A method for inducing haploids through radiation pollination of Camellia oleifera flowers

Abstract

This invention discloses a method for inducing haploids in Camellia oleifera through radiation pollen pollination, belonging to the field of plant tissue culture. The method includes the following steps: irradiating Camellia oleifera pollen; determining the viability and germination rate of the irradiated pollen; performing hybridization pollination using the irradiated pollen; selecting the pollinated embryos and inoculating them into a culture medium for embryo rescue; and screening the plants obtained from the embryo rescue for ploidy identification to identify haploids. This invention creates a new approach for cultivating haploids in Camellia oleifera, applicable to both the Camellia oleifera and Camellia brevicornuate groups, and can be applied to the breeding of Camellia oleifera varieties, accelerating the Camellia oleifera breeding process.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, and relates to the creation of haploid tissue culture seedlings of Camellia oleifera, specifically to a method for inducing haploids through radiation pollen pollination in Camellia oleifera. Background Technology

[0002] Camellia oleifera is a unique woody oilseed tree species native to southern my country. Its kernels contain 45%-60% oil, and the extracted oil is clear, fragrant, and a high-quality edible oil. Its unsaturated fatty acid content can reach over 90%, mainly oleic and linoleic acids. It is also rich in vitamin E, camellia saponins, polyphenols, squalene, and other functional active ingredients. Long-term consumption can lower blood pressure and blood lipids, thus preventing hypertension and cardiovascular diseases, and has health benefits such as improving immunity and delaying aging. In recent years, relevant national departments have attached great importance to the development of the camellia oleifera industry, and it has now been listed as a key woody oilseed tree species for development. Currently, my country's existing camellia oleifera planting area has reached 4.5333 million hectares, forming a production value of hundreds of billions of yuan. However, most of my country's camellia oleifera forests are low- to medium-yield forests, and the area under improved varieties accounts for only 45.7%. The uneven quality of camellia oleifera varieties seriously restricts the rapid development of the camellia oleifera industry. Accelerating the breeding of improved varieties is of paramount importance for the development of the camellia oleifera industry.

[0003] Camellia oleifera is a highly ploid perennial cross-pollinated woody plant. Conventional hybridization and natural selection methods are time-consuming and inefficient. Haploid culture technology can rapidly obtain genetically stable homozygous haploid intermediate materials, saving breeding time and workload, and improving breeding speed and efficiency. Previous researchers conducted extensive studies on Camellia oleifera anther culture, attempting to cultivate haploid Camellia oleifera plants using this method. However, all studies only obtained anther callus tissue and failed to cultivate haploid Camellia oleifera plants. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of existing technologies and provide a method for inducing haploids through radiation pollen pollination in Camellia oleifera. Traditional Camellia oleifera breeding methods are time-consuming and inefficient. Although haploid culture technology can improve efficiency, basic research on Camellia oleifera is weak, and the application of anther culture methods is difficult. This invention utilizes radiation pollen for hybridization pollination and promptly rescues the obtained embryos (early isolation and culture of embryos that are difficult to sow and seedling due to nutritional or physiological reasons or that have aborted or degenerated in the early stages of development), thereby obtaining haploid germplasm of Camellia oleifera. This invention innovates a new approach for haploid breeding of Camellia oleifera. This method has the advantages of short breeding time, low cost, and high efficiency, and can be used to create a high-yield and high-efficiency breeding system for Camellia oleifera.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A method for inducing haploids through pollination with Camellia oleifera radiata pollen includes the following steps:

[0007] (1) Collect pollen, using 60 -Co radiation sources radiate pollen.

[0008] (2) Determine the viability and germination rate of radiated pollen.

[0009] (3) Use highly viable and high-germination-rate radiated pollen to pollinate the female parent, cover it with a bag after pollination, and remove the bag one week after pollination.

[0010] (4) The following year, the fruits were harvested and subjected to low-temperature treatment at 2℃~8℃ for 3~7 days. Immature embryos were then extracted from the seeds and inserted downwards into a germination medium (WPM + 0.5~3.0 mg / L 6-BA + 0~1.0 mg / L NAA + 20~50 g / L sucrose + 7 g / L agar, pH 5.5~6.0). After inoculation, the embryos were cultured at 26~30℃ in darkness for 20~30 days, then transferred to light conditions for 60~80 days to induce seedling formation. In this step, some embryos could directly germinate into seedlings through early maturation, while others would induce embryogenic callus tissue.

[0011] (5) The material that did not germinate prematurely in step (4) was inoculated into embryogenic callus induction medium (WPM + 0.1-2.0 mg / L 6-BA + 0.1-1.0 mg / L NAA + 20-40 g / L sucrose + 7 g / L agar, pH 5.5-5.8) for embryogenic callus induction. The obtained embryogenic callus was inoculated into embryogenic callus proliferation medium (MS + 0-1.0 mg / L 2,4-D + 20-40 g / L sucrose + 7 g / L agar, pH 5.5-5.8) for proliferation culture. Afterwards, the embryos were inoculated onto globular embryo induction medium (MS + 0–1.5 mg / L 2,4-D + 20–40 g / L sucrose + 7 g / L agar, pH 5.5–5.8) and cultured for 20–35 days. Then, they were transferred to heart-shaped embryo induction medium (WPM + 1.0–3.0 mg / L 6-BA + 0.1–1.0 mg / L NAA + 20–40 g / L sucrose + 7 g / L agar, pH 5.5–5.8) and cultured for 5–15 days. Then, they were transferred to cotyledon embryo induction medium (WPM + 1.0–3.0 mg / L 6-BA + 20–40 g / L sucrose + 7 g / L agar, pH 5.5–5.8) and cultured for 15–30 days. Finally, they were transferred to seedling culture medium (MS + 1.0–3.0 mg / L 6-BA + 0.1–1.0 mg / L NAA). Seedlings were induced by culturing on NAA (0.1–3.0 mg / L riboflavin, 20–40 g / L sucrose, 7 g / L agar, pH 5.5–5.8) for 20–40 days.

[0012] (6) The seedlings obtained in steps (4) and (5) are inoculated into seedling proliferation medium for seedling proliferation culture to obtain regenerated plants. The seedling proliferation medium consists of: MS + 1.0-3.0 mg / L 6-BA + 0.5-1.5 mg / L IBA + 20-40 g / L sucrose + 7 g / L agar, pH 5.5-5.8.

[0013] (7) Ploid identification of regenerated plants and screening of haploid plants.

[0014] Step (1) specifically involves: collecting flowers that have not yet released pollen, peeling off the anthers, and drying them until pollen is released, then using 100-1500 Gy of... 60 -Co is subjected to radiation. The drying temperature is 28-30℃.

[0015] In step (2), the pollen viability determination method includes the following steps: adding pollen to the FDA (fluorescein diacetate) assay solution, shaking well, staining in the dark, placing the stained pollen solution on a glass slide, covering it with a coverslip, and observing the pollen viability under a fluorescence microscope. The FDA assay solution is prepared by first preparing a 5 mg / mL FDA stock solution (solvent: acetone), and then diluting it 50 times with a 20% sucrose solution.

[0016] In step (2), the method for determining the pollen germination rate includes the following steps: Pollen is evenly spread on the surface of the germination medium, sealed, and then placed in a constant temperature incubator at 25℃ for 4 hours. The pollen germination is observed under an inverted microscope. The germination medium is 1% agar + 10% sucrose + 0.01% boric acid.

[0017] In step (4), the germination medium is preferably composed of: WPM + 2.0 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar, pH 5.6-5.8.

[0018] In step (5), the preferred composition of the embryogenic callus induction medium is: WPM + 1.0 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.6-5.8; the preferred composition of the embryogenic callus proliferation medium is: MS + 0.7 mg / L 2,4-D + 30 g / L sucrose + 7 g / L agar, pH 5.6-5.8.

[0019] In step (5), the preferred composition of the spherical embryo induction medium is: MS + 0.5 mg / L 2,4-D + 30 g / L sucrose + 7 g / L agar, pH 5.6-5.8.

[0020] In step (5), the preferred composition of the heart-shaped embryo induction medium is: WPM + 2.0 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.6-5.8.

[0021] In step (5), the preferred composition of the cotyledon embryo induction medium is: WPM + 2.0 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar, pH 5.6-5.8.

[0022] In step (5), the preferred composition of the seedling culture medium is: MS + 2.0 mg / L 6-BA + 0.5 mg / L NAA + 1.0 mg / L riboflavin + 30 g / L sucrose + 7 g / L agar, pH 5.6-5.8.

[0023] In step (6), the preferred composition of the seedling proliferation medium is: MS + 2.0 mg / L 6-BA + 1.0 mg / L L BA + 30 g / L sucrose + 7 g / L agar, pH 5.6-5.8.

[0024] The present invention has the following advantages and effects compared with the prior art:

[0025] (1) This invention innovates the method of haploid breeding of Camellia oleifera. Existing research on haploid Camellia oleifera is limited to anther culture and has failed to break through the regeneration bottleneck and successfully cultivate haploid Camellia oleifera. However, this invention breaks through the technical bottleneck of haploid Camellia oleifera regeneration by combining radiation pollination with embryo rescue, and successfully obtains haploid Camellia oleifera plants, laying a technical foundation for haploid breeding of Camellia oleifera.

[0026] (2) This invention is simple and easy to implement. New haploid germplasm of Camellia oleifera can be obtained by pollen radiation, controlled pollination and embryo rescue.

[0027] (3) This invention establishes a set of efficient camellia oleifera embryo rescue technology. About 35% of radiation-pollinated embryos can be directly planted through in vitro early germination, and about 60% of radiation-pollinated embryos can regenerate plants through somatic embryogenesis. The embryo rescue efficiency reaches about 95%.

[0028] This invention addresses the challenge of weak growth in haploid Camellia oleifera seedlings by designing a highly efficient seedling propagation and cultivation method, effectively improving the propagation efficiency of haploid Camellia oleifera seedlings. This invention is applicable to both the Camellia oleifera and Camellia septemlobus groups and can be used for the breeding of Camellia oleifera varieties, accelerating the Camellia oleifera breeding process. Attached Figure Description

[0029] Figure 1These are images of pollen viability and germination effects from Example 1. a: Fluorescence field of view of Huaxin pollen (treated with 150 Gy); b: Natural light field of view of Huaxin pollen (treated with 150 Gy); c: Germination effect of Huaxin pollen (treated with 150 Gy).

[0030] Figure 2 This is a diagram illustrating the in vitro germination process of Camellia oleifera embryos in Example 1. a: Embryo inoculation day 0; b: Embryo inoculation day 20; c: Embryo inoculation day 40; d: Embryo inoculation day 60; e: Embryo inoculation day 70; f: Embryo inoculation day 80.

[0031] Figure 3 This is a flowchart of the somatic cell embryogenesis and regeneration system in Example 1. a: Immature embryo inoculation day 0; b: Embryogenic callus induced by the immature embryo; c: Induced globular embryo; d: Induced heart-shaped embryo; e: Induced torpedo-shaped embryo; f: Induced cotyledonary embryo; g: Cotyledonary embryo germination; h: Cotyledonary embryo induced into seedling; Scale bar g = 1 mm, (b, c, d, e, f) = 0.5 mm.

[0032] Figure 4 This is a diagram of the seedling propagation culture process in Example 1. a: Propagation culture for 0 days; b: Propagation culture for 10 days; c: Propagation culture for 15 days; d: Propagation culture for 30 days; e: Propagation culture for 45 days; f: Propagation culture for 60 days; g-h: Propagation culture after 60 days.

[0033] Figure 5 This is a flow cytometry diagram of 'ASUS' haploid seedlings from Example 1. a: Flow cytometry diagram of 'ASUS' (CK); b: Flow cytometry diagram of 'ASUS' × 'Huaxin' 150Gy triploid seedlings; c: 'ASUS' (CK) hexaploid (2n=6X=90); d: 'ASUS' × 'Huaxin' 150Gy triploid seedlings (2n=3X=45). Detailed Implementation

[0034] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0035] Example 1

[0036] The breeding method for 'ASUS' Camellia oleifera haploid tissue culture seedlings includes the following steps:

[0037] (1) Collect 'Huajin' and 'Huaxin' flowers that are about to open and have not yet released pollen at the beginning of the flowering period. After removing the petals, use tweezers to separate the anthers from the filaments. Place the separated anthers under an 80W light bulb for 1-2 days until they release pollen, and keep the temperature at 28-30℃ during this period.

[0038] (2) Dispense the dried pollen from step (1) into 1.5 mL centrifuge tubes, label them and seal them with sealing film. First, store them in a 4℃ refrigerator for 12 hours, then transfer them to a -20℃ refrigerator for 12 hours, and finally store them in a -80℃ refrigerator.

[0039] (3) The dried and packaged 'Huajin' and 'Huaxin' pollen were sent to the Hunan Irradiation Center (i.e., Hunan Radiation Technology Application Research Center) for irradiation. The radiation source was 60-Co. The radiation doses of 'Huajin' and 'Huaxin' pollen were 150 Gy, 250 Gy, 350 Gy, 450 Gy and 550 Gy, respectively. A portion of the irradiated pollen was taken for viability and germination rate determination, and the remaining pollen was stored in a -80℃ refrigerator.

[0040] (4) The viability of pollen obtained in step (3) with different radiation doses was determined. Figure 1 (a / b) First, prepare a 5 mg / mL FDA (fluorescein diacetate) stock solution using acetone as the solvent. Then, dilute the stock solution 50 times with 20% sucrose solution, resulting in a final FDA assay concentration of 100 μg / mL. Remove large anther particles from the centrifuge tube, leaving only a small amount of pollen adhering to the tube. Add 500 μL of FDA assay solution, mix well, and stain in the dark for 10 min. Pipette 50 μL of the mixture onto a glass slide, cover with a coverslip, and observe and photograph under a fluorescence microscope.

[0041] (5) The germination rate of pollen with different radiation doses obtained in step (3) was determined. Figure 1 c) Prepare a germination medium of 1% agar + 10% sucrose + 0.01% boric acid, pour it into a petri dish and let it cool. Use a clean brush to pick up a small amount of pollen and sprinkle it evenly on the surface of the medium. Seal the petri dish containing pollen and place it in a constant temperature incubator at 25°C for 4 hours. Observe the pollen germination under an inverted microscope and count the pollen. The results of steps (4) and (5) show that the viability and germination rate of the irradiated pollen are significantly reduced (Table 1).

[0042] Table 1 Results of pollen viability and germination rate

[0043]

[0044] (6) Select a sunny, peak flowering period for hybridization pollination. One day before pollination, transfer the Huajin and Huaxin pollen, which has been irradiated (150-550 Gy) and preserved at ultra-low temperature in step (3), to a 4°C refrigerator for later use. Select robust 'Huashuo' parent plants for pollination. Choose flower buds that are about to open but have not yet released pollen, attach pre-marked tags, remove petals and sepals, remove stamens with tweezers, being careful not to damage the pistil, and use a pollination stick to collect pollen from the centrifuge tube and evenly apply it to each stigma. The amount of pollen should be such that pollen is visible to the naked eye on each stigma to ensure pollination quality. After pollination, bag the plants to prevent other pollen from mixing in. Remove the bags one week after pollination. See Table 2 for pollination results.

[0045] Table 2 Pollination combinations and pollination quantities of radiated pollen

[0046]

[0047] Note: Mother parent × Father parent

[0048] (7) Fruits collected in early August of the following year after pollination were cold-treated in a 4℃ refrigerator for 3 days. The cold-treated fruits were then thoroughly immersed in 75% alcohol for 5 seconds, followed by sterilization by burning over an alcohol lamp. After the alcohol had burned completely, the pericarp was cut open with a scalpel to remove the seeds. The seed coat was removed using sterilized tweezers on a sterile culture dish, and the embryos were removed, taking care not to damage them. The embryos were then inserted downwards into a germination medium containing WPM + 2.0 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar, pH 5.6–5.8 (ensuring most of the embryo was inserted into the medium, with the cotyledons not completely submerged). After inoculation, the embryos were cultured at 26–30℃ in the dark for 20–30 days, then transferred to light conditions for 60–80 days to induce seedling formation. Figure 2 ).

[0049] (8) During the process of inducing premature germination of immature embryos, it was found that some immature embryos could be induced to germinate prematurely, while others could be induced to produce embryogenic callus. In order to further improve the efficiency of in vitro culture of immature embryos, seedlings were also induced from the somatic embryogenesis pathway. Therefore, materials that did not germinate prematurely were inoculated into WPM medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 7 g / L agar, and pH 5.6–5.8 to induce embryogenic callus. Then, the embryogenic callus was inoculated into MS medium containing 0.7 mg / L 2,4-D, 30 g / L sucrose, 7 g / L agar, and pH 5.6–5.8 for embryogenic callus proliferation culture.

[0050] (9) The embryogenic callus obtained in step (8) was inoculated onto differentiation medium for inducing globular embryos (MS + 0.5 mg / L 2,4-D + 30 g / L sucrose + 7 g / L agar, pH 5.6–5.8). After culturing for 20–35 days, it was transferred to differentiation medium for inducing heart-shaped embryos (WPM + 2.0 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.6–5.8). After culturing for 5–15 days, it was transferred to differentiation medium for inducing cotyledonary embryos (WPM + 2.0 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar, pH 5.6–5.8). After culturing for 15–30 days, it was transferred to differentiation medium for inducing seedlings (MS + 2.0 mg / L 6-BA + 0.5 mg / L NAA + 1.0 mg / L riboflavin + 30 g / L sucrose + 7 g / L agar, pH 5.6–5.8). Cultured on differentiation medium of 5.6–5.8 for 20–40 days to induce seedling formation. Figure 3 ).

[0051] (10) To further increase the yield of haploid plants, the rootless seedlings obtained in steps (7) and (9) were fed into different culture media as shown in Table 3 for seedling proliferation culture, and the proliferation coefficient reached up to 3.6. Figure 4 The diagram shows the proliferation process in MS medium containing 2.0 mg / L 6-BA, 1.0 mg / L IBA, 30 g / L sucrose, and 7 g / L agar at pH 5.6–5.8.

[0052] Table 3. Effects of different culture media on seedling proliferation coefficient

[0053]

[0054] (11) Cut a 0.5cm piece from the recycled material obtained in steps (7) and (9). 2 Ploidy identification was performed on both sides. The sample to be tested was placed in a plastic petri dish, and 150 μL of UV Precise P cell nuclear extraction solution was added. The sample was chopped with a sharp blade, and another 150 μL of extraction solution was added. The cell suspension was then filtered through a 30 μm microporous membrane into a sample tube, and 1600 μL of UV Precise P staining solution was added. Staining was performed in the dark for 5 minutes, and finally, flow cytometry was used for detection. Young leaves (hexaploid) of 'Asus' Camellia oleifera seedlings were used as a control for detection. Results were automatically generated by FloMax software. Based on the flow cytometry results, haploid materials were obtained by embryo rescue. Vigorous young root tips were collected, and root tip chromosome preparation was performed using a modified Camellia oleifera chromosome preparation technique. Observation and photography were conducted using a microscope (Olympus BX-61, Japan), and the number of chromosomes in metaphase was counted using the analysis and counting function of Adobe Photoshop 2021. Figure 5).

[0055] (12) Ploidy testing showed that: a total of 750 flowers were pollinated in step (6), and 94 fruits were obtained from radiation pollen. Among the 94 fruits, there were 148 embryos. Because some embryos did not develop after radiation pollination, they formed empty seeds. In fact, 55 embryos were rescued from radiation pollen. After steps (7), (8), and (9), 5 regenerated plants, 23 embryoids, and 29 callus tissues were obtained. Ploidy identification of the obtained materials confirmed that 1 'Huashuo' haploid (triploid) plant, 2 'Huashuo' haploid (triploid) embryoids, and 1 'Huashuo' haploid (triploid) embryogenic callus were obtained. In addition, the 'Huashuo' haploid plant and haploid embryogenic callus were propagated and multiplied, resulting in 198 bottles of tissue culture seedlings and 813 bottles of embryogenic callus.

[0056] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for inducing haploids through pollination with Camellia oleifera radiata pollen, characterized in that: Includes the following steps: (1) Collect flowers that have not yet released pollen, peel off the anthers and dry them until pollen is released, then use 100-1500 Gy of water. 60 -Co radiates; (2) Determine the viability and germination rate of radiated pollen; (3) Use highly viable and high-germination-rate radiated pollen to pollinate the female parent, cover it with a bag after pollination, and remove the bag one week after pollination; (4) The following year, the fruits were collected and subjected to low temperature treatment at 2-8℃ for 3-7 days. The embryos were removed from the seeds and inserted downward into the germination medium. After culturing at 26-30℃ in the dark for 20-30 days, the embryos were transferred to light conditions for 60-80 days to induce seedlings. Some embryos may induce embryogenic callus. The germination medium consisted of: WPM + 0.5-3.0 mg / L 6-BA + 20-50 g / L sucrose + 7 g / L agar, pH 5.5-6.

0. (5) The material that did not germinate prematurely in step (4) was inoculated into embryogenic callus induction medium for induction of embryogenic callus. The obtained embryogenic callus was inoculated into embryogenic callus proliferation medium for proliferation culture. Then it was inoculated into spherical embryo induction medium and cultured for 20-35 days. After that, it was transferred to heart-shaped embryo induction medium and cultured for 5-15 days. After that, it was transferred to cotyledon embryo induction medium and cultured for 15-30 days. After that, it was transferred to seedling culture medium and cultured for 20-40 days to induce seedling formation. The embryogenic callus induction medium consists of: WPM + 0.1–2.0 mg / L 6-BA + 0.1–1.0 mg / L NAA + 20–40 g / L sucrose + 7 g / L agar, pH 5.5–5.8; The composition of the embryogenic callus proliferation medium is: MS + 0.7-1.0 mg / L 2,4-D + 20-40 g / L sucrose + 7 g / L agar, pH 5.5-5.8; The composition of the spherical embryo induction medium is: MS + 0.5-1.5 mg / L 2,4-D + 20-40 g / L sucrose + 7 g / L agar, pH 5.5-5.8; The composition of the heart-shaped embryo induction medium is: WPM + 1.0-3.0 mg / L 6-BA + 0.1-1.0 mg / L NAA + 20-40 g / L sucrose + 7 g / L agar, pH 5.5-5.8; The composition of the cotyledon embryo induction medium is: WPM + 1.0-3.0 mg / L 6-BA + 20-40 g / L sucrose + 7 g / L agar, pH 5.5-5.8; The seedling culture medium consists of: MS + 1.0–3.0 mg / L 6-BA + 0.1–1.0 mg / L NAA + 0.1–3.0 mg / L riboflavin + 20–40 g / L sucrose + 7 g / L agar, pH 5.5–5.8; (6) The seedlings obtained in steps (4) and (5) are inoculated into the seedling proliferation medium for seedling proliferation culture to obtain regenerated plants; the seedling proliferation medium is composed of: MS + 2.0 mg / L 6-BA + 1.0 mg / L IBA + 30 g / L sucrose + 7 g / L agar, pH 5.6~5.8; (7) Pluralization of regenerated plants is determined and haploid plants are screened.

2. The method for inducing haploids through radiation pollination of Camellia oleifera according to claim 1, characterized in that: In step (2), the pollen viability determination method includes the following steps: adding pollen to the FDA test solution, shaking well, staining in the dark, placing the stained pollen solution on a glass slide, covering it with a coverslip, and observing the pollen viability under a fluorescence microscope. The method for determining pollen germination rate includes the following steps: evenly spread pollen on the surface of germination medium, seal the container, and incubate it in a constant temperature incubator at 25 ℃ for 4 h. Observe the pollen germination under an inverted microscope.

3. The method for inducing haploids through pollination of Camellia oleifera radiata pollen according to claim 1, characterized in that: In step (4), the germination medium consists of: WPM + 2.0 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar, pH 5.6-5.

8.

4. The method for inducing haploids through pollination of Camellia oleifera radiata pollen according to claim 1, characterized in that: In step (5), the embryogenic callus induction medium consists of: WPM + 1.0 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.6-5.8; the embryogenic callus proliferation medium consists of: MS + 0.7 mg / L 2,4-D + 30 g / L sucrose + 7 g / L agar, pH 5.6-5.

8.

5. The method for inducing haploids through pollination of Camellia oleifera radiata pollen according to claim 1, characterized in that: In step (5), the spherical embryo induction medium is composed of: MS + 0.5 mg / L 2,4-D + 30 g / L sucrose + 7 g / L agar, pH 5.6-5.

8.

6. The method for inducing haploids through pollination of Camellia oleifera radiata pollen according to claim 1, characterized in that: In step (5), the composition of the heart-shaped embryo induction medium is: WPM + 2.0 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.6-5.

8.

7. The method for inducing haploids through pollination of Camellia oleifera radiata pollen according to claim 1, characterized in that: In step (5), the composition of the cotyledon embryo induction medium is: WPM + 2.0 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar, pH 5.6-5.

8.

8. The method for inducing haploids through pollination of Camellia oleifera radiata pollen according to claim 1, characterized in that: In step (5), the seedling culture medium consists of: MS + 2.0 mg / L 6-BA + 0.5 mg / L NAA + 1.0 mg / L riboflavin + 30 g / L sucrose + 7 g / L agar, pH 5.6-5.8.

Images