Method for inducing polyploidy in genus Psoralea

Abstract

The present invention provides a method for inducing polyploidy in a plant of the genus Pleurotus, comprising: (a) germinating seeds of Pleurotus eryngii on a first culture medium to form protocorms; and (b) inoculating the protocorms of Pleurotus eryngii on a second culture medium, and culturing for 1-15 days, wherein the second culture medium contains colchicine at a concentration of 0.01%-0.2%. The present invention uses colchicine to obtain polyploidy of Pleurotus eryngii with a higher mutation rate through a co-cultivation method, and at the same time explores a method for identifying polyploidy of Pleurotus eryngii, including flow cytometry, chromosome compression, morphological observation, stomatal identification, or any combination thereof, which is of great significance for cultivating new germplasm with high ornamental value.

Description

Technical Field

[0001] The invention belongs to the technical field of polyploid breeding, and in particular relates to a method for inducing the protocorms of plants of the genus Henna (such as Henna polyanthus) to produce polyploids by using colchicine. Background Art

[0002] Aerides rosea Lodd.ex Lindl.&paxton belongs to the Orchidaceae family, Aerides genus, and is a monopodial branching orchid. It has pink flowers, large number of flowers, peculiar flower shape, and rich fragrance. It is an orchid with extremely high ornamental value. The flowers are white with purple spots, and the flower shape is like a dove; the flowering period is July, and the fruiting period is from August to May of the following year. It is produced in Guangxi, Guizhou, and Yunnan. In addition to the unique flower shape and bright color, the unique fragrance is also one of the important values ​​of Aerides rosea. It can be hybridized with closely related genera and is an excellent variety for cultivating Phalaenopsis with fragrance and large number of flowers.

[0003] Polyploid induction technology is an important means of orchid breeding at present. The excellent traits of polyploid plants, such as sturdy plants, large flowers, and strong stress resistance, are increasingly valued by breeding experts. Domestic and foreign experts have conducted a lot of research on the induction and identification of orchid polyploids. At present, the tissue culture technology of tropical orchids such as Dendrobium, Phalaenopsis, and Cymbidium grandiflorum, as well as terrestrial orchids such as Spring orchid and Cymbidium orchid has been mature. Polyploid induction is usually carried out by combining tissue culture technology with chromosome doubling, which can also be called in vitro doubling induction. The induction materials for in vitro doubling are selected from seeds, protocorms, protocorms, stem tips, terminal buds, root tips, clustered buds, leaves, etc., and chromosome doubling is induced under tissue culture conditions. Chemical induction is the most commonly used method for polyploid breeding, which promotes plant cell mutation. Auxin, anesthetics, and various plant alkaloids are usually used to induce it. Colchicine is the most commonly used mutagen. The damage caused by the concentration of colchicine to the plant is proportional to the degree of mutation. The lower the concentration, the less damage to the plant and the less mutation. Research has found that compared with other explants, protocorms have a higher reproduction coefficient and can rapidly proliferate to obtain a large number of plants, so protocorms are often used as induction materials for in vitro doubling. Compared with the immersion method, the co-cultivation method has the advantages of low contamination rate and less damage to the protocorms (Fan Wubo, 2007).

[0004] So far, although there are successful examples of using colchicine to induce polyploidy in polyploid breeding of orchids through co-cultivation, there are no reports on polyploidy induction in Cymbidium plants. Moreover, orchids are extremely rich in species, widely distributed, and have high species diversity. The research results between different genera and varieties are often very different, making it difficult to learn from existing research results. For example, Li Han (2005) used colchicine to induce C.iridioides and found that the induction effect was better when colchicine was treated for 72 hours and the concentration was 0.05%. Yang Lijuan (2009) used the co-cultivation method to add 3% colchicine to the culture medium to induce Cymbidium 'Ruby'. The experiment showed that the best effect was achieved after 15 days of treatment, with an induction rate of up to 30%. It can be seen that when using colchicine for co-cultivation, different species of orchids have very different requirements for colchicine concentration, induction time and other conditions, and there is no rule to follow.

[0005] The present invention uses a plant of the genus Tonkinensis (such as Tonkinensis multiflora) as an induction material, adopts a co-cultivation method, and explores suitable conditions for colchicine-induced protocorm doubling and a culture medium suitable for Tonkinensis cultivation by treating it with different concentrations of colchicine for different periods of time. At the same time, the invention also explores the identification method of the polyploid of Tonkinensis, including flow cytometry, chromosome pressing, morphological observation, stomatal identification, etc., so as to provide a reference for improving the polyploid induction technology of orchids and cultivating new germplasms with high ornamental value. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a method for inducing polyploidy in Psoralea corylifolia, comprising the following steps:

[0007] (a) germinating seeds of Orchidaceae to form protocorms on a first culture medium; and

[0008] (b) inoculating the protocorms of the Ungulate Orchid onto a second culture medium and culturing for 1-15 days (e.g., 1-10 days, 2-10 days, 3-10 days, 4-10 days, 5-10 days, 5 days, 6 days, 10 days, 5-8 days, 6-9 days, or any value or range therebetween), wherein the second culture medium contains colchicine at a concentration of 0.01%-0.2% (e.g., 0.01%-0.1%, 0.02%-0.09%, 0.03%-0.08%, 0.04%-0.07%, 0.05%-0.06%, 0.04%, 0.05%, 0.06%, or any value or range therebetween).

[0009] In some embodiments, step (b) comprises culturing the protocorms of the chrysanthemum on a second culture medium for 5-15 days, for example, 5-10 days. In some embodiments, the second culture medium contains colchicine at a concentration of 0.05%-0.2%, for example, 0.05%-0.1%, 0.05%-0.09%, 0.05%-0.08%, 0.05%-0.07%, 0.05%-0.06%. In some embodiments, step (b) comprises culturing the protocorms of the chrysanthemum on a second culture medium for 5-10 days, and the second culture medium contains colchicine at a concentration of greater than or equal to 0.05% and less than 0.1%, for example, 0.05%-0.07%, 0.05%-0.06%.

[0010] In some embodiments, the first culture medium is a liquid culture medium. In some embodiments, the first culture medium comprises 1 / 2MS+0.5g·L-1 agar+20g·L-1 sucrose+100g·L-1 coconut milk, or consists of it. In some embodiments, the first culture medium comprises 1 / 2MS+5g·L-1 carrageenan+100g·L-1 banana puree+20g·L-1 sucrose or consists of it. In some embodiments, the first culture medium comprises 1 / 2MS+0.5g·L-1 agar+100g·L-1 banana puree+20g·L-1 sucrose or consists of it.

[0011] In some embodiments, the second culture medium is a solid culture medium. In some embodiments, the second culture medium comprises 1 / 2MS+1.0mg·L-1 6-benzyladenine+0.2mg·L-1 agar+20g·L-1 sucrose, or consists of 1 / 2MS+1.0mg·L-1 6-benzyladenine+0.2mg·L-1 agar+20g·L-1 sucrose.

[0012] In some embodiments, the method further comprises: (c) transferring the protocorm obtained in step (b) to a third culture medium for subculture. In some embodiments, the third culture medium is a subculture medium, such as those well known in the art (e.g., 1 / 2MS + 1.0 mg / L 6-BA + 0.2 mg·L-1 NAA + 5.0 g·L-1 agar + 20 g·L-1 sucrose). In some embodiments, step (c) is repeated at least once or twice or more times. In some embodiments, step (c) is performed under the following conditions: light intensity: 2000 lx, daily light time: 12 to 14 h, temperature: 25 ± 2 ° C.

[0013] In some embodiments, the method further comprises determining the ploidy of the induced plant by flow cytometry, chromosome compression, morphological observation, stomatal identification, or any combination thereof. In some embodiments, the morphological observation includes but is not limited to observation of the following traits: plant height, stem thickness, growth rate, leaf color, stomatal shape, or any combination thereof.

[0014] In some embodiments, the ploidy of plants induced by colchicine is determined using, for example, flow cytometry.

[0015] In some embodiments, compared to control plants not induced with colchicine, the variant plants exhibit one or more or all of the following morphological shapes: dwarf plants, thick rhizomes, slow growth, dark green leaves, nearly round stomata, or any combination thereof.

[0016] In some embodiments, after colchicine induction, the mutation rate is about 15-45%, such as about 20-45%, 23-43%, 25-40%, 29%, 30%, 35%, 36%, 37%, 36-40%, or any value or range therebetween.

[0017] In some embodiments, the survival rate after colchicine induction is about 16-65%, such as about 16.5-61%, 18-61%, 20-61%, 30-61%, 34-61%, 45-61%, 47-61%, or any value or range therebetween.

[0018] In some embodiments, step (a) comprises collecting seeds of Psoralea corylifolia and sowing them aseptically on the first culture medium, so as to germinate and form protocorms. In some embodiments, the germinated protocorms are green.

[0019] In some embodiments, the method of the present invention further comprises: analyzing the obtained data to determine suitable induction conditions. In some embodiments, the data analysis comprises using Excel 2016 for data analysis, using SPSS2.0 for variance analysis, using LSD multiple comparison method, or any combination thereof.

[0020] In some embodiments, the henna is Henna polyanthus.

[0021] definition

[0022] In the present invention, the terms "induced plants", "variant plants" and "tissue culture seedlings" are sometimes used interchangeably, and generally refer to plants grown from seeds treated by the method described in the present invention, such as Orchidaceae plants grown from seeds treated by the method described in the present invention, unless the context indicates otherwise.

[0023] In the present invention, when the concentration of colchicine is mentioned as 0.05%, 0.1%, 0.2%, for example, it refers to the mass ratio, that is, the weight of colchicine accounts for 0.05%, 0.1%, 0.2% of the total solution mass, respectively.

[0024] In the present invention, when 5d, 10d, 15d, etc. are mentioned, "d" refers to the number of days.

[0025] In the present invention, when "Hydrangea" is mentioned, it generally refers to plants of the genus Hydrangea, such as any species, subspecies or variety of the genus Hydrangea, unless otherwise specified.

[0026] The present invention uses Pseudostella as a material, adopts a co-cultivation method to induce polyploidy to explore the effect of colchicine treatment on the doubling of Pseudostella chromosomes, and obtains suitable induction conditions for doubling Pseudostella chromosomes. The method of the present invention can produce more polyploids, providing data support for the optimization of polyploid breeding technology. The method of the present invention has important value in breeding and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings of the present invention. It should be understood that the drawings described below are only for the purpose of illustrating the present invention, and are not intended to limit the protection scope of the present invention.

[0028] Figure 1 The flowers of Henna multiflora (A), fruit pods (B), and protocorms formed after aseptic sowing according to Example 1 (C) are shown.

[0029] Figure 2 The effects of colchicine treatment concentration and time on the survival rate of Henna protocorms are shown.

[0030] Figure 3 The morphological characteristics of the variant strain of Psoralea corylifolia induced by colchicine according to the embodiment of the present invention are shown. A: control strain; B: variant strain; C: comparison of control (left) and variant leaves (right).

[0031] Figure 4 The stomatal characteristics of the multiflora stomata after colchicine induction according to the embodiment of the present invention are shown: A: stomatal of the control strain (×10); B: stomatal of the mutant strain (×10); C: stomatal of the control strain (×40); D: stomatal of the mutant strain (×40).

[0032] Figure 5The results of chromosome identification of Henna multiflora after colchicine induction according to the embodiment of the present invention are shown. A: peak graph of control plant; B: peak graph of variant strain; C: chromosome status of control plant (×60); D: chromosome status of variant strain (×60). Example

[0033] The present invention will be further described below in conjunction with embodiments.

[0034] 1. Materials

[0035] The seeds of the multi-flowered henna obtained by self-pollination in 2020 were used as materials through the original corms formed by germination after sterile sowing. The experiment was carried out at the Institute of Vegetables and Flowers of the Chinese Academy of Agricultural Sciences. The phenotypic traits are detailed in Figure 1 .

[0036] 2. Statistical methods

[0037] Excel 2016 was used for data analysis, SPSS2.0 was used for variance analysis, and the LSD multiple comparison method was used. The data variation range values ​​(±) in the table are all ± standard deviation

[0038] Example 1: Colchicine induces polyploid plants of Psoralea corylifolia

[0039] 1. Polyploidy Induction

[0040] The collected fruit pods were sown aseptically, and the seeds were germinated on a medium of 1 / 2MS+0.5g·L-1 agar+20g·L-1 sucrose+100g·L-1 coconut juice to form protocorms, and then the germinated green protocorms were transferred to a solid medium containing 0.05%, 0.1%, and 0.2% colchicine (1 / 2MS+1.0mg·L-1 6-benzyladenine (6-BA)+0.2mg·L-1 agar+20g·L-1 sucrose) for 5d, 10d, and 15d, respectively, for a total of 10 treatments, each of which was repeated 5 times. The specific scheme is shown in Table 1 below.

[0041] Table 1 Colchicine treatment plan

[0042]

[0043]

[0044] The protocorms treated with colchicine were transferred to the subculture medium for 2-3 subcultures, and the survival rate was calculated. Culture conditions: light intensity of 2 000 lx, daily light time: 12-14 h, temperature: 25±2℃, pH 5.8.

[0045] Survival rate = surviving plants / inoculated plants × 100%.

[0046] The results showed that the survival rate of the protocorms of P. polyanthus was affected by the concentration and treatment time of colchicine. When the concentration of colchicine increased or the treatment time increased, the survival rate of the protocorms decreased (e.g. Figure 2 ). The concentration of 0.05% caused less damage to the protocorms, and the survival rates were 61%, 47%, and 34.2% respectively; when the concentration of colchicine was 0.2%, the damage to the protocorms was greater, and the survival rates were 16.4%, 10.6%, and 6.6% respectively.

[0047] 2. Morphological Observation

[0048] Referring to the method of Zhuo Xiaokang (2015), when the plants differentiated into seedlings and the leaves were fully expanded, 15 untreated control plants with consistent culture conditions and good growth and 15 treated mutant strains that were successfully identified as doubling were selected to measure the growth, plant height, leaf size and observe color.

[0049] After colchicine treatment, the difference between the mutant and the control strain was large ( Figure 3 AB), mainly characterized by dwarfing of the plant, darkening of the leaf color, roughening of the leaf surface, and widening of the leaf ( Figure 3 B). As shown in Table 2, the length-to-width ratios of the control plant and the variant strains are 3.67 and 1.88, respectively. The leaves of the control plant are relatively narrow and long. Due to the inhibition, the leaves of most variant strains are shorter and rounder ( Figure 3 C). In addition, plant height, leaf length and width showed a decreasing trend compared with the control plant, decreasing by 1.94, 1.03 and 0.04 respectively, while the length-to-width ratio showed a decreasing trend, decreasing by 1.79.

[0050] Table 2 Comparison of morphological indexes of Henna polyphylla mutant strains induced by colchicine

[0051]

[0052] 3. Stomatal Identification

[0053] Since there are differences in stomatal size between chromosome-doubling plants and plants that have not been doubled, comparing the changes in stomatal size can also be used as a preliminary method for identifying doubled plants. Referring to the method of Xiqing Zhang (2020), 10 leaves from the same part of untreated and treated doubled plants were selected. A drop of water was first dripped on the slide to avoid curling after the epidermis was torn off. Then, the lower epidermis of the leaf was torn off with tweezers, placed on a slide, covered with a coverslip and flattened, and placed under a Leica inverted microscope (Beijing Oriental Australia Technology Development Co., Ltd., Fengtai District, Beijing) for observation and photography. Randomly select a field of view with uniform stomata distribution under the microscope, and record the number of stomata per unit area.

[0054] As shown in Table 3, the stomata of the control strain of P. multiflora are densely distributed, with an average of 39 stomata per field of view, while the number of stomata in each field of view of the doubled plant is 21, and the length and width of the stomata are larger than those of the control strain. The stomata of the control strain are oblong, while the stomata of the mutant strain are close to round. The stomata of the control strain are 31μm wide and 34μm long, while the stomata of the mutant strain are 39μm wide and 42μm long. Figure 4 Stomatal comparison between the mutant and control strains is shown.

[0055] Table 3 Comparison of stomatal morphology indices of variant strains of Henna spp. induced by colchicine

[0056]

[0057] 4. Ploidy Analysis by Flow Cytometry

[0058] The plants of Henna multiflora that were not treated with doubling were used as reference to identify the ploidy of the plants treated with colchicine. Using the Sysmex CyStain UV Precise P kit (Beijing Deshida Technology Co., Ltd., Haidian District, Beijing), fresh leaves of the plants to be tested were placed in a petri dish and cut into 0.5 cm 2 The extract was dropped on the leaves and the dye was kept away from light for 2 minutes. Flow cytometry was then used for identification. Each sample was repeated twice. According to the flow cytometer results, the number of mutants was counted and the mutation rate was calculated.

[0059] The results are visible Figure 4 AB. The untreated control strain was identified as diploid, and the induced plants became mutants, but there was a certain amount of chimeras.

[0060] As shown in Table 4, when the treatment time is the same, the concentration is proportional to the mutation rate, and the mutation rate increases with the increase of concentration; the mutation rate of 0.1% colchicine treatment for 15 days and 0.2% colchicine treatment for 15 days decreased by 13.24% and 14.25% respectively compared with the treatment of 10 days. As shown in Table 4, the mutation rate reached a peak of 40% at a concentration of 0.05% for 10 days, and the survival rate was 45%. Secondly, after 5 days of treatment with a concentration of 0.05%, the mutation rate reached 36.36%, but the survival rate was the highest, at 61%.

[0061] Table 4 Effects of colchicine treatment concentration and time on the variation rate of protocorms of Henna

[0062]

[0063] 2.5 Chromosome Squeeze Analysis

[0064] Referring to the tablet pressing method of Zhou Jianjin, Zhuang Donghong, etc., the root tip seedlings of tissue culture were selected between 9:00 and 11:00 in the morning. The specific operation method is as follows:

[0065] (1) Select the root tip of the tissue culture seedling, cut it to 1-2 cm, scrape off its epidermis with a blade, and then soak it in a prepared 0.002 mol / L 8-hydroxyquinoline solution for 7-8 hours;

[0066] (2) After washing with sterile water, place in a newly prepared Carnoy's fixative and soak for 24 hours for fixation. After treatment, the chromosomes can be directly pressed or stored in 70% ethanol for later use;

[0067] (3) Treat the treated material with 1 mol / L hydrochloric acid at room temperature for 5-6 min and soak it in a constant temperature water bath at 60°C for 7-8 min;

[0068] (4) dropping modified phenol fuchsin staining solution on the treated root tip for staining;

[0069] (5) After 10 minutes, crush the root tip with tweezers to remove the residue and cover with a coverslip; tap the coverslip with a pencil with an eraser to ensure that the chromosomes are evenly distributed;

[0070] (6) Observe and count the number of chromosomes under a microscope. Select five areas of cells with better field of view and more evenly dispersed chromosomes, count the number of chromosomes and take photos.

[0071] Flow cytometry can be used to identify the ploidy of a large number of plants, but the results of chromosome squeezing are the most intuitive and accurate ( Figure 4 CD). After chromosome pressing, it was found that the number of chromosomes in the control strain was 2n=2x=36 ( Figure 4C), while the chromosome number of the mutant after induction treatment is 2n=4x=74( Figure 4 D), and there are still cells in the variant strain that have not successfully doubled.

[0072] Example 2: Screening of additives in seed germination medium

[0073] In order to verify the effect of different additives on seed germination rate, 1 / 2MS+5g·L-1 carrageenan+20g·L-1 sucrose was used as the basic culture medium, and 100g·L-1 corn juice, banana puree, and coconut juice were added to it respectively. The culture medium without additives was used as a control, and the seeds of multi-flowered henna were aseptically sown and observed. Under the same culture time, the seeds of multi-flowered henna showed different development conditions due to different additives. Some seeds have developed into healthy green protocorms, and even small seedlings have grown; the protocorms developed by some seeds are mostly yellow. Among them, the germination rate was the highest in the culture medium 1 / 2MS+5g·L-1 carrageenan+100g·L-1 banana puree+20g·L-1 sucrose, and the size was full (Table 5).

[0074] Table 5 Effects of additives on germination of Henna seeds

[0075]

[0076] This study shows that by combining chromosome doubling technology with plant tissue culture, a large number of mutant plants can be obtained in a short period of time. Since there are few studies on Henna, the doubling induction experiment on Henna has important research significance. Colchicine was used to induce protocorms and quasi-protocorms in vitro, and the induction effect was the best. In this study, the mutation rate of plants treated for 15 days was reduced when the concentration was 0.1% and 0.2%. Therefore, it is believed that the induction effect is better when the co-cultivation method is used to induce the protocorms, the treatment time is less than 15 days, and the concentration is within the range of <0.1%.

[0077] In terms of morphological characteristics, the mutants all showed dwarf plants, thick rhizomes, slow growth, and dark green leaves. At the same time, the leaves of the control strain were narrow and long, while the leaves of the mutant strain were short and round. In terms of stomata, the stomata of the mutant strain were close to round, while the stomata of the control strain were close to oblong. Therefore, plant morphology and stomatal characteristics can be used as an indirect method to identify the ploidy of the nail orchid.

[0078] Compared with the immersion method for inducing polyploidy, the co-cultivation method has the advantages of high induction rate and low contamination rate, but there are a certain amount of chimeras after induction. This study used the co-cultivation method to induce polyploidy in protocorms, compared the concentration of colchicine and the treatment time, obtained a better treatment combination, and obtained variant strains. At the same time, a large number of protocorms after treatment can be screened using flow cytometry to obtain polyploids, thereby obtaining polyploid plants.

[0079] References

[0080] [1] Flora of China Editorial Committee, Chinese Academy of Sciences. Flora of China[M]. Beijing: Science Press, 1993, (19): 384.

[0081] [2] Li Han, Zheng Sixiang, Long Chunlin. Preliminary study on the induction of polyploidy in Dendrobium dendrobium[J]. Yunnan Botanical Research, 2005, 27(5):552-556.

[0082] [3]Xiqing Zhang,Jiangyun Gao.Colchicine-induced tetrapoloidy inDendrobium carniferum and ite effect on plantet morphology,anatomy and genomesize[J].Original Article,2020.

[0083] [4] Zhuo Xiaokang. Construction of rapid propagation system and polyploid induction of Dendrobium macrobracte[D]. Fujian Agriculture and Forestry University, 2015.

[0084] [5] Zhou Jianjin, Zeng Ruizhen, Liu Fang, Yi Maosheng, et al. Study on chromosome ploidy of hybrid offspring of Phalaenopsis with different ploidy[J]. Acta Horticulturae Sinica, 2009, 36(10): 1491-1497.

[0085] [6] Zhuang Donghong, Qu Ying, Xu Daxiong, Li Jun, Chen Zhiling. 2007. Analysis of chromosome number and morphology of several varieties (lines) of Phalaenopsis. Acta Horticulturae Sinica, (05): 1257–1262.

[0086] [7] Yang Lijuan, Gao Suping, Zou Zonglan, Chen Guo. Experiment on induction of polyploidy in Cymbidium grandiflorum by colchicine in vitro[J]. Northern Horticulture, 2009(6): 51-53.

Claims

1. A method for inducing Aerides rosea to produce polyploidy, comprising the following steps: (a) germinating seeds of Henna scabra to form protocorms on a first culture medium, wherein the first culture medium is a liquid culture medium, and the first culture medium consists of 1 / 2MS+5g·L-1 carrageenan+100g·L-1 banana puree+20g·L-1 sucrose; and (b) inoculating the protocorms of Henna serrata onto a second culture medium and culturing for 5-10 days, wherein the second culture medium contains colchicine at a concentration of 0.05%-0.1%, wherein the concentration of colchicine refers to the mass ratio.

2. The method according to claim 1, further comprising: (c) transferring the protocorm obtained in step (b) to a third culture medium for subculture.

3. The method according to any one of claims 1-2, wherein the second culture medium is a solid culture medium, and / or the second culture medium comprises 1 / 2MS+1.0 mg·L-1 6-benzyladenine+0.2 mg·L-1 agar+20 g·L-1 sucrose.

4. The method according to claim 2, comprising repeating step (c) at least once or twice, and / or, step (c) is carried out under the following conditions: light intensity: 2 000 lx, daily light time: 12 to 14 h, temperature: 25 ± 2 °C.

5. The method according to any one of claims 1 to 2, wherein step (b) comprises culturing the protocorms of Psoralea corylifolia on the second culture medium for 10 days.

6. The method according to any one of claims 1-2, wherein the second culture medium contains colchicine at a concentration of greater than or equal to 0.05% and less than 0.1%.

7. The method according to any one of claims 1-2, wherein step (b) comprises culturing the protocorms of Henna on a second culture medium for 5-10 days, and the second culture medium contains colchicine at a concentration of 0.05%.

8. The method according to any one of claims 1-2, further comprising determining the ploidy of the induced plant by flow cytometry, chromosome squashing, morphological observation, stomatal identification, or any combination thereof.

9. The method according to claim 8, wherein the morphological observation includes but is not limited to observation of the following traits: plant height, stem thickness, growth rate, leaf color, stoma shape, or any combination thereof.

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