Isatis indigotica flower tissue culture chromosome doubling method

Abstract

The present application relates to the technical field of crops, and is a kind of isatis tinctoria flower anther culture chromosome doubling method, the isatis tinctoria flower anther culture chromosome doubling method, including screening, pretreatment, anther induction callus, induction callus differentiation adventitious bud, induce adventitious bud to produce regenerated plant, induce chromosome doubling and transplanting planting.The present application generates haploid callus and haploid regenerated plant by a series of processing steps using isatis tinctoria flower bud culture in the mononuclear period of isatis tinctoria flower development period, then, doubling regenerated plant is obtained by chromosome doubling, pure line is obtained, the success rate of chromosome doubling is high, which provides pure line parent for isatis tinctoria hybrid breeding, shortens the growth cycle, improves isatis tinctoria breeding efficiency, has strong practicability and economy.

Description

Technical Field

[0001] This invention relates to the field of haploid breeding technology, specifically a method for chromosome doubling in the culture of Isatis indigotica anthers. Background Technology

[0002] Isatis tinctoria L. is a biennial herb belonging to the genus Isatis in the family Brassicaceae. It is an important medicinal plant. Its root is used as medicine as Banlangen (Isatis root), and its leaves are used as Daqingye (Isatis leaf). It has the effects of clearing heat and detoxifying, cooling blood and eliminating spots, and relieving sore throat and pain. In particular, Banlangen has good antiviral effects and has played a good role in controlling the spread of the novel coronavirus.

[0003] Isatis indigotica is cultivated in Xinjiang, Gansu, Shanxi, Henan, Anhui and other places. In recent years, the germplasm has degenerated. In the long-term cultivation in old production areas, the crop rotation cycle has been relatively short. Diseases such as root rot, downy mildew and sclerotinia rot have gradually become more serious, resulting in a decline in both yield and quality.

[0004] Haploid breeding is a plant breeding technique that uses haploid plants induced through haploid induction to double their chromosomes, resulting in pure-line plants. This method significantly shortens the breeding cycle and produces offspring that do not segregate and exhibit uniform phenotypes. The principle of haploid breeding is to use plant tissue culture techniques (such as anther culture) to induce haploid plants, and then use some means (such as colchicine treatment) to double the chromosome set, thereby restoring the plant to a normal chromosome number.

[0005] Compared with traditional breeding methods, haploid breeding can significantly shorten the breeding time. The offspring of haploid breeding do not separate and have uniform phenotypes, which is conducive to the cultivation of superior varieties. It is not only suitable for breeding self-incompatible plants, but can also be applied to rapid propagation, mutation breeding and transgenic breeding.

[0006] Since Kaul and Zutshi successfully obtained homozygous diploids by treating haploids of buckwheat, barley, and rye with colchicine in 1971, haploid doubling technology has been extensively studied in other families and genera. Currently, the most commonly used chromosome doubling method in agricultural breeding is the colchicine doubling method. Colchicine is a quinolone alkaloid extracted from the bulbs and seeds of the autumn crocus (Colchella asiatica), a member of the lily family, and it can induce mutagenesis in seeds, buds, flower buds, pollen, and tender branches.

[0007] Colchicine can inhibit the formation of the spindle during cell division, preventing chromosomes from reaching the poles and stopping them in metaphase, thus preventing further cell division and producing a nucleus with doubled chromosomes. Plants where colchicine haploid doubling technology is relatively well-established include wheat, barley, corn, tobacco, sugar beets, rice, pears, and rapeseed.

[0008] Currently, there are no reports of breeding woad plants using chromosome doubling technology. Summary of the Invention

[0009] This invention provides a method for chromosome doubling in woad anther culture, overcoming the shortcomings of the prior art. It can obtain doubled regenerated plants through chromosome doubling, resulting in pure lines. The chromosome doubling success rate is high, providing pure parent lines for woad hybridization breeding.

[0010] The technical solution of this invention is achieved through the following measures: a method for chromosome doubling in Isatis indigotica anther culture, performed as follows: S1, Screening: Screening for woad flower buds that are in the uninucleate marginal stage during the development of woad anthers; S2, Pretreatment: The buds of Isatis indigotica in the uninucleate marginal stage of anther development were subjected to low-temperature pretreatment; S3, Anther-induced callus: After sterilization, rinsing, disinfection and rerinsing of the woad flower buds that have undergone low-temperature pretreatment, the woad anthers are inoculated into the basal culture medium for culture to obtain callus tissue; S4, Inducing callus tissue to differentiate into adventitious shoots: The callus tissue was transferred to a differentiation medium for culture to obtain callus tissue that sheds adventitious shoots; S5, Inducing adventitious buds to generate regenerated plants: The callus tissue from which adventitious buds have emerged is transferred to a regeneration medium for culture to obtain regenerated plants. S6, Induced Chromosome Doubling: The regenerated plants were transferred to chromosome doubling medium for culture to obtain doubled regenerated plants.

[0011] The following are further optimizations and / or improvements to the above-mentioned technical solution: In step S1 above, after staining the anthers of Isatis indigotica, the developmental stage of the anthers is observed under an optical microscope, and the buds of Isatis indigotica that are in the uninucleate marginal stage are screened. Specifically, the staining process involves adding the staining agent (acetic carmine solution) to the anthers of Isatis indigotica and placing them in a dark environment for 5 to 10 minutes.

[0012] In step S2 above, during the low-temperature pretreatment, the treatment temperature is 0℃ to 5℃ and the treatment time is 24h to 28h.

[0013] In step S3 above, the composition of the basal culture medium is as follows: 3 g / L to 5 g / L plant gel, 0.4 mg / L to 0.6 mg / L glutamic acid and 25 g / L to 35 g / L sucrose are added to MS medium, the pH is adjusted to 5.8 to 6.0, and then 0.05 mg / L to 1.0 mg / L zeatin (ZT) and 0.05 mg / L to 1.0 mg / L indoleacetic acid (IAA) are added to MS medium.

[0014] In step S3 above, the cultivation conditions are as follows: at a humidity of 65%±2% and a temperature of 25℃±2℃, the woad anthers are first cultured under a light intensity of 100lx to 500lx for 28 to 32 days, and then transferred to a light intensity of 1800lx to 2500lx for 5 to 10 days, with a light intensity of 10 to 12 hours per day.

[0015] In step S4 above, the differentiation medium is composed as follows: 3 g / L to 5 g / L plant gel and 25 g / L to 35 g / L sucrose are added to MS medium, the pH is adjusted to 5.8 to 6.0, and then 0.05 mg / L to 1.0 mg / L zeatin (ZT) and 0.05 mg / L to 1.0 mg / L indoleacetic acid (IAA) are added to MS medium.

[0016] In step S4 above, the cultivation conditions are as follows: light intensity is 1800 lx to 2500 lx, cultivation time is 6 days to 11 days, and the daily light exposure is 10 to 12 hours.

[0017] In step S5 above, the regeneration medium is composed as follows: 3 g / L to 5 g / L plant gel and 25 g / L to 35 g / L sucrose are added to 1 / 2 MS medium, the pH is adjusted to 5.8 to 6.0, and then 0.05 mg / L to 1.0 mg / L indolebutyric acid (IBA) is added to the MS medium.

[0018] In step S5 above, the cultivation conditions are as follows: light intensity is 1800 lx to 2500 lx, cultivation time is 5 days to 10 days, and the daily light exposure is 10 to 12 hours.

[0019] In step S6 above, the composition of the chromosome doubling medium is as follows: 3 g / L to 5 g / L plant gel and 25 g / L to 35 g / L sucrose are added to MS medium, the pH is adjusted to 5.8 to 6.0, and then 40 mg / L to 90 mg / L colchicine is added to MS medium.

[0020] In step S6 above, the cultivation conditions are as follows: light intensity is 1800 lx to 2500 lx, cultivation time is 8 days to 12 days, and the daily light exposure is 10 to 12 hours.

[0021] In summary, this invention utilizes a series of processing steps to cultivate haploid callus and haploid regenerated plants from woad flower buds in the uninucleate marginal stage of anther development. Then, chromosome doubling is used to obtain doubled regenerated plants, resulting in pure lines. The chromosome doubling success rate is high, providing pure-line parents for woad hybridization breeding, shortening the growth cycle, and improving woad breeding efficiency. It has strong practicality and economic benefits. Attached Figure Description

[0022] Figure 1This is a comparison chart of the peak DNA content of the doubled regenerated plants in Example 12 of the present invention. Detailed Implementation

[0023] This invention is not limited to the following embodiments, and specific implementation methods can be determined according to the technical solutions and actual conditions of this invention. Unless otherwise specified, all chemical reagents and chemicals mentioned in this invention are well-known and commonly used chemical reagents and chemicals in the prior art; unless otherwise specified, all percentages in this invention are mass percentages; unless otherwise specified, all solutions in this invention are aqueous solutions with water as the solvent, for example, hydrochloric acid solution is an aqueous solution of hydrochloric acid; room temperature in this invention generally refers to a temperature between 15°C and 25°C, generally defined as 25°C.

[0024] The present invention will be further described below with reference to embodiments: Example 1: The method for chromosome doubling in Isatis indigotica anther culture is as follows: S1, Screening: Screening for woad flower buds that are in the uninucleate marginal stage during the development of woad anthers; S2, Pretreatment: The buds of Isatis indigotica in the uninucleate marginal stage of anther development were subjected to low-temperature pretreatment; S3, Anther-induced callus: After sterilization, rinsing, disinfection and rerinsing of the woad flower buds that have undergone low-temperature pretreatment, the woad anthers are inoculated into the basal culture medium for culture to obtain callus tissue; S4, Inducing callus tissue to differentiate into adventitious shoots: The callus tissue was transferred to a differentiation medium for culture to obtain callus tissue that sheds adventitious shoots; S5, Inducing adventitious buds to generate regenerated plants: The callus tissue from which adventitious buds have emerged is transferred to a regeneration medium for culture to obtain regenerated plants. S6, Induced Chromosome Doubling: The regenerated plants were transferred to chromosome doubling medium for culture to obtain doubled regenerated plants.

[0025] In this invention, the woad plant is a two-year-old woad (Isatis indigotica) planted in the Chinese medicinal herb planting base in Minle County, Gansu Province.

[0026] Example 2: As an optimization of the above example, in step S1, after the woad anthers are stained, the development stage of the woad anthers is observed under an optical microscope, and woad buds in the uninucleate marginal stage of woad anther development are screened. Specifically, the staining process involves adding staining agent (acetic carmine solution) to the woad anthers and placing them in a dark environment for 5 to 10 minutes.

[0027] Example 3: As an optimization of the above example, in step S2, during the low-temperature pretreatment, the treatment temperature is 0°C to 5°C and the treatment time is 24h to 28h.

[0028] Example 4: As an optimization of the above example, in step S3, the composition of the basal culture medium is as follows: 3 g / L to 5 g / L plant gel, 0.4 mg / L to 0.6 mg / L glutamic acid and 25 g / L to 35 g / L sucrose are added to the MS culture medium, the pH is adjusted to 5.8 to 6.0, and then 0.05 mg / L to 1.0 mg / L zeatin (ZT) and 0.05 mg / L to 1.0 mg / L indoleacetic acid (IAA) are added to the MS culture medium.

[0029] Example 5: As an optimization of the above example, in step S3, the cultivation conditions are as follows: at a humidity of 65%±2% and a temperature of 25℃±2℃, the woad anthers are first cultured under a light intensity of 100lx to 500lx for 28 to 32 days, and then transferred to a light intensity of 1800lx to 2500lx for 5 to 10 days, with a light intensity of 10 to 12 hours per day.

[0030] Example 6: As an optimization of the above example, in step S4, the composition of the differentiation medium is as follows: 3 g / L to 5 g / L plant gel and 25 g / L to 35 g / L sucrose are added to MS medium, the pH is adjusted to 5.8 to 6.0, and then 0.05 mg / L to 1.0 mg / L zeatin (ZT) and 0.05 mg / L to 1.0 mg / L indoleacetic acid (IAA) are added to MS medium.

[0031] Example 7: As an optimization of the above example, in step S4, the cultivation conditions are: light intensity of 1800 lx to 2500 lx, cultivation time of 6 days to 11 days, and daily light exposure of 10 to 12 hours.

[0032] Example 8: As an optimization of the above example, in step S5, the composition of the regeneration medium is as follows: 3 g / L to 5 g / L plant gel and 25 g / L to 35 g / L sucrose are added to 1 / 2 MS medium, the pH is adjusted to 5.8 to 6.0, and then 0.05 mg / L to 1.0 mg / L zeatin (ZT) and 0.05 mg / L to 1.0 mg / L indolebutyric acid (IBA) are added to the MS medium.

[0033] Example 9: As an optimization of the above example, in step S5, the cultivation conditions are: light intensity of 1800 lx to 2500 lx, cultivation time of 5 days to 10 days, and daily light exposure of 10 to 12 hours.

[0034] Example 10: As an optimization of the above example, in step S6, the composition of the chromosome doubling medium is as follows: 3 g / L to 5 g / L plant gel and 25 g / L to 35 g / L sucrose are added to MS medium, the pH is adjusted to 5.8 to 6.0, and then 40 mg / L to 90 mg / L colchicine is added to MS medium.

[0035] Example 11: As an optimization of the above example, in step S6, the cultivation conditions are: light intensity of 1800 lx to 2500 lx, cultivation time of 8 days to 12 days, and daily light exposure of 10 to 12 hours.

[0036] The beneficial effects of this invention are as follows: First, in this invention, the anthers of Isatis indigotica are pretreated. Specifically, during low-temperature pretreatment, the treatment temperature is 0°C to 5°C and the treatment time is 24h to 28h, which can better break the dormancy of the anthers and produce more embryoids. Secondly, in this invention, the anther-induced callus tissue is first induced by inducing callus tissue in the anthers of Isatis indigotica under weak light conditions of 100 lx to 500 lx. After the callus tissue is generated, it is cultured under strong light conditions of 1800 to 2500 lx. This treatment step is conducive to the dedifferentiation of microspores, which in turn enables the callus tissue to differentiate into adventitious buds, which in turn differentiate into adventitious roots (regenerated plants) and promote the growth of Isatis indigotica plants. Third, in this invention, both the differentiation medium and the regeneration medium contain zeatin (ZT) and indoleacetic acid (IAA). The differentiation medium can effectively promote the production of adventitious shoots from callus tissue, while the regeneration medium is mainly for the production of adventitious roots (regenerated plants). The chromosome doubling medium used utilizes colchicine to stimulate chromosome doubling, which can produce doubled regenerated plants, i.e., diploid regenerated plants.

[0037] In summary, this invention utilizes a series of processing steps to cultivate haploid callus and haploid regenerated plants from woad flower buds at the uninucleate marginal stage of anther development. Then, chromosome doubling is used to obtain doubled regenerated plants, resulting in pure lines with a high chromosome doubling success rate. This provides pure-line parents for woad hybridization breeding, shortens the growth cycle, and improves woad breeding efficiency, demonstrating strong practicality and economic viability. Furthermore, it features low production costs, readily available culture media, easily controllable culture environment, rapid multi-generation propagation, and easy expression of recessive genes.

[0038] Example 12: The method for chromosome doubling in the culture of Isatis indigotica anthers is as follows: S1, Screening: Collect woad flower buds of different sizes from healthy, disease-free plants at 7:00 AM on a sunny day. Place the woad anthers on a glass slide, crush them with tweezers, add 2 drops of acetic acid-carmine solution, and let them stand in the dark for 8 minutes. After staining, observe the development stage of the microspores of the woad anthers under an optical microscope and screen woad flower buds in which most of the microspores of the woad anthers are in the uninucleate marginal stage. S2, Pretreatment: Place the buds of Isatis indicus indicus at the uninucleate marginal stage of anther development in a 4℃ refrigerator for 24 hours. Spray water on the buds during the treatment period to prevent them from drying out. S3, Anther-induced callus: After low-temperature pretreatment, the florets of *Isatis tinctoria* were sterilized with 75% alcohol for 30 seconds, rinsed three times with sterile water, and then soaked in 2% sodium hypochlorite solution for 20 minutes. After rinsing five times with sterile water, the florets were opened with tweezers, and the anthers were inoculated into a basal culture medium for culture to obtain callus. The composition of the basal culture medium was as follows: 3 g / L of plant extract was added to MS medium. The medium was prepared with a gel, 0.5 mg / L glutamic acid, and 30 g / L sucrose. The pH was adjusted to 5.8. Then, 0.5 mg / L zeatin (ZT) and 1.0 mg / L indoleacetic acid (IAA) were added to the MS medium. The culture conditions were as follows: the anthers of Isatis indigotica were first cultured at 200 lx for 30 days at a humidity of 65% ± 2% and a temperature of 25℃ ± 2℃. Then, they were cultured at 1800 lx for 5 to 10 days, with a light intensity of 10 h per day. S4, Induction of adventitious shoot differentiation from callus: Callus tissue was transferred to differentiation medium for culture to obtain callus tissue that spawned adventitious shoots; The composition of the differentiation medium was as follows: 3 g / L plant gel and 30 g / L sucrose were added to MS medium, the pH was adjusted to 5.8, and 0.5 mg / L zeatin (ZT) and 0.1 mg / L indoleacetic acid (IAA) were added to MS medium; The culture conditions were: light intensity of 1800 lx, culture time of 6 to 11 days, and 10 h of light per day; S5, Induction of adventitious buds to generate regenerated plants: Callus tissue from which adventitious buds have emerged was transferred to regeneration medium for culture to obtain regenerated plants; The composition of the regeneration medium: 3 g / L plant gel and 30 g / L sucrose were added to 1 / 2 MS medium, the pH was adjusted to 5.8, and 0.5 mg / L indolebutyric acid (IBA) was added to the MS medium; Culture conditions: light intensity of 1800 lx to 2500 lx, culture time of 5 to 10 days, and 10 h of light per day; S6, Inducing Chromosome Doubling: Regenerated plants were transferred to chromosome doubling medium for culture to obtain doubled regenerated plants. The regenerated plants exhibited organ enlargement, showing characteristics of successful doubling. The composition of the chromosome doubling medium was as follows: 3 g / L plant gel and 30 g / L sucrose were added to MS medium, the pH was adjusted to 5.8, and then 70 mg / L colchicine was added to MS medium. Culture conditions: light intensity of 1800 lx, culture time of 8 to 12 days, and 10 h of light per day. S7, ploidy identification of doubled regenerated plants: ploidy of doubled regenerated plants was determined using flow cytometry. The criteria were based on comparing the peak DNA content of the flow cytometer with that of local traditional farm variety diploid control plants and pollen male gametes haploids. If the peak DNA content was the same as that of the traditional farm variety diploid control plants, the plants were considered diploid; if the peak DNA content was the same as that of the pollen male gametes haploids, the plants were considered haploid. S8, Transplanting: Transplant the successfully ploidy-identified regenerated plants. Specifically, when the regenerated plants have 4 to 6 leaves and roots 3 to 5 cm long, open the culture bottle to harden them off, allowing them to gradually adapt to the external environment. Remove the plants from the culture bottle, clean the roots of any remaining culture medium, and transplant them into nutrient pots. Place them at a room temperature of 21℃ to 25℃ for cultivation. Transplant regenerated plants from the same single anther culture into the same plot. When flowering, cover the plot with insect-proof netting to prevent insect pollination. During peak flowering, after most flowers have opened, cut off any unopened flower spikes. Remove the insect-proof netting 3 days later when all flowers have withered. Once the seeds mature, harvest them individually to propagate homozygous lines.

[0039] ploidy of the regenerated Isatis indigotica plants obtained in step S7 was determined using CyFlow Space flow cytometry. Diploid control plants of a local traditional farm variety were used as a control. The ploidy of the regenerated plants was determined based on the peak DNA content observed during flow cytometry. Results are attached. Figure 1 As shown, Figure 1 The horizontal axis represents DNA content, and the vertical axis represents cell number. A represents haploid pollen male gametes, B represents diploid control plants of local traditional farm varieties, and C represents doubled regenerated plants. Figure 1 It can be seen that the sample peak of the pollen male gamete haploid appears at the horizontal axis value of 50, the sample peak of the local traditional farm variety diploid control plant appears at the horizontal axis value of 100, and the sample peak of the doubled regenerated plant appears at the horizontal axis value of 100. This indicates that the ploidy of the doubled regenerated plant obtained in Example 12 of the present invention is the same as that of the local traditional farm variety diploid control plant, that is, it is a diploid plant.

[0040] In Example 12 of this invention, 10 groups of woad flower buds in the uninucleate marginal stage of the anther development of the same single woad plant were screened. Following the method of Example 12 of this invention, the ploidy of the obtained doubled regenerated plants was finally determined by flow cytometry. The peak DNA content of 9 groups of doubled regenerated plants was consistent with that of the diploid control plants of local traditional farm varieties, and the peak DNA content of 1 group of doubled regenerated plants was consistent with that of the haploid male pollen gametes. The chromosome doubling success rate was 90%.

[0041] Example 13: The method for chromosome doubling in the culture of Isatis indigotica anthers is as follows: S1, Screening: Collect woad flower buds of different sizes from healthy, disease-free plants at 7:00 AM on a sunny day. Place the woad anthers on a glass slide, crush them with tweezers, add 1 drop of acetic carmine solution, and let them stand in the dark for 5 minutes. After staining, observe the development stage of the microspores of the woad anthers under an optical microscope and screen woad flower buds in which most of the microspores of the woad anthers are in the uninucleate marginal stage. S2, Pretreatment: Place the buds of Isatis indicus indicus at the uninucleate marginal stage of anther development in a 0℃ refrigerator for 24 hours. Spray water on the buds during the treatment period to prevent water loss and drying. S3, Anther-induced callus: After low-temperature pretreatment, the florets of *Isatis tinctoria* were sterilized with 75% alcohol for 30 seconds, rinsed three times with sterile water, and then soaked in 2% sodium hypochlorite solution for 15 minutes. After rinsing four times with sterile water, the florets were opened with forceps, and the anthers were inoculated into a basal culture medium for culture to obtain callus. The composition of the basal culture medium was as follows: 3 g / L of plant extract was added to MS medium. The medium was prepared with a gel, 0.5 mg / L glutamic acid, and 25 g / L sucrose. The pH was adjusted to 5.8. Then, 0.5 mg / L zeatin (ZT) and 1.0 mg / L indoleacetic acid (IAA) were added to the MS medium. The culture conditions were as follows: the anthers of Isatis indigotica were first cultured at 500 lx for 28 days at a humidity of 65% ± 2% and a temperature of 25℃ ± 2℃. Then, they were cultured at 2500 lx for 5 to 10 days, with a light intensity of 10 h per day. S4, Induction of adventitious shoot differentiation from callus: Callus tissue was transferred to differentiation medium for culture to obtain callus tissue that spawned adventitious shoots; The composition of the differentiation medium was as follows: 3 g / L plant gel and 25 g / L sucrose were added to MS medium, the pH was adjusted to 5.8, and then 0.5 mg / L zeatin (ZT) and 0.1 mg / L indoleacetic acid (IAA) were added to MS medium; The culture conditions were: light intensity of 2500 lx, culture time of 6 to 11 days, and 10 h of light per day; S5, Induction of adventitious buds to generate regenerated plants: Callus tissue from which adventitious buds have emerged was transferred to regeneration medium for culture to obtain regenerated plants; The composition of the regeneration medium: 3 g / L plant gel and 25 g / L sucrose were added to 1 / 2 MS medium, the pH was adjusted to 5.8, and 0.5 mg / L indolebutyric acid (IBA) was added to the MS medium; Culture conditions: light intensity of 2500 lx, culture time of 5 to 10 days, and 10 h of light per day; S6, Inducing Chromosome Doubling: Regenerated plants were transferred to chromosome doubling medium for culture to obtain doubled regenerated plants. The regenerated plants exhibited organ enlargement, showing characteristics of successful doubling. The composition of the chromosome doubling medium was as follows: 3 g / L plant gel and 25 g / L sucrose were added to MS medium, the pH was adjusted to 5.8, and then 80 mg / L colchicine was added to MS medium. Culture conditions: light intensity of 2500 lx, culture time of 8 to 12 days, and 10 h of light per day. S7, ploidy identification of doubled regenerated plants: ploidy of doubled regenerated plants was determined using flow cytometry. The criteria were based on comparing the peak DNA content of the flow cytometer with that of local traditional farm variety diploid control plants and pollen male gametes haploids. If the peak DNA content was the same as that of the traditional farm variety diploid control plants, the plants were considered diploid; if the peak DNA content was the same as that of the pollen male gametes haploids, the plants were considered haploid. S8, Transplanting: Transplant the successfully ploidy-identified regenerated plants. Specifically, when the regenerated plants have 4 to 6 leaves and roots 3 to 5 cm long, open the culture bottle to harden them off, allowing them to gradually adapt to the external environment. Remove the plants from the culture bottle, clean the roots of any remaining culture medium, and transplant them into nutrient pots. Place them at a room temperature of 21℃ to 25℃ for cultivation. Transplant regenerated plants from the same single anther culture into the same plot. When flowering, cover the plot with insect-proof netting to prevent insect pollination. During peak flowering, after most flowers have opened, cut off any unopened flower spikes. Remove the insect-proof netting 3 days later when all flowers have withered. Once the seeds mature, harvest them individually to propagate homozygous lines.

[0042] In Example 13 of this invention, 10 groups of woad flower buds in the uninucleate marginal stage of the same single plant anther were screened. Following the method of Example 13 of this invention, the ploidy of the obtained doubled regenerated plants was finally determined by flow cytometry. The peak DNA content of 9 groups of doubled regenerated plants was consistent with that of the diploid control plants of local traditional farm varieties, and the chromosome doubling success rate was 90%.

[0043] Example 14: The method for chromosome doubling in the culture of Isatis indigotica anthers is as follows: S1, Screening: Collect woad flower buds of different sizes from healthy, disease-free plants at 7:00 AM on a sunny day. Place the woad anthers on a glass slide, crush them with tweezers, add 2 drops of acetic carmine solution, and let them stand in the dark for 10 minutes. After staining, observe the development stage of the microspores of the woad anthers under an optical microscope and screen woad flower buds in which most of the microspores of the woad anthers are in the uninucleate marginal stage. S2, Pretreatment: Place the buds of Isatis indicus indicus at the uninucleate marginal stage of anther development in a 5℃ refrigerator for 28 hours. Spray water on the buds during the treatment period to prevent water loss and drying. S3, Anther-induced callus: After low-temperature pretreatment, the florets of *Isatis tinctoria* were sterilized with 75% alcohol for 30 seconds, rinsed three times with sterile water, and then soaked in 2% sodium hypochlorite solution for 20 minutes. After rinsing five times with sterile water, the florets were opened with tweezers, and the anthers were inoculated into basal culture medium for culture to obtain callus. The composition of the basal culture medium was as follows: 5 g / L of plant extract was added to MS medium. The medium was prepared by adding 0.6 mg / L glutamic acid and 35 g / L sucrose to a gel, adjusting the pH to 6.0, and then adding 0.5 mg / L zeatin (ZT) and 1.0 mg / L indoleacetic acid (IAA) to the MS medium. The culture conditions were as follows: at a humidity of 65% ± 2% and a temperature of 25℃ ± 2℃, the anthers of Isatis indigotica were first cultured at 100 lx for 32 days, and then transferred to 2000 lx for 5 to 10 days, with a light intensity of 10 h per day. S4, Induction of adventitious shoot differentiation from callus: Callus tissue was transferred to differentiation medium for culture to obtain callus tissue that spawned adventitious shoots; The composition of the differentiation medium was as follows: 5 g / L plant gel and 35 g / L sucrose were added to MS medium, the pH was adjusted to 6.0, and then 0.5 mg / L zeatin (ZT) and 0.1 mg / L indoleacetic acid (IAA) were added to MS medium; The culture conditions were: light intensity of 2000 lx, culture time of 6 to 11 days, and 10 h of light per day; S5, Induction of adventitious buds to generate regenerated plants: Callus tissue from which adventitious buds have emerged was transferred to regeneration medium for culture to obtain regenerated plants; The composition of the regeneration medium: 5 g / L plant gel and 35 g / L sucrose were added to 1 / 2 MS medium, the pH was adjusted to 6.0, and 0.5 mg / L indolebutyric acid (IBA) was added to the MS medium; Culture conditions: light intensity of 2000 lx, culture time of 5 to 10 days, and 10 h of light per day; S6, Inducing Chromosome Doubling: Regenerated plants were transferred to chromosome doubling medium for culture to obtain doubled regenerated plants. The regenerated plants exhibited organ enlargement, showing characteristics of successful doubling. The composition of the chromosome doubling medium was as follows: 5 g / L plant gel and 35 g / L sucrose were added to MS medium, the pH was adjusted to 6.0, and then 60 mg / L colchicine was added to MS medium. Culture conditions: light intensity of 2000 lx, culture time of 8 to 12 days, and 10 h of light per day. S7, ploidy identification of doubled regenerated plants: ploidy of doubled regenerated plants was determined using flow cytometry. The criteria were based on comparing the peak DNA content of the flow cytometer with that of local traditional farm variety diploid control plants and pollen male gametes haploids. If the peak DNA content was the same as that of the traditional farm variety diploid control plants, the plants were considered diploid; if the peak DNA content was the same as that of the pollen male gametes haploids, the plants were considered haploid. S8, Transplanting: Transplant the successfully ploidy-identified regenerated plants. Specifically, when the regenerated plants have 4 to 6 leaves and roots 3 to 5 cm long, open the culture bottle to harden them off, allowing them to gradually adapt to the external environment. Remove the plants from the culture bottle, clean the roots of any remaining culture medium, and transplant them into nutrient pots. Place them at a room temperature of 21℃ to 25℃ for cultivation. Transplant regenerated plants from the same single anther culture into the same plot. When flowering, cover the plot with insect-proof netting to prevent insect pollination. During peak flowering, after most flowers have opened, cut off any unopened flower spikes. Remove the insect-proof netting 3 days later when all flowers have withered. Once the seeds mature, harvest them individually to propagate homozygous lines.

[0044] In Example 14 of this invention, 10 groups of woad flower buds in the uninucleate marginal stage of the anther development of the same single woad plant were screened. Following the method of Example 14 of this invention, the ploidy of the obtained doubled regenerated plants was finally determined by flow cytometry. The peak DNA content of 8 groups of doubled regenerated plants was consistent with that of the diploid control plants of local traditional farm varieties, and the peak DNA content of 2 groups of doubled regenerated plants was consistent with that of the haploid male pollen gametes. The chromosome doubling success rate was 80%.

[0045] Comparative example: The difference from Example 12 is that the phrase "Composition of chromosome doubling medium: Add 3 g / L plant gel and 30 g / L sucrose to MS medium, adjust the pH to 5.8, and then add 70 mg / L colchicine to MS medium" is modified to "Composition of chromosome doubling medium: Add 3 g / L plant gel and 30 g / L sucrose to MS medium, adjust the pH to 5.8, and then add 30 mg / L colchicine to MS medium", while the rest of the steps are the same.

[0046] In the comparative examples of this invention, 10 groups of woad flower buds in the uninucleate marginal stage of the anther development of the same single woad plant were screened. Following the method of Comparative Example 1 of this invention, the ploidy of the obtained doubled regenerated plants was determined by flow cytometry. The peak DNA content of 3 groups of doubled regenerated plants was consistent with that of the diploid control plants of local traditional farm varieties, and the peak DNA content of 7 groups of doubled regenerated plants was consistent with that of the haploid male gametes of pollen. The chromosome doubling success rate was 30%.

[0047] Compared with the comparative examples, the chromosome doubling method of Isatis indigotica anther culture according to Examples 12 to 14 of the present invention has confirmed that the doubled regenerated plants are diploid, and the doubling success rate is higher than 80%.

[0048] In summary, this invention utilizes a series of processing steps to cultivate haploid callus and haploid regenerated plants from woad flower buds in the uninucleate marginal stage of anther development. Then, chromosome doubling is used to obtain doubled regenerated plants, resulting in pure lines. The chromosome doubling success rate is high, providing pure-line parents for woad hybridization breeding, shortening the growth cycle, and improving woad breeding efficiency. It has strong practicality and economic benefits.

[0049] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Unnecessary technical features can be added or removed according to actual needs to meet the needs of different situations.

Claims

1. A method for chromosome doubling in Isatis indigotica anther culture, characterized in that... Perform the following steps: S1, Screening: Screening for woad flower buds that are in the uninucleate marginal stage during the development of woad anthers; S2, Pretreatment: The buds of Isatis indigotica in the uninucleate marginal stage of anther development were subjected to low-temperature pretreatment; S3, Anther-induced callus: After sterilization, rinsing, disinfection and rerinsing of the woad flower buds that have undergone low-temperature pretreatment, the woad anthers are inoculated into the basal culture medium for culture to obtain callus. The culture conditions are as follows: at a humidity of 65%±2% and a temperature of 25℃±2℃, the woad anthers are first cultured under 100lx to 500lx light for 28 to 32 days, and then transferred to 1800lx to 2500lx light for 5 to 10 days, with a light intensity of 10 to 12 hours per day. S4, Inducing callus tissue to differentiate into adventitious shoots: The callus tissue was transferred to a differentiation medium for culture to obtain callus tissue that sheds adventitious shoots; S5, Inducing adventitious buds to generate regenerated plants: The callus tissue from which adventitious buds have emerged is transferred to a regeneration medium for culture to obtain regenerated plants. S6, Inducing Chromosome Doubling: The regenerated plants were transferred to chromosome doubling medium for culture to obtain doubled regenerated plants. The chromosome doubling medium consisted of MS medium containing 3 g / L to 5 g / L plant gel and 25 g / L to 35 g / L sucrose, with the pH adjusted to 5.8 to 6.

0. Then, 40 mg / L to 90 mg / L colchicine was added to the MS medium. S7, ploidy identification of doubled regenerated plants: ploidy of doubled regenerated plants was determined using flow cytometry; S8, Transplanting: Transplanting the doubled regenerated plants that have been successfully identified as ploidy.

2. The method for chromosome doubling in Isatis indigotica anther culture according to claim 1, characterized in that... In step S1, after staining the anthers of Isatis indigotica, the developmental stage of the anthers is observed under an optical microscope, and the buds of Isatis indigotica that are in the uninucleate marginal stage are selected. Specifically, the staining process involves adding staining agent to the anthers of Isatis indigotica and placing them in a dark environment for 5 to 10 minutes.

3. The method for chromosome doubling in Isatis indigotica anther culture according to claim 1 or 2, characterized in that... In step S2, during the low-temperature pretreatment, the treatment temperature is 0℃ to 5℃ and the treatment time is 24h to 28h.

4. The method for chromosome doubling in Isatis indigotica anther culture according to claim 3, characterized in that... In step S3, the composition of the basal culture medium is as follows: 3 g / L to 5 g / L plant gel, 0.4 mg / L to 0.6 mg / L glutamic acid and 25 g / L to 35 g / L sucrose are added to MS medium, the pH is adjusted to 5.8 to 6.0, and then 0.05 mg / L to 1.0 mg / L zeatin and 0.05 mg / L to 1.0 mg / L indoleacetic acid are added to MS medium.

5. The method for chromosome doubling in Isatis indigotica anther culture according to any one of claims 1, 2 and 4, characterized in that... In step S4, the differentiation medium is composed as follows: 3 g / L to 5 g / L plant gel and 25 g / L to 35 g / L sucrose are added to MS medium, the pH is adjusted to 5.8 to 6.0, and then 0.05 mg / L to 1.0 mg / L zeatin and 0.05 mg / L to 1.0 mg / L indoleacetic acid are added to MS medium.

6. The method for chromosome doubling in Isatis indigotica anther culture according to claim 5, characterized in that... In step S4, the cultivation conditions are as follows: light intensity is 1800 lx to 2500 lx, cultivation time is 6 days to 11 days, and the daily light exposure is 10 to 12 hours.

7. The method for chromosome doubling in Isatis indigotica anther culture according to any one of claims 1, 2, 4 and 6, characterized in that... In step S5, the composition of the regeneration medium is as follows: Add 3 g / L to 5 g / L plant gel and 25 g / L to 35 g / L sucrose to 1 / 2 MS medium, adjust the pH to 5.8 to 6.0, and then add 0.05 mg / L to 1.0 mg / L indolebutyric acid to the MS medium.

8. The method for chromosome doubling in Isatis indigotica anther culture according to claim 7, characterized in that... In step S5, the cultivation conditions are as follows: light intensity is 1800 lx to 2500 lx, cultivation time is 5 days to 10 days, and the daily light exposure is 10 to 12 hours.

9. The method for chromosome doubling in Isatis indigotica anther culture according to any one of claims 1, 2, 4, 6 and 8, characterized in that... In step S6, the cultivation conditions are as follows: light intensity is 1800 lx to 2500 lx, cultivation time is 8 days to 12 days, and the daily light exposure is 10 to 12 hours.

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