צמחי פלפל המייצרים פירות בעלי תכונות משופרות

The present invention relates to novel plants, in particular to pepper plants capable of producing fruits which can be kept a longer period of time on the plant as well as being stored after harvesting under refrigerated conditions without exhibiting excessive softening. The invention thus refers to pepper plant which produces fruits with significantly increased fruit firmness at the harvesting stage, wherein said increased fruit firmness is controlled by a genetic determinant, monogenic, wherein said increased fruit firmness is defined as a fruit deformation under a 1 kg load force that is lower than that of a fruit from a control pepper plant which does not have the said genetic determinant.

Description

Peppers are an important crop worldwide with an estimated cultivation of more than about 30 millions of tons per year for bell peppers fruits. Peppers are Solenaceas from the genus Capsicum, which includes the species Capsicum annuum and Capsicum frutescens. Commercial peppers are diploids with n=12 chromosomes. Peppers are cultivated and used around the world as sweet peppers such as the bell pepper; or as pungent chili peppers, jalapeno peppers, and TABASCO® peppers; or as a source of dried powders of various colors such as paprika.

Peppers represent a valuable source of vitamins and nutrients associated with their pigments and fruit color, including various antioxidants, carotenoids as well as chlorophyll. In the present trend of consumers looking for fresh and healthy vegetables, pepper fruits constitute a product of choice. Peppers fruits are generally green when immature and turn generally red or yellow or orange once ripe.

In some of the markets, peppers are usually harvested green, i.e. at a non-mature stage. Immature pepper fruits generally exhibit a distinct taste and also are less sweet compared to red-mature-fruits.

For all fruit products, fruit firmness which is fit for purpose is essential to meet and possibly exceed consumer expectations. Consumers will reject products with unacceptable fruit firmness, even though other quality attributes such as flavour and colour are good or excellent. Additionally, the supply chain requires an appropriate level of fruit firmness for the effective delivery of high quality fruit to retail outlets. Improving the quality of the raw materials will also encourage the development of healthier diets.

Fruit firmness in pepper is determined by a number of factors. The major factor in most cases is turgor, mainly associated with water content. In some cases cell wall structure, turgor and cuticle properties. Fruit firmness is reported to be a complex trait supposedly depending on the action of many genes. This has made it difficult to dissect the events determining changes in fruit firmness by focusing on changes in cell wall degrading enzymes.

Harvesting pepper fruit when ripening has set in would make maturity determination easier as it would be based on visible peel color and would assure full quality development. After harvest, if no particular humidity and temperature control is put in place, softening advances, increasing the susceptibility of the fruit to handling damage and limiting the marketing period. Slowing down the ripening and softening stages would allow harvesting, transport and storage of partially ripe, or fully ripe, but firm fruits.

Pepper fruits, also commonly referred to as “peppers”, are highly perishable. After harvesting, they are prone to water loss, softening and shriveling, which renders them unappealing to customers. Pepper crops produce peaks of fruits that are mature around the same time and have to be harvested quickly to avoid losses. This leads to waves and picks of production, followed by periods of low supply. Furthermore, frequent harvest is not very efficient from an economic point of view and it is more favorable to harvest more fruits in a reduced number of harvests that few fruits in a high number of harvests.

In order to address this problem and bring flexibility in the supply chain, substantial efforts have been made to improve the quality of pepper fruits during post-harvest storage. Treatments involving hot water and polyethylene packing (Gonzalez-Aguilar et al (1999) Journal of Food Quality 22: 287-299), application of CO2 (Wang (1977) J. Amer. Soc. Hort. Sci. 102: 808-812) or other chemicals such as chlorine and Imazalil (Miller et al. (1983) Proc. Fla. State Hort. Soc. 96: 345-350) or chitosan (E I Ghaouth et al. (1991) Journal of Food Processing and Preservation 15: 359-368), have been described. However, these treatments require substantial investments and increase costs of production. Moreover, some of them promote fungal growth or undesired off-flavor production (E I Ghaouth et al. (1991) Journal of Food Processing and Preservation 15: 359-368).

Attempts to increase post-harvest shelf-life of pepper fruits have also been made by genetic engineering approaches. For example, U.S. Pat. No. 5,945,580 reports the transformation of Capsicum annuum with DNA sequences of a hemi-cellulase gene. Reduction of hemi-cellulase activity in fruits of transformed plants was measured, leading to a moderate increase in the proportion of acceptable fruits after post-harvest storage at 4 C. However, the commercial viability and public acceptability of genetically modified crops is poor.

An alternative strategy has concentrated on delaying fruit ripening, whereby unripe fruits are usually harvested and let ripen post-harvest. For example, U.S. Pat. No. 4,843,186 discloses peppers comprising the native pepper Rin gene and their delayed ripening.

However, maturation of pepper fruits is a slow process and post-harvest ripening of pepper fruits results in wilted, low-quality fruits.

EP1553817 discloses pepper plant producing fruits with extended storability on the plant as well as after harvesting characterized by the fact that the fruits do not wilt, show 5 of less yellow spot, remain bright and firm for a long period of time. However the fact is that the underlying trait is phenotypically and genetically complex, likely multigenic and consequently difficult to manipulate and to introgress in a broad variety of genetic backgrounds without linkage drag effects. Indeed, fruit quality appears as a quantitative trait involving many genes and yet the identity of the majority of these genes remains obscure.

The formation of the fruit post anthesis is usually following a three steps scheme. First, the fruit reaches the immature green stage wherein the fruit increases its size and weight. Next when it is still green while having reached its fully expanded size it is mature green (at this stage green peppers are usually harvested). This stage is followed by a stage which is called breaker stage corresponding to the period of time when the first sign of coloring of the fruit appears. The breaker stage is followed by the ripe stage where the fruit is fully red, yellow or orange depending of red, yellow or orange genotype, respectively. Usually, fruits for direct marketing are harvested when the colored portion of the fruit reaches more than 90%. In certain cases (very soft or very high price produces) fruits are harvested when 60% is colored and reach their final full color during transportation and storage.

Tipically, the firmness of the pepper fruit has the tendency to rapidly decrease once the fruit has reached the ripe stage while this stage corresponds to its maximum in term of taste and nutritional value. The fruits become soft and flabby with unappealing texture or appearance and lose their typical crunchy and juicy texture.

It is thus apparent from the prior art that there is a need to modify pepper fruit firmness and to delay the point of softening in particular so that the ripe fruits stay on the vine for a longer period of time without affecting fruit firmness. The fruits would then keep the same firmness while accumulating more flavour and taste components and thus exhibit a final better taste compared to fruits that have been harvested earlier. Such increased firmness of the fruit would allow a longer period of time for harvesting since the fruits could be left on the vine without the associated softening which would be detrimental for the steps of handling, packing and delivery to the supermarket shelves for example.

There is an unmet need in the pepper trade to reduce peaks in production and to favor a constant supply of fresh, firm and crispy products, while keeping production costs low. There is also an unmet need for improved pepper plants and for alternative and improved storage methods for pepper fruits and to save labor.

The present invention addresses the need for more constant supply of pepper fruits, which keep their organoleptic properties, particularly texture, for an extended period of time, on the vine and/or once harvested when kept refrigerated or not. This would thus provide flexibility in the peppers supply chain.

SUMMARY OF THE INVENTION

An investigation has been conducted from a population of pepper plants wherein a plant producing fruits exhibiting unusual mechanical properties was identified. Indeed, the fruits of said plant were displaying and unusual extra hard texture becoming firmer during time, cracked surface and non-ripening fruit; ie stayed green and did not develop taste or sweetness. This plant was isolated and it was identified that this trait was monogenic and recessive. It is important here to indicate that a trait—referring to a characteristic or phenotype—may be inherited in a dominant or recessive manner, or in a partial or incomplete-dominant manner. A trait may be monogenic (i.e. determined by a single locus) or polygenic (i.e. determined by more than one locus) or may also result from the mutual interaction among genes or interaction of one or more genes with the environment. A dominant trait results in a complete phenotypic manifestation at heterozygous or homozygous state; a recessive trait manifests itself only when present at homozygous state. Upon selfing of the selected plant, seeds failed to germinate. Then, pollen from this selected plant was collected and used for crossing the said plant with normal plants (originating from the same population but without the trait of extra hard texture and non-ripening fruits) which were used as the female parent. Plants derived from second generation of the crossed material (F2) and other consequent generations were segregating populations where seeds were derived from such single plants, the proportion of non-ripening and extra hard fruits was in agreement with 1:3 ratio, indicating a single recessive Mendelian nature for the trait of cracked surface, extra hard texture and non-ripening fruits. Surprisingly, the heterozygous plants for the said trait which were generated did not show drawback of the homozygous plant, (extra hard texture and non-ripening) but did undergo normal coloring and ripening while exhibiting, to a valuable extend, a trait of enhanced firmness at breaker and ripe stage. The advantages and benefits of this trait when present, particularly at heterozygous state, are detailed below.

When the identified trait is homozygous, the fruits are extra-hard, do not ripe and are cracked. However when the trait is heterozygous, the fruits do ripe normally, are not cracked and exhibit an increased firmness all along the ripening process till harvest and even after. The advantage of having an enhanced firmness at the harvesting stage as well as after the stage of usual harvesting, ie ripe and over ripe, allows the fruit to remain firm well after the stage of full ripening.

The inventors noticed that the plants homozygous for trait do contain seeds that fail to germinate and need to undergo embryo rescue in order to generate plant due to lack of endosperm around the embryo.

Then, when pepper plant homozygous for the extra-hard and non-ripening trait are grown, they can be used to be crossed with other pepper plants, particularly sibling pepper plants, which do not contain the said trait. Upon crossing of the said other plant without the trait of the invention, then 100% of the offspring of such cross is heterozygous for the said trait, thus giving normal ripening fruits but with enhanced firmness at the harvesting stage and which keep essentially the same firmness after this stage while the normal fruits (not having the genetic determinant, locus or trait according to the invention) undergo progressive and irremediable softening.

The plants that are heterozygous or homozygous for the trait (or locus) can be used to transfer the genetic determinant for such a trait into different pepper plant genome by any mean known by the man skilled in the art. Preferably, the plants according to the invention, (homozygous or heterozygous for the trait (or locus) of the invention) are used as female when crossed with normal pepper plant not having the said trait or locus. When the trait genetic nature is kept at heterozygous state, the associated phenotype is positive and beneficial for the grower. Since the trait is monogenic, it is easily transferable and the phenotype associated can easily be bestow upon various genetic backgrounds. On the top of that, assuming that the phenotype of interest is attached to the heterozygous state of the trait, the obtention of any pepper plant with the trait at heterozygous state according to the present invention is very easy. Indeed, one single cross of any pepper plant with a heterozygous plant for the trait according to the present invention allows to obtain 50% of the progeny heterozygous for the trait and thus having the phenotype according to the present invention. When the trait/locus is homozygous, the associated phenotype does also present advantageous benefits, for example in the case of specialty products such as industry peppers for which high firmness is a key criteria while the cracking is not a default. Furthermore, some of the drawbacks of the homozygous state of the locus/trait of the invention, such as cracking may be managed thanks to cultivation methods by closer control of day/night temperature shift which is an environmental factor that may triggers or worsen the cracking of fruits.

The inventors have defined that the trait is associated to a genetic determinant, a locus, likely a gene, and is surprisingly associated to significantly increased fruit firmness in pepper at the harvesting stage, when present at heterozygous state. The said trait is controlled by a genetic determinant that is stable and thus can be stably inherited to progeny plants and introgressed into commercial pepper varieties.

The present invention therefore relates in a first aspect to a pepper plant which produces fruits with significantly increased fruit firmness at the harvesting stage, wherein said increased fruit firmness is controlled by a genetic determinant, wherein said increased fruit firmness is defined as a fruit deformation under a 1 kg load force that is lower than that of a fruit from a control pepper plant which does not have the said genetic determinant.

In one embodiment the pepper fruit deformation of a pepper fruit according to the present invention, under a 1 kg force load represents 50% to 95% of that of a fruit from a control pepper plant which does not have the said genetic determinant.

In another embodiment the pepper fruit deformation of a pepper fruit according to the present invention, under a 1 kg force load, represents 50% to 80% of that of a fruit from a control pepper plant which does not have the said genetic determinant.

In a further embodiment the pepper fruit deformation of a pepper fruit according to the present invention, under a 1 kg force load, represents 50% to 70% of that of a fruit from a control pepper plant which does not have the said genetic determinant.

The pepper fruit firmness is measured thanks to the fruit deformation, measured in mm, when the fruit is put under a 1 kg load. The instrument and methodology as described in Example section have been designed according to Ben-Yehoshua, S. et al (Mode of action of plastic film in extending life of lemon and bell pepper fruits by alleviation of water stress. 1983, Plant Physiology 73: 87-93). Fruit firmness was determined with a compression tester using a 1 kg weight. The deformation was measured 10 s after exerting the force on the fruit. The firmer the fruit, the lower were the reading.

There is also provided a method of producing a pepper plant which provides fruit with increased fruit firmness at the harvesting stage as herein described.

There is also provided a method of producing an offspring pepper plant which produces fruits with increased fruit firmness at the harvesting stage comprising the steps of detecting a genetic determinant associated to increased fruit firmness at the harvesting stage in a pepper donor plant, and transferring a nucleic acid comprising such a genetic determinant thus detected to a recipient pepper plant, wherein said increased fruit firmness at the harvesting stage is measured in fruit from an offspring pepper plant compared to fruit from a control pepper plant. The transfer of nucleic acid can be performed by any of several methods known in the art e.g. transformation, by protoplast fusion, by a doubled haploid technique or by embryo rescue or by introgression through crossing.

There is also provided a pepper plant, or part thereof, obtainable by a method as described herein.

There is also provided a cultivated pepper plant comprising a genetic determinant responsible for increased fruit firmness at the harvesting stage as described hereinabove.

There is also provided a hybrid pepper plant, or part thereof, obtainable by crossing a pepper plant as described hereinabove with a pepper plant that exhibits commercially desirable characteristics.

There is also provided pepper seed that growths into a pepper plant as described hereinabove.

There is also provided pepper seed produced by crossing a pepper plant as described hereinabove with a plant having desirable phenotypic traits to obtain a plant that has significantly increased fruit firmness at harvesting stage compared to a control plant. There is also provided the use of a pepper plant according to the present invention for expanding the harvesting slot of pepper fruit and/or for use in the fresh cut market or for food processing.

There is also provided processed food made from a pepper plant comprising the genetic determinant as described herein.

In one embodiment, the present invention provides a pepper plant growing fruit with increased firmness which are edible and of high quality and suitable of being used as fresh produce, as fresh cut produce, or for processing such as, for example, canning.

The pepper plant according to the invention and as described herein before may grow a sweet pepper including a dolce-type pepper, a bell pepper, a big rectangular pepper, a conical pepper, a long conical pepper or a blocky-type pepper. The fruit of said plant at maturity may be an evergreen, a yellow, orange, ivory, brown, purple, or red fruit. The pepper plants according to the present invention are characterized in that the genetic determinant is homozygous.

In another embodiment, the pepper plants according to the present invention are characterized in that the genetic determinant is heterozygous.

The plant according to the invention and as described herein before may be an inbred, a dihaploid or a hybrid and/or a male sterile, with the provision that the genetic determinant of the invention is homozygous or heterozygous, particularly heterozygous. In the context of an inbred, this means that the said inbred is homozygous for all locus except the locus associated with the trait of the invention, increased fruit firmness, but homozygous for the rest of the loci.

In one embodiment, the pepper plant according to the invention and as described herein before contains a “increased fruit firmness” trait governed by a genetic determinant, which is obtainable from a hybrid pepper Capsicum annuum A13-1517-6 deposited with NCIMB, Aberdeen AB21 9YA, Scotland, UK on Feb. 5, 2015 under accession number NCIMB 42356. Those hybrid peppers plants are heterozygous for the genetic determinant according to the present invention and can be used for bestowing the trait/locus according to the present invention upon any other pepper plant. Preferably, the deposited plant is used as female plant when crossed with another pepper plant that does not contain the trait/locus/genetic determinant according to the present invention.

In the context of the present invention, the terms trait or locus or genetic determinant are used in order to refer to the genetic element which is monogenic as demonstrated herein and is directing the phenotype of the pepper plants according to the present invention, i.e. increased fruit firmness.

In preferred embodiment, the genetic determinant of the pepper plant according to the present invention is monogenic.

In one embodiment of the invention, the “increased fruit firmness” trait (or locus or genetic determinant) or a plant comprising said trait is obtainable from any of the hybrid plants grown from the deposited seeds by growing the F2 progeny of said hybrid. In particular, the “increase firm firmness” trait or a plant comprising said trait is obtainable from any of the deposited hybrid plants by i) germinating seed of said plants and growing a mature, fertile plant therefrom; ii) inducing self-pollination of said plant grown under (i), growing fruits and harvesting the fertile seeds therefrom, and iii) growing plants from the seeds harvested under ii) and selecting plants which grow fruits with increased fruit firmness.

In one embodiment, the invention relates to plant material obtainable from a plant according to the invention and as described herein before including, but without being limited thereto, leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of the plant which still exhibits the increased fruit firmness phenotype according to the invention, particularly when grown into a plant that produces fruits.

The invention further relates to an agronomic method of producing pepper plant producing pepper fruits with increased fruit firmness comprising the steps of:

i) providing a pepper plant according to the invention and as characterized herein before;
ii) multiplicating/propagating said pepper plant
iii) allowing the plant to grow pepper fruits with increased fruit firmness; and
iv) harvesting said pepper fruits.

In one embodiment of the invention, the multiplication or propagation of the pepper plant is done either through seeds or by vegetative propagation.

The invention further relates to a method of producing a pepper plant producing fruits with increased fruit firmness comprising the steps of

i) providing seeds of an F1 hybrid pepper plant according to the present invention;
ii) germinating said seed and growing a mature, fertile plant therefrom;
iii) inducing self-pollination of said plant grown under (ii), growing fruits and harvesting the fertile seeds therefrom, and
iv) growing plants from the seeds harvested under iii) and selecting plants which grow fruits with increased firm firmness.

Due to the heterozygous nature of the trait of increased fruit firmness and the monogenic nature of the trait of F1 hybrid pepper plants used in i) such as the ones deposited under NCIMB 42356, the segregation observed in plants obtained in step iv) above is: 50% of plants according to the present invention, ie increased fruit firmness with normal ripening fruits and non-cracked fruits (heterozygous for the genetic determinant of the trait); 25% of plants that are normal plants with normal ripening fruits and non-cracked fruits but without increased fruits firmness and 25% of plants are plants with non-ripening, heavily cracked, and extra hard texture fruits (homozygous for the genetic determinant of the trait).

In one embodiment, the F1 hybrid seed used in said method according to the invention is the hybrid seed, which is obtainable from a hybrid pepper plant selected from the group of hybrids consisting of Capsicum annuum A13-1517-6 grown from seeds deposited with NCIMB, Aberdeen AB21 9YA, Scotland, UK on Feb. 5, 2015 under accession number NCIMB 42356.

This plant is heterozygous for the trait/locus of the invention, and allows upon selfing to obtain offsprings that segregate for the trait of the invention.

The invention further relates to a method of producing a pepper plant producing fruits with increased fruit firmness comprising the steps of

i) providing seeds of an F1 hybrid pepper plant according to the present invention;
ii) germinating said seed and growing a mature, fertile plant therefrom;
iii)crossing the plant obtained in ii) with a pepper plant which does not have the trait of increased fruit firmness, growing fruits and harvesting the fertile seeds therefrom and
iv) growing plants from the seeds harvested under iii) and selecting plants which grow fruits with increased firm firmness.

Due to the heterozygous nature of the trait of fruit having normal ripening and increased fruit firmness and the monogenic nature of the trait, the segregation observed in plants obtained in step v) above is: 50% of plants according to one embodiment of the present invention, ie increased fruit firmness with normal ripening fruits and non-cracked fruits (heterozygous for the genetic determinant of the trait); 50% of plants that are normal plants with normal ripening fruits and non-cracked fruits but without increased fruits firmness.

In one embodiment, the F1 hybrid seed used in said method according to the invention is the hybrid seed, which is obtainable from a hybrid pepper plant selected from the group of hybrids consisting of Capsicum annuum A13-1517-6.grown from seeds deposited with NCIMB, Aberdeen AB21 9YA, Scotland, UK on Feb. 5, 2015 under accession number NCIMB 42356.

In one embodiment, the pepper plant which does not have the trait of increased fruit firmness which is crossed in step iii) can be any pepper plant variety.

The selected pepper plants in step iv) are heterozygous for the genetic determinant of the trait according to the present invention and can be used for further back-crosses with the second pepper parent plant which does not contain the genetic determinant of increased fruit firmness in order to convert that initial pepper plant into a very same one with the difference of having in its genome the genetic determinant of increased fruit firmness. For each generation 50% of the plants will be heterozygous for the genetic determinant and will exhibit the trait of increased fruit firmness according to the present invention.

Accordingly it is possible to convert any pepper plant into a pepper plant according to the present invention by sexual crossing of such any pepper plant with a pepper plant according to the present invention, i.e. that contains the genetic determinant in heterozygous state. Preferably the female plant used in the cross is plant according to the invention in order to obtain plant, in the first generation that exhibit the phenotype of enhanced fruit firmness. The genetic determinant for the trait of the present invention is obtainable from a hybrid pepper plant selected from the group of hybrids consisting of Capsicum annuum A13-1517-6 grown from seeds deposited with NCIMB, Aberdeen AB21 9YA, Scotland, UK on Feb. 5, 2015 under accession number NCIMB 42356.

In order to do so, the deposited plant may be used as a female parent since the maternal genotype will govern the first generation phenotype of the fruit.

If one uses, the deposited hybrid plant as male parent, i.e. a pollen donor, to be crossed with a normal pepper plant as female that does not contain the locus/trait according to the invention, then the first generation progeny will not exhibit the enhanced fruit firmess since the maternal tissue of the ovary gives rise to the pericarp of the fruit. In such a situation, the first generation progeny has to undergo self-pollination and this will give 25% of homozygous and 75% of heterozygous (for the trait/locus of the invention) plants. Those plants will exhibit the phenotype of enhanced fruit firmess.

Such genetic determinant is inheritable and can be transmitted thru generation by techniques known to the man skilled in the art of plant breeding or plant biotechnology.

צמחי פלפל המייצרים פירות בעלי תכונות משופרות

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