Method for reducing multifold pigment marigold chimera

Abstract

The invention relates to a method for reducing multiploid pigment marigold chimeras, and belongs to the technical field of cell engineering tissue culture. The method comprises the following steps: (1) pretreating seeds for later use; (2) performing polyploidy treatment: transferring the standby seeds into an MS culture medium containing colchicine, and performing polyploidy treatment; (3) sequentially carrying out dark culture and light culture on the seeds subjected to polyploidy treatment; (4) identifying a marigold variant plant; (5) screening and regenerating variant plants; (6) identifying the marigold variant plants again; (7) expanding propagation of variant plants; and (8) rooting culture. According to the application, the purified polyploidy pigment marigold regenerated plant can be obtained by combining colchicine polyploidy treatment with morphological identification and cytological identification as well as tissue culture and cytological identification again, and the proportion of chimera plants is remarkably reduced.

Description

Technical Field

[0001] The present application belongs to the technical field of cell engineering tissue culture, and in particular relates to a method for reducing polyploidized pigment marigold chimeras. Background Art

[0002] Marigold (Tagetes erecta L.) usually has a head-shaped inflorescence, and the flower color is mainly orange. Its flowers are large, with distinct petals and unique shapes, and have high ornamental and medicinal value. Since the 1960s, Towner et al. discovered male sterile plants of pigment marigolds, and the three-line breeding strategy has been widely used in pigment marigolds. Due to the participation of the sterile line, the entire breeding process reduces the risk of self-pollination contamination, simplifies the breeding procedure, and reduces the workload of manual emasculation.

[0003] In the breeding of pigment marigolds, the restorer line is used as the male parent and the sterile line is used as the female parent to produce F1 hybrids with hybrid vigor. At the same time, the sterile and fertile plants are preserved by using male fertile plants as the male parent and sterile plants as the female parent. This strategy effectively utilizes the characteristics of the sterile line and improves the reproduction efficiency and the expression of hybrid vigor.

[0004] Colchicine-induced polyploidy is an effective breeding method that can obtain polyploid plants in a relatively short period of time. Polyploid plants usually have the characteristics of strong stress resistance, high content of metabolites, and high nutritional value. At present, marigold breeding is mainly focused on hybrid breeding, and there are few reports on polyploidy. In the existing marigold chromosome doubling, colchicine is mainly used to treat the stem tip of the seedling to obtain tetraploid plants. This technology has the advantages of short cycle and good effect, and can significantly improve the stress resistance and metabolite content of plants, thereby enhancing their nutritional value. In addition, through chromosome doubling, new germplasm resources can be created, which is helpful for breeding research and variety improvement. After the chromosomes of marigold are doubled, not only the plant height and crown width are reduced, but also the ground diameter is thickened, which improves the resistance of the plant; the leaves are enlarged and the thickness of the palisade tissue is increased, which is conducive to improving the photosynthetic efficiency and the ability to adapt to the environment. However, the use of colchicine doubling treatment is often accompanied by the problem of chimeras. Flow cytometry results show that some plants identified as tetraploid are actually chimeras, that is, there is a mixture of diploid and tetraploid cells in the body. This situation may affect the subsequent phenotypic expression and genetic stability. Genetic stability issues, polyploid plants usually have unstable genetic characteristics, and their offspring may revert to the normal diploid state, which may affect the long-term goal of variety improvement. Summary of the invention

[0005] In order to solve or partially solve the problems existing in the related art, the present application provides a method for reducing the chimeras of polyploidized pigment marigolds. Through colchicine polyploidization treatment combined with morphological identification and cytological identification, as well as tissue culture and re-cytological identification, purified polyploidized pigment marigold regenerated plants can be obtained, which significantly reduces the proportion of chimera plants. This is of great significance for the breeding research and large-scale production of high-quality and high-yield pigment marigolds.

[0006] The method for reducing polyploidized pigment marigold chimera in the present application comprises the following steps: (1) Seed pretreatment: The male sterile dual-purpose seeds of pigment marigold are dried, cleaned, soaked, disinfected, and rinsed in sequence and then set aside; (2) Polyploidization treatment: The spare seeds are transferred to MS medium containing colchicine for polyploidization treatment; the formula of the MS medium containing colchicine is: MS powder 4.4 g / L, 6-BA 1 mg / L, NAA 0.1 mg / L, agar 8 g / L, sugar 30 g / L, pH = 5.8-6.0, colchicine concentration is 0.10wt%; (3) The polyploidized seeds are cultured in dark and light conditions in turn; (4) Identification of marigold mutant plants: When the plants grow to 3-5 cm under light culture, morphological and cytological identification is performed to screen out marigold mutant plants with polyploid characteristics; (5) Screening and regeneration of mutant plants: Leaves of the selected marigold mutant plants were selected as explants and inoculated into the differentiation induction medium, and then light culture was performed to induce differentiation of adventitious buds; (6) Re-identification of marigold mutant plants: The induced adventitious buds are re-identified by cytology, and the adventitious buds of marigold mutant plants that do not have polyploid characteristics are eliminated, and the adventitious buds of marigold mutant plants that still have polyploid characteristics are screened out; (7) Propagation of variant plants: The adventitious buds of the selected variant marigold plants that still have polyploid characteristics are transferred to the subculture medium for propagation and subculture; (8) Rooting culture: The buds elongated by the propagation culture are subjected to rooting culture to obtain purified polyploid pigment marigold regenerated plants.

[0007] Furthermore, in step (3), the culture temperature of the dark culture and the light culture are both 24±2°C, the relative humidity is maintained at 65-70%, the light intensity during light culture is 2500-3000lx, the light time is 16h / d, the light culture cycle is 9-15 days, and the dark culture period is one week.

[0008] Furthermore, the formula of the induction differentiation medium is: MS powder 4.4g / L, 6-BA 5mg / L, IAA 3mg / L, silver nitrate 2mg / L, agar 8g / L, sugar 30g / L, pH=6.0.

[0009] Furthermore, the formula of the subculture medium is: MS powder 4.4 g / L, agar 8 g / L, sugar 30 g / L, pH=6.0; the subculture time is 30-50 days.

[0010] Furthermore, the formula of the rooting culture is: MS powder 4.4 g / L, NAA 0.15 mg / L, agar 8 g / L, sugar 30 g / L, pH=6.0; the rooting culture time is 30 days.

[0011] The beneficial effects of this application are: 1. In the present application, pre-treated pigment marigold male sterile dual-purpose seeds are subjected to polyploidization treatment in MS medium containing colchicine. During the polyploidization treatment, some cells may double successfully while other cells fail to double, thereby resulting in the production of chimeras; even after morphological and cytological identification, chimeras still exist; after preliminary identification, the present application selects mutants for rapid tissue culture induction to differentiate adventitious buds, and cytological identification is performed again on the induced differentiated adventitious buds to further screen out adventitious buds of marigold mutant plants that still have polyploid characteristics, and after subculture and rooting culture, purified polyploidized pigment marigold regenerated plants can be obtained, significantly reducing the proportion of chimera plants.

[0012] (2) This application uses colchicine polyploidization treatment combined with morphological identification and cytological identification, as well as tissue culture and re-cytological identification, which can shorten the breeding time compared to traditional breeding methods. DETAILED DESCRIPTION

[0013] The embodiments of the present application will be described in more detail below with reference to examples. Although the embodiments of the present application are shown in the examples, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present application more thorough and complete, and the scope of the present application can be fully communicated to those skilled in the art.

[0014] Example 1 The method for reducing polyploidized pigment marigold chimeras in this embodiment comprises the following steps: (1) Seed pretreatment Seed drying: Expose the male sterile dual-purpose seeds of pigment marigold to the sun for 3 days to kill the microorganisms on the surface of the seeds and reduce the occurrence of diseases. When drying the seeds, make sure that the seeds are evenly spread and the thickness does not exceed 0.5 mm to ensure that each seed can fully receive sunlight. At the same time, keep the ambient temperature between 22-28℃ to improve the germination rate.

[0015] Cleaning: After drying, rinse the seeds with tap water to remove dust and impurities on the surface. During this process, some damaged or immature seeds will float on the water surface. These seeds will be removed to increase the germination rate.

[0016] Seed soaking: Soak the seeds in warm water (about 20-30℃) for 5 hours. This step can make the seed coat soft and easier to peel off from the seeds, while promoting the seeds to absorb water, which helps the subsequent germination process.

[0017] Peeling: After soaking the seeds, use tweezers to carefully remove the seed coat on the surface. Since the seeds have absorbed water and become soft, be careful at this time to avoid damaging the inside of the seeds.

[0018] Disinfection: Place the peeled seeds in a clean bench for disinfection. First, use 2% sodium hypochlorite solution to shake and disinfect for 10 minutes, and then use 0.1% mercuric chloride solution to shake and disinfect again for 10 minutes to kill pathogens on the surface and inside of the seeds.

[0019] Rinse: After disinfection, rinse the seeds at least four times with sterile water to ensure that residual disinfectant is completely removed to prevent damage to the seeds.

[0020] Standby: Place the processed seeds in a sterile environment to dry. After the surface of the seeds is dry, they can be used for subsequent cultivation and processing.

[0021] (2) Preparation of culture medium Sterilization of culture medium: Prepare the culture medium with MS powder, the formula of which is: MS powder 4.4 g / L, 6-BA 1 mg / L, NAA 0.1 mg / L, agar 8 g / L, sugar 30 g / L. Adjust the culture medium to pH = 6.0 using a pH meter and sterilize at 121°C for 15 min.

[0022] Add colchicine solution: Place the sterilized culture medium at room temperature and wait for it to cool naturally to a temperature that is not too hot to touch. In a sterile operating table, use a colchicine solution that has been sterilized by filtering through a 0.22 micron filter and add it to the cooled culture medium to make the working concentrations 0.05%, 0.10%, 0.15%, and 0.20%, respectively. Mix the colchicine and culture medium thoroughly and dispense them into sterilized tissue culture bottles. This step needs to be performed under strict sterile conditions.

[0023] Store away from light: Since colchicine is sensitive to light, the culture medium after adding the colchicine solution should be immediately stored in a light-proof container to avoid light-induced decomposition of colchicine, and stored in a refrigerator at 4°C for subsequent use.

[0024] (3) Polyploidization of marigold germplasm Sowing: Under sterile conditions, use a clean bench to perform the following operations: Transfer the preliminarily treated seeds one by one to the MS medium containing the above-mentioned different concentrations of colchicine. Place 10 seeds in each culture bottle to ensure that the seeds are evenly distributed and avoid contact with each other to reduce the risk of cross contamination. For statistical and experimental needs, set up 10 replicate bottles for each colchicine concentration, totaling 100 seeds.

[0025] Dark culture: After transferring the seeds, seal the culture bottle with a sterile sealing film to keep the culture medium sterile and prevent excessive evaporation of water. Then, move the sealed culture bottle into the culture room for dark culture for one week. Dark culture helps break the dormancy of seeds and the initial root growth, and can also reduce the decomposition of colchicine.

[0026] Light culture: After one week, transfer the culture bottles from dark culture conditions to light conditions for continued culture. The environmental conditions of the culture room must be strictly controlled: the temperature is 24±2℃, which is the optimal temperature for marigold growth; the light intensity is 2500-3000lx to ensure that the plant cells have enough light energy for photosynthesis; the light time is set to 16 hours a day to simulate the natural light cycle; the relative humidity is maintained at about 65% to prevent the culture medium from losing water too quickly and at the same time not causing diseases due to excessive humidity.

[0027] Observe and record: During the light culture period, regularly observe the germination of seeds and the growth of plants, and record relevant data, such as root length, bud length, number of leaves, etc., in order to subsequently analyze the effects of different colchicine concentrations on plant growth.

[0028] Table 1 shows the induction effect of marigold seeds treated with different colchicine concentrations As shown in Table 1, the number of chromosomes doubled after chromosome doubling. When the concentration of colchicine was 0.1wt%, both the mutation rate and the survival rate were high, so it was a more suitable mutagenic concentration for medicinal marigold. (4) Identification of marigold mutant plants When the marigold plants grew to about 3-5 cm, morphological identification and cytological identification were carried out in sequence under the sterile environment of a clean bench.

[0029] Morphological identification: Mainly observe plant height, branching, crown width, flower diameter, flowering period, flower color, number of flowers per plant, weight of flowers per plant, leaf color, stem color, stem thickness and other indicators.

[0030] Cytological identification: drop a drop of water in the center of the slide, tear off the lower epidermis of the marigold leaf, place it in the water drop, flatten the leaf epidermis and press it into a slice, and observe the stomatal density, palisade tissue thickness, etc. under an upright microscope.

[0031] Chromosome counting: Cut the marigold stem segment and root it in a vase in water at room temperature. When the root system grows to 1-2 cm, take the sample. Use tweezers to pick up the tender white root tip about 1 cm (when the root tip is observed under light, there is a white spot, which is the meristem required for observation and can be taken) and wash it with distilled water. Use 0.002 mol / L 8-hydroxyquinoline solution at 0-4℃ for 2h; or saturated p-dichlorobenzene at 0-4℃ for 4h. Rinse the pretreated root tip with clean water, fix it with Carnoy's fixative (volume ratio of anhydrous ethanol: glacial acetic acid = 3:1) for 24h, and then rinse it with 95% alcohol until there is no glacial acetic acid. Wash the root tip with distilled water, put it in a 1 mol / L hydrochloric acid water bath preheated to 60℃ for dissociation for 10-15min, and then put it in distilled water for hypotonicity for 1h. Place the root tip in a 1.5mL centrifuge tube with a cap, add modified phenol fuchsin staining solution and stain for 2.5h. Take out the root tip and place it on a slide, cut the meristem (i.e., white spot, about 1-2 mm), gently crush it with the handle of a knife, add a drop of modified phenol fuchsin staining solution, cover with a coverslip, and absorb the excess staining solution. Take the metaphase phase with clear chromosome outline, moderate staining, and dispersion without overlapping, observe under a microscope, take pictures; and observe multiple fields of view. At the same time, select representative individual plants for ploidy identification based on flow cytometry technology.

[0032] (5) Screening and regeneration of mutant plants: Leaves of the selected marigold mutant plants were selected as explants and inoculated into the induction differentiation medium, and then light culture was carried out to induce differentiation of adventitious buds; the formula of the induction differentiation medium was: MS powder 4.4g / L, 6-BA 5mg / L, IAA 3mg / L, silver nitrate 2mg / L, agar 8g / L, sugar 30g / L, pH=6.0.

[0033] The light culture conditions were 16-hour light cycle culture at a temperature of 24±2°C and a light intensity of 2500-3000 lx, and the relative humidity was maintained at about 65%.

[0034] (6) Re-identification of marigold mutant plants: The induced adventitious buds were re-identified by cytology. The cytological identification method was the same as above. The adventitious buds of marigold mutant plants that did not have polyploid characteristics were eliminated, and the adventitious buds of marigold mutant plants that still had polyploid characteristics were screened out.

[0035] (7) Propagation of variant plants: The adventitious buds of the selected variant plants with polyploid characteristics are transferred to the subculture medium for propagation and subculture; the formula of the subculture medium is: MS powder 4.4g / L, agar 8g / L, sugar 30g / L, pH = 6.0.

[0036] (8) Rooting culture: The buds elongated by the propagation culture are subjected to rooting culture to obtain purified polyploid pigment marigold regenerated plants.

[0037] (9) The purified polyploidized pigment marigold regenerated plants (i.e., tissue culture seedlings) obtained in step (8) are placed in a greenhouse or indoor environment for seedling hardening before transplanting. This process takes 7 days to allow the seedlings to gradually adapt to the external environment. Peat soil, perlite and leaf mold are mixed in a ratio of 5:3:2 to ensure that the soil is loose, well-drained and rich in organic matter. The soil is exposed to the sun for several days to achieve a disinfection effect. Fill the prepared soil into the potting container, use sterilized tweezers to gently remove the tissue culture seedlings, and use clean water to gently rinse off the excess culture medium to avoid damaging the root system. Place the seedling in the pot and gently press the soil to fix it. Ensure that the root neck (the junction of the root and the stem) is flush with or slightly above the soil surface. Immediately after transplanting, water the plants gently with a watering can, but do not use too much force to avoid breaking up the soil or damaging the seedlings. Keep the soil moist but not waterlogged. Place the transplanted marigolds in a warm, well-lit place, avoiding strong winds and direct sunlight. The temperature is 20-25℃ and the humidity is moderate. Observe the growth of marigolds every day, pay attention to water management and pest control. Fertilize regularly to promote the healthy growth of plants.

[0038] Comparative Example 1 Based on the fact that the mutation rate and the survival rate were both high when the colchicine concentration was 0.1 wt % in Example 1, this colchicine concentration was used to perform polyploidization treatment on the male sterile dual-purpose line seeds of pigment marigold, and the treatment conditions were the same as in the example.

[0039] In addition to the above operations, the difference between Comparative Example 1 and Example 1 is that the re-identification of the marigold mutant plants in step (6) of Example 1 is not performed in Comparative Example 1, and the rest are the same.

[0040] The polyploidized pigment marigold regenerated plants obtained in Example 1 and Comparative Example 1 were subjected to ploidy identification using flow cytometry. The identification results are shown in Table 2.

[0041] Table 2 shows the effects of different treatments on tetraploid yield in Example 1 and Comparative Example 1 As can be seen from Table 2, compared with the scheme in Comparative Example 1 in which no re-identification was performed, in Example 1 of the present application, cytological identification was performed again on the induced adventitious buds, the adventitious buds of marigold variant plants that did not have polyploid characteristics were eliminated, and the adventitious buds of marigold variant plants that still had polyploid characteristics were screened out. After subsequent subculture and rooting culture, purified polyploidized pigment marigold regenerated plants were obtained, which significantly reduced the proportion of chimera plants. The embodiments of the present application have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for reducing polyploidized pigment marigold chimeras, characterized in that: The following steps are involved: (1) Seed pretreatment: The male sterile dual-purpose seeds of pigment marigold are dried, cleaned, soaked, disinfected, and rinsed in sequence and then set aside; (2) Polyploidization treatment: The spare seeds are transferred to MS medium containing colchicine for polyploidization treatment; the formula of the MS medium containing colchicine is: MS powder 4.4 g / L, 6-BA 1 mg / L, NAA 0.1 mg / L, agar 8 g / L, sugar 30 g / L, pH = 5.8-6.0, colchicine concentration is 0.10wt%; (3) The polyploidized seeds are cultured in dark and light conditions in turn; (4) Identification of marigold mutant plants: When the plants grow to 3-5 cm under light culture, morphological and cytological identification is performed to screen out marigold mutant plants with polyploid characteristics; (5) Screening and regeneration of mutant plants: Leaves of the selected marigold mutant plants were selected as explants and inoculated into the differentiation induction medium, and then light culture was performed to induce differentiation of adventitious buds; (6) Re-identification of marigold mutant plants: The induced adventitious buds are re-identified by cytology, and the adventitious buds of marigold mutant plants that do not have polyploid characteristics are eliminated, and the adventitious buds of marigold mutant plants that still have polyploid characteristics are screened out; (7) Propagation of variant plants: The adventitious buds of the selected variant marigold plants that still have polyploid characteristics are transferred to the subculture medium for propagation and subculture; (8) Rooting culture: The buds elongated by the propagation culture are subjected to rooting culture to obtain purified polyploid pigment marigold regenerated plants.

2. A method for reducing polyploidized pigment marigold chimeras according to claim 1, characterized in that: The temperature of the dark culture and light culture in step (3) is 24±2°C, the relative humidity is maintained at 65-70%, the light intensity is 2500-3000lx, the light time is 16h / d, the light culture cycle is 9-15 days, and the dark culture period is one week.

3. A method for reducing polyploidized pigment marigold chimeras according to claim 1, characterized in that: The formula of the induction differentiation medium is: MS powder 4.4g / L, 6-BA 5mg / L, IAA 3mg / L, silver nitrate 2mg / L, agar 8g / L, sugar 30g / L, pH=6.

0.

4. A method for reducing polyploidized pigment marigold chimeras according to claim 1, characterized in that: The formula of the subculture medium is: 4.4 g / L MS powder, 8 g / L agar, 30 g / L sugar, pH=6.0; the subculture time is 30-50 days.

5. A method for reducing polyploidized pigment marigold chimeras according to claim 1, characterized in that: The formula of the rooting culture is: 4.4 g / L MS powder, 0.15 mg / L NAA, 8 g / L agar, 30 g / L sugar, pH=6.0; the rooting culture time is 30 days.

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