HK1234958B - Stay green cucumber plant - Google Patents

Description

Evergreen cucumber plants

Technical Field

The present invention relates to a cucumber plant (cucumber) that provides fruits with improved storage life. In addition, the present invention relates to seeds, plant tissues, fruits, or plant components of cucumber plants (cucumbers). A further aspect of the present invention relates to a method for providing a cucumber plant (cucumber) that provides fruits with improved storage life. According to a further aspect, the present invention relates to nucleic acids and amino acids associated with improved storage life properties.

Background Art

Cucumber plants have been cultivated for at least 3,000 years and have developed several different cultivars that are grown and sold on the global market. Because the mature yellow form usually becomes bitter and sour, the immature green form of cucumber fruits is mainly eaten. Therefore, cucumber fruits are usually harvested when still green and are generally used in the pickling industry and fresh-keeping market, and for farmers, the latter use has the greatest added value. However, due to its relatively short storage life, the storage and transportation of fresh cucumber fruits become difficult and expensive. Since cucumber fruits are not suitable for storage at low temperatures due to freezing, the refrigeration that is usually used to extend the storage life of fresh products cannot be used for cucumber fruits.

It is known, for example, to extend the shelf life of cucumber fruits by wrapping them in sealed foil or storing them under controlled conditions (>10°C). However, this requires additional processing steps or specific storage measures. Furthermore, this only extends the shelf life of the specific cucumbers that have been wrapped in foil or stored under these specific conditions. Furthermore, although wrapping cucumbers in foil extends their shelf life, these wrapped cucumbers turn yellow within a period of approximately two weeks.

Summary of the Invention

For the reasons stated above, there is a need in the art to provide cucumber plants having fruit with an extended storage life.

It is therefore an object of the present invention, among other objects, to provide cucumber plants which provide fruits having an extended storage life.

This object, among other objects, is achieved by providing a cucumber plant according to the appended claim 1 .

In particular, this object is achieved, among other objects, by providing cucumber plants (cucumbers) which provide fruits with improved storage life, wherein the plant comprises a gene or a stay green gene providing improved fruit storage life, wherein the gene or the stay green gene encodes a protein comprising the amino acid sequence as shown in SEQ ID NO. 2, or an amino acid sequence having more than 90% identity, such as more than 91%, 92% or 93%, preferably more than 94% identity, such as more than 95%, more preferably more than 96% identity, such as more than 97%, even more preferably more than 98% identity or more than 99% identity; and wherein the expression of the gene or the stay green gene is reduced compared to the expression of the gene or the stay green gene in cucumber plants that do not provide fruits with improved storage life, or the enzymatic activity of the protein is reduced compared to the enzymatic activity of the protein in cucumber plants that do not provide fruits with improved storage life.

In the studies leading to the present invention, it was surprisingly found that reduced expression of a gene or reduced enzymatic activity of a protein provides fruits with an extended storage life for a period of up to five weeks when cucumbers are stored under standard storage conditions.

As used herein, having improved storage life refers to the improved storage capacity of harvested immature green cucumbers, the cucumbers remaining green for a longer period of time than with comparable standard harvested immature green cucumbers. Preferably, the cucumbers remain green for a period of up to 4 weeks or 5 weeks.

The plants of the present invention can be distinguished from the plants of the prior art by measuring the period of time during which the cucumber fruits remain green under standard storage conditions. Conventional cucumber fruits turn yellow within 1 or 2 weeks; whereas the cucumber fruits according to the present invention turn yellow after a period of at least 4 weeks or at least 5 weeks.

Alternatively, expression levels in the present plant and in the reference plant can be determined using any suitable and generally known molecular biology technique, such as quantitative polymerase chain reaction (PCR) or mRNA hybridization.

According to the present invention, the enzyme activity is reduced compared to the activity of the protein in a cucumber plant that does not provide cucumber fruits with an extended storage life. The term 'cucumber plants that do not provide a prolonged storage life' refers to a storage life determined with an appropriate storage capacity test and an appropriate reference plant (such as a parent plant) that is less than the storage life observed for the cucumber fruits of the present invention. In addition to the parent plant, a suitable reference plant can also be a plant that is typically designated as providing marketable cucumber fruits.

The protein has a chlorophyll decarboxylase function, wherein, among other reactions, a carboxyl group is removed and carbon dioxide is released. Therefore, a test using a measuring compound (a starting compound or a resulting compound of the enzyme reaction) can determine the reduced activity. Alternatively, for example, the protein level can be determined by ELISA or protein hybridization, two techniques commonly known to those skilled in the art, i.e., inherently indicating a reduced activity of the protein.

Cucumber plants of the present invention can be obtained by mutational variation of cucumber plants. For example, mutations in expression or protein levels of these plants can be caused by using mutagenic chemicals such as ethyl methanesulfonate (EMS) or by utilizing gamma rays or fast neutron radiation plant materials. The mutations produced are directed or random. In the random case, mutagenized plants carrying gene mutations can be easily identified by using the TILLING (Targeting Induced Local Lesions IN Genomes) method (McCallum et al. (2000) Targeted screening for induced mutations. Nat. Biotechnol. 18, 455-457, and Henikoff et al. (2004) TILLING. Traditional mutagenesis meets functional genomics. Plant Physiol. 135, 630-636). Briefly, this method is based on PCR amplification of the gene of interest from the genomic DNA of a large number of mutagenized plants collected in the M2 generation. Individual plants with genetic mutations are identified by DNA sequencing or by scanning for point mutations using single-strand specific nucleases such as CEL-I nuclease (Till et al. (2004) Mismatch cleavage by single-strand specific nucleases. Nucleic Acids Res. 32, 2632-2641).

According to another preferred embodiment of the first aspect of the present invention, the storage life of improvement is provided by one or more sudden changes in the gene coding sequence, thereby producing truncated or non-functional protein.Can easily determine truncated protein by analyzing gene transcription product at mRNA or cDNA level, and can determine non-functional protein in enzyme test or using conformation-dependent antibody.For example, the sudden change that can be measured at the transcriptional level is amino acid replacement, frameshift or premature stop codon.

According to another preferred embodiment of the first aspect of the present invention, improved shelf life is provided by one or more mutations in the regulatory region or non-coding sequence of the gene. Examples of regulatory regions of genes are promoter and terminator regions and examples of non-coding regions are introns and especially splicing influencing motifs therein.

In a further preferred embodiment of the present invention, the mutation results in an amino acid substitution comprising leucine or a substitution designated 'L' (Leu) at position 153 of SEQ ID NO. 2. More preferably, the leucine at position 153 of SEQ ID NO. 2 is substituted with phenylalanine or 'F' (Phe). Thus, in a further preferred embodiment, the evergreen gene encodes a protein comprising the amino acid sequence shown in SEQ ID NO. 4. Because an improved storage life of at least four weeks has been observed for fruit from cucumber plants comprising the gene encoding the protein, the protein comprising the amino acid sequence shown in SEQ ID NO. 4 is highly relevant to the improved storage life trait of the present invention.

According to another preferred embodiment of the first aspect of the present invention, reduced expression is provided by one or more mutations in the regulatory region or non-coding sequence of the gene. Examples of regulatory regions of genes are promoter and terminator regions and examples of non-coding regions are introns and in particular splicing-influencing motifs therein.

According to yet another preferred embodiment, the evergreen gene is present in homozygous form, thereby providing improved storage life of the cucumber fruit. Alternatively, the evergreen gene may be present in heterozygous form, thereby providing an intermediate phenotype.

According to a second aspect, the present invention relates to a cucumber plant (cucumber) comprising a never-green gene that provides improved fruit storage life, or alternatively, seeds, plant tissues, fruits or plant components obtainable or derived from the cucumber plant of the present invention, wherein the never-green gene encodes an amino acid sequence as shown in SEQ ID NO. 2 or an amino acid sequence with more than 90% identity, preferably more than 94% identity, more preferably more than 96% identity, even more preferably more than 98% identity; and wherein the expression of the never-green gene is reduced compared to the expression of the never-green gene in cucumber plants that do not provide fruits with improved storage life, or the enzymatic activity of the protein is reduced compared to the enzymatic activity of the protein in cucumber plants that do not provide fruits with improved storage life.

According to a third aspect, the present invention relates to a method for providing a cucumber plant (cucumber) that provides fruits with improved storage life, the method comprising the following steps: introducing a forever green gene encoding a protein comprising the amino acid sequence as shown in SEQ ID No. 2 or a gene having a sequence identity of more than 90%, preferably more than 94%, more preferably more than 96% to SEQ ID No. 2 into a cucumber plant (cucumber); and wherein the expression of the gene is reduced compared to the expression of the gene in a cucumber plant that does not provide fruits with improved storage life, or the enzymatic activity of the protein is reduced compared to the enzymatic activity of the protein in a cucumber plant that does not provide fruits with improved storage life.

According to a fourth aspect, the present invention relates to the use of a gene encoding a protein comprising the amino acid sequence as shown in SEQ ID No. 2 or its cDNA sequence, or a gene encoding a protein having more than 90% sequence identity, preferably more than 94% sequence identity, more preferably more than 96% sequence identity to SEQ ID No. 2, for providing cucumber plants (cucumbers) that provide fruits with improved storage life, or preferably fruits that remain green during storage for a period of at least 4 weeks or 5 weeks.

According to a fifth aspect, the present invention relates to a protein comprising the amino acid sequence as shown in SEQ ID NO 4.

According to a sixth aspect, the present invention relates to a nucleic acid comprising SEQ ID NO 3, preferably an isolated nucleic acid.

According to a seventh aspect, the present invention relates to a gene encoding a protein having an amino acid sequence comprising SEQ ID No. 4 or a forever green gene.

According to an eighth aspect, the present invention relates to a gene comprising a nucleic acid sequence comprising SEQ ID NO. 3 or a forever green gene.

According to the ninth aspect, the present invention relates to the gene or the evergreen gene defined above, that is, the gene or the evergreen gene encodes a protein comprising the amino acid sequence as shown in SEQ ID NO.2 or an amino acid sequence having more than 90% identity, such as more than 91%, 92% or 93%, preferably more than 94% identity, such as more than 95%, more preferably more than 96% identity, such as more than 97%, even more preferably more than 98% identity or more than 99% identity; wherein, compared with the immature gene encoding the protein comprising the amino acid sequence as shown in SEQ ID NO.2, the gene includes a mutation in the coding sequence that causes one or more amino acid substitutions.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further illustrated in the following exemplary embodiments. In the embodiments, reference is made to the accompanying drawings, in which:

FIG1 : shows a picture of cucumbers according to the prior art and cucumbers according to the invention 4 weeks after harvest;

Figure 2: shows cucumbers from the prior art and cucumbers according to the invention at 4 weeks;

Figure 3 shows the average scores of taste evaluations of Kurios, WT, Het, and Hom fruits at 0, 6, 13, and 20 days after harvest;

Figure 4: From left to right: fresh Kurios, Kurios at 28 days, heterozygous SGR at 28 days, homozygous SGR at 28 days;

Figure 5: From left to right: fresh Kurios, Kurios at 28 days (wild type), heterozygous SGR at 28 days, homozygous SGR at 28 days;

Figure 6: shows cotyledons stored for 2 weeks;

Figure 7: Shown from left to right: mature leaves of wild type, heterozygous SGR and homozygous SGR.

DETAILED DESCRIPTION

Example 1

Two thousand seeds of the cucumber (Cucumis sativus) hybrid Kurios were treated with 0.75% (W/V) ethyl methanesulfonate (EMS) for 16 hours. After rinsing the seeds several times with water, they were sown in peat blocks. After the first leaves appeared, the apical meristem was removed to induce the development of lateral meristems. This process was repeated to induce the outgrowth of the lateral meristems of the first lateral branches. Samples were taken from the tips of these developing branches for further molecular analysis, i.e., detecting mutations in candidate genes according to the method described by Van Eijk and Van Tunen in EP 1929039.

Cucumber fruits derived from EMS-treated plants containing SEQ ID NO. 3 were grown in a greenhouse and harvested. The fruit were measured for storage life over a 4-week period using a storage test with the following rating scale: (1) good color (green); (2) acceptable color (somewhat lighter green); (3) unacceptable color (beginning to turn yellow); (4) unacceptable color (turning yellow); and (5) unacceptable color (extremely yellow). Conventional cucumber plants derived from plants not containing SEQ ID NO. 3 were also measured over a 4-week period using the same storage test.

The cucumbers according to the present invention remained green over a 4-week period and had a continuous score of (1). Conventional cucumbers showed a normal yellowish hue due to the degradation of chlorophyll during the 4-week test period, reaching a score of 4 after 4 weeks. FIG1 shows a picture of a conventional cucumber at 4 weeks with a yellowish hue on the lower side and a cucumber at 4 weeks with a full green hue on the upper side according to the present invention. In addition, the presence of SEQ ID NO.3 also affected the fresh color of the cucumber fruit. Compared to the cucumbers harvested from the plants of the present invention (see FIG2 , upper side), conventional cucumbers showed a normal lighter color (see FIG2 , lower side) at 4 weeks.

Example 2

Plants derived from EMS treatment comprising SEQ ID NO. 3 were grown in the greenhouse together with plants derived from untreated seeds (wild type/WT) and the reference variety Kurios. From the plants carrying the evergreen gene, heterozygous (Het) and homozygous (Hom) forms were cultivated.

The fruit of 4 kinds of different plant types was gathered in the crops at 0 day and stored in wicker boxes at 17 ℃ for 4 weeks. The tasting panel has evaluated the fruit taste for freshness, humidity, acidity/sweetness, aftertaste, color and luster, smell and overall impression. Evaluated at 0 day, 6 days, 13 days and 20 days after the gathering of the fruits. In contrast, on the tasting day, the fresh fruit of reference variety Kurios was also gathered in the crops and evaluated. The scoring using a scoring of 1 to 5 is shown in Fig. 3. The conclusion that can be drawn is that, at 4 tasting moments (0 day, 6 days, 13 days, 20 days), there was slight difference in the taste of 4 different types (Kurios, WT, Het, Hom).

After 4 weeks of storage, the fruits were photographed. The pictures are shown in Figures 4 and 5.

Clearly, after 28 days, the exterior color of the homozygous SGR fruit was at least as dark as freshly picked Kurios fruit. Furthermore, the interior color of the homozygous SGR fruit was darker than freshly picked Kurios fruit, wild-type fruit, and heterozygous fruit. Furthermore, from Figures 4 and 5, it can be concluded that the presence of the evergreen gene does not alter other aspects of storage life (e.g., blisters, soft neck).

Example 3

Make the seed growth of the plant with evergreen gene in homozygote or heterozygote form to the cotyledon stage.From seedling, remove cotyledon and be placed in the plastic container that glass plate covers.Can be considered as the inside of container and can not have air exchange between the environment.After storing for 2 weeks, the picture shown in visible Figure 6.

The cotyledons of the plant of seeds harvested from the plant with the homozygous form of the evergreen gene are placed on sieves A1-C11. The cotyledons of the plant of seeds harvested from the plant with the heterozygous form of the evergreen gene are placed on sieves D1-F11. In sieves D1-F11, there are 8 cotyledons expressing the homozygous form of the evergreen gene (such as sieves A1-C11) and 22 cotyledons lacking this expression. These cotyledons may lack the evergreen gene or have the evergreen gene in heterozygous form. The ratio 8/22 confirms the expected ratio of a single recessive gene, which means that the evergreen gene exists in a homozygous form.

Example 4

It's well known that when the lower leaves of a cucumber plant are covered by the upper leaves, they become old, yellow, and begin to degrade. Among other things, this degradation is caused by the inactivity of chlorophyll, the pigment that gives leaves their green color.

Wild-type plants (lacking the evergreen gene), plants with a heterozygous form of the evergreen gene, and plants with a homozygous form of the evergreen gene were placed in a greenhouse. At the plant's maturity stage (i.e., after 10 weeks), the lower leaves were removed and placed next to each other. The results are shown in FIG7 .

Obviously, plants having the homozygous form of the evergreen gene are able to maintain their chlorophyll for a longer period of time. Likewise, it is expected that plants comprising the homozygous form of the gene will be able to be photosynthetically active for a longer period of time.

Claims (2)

1. A method for providing cucumber plants that provide fruit with improved shelf life, the method comprising the steps of: introducing a perennial green gene of SEQ ID No. 3 in a homozygous form into the cucumber plant; and wherein the expression of the perennial green gene is reduced compared to the expression of the perennial green gene in cucumber plants that do not provide fruit with improved shelf life, or the enzymatic activity of the protein encoded by the perennial green gene is reduced compared to the enzymatic activity of the protein encoded by the perennial green gene in cucumber plants that do not provide fruit with improved shelf life. 2. The gene in the homozygous form of SEQ ID No. 3 is used to provide cucumber plants that provide fruit with improved storage life.