Method for carrying out nine-time breeding by utilizing female gametes

Abstract

The invention discloses a method for carrying out nine-fold breeding by utilizing female gametes, and relates to the technical field of biological breeding. Comprising the following steps: step 1, pollinating hexaploid persimmon pollen to a'rattan royal house 'containing 2n eggs; 2, collecting young fruits which are born for 65-70 days after pollination, stripping seeds, putting the seeds into a sterilized triangular flask, treating the seeds for 30 seconds in an ultra-clean workbench by using 70% ethanol, pouring out the seeds, adding a 1% NaClO solution, soaking the seeds for 10 minutes, and shaking the seeds once every 3-4 minutes. According to the method for carrying out nine-fold breeding by utilizing female gametes, in persimmon breeding, compared with a 2n pollen path, the 2n egg path is higher in feasibility, 2n eggs and complete male germplasm with good pollen traits are hybridized, pollination populations are expanded, and polyploidy seedless materials can be obtained.

Description

Technical Field

[0001] This invention relates to the field of biological breeding technology, specifically to a method for nonoploid breeding using female gametes. Background Technology

[0002] China has abundant persimmon varieties, but most cultivated persimmons are hexaploid. Naturally occurring nonploid persimmons have larger fruits and are seedless. Hexaploid persimmons are the main cultivated varieties in my country's persimmon industry. Currently, consumer demand for seedless varieties is increasing year by year, and breeders are actively developing seedless fruits. In fruit trees such as grapes, citrus, and persimmons, fruit deseeding has become an important goal, and nonploid persimmons possess both seedless characteristics and large size. Developing new nonploid sweet persimmon varieties is crucial for the healthy and sustainable development of my country's sweet persimmon industry. Therefore, a method for nonploid breeding using female gametes is proposed to obtain nonploid persimmons. Summary of the Invention

[0003] The purpose of this invention is to provide a method for nonoploid breeding using female gametes, in order to solve the problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a method for nonoploid breeding using female gametes, comprising the following steps: Step 1: Pollinate the hexaploid persimmon flowers with pollen from the "Fujiwara Palace" containing 2n eggs; Step 2: Collect the young fruits that have formed 65-70 days after pollination, remove the seeds and place them in a sterilized Erlenmeyer flask. Treat them with 70% ethanol for 30 seconds in a clean bench, then pour them out and soak them in 1% NaClO solution for 10 minutes, shaking them once every 3-4 minutes. Step 3: Discard the NaClO solution in the Erlenmeyer flask, rinse with sterile distilled water 3-5 times until the water is clear; Step 4: Take out the sterilized seeds and place them on sterilized filter paper. Carefully remove the embryos with a scalpel and tweezers and place them in the primary culture medium for primary culture for 30-35 days. Step 5: Transfer the plants obtained from the primary culture medium to the secondary culture medium for further culture; Step Six: Molecular amplification and identification of the F1 generation of persimmon hybrids were carried out. Individuals of hybrid offspring with low economic benefits were eliminated in the early stage, and finally nonploid plants were selected.

[0005] Furthermore, the hexaploid persimmon in step one is "Fujiwara no Gosho".

[0006] Furthermore, the primary culture medium in step four is MS (1 / 2N) medium, the mass ratio of its components is: polyvinylpyrrolidone 0.5-0.7%, zeatin 0.044-0.1%, indolebutyric acid 0.03-0.056%, activated carbon 3-5%, sucrose 7-10%, agar 0.7-1%, and distilled water 80-90%, pH=5.8-6.1.

[0007] Furthermore, the specific method for the primary culture in step four is to first culture the embryos in the dark for 4-6 days, and then transfer them to 25-30℃ and a photoperiod of 14h light / 10h for primary culture.

[0008] Furthermore, the subculture medium in step five is DKW medium, the composition of which is as follows (by mass): polyvinylpyrrolidone 0.5-0.7%, zeatin 0.1-0.2%, indoleacetic acid 0.01-0.03%, sucrose 3-5%, agar 0.7-1%, and distilled water 83-95%, pH=5.8-6.1.

[0009] Furthermore, the molecular amplification method includes one of the CPCNA-2K labeled PCR program, the DlSx-AF4S labeled PCR program, or the CPCNA-2K and DlSx-AF4S labeled dual PCR program.

[0010] Furthermore, the method for selecting nonploid plants is one or more of the following: morphological identification, chromosome number identification, or flow cytometry identification.

[0011] Compared with the prior art, the beneficial effects of the present invention are: In the process of cultivating nonploid persimmons through 2n pollen pollination, 2n pollen screening is time-consuming and laborious, and pollen viability is easily affected by external factors, reducing the probability of producing nonploid plants. This method of using female gametes for nonploid breeding is more feasible than the 2n pollen route in persimmon breeding. It utilizes 2n eggs to crossbreed with completely male germplasm with good pollen traits and expands the pollination population, which can obtain polyploid seedless materials.

[0012] In addition, this scheme creates new nonploid persimmon materials through unreduced gametes. The ploidy of the new materials is determined by morphological identification, chromosome number identification, and ploidy identification based on flow cytometry. Molecular marker identification is performed on the F1 generation of persimmon hybrids. Individuals with low economic benefits can be eliminated early. By grafting tender branches, the goal of early fruiting and shortening the breeding cycle can be achieved. After years of evaluation in nurseries, the new varieties can be quickly promoted to the industry. Attached Figure Description

[0013] Figure 1This is a morphological diagram of the present invention “Fujiwara Gosho H9-1” 23# (where A is 'Kyoshiro Mizushiki' (hexaploid), and B is “Fujiwara Gosho H9-1” 23#). Figure 2 This is an observation diagram of the stomata on the leaf of the "Fujiwara Gosho H9-1" 23# of the present invention (where C is the stomata of 'Kyojo Mizushiki' and D is the stomata of "Fujiwara Gosho H9-1" 23#). Figure 3 Chromosome observation of root tip of plantlet 23# of "Fujiwara Gosho H9-1" for the present invention (where A is a hexaploid plantlet; B is plantlet 23#). Figure 4 The nuclear DNA of the hybrid offspring of "Fujiwara Gosho H9-1" in this invention was detected by flow cytometry (where A is "Kyoshiro Mizushi"; B is "Hyoshiro no"; C is "Fujiwara Gosho H9-1" 23#; D is "Fujiwara Gosho H9-1" 23# + "Kyoshiro Mizushi"; E is "Fujiwara Gosho H9-1" 23# + "Hyoshiro no"). Detailed Implementation

[0014] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] Unreduced gametes play a crucial role in plant polyploidization. Hybridization of unreduced gametes with normal gametes allows for the cultivation of intraspecific triploids exhibiting both ploidy and heterosis, making it one of the most advantageous ploidy breeding methods in plant breeding. The advantages of using unreduced gametes for breeding are significant: by hybridizing with unreduced gametes, triploids or tetraploids can be obtained from diploid plants, thus accelerating the breeding process; the offspring of hybridization between unreduced and normal gametes exhibit high heterozygosity and significant heterosis, with FDR (First Division Restitution) gametes and SDR (Second Division Restitution) gametes transmitting 80% and 40% heterozygosity to offspring, respectively; and unreduced gametes can overcome incompatibility and the "endosperm balance number" barrier in distant hybridization, resulting in no mixed-ploidy or chimeric offspring.

[0016] This invention provides a technical solution: a method for nonoploid breeding using female gametes, comprising the following steps: Step 1: Pollinate the hexaploid persimmon flowers with the "Fujiwara Palace" containing 2n eggs. The hexaploid persimmon in Step 1 can be the "Fujiwara Palace". Step 2: Collect the young fruits that have formed 65-70 days after pollination, remove the seeds and place them in a sterilized Erlenmeyer flask. Treat them with 70% ethanol for 30 seconds in a clean bench, then pour them out and soak them in 1% NaClO solution for 10 minutes, shaking them once every 3-4 minutes. Step 3: Discard the NaClO solution in the Erlenmeyer flask, rinse with sterile distilled water 3-5 times until the water is clear; Step 4: Remove the sterilized seeds and place them on sterilized filter paper. Carefully remove the embryos with a scalpel and forceps, and place them in the primary culture medium for primary culture for 30-35 days. The primary culture medium is MS (1 / 2N) medium, and its composition by mass ratio is: polyvinylpyrrolidone 0.5-0.7%, zeatin 0.044-0.1%, indolebutyric acid 0.03-0.056%, activated carbon 3-5%, sucrose 7-10%, agar 0.7-1%, and distilled water 80-90%, pH=5.8-6.1. In addition, the specific method of primary culture is to first culture the embryos in the dark for 4-6 days, and then transfer them to 25-30℃ and a photoperiod of 14h light / 10h for primary culture. Step 5: Transfer the plants obtained from the primary culture medium to the subculture medium for further culture. The subculture medium is DKW medium, and its composition by mass ratio is: polyvinylpyrrolidone 0.5-0.7%, zeatin 0.1-0.2%, indoleacetic acid 0.01-0.03%, sucrose 3-5%, agar 0.7-1%, and distilled water 83-95%, pH=5.8-6.1; Step Six: Molecular amplification and identification of the F1 generation of persimmon hybrids were carried out. Individuals of hybrid offspring with low economic benefits were eliminated in the early stage, and finally nonploid plants were selected.

[0017] The molecular amplification methods include one of the CPCNA-2K labeled PCR program, the DlSx-AF4S labeled PCR program, or the CPCNA-2K and DlSx-AF4S labeled dual PCR program.

[0018] In this protocol, the CPCNA-2K labeled PCR program is as follows: 1. Denaturation, 94℃, 0.5-3 min; 2. Annealing, 52-57℃, 30 s; 3. Final extension, 72℃, 75 s. The number of cycles is 34.

[0019] The DlSx-AF4S labeled PCR program is as follows: 1. Denaturation, 94℃, 0.5-3 min; 2. Annealing, 52℃, 30 s; 3. Final extension, 72℃, 75 s. The number of cycles is 34.

[0020] The specific PCR program for CPCNA-2K and DlSx-AF4S labeled dual PCR is as follows: 1. Denaturation, 94℃, 0.5-3 min; 2. Annealing, 54℃, 30 s; 3. Final extension, 72℃, 75 s. The number of cycles is 35.

[0021] In this protocol, all PCR amplification products were detected by 1% agarose gel electrophoresis, and images were taken using a GelDoc Go Imaging System. The labeled primers were synthesized by Qingke Biotechnology Co., Ltd., and the amplification reaction system is shown in Table 1. Table 1 Dual PCR amplification reaction system labeled CPCNA-2K and DlSx-AF4S Components of the reaction solution Sample volume (µL) 2×Es Taq Master Mix (Dye) 5 DlSx-AF4S Primer-F (10µmol / L) 0.25 DlSx-AF4S Primer-R (10µmol / L) 0.25 CPCNA-2K Primer-F (10µmol / L) 0.25 CPCNA-2K Primer-R (10µmol / L) 0.25 DNA (100-130 ng / µL) 1.5 Ultra pure water Up to 10 Primer sequences are shown in Table 2: Table 2 PCR primer sequences used for molecular markers Primer name Primercode Primer sequence (5'-3') Primer source Reference CPCNA-2K DlSx-AF4S F: ACATCCAAAGTTCTGGAG AATCAR: ATTGGTGCT TGGTCA AACATATC Akagi et al., 2014b All PCR amplification products were detected by 1% agarose gel electrophoresis and photographed using a GelDoc Go Imaging System (Bio-RAD, USA).

[0022] In this study, a total of 63 hybrid offspring were obtained. After PCR amplification and detection of the 63 hybrid offspring, 30 hybrid offspring were selected and labeled as 1#, 2#, 3#, ..., 30#.

[0023] Thirty hybrid offspring were screened and subjected to ploidy testing. Among them, nonploid plants were selected by one or more methods, including morphological identification, chromosome number identification, or flow cytometry.

[0024] Morphological identification: Place mature leaves in boiling 5% NaOH solution for 1-2 minutes until the leaves become transparent. Then transfer them to cold water, gently peel off the epidermis and place it on a glass slide. After absorbing the surface moisture, add 1-2 drops of 0.5% toluidine blue for 1-2 minutes. Gently rinse with deionized water and air dry. Measure under an optical microscope using a micrometer. Measure 3 leaves for each sample, with no less than 30 stomata per field of view.

[0025] In terms of plant morphology, compared to the hexaploid plants, plant #23 had darker leaf color, but there was no significant difference in internode length and stem diameter (see reference). Figure 1In the samples A and B, where A is 'Gongcheng Water Persimmon' (hexaploid) and B is 'Fujiwara Gosho H9-1' 23#, the stomatal differences between the two plants were analyzed. The stomatal length of plant 23# was 1.44 times that of the hexaploid plant, and the stomatal width was 1.37 times that of the hexaploid plant (refer to...). Figure 2 In the middle section, C and D, where C represents the stomata of 'Gongcheng Shuishi' and D represents the stomata of 'Fujiwara Gosho H9-1' No. 23, the stomatal density of the hexaploid plant is 1.57 times that of the No. 23 plant, and the results are basically consistent with the characteristics of the nonploid plant.

[0026] The hexaploid 'Gongcheng Water Persimmon' was used as a control, and the measurement results are shown in Table 3.

[0027] Table 3 Plant type Pore ​​density ( / mm2) Stomatal length (μm±SE) Pore ​​width (μm±SE) "Gongcheng Water Persimmon" (hexaploid) 216.54±30.66 27.57±3.11 21.79±3.2 "Fujiwara Imperial Palace H9-1" 23# 137.32±25.14 39.87±3.13 29.96±1.94 Chromosome number identification: 1.0-1.5 cm of milky white root tip from regenerated plants was treated with 0.002 mol / L 8-hydroxyquinoline for 6-7 h, then with 0.075 mol / L KCl for 0.5 h. The root tip was fixed with methanol:acetic acid (3:1) for 10-24 h, then transferred to 70% ethanol and stored at 4℃. During slide preparation, the root tip was washed 2-3 times with ultrapure water, then dissociated with 5 mol / L HCl solution at room temperature for 8 min, followed by dissociation with a mixture of 4% cellulase, 1% pectinase, and 0.075 mol / L KCl at 37℃ for 50-60 min. The slide was stained with propionic acid-chloral hydrate-hematoxylin staining solution for 3-4 h, then pressed and examined under a microscope. Chromosome counting was performed using a Nikon Ecliple 90i microscope. Hexaploid persimmon plants and milky white root tips from plant #23 were also collected for chromosome observation. Normal hexaploid persimmon plants have a chromosome number of 2n=6x=90, while nonploid plants should have 2n=9x=135. It can be observed that plant #23 has a significantly higher chromosome number than the hexaploid plant (refer to...). Figure 3 (Among them, A is a hexaploid plant and B is plant #23).

[0028] Flow cytometry identification: A small amount of leaf samples were placed in a 55 mm diameter plastic culture dish, and 2 mL of LDAPI staining solution and 1% PVP solution were added. The leaf samples were then broken up with a blade to release the cell nuclei. The sample extract was passed through a 30 μm cell filter membrane (Cell-Trics™). Then, 2 mL of DAPI nuclear fluorescence staining solution HR-B was added to the filtrate, and the ploidy was measured using a ploidy analyzer. Data analysis was performed using FCS Express software.

[0029] Specifically, using the hexaploid 'Gongcheng Shuishi' and the nonploid 'Pinghe Wu' as controls, the ploidy of the F1 progeny of 'Fujiwara Gosho H9-1' was determined by flow cytometry. The results showed that the peak fluorescence intensity of the 'Fujiwara Gosho H9-1' F1 progeny was approximately 22300, consistent with the nonploid 'Pinghe Wu', while the peak fluorescence intensity of the hexaploid plant was approximately 14500. The peak fluorescence intensity of progeny 23# of 'Fujiwara Gosho H9-1' F1 was approximately 1.53 times that of the hexaploid plant. When plant 23# was mixed with both hexaploid and nonploid plants for DNA content analysis, the results showed that the mixture of plant 23# and the hexaploid plant produced two distinct peaks, while the mixture of plant 23# and the nonploid plant produced only one peak. Therefore, progeny 23# of this hybrid combination was confirmed to be nonploid. The measurement results are as follows: Figure 4 As shown, A is "Kyojo Mizushi"; B is "Hiraku Mu"; C is "Fujiwara Gosho H9-1" 23#; D is "Fujiwara Gosho H9-1" 23# + "Kyojo Mizushi"; E is "Fujiwara Gosho H9-1" 23# + "Hiraku Mu". In this study, using the "Fujiwara Gosho H9-1" hybrid combination, embryo rescue was performed on the young fruits 65-70 days after pollination, resulting in 63 hybrid offspring. One nonploid plant was identified from these offspring (our team had previously obtained a new nonploid material using the same hybrid combination). Pollen diameter observation of "Fujiwara Gosho H9-1" before pollination revealed no 2n pollen, thus ruling out the influence of 2n pollen. The nonploid plant was obtained as a result of the combination of the 2n egg of "Fujiwara Gosho" with a normal meiotic gamete, ultimately yielding the nonploid persimmon hybrid offspring numbered 23#.

[0030] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended embodiments and their equivalents.

Claims

1. A method for nonoploid breeding using female gametes, characterized in that, Includes the following steps: Step 1: Pollinate the hexaploid persimmon flowers with pollen from the "Fujiwara Palace" containing 2n eggs; Step 2: Collect the young fruits that have formed 65-70 days after pollination, remove the seeds and place them in a sterilized Erlenmeyer flask. Treat them with 70% ethanol for 30 seconds in a clean bench, then pour them out and soak them in 1% NaClO solution for 10 minutes, shaking them once every 3-4 minutes. Step 3: Discard the NaClO solution in the Erlenmeyer flask, rinse with sterile distilled water 3-5 times until the water is clear; Step 4: Take out the sterilized seeds and place them on sterilized filter paper. Carefully remove the embryos with a scalpel and tweezers and place them in the primary culture medium for primary culture for 30-35 days. Step 5: Transfer the plants obtained from the primary culture medium to the secondary culture medium for further culture; Step Six: Molecular amplification and identification of the F1 generation of persimmon hybrids were carried out. Individuals of hybrid offspring with low economic benefits were eliminated in the early stage, and finally nonploid plants were selected.

2. The method for nonoploid breeding using female gametes according to claim 1, characterized in that, The hexaploid persimmon mentioned in step one is "Fujiwara no Gosho".

3. The method for nonoploid breeding using female gametes according to claim 1, characterized in that, The primary culture medium in step four is MS (1 / 2N) medium, with the following composition by mass ratio: polyvinylpyrrolidone 0.5-0.7%, zeatin 0.044-0.1%, indolebutyric acid 0.03-0.056%, activated carbon 3-5%, sucrose 7-10%, agar 0.7-1%, and distilled water 80-90%, pH=5.8-6.

1.

4. The method for nonoploid breeding using female gametes according to claim 1, characterized in that the primary culture method in step four is to first culture the embryos in the dark for 4-6 days, and then transfer them to 25-30℃ and a photoperiod of 14h light / 10h for primary culture.

5. A method for nonoploid breeding using female gametes according to claim 1, characterized in that, The subculture medium in step five is DKW medium, and its composition by mass ratio is: polyvinylpyrrolidone 0.5-0.7%, zeatin 0.1-0.2%, indoleacetic acid 0.01-0.03%, sucrose 3-5%, agar 0.7-1%, and distilled water 83-95%, pH=5.8-6.

1.

6. The method for nonoploid breeding using female gametes according to claim 1, characterized in that, The molecular amplification method includes one of the CPCNA-2K labeled PCR program, the DlSx-AF4S labeled PCR program, or the CPCNA-2K and DlSx-AF4S labeled dual PCR program.

7. A method for nonoploid breeding using female gametes according to claim 6, characterized in that, The method for selecting nonploid plants is one or more of the following: morphological identification, chromosome number identification, or flow cytometry.

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