Method for promoting regeneration of highland barley microspore callus green seedlings

Abstract

The invention provides a method for promoting regeneration of highland barley microspore callus green seedlings, and belongs to the technical field of agricultural biology. The method comprises the following steps: (1) selecting young ears as samples, and carrying out low-temperature pretreatment; (2) putting a sample subjected to low-temperature pretreatment into the extracting solution, carrying out rotary cutting and filtering, and collecting microspores; (3) placing the microspores in an extracting solution for dark treatment, and purifying to obtain purified microspores; (4) culturing the purified microspores in an induction culture medium to form calluses; and (5) transferring the calluses into a differential medium for culturing, and differentiating to form green seedlings. According to the method, the types of curing agents and carbon sources in differential culture media, whether the differential culture media are replaced or not during differentiation and the replacement time are screened, and the result shows that the efficiency of green seedling differentiation of the highland barley microspore calluses can be remarkably improved by adopting the method.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a method for promoting the regeneration of green seedlings of highland barley microspore callus. Background Art

[0002] Plant microspores are haploid sex cells, ideal receptors for cell engineering genetic breeding and genetic transformation, and ideal objects for studying the mechanism of embryogenesis. Under appropriate conditions, isolated microspores can change the original gametophyte development pathway and turn to the sporophyte development pathway, eventually forming a complete plant. Free microspore culture is an effective way to obtain homozygous doubled haploids, and free microspore culture technology is one of the current research hotspots.

[0003] In free microspore culture, the induction and differentiation of callus are the key to successful culture. The culture of free microspores of highland barley can successfully induce callus, but the differentiation stage of callus needs to be improved, that is, the efficiency of callus differentiation into regenerated plants needs to be improved. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for improving the regeneration of green seedlings of highland barley microspore callus. The present invention significantly improves the efficiency of green seedling differentiation of highland barley microspore callus by screening the types of solidifying agents and carbon sources in the differentiation medium and the timing of differentiation medium replacement.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for promoting the regeneration of green seedlings of highland barley microspore callus, comprising the following steps:

[0007] (1) Select young ears as samples and perform low temperature pretreatment;

[0008] (2) placing the sample after low temperature pretreatment in an extraction solution, rotary cutting, filtering, and collecting microspores;

[0009] (3) placing the microspores in an extracting solution for dark treatment and purification to obtain purified microspores;

[0010] (4) culturing the purified microspores in an induction medium to form callus tissue;

[0011] (5) The callus tissue is transferred to a differentiation medium and cultured to differentiate into green seedlings.

[0012] Preferably, the young spikelets are young spikelets in which the microspore development is in the middle to late mononuclear stage.

[0013] Preferably, the temperature of the low-temperature pretreatment is 2 to 10° C., and the time of the low-temperature pretreatment is 7 to 21 days.

[0014] Preferably, the extract uses water as solvent and includes 50-70 g / L mannitol, 0.9-1.3 g / L CaCl2, 0.8-1.1 g / L N-morphine ethane sulfonic acid, and 15-25 mg / L colchicine.

[0015] Preferably, the temperature of the dark treatment is 23-30° C., and the time of the dark treatment is 1-4 days.

[0016] Preferably, the purified solution is a 15% to 25% maltose solution.

[0017] Preferably, the induction medium comprises N6 medium + 40-60 g / L maltose + 0.5-1.5 mg / L kinetin + 1.0-3.0 mg / L 2,4-dichlorophenoxyacetic acid + 0.8-1.1 g / L N-morphine ethane sulfonic acid + 600-800 mg / L glutamine.

[0018] Preferably, the differentiation medium comprises 1 / 2MS+20-40 g / L sugar+0.5-1.5 mg / L 6-benzylaminoadenine+1.5-2.5 mg / L kinetin+0.25-1 mg / L naphthaleneacetic acid+2-6 g / L phytagel.

[0019] Preferably, the sugar is maltose.

[0020] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a method for promoting the regeneration of green seedlings of highland barley microspore callus, and the present invention screens the types of solidifying agents in the differentiation medium, and the results show that the green seedling yield when plant gel is used as the solidifying agent is significantly higher than the green seedling yield when agar powder is used as the solidifying agent; the present invention screens the types of carbon sources in the differentiation medium, and the results show that the green seedling yield when maltose is used as the carbon source in the differentiation medium is significantly higher than that of sucrose; the present invention screens whether to replace the differentiation medium during differentiation and the replacement time, and the results show that replacing the medium once every 2 weeks of differentiation can significantly increase the green seedling yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effect of different solidifying agents in differentiation medium on green seedling yield, where * indicates significant difference at P < 0.05;

[0022] Figure 2 The effect of different carbon sources in differentiation medium on green seedling yield, where * indicates significant difference at P < 0.05;

[0023] Figure 3The effect of the time of changing the differentiation medium on the yield of green seedlings. Different lowercase letters indicate significant differences (P<0.05). DETAILED DESCRIPTION

[0024] The present invention provides a method for promoting the regeneration of green seedlings of highland barley microspore callus, comprising the following steps:

[0025] (1) Select young ears as samples and perform low temperature pretreatment;

[0026] (2) placing the sample after low temperature pretreatment in an extraction solution, rotary cutting, filtering, and collecting microspores;

[0027] (3) placing the microspores in an extracting solution for dark treatment and purification to obtain purified microspores;

[0028] (4) culturing the purified microspores in an induction medium to form callus tissue;

[0029] (5) The callus tissue is transferred to a differentiation medium and cultured to differentiate into green seedlings.

[0030] In the present invention, young ears are selected as samples for low-temperature pretreatment; young ears with microspore development in the middle of the ear in the middle to late mononuclear stage are selected as samples, 1.0 to 1.5 cm of the base of the two upper leaves of the young ears are retained, and the leaves are cut off and placed in a fresh-keeping bag for low-temperature pretreatment; the temperature of the low-temperature pretreatment is 2 to 10°C, preferably 3 to 7°C, and more preferably 5°C, and the time of the low-temperature pretreatment is 7 to 21 days, preferably 10 to 18 days, and more preferably 14 days. The low-temperature pretreatment slows down the development of microspores in anthers and has a low-temperature starvation treatment effect on the microspores, thereby changing the normal developmental pathway of the microspores.

[0031] In the present invention, a sample pretreated at low temperature is placed in an extracting solution, subjected to rotary cutting, filtered, and microspores are collected; the sample pretreated at low temperature is disinfected with 5% to 15% disinfectant for 5 to 15 minutes, washed with sterile water for 4 to 5 times, the sample is introduced into a 50 mL test tube, 10 to 15 mL of the extracting solution is added, a homogenizer is used for high-speed rotary cutting, a suspension is filtered with a 100 to 200 mesh screen, the filtrate is centrifuged at a low speed of 500 to 1000 r / min for 3 to 10 minutes, the process is repeated 3 times, and microspores are collected. The concentration of the disinfectant is preferably 7% to 12%, more preferably 10%, the disinfection time is preferably 7 to 12 minutes, more preferably 10 minutes, the extract uses water as a solvent, and includes mannitol, CaCl2, N-morphine ethane sulfonic acid, and colchicine, wherein the concentration of mannitol is 50 to 70 g / L, preferably 55 to 65 g / L, more preferably 60 g / L, the concentration of CaCl2 is 0.9 to 1.3 g / L, preferably 1.0 to 1.2 g / L, more preferably 1.1 g / L, and the concentration of N-morphine ethane sulfonic acid is 0.8 to 1.1 g / L, preferably 0.9 to 1.0 g / L. / L, more preferably 0.976g / L, the concentration of colchicine is 15-25mg / L, preferably 17-22mg / L, more preferably 20mg / L; the amount of the extract is preferably 11-13mL, more preferably 12mL, the pH of the extract is 5-7, preferably 5.5-6, more preferably 5.8; the mesh number of the sieve is preferably 120-180 mesh, more preferably 150 mesh, the centrifugal speed is preferably 600-800r / min, more preferably 700r / min, and the centrifugal time is preferably 4-7min, more preferably 5min.

[0032] In the present invention, microspores are placed in an extracting solution for dark treatment and purification to obtain purified microspores; the microspores are placed in an extracting solution for dark treatment at 23-30°C for 1-4 days, centrifuged at 500-1000 r / min for 3-10 minutes, the supernatant is removed, 2-8 mL of a maltose solution with a concentration of 15%-25% is added for purification, and centrifuged at 500-1000 r / min for 3-10 minutes, and suspended bands appear, namely, active microspores, and this part of microspores is collected, namely, the purified microspores. The temperature of the dark treatment is preferably 24-28°C, more preferably 26°C, the time of the dark treatment is preferably 2 days, and the dark treatment improves the survival rate of the microspores by changing the normal developmental pathway of the microspores. The concentration of the maltose solution is preferably 18%-23%, more preferably 21%, the amount of the maltose solution added is preferably 3-6mL, more preferably 4mL, the speed of the centrifugation is preferably 600-800r / min, more preferably 700r / min, and the time of the centrifugation is preferably 4-7min, more preferably 5min.

[0033] In the present invention, the purified microspores are placed in an induction medium for culture to form callus tissue; before culture, the purified microspores are washed once with the induction medium, and the microspore density is adjusted to 1.0×10 5 / mL, obtain a microspore suspension, take 0.5-2mL of the obtained microspore suspension and inoculate it in a culture dish, seal it with a sealing film, and dark-treat it at 23-30°C for 15-21 days to obtain callus tissue; the induction culture medium includes N6 culture medium + 40-60g / L maltose + 0.5-1.5mg / L kinetin + 1.0-3.0mg / L 2,4-dichlorophenoxyacetic acid + 0.8-1.1g / L N-morphine ethane sulfonic acid + 600-800mg / L glutamine; preferably N6 culture medium + 45-55g / L maltose + 0.7-1.2mg / L kinetin + 1.5-2.5mg / L 2,4-dichlorophenoxyacetic acid + 0.9-1.0g / L N-morphine ethane sulfonic acid + 700-780 mg / L glutamine, more preferably N6 medium + 50 g / L maltose + 1.0 mg / L kinetin + 2.0 mg / L 2,4-dichlorophenoxyacetic acid + 0.976 g / L N-morphine ethane sulfonic acid + 750 mg / L glutamine; the pH of the induction medium is 5-7, preferably 5.5-6, more preferably 5.8; the inoculation amount of the microspore suspension is preferably 0.7-1.5 mL, more preferably 1 mL, the temperature of the dark treatment is preferably 24-28° C., more preferably 26° C., and the time of the dark treatment is preferably 17-19 d, more preferably 18 d.

[0034] In the present invention, callus tissue is transferred to a differentiation medium for culture and differentiated into green seedlings; the mass ratio of the callus tissue to the differentiation medium is 1:1-4, preferably 1:1.5-3, and more preferably 1:2-2.5, and the differentiation medium includes 1 / 2MS+20-40g / L sugar+0.5-1.5mg / L 6-benzylaminoadenine+1.5-2.5mg / L kinetin+0.25-1mg / L naphthaleneacetic acid+2-6g / L plant gel, preferably 1 / 2MS+25-35g / L sugar+0.7-1.2mg / L 6-benzylaminoadenine+1.7-2.2mg / L kinetin+0.4-0.7mg / L naphthaleneacetic acid+3-5g / L plant gel, and more preferably 1 / 2MS+30g / L sugar+1.0mg / L 6-benzylaminoadenine+2.0 mg / L kinetin+0.5 mg / L naphthaleneacetic acid+4 g / L plant gel, the sugar is maltose, the plant gel is purchased from Beijing Biodi Biotechnology Co., Ltd., the pH of the differentiation medium is 5-7, preferably 5.5-6, and more preferably 5.8.

[0035] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] Preparation of extract: Weigh 60 g of mannitol, 1.1 g of CaCl2, 0.976 g of N-morpholineethane sulfonic acid (MES), and 20 mg of colchicine, dissolve in 1 L of water, adjust the pH to 5.8, and filter sterilize.

[0037] Preparation of induction medium: Use N6 medium as the basic medium, add 50g / L maltose, 1.0mg / L kinetin (KT), 2.0mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.976g / L MES and 750mg / L glutamine, adjust the pH to 5.8, and filter sterilize.

[0038] Preparation of differentiation medium: Use 1 / 2MS as the basic medium, add 30g / L maltose, 1.0mg / L 6-benzylaminopurine (6-BA), 2.0mg / L KT, 0.5mg / L naphthaleneacetic acid (NAA) and 4.0g / L plant gel solidification medium, adjust the pH to 5.8, and sterilize at 0.11Mpa, 121℃ for 15min.

[0039] The extracts, induction medium and differentiation medium used in the subsequent examples were prepared according to the above method.

[0040] Example

[0041] The highland barley material Zangqing 320 was used as a sample and planted at the Nanmen field base of Shanghai Academy of Agricultural Sciences in early November 2022, and the material was collected in early April 2023. The young spikelets with middle microspore development in the middle to late stages of mononuclear were selected from the field, and the base of the two upper leaves was retained 1.0-1.5 cm, the leaves were cut off, and every 40 young spikelets were placed in a fresh-keeping bag as a sample, and placed at 5°C for low-temperature pretreatment for 2 weeks (14 days).

[0042] On a sterile clean bench, disinfect the sample (young spike) to be inoculated with 10% disinfectant for 10 minutes, rinse with sterile water 4 to 5 times, connect the anthers of 5 young spikes to a 50mL test tube, add 12mL of extract, use a homogenizer to high-speed rotary cut, filter the suspension with a 150-mesh screen, and centrifuge the filtrate at a low speed of 700r / min for 5 minutes. Repeat 3 times to collect microspores.

[0043] The collected microspores were treated with extract at 26°C in the dark for 2 days, centrifuged at 700 r / min for 5 minutes, the supernatant was removed, 4 mL of 21% maltose solution was added for purification, and centrifuged at 700 r / min for 5 minutes. Suspended bands appeared, which were active microspores. The microspores collected were the purified microspores.

[0044] After the purified microspores were washed once with the induction medium, the microspore density was adjusted to 1.0×10 5 / mL, to obtain a microspore suspension, take 1.0mL of the microspore suspension and inoculate it into an empty culture dish (35mm×12mm), seal it with a sealing film, and culture it at 26℃ in the dark for 18 days to form callus tissue.

[0045] Weigh 15 mg of the formed callus tissue and transfer it into 35 mL of differentiation medium. Repeat each treatment 3 times to induce the differentiation of callus tissue and the formation of green seedlings. When the green seedlings grow to 2 to 3 cm, pick them out and count the number of green seedlings.

[0046] Comparative Example 1

[0047] The method described in the example was adopted, except that in this comparative example, the differentiation medium was replaced with 1 / 2MS + 30 g / L maltose + 1.0 mg / L 6-benzylaminoadenine + 2.0 mg / L kinetin + 0.5 mg / L naphthaleneacetic acid + 6 g / L agar powder.

[0048] Comparative Example 2

[0049] The method described in the example was adopted, except that, in this comparative example, the differentiation medium was replaced with 1 / 2MS+30g / L sucrose+1.0mg / L 6-benzylaminoadenine+2.0mg / L kinetin+0.5mg / L naphthaleneacetic acid+4g / L phytagel.

[0050] Experimental Example 1

[0051] The number of green seedlings obtained by the method in Example 1 and Comparative Example 1 was counted respectively, and the effect of the type of solidifying agent in the differentiation medium on the yield of green seedlings was analyzed. The results are shown in Figure 1 .

[0052] Green seedling yield: the number of green seedlings differentiated from 100 mg of callus tissue, expressed as plant / 100 mg of callus tissue;

[0053] All experimental results were analyzed using Microsoft Excel 2010, DPS v7.05 data processing system.

[0054] Depend on Figure 1 It can be seen that in the differentiation medium with plant gel as the solidifying agent, an average of 233 green seedlings can be differentiated from every 100 mg of callus tissue, while in the differentiation medium with agar powder as the solidifying agent, an average of 89 green seedlings can be differentiated from every 100 mg of callus tissue. It can be seen that the green seedling yield of callus tissue in the differentiation medium with plant gel as the solidifying agent is significantly better than that in the differentiation medium with agar powder as the solidifying agent.

[0055] Experimental Example 2

[0056] The number of green seedlings obtained by the method in Example 1 and Comparative Example 2 was counted respectively, and the effect of the type of carbon source in the differentiation medium on the yield of green seedlings was analyzed. The results are shown in Figure 2 , the green seedling yield is calculated according to the method in Experimental Example 1.

[0057] Depend on Figure 2 It can be seen that in the differentiation medium with maltose as the carbon source, an average of 233 green seedlings can be differentiated from every 100 mg of callus tissue, while in the differentiation medium with sucrose as the carbon source, an average of 135 green seedlings can be differentiated from every 100 mg of callus tissue. It can be seen that the green seedling yield of callus tissue in maltose is significantly better than that in sucrose.

[0058] Experimental Example 3

[0059] The differentiation medium in the embodiment was used to analyze the effect of the time of changing the differentiation medium on the regeneration of green seedlings of highland barley microspore callus. The callus obtained by highland barley microspore culture was transferred to the differentiation medium in the embodiment. One group did not change the differentiation medium after entering the differentiation, one group replaced the fresh differentiation medium after 2 weeks of differentiation, and one group replaced the fresh differentiation medium after 4 weeks of differentiation. The number of differentiated green seedlings was counted and the green seedling yield was analyzed. The results are shown in Figure 3 .

[0060] Depend on Figure 3 It can be seen that replacing the culture medium during the differentiation process can increase the green seedling yield of callus tissue, and the green seedling yield of timely replacement within 2 weeks is significantly better than the green seedling yield when the culture medium is not replaced or when the culture medium is replaced at 4 weeks.

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for promoting the regeneration of green seedlings from highland barley microspore callus, characterized in that: The steps include: (1) Select young ears as samples and perform low temperature pretreatment; (2) placing the sample after low temperature pretreatment in an extraction solution, rotary cutting, filtering, and collecting microspores; (3) placing the microspores in an extracting solution for dark treatment and purification to obtain purified microspores; (4) culturing the purified microspores in an induction medium to form callus tissue; (5) The callus tissue is transferred to a differentiation medium and cultured to differentiate into green seedlings.

2. The method according to claim 1, characterized in that The young spikelets are young spikelets whose microspore development is in the middle to late mononuclear stage.

3. The method according to claim 1, characterized in that: The temperature of the low temperature pretreatment is 2-10° C., and the time of the low temperature pretreatment is 7-21 days.

4. The method according to claim 1, characterized in that The extract uses water as solvent and includes 50-70 g / L mannitol, 0.9-1.3 g / L CaCl2, 0.8-1.1 g / L N-morphine ethane sulfonic acid, and 15-25 mg / L colchicine.

5. The method according to claim 1, characterized in that The temperature of the dark treatment is 23-30° C., and the time of the dark treatment is 1-4 days.

6. The method according to claim 1, characterized in that The purified solution is a 15% to 25% maltose solution.

7. The method according to claim 1, characterized in that The induction culture medium comprises N6 culture medium+40-60 g / L maltose+0.5-1.5 mg / L kinetin+1.0-3.0 mg / L 2,4-dichlorophenoxyacetic acid+0.8-1.1 g / L N-morphine ethane sulfonic acid+600-800 mg / L glutamine.

8. The method according to claim 1, characterized in that The differentiation culture medium comprises 1 / 2MS+20-40g / L sugar+0.5-1.5mg / L 6-benzylaminoadenine+1.5-2.5mg / L kinetin+0.25-1mg / L naphthaleneacetic acid+2-6g / L plant gel.

9. The method according to claim 8, characterized in that The sugar is maltose.

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