Method for inducing homozygous doubling of chromosomes of small fruit camellia by colchicine

Abstract

The application discloses a method for inducing homozygous doubling of chromosomes of small fruit camellia by colchicine, and belongs to the technical field of plant biotechnology.The method comprises the following steps: (1) after the small fruit camellia young fruit is cleaned, disinfected and peeled, the young embryo is taken and inoculated into embryogenic callus induction culture medium to induce embryogenic callus for 40-60 days; (2) the yellowish-white small granular embryogenic callus obtained in the step (1) is inoculated into induction culture medium containing colchicine, and doubling induction treatment is carried out in a dark environment for 10-20 days; (3) the callus after doubling induction treatment in the step (2) is inoculated into adventitious bud induction culture medium to induce adventitious buds, and after culture in the light condition for 150 days, doubling of the tissue culture seedlings is obtained through ploidy identification.The application takes the small fruit camellia as the material, and establishes a polyploid induction scheme which has high induction efficiency, low chimeric rate and can quickly grow seedlings.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, and relates to the induction of polyploidy in plants, and more specifically to a method for inducing homozygous doubling of chromosomes in Camellia oleifera using colchicine. Background Technology

[0002] Camellia oleifera is an economic forest tree species belonging to the genus Camellia in the family Theaceae. The oil extracted from its mature seeds is considered the best edible oil, with a clear color and fragrant aroma, and an unsaturated fatty acid content exceeding 90%. Long-term consumption of camellia oil is beneficial to human health. However, the low yield and efficiency of camellia oleifera remain unresolved, primarily due to the mixed varieties and low quality of improved varieties in camellia oleifera forests. Broadly speaking, camellia oleifera refers to oil-producing plants in the genus Camellia, with common camellia oleifera being the most widely distributed and highest-yielding. However, its thick pericarp and low seed yield limit its application in production. As a major camellia oleifera producing area, my country possesses many excellent camellia oleifera germplasm varieties in nature. Small-fruited camellia oleifera, a tetraploid, has advantages such as thin pericarp, high seed yield, and high oil content. However, its small fruit size and low yield prevent it from meeting production requirements. Polyploid plants have advantages such as large fruit size, robust growth, high yield, strong adaptability, and strong resistance to adverse conditions. Therefore, polyploid induction of low-ploidy Camellia oleifera germplasm is expected to create new Camellia oleifera germplasm that retains the original advantages while also possessing the advantages of polyploidy, laying the foundation for Camellia oleifera germplasm innovation and accelerating the process of improving Camellia oleifera varieties.

[0003] Colchicine, as the most widely used antimitotic agent for inducing polyploidization in plants, plays an important role in the polyploid breeding of woody plants, achieving success in poplar, citrus, and jujube trees. However, due to the asynchronous nature of cell development, after colchicine induction, only a portion of cells often form polyploids, leading to the formation of chimeras. This is particularly evident when using shoot tips and tissue-cultured seedlings as materials for colchicine doubling. The isolation and purification of chimeras is time-consuming and labor-intensive. Using cell suspensions, embryoids of single-cell origin, or callus tissue as mutagenic materials can effectively reduce the formation of chimeras and increase the proportion of homozygous polyploids. Previous methods of treating Camellia oleifera seeds with colchicine induced polyploidy, but the induction rate of polyploid plants was low, and most were chimeras. While treating Camellia oleifera leaf callus with colchicine yielded polyploid callus, the callus failed to differentiate into seedlings. Tea leaf callus itself does not have embryogenicity and cannot be used to obtain plants through somatic embryogenesis. Therefore, it is difficult to obtain polyploid plants through this method. Summary of the Invention

[0004] To address the aforementioned problems in existing technologies, the present invention aims to provide a method for inducing homozygous doubling of chromosomes in Camellia oleifera using colchicine. This invention utilizes colchicine to treat embryogenic callus tissue of Camellia oleifera and obtains chimeric homozygous doubled plants through the somatic embryogenesis pathway of the embryogenic callus tissue.

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

[0006] A method for inducing homozygous doubling of chromosomes in Camellia oleifera using colchicine includes the following steps:

[0007] (1) After cleaning, disinfecting and peeling the fruit skin, the young fruit of Camellia oleifera is inoculated into embryogenic callus induction medium to induce embryogenic callus for 40-60 days.

[0008] (2) The yellowish-white small granular embryogenic callus obtained in step (1) was inoculated into an induction medium containing colchicine and cultured in the dark for 10-20 days for double induction treatment.

[0009] (3) The callus tissue that has been doubling induction in step (2) is inoculated into adventitious bud induction medium for adventitious bud induction. After culturing under light for 150 days, the doubled tissue culture seedlings are obtained by ploidy identification.

[0010] In step (1), the young Camellia oleifera fruits are preferably refrigerated at 2-6℃ for 2-3 days before being cleaned and disinfected. The preferred cleaning method is as follows: the young Camellia oleifera fruits are rinsed with water to remove surface dirt and impurities, soaked in a solution containing scouring powder for 5-30 minutes, and then rinsed with water to remove any remaining scouring powder. The preferred disinfection method is as follows: the young fruits are soaked in 75% alcohol for sterilization, washed with sterile water, then disinfected with 0.1% HgCl2 solution, and finally washed with sterile water.

[0011] In step (1), the preferred method for inducing embryogenic callus is as follows: young embryos are inoculated into an embryogenic callus induction medium, cultured in the dark for 20–30 days, and then cultured under light for another 20–30 days. The preferred formulation of the induction medium is WPM + 2 mg / L 6-BA + 1 mg / L IBA + 30 g / L sucrose + 7 g / L agar, with the pH adjusted to approximately 5.8. The preferred culture temperature is 28°C. The preferred light conditions are: light intensity of 2000 lux and a photoperiod of 16 h / 8 h.

[0012] In step (2), the colchicine concentration in the colchicine-containing induction medium is preferably 200 mg / L. The preferred formulation of the colchicine-containing induction medium is WPM + 1 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 7 g / L agar + 200 mg / L colchicine, with the pH adjusted to approximately 5.8. The preferred culture conditions are: a culture temperature of 28℃ and a culture time of 15 days.

[0013] In step (3), the preferred formulation of the adventitious bud induction medium is WPM + 2 mg / L 6-BA + 1 mg / L IBA + 30 g / L sucrose + 7 g / L agar, with the pH adjusted to approximately 5.8. The preferred culture conditions are: a culture temperature of 28℃, a light intensity of 2000 lux, a photoperiod of 16 h / 8 h, and replacement of the induction medium every 30 days.

[0014] Compared with the prior art, the method of the present invention has the following beneficial effects:

[0015] (1) This invention uses camellia oleifera embryos as materials for inducing embryogenic callus. The experimental materials are sufficient and easy to obtain, and a large amount of embryogenic callus can be prepared as materials for subsequent doubling experiments.

[0016] (2) This invention uses small, yellowish-white granular embryogenic callus from Camellia oleifera as material. The primary yellowish-white embryogenic callus has strong embryogenicity and low development level, making it easier to proliferate into secondary callus. When small granular embryogenic callus is selected, the primary callus is prone to browning and death, and the secondary embryo is more likely to originate from a single cell, which can achieve 100% chimerism-free homozygous doubling.

[0017] (3) The present invention uses Camellia oleifera embryogenic callus as the material for inducing doubling. Since embryogenic callus can obtain regenerated plants through somatic embryogenesis, it can effectively increase the induction efficiency of polyploid plants.

[0018] (4) The present invention first obtains doubled embryogenic callus after doubling, and the callus is easier to preserve and proliferate, and can be used as experimental material for subsequent genetic transformation, protoplast culture and other experiments.

[0019] (5) This invention uses Camellia oleifera as material to establish a polyploid induction scheme with high induction efficiency, low chimerism rate and rapid seedling growth. The polyploid induction rate can reach up to 43.89% and the chimerism rate is 0. Attached Figure Description

[0020] Figure 1 : The process of inducing embryogenic callus in Camellia oleifera embryos. a: Embryo cultured for 0 days; b: Embryo cultured for 20 days; c: Embryo cultured for 40 days; d: Embryo cultured for 60 days.

[0021] Figure 2 The culture process of colchicine-induced doubling of embryogenic callus. a: Cultured on colchicine-containing medium for 0 days; b: Cultured on colchicine-containing medium for 20 days; c: Cultured on adventitious shoot induction medium without colchicine for 0 days; d: Cultured on adventitious shoot induction medium for 90 days; e: Cultured on adventitious shoot induction medium for 150 days.

[0022] Figure 3Flow cytometry identification and chromosome counting results. a: Flow cytometry identification results of tetraploid Camellia oleifera; b: Flow cytometry identification results of callus tissue of octoploid Camellia oleifera; c: Chromosome counting results of root tip of tetraploid Camellia oleifera; d: Chromosome counting results of root tip of octoploid Camellia oleifera.

[0023] Figure 4 Tissue culture seedlings and leaf morphology of tetraploid and octoploid Camellia oleifera. a: Tetraploid Camellia oleifera plant; b: Octoploid Camellia oleifera plant; c: Leaf morphology.

[0024] Figure 5 Stomatal observation of tetraploid and octoploid Camellia oleifera. a: Stomatal observation of tetraploid Camellia oleifera; b: Stomatal observation of octoploid Camellia oleifera. Detailed Implementation

[0025] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0026] Example 1

[0027] I. Colchicine induces a doubling of embryogenic callus.

[0028] (1) Culture of embryogenic callus from Camellia oleifera: In July, under sunny weather conditions, immature fruits of Camellia oleifera were collected and stored in a 4℃ refrigerator for 3 days before use. The fruits were placed in a wide-mouthed bottle, rinsed with tap water, soaked in laundry detergent for 15 minutes, and then rinsed with tap water again. In a clean bench, the fruits were washed twice with sterile water, then disinfected with 75% alcohol for 30 seconds, washed 4-5 times with sterile water, then disinfected with 0.1% HgCl2 solution for 15 minutes, and finally washed 4-5 times with sterile water. The moisture on the seed surface was absorbed with filter paper, and the pericarp and the seed coat around the embryo were removed with a sterile blade. The immature embryos were then inoculated onto the induction medium (see Figure 1 a) After culturing at 28℃ in darkness for 25 days, the embryos were transferred to light conditions (light intensity of 2000 lux, 16h / 8h photoperiod) and cultured for 20–30 days. After 20 days of in vitro culture, the embryos significantly enlarged. Figure 1 b) After 40 days of in vitro culture, a small amount of callus tissue appeared and granular callus tissue began to appear. Figure 1 c), after 60 days of in vitro culture, a large amount of granular embryogenic callus appeared. Figure 1d). The induction medium formulation was WPM + 2 mg / L 6-BA + 1 mg / L IBA + 30 g / L sucrose + 7 g / L agar. All media were adjusted to pH 5.8 with 1 mol / L HCl and NaOH.

[0029] (2) Chromosome doubling induction: Colchicine was dissolved in a small amount of alcohol to prepare a colchicine stock solution with a concentration of 10 mg / mL. In a clean bench, the colchicine stock solution was filtered through a 0.22 μm sterile microporous membrane and added to an unsolidified solid culture medium to prepare an induction medium containing 100–400 mg / L of colchicine. Different morphological embryogenic callus tissues obtained in (1) were then used to induce chromosome doubling. Figure 2 a) Incubate on a colchicine-containing medium for 10–20 days in a dark incubator. The colchicine-containing induction medium is formulated as follows: WPM + 1 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 7 g / L agar + 100 mg / L colchicine, pH 5.8, and incubation temperature 28°C.

[0030] (3) Adventitious bud induction: In a clean bench, use sterile forceps to gently detach undying callus tissue ( Figure 2 b) After removal, the tissue was placed in an adventitious bud induction medium and cultured in a tissue culture room at 28℃ with a light intensity of 2000 lux and a photoperiod of 16h / 8h. The medium was changed every 30 days. After 30 days of culture, the callus tissue began to proliferate. Figure 2 c) After culturing for 90 days, the formation of adventitious buds can be observed. Figure 2 d), after culturing for 150 days, double the number of tissue culture seedlings were obtained. Figure 2 e). The adventitious bud induction medium is formulated as follows: WPM + 2 mg / L 6-BA + 1 mg / L IBA + 30 g / L sucrose + 7 g / L agar, with a pH of 5.8.

[0031] II. Ploidy Identification

[0032] (1) Flow cytometry identification: Using colchicine-treated callus tissue and leaves of regenerated plants from step (3) as materials, and leaves of tissue culture seedlings not treated with colchicine as controls, ploidy was identified using flow cytometry. First, the cell lysis buffer (Precise-P, Sysmex) and DAPI staining solution (Precise-P, Sysmex) were pre-cooled in an ice box to ensure the reagent temperature was suitable, thereby maintaining cell integrity and staining effect. Then, approximately 0.5 cm of the sample was cut from the callus tissue or young leaf to be tested using a new double-edged blade. 2The samples were collected and placed in disposable culture dishes. Next, 300 μL of dissociation buffer and 10 μL of 28.57% PVP (Polyvinyl Pyrrolidone, Solarbio) aqueous solution were added to the dishes to aid cell dispersion and stability. The samples were then rapidly shredded using a double-edged blade for easier handling and analysis. The samples were then filtered through a 50 μm nylon filter into loading tubes to ensure only a single cell passed through. 1000 μL of PI staining solution was added to the filtrate, and the mixture was incubated in the dark at room temperature for 5 minutes to allow PI to fully bind to DNA and generate a strong fluorescence signal. Subsequently, flow cytometry (Sysmex, Germany) was used to analyze 5000–10000 cells, and the peak DNA content and coefficient of variation (CV, %) for each sample were recorded. Ploidy analysis images of the samples were generated using Flomax software (Sysmex, Germany).

[0033] (2) Chromosome counting: Take the vigorous young root tips of the regenerated plants, and prepare root tip chromosome slides according to the improved Camellia oleifera chromosome preparation technique. Observe and photograph them with a microscope (Olympus BX-61, Japan), and use the analysis and counting function of Adobe Photoshop 2021 to count the number of chromosomes in the metaphase.

[0034] (3) Leaf phenotype observation

[0035] Using the octoploid and tetraploid Camellia oleifera plants identified above as materials, tissue culture seedlings with the same growth time were selected, and the third and fourth mature leaves from the top were cut off. The leaf length, leaf width, and leaf thickness were observed and recorded using vernier calipers.

[0036] (4) Stomatal observation

[0037] Using the seedlings identified by the above ploidy identification as materials, three leaves were taken from each tissue culture seedling for stomatal observation. The nail polish tearing method was used to prepare slides. Five fields of view were randomly selected from each leaf for photography. The size and number of stomata were observed and counted under a 20x objective lens, and the stomatal density was calculated.

[0038] Example 2

[0039] Following the method in Example 1, callus tissues of different embryogenic morphologies at 60 days of embryonic induction were cultured on a medium containing 200 mg / L colchicine for 20 days. This invention investigated the survival rate, induction rate, and chimerism rate of polyploid plants induced from callus tissues of different embryogenic morphologies, setting up four treatments (see Table 1). It can be seen that different callus morphologies all affect the survival rate, induction rate, and chimerism rate of polyploid induction. Among them, small green (diameter less than 0.5 cm) granular callus tissues developed faster and had a higher degree of differentiation; although callus proliferation occurred, no polyploid plants were obtained. Transparent small callus tissues had weak embryogenicity and significant colchicine toxicity, also failing to yield polyploid plants. Yellowish-white callus tissue exhibits strong embryogenicity and low differentiation, making it suitable for inducing polyploid plants. Large, yellowish-white granular callus tissue (diameter greater than 0.5 cm) has a higher cell count and relatively higher survival rate. Newly proliferating secondary callus tissue often originates from multicellular sources, resulting in low homogeneity. Furthermore, the presence of primary callus leads to a high chimerism rate in the resulting callus clusters. Conversely, small, yellowish-white granular callus tissue (diameter less than 0.5 cm) is prone to browning and death of primary callus tissue when exposed to colchicine. The resulting secondary callus tissue is often of single-cell origin, with a low chimerism rate. Figure 2 b). In the following examples, small, yellowish-white granular embryogenic callus tissue was selected for colchicine induction.

[0040] Table 1: Effects of callus morphology on polyploidization induction

[0041]

[0042]

[0043] Example 3

[0044] Following the method described in Example 1, the obtained embryogenic callus was cultured in both colchicine aqueous solution and a solid culture medium containing colchicine (solid-state mixed culture method). This invention investigated the treatment methods of colchicine on embryogenic callus. Since the colchicine aqueous solution soaking method has a dual effect on explant toxicity, the embryogenic callus was soaked in colchicine aqueous solution at a concentration of 100 mg / L for 12 hours. The results (Table 2) show that the low-concentration, short-duration soaking method was superior to the solid-state method in affecting explant survival rate, but it failed to induce polyploid plants. Although the solid-state mixed culture method had a certain toxic effect on explants, its inducing effect was significant.

[0045] Table 2: Effects of different induction methods on polyploid induction

[0046]

[0047] Example 4

[0048] Following the method in Example 1, the obtained embryogenic callus was cultured on a medium containing colchicine. This invention investigated the effect of colchicine on callus survival rate, setting up nine treatments (see Table 3). It was found that colchicine has a toxic effect on callus. Generally, the higher the concentration of colchicine and the longer the treatment time, the more pronounced the toxic effect. The mortality rate of the high-concentration, long-term colchicine treatment group was significantly higher than that of the low-concentration, short-term treatment group. 100 mg / L colchicine caused relatively little damage to embryogenic callus, with survival rates of 77.78%, 58.33%, and 41.67% after treatment for different times (10d, 15d, and 20d), respectively. The 400 mg / L colchicine mixed culture treatment caused greater damage to embryogenic callus, with survival rates of 38.89%, 30.56%, and 13.89% after treatment for different times (10d, 15d, and 20d), respectively.

[0049] Table 3: Effects of colchicine on callus survival

[0050]

[0051]

[0052] Example 5

[0053] Following the method in Example 1, colchicine-treated callus tissue was used as material, and ploidy was determined using flow cytometry. Figure 3 As shown, the horizontal axis represents the fluorescence intensity of the cell nucleus, and the vertical axis represents the number of cell nuclei, used to determine whether doubling has occurred. For example, if the fluorescence intensity of the tetraploid control is around 100, then the fluorescence intensity of the octoploid should be around 200, with a similar cell number. This invention investigated the mutation rate of colchicine-induced callus tissue, setting up nine treatments (see Table 4). It can be seen that with the increase of colchicine concentration and time, the mutation rate of callus tissue generally showed an upward trend. The mutation rate of the 100 mg / L colchicine treatment was relatively low, at 5.56%, 11.11%, and 11.11% after treatment at different times (10d, 15d, and 20d), respectively. The mutation rates of 200 mg / L and 400 mg / L colchicine were not significantly different. Among them, the mutation rate of the 200 mg / L colchicine treatment for 20 days was the highest, reaching 38.89%.

[0054] Table 4: Effects of colchicine on callus variation rate

[0055]

[0056] After identifying the mutated embryogenic callus, the callus was induced to sprout, resulting in 204 regenerated plants. Based on flow cytometry results, vigorous young root tips from tetraploid and octoploid plants were collected according to the method described in Example 1. Chromosome preparations were performed using a modified Camellia oleifera chromosome preparation technique, and the chromosome numbers were counted. The results showed that the chromosome number of the undoubted tetraploid plants was 60 (2n = 4X = 60). Figure 3 c) The number of chromosomes in the doubled plants was 120 (2n = 8X = 120). Figure 3 d) further confirmed that the obtained doubled plants were octoploids.

[0057] Example 6

[0058] Following the method described in Example 1, tetraploid plants and induced octoploid plants were used as materials to determine their leaf phenotype and stomatal characteristics (see Tables 5 and 6). Compared with tetraploid plants, octoploid plants showed decreased leaf length, increased leaf width, increased leaf thickness, and decreased leaf length-to-width ratio. Figure 4 Furthermore, the stomatal density of octoploids is significantly lower than that of tetraploids, while the stomatal length and width of octoploids are significantly greater than those of tetraploids. Figure 5 It is evident that leaf phenotype and stomatal characteristics can be used for the identification of polyploid Camellia oleifera plants.

[0059] Table 5: Comparison of leaf phenotypic characteristics between tetraploid and octoploid Camellia oleifera

[0060]

[0061] Table 6: Stomatal density and size of leaves in some octoploid Camellia oleifera plants

[0062]

[0063] Example 7

[0064] In plant polyploid induction, colchicine-induced doubling of tissue-cultured seedlings is a common method, but the resulting polyploids are mostly chimeric. Referring to the method of Li Yaxuan et al. (Maintenance and Changes of Chromosomal Ploidy under Different Camellia Germplasm Tissue Culture Conditions. Guangxi Plants, 2024, DOI:10.11931 / guihaia.gxzw202305077), polyploid induction was performed on tissue-cultured seedlings of different genotypes of Camellia oleifera using colchicine, but only NR-4 yielded 3 doubled plants, with a polyploid induction rate of only 1.67%, and all the obtained doubled plants were chimeric. In contrast, this invention uses embryogenic callus tissue as explants for polyploid induction, obtaining a large number of polyploid plants in three genotypes: NR-4, DF24, and LYC, with induction rates all above 30%, reaching a maximum of 43.89%, and all obtained doubled plants were homozygous, with no chimeras (see Table 7).

[0065] Table 7: Effects of different explants on the induction of polyploid plants

[0066]

[0067]

[0068] The results of the above examples show that, according to the method in Example 1, when the yellowish-white small granular embryogenic callus is treated with solid mixed culture and the colchicine concentration is 200 mg / L for 20 days, the embryogenic callus variation rate of Camellia oleifera can reach the highest level of 43.89%, and the polyploid induction effect is the best. Furthermore, the flow cytometry, chromosome counting, leaf phenotypic determination, and stomatal identification methods have verified that all polyploid plants are homozygous doubling plants and no chimeras exist.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for inducing homozygous doubling of chromosomes in Camellia oleifera using colchicine, characterized in that, Includes the following steps: (1) After cleaning, disinfecting and peeling the fruit skin, the young fruit of Camellia oleifera is inoculated into embryogenic callus induction medium to induce embryogenic callus for 40-60 days; the formula of the induction medium is WPM + 2mg / L 6-BA + 1mg / L IBA + 30g / L sucrose + 7g / L agar. (2) The yellowish-white small granular embryogenic callus obtained in step (1) is inoculated into an induction medium containing colchicine and cultured in the dark for 10-20 days for double induction treatment; the formula of the induction medium containing colchicine is: WPM + 1 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 7 g / L agar + 200 mg / L colchicine; (3) The callus tissue after doubling induction treatment in step (2) is inoculated into adventitious bud induction medium for adventitious bud induction. After culturing under light conditions for 150 days, the doubled tissue culture seedlings are obtained by ploidy identification. The formula of the adventitious bud induction medium is: WPM + 2mg / L 6-BA + 1mg / L IBA + 30g / L sucrose + 7g / L agar.

2. The method for inducing homozygous doubling of chromosomes in Camellia oleifera using colchicine according to claim 1, characterized in that: In step (1), the young camellia oleifera fruits are first refrigerated at 2-6℃ for 2-3 days, and then cleaned and disinfected.

3. The method for inducing homozygous doubling of chromosomes in Camellia oleifera using colchicine according to claim 1, characterized in that: In step (1), the method for cleaning the young Camellia oleifera fruit is as follows: the young Camellia oleifera fruit is rinsed with water to remove surface dirt and impurities, soaked in a solution containing detergent powder for 5 to 30 minutes, and then rinsed with water to remove detergent powder residue.

4. The method for inducing homozygous doubling of chromosomes in Camellia oleifera using colchicine according to claim 1, characterized in that: In step (1), the method for disinfecting the young Camellia oleifera fruit is as follows: the young fruit is soaked in 75% alcohol for sterilization, washed with sterile water, then disinfected with 0.1% HgCl2 solution, and after disinfection, washed with sterile water again.

5. The method for inducing homozygous doubling of chromosomes in Camellia oleifera using colchicine according to claim 1, characterized in that: In step (1), the method for inducing embryonic callus is as follows: the embryonic embryo is inoculated into the embryonic callus induction medium, cultured in the dark for 20-30 days, and then cultured under light for 20-30 days.

6. The method for inducing homozygous doubling of chromosomes in Camellia oleifera using colchicine according to claim 1, characterized in that: In step (2), the culture time is 15 days.

7. The method for inducing homozygous doubling of chromosomes in Camellia oleifera using colchicine according to claim 1, characterized in that: In step (3), the illumination conditions are an illumination intensity of 2000 lux and a light cycle of 16h / 8h.

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