without loosing its organoleptic characteristics and aroma. In one embodiment, the pH of a fruit of the instant invention remains stable after a fruit reaches maturity. In one embodiment, the citric acid content of a fruit of the present invention remains stable after a fruit reaches maturity. In one embodiment, the malic acid content of a fruit of the present invention remains stable after a fruit reaches maturity. In one embodiment, the ratio citric acid to malic acid of a fruit of the present invention remains stable after a fruit reaches maturity. In one embodiment, the pH and organic acid contents and composition of a fruit of the present invention remain stable after a fruit reaches maturity. In one embodiment, such characteristics remain within the ranges described herein after a fruit reaches maturity. In one embodiment, the present invention discloses a plant capable of producing a fruit, the characteristics of which remain stable for at least 2 days when the fruit is kept on the plant, in one embodiment for at least 3 days when the fruit is kept on the plant, in one embodiment for at least 4 days when the fruit is kept on the plant. In one embodiment, the present invention discloses a plant capable of producing a fruit, the characteristics of which remain stable for at least 5 days when kept in storage at 20° C., in one embodiment for at least 7 days when kept in storage at 20° C., in one embodiment for at least 9 days when kept in storage at 20° C. In one embodiment, the present invention discloses a plant capable of producing a fruit, the characteristics of which remain stable for at least 7 days when kept in storage at 8-12° C. followed by at least 2 days at 20° C., in one embodiment for at least 12 days when kept in storage at 8-12° C. followed by at least 2 days at 20° C., in one embodiment for at least 26 days when kept in storage at 8-12° C. followed by at least 2 days at 20° C. In one embodiment, a plant of the instant invention is capable of producing a long shelf-life fruit (LSL) or a medium shelf-life fruit (MSL). In one embodiment, a plant of the instant invention is capable of producing a non-turning fruit or low turning fruit. In one embodiment, a plant of the instant invention is capable of producing a non-climacteric fruit or a low climacteric fruit.
The instant invention further discloses seeds of a C. melo plant of the present invention, and seeds of the progeny thereof, wherein said progeny seed is capable of producing a plant of the present invention. The instant invention further discloses parts of a C. melo plant of the present invention, e.g. ovules or pollen, and fruits of a C. melo plant of the present invention. The instant invention further discloses the flesh of a fruit of a C. melo plant of the present invention. The instant invention further discloses the juice of a fruit of a C. melo plant of the present invention.
The present invention further discloses the use of the flesh of a fruit according to the present invention in a fresh cut product. The present invention further discloses the use of the juice of a fruit according to the present invention in a soft drink.
The present invention further discloses methods of increasing the citric acid content of a plant comprising obtaining a first C. melo plant; crossing said first C. melo plant with a second C. melo plant comprising a low pH trait, obtaining a progeny C. melo plant, determining the pH and citric acid content of a fruit of said progeny plant, selecting a fruit of said progeny C. melo plant which has increased citric acid content, when compared to a fruit of said first C. melo plant. In one embodiment, said progeny C. melo plant has a lower pH, when compared to a fruit of said first C. melo plant. In one embodiment, the method comprises detecting a DNA fragment described herein using the primers described herein. The present invention further discloses the use of a melon plant comprising a low pH trait to obtain a melon plant of the present invention. In one embodiment, the melon plant comprising a low pH trait further has the ability of accumulating relevant levels of sugars, for example sucrose. In one embodiment, the melon plant comprising a low pH accumulates high levels of citric acid. In one embodiment, the melon plant comprising a low pH accumulates low levels of malic acid. In one embodiment, the melon plant comprising a low pH trait further has the ability of accumulating relevant levels of sugars, for example sucrose and of accumulating high levels of citric acid and of accumulating low levels of malic acid. In one embodiment, the melon plant comprising a low pH is a plant of line IND-35 or a descendent thereof.
The present invention further discloses a method to produce seed of a plant according to the instant invention comprising obtaining a plant of the present invention, self-pollinating said plant or crossing said plant with another melon plant, and harvesting progeny seed. The present invention further discloses a method to vegetatively propagate a melon plant according to the present invention. The present invention further discloses a method for producing a fruit comprising planting a plant according to the present invention, growing said plant and harvesting a fruit, wherein said fruit comprises the characteristics described herein. The method further comprises storing said fruit, for example as described herein. The method further comprises shipping said fruit. In one embodiment, the characteristics of said fruit described herein remain stable during the storage of said fruit. In one embodiment, the characteristics of said fruit described herein remain stable during the storage of said fruit. In one embodiment, a plant of the present invention is an inbred line, a hybrid, a dihaploid, or a vegetatively propagated clone.
The present invention thus provides melon fruits offering a pleasant acid component but avoiding an astringent taste. This enhances or complements melon flavors to their maximum potential. These combinations of organic acid content and low pH with high sugars provide new ranges and classes of appealing tastes for fresh consumption or the fresh cut or fresh juice industry. Low pH in fruit flesh also prevents bacterial contamination in fresh cut and juice industrial processes.
DEFINITIONS
Trait: characteristic or phenotype. For example, in the context of the present invention a low pH trait confers a low pH, for example from about 4.2 to about 5.6, to the flesh of a melon fruit. A trait may be inherited in a dominant or recessive manner, or in a partial or incomplete-dominant manner. A trait may be monogenic or polygenic, or may also result from the interaction of one or more genes with the environment.
Monogenic: determined by a single locus.
Polygenic: determined by more than one locus.
Dominant: results in a complete phenotypic manifestation at heterozygous or homozygous state.
Recessive: manifests itself only when present at homozygous state.
Partial or incomplete-dominance: when present at the heterozygous stage determines a phenotype that is intermediate to that of the homozygous stage or when the trait is absent.
Backcrossing: backcrossing is a process in which a hybrid progeny is repeatedly crossed back to one of the parents.
Locus: region on a chromosome, which comprises a gene contributing to a trait.
Genetic linkage: association of characters in inheritance due to location of genes in proximity on the same chromosome. Measured by percent recombination between loci (centi-Morgan, cM).
Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer to genetic loci that control to some degree numerically representable traits that are usually continuously distributed.
Isogenic: plants, which are genetically identical, except that they may differ by the presence or absence of a gene, a locus conferring a trait or heterologous DNA sequence.
Marker assisted selection: refers to the process of selecting a desired trait or desired traits in a plant or plants by detecting one or more nucleic acids from the plant, where the nucleic acid is associated with the desired trait.
Dihaploid: doubling of haploid (single chromosome) status of the genome (e.g. through another culture or microspore culture) giving a complete homozygous plant.
“Tester” plant: plant used to characterize genetically a trait in a plant to be tested. Typically, the plant to be tested is crossed with a “tester” plant and the segregation ratio of the trait in the progeny of the cross is scored.
Gene: Unit of inheritance. Genes are located at fixed loci in chromosomes and can exist in a series of alternative forms called alleles.
Allele: One of a pair or series of forms of a gene, which are alternative in inheritance because they are situated at the same locus in homologous chromosomes.
Homozygous: Having like alleles at one or more corresponding loci on homologous chromosomes.
Heterozygous: Having unlike alleles at one or more corresponding loci on homologous chromosomes.
Low pH gene: gene, which when present in the genome of a plant leads to a lower pH of the flesh of a fruit of said plant, when compared to a plant not comprising said gene.
Low pH melon plant: melon plant comprising a low pH trait. In one embodiment, the pH of a fruit of a low pH melon plant is from about 4.2 to about 5.6.
Cucumis melo L: also referred herein to as C. melo or melon.
Cavity: refers to the center of the melon fruit containing seeds and maternal tissues.
Soluble Solids: refers to the percent of solid material found in the fruit tissue, the vast majority of which is sugars.
Climacteric/non-climacteric: as for example defined in Watkins (2002) “Ethylene synthesis, mode of action, consequences and control” In: Michael Knee (ed) “Fruit Quality and its Biological Basis”. Sheffield Academic Press, Sheffield, UK. Chapter 8 pp. 180-224, in particular at page 181, section 8.2.1, first two paragraphs.
Turning melon: turning refers to the marked change in rind color of a melon fruit when it reaches maturity, for example from green to yellow rind in Galia types, or from gray to creamy-yellow rind in Charentais types. This change of color is related to the degradation of green pigments occurring when the fruit reaches maturity.
Non-turning/low-turning melon: the rind color of a fruit of a non-turning or low turning melon does not dramatically change upon maturation. In the rind of the fruit of non-turning or low turning melon, there may be a continuous slight increase in yellow component in the stable background of the rind, as for example in Piel de Sapo type or in honeydew types. In low-turning melons, a slight pigment degradation may also be observed in connection with senescence of the fruit rather than with reaching maturity, which occurs earlier.


DETAILED DESCRIPTION OF THE INVENTION
The present invention provides C. melo plants capable of producing fruits with novel tastes. In particular, the present invention provides melon plants capable of producing fruits with novel combinations of organic acid contents, pH and sugar contents. The inventors of the instant application have identified that melon fruits contain various levels of organic acid, e.g. citric acid. The inventors of the instant application also have identified that melon fruits contain various relative contents citric acid and malic acid. Moreover, the inventors of the instant application have determined that by introducing a low pH trait into a sweet melon background, a reduction in pH and an increase in the citric acid content in the fruit are obtained, further expanding the ability to manipulate the taste of a melon fruit. In one embodiment, melon fruits of the present invention have low contents of malic acid. Accordingly, the present invention discloses melon plants producing fruits having elevated citric acid contents and lower pH, as described herein. In one embodiment, sugar levels observed in fruits of plants of the instant invention are maintained at levels present in currently available sweet melons or increased. Accordingly, plants of the present invention are capable of producing fruits with novel, pleasant tastes. Measurements of pH, citric acid and malic acid contents, and sugar contents described herein were carried out are shown in Examples 1 to 5. Tables 1A and 1B herein disclose representative melon plants according to the present invention and their fruits.
In one embodiment, the melon plants of the instant invention are capable of producing fruit with citric acid content equal or higher than about 400 mg per 100 g fresh weight (fwt). In one embodiment, melon plants of the instant invention are capable of producing fruit with a pH of about 4.2 to about 5.6. In one embodiment, melon plants of the instant invention are capable of producing fruit with a sugar content equal or higher that about 5.0 g per 100 g fwt. In one embodiment, the present invention discloses melon plants capable of producing fruit with citric acid content equal or higher than about 400 mg per 100 g fwt, pH of about 4.2 to about 5.6, and sugar content equal or higher that about 5.0 g per 100 g fwt.
In one embodiment, the present invention discloses a C. melo plant capable of producing a fruit comprising at maturity:

    
    
        a) about 400 mg to about 1,200 mg citric acid per 100 g fwt;
        b) pH of about 4.2 to about 5.6; and
        c) about 5.0 g to about 15.0 g sugar per 100 g fwt.
    
    


In one embodiment, the fruit comprises about 400 mg to about 1,000 mg citric acid per 100 g fwt. In one embodiment, the fruit comprises about 450 mg to about 950 mg citric acid per 100 g fwt, in one embodiment about 475 mg to about 900 mg citric acid per 100 g fwt. In one embodiment, the fruit has a pH of about 4.3 to about 5.4, in one embodiment about 4.4 to about 5.1. In one embodiment, the fruit comprises about 5.5 g to about 13.0 g sugar per 100 g fwt. In one embodiment, the fruit comprises about 7.0 g to about 15.0 g sugar per 100 g fwt.
In one embodiment, the ratio citric acid to malic acid in a fruit of said plant is greater than 4.4, in one embodiment greater than 5, in one embodiment greater than 10. In one embodiment, the ratio citric acid to malic acid in a fruit of said plant is less than 450, in one embodiment less than 200, in one embodiment less than 150. In one embodiment, a fruit of said plant comprises less than about 85 mg malic acid per 100 g fwt, in one embodiment less than about 75 mg malic acid per 100 g fwt.
In one embodiment, the ratio sucrose to hexoses in a fruit of said plant is about 1:1, in one embodiment between about 1:1 and about 1:2, in one embodiment between about 1:1 and about 2:1.
In one embodiment, the flesh of a fruit of said plant is orange, white, green or yellow. In one embodiment, said fruit is edible in fresh consumption. In one embodiment, the mesocarp of a fruit of said plant represents more than 50% of the total fresh fruit weight.
In one embodiment, the C. melo plant comprises a low pH trait. In one embodiment, the low pH trait is obtainable from a plant of line IND-35, representative seeds of which is deposited under Accession number NCIMB 41202, or a descendent of said line IND-35. In one embodiment, the low pH trait is homozygous or heterozygous in said plant.
In one embodiment of the instant invention, melon plants referred to as “Citric+” plants are disclosed. Such plants are capable of producing a very sweet juicy fruit with tart-refreshing sour taste. Such fruits comprise high contents of citric acid and low pH, increasing the acid taste and providing a tart perception. This is compensated by a high sugar content, in one embodiment by elevated levels of reducing sugars (glucose and fructose). The high reducing sugar content is particularly pronounced in early mature stages. The combination of tart and sweet tastes, which cannot be obtained by currently available sweet melon, is appreciated by consumers. The tart-refreshing sour sweet melon combination satisfies this need. This fruity tart perception also cover still too flat and poor flavors of early mature fruits. In one embodiment, Citric+plants are generally obtained by introducing a low pH trait, for example from line IND-35, into an oriental melon background. In one embodiment, the oriental melon background is selected for one or more of the following criteria: high citric acid content, low malic acid content, high citric acid to malic acid content, high sugar content, high hexose content, high ratio hexoses to sucrose, high juiciness. During the introduction of the low pH trait, careful selection for the above characteristics is maintained until a desired progeny is obtained.
For example, Examples 9, 10 and 11 describe the construction of such plants. Alternative C. melo plants can also be screened for desired characteristics and are used as starting materials as described herein.
In one embodiment, a fruit of a Citric+plant of the present invention at maturity comprises:

    
    
        a) about 600 to about 1,200 mg citric acid per 100 g fwt;
        b) pH of about 4.2 to about 5.1; and
        c) about 5.0 g to about 15.0 g sugar per 100 g fwt.
    
    


In one embodiment, the fruit comprises about 600 to about 1,000 mg citric acid per 100 g fwt. In one embodiment, the fruit comprises about 650 to about 950 mg citric acid per 100 g fwt.
In one embodiment, the pH of the fruit is about 4.4 to about 5.0.
In one embodiment, said fruit comprises about 7.0 g to about 13.0 g sugar per 100 g fwt. In one embodiment, the ratio citric acid to malic acid in a fruit of said plant is greater than 6, in one embodiment greater than 7, in one embodiment greater than 10. In one embodiment, the ratio citric acid to malic acid in a fruit of said plant is less than 450, in one embodiment less than 200. In one embodiment, a fruit of said plant comprising less than about 85 mg malic acid per 100 g fwt, in one embodiment less than about 75 mg malic acid per 100 g fwt, in one embodiment less than about 60 mg malic acid per 100 g fwt.
In one embodiment, a fruit of a plant of the present invention has a green or white flesh and at maturity comprises:

    
    
        a) about 600 to about 1,200 mg citric acid per 100 g fwt;
        b) pH of about 4.2 to about 5.1; and
        c) about 5.0 g to about 15.0 g sugar per 100 g fwt.
    
    


In one embodiment, the fruit comprises about 600 to about 1,000 mg citric acid per 100 g fwt. In one embodiment, said fruit with green or white flesh comprises at maturity about 8.0 g to about 12.0 g sugar per 100 g fwt. In one embodiment, said fruit with green or white flesh has at maturity a ratio citric acid to malic acid between about 25 and 200. In one embodiment the malic acid content of said fruit is less than about 50 mg malic acid per 100 g fwt, in one embodiment less than about 30 mg malic acid per 100 g fwt. In one embodiment, the ratio sucrose to hexoses in a fruit of said plant is about 1:1, in one embodiment between about 1:1 and about 2:1.
In one embodiment, a fruit of a plant of the present invention has orange flesh and comprises at maturity:

    
    
        a) about 600 mg to about 750 mg citric acid per 100 g fwt;
        b) pH of about 4.5 to about 5.1; and
        c) about 6.0 g to about 13.0 g sugar per 100 g fwt.
    
    


In one embodiment, the ratio citric acid to malic acid in said fruit is between about 4.4 and 30. In one embodiment, the fruit comprises about 7.0 g to about 13.0 g sugar per 100 g fwt.
In one embodiment, the present invention discloses a melon plant capable of producing a sweet aromatic fruit with mild-fruity sour taste, referred herein as “Citric-” melon plant or fruit. Such fruits comprise relatively high contents of citric acid and mildly low pH. This mild low pH makes a fruity sour perception that cover other still too flat and poor flavors of early mature fruits and without affecting the full expression of fruit flesh flavors and colors of further fully mature fruits. Further during ripening (early to late mature), fruits follow relevant increases of pH, decrease of acidity, in line with the weak buffer potential from the low citric acid content. This provides fully mature fruits with a very slight sour note to the full expression of very sweet and aromatic melon flavors. Melon flavor is based primarily on sweetness and aromas that usually get the full expression on really advanced fruit ripening stages. Early ripe melons are often described as unpleasant tasteless as sugars and aromas are below expectation and not any other component of flavor is present. In other fruits, such as strawberry, peach, or oranges, acid taste makes a well appreciated complement in this early ripe stages. The mild-fruity sour and sweet melon combination avoids or limits the tasteless risk on early ripe melons.
In one embodiment, Citric-plants are obtained by introducing a low pH trait, for example from line IND-35, into a Charentais melon background. In one embodiment, the Charentais melon background is selected for one or more of the following criteria: low citric acid content, low malic acid content, relatively high citric acid to malic acid content, high sugar content, high sucrose content. During the introduction of the low pH trait, careful selection for the above characteristics is maintained until a desired progeny is obtained.
For example, Example 11 describes the preparation of such plants. Alternative C. melo plants can also be screened for desired characteristics and can be used as starting materials as described herein.
In one embodiment, the fruit of a Citric-plant of the present invention at maturity comprises:

    
    
        a) about 400 to about 650 mg citric acid per 100 g fwt;
        b) pH of about 4.6 to about 5.6; and
        c) about 5.0 g to about 13.0 g sugar per 100 g fwt.
    
    


In one embodiment, the fruit comprises about 450 to about 600 mg citric acid per 100 g fwt.
In one embodiment, the fruit comprises a pH of about 4.8 to about 5.4, in one embodiment to about 5.2. In one embodiment, the fruit comprises about 5.0 g to about 13.0 g sugar per 100 g fwt, in one embodiment about 6.0 g to about 12.0 g sugar per 100 g fwt. In one embodiment, the fruit comprises about 7.0 g to about 13.0 g sugar per 100 g fwt.
In one embodiment, a fruit of a plant of the present invention has orange flesh and comprises at maturity:

    
    
        a) about 400 mg to about 550 mg citric acid per 100 g fwt;
        b) pH of about 4.8 to about 5.6; and
        c) about 5.0 g to about 11.0 g sugar per 100 g fwt.
    
    


In one embodiment, the ratio citric acid to malic acid in said fruit is between about 4.4 and 10. In one embodiment, the fruit comprises about 7.0 g to about 11.0 g sugar per 100 g fwt.







TABLE 1A







Summary of data for lines


Table 1A discloses representative lines of the present invention. The data shown in Table 1A


are averages based on measurements from individual trials. The data for the individual trials are


disclosed in Tables 9-13 in the Examples below.


















tot. sugar
sucrose
hexose
hex/suc

citric

malic


LINES (averages)

avg
avg
avg
inv
pH
avg
citric/
avg


Plastic house data
nr. frt
g/100 g
g/100 g
g/100 g
activity
avg
mg/100 g
malic
mg/100 g





















Green
YUSOL
High pH
34
9.85
4.50
5.34
1.1
6.05
265
24
11


Flesh
YUSAZ A
Low pH, Citric−
246
8.92
3.24
5.68
1.8
4.67
577
19
31



YUSAZ B
Low pH, Citric+
30
8.70
2.16
6.54
3.0
4.59
832
16
53


White
SOLAZ/1
Line
71
6.97
2.16
4.81
2.2
4.59
579
23
25


Flesh
SOLAZ/2
Line
43
6.19
1.92
4.27
2.2
4.64
590
47
13


Orange
L53AZ A
Low pH, Citric−
76
7.09
2.51
4.42
1.8
4.89
509
3
153


Flesh
L53AZ B
Low pH, Citric+
52
8.11
4.24
3.87
0.9
4.81
701
10
67



L53
High pH
10
8.29
5.05
3.23
0.6
6.67
92
1
93
















TABLE 1B







Summary of data for hybrids


Table 1B discloses representative hybrids of the present invention. The data shown in Table 1B are averages


based on measurements from individual trials. The data for the individual trials are disclosed in Tables 9-13 in


the Examples below. Mehari means the female parent of hybrid MEHARI.



















tot.












sugar
sucrose
hexose
hex/suc

citric

malic


HYBRIDS (average)
nr.
avg
avg
avg
inv
pH
avg
citric/
avg


Plastic house (PS) & Open Field (OP) data
frt
g/100 g
g/100 g
g/100 g
activity
avg
mg/100 g
malic
mg/100 g






















Green
YUSAZ A X YUSOL
Low pH, Citric+
17
9.70
4.46
5.24
1.2
4.77
692
46
15
PS


Flesh
MILENIUM-DENEV
High pH
12
7.61
3.47
4.13
1.4
5.88
444
X
0
OP



F1


White
SOLAZ/1 X YUSOL
Solaz1 x Yusol
26
9.71
6.40
3.31
0.5
4.75
760
58
13
OP


Flesh
SOLAZ/2 X YUSOL
Solaz2 x Yusol
13
9.08
6.26
2.82
0.5
4.70
782
162
6
OP



SOLAR F1
High pH
8
8.93
4.39
4.54
1.2
6.48
305
35
9
PS


Orange
Mehari/L53AZ A
Low pH, Citric−
13
7.34
2.24
5.10
2.3
4.98
487
6
78
PS


Flesh
Mehari/L53AZ B
Low pH, Citric+
17
8.35
3.37
4.97
1.5
4.76
663
11
60
OP



Mehari/L53
High pH
14
8.80
3.96
4.84
1.2
6.31
274
5
54
PS









The instant invention demonstrates that the manipulation of the degree of sourness in a fruit produce valuable taste variations. Sourness is a result of the interaction between several parameters, pH being one of the most important ones along with organic acids contents and compositions. The pH greatly influences the degree of dissociation of acids involved in taste. Each acid has a different dissociation constant (pKa), which refers to the pH, at which 50% of the acid is dissociated in its relative ions and H+. A lower pH results in more of the acid that is not in dissociated form. The sourness perception comes mainly from the undissociated form of the acid. This explains why, at higher pH levels, weaker acids such as organic acids with a higher pKa are perceived as sourer than stronger acids.
The pH of a solution is correlated with the concentration and pKa of the acid. Equinormal solutions of stronger acids are sourer than weaker ones, since they have a much lower pH. The higher the concentration of the acid (titratable acidity), the more sour the acid will be perceived. Sourness is also correlated with the chemical structure of the acidulant, the number of carboxylic groups, the molecular weight, and polarity of the molecule (see e.g. PURAC Biochem, Gorinchen, The Netherlands, “Flavor Special”, www.Purac.com). Besides sourness, each food acid has its own flavor characteristics in terms of lasting time, flavor intensity and the contribution to other non-sour flavor notes such as astringency, bitterness and sweetness.
The major organic acids in melon fruits are succinic acid, malic acid and citric acid (Wang et al. (1996) J. Agric. Food Chem. 44: 210-216). Malic acid tends to be associated either with very unripe phases or with senescence and degenerative phases of fruit over-ripening, and its dominance in the taste is generally not preferred. In the fruit flesh, gradients of increasing malic acid contents are observed from close to the seed cavity to close to the fruit rind. Citric acid has a lower pKa than malic acid. Citric acid has more tart and a clean effect, which often overpowers other taste or aromas notes. In contrast to malic acid, fruit flesh gradients of decreasing citric acid contents are observed from more ripe areas close to seed cavity to areas close to the fruit rind. Such a gradient is also observed for sugars.
Thus, in melon fruits of the instant invention, higher sugar contents and high citric acid contents tend to be associated in the fruit flesh to provide high contents of both in the most flavorful areas of the fruit, resulting in a very pleasant new taste.
Also, in the melon fruits of the instant invention, the sour perception is higher when the pH is low and the citric acid content high.
In one embodiment, fruits according to the instant invention were tested by an Expert panel of trained tasters. Sensory characteristics of the fruits were measured on a scale of quotation from 0 to 9. The pH, organic acid contents and sugar contents of the fruits were also measured. The results of the Expert panel are described in Example 12, Tables 14 and 15. The sensory analysis shows that the acid savor in fruits of the instant invention is a function of the pH and of the citric acid content in the fruit. Based on data gathered from mature and unripe fruits, the Expert panel determined the following formula for the correlation between the acid savor and pH and citric acid contents in a fruit: Acid savor=13, 12−(2.97× pH)+(0,00587× citric acid content (mg/100 g fwt)), with r2=0.70.
Accordingly, in one embodiment, the present invention discloses a C. melo plant capable of producing fruit comprising an acid savor above about 0.5 as determined by an Expert panel. In one embodiment, the present invention discloses a C. melo plant capable of producing fruit comprising an acid savor of about 1.6 or above. In one embodiment, the present invention discloses a C. melo plant capable of producing fruit comprising an acid savor above about 0.5 and a sugar savor of about 4.3 or above. In one embodiment, the present invention discloses a C. melo plant capable of producing fruit comprising an acid savor of about 1.6 to about 3.8 and a sugar savor of about 4.3 to about 5.8.
In one embodiment, the present invention discloses a C. melo plant capable of producing fruit comprising an acid savor of about 2.5 to about 3.8 and a sugar savor of about 4.3 to about 5.6. Hybrids YUSOL X SOLAZ and Mehari X L53 AZ B are representative examples of such plant.
In one embodiment, such fruit comprises a pH and citric acid content as described herein. In one embodiment, the present invention discloses a C. melo plant capable of producing fruit comprising an acid savor of about 1.6 to about 3.0 and a sugar savor of about 5.2 to about 5.8. Hybrids YUSAZ X YUSOL, Mehari X L53 AZ A and TD X L53 AZ A are representative examples of such plant.
In one embodiment, such fruit comprises a pH and citric acid content as described herein. In one embodiment, such fruit comprises a pH, organic acid contents and compositions and sugar contents and compositions as described herein.
It is understood that values of Expert panels vary depending on the pH, citric acid content and sugar content of a fruit.
The inventors of the present invention have screened melon entries and accessions for the content in citric acid and sugars and compositions and have determined variations within C. melo types (see Table 2). For example, some Charentais-type melons were found to have low citric acid contents (for example Lunastar in Table 2). Some Charentais type fruits were also found to have a relatively low citric acid to malic acid ratio and a high ratio of sucrose to hexoses. On the other hand, Oriental-type melons, such as Japanese rocky types melon, were found to have higher contents in citric acid (for example YUCA in Table 2). A representative of YUCA has been deposited with NCIMB, Aberdeen, AB243RY, Scotland on Dec. 17, 2003 as YUC-15 under Accession number NCIMB 41203. These melons were also found to have high ratios of citric acid to malic acid and high ratios of hexose to sucrose. Some Galia type melons, such as MG 755, were also found to have high citric acid contents. Surprisingly, according to the present invention, in a cross between a Japanese-type melon and a Charentais-type melon (Prince PF in Table 2), the high citric acid content of the Japanese-type melon could be disassociated from the high hexose to sucrose ratio and combined with the high sucrose to hexose ratio of the Charentais-type melon. The invention further recognized that variations in citric acid contents also exist among non-sweet accessions such as Faqqous and IND-35 (low citric acid content in Faqqous when compared to IND-35, table 2).
Also, although it was generally assumed in the art that low pH and high sugar accumulation were not frequent or even compatible in the same melon fruit (Stepanski et al (1999)), the inventors of the present invention have been able to combine that characteristics of pH and organic acid described above with desired sugars contents in the fruits (see e.g. Table 3).







TABLE 2







Variation in citric acid contents in various melon types and varieties


(Fruit analysis data trial Sarrians August 1997)

















No.


Citric acid
Sucrose
Hexoses
Glucose
Fructose
Total Sugar


Name
fruits
Ri
pH
(mg/100 g)
(g/100 g)
Glucose
(g/100 g)
(g/100 g)
(g/100 g)



















Lunastar (Charentais)
6
11.8
6.8
159
6.7
3.2
1.6
1.6
9.2


Yuca-18-3-8
8
10.5
6.7
286
3.8
4.2
2.3
1.9
8.0


MG 755 OA
4

6.8
294
11.4
3.7
2.1
1.7
15.1


Prince PF (Japanese X
5

6.1
438
10.0
3.3
1.2
2.1
13.3


Charentais)


Sancho
4


409
3.7
4.1
2.1
1.9
7.8


Fagouss Egypt
10
3.8
5.6
35
0.1
1.9
0.9
1.0
1.9


Fagouss Jim MC Greigt
6
3.3
5.2
62
0.1
2.1
0.9
1.2
2.2


Fagouss Jordany
11
3.7
5.1
125
0.1
2.1
0.9
1.2
2.2


IND35-1
8
4.5
4.9
340
0.6
3.4
1.8
1.7
4.0





Ri: Brix, refractometer index, measuring soluble solids













TABLE 3







Sugar accumulation vs. pH


Statistical analysis of sugar accumulation (brix) versus pH in


F2 populations of a cross between IND-35 and YUCA or OGEL


(Agadir'98). The analysis shows no negative correlation between


brix values and the pH.











pH < 5.5
pH > 5.5
ANOVA













RI (Brix)
pH
RI (Brix)
pH
P = 0.05

















IND35/YUCA-15
NR plts
68
68
22
22
P = 0.98


F2
Maximum
13.00
5.40
13.60
7.00
No Significant



Average
8.03
4.53
8.01
5.85
Difference



Std. Deviation
2.34
0.30
2.72
0.37
in brix



Minimum
2.40
3.80
2.40
5.50
Populations


IND35/OGEL-17
NR plts
98
98
39
39
P = 0.65


F2
Maximum
13.00
5.45
12.20
7.50
No Significant



Average
7.75
4.70
7.93
5.97
Difference



Std. Deviation
2.16
0.33
1.89
0.35
in brix



Minimum
2.80
3.90
4.20
5.50
Populations


IND.35
NR plts
29
29



Maximum
7.60
5.40



Average
5.45
4.63



Std. Deviation
1.18
0.31



Minimum
3.00
4.10









The present invention further discloses the incorporation of a low pH trait in sweet C. melo backgrounds. The presence of the low pH trait in sweet C. melo backgrounds allowed to lower the pH and increase concentrations of organic acids in the fruit flesh and to combine desirable pH and citric acid contents with appropriate sugar concentrations and contents, resulting in pleasant, new tastes.
In one embodiment, a low pH trait is determined by a low pH gene. In one embodiment, a low pH gene is obtained from a wild melon accession or cultivar. In one embodiment, a wild melon accession or cultivar used as donor for the low pH gene comprises traits that facilitate the construction of commercial melons with acceptable agronomic characteristics and producing fruits with desirable taste. In one embodiment, such donor has the ability to accumulate relevant levels of sugars, such as sucrose. In one embodiment, such wild melon accession or cultivar has at least one of the following characteristics: non climacteric behavior, relative bigger fruit size and mesocarp component, crispy flesh.
In one embodiment, the low pH trait is obtained from line IND-35, representative seeds of which was deposited with NCIMB, Aberdeen, AB243RY, Scotland on Dec. 17, 2003 under Accession Number NCIMB 41202. This is a C. melo accession from India, which could be classified within botanical Var. chito, but it may be better classified as var. acidulus (Naudin, Pitrat et al.) because of its bigger size.
Citric acid is the main organic acid in the fruits of line IND-35 (up to 911 mg citric acid per 100 g fwt), while malic acid contents are below 50 mg per 100 g fwt. Surprisingly, fruits of IND-35 also have the ability to accumulate relevant levels of sucrose (up to 1.7 g sucrose per 100 g fwt and 5.6 g total sugars 100 g fwt after long fruit cycles, i.e after late harvest 50 or 53 days after fruit set). Table 2 also reports analysis of fruits of IND-35 showing a pH of about 4.9, a citric acid content of about 340 mg/100 g fresh weight (fwt), and a sugar content of about 4.0 g/100 g fwt.
In one embodiment, the low pH trait co-segregates with a molecular marker. In one embodiment, a molecular marker is a DNA fragment amplified by PCR, e.g. a SSR marker or a RAPDS marker. In one embodiment, the presence or absence of an amplified DNA fragment is indicative of the presence or absence of the trait itself or of a particular allele of the trait. In one embodiment, a difference in the length of an amplified DNA fragment is indicative of the presence of a particular allele of a trait, and thus enables to distinguish between different alleles of a trait. In one embodiment, the instant invention discloses markers, which distinguish between different sources of low pH trait, and for the presence or absence of a low pH trait in a plant. For example, such marker is CMAT 141, described in Danin-Poleg et al. (2001) Theor. Appl. Genet. 102: 61-72 and Danin-Poleg et al. (2002) Euphytica 125: 373-384. Other examples of molecular markers are NE0585 and NE1746 disclosed herein (see Example 13). These markers are closely linked to the locus of the pH gene. Markers CMAT141 and NE0585 are on one side of the low pH gene, while marker NE1746 is on the other side of the low pH gene.
In one embodiment, the inventors of the instant invention have determined that various sources of low pH trait amplify DNA fragments of different length when the primers for the markers are used (acid fragments, see Example 14, Table 16). For example, in plants of Faggous as fragment of about 176 bp is amplified using for CMAT141. In plants of accessions PI414723, PI414724, PI161375 and PI124112 a fragment of about 175 bp is amplified. In contrast, in plants of IND-35, described herein, unique fragments of about 168 bp and of about 173 bp are amplified. Using marker NE0585, unique fragments of about 230 bp and about 232 bp are amplified for IND-35. Using marker NE1746, a unique fragments of about 127 bp is amplified for IND-35. Another fragment of about 124 bp is amplified for IND-35, which is for example absent in Faggous.
In one embodiment, plants not comprising the low pH trait were also analyzed using the primers described herein. A number of DNA fragments associated with the absence of the low pH trait were determined (basic fragments). For example, using the primers of CMAT141, fragments of about 169 bp, about 172 bp and about 178 bp long were detected in plants not comprising a low pH trait. Using the primers of NE0585, fragments of about 218 bp, about 229 bp, about 234 bp and about 239 bp long were detected. Using the primers of NE1746, fragments of about 133 bp, about 142 bp and about 145 bp long were detected. The skilled person would know how to analyze further plants not comprising the low pH trait and determine additional DNA fragments associated with the absence of the low pH trait.
The indicated sizes (in bp) are not absolute but relative to the other size products detected with the same primer pair. The real (exact) size of the amplified fragments (e.g. determined by sequencing) could be slightly different (+/−1 bp) of those indicated herein.
Accordingly, in one embodiment, the present invention discloses a C. melo plant comprising a DNA sequence, which is a template for amplification of a DNA fragment described herein using the primers described herein. In one embodiment, a DNA fragment of about 168 bp to about 178 bp is amplified from the DNA of said plant when the primers capable of identifying the CMAT141 marker are used. In one embodiment, a DNA fragment of 168 bp, 173 bp, 169 bp, 172 bp or 178 bp is amplified when the primers capable of identifying the CMAT141 marker are used. In one embodiment, a DNA fragment of less than 176 bp is amplified when the primers capable of identifying the CMAT141 marker are used. In one embodiment, a DNA fragment of less than 175 bp is amplified when the primers capable of identifying the CMAT141 marker are used. In one embodiment, a DNA fragment of about 218 bp to about 253 bp is amplified when the primers capable of identifying the NE0585 marker are used. In one embodiment, a DNA fragment of 230 bp, 232 bp, 218 bp, 229 bp, 234 bp or 239 bp is amplified when the primers capable of identifying the NE0585 marker are used. In one embodiment, a DNA fragment of about 121 bp to about 145 bp is amplified when the primers capable of identifying the NE1746 marker are used. In one embodiment, a DNA fragment of 124 bp, 127 bp, 133 bp, 142 bp or 145 bp is amplified when the primers capable of identifying the NE1746 marker are used.
In one embodiment, the inventors of the instant invention have separated the link between a DNA fragment indicative of a low pH trait and a low pH gene. In this case, a DNA sequence, which is a template for amplification of a basic fragment, is linked to a low pH trait. Accordingly, in one embodiment, the instant invention discloses a plant comprising a low pH trait co-segregating with markers indicative for a basic allele, in particular when using the markers disclosed herein. In one embodiment, a marker on one side of the pH gene is basic. In one embodiment, markers on both sides of the pH gene are basic. In one embodiment, fruits of such a plant comprise a pH within the ranges disclosed herein. In one embodiment, fruits of such a plant comprise the contents and compositions of sugars as described herein. In one embodiment, fruits of such a plant comprise the contents and compositions of organic acids as described herein. In one embodiment, fruits of such a plant comprise the pH, contents and compositions of sugars and contents and compositions of organic acids as described herein.
Accordingly, in one embodiment, the present invention discloses a C. melo plant comprising a DNA sequence, which is a template for amplification of a basic DNA fragment, linked to a low pH gene. In one embodiment, the present invention discloses a C. melo plant comprising a chromosome fragment comprising a DNA sequence, which is a template for amplification of a basic DNA fragment and a low pH gene. In one embodiment, such chromosome fragment is heterozygous or homozygous in said plant. Line SOLAZ/2 described herein is a representative example of such a C. melo plant. In SOLAZ/2, fragments of about 172 bp, about 229 bp and about 124 bp are amplified using the primers of markers CMAT141, NE0585 and NE1746, respectively.
In one embodiment, the present invention discloses a C. melo plant comprising a DNA sequence, which is a template for amplification of a basic DNA fragment, on one side of a low pH gene, a low pH gene, and a DNA sequence, which is a template for amplification of a basic DNA fragment, on the other side of the low pH gene, wherein both DNA sequence are linked to the pH gene in said plant. In one embodiment, the present invention discloses a C. melo plant comprising a chromosome fragment comprising a DNA sequence, which is a template for amplification of a basic DNA fragment, on one side of a low pH gene, the low pH gene and a DNA sequence, which is a template for amplification of a basic DNA fragment, on the other side of the low pH gene. In one embodiment, such chromosome fragment is heterozygous or homozygous in said plant. Line SOLAZ/1 described herein is a representative example of such a C. melo plant. In SOLAZ/1, fragments of about 172 bp, about 239 bp and about 142 bp are amplified using the primers of markers CMAT141, NE0585 and NE1746, respectively. Line YUSOL/3 described herein is another representative example of such a C. melo plant. In YUSOL/3, fragments of about 172 bp, about 239 bp and about 145 bp are amplified using the primers of markers CMAT141, NE0585 and NE1746, respectively.
Accordingly, the present invention discloses the use of a melon plant comprising a low pH trait to obtain a melon plant of the present invention. In one embodiment, the melon plant comprising a low pH trait further has the ability of accumulating relevant levels of sugars, for example sucrose. In one embodiment, the melon plant comprising a low pH accumulates high levels of citric acid. In one embodiment, the melon plant comprising a low pH accumulates low levels of malic acid. In one embodiment, the melon plant comprising a low pH is a plant of line IND-35 or a descendent thereof.
In one embodiment, the low pH trait is obtained from a descendent of said line IND-35. The skilled person recognizes that a low pH trait can also be obtained from other sources. In one embodiment, the low pH trait in such other sources is allelic to the low pH trait in IND-35. In one embodiment, a test is conducted to determine whether a line to be tested for a low pH gene comprises an allelic gene to that of line IND-35. Line IND-35 is used as a tester line in a cross with a line to be tested and the segregation ratio of the low pH phenotype is determined in the resulting progeny.
In one embodiment, a plant of line IND-35 is crossed with breeding melon lines, preferably having high citric acid contents and high levels of sugar. After each cross plants producing fruits having low pH are selected. Selection is also carried out for increased citric acid contents and high sugar contents. Examples of the introduction of the low pH trait into elite lines are disclosed in Examples 7-11.
In one embodiment, a molecular marker as disclosed hereinabove is used to transfer the pH trait in a desired background, in particular in method of increasing the citric acid content of a fruit of a melon plants as described herein. In one embodiment, plants for which a fragment corresponding to a low pH trait is amplified are selected and further used.
In one embodiment, a reduction of about 1 to about 2.5 pH units, in one embodiment about 1.5 to about 2.0 pH units, is obtained after the introduction of the low pH trait in a sweet C. melo plant, when compared to fruits of a melon not comprising the low pH trait, for example when compared to an isogenic or near-isogenic line not comprising the low pH trait. In one embodiment, the citric acid content in the flesh of a melon plant is multiplied by a factor of about 1.5 to about 3 upon introduction of a low pH trait in said melon plant, when compared to fruits of a melon not comprising the low pH trait, for example when compared to an isogenic or near-isogenic line not comprising the low pH trait. Mature fruits of the various plants are compared. For example, Table 1A and 1B show comparisons between melon comprising a low pH trait and melons not comprising a low pH trait, for example between YUSAZ X YUSOL and MILENIUM-DENEV F1, SOLAZ X YUSOL and SOLAR F1, Mehari/L53-L53 AZ A-L53 AZ B.
Accordingly, melon fruits of the present invention have higher concentrations of organic acids than comparable currently available melons not comprising the low pH trait (titratable acidity). In melon fruits of the present invention, variations in pH are more correlated with the composition (pKa of dominant acids) than with total concentration of organic acids. An increase in pH is generally observed during the late ripening process, and is concurrent with sucrose accumulation. This increase in pH is buffered by the content in organic acids. Low contents in organic acids result in high increases in pH and reduction in sour taste perception. For example, in melons with low citric content as some currently available Charentais-type melons, for example of climacteric turning type, this increase in pH can be over 1.0 pH unit (from pH 6.0 to 7.0). By contrast, higher contents in organic acids lead to reduced pH increases during the ripening process, and thus prevent or reduce the decline in sour taste perception, as for example in less climacteric, less turning melons.
Accordingly, in one embodiment, the present invention discloses melons fruits with more stable sour tart taste based on stable low pH and high citric acid content. In one embodiment, the present invention discloses melons fruits with an improved stability of the taste after ripening or post-harvest. The present invention also discloses a method of delaying or reducing the increase in pH in the fruit of a melon plant comprising increasing the content of organic acids in said fruit and introducing a low pH trait in a melon plant. In one embodiment, such method comprises introducing the low pH trait in a plant with low or non climacteric behaviour. In one embodiment, such method comprises introducing the low pH trait in a plant capable of producing a low or non turning fruit.
In one embodiment, the present invention also discloses melons fruits, which mature from an initial early ripe mild sour taste to a fruity and full flavored taste based on a moderate increase of pH (up to 0.5 pH units) and moderately elevated citric acid contents.
According to the present invention, a low pH trait is introduced in melon plants producing fruits with various contents and composition of sugars. In one embodiment, plants from the Japanese-Oriental type, such as YUCA, are used as a source of sugars. It is generally observed that accumulation of sugars, particularly sucrose, is a question of fruit cycle time understood as days after pollination, from fruit setting to fruit ripening. In one embodiment, melon plants producing fruits having an early and relevant sugar accumulation are used in the instant invention. For example, plants of the Galia type, such as cultivar OGEL, are used as a source of sugars. In one embodiment, melon plants producing fruits having potential for a high and fast accumulation of sucrose independently from the initial levels of hexoses are used in the instant invention. For example, plants of the Charentais type, such as L53, are used as a source of such a potential for sucrose accumulation.
Examples of the transfer of a low pH trait to elite lines are disclosed in the Examples below. Table 8 below also discloses melon plants obtained during the transfer to elite lines. However, other types of melons or other melons cultivars or varieties of the types mentioned above are used in the context of the present invention to construct melon plants according to the instant invention.
In one embodiment, a C. melo plant comprising a low pH trait is crossed to a C. melo plant of the Oriental type or of the Galia-type. For example the C. melo plant comprising a low pH gene is line IND-35. For example, the C. melo plant of the Oriental type is YUCA, and the C. melo plant of the Galia-type is OGEL, as described in Example 7 below. The resulting progenies are for example further crossed to C. melo plants of the Oriental type or of the Galia-type to obtain desired pH and contents and compositions of organic acids and sugars. This process is assisted by measurement of pH, organic acids and sugars, as disclosed herein. Alternative C. melo plants of the Oriental type or of the Galia-type can also be screened for desired characteristics and used as starting materials to obtain C. melo plants as described herein.
In one embodiment, a C. melo plant comprising a low pH trait is crossed to a C. melo plant of the Charentais-type, such as L53 as described in Example 11. Alternative C. melo plants of the Charentais-type can also be screened for desired characteristics and used as starting materials to obtain C. melo plants as described herein.
Other types of C. melo plants are also crossed to a C. melo plant comprising a low pH trait to obtain melo plants as described herein.
In one embodiment, the present invention discloses a plant capable of producing a fruit, the characteristics of which remain stable after a fruit reaches maturity and is kept on the plant or when the fruit is harvested and kept in storage. In one embodiment, the characteristics of a fruit described herein remain stable during the commercial post-harvest life of the fruit. This allows storing or shipping a fruit of the present invention for extended periods of time without loosing its organoleptic characteristics and aromas.
In one embodiment, the pH of a fruit of the instant invention remains stable after a fruit reaches maturity. In one embodiment, the pH of a fruit of the instant invention remains within a range of about 4.2 to about 5.6 after a fruit reaches maturity. In one embodiment, the citric acid content of a fruit of the present invention remains stable after a fruit reaches maturity. In one embodiment, the citric acid content of a fruit of the present invention remains at or above 400 mg per 100 g fwt after a fruit reaches maturity. In one embodiment, the malic acid content of a fruit of the present invention remains stable after a fruit reaches maturity. In one embodiment, the ratio citric acid to malic acid of a fruit of the present invention remains stable after a fruit reaches maturity. In one embodiment, the ratio citric acid to malic acid remains greater than 4.4 after a fruit reaches maturity. In one embodiment, such characteristics remain with the ranges described herein after a fruit reaches maturity. In one embodiment, these characteristics remain on within about 70% to about 130% of the values of the characteristics measured when the fruit reaches maturity, in one embodiment within about 80% to about 120% of the values of the characteristics measured when the fruit reaches maturity, in one embodiment within about 90% to about 110% of the values of the characteristics measured when the fruit reaches maturity. In one embodiment, the present invention discloses a plant capable of producing a fruit, the characteristics of which remain stable after a fruit reaches maturity when kept on the plant, or when harvested and kept in storage In one embodiment, the present invention discloses a plant capable of producing a fruit, the characteristics of which remain stable for at least 2 days when the fruit is kept on the plant, in one embodiment for at least 3 days when the fruit is kept on the plant, in one embodiment for at least 4 days when the fruit is kept on the plant. In one embodiment, the present invention discloses a plant capable of producing a fruit, the characteristics of which remain stable for at least 5 days when kept in storage at 20° C., in one embodiment for at least 7 days when kept in storage at 20° C., in one embodiment for at least 9 days when kept in storage at 20° C. In one embodiment, the present invention discloses a plant capable of producing a fruit, the characteristics of which remain stable for at least 7 days when kept in storage at 8-12° C. followed by at least 2 days at 20° C., in one embodiment for at least 12 days when kept in storage at 8-12° C. followed by at least 2 days at 20° C., in one embodiment for at least 26 days when kept in storage at 8-12° C. followed by at least 2 days at 20° C. Typically, after its harvest a fruit may remain under field conditions for several hours until its is stored in the conditions set forth herein.
Examples of the evolution of the characteristics of a fruit of a plant of the present invention when kept on the plant are shown in Example 15, Table 17. Examples of the evolution of the characteristics of a fruit of a plant of the present invention after post harvest storage are shown in Example 16, Tables 18 and 19.
In one embodiment, melons can be described as short shelf life (SLS), medium shelf life (MSL) or long shelf life (LSL). Examples of LSL melons are Milenium, Piel de Sapo, Italo and non turning Charentais LSL. Examples of MSL melons are Galia and turning Charentais MSL. Examples of short shelf life (SSL) melons are classical Charentais.
Typically, the life of a fruit of a SSL melon on the plant is about 1 to about 2 days. This means that fruits have to be harvested about every 1 to 2 days to avoid losses. Typically, the life of a fruit of a MSL melon on the plant is about 3 to about 4 days. This means that fruits are harvested about every 3 to 4 days to avoid losses. Typically, the life of a fruit of a LSL melon on the plant is more than about 5 days Typically a SSL melon can be stored for about 4 to about 7 days at 8-12° C. followed by an additional 2 days at 20° C., or for about 3 to 4 days at 20° C. Typically a MSL melon can be stored for about 7 to about 12 days at 8-12° C. followed by an additional 2 days at 20° C., or for about 5 to 10 days at 20° C. Typically a LSL melon can be stored for more than about 12 days at 8-12° C. followed by an additional 2 days at 20° C., or for more than about 10 days at 20° C.
In one embodiment, a plant of the instant invention is capable of producing a long shelf-life fruit (LSL) or a medium shelf-life fruit (MSL).
The inventors of the present invention have determined that the low pH trait and high citric accelerates the climacteric rise in a melon fruit after a fruit reaches maturity, especially in turning and climacteric melons. This leads to degradation of the fruit for example shown by a rapid increase in pH and malic acid content and a decrease in citric acid content. This is also shown by the appearance of mealy texture and alcoholic degradation. In turning and climacteric melons, the shelf life of the fruit is reduced, while this phenomenon is less perceptible in non-turning or/and non-climacteric melons. Accordingly, in one embodiment, a plant of the present invention is of a non-turning or low turning melon genotype. In one embodiment, a plant of the present invention is capable of producing a non-climacteric or low-climacteric fruit.
Accordingly, in one embodiment, the present invention discloses a C. melo plant with low or non-climacteric behaviour comprising a low pH gene. In one embodiment, a fruit of such C. melo plant further comprises the characteristic of sugar contents and compositions described herein. In one embodiment, a fruit of such C. melo plant further comprises the characteristic of organic acid contents and compositions described herein. In one embodiment, a fruit of such C. melo plant comprises a ratio of citric acid to malic acid as described herein. In one embodiment, a fruit of such plant further comprises a pH as described herein.
In one embodiment, the present invention discloses a C. melo plant capable of producing a low-turning or non-turning fruit, wherein said plant comprises a low pH gene. In one embodiment, a fruit of such C. melo plant further comprises the characteristic of sugar contents and compositions described herein. In one embodiment, a fruit of such C. melo plant further comprises the characteristic of organic acid contents and compositions described herein. In one embodiment, a fruit of such C. melo plant comprises a ratio of citric acid to malic acid as described herein. In one embodiment, a fruit of such plant further comprises a pH as described herein.
In one embodiment, the inventors of the instant invention have identified that a low pH in the flesh of a melon fruit is associated with a very poor color intensity of the flesh of fruits with orange flesh. In particular, in the case of melon plants producing fruit with orange flesh, a slightly orange or pale orange color was observed (see e.g. Table 4). Surprisingly, the inventors of the present invention have been able to combine low pH and deep orange color in the fruit of a melon plant.
Table 4 shows that the color intensity of the fruits of line L53, a parent of Syngenta hybrid MEHARI, is deep to very deep orange (average of 5.17). Plants originating from a back-cross program to introduce the low pH trait in L53 but not comprising the low pH trait (L53*High pH) showed a slight decrease in both color intensity and pH (4.71 and 6.46). Plants originating from the back-cross program and comprising the low pH trait showed a further decrease in color intensity and pH. Plants with the low pH trait in homozygote stage (L53*Low pH) had the lowest rate for orange flesh color: 4.04. Plants with the low pH trait in heterozygote stage (L53*acid) have the intermediate rate for orange flesh color: 4.36. This indicates a linkage or phenotypic effect between low pH and pale orange flesh color. Plants were therefore selected for intense orange flesh color in presence of the low pH trait (rated in one embodiment at 4 or higher in the scale below, in one embodiment at 5 and higher, in one embodiment at 6).
In one embodiment, the present invention discloses a C. melo plant capable of producing fruit with pH of about 4.5 to about 5.6, wherein said fruit has orange flesh rated 4 or higher. In one embodiment, the orange color of said fruits is rated 5 and higher, in one embodiment 6. In one embodiment, the pH of said fruit is about 4.5 to about 5.4, in one embodiment about 4.8 to about 5.2. In one embodiment, said fruit comprises about 400 mg to about 900 mg citric acid per 100 g fwt. In one embodiment, the fruit comprises about 450 mg to about 750 mg citric acid per 100 g fwt. In one embodiment said fruit comprises a sugar content equal or above about 5 g per 100 g fwt, in one embodiment about 5.0 g to about 13.0 g sugar per 100 g fwt. In one embodiment, the fruit comprises about 6.0 g to about 12.0 g sugar per 100 g fwt. In one embodiment, the ratio citric acid to malic acid in said fruit is between about 4 and 30, in one embodiment between about 5 and about 15. In one embodiment, the ratio sucrose to hexoses in a fruit of said plant is about 1:1 and about 1:2.
A scale of: 1: white, 2: slightly orange, 3: pale orange, 4: orange, 5: deep orange, 6: very deep orange was used for the color rating (see table 4). In one embodiment, the color of the fruit is assessed using a spectrophotometer, such as a Minolta CM-2500d spectrophotometer.














TABLE 4







Pale



Deep



Fruits

Orange



Orange


Nr.

2
3
4
5
6















% of flesh colour intensity class per





















Col avg


5
True L53



83%
17% 
5.17


14
L53* High pH


29%
71%

4.71


91
L53* acid
0%
 7%
55%
34%
4%
4.36


244
L53* low
2%
19%
48%
27%
3%
4.04



pH







pH average per flesh colour intensity class





















pH avg


5
True L53



6.90
6.64
6.85


14
L53* High pH


6.28
6.53

6.46


91
L53* acid

4.75
4.81
4.87
4.94
4.83


244
L53* low
4.53
4.64
4.76
4.73
4.65
4.72



pH





“L53* acid”: means acid phenotype excluded proved fix Low pH homozigotes






In one embodiment, characteristics of melon fruits of the present invention are measured on fruits harvested at maturity, i.e. mature or ripe fruits. The concrete composition of a melon fruit, and therefore its taste, is affected by the ripening stage, at which it is harvested. Sugars and organic acids accumulation in melon fruits are dynamic processes. As the fruit is approaching maturity their accumulation starts. As the ripening process advances, each of these compounds follows a time-specific pattern of accumulation or degradation, which is also affected by environmental and growth conditions. The person skilled in the art knows how to recognize a mature melon fruit and understand criteria defining the maturity of a melon fruit.
In one embodiment, one of the following external maturity markers is used for identification of ripening in sweet melon:

    
    
        Senescence of the side fruit leaf (the melon fruit sets in flower axillar to the peduncle insertion of one leaf named side leaf fruit). The side fruit leaf becomes necrotic.
        Fruit skin color changes (turning color green to yellow in Galia type, gray to creamy-yellow in Charentais type, or increase in yellow component in Piel de Sapo type).
        Peduncle dehiscence (specially for Shipper melon, less for Charentais, less for Galia).
        Decline in fruit shell firmness, particularly in the blossom end area.
    
    


In one embodiment, “maturity” includes the ripening physiological process between the stages identified as “2” for “Early—Partial mature” and “4” for “Late—Fully mature”.
In one embodiment, the Early-Partial mature stage in melons is identified with the initial accumulation of sucrose contents at or over 2 g per 100 g fwt of sucrose. In one embodiment, this is associated with the peaking of citric acid and reducing sugars (glucose plus fructose) contents, and a typical ripe fruit flesh pigments and texture. In one embodiment, the Late—Fully mature stage in melons is defined with the end of peaking sucrose sugars contents. It is also defined as before the degradation process starts, such as loss of fruit flesh consistency, fast declines in glucose, citric acid contents or increases in malic acid contents. Accordingly, in one embodiment, maturity of a fruit starts when the sucrose content in the fruit reaches 2 g per 100 g fwt. In one embodiment, maturity lasts until no more increase in sucrose content is observed.
The measurements disclosed herein are usually averages of measurements or data taken from a number of fruits. It is understood that, in any sample, individual fruits of a plant or fruits from individual plants do not lie within the ranges described, because of variations generally observed while growing melon plants. In one embodiment, the characteristics of melon fruits described herein are measured using fruits grown in the conditions described herein or under similar conditions (e.g. in Example 6, Tables 5-7 below). In one embodiment, a figure for a characteristic according to the instant invention is an average taken from fruits grown from plastic house staked plants (one fruit per plant in staked plants).
In one embodiment, a plant of the instant invention is an inbred line, a dihaploid or a hybrid. In one embodiment, an inbred line comprises a low pH trait and the characteristics of organic acids, pH and sugars described herein. In this case, such inbred line is crossed with another melon plant, preferably another inbred line, to obtain a hybrid plant according to the instant invention. In one embodiment, the other inbred line in the cross is also capable of producing fruit having high citric acid contents and/or high sugar contents. Representative inbred lines according to the present invention are disclosed in Table 1A.
In one embodiment, a plant of the instant invention is a hybrid plant. In this case, some of the characteristics of low pH, high citric acid content and high sugar content are contributed from one of the parent, while the remaining ones are contributed by the other parent. In a one embodiment, one parent in the cross produces fruit having high citric acid content, high sugar content but having high pH (e.g. around pH 6.5), while the other parent in the cross produces fruit having low pH. In one embodiment, fruits of the other parent also have high citric acid content. Representative hybrids according to the present invention are disclosed in Table 1B. As used herein, the term “plant” includes plant cells, plant protoplasts, plant cell of tissue culture from which melon plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants, such as pollen, flowers, leaves, stalks, and the like. In one embodiment, a plant of the instant invention is capable of producing edible melon fruits. The mesocarp represents the edible part of the melon fruit (flesh). The mesocarp surrounds the seed cavity, which is itself surrounded by the rind (or shell). The mesocarp of a fruit according to the present invention preferably has a thickness of more than 2 cm and preferably represents more than 50% of the total fruit fresh weight. In one embodiment, the flesh of a fruit of a melon plant of the present invention has a green, white, yellow or orange flesh.
In one embodiment, the present invention provides regenerable cells for use in tissue culture of a plant of the present invention. The tissue culture is capable of regenerating plants having the characteristics of a plant of the present invention. Preferably, the regenerable cells in such tissue cultures are immature embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, or flowers. Still further, the present invention provides melon plants regenerated from the tissue cultures of the invention. Examples of regeneration protocols are disclosed in U.S. Pat. No. 6,420,631. The present invention further provides a method of asexually propagating a plant of the present invention comprising collecting a tissue of a plant of the present invention, cultivating said tissue to obtain proliferated shoots, rooting said proliferated shoots to obtain rooted plantlets. The present invention further discloses a method to produce seed of a plant according to the instant invention comprising obtaining a plant of the present invention, self-pollinating said plant or crossing said plant with another melon plant, and harvesting progeny seed. The present invention further discloses a method for producing a fruit comprising planting a plant according to the present invention, growing said plant and harvesting a fruit, wherein said fruit comprises the characteristics described herein. The method further comprises storing said fruit, for example as described herein. The method further comprises shipping said fruit. In one embodiment, the characteristics of said fruit described herein remain stable during the storage of said fruit. In one embodiment, the characteristics of said fruit described herein remain stable during the storage of said fruit.
Some characteristics of a number of C. melo types and lines are described below. These characteristics are examples for the various melon types and lines, and are not meant to be limiting but are illustrative of the various melon types and lines. Variations from these characteristics may occur.
Melon Type Charentais:
Fruit: Shape round to hight round; Size 600 to 1200 grs; Rind: White-Gray with Green-Gray sutures, smooth skin (smooth or slightly netted skin). Orange, melty, very aromatic sweet flesh, climacteric. Some Charentais varieties have reduced climateric phase and not turning rind colour.
Var LUNASTAR: Lunastar is monoecious hybrid from Nunhems in Charentais type.
Melon Type Japanese:
Fruit: Shape round to hight round; Size 700 to 1500 grs; Rind: White-Gray netted skin, (exceptionally sutured). Green-Yellow (exceptionally orange), from cryspy to melty and few aromatic and very sweet flesh, only in cases. Long cycle non climacteric.
Var YUCA: Andromonoecious hybrid in Japanese type green flesh.
Var PRINCE: Andromonoecious hybrid in Japanese type orange flesh.
Melon Type Galia:
Fruit: Shape round to hight round; Size 600 to 1500 grs; Rind: Green tuning yellow netted skin. Green-White, melty, aromatic sweet flesh. Short cycle Medium shelf life, climacteric. Var MG.755: Andromonoecious hybrid in Galia type. Very short fruit cycle with high brix.
Melon Type Piel De Sapo:
Fruit: Shape Ovoid to oblong-eliptic; Size 2 to 5 Kgrs; Rind: Golden Green spotted, few longitudinal netted skin. White, crispy, juicy and aromatic sweet flesh. Medium fruit cycle Long shelf life, Non climacteric.
Var Sancho: Andromonoecious hybrid in Piel de sapo type. Golden rind with high brix.
Melon type Fagous:
Fruit: Shape Medium short cylindrical; Size 300 to 1000 grs; Rind: Green skin. White, crispy, non sweet flesh. For consumption green in salad. Monoecious
IND 35: Fruit: Shape Long pear—ovoid; Size 300 to 1000 grs; Rind: Light Green spotted skin yellow at maturity. White, crispy, non sweet flesh. For consumption green in salad. Monoecious. Syngenta Seeds line, non climacteric.
YUSOL: Shape round to hight round; Size 600 to 1300 grs; Rind: Yellow few netted with light green sutures skin. Green-white, melty, few aromatic sweet non acid flesh. Short cycle Medium shelf life, non turning low climacteric. Andromonoecious Syngenta Seeds Line.
YUSAZ A: Shape round to hight round; Size 600 to 1300 grs; Rind: White-gray few netted with Green-Gray sutures skin. Green, specially crispy, few aromatic, sweet and acid flesh. Short cycle Medium-long self life, non turning, low to medium climacteric. Andromonoecious Syngenta Seeds Line.
YUSAZ B: Shape round to hight round; Size 600 to 1300 grs; Rind: Yellow netted with Green-Gray sutures skin. Green, specially crispy, few aromatic, sweet and acid flesh. Long cycle, long self life, non turning low climacteric. Andromonoecious Syngenta Seeds Line.
MILENIUN-DENEV: Shape round to hight round; Size 600 to 1500 grs; Rind: Yellow netted non sutured skin. Green-White, crispy, non aromatic, non acid and sweet flesh. Long cycle, non turning, long self life, non climacteric. Andromonoecious Syngenta Seeds Hybrid.
SOLAZ/1: Shape round to hight round; Size 700 to 1400 grs; Rind: Yellow few netted with light green sutures skin. White, crispy, few aromatic sweet and acid flesh. Short cycle, long self life, non turning, very low climacteric. Andromonoecious Syngenta Seeds Line.
SOLAZ/2: Shape round to hight round; Size 600 to 1300 grs; Rind: Yellow few netted with light green sutures skin. White, crispy, few aromatic sweet and acid flesh. Short cycle long self life, non turning, very low climacteric. Andromonoecious Syngenta Seeds Line.
MEHARI: monoecious hybrid from Syngenta in Charentais type. Turning medium shelf life.
L53: andromonoecious line from Syngenta in Charentais type. Turning medium shelf life. Small size round flat shape. Derived from var LUNASTAR, a monoecious hybrid of Nunhems in turning Charentais type.
TD: monoecious line from Syngenta in Charentais type. Non turning and long shelf life. Derived from var TORNADO, a monoecious hybrid from Limagrain in non turning Charentais type.
All references cited herein are incorporated by reference in the application in their entireties. When ranges are disclosed herein, it is understood that all the individual numbers falling within these ranges are also part of the invention.
The following examples are intended to provide illustrations of the application of the present invention. The following examples are not intended to completely define or otherwise limit the scope of the invention.
EXAMPLES
Example 1
Preparation of Melon Extracts
A wedge of ca. 400 g was taken from a melon fruit, the seeds and the skin (1 cm thickness) were removed. The flesh was cut in small parts, which were blended for 30 seconds in a Warring blender until a smooth slurry was obtained. The slurry was filtrated over a Whatman paper filter, the juice centrifugated in an Eppendorf centrifuge at 10.000 g and stored at −20 degrees Celcius.
Example 2
Determination of Citric Acid Content
Samples as prepared in Example 1 were incubated with citrate lyase (CL) to convert citric acid to oxaloactate and acetate. In the presence of the enzymes malate dehydrogenase (MDH) and lactate dehydrogenase (LDH), oxaloacetate and its decarboxylated derivative pyruvate were reduced with NADH to respectively L-malate and L-lactate. The decrease of NADH is proportional with the quantity of citric acid in the sample and can be determined at 340 nm. The assay was conducted in microplates. 20 μl of diluted sample was added to a microtiterplate. 200 μl of assay mixture containing NADH, MDH and LDH was added and mixed to the plate. The reaction was started with 15 μl of start-solution containing CL. The plate was mixed and let the reaction proceed for 1 hour. Absorbance values were measured at 340 nm with a microtiterplate reader (Biotek EL808 reader with KCJunior software and computer). A calibration curve was used to calculate the concentration citric acid in the samples. The enzymes were purchased from Roche Diagnostics.
Example 3
Determination of Malic Acid Content
Samples as prepared in Example 1 were incubated with L-malate dehydrogenase (MDH) and NAD+ to convert L-malate to oxaloacetate. The equilibrium of the reaction is on the side of malate but was forced to the side of oxaloacetate with hydrazine by means of derivatization. NADH formed during the assay is proportional with the malic acid content in the samples. NADH can be determined at 340 nm as a measure for malic acid.
The assay was conducted in microplates. 20 μl of diluted sample was added to a microtiterplate. The reaction was started by adding 200 μl of assay mixture containing hydrazine, MDH and NAD+ at pH 10. The plate was mixed and the reaction was allowed to proceed for 1 hour. Absorbance values were measured at 340 nm with a microtiterplate reader (Biotek EL808 reader with KCJunior software and computer). A calibration curve was used to calculate the concentration malic acid in the samples. MDH was purchased from Roche Diagnostics.
Example 4
Determination of Glucose, Fructose and Sucrose Contents
Glucose:
Glucose was determined with the enzymes hexokinase and glucose-6-phosphate dehydrogenase (G-6-PDH). Glucose was phosphorylated with hexokinase to glucose-6-phosphate (G-6-P) and subsequently dehydrated to 6-phosphogluconate with the aid of NADP and G-6-PDH (reactions 1 and 2, respectively). The concentration of formed NADPH (the H-acceptor) is quantitatively related to the initial glucose concentration and was measured at 340 nm in the UV range of the light spectrum.
Fructose:
Fructose was determined in the same assay. Fructose was phosphorylated to fructose-6-phosphate (F-6-P) with the enzyme hexokinase. F-6-P was converted to G-6-P with the enzyme phosphoglucose-isomerase (PGI) and subsequently in 6-PG as described in reaction 2 above.
Sucrose:
Sucrose was converted to glucose and fructose with the enzyme β-fructosidase. Formed glucose was determined according to the reactions 1 and 2 as described above. The determinations were conducted in microtiterplates, absorbance values are measured with a microtiterplate reader (Biotek EL×808 with data collection software and computer). Enzymes were purchased from Roche Diagnostics.
Example 5
Measurements of pH
The pH of samples as described in Example 1 was determined using a CRIMSON GLP21 pH meter calibrated to pH4 and pH7 with standard solutions.
Example 6
Growth Conditions of Melons Plants
Melon plants were grown under different conditions at different locations (see table 5 below). The dates of the trials are shown in Table 6 and the growing conditions in the different trial are described in Table 7.







TABLE 5







Description of trials












LOCATION IN SPAIN
CROP TYPE
CONDITIONS
















TORREPACHECO
EL EJIDO
VINE BEES
STACKED HAND

PLASTIC-
SUBSTRATE
















T. St
T. St.
POLLINATION
POLLINATION
OPEN FIELD
HOUSE
SOIL
HYDROPONIC



















FA02PS

X

X

X

X


SP03PV

X
X


X
X


SP03PS

X

X

X

X


SP03OF
X

X

X

X


SU03PS

X

X

X

X


FA03PS

X

X

X

X





FA: Fall,


SP: Spring,


SU: Summer,


PS: Plastic house staked crop,


PV: Plastic house vine crop













TABLE 6







Trial dates


TRIAL DATES















MEDIUM



SOWING
TRANSPLANTING
1ST POLLINATION
HARVEST















FA02PS
Aug. 1, 2002
Aug. 19, 2002
Sep. 1, 2002
Oct. 30, 2003


SP03PV
Jan. 18, 2003
Feb. 18, 2003
Apr. 4, 2003
May 15, 2003


SP03PS
Feb. 18, 2003
Mar. 18, 2003
Apr. 18, 2003
May 30, 2003


SP03OF
Mar. 10, 2003
Apr. 11, 2003
May 20, 2003
Jun. 28, 2003


SU03PS
Jul. 24, 2003
Aug. 8, 2003
Aug. 20, 2003
Oct. 6, 2003


FA03PS
Aug. 7, 2003
Aug. 22, 2003
Sep. 8, 2003
Nov. 3, 2003





Herein, SP03OF1 refers to analytical data of fruits harvested on Jun. 20, 2003. SP03OF2 refers to fruits harvested Jul. 4, 2003.













TABLE 7A







Growing conditions for FA02PS


Location: EL EJIDO T. St.


PLASTIC-HOUSE









Week no. 2002













29/30/
33/34/
37/
41/42/
45/46/



31/32
35/36
38/39/40
43/44
47/48
















temperature-day
31.5
31
28.6
22.4
18.4


temperature-night
25
23.1
22.4
17.8
14.4


temperature-24 hours
28.8
27.4
25.5
19.8
18


R. Humidity-day %
51
47
55
78
84


R. Humidity-night %
73
72
78
89
90


R. Humidity-24 h. %
60
69
66
84
88





Weeks are numbered starting on January 1, week 29 being the 29th week of the year.













TABLE 7B





Growing conditions for SP03PV, SP03PS, SU03PS, FA03PS


Location: EL EJIDO T. St., PLASTIC-HOUSE

















Week no. 2003
















1/2/3/4
5/6/7/8
9/10/11/12
13/14/15/16
17/18/19/20
21/22/23/24
25/26/27/28
29/30/31/32





temperature-day
15.9
16.7
25.1
26.9
26.9
31.2
37.4
37.1


temperature-night
10.4
11.1
17.9
20.3
21.9
25.6
28.6
28.5


temperature-24 hours
12.6
13.5
21.4
23.8
24.8
28.9
33.9
33.5


R. Humidity-day %
71
59
46
53
63
54
38
32


R. Humidity-night %
91
83
64
68
73
67
57
46


R. Humidity-24 h. %
83
73
55
61
67
59
45
38













Week no. 2003















33/34/35/36
37/38/39/40
41/42/43/44
45/46/47/48







temperature-day
30.9
25.3
20.8
19



temperature-night
25.1
21.8
17.9
15



temperature-24 hours
28.3
23.6
19.2
16.7



R. Humidity-day %
60
82
89
86



R. Humidity-night %
80
89
96
99



R. Humidity-24 h. %
69
86
92
93

















TABLE 7C







Growing conditions for SP03OF


TORREPACHECO T. St.


OPEN FIELD









Week no. 2003














13/14/
17/18/
21/22
25/26/



9/10/11/12
15/16
19/20
/23/24
27/28
















Temperature Max
28.0
36.0
34.0
33.0
37.0


Temperature avg. Max
19.9
26.2
27.0
28.0
34.5


Temperature Med
14.3
20.1
19.9
22.1
27.1


Temperature avg. Min.
8.8
14.0
12.7
16.2
19.7


Temperature Min.
5.0
8.0
9.0
12.0
17.0









Example 7
Transfer of the Low pH Trait to Elite Lines
1. Line IND-35 was crossed with a selected set of Syngenta elite lines in the El Ejido Trial Station (Spain).
The selected lines were:

    a).—YUCA-15: Selected because of high potential for sugars accumulation in medium long cycle (time from fruit set to fruit ripening). YUCA is a proprietary Syngenta Seeds line obtained through 5 generations of self-pollination. It is a Japanese Rocky melon type, non turning and very low climacteric.
    b).—OGEL-17: Selected because of medium short cycle (time from fruit set to fruit ripening) and medium high potential for sugars accumulation. This line is a proprietary Syngenta Seeds line obtained through 10 generations of self-pollination from a breeding F1 from cross between parental lines of commercial hybrids GUSTAL and RADICAL (Syngenta Seeds). It is a GALIA melon type, turning and medium climacteric.


F1 crosses produced self-pollinated F2 descendent generation. The F2 fruits were selected for low pH and sugar (Table 8A).
2. Both F2 populations were grown and cross pollinated plant by plant with a new selected set of Syngenta elite lines:

    a).—IND35/YUCA-15 F2 population was crossed with MG.755-68 (755), a proprietary Syngenta Seeds line obtained through 5 generations of self-pollination from a commercial hybrid BETULO (Syngenta Seeds). It is a GALIA turning and medium climacteric melon type selected for the purpose because of green flesh, high sugars, short cycle and relatively high citric acid content.
    b).—IND35/OGEL-17 F2 population was crossed with SEN19C8 (SN8). This line is a proprietary Syngenta Seeds line, non turning and very low climacteric, obtained through 6 generations of self-pollination of a commercial hybrid EARLS SEINU (Yae Nogey Seeds Co. Isahaya, Nagasaki, Japan). It is a Japanese Rocky melon type selected for the purpose because of high sugars and netting.


In these populations, the single dominance genetic regulation for the Low pH trait was verified and that no linkage existed between sugar accumulation and said Low pH. (Table 3). From each populations (150 plants each), recombinants including the higher R.I. Brix degree and the lowest pH were selected. Cross-pollinated seed progeny was collected.
The cross progenies selected from F2 population a) included the plants identified as:

    755YUCIND-19, 755YUCIND-49, 755YUCIND-75.


The one selected from F2 population b) included the plant identified as:

    SN80GLIND-03.


755YUCIND lines were selected for sucrose accumulation. SN80GLIND was selected for citric acid accumulation. These cross progenies were grown and produced data for pH, sugars and organic acid contents. Lines combining high sugars, low pH and high citric acid content were selected. The analysis of the selected plants is shown in Table 8B.
Table 8: Analysis of intermediates







TABLE 8A







Analytical Data extract for selected plants in F2


(Agadir Autumn 1998)















Geno



FEMALE
pH
BRIX
type







YUCA-15.071.xIND35-1)-49.
4.30
13.0
Aa



YUCA-15.071.xIND35-1)-75.
4.10
12.4
Aa



YUCA-15.071.xIND35-1)-19.
4.05
10.4
AA



OGEL-17.974.xIND35-1)-03.
4.57
11.8
Aa

















TABLE 8B







Analytical Data extract for progeny B1F1 (El Ejido Spring 1999)




















Citric acid
Malic acid
Glucose
Fructose
Sucrose
Total sugar
Brix
Nr.


MALE
FEMALE
pH
(mg/100 g)
(mg/100 g)
(g/100 g)
(g/100 g)
(g/100 g)
(g/100 g)
R.I.
Fruits






















4.7
773
52
2.3
2.4
1.7
6.3
10.0





4.5
930
48
1.8
2.0
4.4
8.2
12.0


SN8
OGLIND-03.
4.7
809
56
2.0
2.2
2.4
6.6
10.2
5




5.0
540
45
2.6
2.7
1.7
6.9
10.0




5.0
619
39
1.7
2.4
3.3
7.4
11.5


755
YUCIND-49.
4.8
535
67
2.0
2.5
1.8
6.3
9.9
5




5.0
549
38
2.5
2.7
2.7
7.9
12.0




5.4
564
52
2.6
2.7
2.2
7.4
11.0


755
YUCIND-75.
5.3
568
40
2.3
2.5
2.6
7.5
10.7
3




5.5
624
44
1.5
2.2
4.5
8.2
12.0




4.7
648
53
1.9
2.0
3.7
7.6
13.0


755
YUCIND-19.
5.1
569
44
1.7
2.1
3.3
7.2
11.1
8
















TABLE 8C







Analytical Data extract for progeny B2&3F1 (El Ejido Autumn 1999)



















Citric acid
Glucose
Fructose
Sucrose
Total Sugar
Brix
Nr.


MALE
FEMALE
pH
(mg/100 g)
(g/100 g)
(g/100 g)
(g/100 g)
(g/100 g)
R.I.
Fruits























4.7
802
2.5
2.5
2.9
7.9
12.5






4.7
1036
2.3
2.4
3.7
8.4
14.5





5.1
594
2.5
2.6
2.4
7.4
12.0


IOTYU
SN8
OGLIND-03.
4.7
771
2.3
2.4
2.4
7.1
12.6
9


755
755
YUCIND-19.


YUCA64
755)2
YUCIND-19.
















TABLE 8D







Analytical Data extract for progeny B3F1&2 (Torrepacheco Spring 2000)



















Citric acid
Glucose
Fructose
Sucrose
Total Sugar
Brix
Nr.


MALE
FEMALE
pH
(mg/100 g)
(g/100 g)
(g/100 g)
(g/100 g)
(g/100 g)
R.I.
Fruits





















SOLAR-19
IOTYU
SN8
OGLIND-03.










SOLAR-19
IOTYU
SN8
OGLIND-03.
4.14
725
1.9
1.6


15.0
13


IOTYU
IOTYU
SN8
OGLIND-03.
4.73
523
1.8
1.2


14.3
3



YUCA64
755)2
YUCIND-19.
4.87
566
2.3
1.7


14.7
3
















TABLE 8E







Analytical Data extract (El Ejido Autumn 2000)



















Citric acid
Glucose
Fructose
Sucrose
Total Sugar
Brix
Nr.


MALE
FEMALE
pH
(mg/100 g)
(g/100 g)
(g/100 g)
(g/100 g)
(g/100 g)
R.I.
Fruits






















SOLAZ4.4.4


4.32
573
3.0
1.2


7.0




SOLAZ4.7.2.


4.44
564
3.3
1.1


11.0



SOLAZ2.4.2


4.52
494
2.9
1.4


11.0



SOLAZ2.4.5


4.43
474
2.7
1.0


10.0


YUCA-40
IOTYU)2
SN8
OGLIND-03.
4.48
453
2.5
1.0


12.0



YUCA64
755)2
YUCIND-19.
4.79
904
4.8
1.8


13.0
















TABLE 8F







Analytical Data extract (El Ejido 2001)



















Citric acid
Glucose
Fructose
Sucrose
Total Sugar




MALE
FEMALE
pH
(mg/100 g)
(g/100 g)
(g/100 g)
(g/100 g)
(g/100 g)
Brix R.I.
Nr. Fruits






















SOLAZ 1


4.59
940
2.3
2.9
4.1
9.4
11.0




SOLAZ 2


4.63
770
2.5
2.7
1.3
6.6
8.0


YUSOL27
YUCA-40
IOTYU)2
SNC8OGLIND-
4.6
741
3.4
2.5
4.6
10.4
14.0





03.


YUSOL27
YUCA-64
755)2
YUCIND-19.
5.05
467
2.5
2.6
5.0
10.1
12.0


L53

755
YUCIND-75.
4.80
718
1.3
2.2
1.6
5.1
7.0


L53

755
YUCIND-49.
4.68
673
2.5
2.7
2.3
7.6
9.0









Example 8
Trait Introgresion into SOLAZ
The Low pH trait was introgressed in white flesh non climacteric LSL melons with round shape and yellow sutured skin.
1. Selected SN80GLIND-103 progeny plants were crossed with IOTYU, a proprietary Syngenta Seeds line obtained through generations of self-pollination from the traditional open pollinated Japanese variety Makuwauri EIJYU (Nanto seed Co. Ltd, Kashiwara, Nara, Japan). It was selected because of non-climacteric, non turning, very high sugar content, high citric acid content, yellow rind.
2. Selected plants of the progeny from previous cross were crossed with SOLAR-19 a proprietary Syngenta Seeds line obtained through 5 generations of self-pollination from a commercial hybrid SOLARKING F1 (Nunhems Zaden B V, Haelen, Holland). It is a long shelf life, non turning Galia melon type selected for the purpose because of non climacteric, very high sugar content, white flesh, yellow and netted rind.
Seven cycles of self-pollination were carried out from these cross progenies. The selection and fixation on the self-pollination progenies was done with analytical data for pH, sugars and organic acid contents (Table 8C and 8D). Fixed lines progenies were selected:

    A) SOLAZ 1 selected as Low pH in combination for high sugar accumulation, powdery mildew tolerance, and plant with Summer growing adaptation (Table 8E and F).
    B) SOLAZ 2 selected as Low pH with combination for medium sugar accumulation, and plant with Spring growing adaptation (Table 8E and F).


3. Progenies SOLAZ 1 and SOLAZ 2 were used as males pollinators for crosses with three proprietary Syngenta Seeds lines, YUSOL 1, 2 & 3, obtained through 6 generations of self-pollination from a breeding hybrid MD.997 F1 from cross YUCA-15×SOLAR-48 both proprietary Syngenta Lines with origin indicated before.
The following F1 combinations were obtained:

    A) YUSOL 1/SOLAZ 1; when using SOLAZ 1 as pollinator of lines YUSOL 1.
    B) YUSOL 2/SOLAZ 1; when using SOLAZ 1 as pollinator of lines YUSOL 2.
    C) YUSOL 3/SOLAZ 2; when using SOLAZ 2 as pollinator of lines YUSOL 3.


The analysis of these selected plants and commercially available controls is shown in Tables 9 and 10. The plants were grown under the conditions described in Example 6. The number of fruits tested in each experiment is shown (nr. Frt). The measurements were carried out as described in Example 1-5. The figures for sucrose (suc), hexoses (hex), and total sugars are in g per 100 g fresh weight (fwt). The figures for citric acid and malic acid are in mg per 100 g fresh weight (fwt).







TABLE 9A







SOLAZ






























tot.
tot.
hex/suc










Growing
nr
suc
suc
hex
hex
sugar
sugar
inv
pH
pH
citric
citric
citric/
malic
malic



conditions
frt
avg
stdev
avg
stdev
avg
stdev
activity
avg
stdev
avg
stdev
malic
avg
stdev



























SOLAZ/2
SP03PS
6
2.41
1.70
3.41
0.34
5.82

1.42
4.6
0.2
605
98
42
14
21



FA03PS
37
1.43
0.81
5.12
0.36
6.55
1.34
3.58
4.7
0.2
576
86
52
11
15


Average

43
1.92

4.27

6.19

2.50
4.6

590

47
13


SOLAZ/1
SP03PS
4
2.90
0.86
3.93
0.42
6.83

1.35
4.6
0.0
549
81
16
33
15



FA03PS
35
1.85
1.18
5.88
0.33
7.73
1.60
3.17
4.5
0.1
663
107
35
19
17.5


SOLAZ/1
SP03PS
4
2.01
0.22
3.82
0.20
5.83

1.90
4.6
0.1
513
53
20
26
11



FA03PS
16
1.80
0.74
5.32
0.27
7.13
1.25
2.95
4.5
0.1
591
83
52
11
10


SOLAZ/1
SP03PS
5
2.35
0.88
3.79
0.37
6.14

1.61
4.8
0.2
497
58
14
36
24



FA03PS
7
2.02
1.36
6.14
0.35
8.15
1.65
3.04
4.5
0.1
664
86
28
24
24


Average

71
2.16

4.81

6.97

2.34
4.6

579

27
25






















average SOLAZ/1 and /2
114
2.10

4.68

6.77


4.6

582

27
22
















TABLE 9B







YUSOL






























tot.
tot.
hex/suc










Growing
nr
suc
suc
hex
hex
sugar
sugar
inv
pH
pH
citric
citric
citric/
malic
malic



conditions
frt
avg
stdev
avg
stdev
avg
stdev
activity
avg
stdev
avg
stdev
malic
avg
stdev



























YUSOL/1
SP03PS
8
4.25
1.18
4.77
0.82
9.02

1.12
6.0
0.3
295
85
47
6
10



FA03PS
8
5.01
0.58
5.33
0.85
10.34
1.16
1.07
6.3
0.1
214
49
13
16
10


Average/1

16
4.63

5.05

9.68

1.09
6.1

255

30
11


YUSOL/2
SP03PS
6
2.58
0.62
4.99
0.81
7.57

1.94
5.8
0.1
297
83
424
1
3



FA03PS
1
3.92

7.30

11.21

0.54
5.9

344

X
0


Average/2

7
3.25

6.14

9.39

1.24
5.9

320


1


YUSOL/3
SP03PS
6
5.04
1.43
4.04
0.81
9.08

0.80
6.1
0.2
225
97
12
18
21



FA03PS
5
6.23
0.67
5.64
0.47
11.87
0.54
0.91
6.2
0.2
218
62
9
24
10


Average/3

11
5.63

4.84

10.47

0.85
6.2

221

11
21


Average 1, 2, 3

34
4.50

5.34

9.85

1.06
6.05

265

24
11
















TABLE 10A







Hybrid SOLAZ X YUSOL





























tot.
hex/suc










Growing
nr
suc
suc
hex
hex
sugar
inv
pH
pH
citric
citric
citric/
malic
malic



conditions
frt
avg
stdev
avg
stdev
avg
activity
avg
stdev
avg
stdev
malic
avg
stdev


























SOLAZ/1 X
SP03PV
4
5.58
1.07
3.38
0.51
8.95
0.61
4.8
0.1
845
87
11240
0
2


YUSOL/1
SP03OF1
5
6.25
0.43
3.71
0.70
9.96
0.59
4.7
0.1
794
25
76
10
12



SP03OF2
5
6.90
0.60
2.55
0.19
9.44
0.37
4.8
0.1
679
50
47
14
8


average/1

14
6.24

3.21

9.45
0.52
4.8

773

97
8


SOLAZ/1 X
SP03PS
3
3.45
0.24
4.68
0.52
8.14
1.36
4.7
0.1
697
32
45
16
12


YUSOL/2
SP03OF1
5
9.20
0.34
2.85
0.19
12.05
0.31
4.7
0.1
800
27
42
19
7



SP03OF2
4
7.01
1.30
2.69
0.40
9.70
0.38
4.8
0.1
744
46
39
19
4


average/2

12
6.56

3.41

9.97
0.68
4.7

747

42
18


SOLAZ/2 X
SP03PV
4
4.41
0.72
3.20
0.44
7.61
0.73
4.7
0.2
863
62
100
9
18


YUSOL/3
SP03OF1
4
4.94
2.05
3.23
0.29
8.17
0.65
4.5
0.1
789
85
277
3
4



SP03OF2
5
9.42
1.09
2.04
0.37
11.46
0.22
4.9
0.1
694
63
109
6
2


average/3

13
6.26

2.82

9.08
0.53
4.7

782

162
6


Average

39
6.35

3.15

9.50
0.58
4.7

767

70
11
















TABLE 10B







Controls






























tot.
tot.
hex/suc










Growing
nr
suc
suc
hex
hex
sugar
sugar
inv
pH
pH
citric
citric
citric/
malic
malic



conditions
frt
avg
stdev
avg
stdev
avg
stdev
activity
avg
stdev
avg
stdev
malic
avg
stdev



























SOLAR F1
SP03PV
6
5.28
0.93
2.52
0.32
7.80
0.92
0.48
6.1
0.08
500
51
28
18
26



FA03PS
2
3.50
0.05
6.55
0.17
10.06
0.22
1.87
6.9
0.04
109
17
X
0
0


Average

8
4.39

4.54

8.93

1.17
6.48

305

35
9


MILENIUM
SP03PV
4
2.99
1.23
4.94
0.67
7.93
1.57
1.65
5.8
0.1
546
25
X
0
0



SP03OF1
5
2.29
0.15
4.16
0.10
6.44
0.15
1.82
5.8
0.05
441
23
X
0
0



SP03OF2
3
5.15
3.82
3.30
0.99
8.45
4.81
0.64
6.1
0.4
345
40
X
0
0


Average

12
3.47

4.13

7.61

1.37
5.88

444

X
0









Example 9
Trait Introgresion into YUSAZ A
The Low pH trait was introgressed in green flesh non climacteric LSL melons with round shape and yellow sutured skin.
1. Direct back-cross was made from line 755YUCIND-19 using as recurrent parent, line MG.755-68 indicated above.
Selected plants of the progeny from previous cross were crossed with YUCA-64 a proprietary Syngenta Seeds line obtained through 5 generations of self-pollination from a same origin as the sister line indicated above.
This line was selected because of green flesh, non turning, non climacteric long-shelf life, very high sugars very crispy watermelon texture and sutures.
Three cycles of self-pollination from this cross progenies were carried out. The selection and fixation on the self-pollination progenies was made with analytical data for pH, sugars and organic acid contents (Table 8C-E).
2. Selected plants from progeny generated above were crossed with YUSOL.3 a proprietary Syngenta Seeds line described above. This line was selected because of green flesh, shorter cycle, very high sugars, yellow rind and sutures.
Five cycles of self-pollination from the previous cross progenies was made. Selection and fixation breeding process concentrated on the self-pollination progenies for short cycle, green flesh, crispy watermelon texture and yellow rind. Selection and fixation was assisted with analytical data for pH, sugars and organic acid contents. There were so selected and fixed lines progeny YUSAZ A.
3. YUSAZ A lines were used as males pollinators for crosses with three proprietary Syngenta Seeds lines, YUSOL 1 a proprietary Syngenta Seeds line described above. The F1 combination: YUSOL 1/YUSAZ A was obtained.
The analysis of these selected plants is shown in Table 11. The plants were grown under the conditions described in Example 6. The number of fruits tested in each experiment is shown (nr. Frt). The measurements were carried out as described in Example 1-5. The figures for sucrose (suc), hexoses (hex), and total sugars are in g per 100 g fresh weight (fwt). The figures for citric acid and malic acid are in mg per 100 g fresh weight (fwt).







TABLE 11A







YUSAZ A






























tot.
tot.
hex/suc










Growing
Nr
suc
suc
hex
hex
sugar
sugar
inv
pH
pH
citric
citric
citric/
malic
malic



conditions
frt
avg
stdev
avg
stdev
avg
stdev
activity
avg
stdev
avg
stdev
malic
avg
stdev



























YUSAZ A/1
SP03PS
16
3.01
1.28
5.36
0.67
8.37

1.78
4.6
0.2
602
90
28
22
13



FA02PS
3
2.05
0.43
5.62
0.37
7.67

2.74
4.6
0.1
655
135
21
31
1



SU03PS
6
3.70
1.00
5.76
0.53
9.46
1.92
1.55
4.6
0.2
514
115
23
23
18



FA03PS
43
2.10
0.73
7.01
0.44
9.11
1.25
3.34
4.6
0.2
558
82
44
13
18


Average

68
2.72

5.94

8.65

2.35
4.6

582

29
22


YUSAZ A/2
SP03PS
25
3.81
0.99
5.03
0.54
8.84

1.32
4.8
0.2
586
55
17
35
14



FA02PS
6
3.53
0.83
5.74
0.38
9.27

1.63
4.6
0.1
582
70
10
56
15



SU03PS
95
3.99
1.42
4.82
0.41
8.80
1.72
1.21
4.8
0.3
543
80
16
33
19



FA03PS
52
3.70
1.30
6.12
0.35
9.81
1.28
1.66
4.7
0.1
578
63
18
33
13


Average

178
3.76

5.43

9.18

1.46
4.7

572

15
39






















average YUSAZ A/1, /2
246
3.24

5.68

8.92

1.90
4.7

577

19
31
















TABLE 11B







F1 Hybrids using YUSAZ A






























tot.
tot.
hex/suc










Growing
Nr
suc
suc
hex
hex
sugar
sugar
inv
pH
pH
citric
citric
citric/
malic
malic



conditions
frt
avg
stdev
avg
stdev
avg
stdev
activity
avg
stdev
avg
stdev
malic
avg
stdev



























F1 Hybrid
SP03PS
12
5.24
1.83
5.09
0.99
10.33

0.97
4.9
0.2
659
113
69
10
7


YUSAZ A1/


YUSOL


F1 Hybrid
SP03PS
5
3.68
0.46
5.40
0.63
9.08

1.47
4.6
0.1
724
87
35
21
8


YUSAZ A2/


YUSOL


Average

17
4.46

5.24

9.70

1.22
4.8

692

46
15





Characteristics of line YUSAZ A are shown. YUSAZ A/1 and YUSAZ A/2 represent two sister lines. The F1 hybrids result from a cross with line YUSOL.






Example 10
Trait Introgresion into YUSAZ B
The Low pH trait was introgressed in green flesh non climacteric LSL melons with round shape and yellow sutured skin.
1. Selected plants of cross SN80GLIND-103×IOTYU progeny indicated above were back-crossed again with IOTYU, non turning and very low climacteric, green flesh melon (see Table 8D).
Selected plants of the progeny from previous cross were crossed with YUCA-40 a proprietary Syngenta Seeds line obtained through 5 generations of self-pollination from a same origin as the sister line indicated above (see Table 8E).
This line was selected because of green flesh, non climacteric long-shelf life, very high sugars very crispy watermelon texture and sutures.
2. Selected plants from progeny generated after one cycle of self-pollination from the one before were crossed with YUSOL.3 a proprietary Syngenta Seeds line described above. This line was selected because of green flesh, shorter cycle, very high sugars, yellow rind and sutures (see Table 8F).
Five cycles of self-pollination from the previous cross progenies were carried out. Self-pollination progenies were selected for green flesh, crispy watermelon texture, high citric acid content and yellow rind.
3. The selection and fixation was assisted with analytical data for pH, sugars and organic acid contents. Fixed lines progeny YUSAZ B were selected (Table 12).
YUSAZ B is an example of a “Citric+” melon plant.
YUSAZ B lines were used as males pollinators for crosses with three proprietary Syngenta Seeds lines, YUSOL 1 a proprietary Syngenta Seeds line described above. The F1 combination: YUSOL 1/YUSAZ B was obtained (Table 12).
The plants were grown under the conditions described in Example 6. The number of fruits tested in each experiment is shown (nr. Frt). The measurements were carried out as described in Example 1-5. The figures for sucrose (suc), hexoses (hex), and total sugars are in g per 100 g fresh weight (fwt). The figures for citric acid and malic acid are in mg per 100 g fresh weight (fwt).







TABLE 12







YUSAZ B






























tot.
tot.
hex/suc










Growing
nr
sucr
suc
hex
hex
sugar
sugar
inv
pH
pH
citric
citric
citric/
malic
malic



conditions
frt
avg
stdev
avg
stdev
avg
stdev
activity
avg
stdev
avg
stdev
malic
avg
stdev



























YUSAZ B/0
SP03PS
7
3.16
0.66
4.99
0.60
8.14

1.58
4.7
0.1
891
73
17
73
12



FA02PS
2
2.46
0.01
5.75
0.42
8.20

2.34
4.3
0.0
848
14
11
75
2



FA03PS
6
1.61
0.61
6.63
0.39
8.24
1.02
4.12
4.4
0.1
878
169
11
78
57


Average

15
2.41

5.79

8.19

2.68
4.5

872

13
75


YUSAZ B/1
SP03PS
6
1.81
1.20
6.74
0.40
8.55

3.71
4.7
0.2
696
133
39
18
17



FA02PS
1
2.72

6.76

9.48

2.48
4.7

739

18
41



FA03PS
8
1.21
0.45
8.39
0.20
9.60
0.69
6.94
4.7
0.1
939
37
26
36
16


Average

15
1.91

7.30

9.21

4.38
4.7

791

28
32






















average YUSAZ B/0, /1
30
2.16

6.54

8.70

3.53
4.6

832

16
53





Characteristics of line YUSAZ B are shown. YUSAZ B/1 and YUSAZ B/2 represent two sister lines. The F1 hybrids result from a cross with line YUSOL.






Example 11
Trait Introgresion to MEHARI F1
The low pH trait was introgressed in orange flesh melons. The selected target was the conversion of the commercial hybrid MEHARI (Syngenta Seeds) into two low pH versions, one with higher citric acid content and one with lower citric acid content.
1. Direct backcrosses from lines 755YUCIND-49 and 755YUCIND-75 were conducted using as recurrent parent line L53, a proprietary Syngenta Seeds. L53 is a Charentais melon type selected for the purpose because of high sugars, low citric content. L53 is a parent of the commercial hybrid MEHARI (see Table 8F).
During the backcross process and within the progeny of each cross plants with low pH and more or less acid perception (higher or lower citric content) were selected, in addition to other traits to be closer to the recurrent L53.
After the second back-cross (third cross) the following progenies were obtained:

    a) L53)3)×755YUCIND75-15/03/06/, selected for low pH and low citric acid content
    b) L53)3+)×755YUCIND49-8/2/03/05/, selected for low pH and high citric acid content.


Four cycles of self-pollination from these cross progenies and one further back-cross with L53 were carried out. The selection and fixation breeding process on the self-pollination progenies was assisted with analytical data for pH, sugars and organic acid contents.
2. Fixed lines progenies were selected:

    A) L53AZ A selected from previous population a) and fixed for low citric acid content and low pH and, for other traits, close to the recurrent L53.


Line L53AZ A is an example of a “Citric-” melon plant.

    B) L53AZ B selected from previous population b) and fixed for low pH and high citric acid content and, for other traits, close to the recurrent L53.


Plants were also selected for intense orange flesh color in presence of the low pH trait. The intensity of the orange color in the flesh had to be particularly selected for, as a generally poor orange color intensity tended to be associated within low pH acid in the fruit flesh (see Table 4).
Progenies lines L53AZ A and L53AZ B, and line L53 were used in crosses with the other parent line of MEHARI to obtain MEHARI AZ A, MEHARI AZ B and MEHARI the current commercial F1, respectively.
All these products, lines and crosses were tested in agronomic trials and analytical data were collected from fruits (Table 13).
The plants were grown under the conditions described in Example 6. The number of fruits tested in each experiment is shown (nr. Frt). The measurements were carried out as described in Example 1-5. The figures for sucrose (suc), hexoses (hex), and total sugars are in g per 100 g fresh weight (fwt). The figures for citric acid and malic acid are in mg per 100 g fresh weight (fwt).







TABLE 13A







MEHARI, low pH/high pH






























tot.
tot.
Hex/suc










Growing
nr
suc
Suc
Hex
hex
sugar
sugar
inv
pH
pH
citric
citric
citric/
Malic
malic



conditions
frt
avg
Stdev
Avg
stdev
avg
stdev
activity
avg
stdev
avg
stdev
malic
avg
stdev



























Male high pH
FA02PS
4
4.11
1.66
2.93
0.67
7.04

0.71
6.9
0.2
65
26
1
114
38


cit/—(L53)
SP03PS
3
1.98
0.85
2.70
2.05
4.68

1.36
6.2
0
52
11
1
55
26



FA03PS
6
9.07
0.67
4.07
0.40
13.14
1.06
0.45
6.9
0.2
159
40
1
109
18






















Average/High pH−
10
5.05

3.23

8.29


6.7

92

1
93
























Male acid cit/—
SP03PS
4
2.75
1.65
3.85
0.38
6.61

1.40
5.0
0.3
442
168
4
109
51


(L53AZ A)
SU03PS
24
2.03
1.40
3.35
1.11
5.82
1.83
1.65
4.8
0.3
484
132
3
150
64



FA03PS
48
2.76
1.85
6.07
0.62
8.82
2.19
2.20
4.8
0.4
600
158
3
200
130






















Average/low pH, citric−
76
2.51

4.42

7.09


4.9

509

3
153
























Male acid
SP03PS
3
4.82
1.37
2.72
0.30
7.54

0.56
5.0
0.1
654
86
9
75
23


(L53AZ B)
SU03PS
12
3.74
1.50
3.33
0.89
7.08
1.64
0.89
4.7
0.1
670
95
10
64
26



FA03PS
37
4.14
1.64
5.57
0.70
9.71
1.76
1.35
4.7
0.2
779
74
12
63
20






















Average/low pH, citric+
52
4.24

3.87

8.11


4.8

701

11
67
















TABLE 13B







Hybrids of MEHARI, low pH/high pH






























Tot.
tot.
Hex/suc










Growing
nr
suc
Suc
hex
hex
Sugar
sugar
Inv
pH
pH
citric
citric
citric/
malic
malic



conditions
frt
avg
Stdev
avg
stdev
Avg
stdev
activity
avg
stdev
avg
stdev
malic
avg
stdev



























F1 Hybrid
SP03PS
7
5.06
1.26
3.15
0.69
8.21

0.62
6.6
0.2
233
70
3
73
10


MEHARI
FA03PS
7
2.86
0.80
6.53
1.42
9.40
1.70
2.28
6.0
0.2
316
137
9
34
23


High pH


Average

14
3.96

4.84

8.80

1.22
6.3

274

5
54


F1 Hybrid
SP03PS
7
3.10
1.42
4.46
0.53
7.55

1.44
5.4
0.2
447
85
6
79
22


low pH, citric−
FA03PS
6
1.39
1.46
5.75
0.66
7.14
1.99
4.14
4.6
0.2
527
80
7
76
27


Average

13
2.24

5.10

7.34

2.27
5.0

487

6
78


F1 Hybrid
SP03PS
7
3.19
1.22
4.56
0.67
7.76

1.43
4.8
0.1
619
78
12
53
23


Low pH, citric+
SP03OF1
3
5.82
1.55
3.63
0.76
9.45
0.86
0.62
4.9
0.2
718
57
20
37
7



FA03PS
7
1.12
0.52
6.73
0.56
7.84
0.88
6.02
4.5
0.1
654
128
7
92
69


Average

17
3.37

4.97

8.35

1.47
4.76

663

11
60









Example 12
Sensory Analysis
Sensory analysis of fruits of plants of the instant invention was performed by an Expert panel. The panel was composed of 12 persons, especially trained to describe melons texture, sugar and acid savours. Six sessions of training were carried out before the expert sessions. The training program of the study was:

    
    
        Training 1: Reading of the lexicon of tasting. Notation of 6 products in the assembly room and discussion in order to see which descriptor is difficult to agree about.
        Training 2: Notation of 2 products+2 repeated in laboratory of sensory analysis to see how the panel evaluate the descriptors personally.
        Training 3: Results of the training 2 are presented to the panel. Notation of 2 products+2 repeated in the assembly room and discussion. Notation of 2 products in the individual cabins.
        Training 4: Works on sugar and acid solutions at different concentrations to evaluate the difficulties of the panel on these descriptors. Results of the training 3 are presented to the panel. Notation of 3 products+1 repeated in the assembly room and discussion. Notation of 3 products+1 repeated in individual cabins.
        Training 5. Notation of 4 products in the assembly room and notation of 2 products+2 repeated in the individual cabins. It allows us to check the repeatability of the tasters inside the session. Works on acid solutions.
        Training 6: Notation of 3 products in the assembly room and notation of 2 products+2 repeated in the individual cabins to check the repeatability of the tasters inside the session.
    
    


The sensory analysis was carried out in an air-conditioned sensory analysis laboratory equipped with individual cabins. A structured scale of quotation in 10 points (from 0 to 9) was used with the following descriptors:
Sweet Savor
Definition: It is about the perception of the sweet savour perceived in mouth. Mode of evaluation: Chew the product until its disappearance. Estimate the intensity of the sweet savour. Notation: 0=not sweet, 9=very sweet
Acid Savor
Definition: It is about the perception of the acid savour perceived in mouth. Mode of evaluation: Chew the product until its disappearance. Estimate the intensity of the acid savour. Notation: 0=not acid, 9=very acid.
Protocol of Preparation


    
    
        Wash of melons in the cold water
        Longitudinal cutting of the melon
        Removing of the pulp and the seeds
        Cutting each half of melons in 3 parts (longitudinal cutting)
        Removing of the extremities of 3 parts
        Cutting each part in 2 in order to have 6 parts on the half of the melon

Presentation of the Products

        Samples are presented coded with random numbers and according to a plan of presentation (Latin Square) avoiding the effects of presentation order.
        The products are given one after the other.
    
    


Tables 14 and 15 show the results of two sensory analysis conducted on different fruits.
In Table 14, plants were sown in early March and transplanted to open protected fields as vine crops in early April. Fruits were harvested in early July. In Table 14 A, fruits were stored for 7 to 12 days at 10° C. followed by 2 days at 20° C. In Table 14 B, fruits were stored for 4 to 8 days at 10° C. followed by 2 days at 20° C.
In Table 15, plants were sown in early August and transplanted to plastic houses as staked crops in late August. Fruits were harvested in early November. Fruits were stored for 4 to 8 days 5° C. followed by 3 days at 20° C.
Sensory analysis was performed on the number of fruits indicated in the Tables. Each fruit was tested by the 12 qualified tasters. The figures for acid and sugar savors for each line represent the average of the number of fruit times 12 (for the 12 tasters). Tables 14 and 15 also show measurements of the pH and contents of sugars and organic acid for the fruits tested.



















TABLE 14A







FRT


GLU
SUC
FRU
Tot sug
Citric Ac
Malic Ac
ACID
SUGAR



nr
Brix
pH
g/100 g
g/100 g
g/100 g
g/100 g
mg/100 g
mg/100 g
savor
savor




























YUSOL/1 X
Avg
10
11.7
6.24
1.2
7.2
2.1
10.6
309
4
0.5
5.6


SOLAZ/1 basic
Std Dev

2.1
0.30
0.3
2.3
0.3
1.9
121
10
0.3
1.0


YUSAZ A
Avg
6
12.7
4.79
1.1
7.7
2.0
10.7
808
0
2.1
5.4


X YUSOL
Std Dev

0.5
0.09
0.2
0.6
0.2
0.5
17
0
0.7
0.5


SOLAZ/2
Avg
10
13.5
4.68
1.5
7.9
2.0
11.3
832
18
3.6
4.7


X YUSOL/3
Std Dev

1.4
0.17
0.5
1.0
0.3
1.0
37
20
0.5
0.3


SOLAZ/1
Avg
10
13.2
4.58
1.8
7.2
2.4
11.4
816
41
3.1
4.7


X YUSOL/1
Std Dev

0.8
0.22
0.6
1.2
0.4
0.6
37
32
1.2
0.6


















Average SOLAZ
20
13.4
4.63
1.7
7.6
2.2
11.4
824
30
3.4
4.7


X YUSOL




























TABLE 14B







FRT


GLU
SUC
FRU
SUG
Citric Ac
Malic
ACID
SUGAR



nr
Brix
pH
g/100 g
g/100 g
g/100 g
Tot
mg/100 g
mg/100 g
savor
savor




























Female Mehari
Avg
6
13.2
6.64
1.0
8.5
1.7
11.2
272
63
0.5
5.8


X L53
Std Dev

1.7
0.09
0.1
1.5
0.2
1.5
54
32
0.2
0.6


Female Mehari
Avg
7
13.4
5.20
1.1
7.8
1.9
10.8
652
88
1.7
5.2


X L53 AZ A/1
Std Dev

0.7
0.11
0.2
0.8
0.3
0.9
87
69
0.4
0.4


Female Mehari
Avg
5
13.1
5.28
1.2
8.1
2.0
11.3
581
107
1.6
5.3


X L53 AZ A/2
Std Dev

0.8
0.08
0.2
0.9
0.1
0.8
50
40
0.4
0.6


















AVG Female Mehari
12
13.3
5.24
1.2
8.0
2.0
11.1
617
98
1.7
5.3


X L53 AZ A



















Female Mehari
Avg
6
12.2
4.98
1.2
7.1
1.9
10.2
708
107
3.2
4.6


X L53 AZ B
Std Dev

1.0
0.21
0.1
0.7
0.2
0.9
78
44
0.6
0.3




























TABLE 15







FRT


GLU
SUC
FRU
Tot sug
Citric Ac
Malic Ac
ACID
SUGAR



nr
Brix
pH
g/100 g
g/100 g
g/100 g
g/100 g
mg/100 g
mg/100 g
savor
savor




























SOLAZ/2
Avg
3
9.60
4.53
1.97
2.98
2.47
7.41
717
0
3.78
4.31


X YUSOL/3
Std Dev

0.53
0.06
0.36
0.30
0.36
0.94
39
0
0.84
0.55


SOLAZ/1
Avg
2
11.00
4.74
2.14
4.04
3.05
9.24
574
0
2.54
5.63


X YUSOL/1
Std Dev

0.85
0.09
0.35
0.00
0.09
0.44
169
0
1.20
0.06


















Average SOLAZ
5
10.2
4.6
2.0
3.4
2.7
8.1
660
0
3.3
4.8


X YUSOL



















YUSOL X
Avg
5
12.40
4.71
3.04
4.02
3.59
10.65
713
18
2.95
5.47


YUSAZ A
Std Dev

1.04
0.17
0.56
1.04
0.67
0.68
42
15
0.40
0.75


TD X L53
Avg
8
12.13
4.97
1.23
5.53
2.06
8.82
696
74
2.56
5.72


AZ/A
Std Dev

1.13
0.14
0.36
1.13
0.32
0.80
83
41
0.85
0.39


Mehari female
Avg
3
9.50
5.10
1.48
3.48
2.52
7.48
632
62
2.17
5.50


X L53 AZ/A
Std Dev

1.32
0.27
0.17
0.70
0.37
1.17
79
25
0.43
0.55









Example 13
Molecular Marker Analysis
DNA was extracted from young leaves (15 day-old seedlings). Leaves were freeze-dried and DNA was extracted following the method of Dellaporta (Dellaporta 1983).
PCR cycling conditions were: 15 s denaturation at 94° C. followed by 15 s annealing at 54° C. and 30 s extension at 72° C. for 40 cycles. Sample's DNA was initially denatured for 2 minutes at 94° C. and extended for 2 min at 72° C. after PCR.
The PCR mix contained 1.65 mM MgCl2, 60 mM of each deoxyribonucleotide, 1× Taq Buffer, 0.2 unit Taq polymerase, 15-20 ng template DNA and 400 nM of each non-fluorescent primer or 200 nM of each fluorescently labeled primer. Fluorescent primers were labeled with 6-FAM, NED or HEX. Fluorescent PCR products were separated on an ABI3700 capillary sequencer and their sizes measured using Applied Biosystem's Genescan and Genotyper fragment analysis software.
Non-fluorescent PCR products were separated by electrophoresis in 3% agarose gels (Resophor, Eurobio) at 400V with cooling system. Gels were stained with ethidium bromide. Three markers were used, CMAT141 (described in Danin-Poleg et al. (2001) Theor. Appl. Genet. 102: 61-72 and Danin-Poleg et al. (2002) Euphytica 125: 373-384) and NE0585 and NE1746. The primers for these markers are shown below.














Forward Primer
Reverse Primer



(5′-3′)
(5′-3′)


















CMAT141
AAGCACACCACCACCCGTAA
GTGAATGGTATGTTATCCTTG



(SEQ ID NO: 1)
(SEQ ID NO: 2)


 


NE0585
GTATCATGTCGGAGAAACG
CCTTTATCCCCACTTTTTC



(SEQ ID NO: 3)
(SEQ ID NO: 4)


 


NE1746
TTCTCCGATGTGTCCTCTC
GTCGCTTGGAATATATCGG



(SEQ ID NO: 5)
(SEQ ID NO: 6)









Example 14
Analysis of Accessions
A number of melon accessions comprising a low pH trait were analyzed using the markers above. The sizes of the amplified fragments are reported in Table 16 below. The indicated sizes (in bp) are not absolute but relative to the other size products detected with the same primer pair. The real (exact) size of the amplified fragments (e.g. determined by sequencing) could be slightly different (+/−1 bp) of those indicated herein.











TABLE 16







CMAT141
NE0585
NE1746





























IND35-1.2.

173



230





127



IND35-2.3.
168





232



124


FAGOUS2:4-



176
228



253
121


FAGOUS2:5-



176





121


142


FAGOUS2:7-



176




253



142


PI414723


175

228





124


PI414724


175







124


PI161375


175

228





124


PI124112


175




239


124









Example 15
Evolution of Fruit Characteristics of Fruits Kept on the Plant
Plants were grown as for the plants in Table 15 above. Fruits were harvested at different maturation times points (cycle) calculated by the difference between the harvest date (HDT) and the pollination date (PDT). Maturity was considered as reached at a particular time point in the cycle, when the average of the sucrose content for fruits at the particular time point in the cycle reached 2.0 g sucrose per 100 g fwt and using other ripening signs such as side leaf senescence. The results are shown in Table 17.
The results are based on individual fruit measurements and it is understood that some fruits may yield deviating values because of damages, aberrant development or other environmental factors.





















TABLE 17












Flesh
















Orange



FRT



Colour


GLU
SUC
FRU
HEX
Tot sug
Citric Ac
Malic Ac



nr
PDT
HDT
Cycle
1 to 5
pH
Brix
g/100 g
g/100 g
g/100 g
g/100 g
g/100 g
mg/100 g
mg/100 g





TD X
1
17
37
20
1
5.09
5.0
1.6
0.0
1.4
2.6
3.0
169
460


L53 AZ A/1
1
16
37
21
1
4.83
5.4
1.8
0.0
1.7
3.9
3.5
167
273


Maturity
1
14
37
23
1
4.94
4.6
1.4
0.0
1.4
2.7
2.8
128
243


reached between
1
16
40
24
1
4.55
4.8
1.4
0.0
1.2
2.8
2.6
387
314


cycle 33 and 40
1
16
40
24
1
4.66
5.0
1.2
0.0
1.7
2.9
2.9
387
114



1
14
40
26
2
4.88
6.5
1.9
0.0
1.4
2.3
3.3
498
255



1
14
40
26
2
4.44
7.0
1.4
0.6
2.7
4.1
4.7
852
119



1
14
44
30
2
4.36
6.7
2.3
0.4
2.0
3.2
4.6
776
178



1
14
44
30
2
4.47
7.0
1.7
0.5
2.2
3.9
4.3
528
77



1
14
44
30
2
4.70
7.0
2.3
0.2
2.4
4.7
5.0
649
11



1
14
47
33
2
4.37
6.5
2.0
0.0
1.6
3.2
3.6
730
174



1
14
47
33
2
4.59
7.0
1.2
1.0
1.7
2.9
3.9
453
117



1
14
47
33
3
4.85
13.6
2.3
3.9
1.9
4.0
8.2
876
199



1
14
54
40
3
5.02
13.0
1.0
7.3
2.2
3.2
10.5
663
89



1
17
57
40
3
4.83
11.5
1.5
4.5
2.4
3.9
8.4
590
155



1
16
57
41
3
4.79
14.0
1.8
6.9
2.1
3.9
10.9
890
31



1
16
58
42
4
4.75
13.3
2.1
5.2
2.0
3.1
9.2
985
104



1
14
57
43
3
4.96
12.7
1.7
5.5
1.9
4.3
9.0
893
167



1
14
57
43
4
5.24
14.0
1.4
8.4
2.3
3.7
12.1
648
40



1
14
57
43
4
5.06
12.5
1.0
5.9
1.8
2.8
8.7
623
48



1
14
57
43
4
5.00
12.5
1.3
5.7
2.2
3.4
9.2
758
29


TD X
1
17
37
20
1
4.45
4.8
1.7
0.1
1.8
3.5
3.5
307
319


L53 AZ A/2
1
17
37
20
1
4.53
5.2
1.9
0.0
2.0
5.0
3.9
331
332


Maturity
1
14
37
23
1
4.43
4.1
1.0
0.0
1.1
2.3
2.1
572
323


reached between
1
17
40
23
3
4.55
7.0
1.3
0.8
1.7
3.0
3.8
682
120


cycle 34 and 38
1
14
40
26
2
4.48
6.0
1.3
0.3
1.8
3.2
3.5
590
136



1
14
40
26
3
4.44
6.2
1.9
0.0
1.9
1.5
3.8
594
206



1
17
44
27
2
4.36
4.9
1.5
0.0
1.3
2.8
2.9
640
295



1
19
47
28
3
4.33
8.9
2.8
0.0
2.7
4.4
5.5
977
156



1
14
44
30
2
4.65
6.0
1.0
1.3
2.4
3.4
4.8
715
56



1
17
51
34
3
4.43
10.1
2.9
1.0
2.9
5.9
6.8
989
141



1
17
51
34
3
4.77
11.0
1.9
3.5
2.5
4.4
7.9
836
31



1
17
51
34
3
4.91
10.5
1.3
5.3
1.9
3.2
8.5
753
96



1
16
51
35
3
4.31
8.6
2.2
0.9
2.1
5.0
5.3
970
216



1
16
54
38
3
4.76
14.1
2.3
5.5
2.2
3.8
10.1
983
164



1
14
54
40
4
4.80
14.8
2.1
7.0
2.1
4.0
11.3
992
166



1
14
54
40
4
5.03
15.0
0.8
9.0
1.8
2.6
11.6
843
5



1
17
58
41
3
4.99
14.5
1.6
8.0
2.1
3.6
11.6
909
75



1
14
57
43
4
4.97
12.0
0.8
5.8
1.7
2.5
8.2
635
76



1
14
57
43
5
5.20
14.0
1.2
6.3
1.7
2.9
9.2
711
64



1
14
58
44
4
5.06
16.0
1.6
8.5
1.7
3.4
11.9
986
50

























FRT






GLU
SUC
FRU
HEX
Tot sug
Citric Ac
Malic Ac



nr
PDT
HDT
Cycle
Colour
pH
Brix
g/100 g
g/100 g
g/100 g
g/100 g
g/100 g
mg/100 g
mg/100 g





Female Mehari
1
21
40
20
1
4.45
4.7
1.5
0.0
1.3
1.3

437
292


X L53 AZ A/1
1
17
37
20
1
4.62
4.5
1.1
0.1
1.4
2.5
2.6
295
98


Maturity
1
17
40
23
1
4.78
5.0
1.0
0.2
1.9
2.9
3.1
482
71


reached between
1
14
40
26
2
4.54
5.0
1.1
0.1
1.6
2.7
2.8
397
81


cycle 36 and 38
1
17
44
27
2
4.46
5.0
1.2
0.4
1.9
3.1
3.4
547
38



1
17
44
27
2
4.43
4.9
1.6
0.0
1.4
1.4
3.0
561
187



1
17
44
27
2
4.52
6.0
1.5
0.7
1.9
3.4
4.1
505
18



1
19
51
32
4
4.81
8.0
1.3
2.7
2.2
3.5
6.2
575
76



1
19
51
32
2
4.50
6.2
2.0
0.0
1.6
2.8
3.6
592
75



1
19
51
32
3
4.76
7.0
1.5
1.4
2.0
3.5
4.9
465
105



1
14
50
36
3
5.99
8.6
1.5
2.5
1.6
2.6
5.6
414
259



1
16
53
37
5
6.28
9.0
1.4
3.0
1.8
3.3
6.3
297
166



1
17
54
37
4
5.57
8.5
1.7
2.6
2.6
4.3
6.9
309
261



1
17
54
37
2
5.68
8.4
1.6
1.7
1.4
2.6
4.8
339
276



1
14
51
37
3
4.86
5.8
1.3
0.4
1.4
3.9
3.1
446
339



1
14
51
37
4
6.36
9.0
1.5
2.7
1.9
3.4
6.1
276
167



1
17
54
37
4
5.21
12.0
1.6
4.5
1.9
3.5
8.0
532
56



1
16
54
38
5
5.79
9.5
1.4
3.8
2.7
4.1
7.9
351
264



1
17
57
40
2
5.47
9.0
1.7
1.5
1.4
4.0
4.6
410
312



1
17
57
40
4
4.63
10.9
2.4
3.0
2.3
3.8
7.7
894
111



1
17
57
40
5
6.22
9.5
1.5
3.8
2.1
3.6
7.4
360
277


Female Mehari
1
17
37
20
1
4.84
4.5
0.7
0.3
1.7
2.5
2.7
368
33


X L53 AZ A/2
1
17
37
20
1
5.25
4.7
1.6
0.0
1.5
2.5
3.1
210
375


Maturity
1
17
37
20
1
5.31
4.3
1.4
0.0
1.2
2.6
2.6
200
288


reached between
1
17
44
27
2
4.52
4.9
1.7
0.0
1.4
2.4
3.1
320
190


cycle 35 and 37
1
17
44
27
2
4.49
5.0
0.8
0.3
1.7
2.5
2.8
423
5



1
17
44
27
2
6.69
5.3
1.5
0.0
0.8
3.4
2.4
238
160



1
17
44
27
2
4.47
6.0
2.1
0.0
1.7
7.5
3.8
437
183



1
21
51
30
3
4.63
9.5
2.6
1.4
2.4
4.3
6.4
548
150



1
17
47
30
2
4.53
7.4
2.4
0.1
2.1
4.0
4.6
672
126



1
17
47
30
3
4.60
6.5
1.5
1.0
2.3
3.8
4.8
562
44



1
17
51
34
2
4.68
6.5
2.0
0.2
1.9
2.6
4.1
713
99



1
17
51
34
3
4.50
9.9
2.7
1.5
2.5
4.9
6.7
944
118



1
16
51
35
4
5.35
10.5
1.7
4.0
2.9
4.6
8.6
723
32



1
16
51
35
4
4.77
11.7
2.2
3.2
2.2
3.7
7.6
904
133



1
14
50
36
2
5.84
7.1
1.4
0.6
1.5
2.6
3.5
224
168



1
14
50
36
4
6.09
7.0
1.1
2.1
2.1
3.2
5.3
273
201



1
14
50
36
2
5.95
8.3
1.6
1.7
1.8
3.7
5.1
279
235



1
14
50
36
5
6.51
9.5
1.7
2.9
2.7
4.4
7.3
336
202



1
14
51
37
4
5.13
10.0
1.5
3.8
2.4
3.9
7.7
599
77



1
19
57
38
3
5.60
13.1
2.3
4.1
2.2
3.3
8.6
613
150



1
19
57
38
3
4.62
12.2
3.0
3.7
2.7
5.2
9.4
1011
63
























FRT





GLU
SUC
FRU
HEX
Tot sug
Citric Ac
Malic Ac



nr
PDT
HDT
Cycle
pH
Brix
g/100 g
g/100 g
g/100 g
g/100 g
g/100 g
mg/100 g
mg/100 g





SOLAZ/2
x2
9
37
28
4.23
5.0
1.6
0.1
1.7
4.0
3.3
664
116


X YUSOL/3
x2
9
37
28
4.30
5.1
1.8
0.0
1.9
4.8
3.7
496
123


Maturity
x2
9
37
28
4.21
5.9
2.4
0.0
2.0
3.7
4.4
674
77


reached between
1
9
37
28
4.53
6.0
2.3
1.0
2.6
4.8
5.9
675
102


cycle 38 and 41
x2
9
37
28
4.19
5.3
1.6
0.1
1.8
3.9
3.5
703
123



x2
9
37
28
4.37
4.6
1.4
0.0
1.4
2.9
2.8
509
264



x2
10
40
30
4.18
5.5
2.0
0.0
1.9
3.2
3.9
545
94



x2
9
40
31
4.21
6.4
2.1
0.0
2.1
3.5
4.1
795
71



1x
9
40
31
4.25
7.2
2.9
0.0
2.9
4.8
5.8
723
186



1
9
40
31
4.37
6.8
2.3
0.3
2.8
5.1
5.4
667
55



x2
9
40
31
4.15
5.5
1.9
0.0
2.0
4.7
3.9
728
146



1x
9
44
35
4.21
8.6
2.8
0.0
3.3
6.4
6.2
990
34



x2
9
44
35
4.70
7.0
2.1
1.7
3.0
5.0
6.7
706
0



x2
9
44
35
4.18
7.3
2.3
0.0
2.8
5.3
5.1
984
32



x2
9
44
35
4.17
7.3
2.1
0.0
2.7
5.8
4.8
992
28



x2
9
44
35
4.06
6.5
2.3
0.0
2.3
4.9
4.6
990
67



1
9
44
35
4.34
7.2
1.9
0.5
2.3
4.2
4.7
736
14



1
9
47
38
4.49
10.2
2.4
3.3
2.8
5.2
8.5
761
0



x2
9
47
38
4.24
9.0
2.6
0.6
3.1
6.7
6.2
782
15



x2
9
47
38
4.21
10.5
3.1
0.9
3.4
7.3
7.5
834
41



1
9
47
38
4.60
9.2
1.7
2.7
2.5
4.2
6.9
695
0



x2
9
47
38
4.21
10.3
2.9
1.2
3.0
6.2
7.1
1052
53



x2
9
50
41
4.44
9.2
2.2
2.0
2.4
4.7
6.6
701
24



x2
9
50
41
4.69
10.5





803
66



x2
9
50
41
4.76
10.2





853
37



x2
9
50
41
4.77
10.4
1.8
3.6
2.8
4.6
8.2
637
1



x2
9
50
41
4.56
12.2





943
0



1
9
50
41
4.51
9.4
1.8
2.9
2.1
3.9
6.9
694
0



x2
9
51
42
4.55
10.5





887
0



x2
9
54
45
4.28
12.4
2.7
3.3
2.6
5.0
8.7
982
48



x2
9
54
45
4.23
10.8
2.4
3.0
2.6
4.7
8.0
1026
61



1
9
54
45
4.44
12.9
2.7
4.7
2.4
3.6
9.8
997
52


SOLAZ/1
x2
10
37
27
4.36
4.8
1.7
0.0
1.7
3.7
3.3
526
136


X YUSOL/1
1
10
37
27
4.84
4.0
1.4
0.4
2.1
3.5
3.9
428
27


Maturity
1
9
37
28
4.51
4.5
2.1
0.0
2.1
4.2
4.2
476
86


reached between
1
9
37
28
4.49
5.4
2.1
1.1
2.4
4.5
5.6
524
55


cycle 38 and 41
1
9
40
31
4.42
6.2
1.8
0.4
2.8
4.6
5.1
598
62



1
9
40
31
4.38
5.0
1.6
0.1
2.0
3.6
3.7
546
94



1
9
40
31
4.25
5.8
1.9
0.0
2.1
3.6
3.9
642
91



1
9
44
35
4.53
7.0
2.2
1.6
3.2
5.4
7.0
654
0



1
9
44
35
4.24
8.7
2.7
0.5
3.0
5.3
6.1
977
70



1
9
44
35
4.44
7.6
2.0
0.8
2.6
4.6
5.4
498
0



1
9
44
35
4.38
8.6
2.3
1.4
3.1
5.5
6.9
640
6



1
9
47
38
4.42
10.4
1.8
2.4
2.6
4.4
6.9
733
0



1
9
47
38
4.53
8.0
2.0
1.8
3.0
4.9
6.7
540
0



1
9
47
38
4.74
8.5
2.2
1.6
2.7
5.4
6.5
600
3



1
9
47
38
4.50
10.1
2.3
1.8
2.5
3.4
6.7
568
15



1
9
50
41
4.45
10.2
1.7
2.6
2.2
4.0
6.6
676
8



1
9
50
41
4.80
10.4
1.9
4.0
3.0
4.9
8.9
455
0



1
9
50
41
4.67
11.6
2.4
4.0
3.1
5.5
9.5
694
0



x2
9
51
42
4.42
11.1





796
28



1
9
51
42
4.75
8.6





767
8



1
9
54
45
4.40
11.2
2.2
2.5
2.4
4.4
7.1
745
19



1
9
54
45
4.47
8.5
1.8
1.3
1.9
3.8
5.1
705
11


YUSAZ A/1
1
14
37
25
5.22
7.1
2.5
0.0
2.6
5.1
5.1
284
406


X YUSOL/1
x2
10
37
27
4.38
5.9
2.1
0.0
1.8
6.7
3.9
513
146


Maturity
x2
9
37
28
4.67
5.5
2.0
0.0
2.7
4.7
4.7
530
93


reached between
x2
9
37
28
4.37
6.2
2.1
0.0
2.0
4.0
4.1
695
104


cycle 35 and 42
1
19
47
28
5.23
5.8
1.9
0.6
3.1
5.0
5.6
410
0



1
9
37
28
4.65
7.2
2.8
0.0
3.4
6.2
6.2
594
22



x2
9
40
31
4.66
7.0
2.0
0.4
2.8
4.8
5.2
490
12



1
9
40
31
4.50
7.4
2.3
0.1
2.8
5.1
5.2
635
36



x2
9
40
31
4.32
5.9
2.2
0.0
2.1
4.0
4.3
495
102



x2
9
44
35
4.39
7.3
2.7
0.3
2.4
4.2
5.4
579
55



1
9
44
35
4.62
9.2
2.5
1.2
3.0
5.5
6.7
689
0



1
9
44
35
4.39
8.4
3.0
0.0
3.5
6.7
6.6
685
55



x2
9
44
35
4.41
8.8
3.0
0.3
3.0
5.4
6.3
702
27



1
9
44
35
4.77
9.6
2.7
1.6
3.4
6.2
7.8
736
4



x2
9
44
35
5.85
8.0
1.9
3.1
3.4
5.3
8.4
387
0



x2
9
47
38
4.46
8.9
3.1
0.3
3.6
6.9
7.1
653
37



x2
9
47
38
4.85
11.4
3.9
2.4
4.7
8.6
11.0
776
17



1
9
47
38
6.46
9.4
1.3
3.6
2.8
4.1
7.8
274
0



x2
9
47
38
4.75
9.2
2.8
1.4
3.1
5.9
7.3
525
0



1
9
51
42
4.87
12.4
2.8
4.3
3.1
5.9
10.2
696
19



x2
9
51
42
4.45
11.4
2.6
3.9
3.2
5.8
9.7
678
11



x2
9
51
42
4.57
14.4





791
47



x2
9
51
42
4.41
11.6
2.7
3.0
2.6
5.1
8.3
730
52



x2
9
51
42
4.73
13.8
2.5
5.3
3.5
6.0
11.3
679
0



1
10
53
43
6.24
11.0





410
47



x2
10
53
43
6.18
10.5





350
48



1
9
54
45
4.58
13.5
2.7
4.8
2.8
4.8
10.4
780
62



1x
9
54
45
4.54
13.2
2.5
4.2
2.3
4.4
9.0
751
34


YUSAZ A/2
x2
10
37
27
4.55
7.0
2.7
0.0
3.1
5.7
5.8
543
24


X YUSOL/3
x2
10
37
27
4.52
7.6
2.8
0.0
3.1
6.0
5.9
640
64


Maturity
1
9
37
28
5.46
8.3
2.2
1.1
2.9
4.9
6.2
457
166


reached between
x2
9
37
28
4.40
6.1
2.5
0.0
2.5
4.4
5.0
630
64


cycle 34 to 38
1
9
37
28
4.67
9.4
2.9
2.5
4.1
7.0
9.5
618
30



1
10
40
30
4.71
9.5
3.1
1.3
3.3
6.4
7.7
610
54



x2
9
40
31
4.51
6.6
2.4
0.0
2.7
5.2
5.1
460
64



x2
9
40
31
4.62
7.7
3.0
0.0
3.2
6.1
6.1
569
50



x2
9
40
31
4.58
7.6
2.7
0.0
3.2
5.2
6.0
655
41



x2
9
40
31
4.48
8.3
2.9
0.0
3.4
2.6
6.3
721
48



1
10
44
34
4.76
11.2
3.4
1.0
4.0
7.2
8.5
844
74



1
10
44
34
4.69
13.1
3.1
3.1
3.9
9.7
10.0
825
90



1
17
51
34
4.72
10.1





612
22



x2
10
44
34
4.48
12.1
3.4
2.0
4.0
6.7
9.4
683
64



1
9
44
35
4.66
13.0
3.3
4.2
3.5
6.8
11.0
733
42



1
9
47
38
5.22
14.4
2.6
5.9
3.0
5.6
11.4
514
87



1
9
47
38
4.50
13.2
3.1
4.1
3.1
5.8
10.3
899
80



x2
9
47
38
4.89
12.4
2.2
4.1
3.2
6.7
9.5
696
21



1
9
47
38
4.57
14.2
3.3
4.3
3.3
6.2
10.9
725
66



1
9
47
38
4.66
15.2
2.8
5.7
2.9
4.8
11.4
774
141



x2
10
51
41
4.67
15.2





818
44



x2
9
51
42
4.74
13.6





754
65



1
9
51
42
4.97
15.4
2.1
7.0
2.6
4.7
11.7
507
1



1
9
51
42
5.60
15.2
2.0
8.6
2.3
4.3
12.9
462
97



1
11
54
43
4.94
15.8
2.9
6.0
2.8
8.1
11.7
680
66



x2
9
53
44
4.93
15.1





881
221



x2
9
54
45
4.80
15.3
2.3
7.4
2.7
5.1
12.4
831
103



x2
9
54
45
4.53
14.1
2.7
5.2
2.8
5.2
10.8
876
64



1
9
54
45
5.00
15.8
2.8
6.3
2.5
4.8
11.6
641
188









Example 16
Post Harvest Evolution of Fruit Characteristics
A. Fruits of plants of trial SP030F (see Tables 6 and 7C) were harvested and stored for 1 week at 12° C. followed by 3 days at 20° C. or for 6 weeks at 12° C. followed by 2 days at 20° C. The characteristics of the fruits were measured after storage and are shown in Table 18.
B. Plants were grown as for the plants in Table 14 above. Fruits were harvested and stored for 1 week at 12° C. followed by 3 days at 20° C. The characteristics of the fruits were measured after storage and are shown in Table 19.




















TABLE 18









FRT


Glu
Suc
Fru
Hex
Total
Citric
Malic



Post harvest protocol
Hv
Nr
Brix
pH
g/100 g
g/100 g
g/100 g
g/100 g
Sugar
mg/100 g
mg/100 g




























Millenium
1 w 12° + 3 d 20°
Avg
5
8.4
5.75
2.0
2.3
2.2
4.2
6.4
441
−8




Stdev

0.3
0.05
0.1
0.2
0.0
0.1
0.2
25
4



6 w 12° + 2 d 20°
Avg
2
10.1
5.86
1.5
3.9
2.2
3.7
7.6
487




Stdev

0.6
0.03
0.3
0.9
0.2
0.5
0.4
4


YUSOL/1&2
1 w 12° + 3 d 20°
Avg
4
12.0
4.71
1.0
6.7
2.1
3.1
9.8
802
−1


X SOLAZ/1

Stdev

0.7
0.05
0.1
0.7
0.1
0.2
0.7
33
6



6 w 12° + 2 d 20°
Avg
2
13.2
4.77
0.5
8.1
1.8
2.3
10.4
808




Stdev

0.5
0.07
0.1
0.6
0.0
0.1
0.5
36


YUSOL/3
6 w 12° + 2 d 20°
Avg
4
11.9
4.89
1.1
6.2
1.9
3.0
9.2
638


X SOLAZ/1

Stdev

0.9
0.07
0.4
1.6
0.3
0.7
1.1
72


YUSOL/1
1 w 12° + 3 d 20°
Avg
9
9.5
4.46
1.0
4.5
1.9
2.9
7.4
773
−5


X SOLAZ/2

Stdev

1.4
0.12
0.2
1.5
0.2
0.4
1.2
85
5



6 w 12° + 2 d 20°
Avg
4
9.5
4.65
1.1
3.7
2.0
3.1
6.7
669




Stdev

1.4
0.17
0.2
1.4
0.3
0.5
1.0
70


YUSOL/2
1 w 12° + 3 d 20°
Avg
5
10.1
4.53
1.1
4.9
2.1
3.2
8.2
789
3


X SOLAZ/2

Stdev

2.3
0.13
0.2
2.3
0.2
0.3
2.1
95



6 w 12° + 2 d 20°
Avg
2
10.6
4.70
1.1
5.5
2.0
3.1
8.6
697




Stdev

0.7
0.02
0.1
0.6
0.1
0.2
0.4
51


YUSOL/3
1 w 12° + 3 d 20°
Avg
12
10.4
4.50
0.9
5.8
1.8
2.7
8.4
777
−4


X SOLAZ/2

Stdev

0.8
0.09
0.2
0.9
0.1
0.3
0.8
37
6



6 w 12° + 2 d 20°
Avg
6
11.0
4.85
0.6
6.2
1.5
2.1
8.3
656




Stdev

0.8
0.14
0.3
1.1
0.3
0.6
0.7
46



























TABLE 19







FRT


GLU
SUC
FRU
Tot sug
Citric Ac
Malic Ac
ratio



Nr
Brix
pH
g/100 g
g/100 g
g/100 g
g/100 g
mg/100 g
mg/100 g
Citric Ac/Malic Ac


























SOLAZ/2 X YUSOL/3
51
13.1
4.75
1.4
7.2
2.0
10.6
771
23
35


SOLAZ/1 X YUSOL/1
51
13.8
4.71
1.8
7.9
2.3
12.0
778
21
53


YUSOL/1 X
51
13.4
6.14
1.4
8.0
2.3
11.7
354
14
31


SOLAZ/1 basic


MEHARY
10
14.8
6.51
0.5
10.4
1.3
12.2
289
73
4


Female MEHARI
59
12.4
5.45
0.8
7.4
1.4
9.7
491
211
2


X L53 AZ/A


TD x L53AZ/A
49
13.9
5.16
0.6
9.0
1.3
10.9
579
69
11

## Claims

1. A method of increasing the citric acid content of a fruit of a first C. melo plant comprising:
a) obtaining a first C. melo plant;
b) crossing said first C. melo plant with a second C. melo plant, wherein said second C. melo plant is a plant of line IND-35, representative seed of which is deposited under Accession number NCIMB 41202; and
c) selecting a progeny C. melo plant comprising in its genome the molecular markers NE0585 and NE1746, and producing a fruit having:
i) increased citric acid content and lower pH, when compared to a fruit of said first C. melo plant grown under similar conditions, and
ii) a ratio of citric acid to malic acid that is greater than 5,
wherein selecting comprises detecting at least one amplified DNA fragment of a length of 228 bp, 230 bp, 232 bp, and/or 253 bp when amplifying in the molecular marker NEO585 with an amplification primer pair of SEQ ID NO:3 and SEQ ID NO:4, or 121 bp, 124 bp, and/or 127 bp when amplifying the molecular marker NE1746 with an amplification primer pair of SEQ ID NO:5 and SEQ ID NO:6.