Isatis tinctoria anther culture chromosome doubling method

Abstract

The invention relates to the technical field of crops, in particular to a isatis tinctoria anther culture chromosome doubling method which comprises the steps of screening, pretreatment, callus induction by anther, adventitious bud differentiation induction by the callus, regeneration plant generation by the adventitious bud induction, chromosome doubling induction and transplanting. Through a series of treatment steps, the isatis tinctoria buds in the anther development period of isatis tinctoria are used for culturing to generate haploid calluses and haploid regenerated plants, then the doubled regenerated plants are obtained through chromosome doubling, the pure line is obtained, the success rate of chromosome doubling is high, pure line parents are provided for isatis tinctoria cross breeding, and the method is suitable for industrial production. The growth cycle is shortened, the isatis tinctoria breeding efficiency is improved, and high practicability and economical efficiency are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of haploid breeding, and is a chromosome doubling method for anther culture of Isatis indigofera. Background Art

[0002] Isatis tinctoria L. is a biennial herb of the genus Isatis in the family Cruciferae. It is an important medicinal plant. Its root is used as Radix Isatidis and its leaves are used as Folium Isatidis. They have the effects of clearing away heat and detoxifying, cooling blood and removing spots, and relieving sore throat and pain. In particular, Radix Isatis has good antiviral effects and has played a good role in controlling the epidemic of the new coronavirus.

[0003] Isatis indigotica is cultivated in Xinjiang, Gansu, Shanxi, Henan, Anhui and other places. In recent years, the germplasm has degraded. The rotation cycle is relatively short during the long-term cultivation in old production areas. Diseases such as isatis indigotica root rot, downy mildew, and sclerotinia have gradually become more serious, causing both yield and quality to show a downward trend.

[0004] Haploid breeding is a method of plant breeding. Haploid plants obtained through haploid induction can be used to obtain pure line plants after chromosome doubling. This method can significantly shorten the breeding period, and the offspring obtained will not separate and have uniform phenotypes. The principle of haploid breeding is to use plant tissue culture technology (such as anther in vitro culture) to induce haploid plants, and then double the chromosome set through some means (such as colchicine treatment), so that the plant can restore the 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 the phenotype is uniform, which is conducive to the cultivation of excellent varieties. It is not only suitable for the breeding of self-incompatible plants, but also can be applied to rapid reproduction, mutation breeding and transgenic breeding.

[0006] Since Kaul and Zutshi used colchicine to treat wheat, barley and rye haploids in 1971 and successfully obtained homozygous diploids, haploid doubling technology has also been widely studied in other families. At present, the most commonly used chromosome doubling method in agricultural breeding is the colchicine doubling method. Colchicine is a tropolone alkaloid extracted from the bulbs and seeds of Colchicum of the Liliaceae family, which can produce mutagenic effects on seeds, young shoots, flower buds, pollen and young branches.

[0007] Colchicine can inhibit the formation of spindles during cell division, so that chromosomes do not move to the two poles, but are blocked in the metaphase, causing the cell to be unable to continue dividing, thus producing a nucleus with doubled chromosomes. The plant species that have been more mature in the application of colchicine haploid doubling technology mainly include wheat, barley, corn, tobacco, sugar beet, rice, pear, rapeseed, etc.

[0008] At present, there are no reports on the breeding of Isatis indigotica plants using chromosome doubling technology. Summary of the invention

[0009] The invention provides a chromosome doubling method for anther culture of Isatis indigotica, which overcomes the shortcomings of the prior art. The method can obtain doubled regenerated plants and pure lines through chromosome doubling, and has a high chromosome doubling success rate, thereby providing pure line parents for Isatis indigotica hybrid breeding.

[0010] The technical solution of the present invention is achieved by the following measures: A method for chromosome doubling in anther culture of Isatis indigofera is carried out according to the following method: S1, screening: screening the buds of Isatis indigofera that are in the mononuclear marginal stage during the anther development period; S2, pretreatment: subjecting the buds of Isatis indigofera indigofera in the mononuclear marginal stage during the anther development period to low temperature pretreatment; S3, inducing callus tissue from anthers: sterilizing, washing, disinfecting and re-washing the buds of Isatis indigo plant that have been pretreated at low temperature, and then inoculating the anthers of Isatis indigo plant into a basic culture medium for culture to obtain callus tissue; S4, inducing callus to differentiate into adventitious buds: transferring the callus to a differentiation medium for culture to obtain callus that has differentiated into adventitious buds; S5, inducing adventitious buds to produce regenerated plants: transferring the callus tissue that has produced adventitious buds to a regeneration medium for culture to obtain regenerated plants; S6, inducing chromosome doubling: transferring the regenerated plants to a chromosome doubling medium for culture to obtain doubled regenerated plants.

[0011] The following are further optimizations and / or improvements to the above technical solutions: In the above step S1, after dyeing the anthers of woad, the development period of the anthers is observed under an optical microscope, and the woad flower buds in the mononuclear marginal stage of the anther development period are selected, wherein the dyeing process specifically includes adding a dye (acetic acid magenta solution) to the anthers of woad and placing them in a dark environment for 5 to 10 minutes.

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

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

[0014] In the above step S3, the culture conditions are: at a humidity of 65%±2% and a temperature of 25℃±2℃, the anthers of Isatis tinctoria are first cultured under 100lx to 500lx light for 28d to 32d, and then transferred to 1800lx to 2500lx light for 5d to 10d, with light culture for 10h to 12h every day.

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

[0016] In the above step S4, the culture conditions are: light intensity is 1800lx to 2500lx, culture time is 6d to 11d, and light culture is performed for 10h to 12h every day.

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

[0018] In the above step S5, the culture conditions are: the light intensity is 1800lx to 2500lx, the culture time is 5d to 10d, and the light culture is 10h to 12h per day.

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

[0020] In the above step S6, the culture conditions are: light intensity is 1800lx to 2500lx, culture time is 8d to 12d, and light culture is performed for 10h to 12h every day.

[0021] In summary, the present invention uses a series of processing steps to culture the buds of Isatis in the mononuclear marginal stage during the anther development period of Isatis to produce haploid callus tissue and haploid regenerated plants, and then obtains the doubly regenerated plants through chromosome doubling to obtain pure lines. The chromosome doubling success rate is high, and pure line parents are provided for Isatis hybrid breeding, the growth cycle is shortened, and the breeding efficiency of Isatis is improved. The method has strong practicality and economy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1This is a comparison chart of the DNA content peak values ​​of the doubly regenerated plants in Example 12 of the present invention. DETAILED DESCRIPTION

[0023] The present invention is not limited by the following embodiments, and the specific implementation method can be determined according to the technical scheme of the present invention and the actual situation. Unless otherwise specified, the various chemical reagents and chemicals mentioned in the present invention are all chemical reagents and chemicals known and used in the prior art; unless otherwise specified, the percentages in the present invention are all mass percentages; unless otherwise specified, the solutions in the present invention are all aqueous solutions with water as the solvent, for example, hydrochloric acid solution is a hydrochloric acid aqueous solution; the normal temperature and room temperature in the present invention generally refer to a temperature of 15°C to 25°C, and are generally defined as 25°C.

[0024] The present invention will be further described below in conjunction with embodiments: Example 1: The chromosome doubling method of Isatis indigofera anther culture is carried out as follows: S1, screening: screening the buds of Isatis indigofera that are in the mononuclear marginal stage during the anther development period; S2, pretreatment: subjecting the buds of Isatis indigofera indigofera in the mononuclear marginal stage during the anther development period to low temperature pretreatment; S3, inducing callus tissue from anthers: sterilizing, washing, disinfecting and re-washing the buds of Isatis indigo plant that have been pretreated at low temperature, and then inoculating the anthers of Isatis indigo plant into a basic culture medium for culture to obtain callus tissue; S4, inducing callus to differentiate into adventitious buds: transferring the callus to a differentiation medium for culture to obtain callus that has differentiated into adventitious buds; S5, inducing adventitious buds to produce regenerated plants: transferring the callus tissue that has produced adventitious buds to a regeneration medium for culture to obtain regenerated plants; S6, inducing chromosome doubling: transferring the regenerated plants to a chromosome doubling medium for culture to obtain doubled regenerated plants.

[0025] In the present invention, the Isatis indigotica plant is a 2-year-old Isatis indigotica planted in a traditional Chinese medicine planting base in Minle County, Gansu Province.

[0026] Example 2: As an optimization of the above example, in step S1, after staining, the anthers of woad are taken, and the development period of the anthers is observed under an optical microscope, and the woad flower buds in the mononuclear marginal stage of the anther development period are screened, wherein the staining process is specifically to add a dye (acetic acid magenta solution) to the anthers of woad and place them in a dark environment for 5 to 10 minutes.

[0027] Embodiment 3: As an optimization of the above embodiment, 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: 3g / L to 5g / L of plant gel, 0.4mg / L to 0.6mg / L of glutamic acid and 25g / L to 35g / L of sucrose are added to the MS culture medium, the pH value is adjusted to 5.8 to 6.0, and then 0.05mg / L to 1.0mg / L of zeatin (ZT) and 0.05mg / L to 1.0mg / L of indoleacetic acid (IAA) are added to the MS culture medium.

[0029] Example 5: As an optimization of the above example, in step S3, the culture conditions are: at a humidity of 65%±2% and a temperature of 25℃±2℃, the Isatis anthers are first cultured under 100lx to 500lx light for 28d to 32d, and then transferred to 1800lx to 2500lx light for 5d to 10d, with light culture for 10h to 12h every day.

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

[0031] Example 7: As an optimization of the above example, in step S4, the culture conditions are: light intensity is 1800lx to 2500lx, culture time is 6d to 11d, and light culture is performed for 10h to 12h every day.

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

[0033] Example 9: As an optimization of the above example, in step S5, the culture conditions are: light intensity is 1800lx to 2500lx, culture time is 5d to 10d, and light culture is performed for 10h to 12h every day.

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

[0035] Example 11: As an optimization of the above example, in step S6, the culture conditions are: light intensity is 1800lx to 2500lx, culture time is 8d to 12d, and light culture is performed for 10h to 12h every day.

[0036] The beneficial effects of the present invention are: First, in the present invention, the anthers of Isatis indigotica are pretreated, and specifically, during the 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 too many embryoids; Second, in the present invention, the anthers are induced to induce callus tissue, firstly, the anthers of Isatis indigotica are cultured under weak light conditions of 100lx to 500lx to induce callus tissue, and after the callus tissue is produced, it is cultured under strong light conditions of 1800 to 2500lx. This treatment step is conducive to the dedifferentiation of microspores, and then the callus tissue can differentiate into adventitious buds, and the adventitious buds can be further differentiated into adventitious roots (regenerated plants), and the growth of Isatis indigotica plants is promoted; Third, in the present invention, zeatin (ZT) and indoleacetic acid (IAA) are added to the differentiation medium and the regeneration medium. The differentiation medium can effectively promote the callus tissue to produce adventitious buds, and the regeneration medium is mainly for producing adventitious roots (regenerated plants). The chromosome doubling medium used utilizes colchicine to stimulate chromosome doubling, and can obtain doubled regenerated plants, that is, diploid regenerated plants.

[0037] In summary, the present invention uses a series of processing steps to culture the buds of Isatis indigofera in the mononuclear marginal stage during the anther development period to produce haploid callus and haploid regenerated plants, then obtains the doubly regenerated plants through chromosome doubling, obtains pure lines, and has a high chromosome doubling success rate, provides pure line parents for Isatis indigofera hybrid breeding, shortens the growth cycle, improves Isatis indigofera breeding efficiency, and has strong practicality and economy. At the same time, it has the characteristics of low production cost, easy acquisition of culture medium, easy control of culture environment, short-term realization of multi-generation reproduction, easy expression of recessive genes, etc.

[0038] Embodiment 12: The chromosome doubling method for woad anther culture is carried out according to the following method: S1, screening: at 7 am on a sunny day, collect woad buds of different sizes from healthy and disease-free plants, place the woad anthers on a glass slide, crush them with tweezers, add 2 drops of acetic acid magenta solution, and let them stand in a dark environment for 8 minutes. After staining, observe the development period of microspores in the woad anthers under an optical microscope, and screen the woad buds in which most of the microspores in the woad anthers are in the mononuclear marginal stage; S2, pretreatment: placing the buds of Isatis indigo plant at the mononuclear marginal stage during the anther development period in a 4°C refrigerator for 24 hours, spraying the buds with water during the treatment period to prevent dehydration and drying; S3, anther induction callus: sterilize the buds of Isatis indigo after low temperature pretreatment with 75% alcohol for 30 seconds, rinse with sterile water for 3 times, soak and disinfect with 2% sodium hypochlorite for 20 minutes, rinse with sterile water for 5 times, peel the buds of Isatis indigo with tweezers, take the anthers of Isatis indigo and inoculate them in the basic culture medium for culture to obtain callus, wherein the composition of the basic culture medium is as follows: add 3g / L plant culture medium to the MS medium Biogel, 0.5mg / L glutamic acid, 30g / L sucrose, adjust the pH value to 5.8, and then add 0.5mg / L zeatin (ZT) and 1.0mg / L indoleacetic acid (IAA) to the MS medium; culture conditions: at a humidity of 65%±2% and a temperature of 25℃±2℃, first culture the anthers of Isatis indigo at 200lx for 30 days, then transfer to 1800lx for 5 to 10 days, and culture for 10 hours every day; S4, inducing callus to differentiate into adventitious buds: transferring the callus to a differentiation medium for culture to obtain callus that has differentiated into adventitious buds; wherein the composition of the differentiation medium is: adding 3 g / L of plant gel and 30 g / L of sucrose to the MS medium, adjusting the pH value to 5.8, and then adding 0.5 mg / L of zeatin (ZT) and 0.1 mg / L of indoleacetic acid (IAA) to the MS medium; culture conditions: light intensity of 1800 lx, culture time of 6 to 11 days, and light culture for 10 hours per day; S5, inducing adventitious buds to produce regenerated plants: transferring the callus tissue that produced adventitious buds to the regeneration medium for culture to obtain regenerated plants; wherein the composition of the regeneration medium: adding 3g / L plant gel and 30g / L sucrose to 1 / 2 MS medium, adjusting the pH value to 5.8, and then adding 0.5mg / L indolebutyric acid (IBA) to the MS medium; culture conditions: light intensity of 1800lx to 2500lx, culture time of 5d to 10d, and light culture for 10h per day; S6, inducing chromosome doubling: transferring the regenerated plants to a chromosome doubling medium for culture to obtain doubled regenerated plants, which have enlarged organs and doubling success characteristics; wherein the composition of the chromosome doubling medium is: adding 3g / L plant gel and 30g / L sucrose to the MS medium, adjusting the pH value to 5.8, and then adding 70mg / L colchicine to the MS medium; the culture conditions are: the light intensity is 1800lx, the culture time is 8d to 12d, and the light culture is 10h per day; S7, ploidy identification of doubling regenerated plants: Flow cytometry was used to identify the ploidy of doubling regenerated plants. The judgment standard was to compare the peak DNA content of the flow cytometer with the diploid control plants of local traditional farmer varieties and the haploid male gametes of pollen. If the peak DNA content was consistent with the diploid control plants of traditional farmer varieties, it was determined to be diploid, and if the peak DNA content was consistent with the haploid male gametes of pollen, it was haploid; S8, transplanting and planting: transplanting and planting the doubled regenerated plants with successful ploidy identification, specifically, when the doubled regenerated plants grow 4 to 6 leaves and the roots are 3 cm to 5 cm long, open the mouth of the culture bottle for seedling hardening, so that the doubled regenerated plants gradually adapt to the external environment, take out the doubled regenerated plants from the culture bottle, clean the culture medium remaining on the roots, transplant them into the nutrient pot, and place them at room temperature of 21°C to 25°C for cultivation. Transplant the doubled regenerated plants cultured from the same single anther to the same plot, and when flowering, cover the plot with an insect-proof net to prevent insect pollination. When the flowering period is in full bloom, cut off the undeveloped flower spikes after most of the flowers bloom, remove the insect-proof net when all the flowers wither after 3 days, and harvest the seeds separately after the seeds mature to reproduce into homozygous strains.

[0039] The ploidy of the regenerated plants of Isatis indigotica obtained in step S7 was identified using the CyFlow Space flow cytometer. The diploid control plants of the local traditional farm variety were used as controls. The ploidy of the regenerated plants was determined based on the peak DNA content of the flow cytometer. The results are shown in the attached figure. Figure 1 As shown, Figure 1 The horizontal axis represents the DNA content, the vertical axis represents the number of cells, A is the pollen male gamete haploid, B is the local traditional farmer variety diploid control plant, C is the doubled regeneration plant, 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 diploid control plant of the local traditional farm variety appears at the horizontal axis value of 100, and the sample peak of the doubly regenerated plant appears at the horizontal axis value of 100, indicating that the ploidy of the doubly regenerated plant obtained in Example 12 of the present invention is the same as the ploidy of the diploid control plant of the local traditional farm variety, that is, it is a diploid plant.

[0040] In Example 12 of the present invention, 10 groups of Isatis indigotica flower buds whose anther development period was in the mononuclear marginal stage of the same single Isatis indigotica plant were screened, and according to the method of Example 12 of the present invention, finally, the obtained doubly regenerated plants were subjected to ploidy identification using a flow cytometer, and the DNA content peak values ​​of 9 groups of doubly regenerated plants were consistent with those of the local traditional farm variety diploid control plants, and the DNA content peak value of 1 group of doubly regenerated plants was consistent with the pollen male gamete haploid, and the chromosome doubling success rate was 90%.

[0041] Embodiment 13: The chromosome doubling method for woad anther culture is carried out according to the following method: S1, screening: at 7 am on a sunny day, collect woad buds of different sizes from healthy and disease-free plants, place the woad anthers on a glass slide, crush them with tweezers, add 1 drop of acetic acid magenta solution, and let them stand in a dark environment for 5 minutes. After staining, observe the development period of microspores in the woad anthers under an optical microscope, and screen the woad buds in which most of the microspores in the woad anthers are in the mononuclear marginal stage; S2, pretreatment: placing the buds of Isatis indigo plant at the mononuclear marginal stage during the anther development period in a 0°C refrigerator for 24 hours, spraying the buds with water during the treatment period to prevent dehydration and drying; S3, anther induction callus: sterilize the buds of Isatis indigo after low temperature pretreatment with 75% alcohol for 30 seconds, rinse with sterile water for 3 times, soak and disinfect with 2% sodium hypochlorite for 15 minutes, and then rinse with sterile water for 4 times. Then, peel the buds of Isatis indigo with tweezers, take the anthers of Isatis indigo and inoculate them in the basic culture medium for culture to obtain callus, wherein the composition of the basic culture medium is as follows: add 3g / L plant culture medium to the MS medium Biogel, 0.5 mg / L glutamic acid, 25 g / L sucrose, adjust the pH value to 5.8, and then add 0.5 mg / L zeatin (ZT) and 1.0 mg / L indoleacetic acid (IAA) to the MS medium; culture conditions: at a humidity of 65% ± 2%, a temperature of 25 ° C ± 2 ° C, first place the anthers of Isatis indigo at 500 lx for 28 days, then transfer to 2500 lx for 5 to 10 days, and light culture for 10 hours every day; S4, inducing callus to differentiate into adventitious buds: transferring the callus to a differentiation medium for culture to obtain callus that has differentiated into adventitious buds; wherein the composition of the differentiation medium is: adding 3 g / L of plant gel and 25 g / L of sucrose to the MS medium, adjusting the pH value to 5.8, and then adding 0.5 mg / L of zeatin (ZT) and 0.1 mg / L of indoleacetic acid (IAA) to the MS medium; culture conditions: light intensity of 2500 lx, culture time of 6 to 11 days, and light culture for 10 hours per day; S5, inducing adventitious buds to produce regenerated plants: transferring the callus tissue that produced adventitious buds to the regeneration medium for culture to obtain regenerated plants; wherein the composition of the regeneration medium is: adding 3g / L plant gel and 25g / L sucrose to 1 / 2 MS medium, adjusting the pH value to 5.8, and then adding 0.5mg / L indolebutyric acid (IBA) to the MS medium; culture conditions: light intensity of 2500lx, culture time of 5d to 10d, and light culture for 10h per day; S6, inducing chromosome doubling: transferring the regenerated plants to a chromosome doubling medium for culture to obtain doubled regenerated plants, which showed organ enlargement and doubling success characteristics; wherein the composition of the chromosome doubling medium was as follows: adding 3 g / L of plant gel and 25 g / L of sucrose to the MS medium, adjusting the pH value to 5.8, and then adding 80 mg / L of colchicine to the MS medium; the culture conditions were as follows: the light intensity was 2500 lx, the culture time was 8 to 12 days, and the light culture was performed for 10 hours every day; S7, ploidy identification of doubling regenerated plants: Flow cytometry was used to identify the ploidy of doubling regenerated plants. The judgment standard was to compare the peak DNA content of the flow cytometer with the diploid control plants of local traditional farmer varieties and the haploid male gametes of pollen. If the peak DNA content was consistent with the diploid control plants of traditional farmer varieties, it was determined to be diploid, and if the peak DNA content was consistent with the haploid male gametes of pollen, it was haploid; S8, transplanting and planting: transplanting and planting the doubled regenerated plants with successful ploidy identification, specifically, when the doubled regenerated plants grow 4 to 6 leaves and the roots are 3 cm to 5 cm long, open the mouth of the culture bottle for seedling hardening, so that the doubled regenerated plants gradually adapt to the external environment, take out the doubled regenerated plants from the culture bottle, clean the culture medium remaining on the roots, transplant them into the nutrient pot, and place them at room temperature of 21°C to 25°C for cultivation. Transplant the doubled regenerated plants cultured from the same single anther to the same plot, and when flowering, cover the plot with an insect-proof net to prevent insect pollination. When the flowering period is in full bloom, cut off the undeveloped flower spikes after most of the flowers bloom, remove the insect-proof net when all the flowers wither after 3 days, and harvest the seeds separately after the seeds mature to reproduce into homozygous strains.

[0042] In Example 13 of the present invention, 10 groups of Isatis indigotica flower buds whose anther development period was in the mononuclear marginal stage of the same single Isatis indigotica plant were screened, and according to the method of Example 13 of the present invention, finally, the obtained doubly regenerated plants were subjected to ploidy identification using a flow cytometer, and the DNA content peak values ​​of the 9 groups of doubly regenerated plants were consistent with those of the local traditional farm variety diploid control plants, and the chromosome doubling success rate was 90%.

[0043] Embodiment 14: The chromosome doubling method for woad anther culture is carried out according to the following method: S1, screening: at 7 am on a sunny day, collect woad buds of different sizes from healthy and disease-free plants, place the woad anthers on a glass slide, crush them with tweezers, add 2 drops of acetic acid magenta solution, and let them stand in a dark environment for 10 minutes. After staining, observe the development period of the microspores of the woad anthers under an optical microscope, and screen the woad buds whose development period of most microspores of the woad anthers is in the mononuclear marginal stage; S2, pretreatment: placing the buds of Isatis indigo plant at the mononuclear marginal stage during the anther development period in a 5°C refrigerator for 28 hours, spraying the buds with water during the treatment period to prevent dehydration and drying; S3, anther induction callus: sterilize the buds of Isatis indigo after low temperature pretreatment with 75% alcohol for 30 seconds, rinse with sterile water for 3 times, soak and disinfect with 2% sodium hypochlorite for 20 minutes, rinse with sterile water for 5 times, peel the buds of Isatis indigo with tweezers, take the anthers of Isatis indigo and inoculate them in the basic culture medium for culture to obtain callus, wherein the composition of the basic culture medium is as follows: add 5g / L plant culture medium to the MS medium Biogel, 0.6mg / L glutamic acid, 35g / L sucrose, adjust the pH value to 6.0, and then add 0.5mg / L zeatin (ZT) and 1.0mg / L indoleacetic acid (IAA) to the MS medium; culture conditions: at a humidity of 65%±2% and a temperature of 25℃±2℃, first culture the anthers of Isatis indigo at 100lx for 32 days, then transfer to 2000lx for 5 to 10 days, and culture for 10 hours every day; S4, inducing callus to differentiate into adventitious buds: transferring the callus to a differentiation medium for culture to obtain callus that has differentiated into adventitious buds; wherein the composition of the differentiation medium is: adding 5 g / L of plant gel and 35 g / L of sucrose to the MS medium, adjusting the pH value to 6.0, and then adding 0.5 mg / L of zeatin (ZT) and 0.1 mg / L of indoleacetic acid (IAA) to the MS medium; the culture conditions are: the light intensity is 2000 lx, the culture time is 6 days to 11 days, and the light culture is carried out for 10 hours every day; S5, inducing adventitious buds to produce regenerated plants: transferring the callus tissue that produced adventitious buds to the regeneration medium for culture to obtain regenerated plants; wherein the composition of the regeneration medium is: adding 5g / L plant gel and 35g / L sucrose to 1 / 2 MS medium, adjusting the pH value to 6.0, and then adding 0.5mg / L indolebutyric acid (IBA) to the MS medium; culture conditions: light intensity of 2000lx, culture time of 5d to 10d, and light culture for 10h per day; S6, inducing chromosome doubling: transferring the regenerated plants to a chromosome doubling medium for culture to obtain doubled regenerated plants, the regenerated plants show organ enlargement, and show the characteristics of successful doubling; wherein the composition of the chromosome doubling medium is: adding 5g / L plant gel and 35g / L sucrose to the MS medium, adjusting the pH value to 6.0, and then adding 60mg / L colchicine to the MS medium; the culture conditions are: the light intensity is 2000lx, the culture time is 8d to 12d, and the light culture is 10h per day; S7, ploidy identification of doubling regenerated plants: Flow cytometry was used to identify the ploidy of doubling regenerated plants. The judgment standard was to compare the peak DNA content of the flow cytometer with the diploid control plants of local traditional farmer varieties and the haploid male gametes of pollen. If the peak DNA content was consistent with the diploid control plants of traditional farmer varieties, it was determined to be diploid, and if the peak DNA content was consistent with the haploid male gametes of pollen, it was haploid; S8, transplanting and planting: transplanting and planting the doubled regenerated plants with successful ploidy identification, specifically, when the doubled regenerated plants grow 4 to 6 leaves and the roots are 3 cm to 5 cm long, open the mouth of the culture bottle for seedling hardening, so that the doubled regenerated plants gradually adapt to the external environment, take out the doubled regenerated plants from the culture bottle, clean the culture medium remaining on the roots, transplant them into the nutrient pot, and place them at room temperature of 21°C to 25°C for cultivation. Transplant the doubled regenerated plants cultured from the same single anther to the same plot, and when flowering, cover the plot with an insect-proof net to prevent insect pollination. When the flowering period is in full bloom, cut off the undeveloped flower spikes after most of the flowers bloom, remove the insect-proof net when all the flowers wither after 3 days, and harvest the seeds separately after the seeds mature to reproduce into homozygous strains.

[0044] In Example 14 of the present invention, 10 groups of Isatis indigotica flower buds whose anther development period was in the mononuclear marginal stage of the same single Isatis indigotica plant were screened, and according to the method of Example 14 of the present invention, finally, the obtained doubly regenerated plants were subjected to ploidy identification using a flow cytometer, and the DNA content peak values ​​of 8 groups of doubly regenerated plants were consistent with those of the local traditional farm variety diploid control plants, and the DNA content peak values ​​of 2 groups of doubly regenerated plants were consistent with the pollen male gamete haploid, and the chromosome doubling success rate was 80%.

[0045] Comparative Example: The difference from Example 12 is that “the composition of the chromosome doubling medium: add 3g / L plant gel and 30g / L sucrose to the MS medium, adjust the pH value to 5.8, and then add 70mg / L colchicine to the MS medium” is changed to “the composition of the chromosome doubling medium: add 3g / L plant gel and 30g / L sucrose to the MS medium, adjust the pH value to 5.8, and then add 30mg / L colchicine to the MS medium”, and the remaining steps are the same.

[0046] In the comparative example of the present invention, 10 groups of Isatis indigotica flower buds whose anther development period of the same single Isatis indigotica plant was in the mononuclear marginal stage were screened, and the obtained doubly regenerated plants were identified for ploidy using a flow cytometer according to the method of comparative example 1 of the present invention. The peak values ​​of DNA contents of 3 groups of doubly regenerated plants were consistent with those of diploid control plants of local traditional farm varieties, and the peak values ​​of DNA contents of 7 groups of doubly regenerated plants were consistent with the haploid pollen male gametes, and the chromosome doubling success rate was 30%.

[0047] Compared with the comparative example, through the chromosome doubling method of Isatis indigotica anther culture of Examples 12 to 14 of the present invention, the doubly regenerated plants were confirmed to be diploid, and the doubling success rate was higher than 80%.

[0048] In summary, the present invention uses a series of processing steps to culture the buds of Isatis in the mononuclear marginal stage during the anther development period of Isatis to produce haploid callus tissue and haploid regenerated plants, and then obtains the doubly regenerated plants through chromosome doubling to obtain pure lines. The chromosome doubling success rate is high, and pure line parents are provided for Isatis hybrid breeding, the growth cycle is shortened, and the breeding efficiency of Isatis is improved. The method has strong practicality and economy.

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

Claims

1. A method for doubling chromosomes in anther culture of Isatis indigofera, characterized in that Proceed as follows: S1, screening: screening the buds of Isatis indigofera that are in the mononuclear marginal stage during the anther development period; S2, pretreatment: subjecting the buds of Isatis indigofera indigofera in the mononuclear marginal stage during the anther development period to low temperature pretreatment; S3, inducing callus tissue from anthers: sterilizing, washing, disinfecting and re-washing the buds of Isatis indigo plant that have been pretreated at low temperature, and then inoculating the anthers of Isatis indigo plant into a basic culture medium for culture to obtain callus tissue; S4, inducing callus to differentiate into adventitious buds: transferring the callus to a differentiation medium for culture to obtain callus that has differentiated into adventitious buds; S5, inducing adventitious buds to produce regenerated plants: transferring the callus tissue that has produced adventitious buds to a regeneration medium for culture to obtain regenerated plants; S6, inducing chromosome doubling: transferring the regenerated plants to a chromosome doubling medium for culture to obtain doubled regenerated plants; S7, ploidy identification of doubling regenerated plants: ploidy identification of doubling regenerated plants was performed using flow cytometry; S8, Transplanting and Planting: Transplant and plant the doubled regenerated plants that have been successfully identified for ploidy.

2. The method for chromosome doubling in anther culture of Isatis indigotica according to claim 1, characterized in that In step S1, after staining, the anthers of woad are observed under an optical microscope to select woad flower buds in the mononuclear marginal stage of the anther development period, wherein the staining process specifically comprises adding a stain to the anthers of woad and placing them in a dark environment for 5 to 10 minutes.

3. The method for chromosome doubling in anther culture of Isatis indigotica according to claim 1 or 2, characterized in that 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.

4. The method for chromosome doubling in anther culture of Isatis indigotica according to any one of claims 1 to 3, characterized in that In step S3, the composition of the basal culture medium is as follows: 3g / L to 5g / L of plant gel, 0.4mg / L to 0.6mg / L of glutamic acid and 25g / L to 35g / L of sucrose are added to the MS culture medium, the pH value is adjusted to 5.8 to 6.0, and then 0.05mg / L to 1.0mg / L of zeatin and 0.05mg / L to 1.0mg / L of indoleacetic acid are added to the MS culture medium.

5. The method for chromosome doubling in anther culture of Isatis indigotica according to any one of claims 1 to 4, characterized in that In step S3, the culture conditions are as follows: at a humidity of 65%±2% and a temperature of 25°C±2°C, the anthers of Isatis tinctoria are first cultured under 100lx to 500lx light for 28d to 32d, and then transferred to 1800lx to 2500lx light for 5d to 10d, with light culture for 10h to 12h every day.

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

7. The method for chromosome doubling in anther culture of Isatis indigotica according to any one of claims 1 to 6, characterized in that In step S4, the culture conditions are: light intensity is 1800lx to 2500lx, culture time is 6d to 11d, and light culture is performed for 10h to 12h every day.

8. The method for chromosome doubling in anther culture of Isatis indigotica according to any one of claims 1 to 7, characterized in that In step S5, the composition of the regeneration medium is: 3 g / L to 5 g / L of phytagel and 25 g / L to 35 g / L of sucrose were added to 1 / 2 MS medium, the pH value was adjusted to 5.8 to 6.0, and 0.05 mg / L to 1.0 mg / L of indolebutyric acid was added to the MS medium.

9. The method for chromosome doubling in anther culture of Isatis indigotica according to any one of claims 1 to 8, characterized in that In step S5, the culture conditions are as follows: the light intensity is 1800 lx to 2500 lx, the culture time is 5 d to 10 d, and the light culture is 10 h to 12 h per day.

10. The method for chromosome doubling in anther culture of Isatis indigotica according to any one of claims 1 to 9, characterized in that In step S6, the composition of the chromosome doubling medium is: adding 3g / L to 5g / L plant gel and 25g / L to 35g / L sucrose to the MS medium, adjusting the pH value to 5.8 to 6.0, and then adding 40mg / L to 90mg / L colchicine to the MS medium; or / and, in step S6, the culture conditions are: the light intensity is 1800lx to 2500lx, the culture time is 8d to 12d, and the light culture is 10h to 12h every day.

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