Method for obtaining leymus chinensis mutant by using heavy ion beam to irradiate seed callus

Abstract

The invention discloses a method for obtaining leymus chinensis mutants by using heavy ion beams to irradiate seed calluses, relates to the technical field of leymus chinensis breeding biology, and solves the three-low problems of low heading rate, low maturing rate and low germination rate of leymus chinensis seeds. After the leymus chinensis callus is subjected to induction culture, selecting faint yellow granular callus obtained by induction culture, and transferring the faint yellow granular callus into a culture dish filled with an induction culture medium for subculture; carrying out irradiation treatment by using a 5Gy carbon ion beam, transferring the callus subjected to irradiation treatment into a sterile culture bottle filled with a differential culture medium, and culturing to obtain adventitious buds; cutting off callus adhered to the base under a sterile condition, and inserting into a rooting culture medium for rooting culture after a rooting point is exposed; and after obvious root systems grow out, carrying out potting culture to obtain the leymus chinensis mutant. According to the invention, new genetic variation can be created, early in-vitro selection is realized, more effective mutants are obtained as new germplasm resources of leymus chinensis, and the breeding process of leymus chinensis is accelerated.

Description

Technical Field

[0001] The invention relates to the field of leymus chinensis breeding biotechnology, and in particular to a method for obtaining leymus chinensis mutants by irradiating seed callus tissue with heavy ion beams. Background Art

[0002] Leymus chinensis (Trin.) Tzvel. is a perennial rhizome grass of the genus Leymus in the Poaceae family. It has the advantages of salt-alkali resistance, barrenness resistance, high crude protein content, and good palatability. However, its seeds still have the "three lows" problem of low heading rate, low fruiting rate, and low germination rate, which restricts the promotion and application of Leymus chinensis to improve grassland ecosystems and provide high-quality forage. In addition, Leymus chinensis is a perennial plant with a long growth cycle. It takes a long time from sowing to maturity, which prolongs the breeding process and increases costs.

[0003] Plant tissue culture is a method that utilizes the omnipotence of plant cells, separates plant organs or tissues through sterile operation as explants to obtain callus tissue, and then obtains regenerated plants through differentiation. Leymus tissue culture uses seeds as explants, and it takes 3 to 4 months to quickly and efficiently obtain a large number of regenerated plants. It has a fast reproduction speed and is not affected by seasons.

[0004] During the rapid proliferation of sheep fescue callus, cells divide quickly and genetic material replicates rapidly. During this process, mild physical or chemical mutagenesis can produce a large number of effective mutations, and regenerated plants with a high mutation frequency can be obtained in a short period of time.

[0005] Therefore, how to utilize the research and application of mutagenesis technology to obtain more effective mutants as new germplasm resources of sheep fescue and accelerate the breeding process of sheep fescue is not only a hot topic in this field, but also a technical problem that needs to be solved urgently. It is of great significance to promote the progress of sheep fescue breeding and promote the restoration of grassland ecosystems. Summary of the invention

[0006] In order to obtain more effective mutants as new germplasm resources of Leymus chinensis and accelerate the breeding process of Leymus chinensis, the present invention proposes a method for obtaining Leymus chinensis mutants by irradiating seed callus tissue with heavy ion beams.

[0007] The technical solution of the present invention is as follows:

[0008] A method for obtaining a mutant of Leymus chinensis by irradiating seed callus tissue with heavy ion beams comprises the following steps:

[0009] Step 1: Induction culture of Leymus chinensis callus:

[0010] The lemma-removed Leymus chinensis seeds are sterilized and cleaned, 1 / 4 to 1 / 3 of the endosperm is cut off under a sterile environment, and then the seeds are placed flat in a culture dish containing an induction medium, and then sealed and placed in a dark environment at a temperature of 27±1°C for induction culture for 30 to 40 days;

[0011] Step 2: Subculture of callus tissue:

[0012] The light yellow granular callus obtained by induction culture was selected and transferred to a culture dish containing induction medium, which was sealed and placed in a dark environment at a temperature of 27±1°C for subculture for 28 days;

[0013] Step 3: Callus irradiation mutagenesis:

[0014] Using carbon ion beam 12 C 6+ The irradiation treatment was performed with an initial energy of 60 MeV / u and an irradiation dose of 5 Gy;

[0015] Step 4: Irradiated callus differentiation culture:

[0016] The irradiated callus was transferred to a sterile culture bottle containing differentiation medium and cultured at a constant temperature of 27±1°C for 40 days under the conditions of 16h light / 8h dark alternation to obtain adventitious buds;

[0017] Step 5: Rooting culture of adventitious buds:

[0018] After the adventitious buds were taken out, the callus tissue attached to the base was cut off under sterile conditions, and the rooting point was exposed and inserted into the rooting medium with an insertion depth of 0.3-0.5 cm. The rooting culture was carried out at a constant temperature of 27±1℃ under the conditions of 16h light / 8h dark alternation;

[0019] Step 6: Transplanting of regenerated seedlings:

[0020] After the adventitious buds grow obvious root systems, the regenerated seedlings are potted and cultured after the height reaches 3 to 5 cm to obtain the sheepgrass mutants.

[0021] Preferably, the induction medium and the subculture medium have the same formula, both are prepared by MS+2.0 mg / L 2,4-D+30 g / L sucrose+7 g / L agar, and the pH is 5.8.

[0022] Preferably, the genotype number of the Leymus chinensis seeds is Dongdi No. 4, Dongdi No. 5 or Jingmu No. 3.

[0023] Preferably, the disinfection and cleaning process is specifically as follows:

[0024] After disinfecting with 75% alcohol for 30 seconds, wash it 2 to 3 times with sterile distilled water, then soak it in 0.1% HgCl2 for 8 minutes, and then wash it 3 to 5 times with sterile distilled water.

[0025] Preferably, the differentiation medium is prepared according to the formula MS+1.0 mg / L 6-BA+0.15 mg / L NAA+1.0 mg / L KT+1.0 mg / L TDZ+30 g / L sucrose+8 g / L agar, with a pH of 5.8.

[0026] Preferably, the rooting medium is prepared according to the formula MS+30 g / L sucrose+8 g / L agar, and the pH is 5.8.

[0027] Preferably, the specific method of potted cultivation is:

[0028] First, mix peat soil and vermiculite in a volume ratio of 1:1 and place it in a pot. Then take the regenerated seedlings out of the sterile bottle and thoroughly wash the culture medium at the roots. Divide the clumps of regenerated seedlings into single plants and transplant them into pots with a transplanting depth of 0.5-1.0 cm. Write the genotype number and treatment name. Water regularly to keep the soil moist. The indoor culture temperature is 26°C and the light is 12 hours a day.

[0029] Preferably, the carbon ion beam 12 C 6+ Provided by the Lanzhou Heavy Ion Accelerator of the China Institute of Modern Physics.

[0030] Preferably, the carbon ion beam 12 C 6+ Before irradiation treatment, the subculture medium was dispensed into a small culture dish with a diameter of 5.4 cm, and the proliferated callus tissue was densely placed in the small culture dish.

[0031] Compared with the prior art, the specific beneficial effects of the present invention are:

[0032] The present invention combines tissue culture technology and heavy ion beam irradiation mutagenesis technology, uses callus cell groups that are more sensitive to irradiation as experimental materials, irradiates callus obtained by induced culture of leymus chinensis seeds with heavy ion beams, obtains embryonic callus with a large number of genetic material mutations, and finally redifferentiates and cultures to obtain new leymus chinensis mutants. Not only can it process large groups for synchronous culture with high consistency, reduce costs, and save irradiation space, it can also create new genetic variations, achieve early in vitro selection, and easily expand the selected mutants, obtain more effective mutants as new leymus chinensis germplasm resources, reduce the breeding period of leymus chinensis, and accelerate the breeding process of leymus chinensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1The photos are of the callus induction culture process of Leymus chinensis in a specific implementation method;

[0034] Figure 2 It is a photo of the subculture process of Leymus chinensis callus in a specific implementation method;

[0035] Figure 3 This is a photo of the callus tissue of Jingmu No. 3 Leymus chinensis seeds after heavy ion beam irradiation in a specific implementation method;

[0036] Figure 4 This is a photo of differentiation culture of callus tissue of Leymus chinensis No. 5 in Zhongdongdi after irradiation with a dose of 5Gy in a specific implementation method;

[0037] Figure 5 This is a photo of rooting culture of callus of Leymus chinensis No. 5 in Dongdi after irradiation with a dose of 5Gy in a specific implementation method;

[0038] Figure 6 This is the phenotypic difference of the regenerated mutant of Dongdi Leymus 5 callus after irradiation with 0Gy and 5Gy doses for 30 days. DETAILED DESCRIPTION

[0039] In order to make the technical solution of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the specification of the present invention. It should be noted that the following embodiments are only used to better understand the technical solution of the present invention and should not be understood as a limitation to the present invention.

[0040] The following experiment was conducted from February to October 2024, using callus tissue of Dongdi No. 4, Dongdi No. 5 and Jingmu No. 3 seeds as the research object, using carbon ion beam 12 C 6+ Radiation mutagenesis, the experiment was carried out according to the following steps:

[0041] 1. Callus induction culture of Leymus chinensis:

[0042] Prepare the induction medium according to the formula: MS+2.0mg / L 2,4-D+30g / L sucrose+7g / L agar, adjust the pH to 5.8, sterilize at 121℃ for 20min, and then divide into sterile culture dishes, cool and set aside. Select clean and full Dongdi No. 4, Dongdi No. 5 and Jingmu No. 3 sheepgrass seeds, remove the seed lemma and disinfect with 75% alcohol for 30s, wash with sterilized distilled water 3 times, then soak with 0.1% HgCl2 for 8min, and wash with sterilized distilled water 5 times. Place the sterilized sheepgrass seeds on sterilized filter paper, cut off 1 / 4 to 1 / 3 of the endosperm with a scalpel and place it flat on the induction medium, and place 14 sheepgrass seeds in each culture dish. After sealing with a sealing film, place the culture dish in the dark at 27±1℃.

[0043] 2. Callus subculture:

[0044] After 30-40 days of induction culture, the light yellow granular callus was selected and transferred to the subculture medium for further culture. The initial size of each callus group was about 0.5 cm. The subculture medium formula and culture conditions were exactly the same as those of the induction culture. After 28 days of subculture, the diameter of the callus group increased significantly.

[0045] 3. Callus irradiation mutagenesis:

[0046] Prepare the subculture medium and dispense it into a small culture dish with a diameter of 5.4 cm. Place the proliferated callus tissue tightly in the small culture dish to make full use of the irradiation space. Seal it with a sealing film and then wrap it with tin foil and keep it in the dark. Use the carbon ion beam provided by the Lanzhou Heavy Ion Accelerator of the Institute of Modern Physics of China 12 C 6+ The irradiation treatment was carried out with an initial energy of 60 MeV / u. The irradiation doses were 0, 5 Gy, 15 Gy and 25 Gy, respectively. Three replicates were set for each genotype and each irradiation dose, and 80 to 100 calli were placed in each replicate.

[0047] 4. Irradiated callus differentiation culture:

[0048] Prepare differentiation medium according to the formula MS+1.0mg / L 6-BA+0.15mg / L NAA+1.0mg / L KT+1.0mg / LTDZ+30g / L sucrose+8g / L agar (pH 5.8), and dispense into sterile culture bottles with a diameter of 8.0cm and a height of 6.7cm while hot. Transfer most of the irradiated callus to the culture bottles, place 25 to 30 callus groups with a diameter of about 0.5cm in each culture bottle, and culture at a constant temperature of 27±1℃ for 40 days under 16h light / 8h dark conditions. Replace the remaining small part with subculture medium and continue subculture to observe the cell proliferation ability.

[0049] 5. Adventitious bud rooting culture:

[0050] Prepare the rooting medium according to the formula MS + 30g / L sucrose + 8g / L agar (pH 5.8), and dispense it into sterile culture bottles with a diameter of 6.9cm and a height of 9.0cm while hot. Take the adventitious buds out of the differentiation medium and place them on a sterile culture dish. Cut off the callus tissue attached to the base of the adventitious buds with a scalpel, expose the rooting point, and insert it into the rooting medium to a depth of 0.3 to 0.5cm. The rooting culture conditions are the same as those for differentiation culture.

[0051] 6. Transplanting of regenerated seedlings:

[0052] After 40 days of rooting culture, the adventitious buds can be seen to grow obvious roots. When the height of the regenerated seedlings reaches 3-5 cm, potting culture can be carried out. The specific method is: first, mix the peat soil and vermiculite in a volume ratio of 1:1 and put them into a two-color pot with a diameter of 12 cm, then take the regenerated seedlings out of the sterile bottle and thoroughly wash the culture medium at the roots, divide the clustered regenerated seedlings into individual plants and transplant them into the two-color pots, the transplanting depth is 0.5-1.0 cm, write the genotype number and treatment name, water regularly to keep the soil moist, the indoor culture temperature is 26℃, the light is 12 hours a day, and the survival rate of the seedlings is counted after 7-10 days of indoor culture.

[0053] 7. Investigate the growth characteristics of mutants:

[0054] After 30 days of cultivation in pots, the plant height and tiller number of all regenerated plantlets obtained from irradiated and non-irradiated callus were investigated. The number of mutants with plant height and tiller number higher than those of regenerated plantlets obtained from non-irradiated callus was counted.

[0055] 8. Statistical results:

[0056] (1) The specific statistical results of the Dongdi 4 Leymus chinensis mutant are as follows:

[0057] The average plant height of the regenerated plants of the control (0Gy) of the callus tissue of Dongdi No. 4 Leymus chinensis that had not been irradiated was 32.2cm, and the average number of tillers was 2.

[0058] After 5Gy heavy ion beam irradiation, 17 mutants were finally obtained from the callus of Dongdi No. 4 Leymus chinensis. Among them, the plant height of 2 mutants was higher than that of the control, accounting for 11.8% of the total number of mutants, and the highest mutant plant height reached 39.5cm; the plant height of the remaining 15 mutants was lower than that of the control, accounting for 88.2% of the total number of mutants, and the lowest mutant plant height was only 8cm. Among them, the number of tillers of 1 mutant was higher than that of the control, accounting for 5.9% of the total number of mutants, and the maximum number of tillers of the mutant was 3; the number of tillers of the remaining 15 mutants was lower than or equal to that of the control, accounting for 94.1% of the total number of mutants, and the minimum number of mutants was only 1 tiller.

[0059] The callus of Dongdi No. 4 Leymus chinensis failed to differentiate and culture to obtain regenerative mutants after 15Gy and 25Gy heavy ion beam irradiation mutagenesis. The callus irradiated with 15Gy and 25Gy was subcultured for 28 days, and the callus turned brown. The difference is that the brown callus after 15Gy irradiation grew new light yellow granular callus, but the new callus also failed to differentiate into adventitious buds and lost its regeneration ability.

[0060] (2) The specific statistical results of the Dongdi 5 Leymus chinensis mutant are as follows:

[0061] The average plant height of the regenerated plants of the control (0Gy) of the callus tissue of Dongdi No. 5 Leymus chinensis that had not been irradiated was 36.5cm, and the average tiller number was 5.

[0062] After 5Gy heavy ion beam irradiation, 76 mutants were finally obtained from the callus of Dongdi No. 5 Leymus chinensis. Among them, 13 mutants had a higher plant height than the control, accounting for 17.1% of the total number of mutants, and the highest mutant plant height reached 46.6cm; the remaining 63 mutants had a lower plant height than the control, accounting for 82.9% of the total number of mutants, and the lowest mutant plant height was only 7.8cm. Among them, 20 mutants had a higher number of tillers than the control, accounting for 26.3% of the total number of mutants, and the maximum number of tillers of the mutants was 12; the remaining 56 mutants had a tiller number less than or equal to the control, accounting for 73.7% of the total number of mutants, and the minimum mutant had only 1 tiller.

[0063] The callus of Leymus chinensis Dongdi No. 5 failed to differentiate and culture to obtain regenerative mutants after 15Gy and 25Gy heavy ion beam irradiation mutagenesis. The callus irradiated with 15Gy and 25Gy was subcultured for 28 days. The callus irradiated with 25Gy gradually turned brown, while the callus irradiated with 15Gy did not turn brown and could continue to proliferate and grow new callus. However, the original callus and the new callus failed to differentiate into adventitious buds, and the callus lost its regeneration ability.

[0064] (3) The specific statistical results of the Jingmu 3 Leymus chinensis mutant are as follows:

[0065] The average plant height of the regenerated plants of the control (0Gy) of the callus tissue of Jingmu No. 3 Leymus chinensis that had not been irradiated was 34.3cm, and the average tiller number was 4.

[0066] After 5Gy heavy ion beam irradiation, 24 mutants were finally obtained from the callus of Jingmu No. 3 Leymus chinensis. Among them, the plant height of 3 mutants was higher than that of the control, accounting for 12.5% ​​of the total number of mutants, and the highest mutant plant height reached 40.2cm; the plant height of the remaining 21 mutants was lower than that of the control, accounting for 87.5% of the total number of mutants, and the lowest mutant plant height was only 8.8cm. Among them, the number of tillers of 5 mutants was higher than that of the control, accounting for 20.8% of the total number of mutants, and the maximum number of tillers of the mutants was 7; the number of tillers of the remaining 19 mutants was lower than or equal to that of the control, accounting for 79.2% of the total number of mutants, and the minimum number of mutants was only 1 tiller.

[0067] The callus of Leymus chinensis No. 3 failed to differentiate and culture to obtain regenerative mutants after 15Gy and 25Gy heavy ion beam irradiation mutagenesis. The callus irradiated with 15Gy and 25Gy was subcultured for 28 days. The callus irradiated with 25Gy gradually turned brown, while the callus irradiated with 15Gy did not turn brown and could continue to proliferate and grow new callus. However, the original callus and the new callus failed to differentiate into adventitious buds, and the callus lost its regeneration ability.

[0068] The above statistical results show that after 5Gy heavy ion beam irradiation mutagenesis, embryonic callus with genetic material mutations can be obtained from callus of each genotype, and new mutants of Leymus chinensis can be obtained by differentiation culture. The method provided in this application can create new genetic variations, realize early in vitro selection, obtain more effective mutants as new germplasm resources of Leymus chinensis after screening, reduce the breeding period of Leymus chinensis, and accelerate the breeding process of Leymus chinensis.

[0069] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, and these simple modifications all belong to the protection scope of the present invention. It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for obtaining a mutant of Leymus chinensis by irradiating seed callus tissue with heavy ion beams, characterized in that: The following steps are included: Step 1: Induction culture of callus tissue of Leymus chinensis: The lemma-removed Leymus chinensis seeds are sterilized and cleaned, 1 / 4 to 1 / 3 of the endosperm is cut off under a sterile environment, and then the seeds are placed flat in a culture dish containing an induction medium, and then sealed and placed in a dark environment at a temperature of 27±1°C for induction culture for 30 to 40 days; Step 2: Subculture of callus tissue: The light yellow granular callus obtained by induction culture was selected and transferred to a culture dish containing induction medium, and then sealed and placed in a dark condition at a temperature of 27±1°C for subculture for 28 days; Step 3: Callus irradiation mutagenesis: Using carbon ion beam 12 C 6+ The irradiation treatment was performed with an initial energy of 60 MeV / u and an irradiation dose of 5 Gy; Step 4: Irradiated callus differentiation culture: The irradiated callus was transferred to a sterile culture bottle containing differentiation medium and cultured at a constant temperature of 27±1°C for 40 days under the conditions of 16h light / 8h dark alternation to obtain adventitious buds; Step 5: Rooting culture of adventitious buds: After the adventitious buds were taken out, the callus tissue attached to the base was cut off under sterile conditions, and the rooting point was exposed and inserted into the rooting medium with an insertion depth of 0.3-0.5 cm. The rooting culture was carried out at a constant temperature of 27±1℃ under the conditions of 16h light / 8h dark alternation; Step 6: Transplanting of regenerated seedlings: After the adventitious buds grow obvious root systems, the regenerated seedlings are potted and cultured after the height reaches 3 to 5 cm to obtain the sheepgrass mutants.

2. The method for obtaining a mutant of Leymus chinensis by irradiating seed callus with heavy ion beams according to claim 1, characterized in that: The induction medium and the subculture medium have the same formula, both of which are prepared by MS+2.0 mg / L 2,4-D+30 g / L sucrose+7 g / L agar, and the pH is 5.

8.

3. The method for obtaining a mutant of Leymus chinensis by irradiating seed callus with heavy ion beams according to claim 1, characterized in that: The genotype number of the sheep fescue seeds is Dongdi No. 4, Dongdi No. 5 or Jingmu No.

3.

4. The method for obtaining a mutant of Leymus chinensis by irradiating seed callus with heavy ion beams according to claim 1, characterized in that: The disinfection and cleaning process is specifically as follows: After disinfecting with 75% alcohol for 30 seconds, wash it 2 to 3 times with sterile distilled water, then soak it in 0.1% HgCl2 for 8 minutes, and then wash it 3 to 5 times with sterile distilled water.

5. The method for obtaining a mutant of Leymus chinensis by irradiating seed callus with heavy ion beams according to claim 1, characterized in that: The differentiation medium was prepared according to the formula of MS+1.0 mg / L 6-BA+0.15 mg / L NAA+1.0 mg / L KT+1.0 mg / LTDZ+30 g / L sucrose+8 g / L agar, with a pH of 5.

8.

6. The method for obtaining a mutant of Leymus chinensis by irradiating seed callus with heavy ion beams according to claim 1, characterized in that: The rooting medium was prepared according to the formula MS+30 g / L sucrose+8 g / L agar, and the pH value was 5.

8.

7. The method for obtaining a mutant of Leymus chinensis by irradiating seed callus with heavy ion beams according to claim 1, characterized in that: The specific method of the potted culture is: First, mix peat soil and vermiculite in a volume ratio of 1:1 and place it in a pot. Then take the regenerated seedlings out of the sterile bottle and thoroughly wash the culture medium at the roots. Divide the clumps of regenerated seedlings into single plants and transplant them into pots with a transplanting depth of 0.5-1.0 cm. Write the genotype number and treatment name. Water regularly to keep the soil moist. The indoor culture temperature is 26°C and the light is 12 hours a day.

8. The method for obtaining a mutant of Leymus chinensis by irradiating seed callus with heavy ion beams according to claim 1, characterized in that: The carbon ion beam 12 C 6+ Provided by the Lanzhou Heavy Ion Accelerator of the China Institute of Modern Physics.

9. The method for obtaining a mutant of Leymus chinensis by irradiating seed callus with heavy ion beams according to claim 1, characterized in that: The use of carbon ion beam 12 C 6+ Before irradiation treatment, the subculture medium was dispensed into a small culture dish with a diameter of 5.4 cm, and the proliferated callus tissue was densely placed in the small culture dish.

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