Method for treating poa pratensis seeds with colchicine

Abstract

Provided is a method for treating Poa pratensis seeds with colchicine, comprising the following steps: selecting plump Poa pratensis seeds and soaking same in sterile water for 23-25 h, picking out seeds which do not swell, and then re-drying the remaining seeds for 23-25 hours; treating the re-dried seeds with 75% absolute ethanol for 50-70 s, and rinsing the seeds with sterile water for 30 s for 2-3 times; soaking the primarily disinfected seeds in a 10% sodium hypochlorite solution for 24-26 min, rinsing same with sterile water for 30 s for 4-6 times, and drying with sterile filter paper; placing the secondarily disinfected seeds into a germination box for culture and germination; transferring the cultured and germinated seeds into a 0.04-0.06% colchicine solution for soaking for 5.5-6.5 h, rinsing same with sterile water for 2-4 times, disinfecting the seeds with a mercuric chloride solution, rinsing same with sterile water for 2-4 times, drying with sterile filter paper, and inoculating the seeds into an MS culture medium for culture. When performing chemical mutation breeding, the contamination rate of Poa pratensis seeds is low, and the survival rate of tissue culture seedlings after mutation is high.

Description

Method for treating Kentucky bluegrass seeds with colchicine Technical Field

[0001] The invention belongs to the field of plant biotechnology and mainly relates to a method for treating Kentucky bluegrass seeds with colchicine. Background Art

[0002] Kentucky bluegrass is a plant of the Poaceae family. As a commonly used cool-season lawn grass species in northern my country, it has the characteristics of fast lawn formation, soft leaves, dark green leaf color, and long sustainable green period. It is an excellent lawn greening plant. However, the seed germination rate of Kentucky bluegrass is low, the contamination rate is high, and the germination speed is slow, which limits the promotion and application of Kentucky bluegrass. At present, the number of Kentucky bluegrass varieties in my country is very small, and there are only 9 varieties that have passed the approval and registration. Most of the varieties that can be used for lawn construction are introduced varieties. Therefore, carrying out the selection and breeding of Kentucky bluegrass varieties to provide high-quality seed sources for lawn construction in northern my country has become an urgent problem to be solved in my country's lawn construction. Summary of the Invention

[0003] The present invention provides a method for treating Kentucky bluegrass seeds with colchicine to improve the efficiency of mutagenesis of Kentucky bluegrass using colchicine. When the present invention is used for chemical mutagenesis breeding, the contamination rate of Kentucky bluegrass seeds is low and the survival rate of tissue culture seedlings after mutagenesis is high. The specific steps of the method are as follows:

[0004] S1. Soaking: Select plump Kentucky bluegrass seeds and soak them in sterile water for 23-25 ​​hours. Pick out the unswelled seeds and place them on filter paper to dry for 23-25 ​​hours before use.

[0005] S2. Primary disinfection: Treat the dried seeds with 75% anhydrous ethanol for 50-70 seconds, rinse the seeds with sterile water for 30 seconds, and rinse 2-3 times;

[0006] S3. Secondary disinfection: Soak the sterilized seeds in 10% sodium hypochlorite solution for 24-26 minutes, rinse with sterile water for 30 seconds, rinse 4-6 times, and then dry with sterile filter paper;

[0007] S4. Germination: Place the seeds that have been sterilized twice in a germination box and culture them in the light for 15-17 hours and in the dark for 7-9 hours at a temperature of 19-22°C.

[0008] S5. Colchicine treatment: The germinated seeds were transferred to a 0.04%-0.06% colchicine solution containing 2% DMSO and soaked for 5.5-6.5 hours.

[0009] S6. Triple disinfection: Dissolve 0.1 g HgCl2 in 100 mL distilled water to prepare a 0.1% mercuric chloride solution. Rinse the seeds soaked in colchicine solution with sterile water 2-4 times, and then disinfect with 0.1% mercuric chloride solution for 1.5-2.5 min.

[0010] S7. Inoculation: Rinse the seeds after three disinfections with sterile water for 2-4 times, dry them with sterile filter paper, and inoculate them into MS culture medium for cultivation.

[0011] Furthermore, a method for treating Kentucky bluegrass seeds with colchicine comprises the following steps:

[0012] S1. Soaking: Select plump Kentucky bluegrass seeds and soak them in sterile water for 24 hours. Pick out the unswelled seeds and place them on filter paper to dry for 24 hours before use.

[0013] S2. Primary disinfection: Treat the dried seeds with 75% anhydrous ethanol for 60 seconds, rinse the seeds with sterile water for 30 seconds, and rinse three times;

[0014] S3. Secondary disinfection: Soak the sterilized seeds in 10% sodium hypochlorite solution for 25 minutes, rinse with sterile water for 30 seconds, rinse five times, and then dry with sterile filter paper;

[0015] S4, germination cultivation: Place the seeds that have been disinfected twice into a germination box for germination cultivation;

[0016] S5. Colchicine treatment: The germinated seeds were transferred to a 0.05% colchicine solution containing 2% DMSO and soaked for 6 h.

[0017] S6. Three-time disinfection: rinse the seeds soaked in colchicine solution with sterile water three times, and then disinfect with mercuric chloride solution;

[0018] S7. Inoculation: Rinse the seeds disinfected three times with sterile water three times, dry them with sterile filter paper, and inoculate them into MS culture medium for cultivation.

[0019] Moreover, the colchicine solution is prepared by adding 0.14g-0.16g of colchicine powder to 4.5mL of DMSO, then adding distilled water to make the volume to 300mL, and stirring evenly.

[0020] Furthermore, in step S4, the germination box is disinfected with alcohol and then covered with two layers of sterile filter paper.

[0021] Moreover, in step S4, during the germination period, water is added every 24 hours, and moldy seeds are picked out.

[0022] Furthermore, in step S5, the germinated seeds are imbibed and whitened seeds.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention provides a method for treating Kentucky bluegrass seeds with colchicine, which improves the efficiency of colchicine in inducing mutation of Kentucky bluegrass. When the present invention is used for chemical mutagenesis breeding, the contamination rate of Kentucky bluegrass seeds is low and the survival rate of tissue culture seedlings after mutation is high.

[0025] 2. The present invention treats Kentucky bluegrass seeds with 75% anhydrous ethanol for 50-70 seconds, rinses the seeds with sterile water for 2-3 times, then adds the seeds into a 10% sodium hypochlorite solution for immersion for 24-26 minutes, and rinses them with sterile water for 4-6 times. The contamination rate of the treated seeds is only 5.1%, and the germination rate reaches 83.5%. The contamination rate of the Kentucky bluegrass seeds is reduced, the germination rate is increased, and favorable conditions are provided for the reproduction of Kentucky bluegrass.

[0026] 3. On the basis of reducing pollution of Kentucky bluegrass, the present invention further soaks the seeds in 0.04%-0.06% colchicine solution for 5.5-6.5 hours, so that the survival rate of Kentucky bluegrass reaches 95.72%, which greatly improves the survival rate of Kentucky bluegrass and lays a foundation for the promotion and application of Kentucky bluegrass. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Fig. 1 is a germination diagram of Example 3 of the present invention;

[0028] FIG2 is a diagram showing the germination of Comparative Example 3 of the present invention;

[0029] FIG3 shows tissue culture seedlings of the second group of experiments of Kentucky bluegrass in the experimental part of the present invention;

[0030] FIG4 shows tissue culture seedlings of the third group of experiments of Kentucky bluegrass in the experimental part of the present invention. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0032] Example 1

[0033] A method for treating Kentucky bluegrass seeds with colchicine comprises the following steps:

[0034] S1. Soaking: Select plump Kentucky bluegrass seeds and soak them in sterile water for 23 hours. Pick out the unswelled seeds and place them on filter paper to dry for 23 hours before use.

[0035] S2. Primary disinfection: Treat the dried seeds with 75% anhydrous ethanol for 50 seconds, rinse the seeds with sterile water for 30 seconds, and rinse twice;

[0036] S3. Secondary disinfection: Soak the sterilized seeds in 10% sodium hypochlorite solution for 24 minutes, rinse with sterile water for 30 seconds, rinse four times, and then dry with sterile filter paper;

[0037] S4. Germination: Place the seeds that have been sterilized twice in a germination box for germination. The germination method is: culture under light conditions for 15 hours and dark conditions for 7 hours at a temperature of 19°C.

[0038] S5. Colchicine treatment: The germinated seeds were transferred to a 0.04% colchicine solution containing 2% DMSO and soaked for 5.5 h.

[0039] S6. Three-stage disinfection: rinse the seeds soaked in colchicine solution twice with sterile water and then disinfect with mercuric chloride solution;

[0040] S7. Inoculation: Rinse the seeds disinfected three times with sterile water twice, dry them with sterile filter paper, and inoculate them into MS culture medium for cultivation.

[0041] Furthermore, the colchicine solution is prepared by adding 0.14 g of colchicine powder to 4.5 mL of DMSO, then adding distilled water to make the volume to 300 mL, and stirring evenly.

[0042] Furthermore, in step S4, the germination box is disinfected with alcohol and then covered with two layers of sterile filter paper.

[0043] Furthermore, in step S4, during the germination period, water is added every 24 hours, and moldy seeds are picked out.

[0044] Furthermore, in step S5, the germinated seeds are imbibed and whitened seeds.

[0045] Furthermore, in step S6, the mercuric chloride solution is prepared by dissolving 0.1 g HgCl2 in 100 mL distilled water to prepare a 0.1% mercuric chloride solution, and the disinfection time is 1.5 minutes.

[0046] Example 2

[0047] A method for treating Kentucky bluegrass seeds with colchicine comprises the following steps:

[0048] S1. Soaking: Select plump Kentucky bluegrass seeds and soak them in sterile water for 25 hours. Pick out the unswelled seeds and place them on filter paper to dry for 25 hours before use.

[0049] S2. Primary disinfection: Treat the dried seeds with 75% anhydrous ethanol for 70 seconds, rinse the seeds with sterile water for 30 seconds, and rinse three times;

[0050] S3. Secondary disinfection: Soak the sterilized seeds in 10% sodium hypochlorite solution for 26 minutes, rinse with sterile water for 30 seconds, rinse six times, and then dry with sterile filter paper;

[0051] S4, germination cultivation: Place the seeds that have been disinfected twice into a germination box for germination cultivation;

[0052] S5. Colchicine treatment: The germinated seeds were transferred to a 0.06% colchicine solution containing 2% DMSO and soaked for 6.5 h.

[0053] S6. Three-stage disinfection: rinse the seeds soaked in colchicine solution with sterile water four times and then disinfect with mercuric chloride solution;

[0054] S7. Inoculation: Rinse the seeds disinfected three times with sterile water for 4 times, dry them with sterile filter paper, and inoculate them into MS culture medium for cultivation.

[0055] Furthermore, the colchicine solution is prepared by adding 0.16 g of colchicine powder to 4.5 mL of DMSO, then adding distilled water to make the volume to 300 mL, and stirring evenly.

[0056] Furthermore, in step S4, the germination box is disinfected with alcohol and then covered with two layers of sterile filter paper.

[0057] Furthermore, in step S4, during the germination period, water is added every 24 hours, and moldy seeds are picked out.

[0058] Furthermore, in step S5, the germinated seeds are imbibed and whitened seeds.

[0059] Furthermore, in step S6, the mercuric chloride solution is prepared by dissolving 0.1 g HgCl2 in 100 mL distilled water to prepare a 0.1% mercuric chloride solution, and the disinfection time is 2.5 minutes.

[0060] Furthermore, in step S4, the method of culturing germination is: culturing under light for 17 hours and culturing in darkness for 9 hours at a temperature of 22°C.

[0061] Example 3

[0062] A method for treating Kentucky bluegrass seeds with colchicine comprises the following steps:

[0063] S1. Soaking: Select plump Kentucky bluegrass seeds and soak them in sterile water for 24 hours. Pick out the unswelled seeds and place them on filter paper to dry for 24 hours before use.

[0064] S2. Primary disinfection: Treat the dried seeds with 75% anhydrous ethanol for 60 seconds, rinse the seeds with sterile water for 30 seconds, and rinse three times;

[0065] S3. Secondary disinfection: Soak the sterilized seeds in 10% sodium hypochlorite solution for 25 minutes, rinse with sterile water for 30 seconds, rinse five times, and then dry with sterile filter paper;

[0066] S4. Germination: Place the seeds that have been sterilized twice in a germination box covered with two layers of sterile filter paper for germination.

[0067] S5. Colchicine treatment: The germinated seeds were transferred to a 0.05% colchicine solution containing 2% DMSO and soaked for 5 h.

[0068] S6. Three-time disinfection: rinse the seeds soaked in colchicine solution with sterile water three times, and then disinfect with mercuric chloride solution;

[0069] S7. Inoculation: Rinse the seeds disinfected three times with sterile water three times, dry them with sterile filter paper, and inoculate them into MS culture medium for cultivation.

[0070] Furthermore, the colchicine solution is prepared by adding 0.15 g of colchicine powder to 4.5 mL of DMSO, then adding distilled water to make the volume to 300 mL, and stirring evenly.

[0071] Furthermore, in step S4, the germination box is disinfected with alcohol and then covered with two layers of sterile filter paper.

[0072] Furthermore, in step S4, during the germination period, water is added every 24 hours, and moldy seeds are picked out.

[0073] Furthermore, in step S5, the germinated seeds are imbibed and whitened seeds.

[0074] Furthermore, in step S6, the mercuric chloride solution is prepared by dissolving 0.1 g HgCl2 in 100 mL distilled water to prepare a 0.1% mercuric chloride solution, and the disinfection time is 2 minutes.

[0075] Furthermore, in step S4, the method of culturing germination is: culturing under light for 16 hours and culturing in darkness for 8 hours at a temperature of 20°C.

[0076] Comparative Example 1

[0077] Step 1: Select plump Kentucky bluegrass seeds and soak them in sterile water for 24 hours, pick out the unswelled seeds, and then place them on filter paper to dry for 24 hours for later use;

[0078] Step 2: Treat the dried seeds with 75% anhydrous ethanol for 30 seconds, rinse the seeds with sterile water for 30 seconds, and rinse three times;

[0079] Step 3: Soak the seeds in 10% sodium hypochlorite solution for 25 minutes, rinse with sterile water for 30 seconds, rinse five times, and dry with sterile filter paper;

[0080] Step 4: Place the seeds in a germination box for germination. The germination method is as follows: culture under light conditions of 20°C for 16 hours and in darkness for 8 hours. Count the seed germination rate and contamination rate at 13 days and 20 days, respectively. Take the average of the two measured data. See Table 1 for details.

[0081] Comparative Example 2

[0082] Step 1: Select plump Kentucky bluegrass seeds and soak them in sterile water for 24 hours, pick out the unswelled seeds, and then place them on filter paper to dry for 24 hours for later use;

[0083] Step 2: Treat the dried seeds with 75% anhydrous ethanol for 60 seconds, rinse the seeds with sterile water for 30 seconds, and rinse three times;

[0084] Step 3: Soak the seeds in 10% sodium hypochlorite solution for 30 minutes, rinse with sterile water for 30 seconds, rinse five times, and dry with sterile filter paper;

[0085] Step 4: Place the seeds in a germination box for germination. The germination method is as follows: culture under light conditions of 20°C for 16 hours and in darkness for 8 hours. Count the seed germination rate and contamination rate at 13 days and 20 days, respectively. Take the average of the two measured data. See Table 1 for details.

[0086] Comparative Example 3

[0087] This comparative example is the same as comparative example 2 except that the seeds are placed in a 10% sodium hypochlorite solution and soaked for 15 minutes in step 3. See Table 1 for details.

[0088] Comparative Example 4

[0089] In this comparative example, the 75% anhydrous ethanol and 10% sodium hypochlorite solution in comparative example 3 were replaced with sterile water. Other aspects were the same as those in comparative example 3. See Table 1 for details.

[0090] Table 1 Contamination rate and germination rate under different disinfection treatments

[0091]

[0092] It can be seen from Table 1 that Comparative Example 1 uses 75% anhydrous ethanol for disinfection for 30s, the contamination rate is 15.3%, and the germination rate is only 40.7%. The technical solution of the present invention is 75% anhydrous ethanol for disinfection for 50s-70s, the contamination rate is greatly reduced, the lowest is only 5.1%, the germination rate is also greatly improved, up to 83.5%, and Comparative Example 2 uses 10% NaClO for disinfection time increase. Although the contamination rate is really reduced, the effect on seeds is too serious, and the seed germination rate is only 25.4%. Comparative Example 3 has too short disinfection time, high contamination rate and low germination rate, and all fail to achieve the desired effect. Comparative Example 4 does not use medicaments, but simply rinses with sterile water. The contamination rate is as high as 40.3%, and the germination rate is only 28.7%. This also proves the effectiveness of the technical solution of the present invention for selecting medicaments.

[0093] Experimental part

[0094] In order to prove the effect of the present invention, experiments were carried out, which are as follows:

[0095] The Kentucky bluegrass seeds obtained in Example 3 that germinated, imbibed, and turned white after culture in step S4 were immersed in colchicine solutions of different concentrations. The specific treatment method is as follows:

[0096] Method 1: 0.05% colchicine (containing 2% DMSO) soaking for 4 h, 6 h, and 8 h (Groups 1-3);

[0097] Method 2: 0.02% colchicine (containing 2% DMSO) soaked for 6 h (Group 4);

[0098] Method 3: 0.15% colchicine (containing 2% DMSO) was soaked for 6 h (Group 5).

[0099] The treated seeds were rinsed three times with sterile water, disinfected with 0.1% mercuric chloride solution for 2 min, and then rinsed three times with sterile water. The water was absorbed with sterile filter paper and inoculated into MS medium for culture. The survival rates were recorded respectively. The specific combinations and survival rates are shown in Table 2.

[0100] Table 2 Survival rate of tissue culture seedlings under different colchicine treatments

[0101]

[0102] It can be seen from Table 2 that the treatment method of Group 2, which adopted the technical solution of the present invention, had a survival rate of 95.72% after being treated with 0.05% colchicine (containing 2% DMSO) for 6 hours. In the experiments of Groups 1 and 3, although 0.05% colchicine (containing 2% DMSO) was also used, the survival rates decreased significantly if the immersion time was too long or too short. In the experiments of Groups 4 and 5, although the treatment time was 6 hours, the concentrations of colchicine (containing 2% DMSO) used were 0.02% and 0.15%, respectively, and the survival rates also decreased significantly.

[0103] In summary, the method for treating Kentucky bluegrass of the present invention, after being treated with 75% anhydrous ethanol for 50-70 seconds, was then immersed in a 10% sodium hypochlorite solution for 28-33 minutes, which significantly reduced the contamination rate of Kentucky bluegrass and increased the germination rate of Kentucky bluegrass. Furthermore, after the first and second disinfection treatments, on the basis of reducing the contamination rate of Kentucky bluegrass and increasing the germination rate, it was treated with 0.04%-0.06% colchicine solution (containing 2% DMSO) for 5.5-6.5 hours, which further improved the efficiency of Kentucky bluegrass mutagenesis and the survival rate of Kentucky bluegrass tissue culture seedlings.

Claims

1. A method for treating Kentucky bluegrass seeds with colchicine, characterized in that: The steps include: S1. Soaking: Select plump Kentucky bluegrass seeds and soak them in sterile water for 23-25 ​​hours, pick out the seeds that have not been absorbed, and then put them in filter paper to dry for 23-25 ​​hours for later use; S2. Primary disinfection: treat the dried seeds with 75% ethanol for 50-70 seconds, rinse the seeds with sterile water for 30 seconds, and rinse 2-3 times; S3. Secondary disinfection: Soak the seeds that have been disinfected once in a 10% sodium hypochlorite solution for 24-26 minutes, rinse with sterile water for 30 seconds, rinse 4-6 times, and then dry with sterile filter paper; S4, germination cultivation: placing the seeds that have been disinfected twice into a germination box for germination cultivation; S5. Colchicine treatment: The cultured and germinated seeds were transferred into a 0.04%-0.06% colchicine solution containing 2% DMSO and soaked for 5.5-6.5 hours; The 0.04%-0.06% colchicine solution is prepared by adding 0.14g-0.16g of colchicine powder to 4.5mL of DMSO, then adding distilled water to make up to 300mL, and stirring evenly; S6. Three-time disinfection: wash the seeds soaked in colchicine solution with sterile water 2-4 times, and then disinfect with mercuric chloride solution; S7. Inoculation: Rinse the seeds disinfected three times with sterile water for 2-4 times, absorb the moisture with sterile filter paper, and inoculate them into MS culture medium for cultivation.

2. The method for treating Kentucky bluegrass seeds with colchicine according to claim 1, characterized in that: The steps include: S1. Soaking: Select plump Kentucky bluegrass seeds and soak them in sterile water for 24 hours, pick out the seeds that have not been absorbed, and then put them in filter paper to dry for 24 hours for later use; S2. Primary disinfection: treat the dried seeds with 75% ethanol for 60 seconds, rinse the seeds with sterile water for 30 seconds, and rinse three times; S3. Secondary disinfection: Soak the seeds that have been disinfected once in a 10% sodium hypochlorite solution for 25 minutes, rinse with sterile water for 30 seconds, rinse five times, and then dry with sterile filter paper; S4, germination cultivation: placing the seeds that have been disinfected twice into a germination box for germination cultivation; S5. Colchicine treatment: The cultured and germinated seeds were transferred into a 0.05% colchicine solution containing 2% DMSO and soaked for 6 h. S6. Three-time disinfection: wash the seeds soaked in colchicine solution with sterile water three times, and then disinfect with mercuric chloride solution; S7. Inoculation: Rinse the seeds disinfected three times with sterile water, dry them with sterile filter paper, and inoculate them into MS medium for cultivation.

3. The method for treating Kentucky bluegrass seeds with colchicine according to claim 1, characterized in that: In step S4, the germination box is disinfected with alcohol and then covered with two layers of sterile filter paper.

4. The method for treating Kentucky bluegrass seeds with colchicine according to claim 1, characterized in that: In step S4, during the germination period, water is added every 24 hours and moldy seeds are picked out.

5. The method for treating Kentucky bluegrass seeds with colchicine according to claim 1, characterized in that: In step S5, the germinated seeds are cultured to be seeds that have imbibed and turned white.

6. The method for treating Kentucky bluegrass seeds with colchicine according to claim 1, characterized in that: In step S6, the mercuric chloride solution is prepared by dissolving 0.1 g HgCl2 in 100 mL distilled water to prepare a 0.1% mercuric chloride solution, and the disinfection time is 1.5-2.5 min.

7. The method for treating Kentucky bluegrass seeds with colchicine according to claim 1, characterized in that: In step S4, the method of culturing germination is: culturing under light for 15-17 hours and culturing in the dark for 7-9 hours at a temperature of 19° C.-22° C.

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