Method for creating flavored sweet melon by intergeneric hybridization of thick-skinned melon and cucumber

Abstract

This invention discloses a method for creating a flavorful sweet and sour melon through interspecific hybridization of thick-skinned melon and cucumber. Using the thick-skinned melon inbred line 'Baipicui' as the female parent and the cucumber inbred line 'Jinyou 406' as the male parent, artificial pollination and hybridization are performed to obtain interspecific hybrid offspring. These offspring are then subjected to several generations of self-pollination and selection to obtain the flavorful sweet and sour melon. This invention is the first to utilize interspecific hybridization between high-quality thick-skinned melon and cucumber to create a new high-quality flavorful sweet and sour melon germplasm. This new germplasm has a moderate sweet and sour taste, a well-proportioned fruit shape, and green flesh, and can be directly used for melon variety improvement.

Description

Technical Field

[0001] This invention relates to the field of hybridization breeding technology, and in particular to a method for creating flavorful sweet and sour melons through interspecific hybridization of thick-skinned melons and cucumbers. Background Technology

[0002] Muskmelon (Cucumis melo L.) is an annual vine belonging to the genus Cucumis in the family Cucurbitaceae. Its fruit is sweet and delicious, making it one of China's traditional summer fruits. With the improvement of people's living standards, the demand for muskmelon quality has shifted from pursuing high sugar content to pursuing low sugar and healthier options, especially for people with high blood pressure, high cholesterol, and high blood sugar. The flavor quality of muskmelon is mainly determined by the content of soluble sugars and organic acids; an appropriate sugar-acid ratio helps improve the fruit's flavor quality and produce the best taste. Due to the historical emphasis on high sugar content in artificial domestication and selection, there is a severe lack of low-sugar, low-acid muskmelon resources among existing cultivated muskmelon resources. Summary of the Invention

[0003] In view of the above-mentioned defects in the prior art, the present invention aims to provide a method for creating flavorful sweet and sour melons through interspecific hybridization of thick-skinned melons and cucumbers, with the purpose of improving the organic acid content of melons.

[0004] The technical solution of this invention is as follows: A method for creating flavorful sweet and sour melons through interspecific hybridization of thick-skinned melon and cucumber, using the thick-skinned melon inbred line 'Baipicui' as the female parent and the cucumber inbred line 'Jinyou 406' as the male parent, to carry out artificial pollination and hybridization to obtain offspring of interspecific hybrid plants, and to conduct several generations of self-pollination and screening of the offspring of hybrid plants to obtain flavorful sweet and sour melons.

[0005] Furthermore, the specific steps include:

[0006] Step 1: Collect pollen from cucumber 'Jinyou 406' and pollinate it on the stigma of the female parent, thick-skinned melon 'Baipicui'. Before pollinating the female parent, the ovary needs to be emasculated and bagged for isolation until the fruit begins to swell.

[0007] Step 2: After pollination, spray the ovary of the fruit with chlorpyrifos solution and thiamethoxam solution.

[0008] Furthermore, the spraying of chlorpyrifos solution and thiamethoxam solution on the ovary of the fruit after pollination involves spraying the female flower ovary with chlorpyrifos solution on the day of pollination and spraying the female flower ovary with thiamethoxam solution on the day following pollination.

[0009] Further, the concentration of the chlorpyrifos solution in the step is 0.001% to 0.002%.

[0010] Furthermore, the concentration of the thiamethoxam solution is 0.01–0.02 mg / L.

[0011] Furthermore, the self-pollination screening of the hybrid plant offspring for several generations is no less than four generations of self-pollination screening.

[0012] Furthermore, in step 3, when performing self-pollination screening, offspring with a sweet and sour taste are selected for self-pollination.

[0013] The advantages of this invention compared to the prior art are:

[0014] The method of obtaining new high-quality sweet and sour melon germplasm through interspecific hybridization and four generations of self-pollination in this invention belongs to interspecific variation, breaks reproductive isolation, uses cultivated cucumber of the genus Cucumis as an exogenous gene pool, and utilizes the characteristics of organic acid content in it to improve cultivated melons to obtain more stable and superior traits.

[0015] The new high-quality sweet and sour melon variety cultivated in this invention has a moderately sweet and sour taste, a regular fruit shape, and green flesh, and can be directly used for melon variety improvement. Attached Figure Description

[0016] Figure 1 The results are the SSR identification results of the parents and the F1 to F4 generations of BJ-5.

[0017] Figure 2 Longitudinal sections of the fruits of 'White-skinned Crisp' and BJ-5 from F1 to F4 generations.

[0018] Figure 3 This refers to the soluble solids content of the fruit.

[0019] Figure 4 This refers to the soluble sugar content of the fruit.

[0020] Figure 5 This refers to the acidity (pH) content of the fruit.

[0021] Figure 6 This refers to the citric acid content of the fruit.

[0022] Figure 7 This refers to the malic acid content of the fruit.

[0023] Figure 8 This refers to the total acid content of the fruit.

[0024] Figure 9 The size of the fruit is determined by its sugar-acid ratio. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. The illustrative embodiments and descriptions of this invention are used to explain the invention, but are not intended to limit the invention.

[0026] An example of a method for creating a new sweet and sour melon germplasm through interspecific hybridization of thick-skinned melon and cucumber involves using the thick-skinned melon inbred line 'Baipicui' as the female parent and the cucumber inbred line 'Jinyou 406' as the male parent for artificial pollination and hybridization to obtain interspecific hybrid offspring. After multiple generations of self-pollination and selection, a new sweet and sour melon germplasm is obtained. The specific steps are as follows:

[0027] Step 1: One day before flowering, select healthy and vigorous female flowers from the 'White Skin Crisp' melon mother plant. Use tweezers to cut open the calyx and corolla of the female flower, then remove all stamens (usually 3-4) with tweezers. Isolate the flower by bagging it. Also, isolate the healthy male flowers of the 'Jinyou 406' cucumber father plant that will flower the next day by bagging it. On the day of flowering, between 9:00 and 10:00 AM, use 2-3 male flowers from the father plant to pollinate the female flowers of the mother plant. After pollination, continue to isolate the flowers by bagging them to prevent pollen contamination. At the same time, spray the ovary of the female flower with a 0.001%-0.002% chlorpyrifos solution. Then, label the flowers with the hybridization combination, pollination date, and specific treatment details. The next morning, between 9:00 and 10:00 AM, spray the ovary of the pollinated female flower with a 0.01-0.02 mg / L thiamethoxam solution.

[0028] Step 2: After the fruit matures, collect the seeds. In the end, a total of 7 plump hybrid offspring seeds were obtained.

[0029] Step 3: Germinate and sow the 7 harvested hybrid seeds and transplant them to the Baima Research and Teaching Base of Nanjing Agricultural University. Cultivate and manage them in a plastic greenhouse in the field. The 7 F1 plants are numbered BJ-1, BJ-2, ​​BJ-3, BJ-4, BJ-5, BJ-6, and BJ-7 in sequence. Observe the fruit characteristics of the offspring F1 plants.

[0030] Step 4: After tasting and screening, among the 7 F1 plants, only the fruit of BJ-5 tasted sour, while the fruit of the other 6 plants was normal.

[0031] Step 5: Self-pollinate 7 F1 plants to obtain F2 plants. Germinate and sow each F2 plant and transplant them to the Baima Research and Teaching Base of Nanjing Agricultural University. Cultivate and manage them in plastic greenhouses in the field. After tasting and screening, none of the F2 plants of 6 materials, namely BJ-1, BJ-2, ​​BJ-3, BJ-4, BJ-6, and BJ-7, had a sour taste. Among the F2 plants of BJ-5, the ratio of sour (or sweet and sour) to non-sour (sweet) fruits was 1:2.

[0032] Step 6: Self-pollinate the sour-tasting individual plants of F2 from BJ-5, and continue to screen them according to whether they taste sour. From the F4 generation materials obtained, four new varieties of sweet and sour melon with excellent fruit shape and stable genetics were selected and numbered BJ-5F4-1, BJ-5F4-2, BJ-5F4-3, and BJ-5F4-4 respectively.

[0033] The identification of the new germplasm materials of flavored melons obtained in the above embodiments mainly includes: morphological identification, molecular marker identification and nutritional quality identification of the obtained progeny materials.

[0034] The molecular marker identification method for new germplasm materials of flavorful melons, with the following specific steps:

[0035] Leaves from the female parent melon 'Baipicui', the male parent cucumber 'Jinyou 406', and F1, F2, F3, and F4 plants were collected and improved according to the method of Luan et al. (2008). DNA was extracted from the parent plants and their hybrid offspring.

[0036] Young leaves from generations F1 to F4 of BJ-5 were subjected to next-generation sequencing, which was performed by Shanghai Meiji Biotechnology Co., Ltd. 98 pairs of specific primers for the paternal parent 'Jinyou 406' were designed and amplified for both parents. The results showed that the 98 pairs of primers had good polymorphism.

[0037] Take a 96-well PCR plate and add the following reagents in sequence:

[0038] Mix was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0039]

[0040] PCR amplification procedure:

[0041]

[0042] Electrophoresis results analysis showed that simultaneous amplification of both parents and F1, F2, F3, and F4 hybrids yielded 23 primer pairs with varying amplification results. Of these, 20 primer pairs only amplified the paternal characteristic band in F1 hybrids, while the remaining materials showed no band. Two primer pairs only amplified the paternal characteristic band in F4 hybrids. One primer pair showed no band in F4 hybrids, while both parents showed bands. Figure 1 As shown.

[0043] The specific steps for plant morphological identification of new germplasm materials of flavorful melons are as follows:

[0044] In September 2023, four new germplasm materials of 'White Skin Crisp' and flavored melon were planted at the Baima Scientific Research and Teaching Base of Nanjing Agricultural University. Following the methods of the "Description Specification and Data Standard of Melon Germplasm Resources", traits such as fruit stalk length, fruit longitudinal diameter, fruit transverse diameter and flesh thickness were observed and counted in mid-November.

[0045] By observing and comparing with the control parent, it was found that the four new flavor melon germplasms exhibited diversity in several traits. The fruit stalks of BJ-5F4-1 and BJ-5F4-4 were significantly shorter than those of the control parent, while the fruit diameter and flesh thickness of BJ-5F4-1, BJ-5F4-2, BJ-5F4-3, and BJ-5F4-4 were significantly wider than those of the control parent. This resulted in the four new flavor melon germplasms having attractive fruit shapes and thicker flesh (Table 1). The control parent, 'White Skin Crisp', had oval-shaped fruits with green striped markings and orange flesh; while BJ-5F4-3 and BJ-5F4-4 had round fruits. BJ-5F4-4 lacked striped markings and had green flesh, while the flesh of BJ-5F4-1, BJ-5F4-2, and BJ-5F4-3 was whitish-green. Figure 2 As shown.

[0046] Table 1 Fruit appearance characteristics

[0047] Material Fruit stalk length / cm Fruit longitudinal diameter / cm Fruit diameter (cm) Fruit shape index The flesh is cm thick taste White crispy 4.70±0.36 12.80±0.80 9.00±1.31 1.43±0.12 2.07±0.21 sweet <![CDATA[BJ-5F4-1]]> <![CDATA[2.29±0.41 ** ]]> 14.75±1.04 <![CDATA[9.90±0.73 ** ]]> 1.50±0.15 <![CDATA[2.50±0.19 ** ]]> Sour / Sweet and sour <![CDATA[BJ-5F4-1]]> 4.98±0.70 14.60±0.99 <![CDATA[10.40±0.87 ** ]]> 1.41±0.15 <![CDATA[2.20±0.34 ** ]]> Sour / Sweet and sour <![CDATA[BJ-5F4-1]]> 4.31±0.78 12.54±1.73 <![CDATA[10.36±1.15 ** ]]> 1.22±0.20 <![CDATA[2.37±0.43 ** ]]> acid <![CDATA[BJ-5F4-1]]> <![CDATA[1.47±0.21 ** ]]> 10.53±0.21 <![CDATA[9.97±0.21 ** ]]> 1.06±0.01 <![CDATA[2.13±0.15 ** ]]> acid

[0048] The specific steps for identifying the nutritional quality of new germplasm materials for flavorful melons are as follows:

[0049] The pulp of the parent variety 'Baipicui' and four new varieties of flavored melon were used as test materials. The soluble solids content, acidity (pH), soluble sugar content, citric acid content and malic acid content were determined, and the total acid content and sugar-acid ratio were calculated.

[0050] Formula: Total acid content = Citric acid content + Malic acid content

[0051] Formula for sugar-acid ratio: Total content / Total sugar content (soluble sugar content)

[0052] (1) Determination of soluble solids content

[0053] The soluble solids content was determined using a portable digital refractometer PAL-1: A. Zero the saccharimeter; B. Clean the refracting prism, then crush the pulp of a ripe melon to extract the juice, drop a few drops of the juice onto the refracting prism surface, cover with the lid, and press the measurement button to obtain the soluble solids content of the solution. The portable digital refractometer PAL-1 was purchased from ATAGO Scientific Instruments Co., Ltd.

[0054] (2) Acidity (pH) measurement

[0055] pH was measured using a PH-100pro pen-type pH meter: A. Instrument calibration: 6.86 pH and 4.00 pH standard buffer solutions were used for calibration; B. Ripe melon pulp was crushed to extract juice. The pH electrode was placed in the melon juice, and the reading on the display stabilized; this reading is the acidity (pH) of the solution. The PH-100pro pen-type pH meter was purchased from Shanghai Lichen Instrument Technology Co., Ltd.

[0056] (3) Determination of soluble sugar content

[0057] Soluble sugar content was determined using a kit and an ELISA reader. Three portions of pulp from mature melons of each strain were taken and the assay was performed in triplicate. The kit was purchased from Nanjing Jiancheng Bioengineering Institute, and the ELISA reader was a Cytation 3 multi-functional ELISA reader.

[0058] (4) Determination of citric acid and malic acid content

[0059] The contents of citric acid and malic acid were determined by liquid chromatography-mass spectrometry (LC-MS). Three portions of pulp from mature melons of each parent and flavor acid population were taken and replicated three times. The analysis was performed by Nanjing Hanguang Testing Technology Co., Ltd. The instrument used was an Agilent 1260 high-performance liquid chromatograph connected in series with a G6420A mass spectrometer.

[0060] Compared with the control parent, the nutritional quality of the four new flavored melon germplasms showed significant differences. The soluble solids content of all four new flavored melon germplasms was lower than that of the control parent, with BJ-5F4-3 and BJ-5F4-4 showing significantly lower contents. Figure 3 As shown; the soluble sugar content of BJ-5F4-1 and BJ-5F4-2 was lower than that of the control parent, while the content of BJ-5F4-3 and BJ-5F4-4 was significantly higher than that of the control parent. The taste of these two materials was "sweet and sour," as... Figure 4 As shown; the acidity (pH) of BJ-5F4-1, BJ-5F4-2, BJ-5F4-3, and BJ-5F4-4 was significantly lower than that of the control parent plant, such as Figure 5 As shown; the citric acid content of the four new flavor melon varieties was higher than that of the control parent, with highly significant differences observed in the content of BJ-5F4-1, BJ-5F4-2, and BJ-5F4-3. Figure 6 As shown; the malic acid content of BJ-5F4-1, BJ-5F4-2, and BJ-5F4-4 was higher than that of the control parent parent, such as Figure 7 As shown; the total acid content of all four new flavor melon germplasms was higher than that of the control parent, such as Figure 8 As shown; the sugar-acid ratios of the four new flavor melon varieties were all significantly lower than those of the control. Among them, BJ-5F4-2, BJ-5F4-3, and BJ-5F4-4 had moderate sugar-acid ratios. Figure 9 As shown.

Claims

1. A method for creating a flavored sweet melon by intergeneric cross between Cucumis cantutlus and Cucumis sativus, characterized by, Using the thick-skinned melon inbred line 'Baipicui' as the female parent and the cucumber inbred line 'Jinyou 406' as the male parent, artificial pollination and hybridization were carried out to obtain interspecific hybrid offspring. Several generations of self-pollination and selection were then performed on these hybrid offspring to obtain a sweet and sour melon with a specific flavor. The method specifically includes the following steps: Step 1: Collect pollen from cucumber 'Jinyou 406' and pollinate it on the stigma of the female parent, thick-skinned melon 'Baipicui'. Before pollinating the female parent, the ovary needs to be emasculated and bagged for isolation until the fruit begins to swell. Step 2: After pollination, spray the ovary of the fruit with chlorpyrifos solution and thiamethoxam solution. Specifically, spray the ovary of the female flower with chlorpyrifos solution on the day of pollination, and spray the ovary of the female flower with thiamethoxam solution on the day after pollination. The concentration of the chlorpyrifos solution in this step is 0.001% to 0.002%, and the concentration of the thiamethoxam solution is 0.01 to 0.02 mg / L. Step 3: After the fruit matures, collect the seeds and perform self-pollination selection.

2. The method for creating a flavorful sweet and sour melon through interspecific hybridization of thick-skinned melon and cucumber according to claim 1, characterized in that, The self-pollination screening of the hybrid plant offspring for several generations shall be no less than four generations of self-pollination screening.

3. The method for creating a flavorful sweet and sour melon through interspecific hybridization of thick-skinned melon and cucumber according to claim 1, characterized in that, In step 3, when performing self-pollination screening, offspring with a sweet and sour taste are selected for self-pollination.

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