Lysimachia christinae Hance variation induction method
By optimizing the treatment conditions of amisulfolin, triploid pass yellow variants were successfully induced, which solved the problems of low breeding efficiency and high toxicity in the prior art, and achieved efficient triploid induction and breeding effects.
Patent Information
- Application Number
- CN202510063186.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-15
- Publication Date
- 2025-03-04
AI Technical Summary
The prior art is difficult to effectively induce triploid through yellow. Traditional methods such as the use of colchicine have problems of toxicity and low breeding efficiency.
Amosulforin was used to induce the pass yellow variation, and by optimizing the concentration, treatment time and explant selection of amosulforin, the variation rate and triploid rate of triploid were significantly improved, reaching nearly 100%.
The triploid pass yellow variant was successfully induced, showing stronger stress resistance, adaptability and ornamental value, and its breeding efficiency was higher than that of tetraploid.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of plant breeding, and in particular to a method for inducing mutation of Rhizoma Cibotii. Background Art
[0002] Lysimachia xiangxiensis is a perennial herb belonging to the Primulaceae family, widely distributed in China and other parts of Asia. Due to its rich medicinal value and unique ornamental characteristics, Lysimachia xiangxiensis has high application potential in horticulture and ecological restoration. Although it is aesthetically attractive, the morphological uniformity of Lysimachia xiangxiensis limits its adaptability and resistance in different environments, especially in the face of external pressures such as climate change, pests and diseases, showing a certain degree of vulnerability. Therefore, the development of new Lysimachia xiangxiensis varieties with stronger environmental adaptability and better resistance has become the focus of current breeding work.
[0003] Traditional breeding methods for Rhizoma Dioscoreae, such as hybridization and selective breeding, usually rely on natural environment and seasonal changes, with long breeding cycles and low efficiency. With the advancement of biotechnology, polyploid induction in plants has become a more efficient breeding method. Polyploid plants usually show characteristics such as stronger genetic plasticity, larger organs, and stronger stress resistance. They can adapt to more diverse growth environments and show better tolerance in terms of drought resistance and salt-alkali resistance. Therefore, polyploid induction has become one of the important methods for improving traits in plant breeding. By inducing polyploidy in vitro, not only can the breeding process be accelerated, but also the limitations of seasonal and climatic conditions can be broken through.
[0004] At present, commonly used anti-mitotic agents such as colchicine have been widely used to induce plant mutations. Although colchicine has a strong ploidy induction effect, its toxicity is relatively high, which limits its application in plant breeding. And colchicine, as a mutagen, usually doubles the number of chromosomes in diploid plants to produce tetraploids, but tetraploids are usually accompanied by slow growth characteristics, and their breeding efficiency and commercial value are lower than those of triploids. Therefore, the use of colchicine to induce Rhizoma Cibotii also does not have a breeding advantage.
[0005] Therefore, there is an urgent need for an induction method for triploid R. passerine. Summary of the invention
[0006] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for inducing mutation of Rhizoma Citri Reticulatae. The present invention is the first to carry out induced breeding for Rhizoma Citri Reticulatae species, creatively using amoxicillin to induce mutation of Rhizoma Citri Reticulatae, and unexpectedly found that the mutation of Rhizoma Citri Reticulatae produced triploid plants, whose breeding efficiency is much higher than that of tetraploid Rhizoma Citri Reticulatae; and the ornamental value and stress resistance / adaptability are better than those of diploid plants, so it has obvious breeding advantages. In addition, the present invention further optimizes the concentration of amoxicillin, the treatment time and the selection of induced explants, thereby significantly improving the mutation rate and triploid rate of triploid plants to nearly 100%.
[0007] The explants of Rhizoma Cibotii, especially the stem segments, are treated with amoxicillin, which can not only improve the reproduction efficiency, but also change the genetic background by inducing polyploidy, improve the morphology, and enhance stress resistance and adaptability, thereby providing a new material basis and technical support for the breeding of Rhizoma Cibotii. By optimizing the concentration and treatment time of amoxicillin, the variation induction effect of Rhizoma Cibotii can be effectively improved, thereby providing a new breakthrough point for the variety improvement and resistance research of Rhizoma Cibotii.
[0008] The purpose of the present invention can be achieved by the following scheme:
[0009] In a first aspect, the present invention provides a method for inducing triploid Lycopodiella vulgaris, the induction method comprising the following steps:
[0010] S1, soaking the stem segments of Psoralea corylifolia in a solution of amoxicillin for mutagenesis;
[0011] S2. The mutagenized L. lycopersicum stem segments are inoculated into a culture medium for cultivation, and then regenerated and cultured under light conditions, and then acclimated and transplanted to obtain the triploid L. lycopersicum.
[0012] As a low-toxic antimitotic agent, the amosulfuron in the present invention can prevent chromosome distribution by interfering with microtubule formation during cell division, thereby promoting chromosome doubling and further increasing the mutation incidence of plants.
[0013] In some embodiments, the amisulaline solution is prepared on a clean bench and sterilized by filtration.
[0014] As an embodiment of the present invention, in step S1, the variety of the Rhizoma Cibotii is Lysimachia xiangxiensis.
[0015] As an embodiment of the present invention, in step S1, the stem segments of the Rhizoma Cibotii are cut Rhizoma Cibotii stem segments, and the cut length is 3-5 cm. The present invention selects the stem segments of the Rhizoma Cibotii with uniform growth as the mutagenesis explants.
[0016] As an embodiment of the present invention, in step S1, the mass concentration of the amisulaline solution is 0.001-0.003%.
[0017] Preferably, the mass concentration of the amisulaline solution is 0.001%.
[0018] As an embodiment of the present invention, in step S1, the time of the mutagenesis treatment is 2-6 days.
[0019] Preferably, the mutagenesis treatment is carried out for 4 days.
[0020] As an embodiment of the present invention, in step S1, the mutagenesis treatment is carried out by oscillation treatment at a rate of 100-200 r / min.
[0021] As an embodiment of the present invention, in step S2, before inoculation, the moisture on the surface of the stem segment of Rhizoma Cibotii is absorbed with sterile filter paper.
[0022] As an embodiment of the present invention, in step S2, the culture medium is an MS culture medium containing 3-4% sucrose and 0.65-0.7% agar, with a pH of 5.6-5.8. The culture is performed until plants are regenerated on the culture medium.
[0023] As an embodiment of the present invention, in step S2, the illumination intensity is 1200-1400 lx, the illumination time is 14-15 h / d, the regeneration culture time is 3-5 months, and the regeneration culture temperature is 22-27° C. In some embodiments, the regeneration culture is performed in a plug tray.
[0024] As an embodiment of the present invention, in step S2, the time for acclimatization and transplanting is 7-10 months.
[0025] In the present invention, the triploid Passerella obtained in step S2 is used to identify the ploidy using a flow cytometer.
[0026] Compared with the prior art, the present invention has the following beneficial effects:
[0027] 1. The present invention is the first to carry out induced breeding on the species of Rhizoma Cibotii, creatively uses amoxicillin to treat Rhizoma Cibotii, identifies the ploidy of the regenerated plants through chromosome analysis and flow cytometry, successfully induces mutation of Rhizoma Cibotii, and unexpectedly finds that the induced triploid Rhizoma Cibotii is produced, which provides effective germplasm material for Rhizoma Cibotii variety improvement and polyploid induction.
[0028] 2. The present invention induces the explants (stem segments) of Rhizoma Cibotii by optimizing the mass concentration of amosulfuron (0.001%) and the treatment time (4 days), thereby significantly improving the mutation rate and triploid rate of the triploid Rhizoma Cibotii plants to nearly 100%, and has extremely high practicality.
[0029] 3. The present invention successfully induced a triploid variant of R. cyrtonema, which is significantly different from the wild type in morphology, with thicker stems, larger and thicker leaves, stronger overall growth potential, better stress resistance, and wider adaptability; it has a higher breeding efficiency than tetraploid plants. The variants obtained by the present invention can significantly improve the ornamental value, ecological adaptability and breeding efficiency of R. cyrtonema, and provide important germplasm materials and technical support for future R. cyrtonema breeding. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:
[0031] Figure 1 Figure 3 is a diagram of the regeneration process of the stem segments of the Rhizoma Cibotii after induction treatment in Example 3; wherein A is the growth observed after 15 days of culture on the MS basal medium, B is the growth after 80 days of culture on the MS basal medium, C is the growth of the regenerated plants after 45 days of culture in the plug tray, and D is the growth of the plants after 10 months of transplantation into the flower pot;
[0032] Figure 2 The flow cytometry ploidy identification results of the leaves of Psoralea corylifolia in Example 2;
[0033] Figure 3 Figure 2 is a morphological characteristic diagram of the mutant plants of Rhizoma Cibotii in Example 2; wherein A is the growth of the wild type (WT) transplanted into a flower pot and cultured for 18 months, B is the growth of VT1 transplanted into a flower pot and cultured for 18 months, C is the growth of VT2 transplanted into a flower pot and cultured for 18 months, D is the growth of VT3 transplanted into a flower pot and cultured for 18 months, and E is the leaf morphology of WT and VT1-3 transplanted into a flower pot and cultured for 18 months;
[0034] Figure 4 The figure is the result of counting the passing yellow chromosomes in Example 2; wherein A is the somatic chromosome number of wild-type (WT) root tip cells, B is the somatic chromosome number of VT1 root tip cells, C is the somatic chromosome number of VT2 root tip cells, and D is the somatic chromosome number of VT3 root tip cells;
[0035] Figure 5 This is the flow cytometry ploidy identification result of Badong Passiflora in Comparative Example 1. DETAILED DESCRIPTION
[0036] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples are implemented on the premise of the technical solution of the present invention, provide detailed implementation methods and specific operation processes, and will help those skilled in the art to further understand the present invention. It should be pointed out that the protection scope of the present invention is not limited to the following embodiments, and several adjustments and improvements made under the premise of the concept of the present invention all belong to the protection scope of the present invention.
[0037] Example 1 Optimization of the concentration of amosulfuron solution
[0038] 1. Induction of mutation of Rhizoma Citri Reticulatae
[0039] (1) A 0.001%, 0.002% and 0.003% amoxicillin solution was prepared on a clean bench and sterilized by filtration. Uniformly grown stem segments of Lysimachia xiangxiensis seedlings were selected as explants, cut into 3-5 cm lengths, immersed in different concentrations of amoxicillin solution, and placed on a shaker (100 r / min) for 4 days.
[0040] (2) After the surface moisture of the stem segments was absorbed with sterile filter paper, they were inoculated into MS medium (3% sucrose + 0.65% agar powder, pH 5.6-5.8). Three groups were set up for each treatment concentration, and 9 to 12 explants were inoculated in each group and cultured in a culture room; after the plants were regenerated on the MS medium, they were individually transplanted into plug trays (each hole size was 6×6×11 cm) filled with commercial nursery soil (Nursery Soil Mixture, Yantai Jimmy Sunshine Environmental Technology Co., Ltd., China) and cultured in an artificial light incubator at a temperature of (25±1)°C, with a light intensity of 1200 lx and a light duration of 14 h / d for 3 months, and then acclimated and transplanted for 10 months.
[0041] (3) Ploidy identification of leaves of regenerated plants, including flow cytometry analysis and chromosome counting.
[0042] 2. Detection of mutant plants of Rhizoma Cibotii
[0043] (1) Flow cytometric analysis.
[0044] The steps are as follows: 50-100 mg of fresh leaves were collected from each surviving P. cyrtonema plant and placed in a pre-cooled 60 mm culture dish. 1.5 mL of Galbraith's buffer was added, and the leaf tissue was minced thoroughly with a sharp blade to release the nuclei. The homogenate was filtered through a 30 μm nylon mesh to obtain a nuclear suspension. About 500 μL of the nuclear suspension was transferred to a new centrifuge tube, 2.5 μL of RNase A solution (concentration 10 mg / mL) was added, and incubated on ice for 15 minutes. Propidium iodide (PI) was added to a final concentration of 50 μg / mL to stain the nuclei. A Cytek Aurora flow cytometer (Cytek Biosciences, USA) was used to detect the fluorescence signal using the B4 fluorescence channel. Each sample was measured three times, and at least 1000 nuclear signals were analyzed each time. The data were processed using FlowJo 10.9 software to calculate the ploidy level.
[0045] (2) Chromosome counting and identification.
[0046] The steps are as follows: fresh root tips (length 1-2 cm) of the mutants were collected between 9:00 and 10:00 in the morning, and the root tips were immersed in 0.1% colchicine solution at 4°C for 3 hours. After rinsing the root tips with distilled water 3 times, they were placed in a freshly prepared ethanol and glacial acetic acid (3:1, v / v) solution and fixed at room temperature for 24 hours. The root tips were removed from the fixative, washed with distilled water 3 times, placed in a 1mol / L HCl solution, and hydrolyzed in a 60°C water bath for 10 minutes. Subsequently, Carbofuchsin stain (Solarbio) was used for staining at room temperature for 30 minutes. The stained root tips were placed on a slide and gently squeezed with a coverslip to disperse the cells. The chromosomes in the metaphase of cell division were observed and photographed at a magnification of 1000 times using a bright field microscope (Panthera L, Motic, China).
[0047] 3. Test results
[0048] From Table 1 we can see that:
[0049] Under the treatment condition of 0.001%, the mutation rate and triploid rate both reached the highest value (100%), and the survival rate was 11.11%. Under the treatment condition of 0.002% and 0.003%, triploid was not induced successfully, and the survival rate showed a downward trend. Considering the mutation rate and triploid rate comprehensively, 0.001% is the best induction concentration of amosulfuron solution.
[0050] Example 2 Optimization of processing time
[0051] 1. Induction of mutation of Rhizoma Citri Reticulatae
[0052] (1) A 0.001% amoxicillin solution was prepared on a clean bench and sterilized by filtration. Uniformly grown stem segments of Lysimachia xiangxiensis (Lysimachia xiangxiensis) were selected and cut into 3-5 cm lengths. The stem segments were immersed in the solution and placed on a shaker (100 r / min) for 2, 4 and 6 days.
[0053] (2) After the surface moisture of the stem segment was absorbed with sterile filter paper, it was inoculated into MS medium (3% sucrose + 0.65% agar powder, pH 5.6-5.8). Three groups were set up for each treatment time, and 9 to 12 explants were inoculated in each group and cultured in a culture room; after the plants were regenerated on the MS medium, they were individually transplanted into a plug tray (each hole size was 6×6×11 cm) filled with commercial nursery soil (Nursery Soil Mixture, Yantai Jimmy Sunshine Environmental Technology Co., Ltd., China) and cultured in an artificial light incubator at a temperature of (25±1)°C, a light intensity of 1200 lx, and a light duration of 14 h / d for 3 months, and then acclimated and transplanted for 10 months.
[0054] (3) Ploidy identification of leaves of regenerated plants, including flow cytometry analysis and chromosome counting.
[0055] 2. Detection of mutant plants of Rhizoma Cibotii
[0056] (1) Flow cytometric analysis.
[0057] The steps are as follows: 50-100 mg of fresh leaves were collected from each surviving P. cyrtonema plant and placed in a pre-cooled 60 mm culture dish. 1.5 mL of Galbraith's buffer was added, and the leaf tissue was minced thoroughly with a sharp blade to release the nuclei. The homogenate was filtered through a 30 μm nylon mesh to obtain a nuclear suspension. About 500 μL of the nuclear suspension was transferred to a new centrifuge tube, 2.5 μL of RNase A solution (concentration 10 mg / mL) was added, and incubated on ice for 15 minutes. Propidium iodide (PI) was added to a final concentration of 50 μg / mL to stain the nuclei. A Cytek Aurora flow cytometer (Cytek Biosciences, USA) was used to detect the fluorescence signal using the B4 fluorescence channel. Each sample was measured three times, and at least 1000 nuclear signals were analyzed each time. The data were processed using FlowJo 10.9 software to calculate the ploidy level.
[0058] (2) Chromosome counting and identification.
[0059] The steps are as follows: fresh root tips (length 1-2 cm) of the mutants were collected between 9:00 and 10:00 in the morning, and the root tips were immersed in 0.1% colchicine solution at 4°C for 3 hours. After rinsing the root tips with distilled water 3 times, they were placed in a freshly prepared ethanol and glacial acetic acid (3:1, v / v) solution and fixed at room temperature for 24 hours. The root tips were removed from the fixative, washed with distilled water 3 times, placed in a 1mol / L HCl solution, and hydrolyzed in a 60°C water bath for 10 minutes. Subsequently, Carbofuchsin stain (Solarbio) was used for staining at room temperature for 30 minutes. The stained root tips were placed on a slide and gently squeezed with a coverslip to disperse the cells. The chromosomes in the metaphase of cell division were observed and photographed at a magnification of 1000 times using a bright field microscope (Panthera L, Motic, China).
[0060] 3. Test results
[0061] From Table 1 we can see that:
[0062] When treated for 2 days, the survival rate was higher, but the triploid rate was only 20%.
[0063] After 6 days of treatment, the survival rate was 38.89%, but the mutation rate and triploid rate decreased significantly. The DNA content of plant leaves was analyzed by flow cytometry, and it was found that compared with WT and VT1, some plants (VT2, VT3) showed higher DNA fluorescence intensity ( Figure 2 is the result of flow cytometry ploidy identification), indicating that its ploidy is high; further chromosome counting method ( Figure 4 are chromosome counting results, scale bar = 5 μm), respectively. Figure 4 As shown in AD, it was confirmed that the wild type (WT, 2n = 2x = 24) was diploid, VT1 (2n = 2x-2 = 22) and VT2 (2n = 2x + 2 = 26) were diploid aneuploids, and VT3 was triploid (3n = 3x = 36). The experiment showed that the ploidy of the Rhizoma Dioscoreae plants can be effectively identified by combining flow cytometry and chromosome counting methods.
[0064] It was observed that some stem segments began to produce adventitious buds on the 30th day after treatment. After 10 months of cultivation, the regenerated plants were successfully cultured and several variants (VT1, VT2, VT3) were finally obtained. After the regenerated plants were transplanted into pots and cultured for 18 months, these plants were significantly different from the wild type in morphology ( Figure 3 Figure 2 shows the morphological characteristics of mutant plants after being transplanted into pots for 18 months (scale bar = 2 cm). The diploid plants showed leaf spots and leaf color variation, while the triploid plants showed leaf enlargement and thickened stems in addition to leaf spots and leaf color variation. The growth conditions of WT, VT1, VT2 and VT3 are shown in Figure 2. Figure 3As shown in AD, Figure 3 E is the leaf morphology of the above plant.
[0065] When the treatment time was 4 days, the mutation rate and triploid rate both reached the highest value (100%), but the survival rate was relatively low (11.11%). Taking the mutation rate and triploid rate into consideration, 4 days was the best treatment time.
[0066] Table 1 shows the effects of different concentrations and treatment times of amoxicillin solution on the survival rate, mutation rate and triploid rate of regenerated plants from stem segments of Rhizoma Cibotii.
[0067] Table 1
[0068]
[0069] illustrate:
[0070] 1. Survival rate (%) = number of surviving samples / total number of processed samples × 100;
[0071] 2. Mutation rate (%) = number of diploid, aneuploid and triploid / total number of surviving samples × 100;
[0072] 3. Triploid rate (%) = number of triploids / number of surviving samples × 100.
[0073] Through the above experimental optimization, the treatment with 0.001% amoxicillin solution for 4 days can significantly increase the mutation rate and triploid induction rate of R. passerine, providing a reliable methodological basis for further breeding and resistance research.
[0074] Example 3
[0075] 1. Induction of mutation of Rhizoma Citri Reticulatae
[0076] (1) A 0.001% amoxicillin solution was prepared on a clean bench and filtered for sterilization. Stem segments of uniformly grown Lysimachia xiangxiensis tissue culture seedlings (Lysimachia xiangxiensis) were selected as explants, cut into 3-5 cm long pieces, immersed in the amoxicillin solution, and placed on a shaker (100 r / min) for 4 days;
[0077] (2) Regeneration process Figure 1 As shown (the scale bar is 2 cm); the moisture on the surface of the leaves treated in step (1) was absorbed with sterile filter paper, and inoculated into MS medium + 3% sucrose + 0.65% agar powder, pH 5.6-□5.8, and repeated 3 groups were set up, each group was inoculated with 9 to 12 explants, and placed in the culture room for culture (the growth conditions of 15 days and 80 days of culture on MS basic medium are shown in Figure 2). Figure 1 A. Figure 1After the plants were regenerated on the MS medium, they were transplanted individually into plug trays (each hole was 6×6×11 cm) filled with commercial nursery soil (Nursery Soil Mixture, Yantai Jimmy Sunshine Environmental Technology Co., Ltd., China) and cultured in an artificial light incubator at a temperature of (25±1)°C, with a light intensity of 1200 lx and a light duration of 14 h / d for 3 months (the growth after 45 days of culture is shown in Figure 2). Figure 1 C), and then acclimate and transplant for 10 months (growth conditions are shown in Figure 1 D).
[0078] (3) Take the plant leaves obtained in step (2) and use flow cytometry and chromosome counting to identify the ploidy.
[0079] 2. Detection of mutant plants of Rhizoma Cibotii
[0080] (1) Flow cytometric analysis.
[0081] The steps are as follows: 50-100 mg of fresh leaves were collected from each surviving P. cyrtonema plant and placed in a pre-cooled 60 mm culture dish. 1.5 mL of Galbraith's buffer was added, and the leaf tissue was minced thoroughly with a sharp blade to release the nuclei. The homogenate was filtered through a 30 μm nylon mesh to obtain a nuclear suspension. About 500 μL of the nuclear suspension was transferred to a new centrifuge tube, 2.5 μL of RNase A solution (concentration 10 mg / mL) was added, and incubated on ice for 15 minutes. Propidium iodide (PI) was added to a final concentration of 50 μg / mL to stain the nuclei. A Cytek Aurora flow cytometer (Cytek Biosciences, USA) was used to detect the fluorescence signal using the B4 fluorescence channel. Each sample was measured three times, and at least 1000 nuclear signals were analyzed each time. The data were processed using FlowJo 10.9 software to calculate the ploidy level.
[0082] (2) Chromosome counting and identification.
[0083] The steps are as follows: fresh root tips (length 1-2 cm) of the mutants were collected between 9:00 and 10:00 in the morning, and the root tips were immersed in 0.1% colchicine solution at 4°C for 3 hours. After rinsing the root tips with distilled water 3 times, they were placed in a freshly prepared ethanol and glacial acetic acid (3:1, v / v) solution and fixed at room temperature for 24 hours. The root tips were removed from the fixative, washed with distilled water 3 times, placed in a 1mol / L HCl solution, and hydrolyzed in a 60°C water bath for 10 minutes. Subsequently, Carbofuchsin stain (Solarbio) was used for staining at room temperature for 30 minutes. The stained root tips were placed on a slide and gently squeezed with a coverslip to disperse the cells. The chromosomes in the metaphase of cell division were observed and photographed at a magnification of 1000 times using a bright field microscope (Panthera L, Motic, China).
[0084] 3. Test results
[0085] According to the induction scheme of the present invention, 0.001% amoxicillin is used for treatment for 4 days, and the mutation induction rate of the Psoralea corylifolia plants is 100%, and the triploid rate is 100%.
[0086] The present invention provides a method for inducing mutation of Rhizoma Cibotii, which can induce Rhizoma Cibotii to produce polyploidy and other morphological mutations by amoxicillin, and can provide a solid foundation for breeding, variety improvement and resistance research of Rhizoma Cibotii. The method is simple and easy to implement, and has high breeding value and application prospects.
[0087] Comparative Example 1
[0088] The induction method of this comparative example is basically the same as that of Example 3, except that Lysimachia xiangxiensis is replaced by Lysimachia badongensis. The specific process is as follows:
[0089] The stem segments of Padang Passiflora were used as materials and subjected to the same amoxicillin induction treatment as in the present invention. The treatment conditions used in the experiment were the same as those of Xiangxi Passiflora, specifically: 0.001% amoxicillin solution, and the soaking treatment time was 4 days. The experimental results showed that the induction rate of Padang Passiflora under the treatment conditions was 0, and polyploid plants could not be effectively induced. Specifically, after the Padang Passiflora was treated and grown, no obvious morphological variation was observed, and no characteristics related to polyploidy (such as enlarged leaves, thickened stems, etc.) appeared. According to the results of flow cytometry analysis, the Padang Passiflora plants did not show a significant increase in DNA ploidy (results as shown in Figure 2). Figure 5 shown).
[0090] In contrast, the Xiangxi radix scutellariae showed significant responsiveness under the same treatment conditions, with an induction rate of 100% and a triploid rate of 100%. Through flow cytometry and chromosome counting analysis, all treated plants showed a significant increase in ploidy, and the morphological characteristics of some plants (such as enlarged leaves, thickened stems, etc.) were consistent with the characteristics of triploid plants.
[0091] These comparison results clearly show that Badong Passiflora has poor responsiveness or does not have good induction potential under this induction condition, while Xiangxi Passiflora has stronger variation induction ability and good triploid induction effect under the induction conditions of the present invention. This experimental result further proves the advantage of Xiangxi Passiflora in this technical solution, confirms the effect of the amoxicillin induction method adopted by the present invention on the variation induction of Passiflora, and demonstrates its use as an effective tool for breeding and variety improvement.
[0092] It should be noted that the experimental methods used in the examples of the present invention, unless otherwise specified, are all conventional techniques; the materials and reagents used in the examples of the present invention, unless otherwise specified, can be obtained through commercial channels.
[0093] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.
Claims
1. A method for inducing triploid Lycopodiella vulgaris, characterized in that: The induction method comprises the following steps: S1, soaking the stem segments of Psoralea corylifolia in a solution of amoxicillin for mutagenesis; The variety of the Lysimachia xiangxiensis is Lysimachia xiangxiensis; S2. The mutagenized L. lycopersicum stem segments are inoculated into a culture medium for cultivation, and then regenerated and cultured under light conditions, and then acclimated and transplanted to obtain the triploid L. lycopersicum.
2. The induction method according to claim 1, characterized in that: In step S1, the mass concentration of the amisulaline solution is 0.001-0.003%.
3. The induction method according to claim 2, characterized in that: The mass concentration of the amisulaline solution is 0.001%.
4. The induction method according to claim 1, characterized in that: In step S1, the time of the mutagenesis treatment is 2-6 days.
5. The induction method according to claim 4, characterized in that: The duration of the mutagenesis treatment was 4 days.
6. The induction method according to claim 1, characterized in that: In step S1, the mutagenesis treatment is carried out by oscillation, and the rate of the oscillation treatment is 100-200 r / min.
7. The induction method according to claim 1, characterized in that: In step S2, the culture medium is MS culture medium containing 3-4% sucrose and 0.65-0.7% agar, and the pH value is 5.6-5.
8.
8. The induction method according to claim 1, characterized in that: In step S2, the illumination intensity is 1200-1400 lx, and the illumination time is 14-15 h / d.
9. The induction method according to claim 1, characterized in that: In step S2, the regeneration culture time is 3-5 months, the regeneration culture temperature is 22-27° C.; the acclimatization and transplanting time is 7-10 months.
10. A triploid Lycopodiella vulgaris obtained by the induction method according to any one of claims 1 to 9.