Application of glutathione solution in increasing yield of highland barley microspore calluses

Abstract

The invention provides application of a glutathione solution in increasing the yield of highland barley microspore calluses, and belongs to the technical field of plant breeding. According to the method disclosed by the invention, the yield of the highland barley microspore calluses can be obviously improved by soaking highland barley young ears in a glutathione (GSH) aqueous solution or adding glutathione (GSH) into a highland barley microspore callus induction culture medium.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and particularly relates to application of glutathione solution in improving the yield of highland barley microspore callus. Background Art

[0002] Highland barley is the main food crop and cash crop for Tibetan compatriots. However, the germplasm resources of highland barley are scarce and there are few new varieties, so the genetic improvement of highland barley is particularly important. Plant microspores are haploid sex cells. When given appropriate culture conditions in vitro, they can change their original gametophyte development pathway and turn to the sporophyte development pathway, eventually forming a complete plant. Microspore haploid breeding technology can provide an efficient and stable method for the genetic improvement of highland barley.

[0003] The current microspore haploid breeding technology has been successful in a few highland barley varieties, but there are problems such as genotype dependence, low callus induction rate, difficulty in differentiating green seedlings, and serious albino seedling phenomenon, which greatly limit the application of microspore culture technology in highland barley genetic breeding research. Summary of the invention

[0004] In view of this, the object of the present invention is to provide an application of glutathione solution in improving the yield of highland barley microspore callus, so as to solve the problems of genotype dependence, low callus induction rate, difficulty in green seedling differentiation, serious albino seedling phenomenon, etc. in existing highland barley breeding methods.

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

[0006] The invention provides application of a glutathione solution in improving the yield of highland barley microspore callus tissue. The concentration of the glutathione solution is 80-100 mg / L.

[0007] Preferably, the young highland barley ears are soaked in a glutathione solution at low temperature.

[0008] Preferably, the method specifically comprises the following steps:

[0009] 1) Select young highland barley spikelets with microspore development in the middle of the spikelet at the middle to late stages of mononuclear development, pre-treat them at low temperature, and then soak them in a glutathione solution at low temperature;

[0010] 2) taking anthers on young spikelets of highland barley soaked at low temperature in step 1), adding an extract and rotary cutting to obtain a suspension, filtering and centrifuging the suspension to obtain free microspores;

[0011] 3) pre-treating the free microspores with an extract, and then sequentially performing purification culture and induction culture. Preferably, the temperature of the low-temperature pre-treatment and low-temperature soaking in step 1) is 3-7°C, and the time of the low-temperature pre-treatment and low-temperature soaking is 5-9 days;

[0012] Step 2) The extract comprises the following components in concentrations: 50-70 g / L mannitol, 0.8-1.5 g / L CaCl2, 0.8-1.2 g / L N-morphine ethane sulfonic acid and 8-12 mg / L colchicine;

[0013] The rotation speed of the peeling is 2500-3500 rpm, the number of the peeling is 2-4 times, and the peeling time is 8-12 seconds / time;

[0014] The centrifugal speed is 600-800 rpm, the number of centrifugation is 2-4 times, and the centrifugal time is 3-8 min / time.

[0015] Preferably, the induction culture in step 3) uses an induction medium, wherein the induction medium uses N6 medium as a basic medium and comprises the following components in concentrations: 80-100 g / L maltose, 0.3-0.8 mg / L kinetin, 0.5-1.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.2-0.6 g / L hydrolyzed casein, 0.8-1.2 g / L MES and 1-2 g / L glutamine;

[0016] The induction culture is dark culture, the temperature of the induction culture is 23-28° C., and the time of the induction culture is 17-21 days.

[0017] Preferably, glutathione is added to the highland barley microspore callus induction medium.

[0018] The invention also provides a highland barley microspore callus induction culture medium, which uses N6 culture medium as a basic culture medium and comprises the following components in concentrations: 80-100 mg / L glutathione, 80-100 g / L maltose, 0.3-0.8 mg / L kinetin, 0.5-1.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.2-0.6 g / L hydrolyzed casein, 0.8-1.2 g / L MES and 1-2 g / L glutamine.

[0019] Preferably, the method specifically comprises the following steps:

[0020] 1) Select young highland barley ears whose microspore development in the middle of the ear is in the middle to late mononuclear stage, and perform low temperature pretreatment;

[0021] 2) taking anthers on young spikelets of highland barley pretreated at low temperature in step 1), adding the extract and rotary cutting to obtain a suspension, filtering and centrifuging the suspension to obtain free microspores;

[0022] 3) pre-treating the free microspores with an extract, purifying and culturing them, and then inducing them with the highland barley microspore callus induction medium.

[0023] Preferably, the temperature of the low-temperature pretreatment in step 1) is 3 to 7°C, and the time of the low-temperature pretreatment is 5 to 9 days;

[0024] Step 3) The induction culture is dark culture, the temperature of the induction culture is 23-28° C., and the time of the induction culture is 17-21 days.

[0025] Preferably, the extract in step 2) comprises the following components in concentrations: 50-70 g / L mannitol, 0.8-1.5 g / L CaCl2, 0.8-1.2 g / L N-morphine ethane sulfonic acid and 8-12 mg / L colchicine;

[0026] The rotation speed of the peeling is 2500-3500 rpm, the number of the peeling is 2-4 times, and the peeling time is 8-12 seconds / time;

[0027] The centrifugal speed is 600-800 rpm, the number of centrifugation is 2-4 times, and the centrifugal time is 3-8 min / time.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The invention can significantly increase the yield of highland barley microspore callus by soaking highland barley spikelets in a glutathione (GSH) aqueous solution or adding glutathione (GSH) to a highland barley microspore callus induction medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The effect of GSH aqueous solution treatment on the callus yield of microspore culture of highland barley variety 'Dongqing 19';

[0031] Figure 2 The effect of adding GSH to the highland barley microspore callus induction medium on the microspore callus yield of highland barley varieties 'Dongqing 19', 'Xila 22' and 'Kunlun 14'. DETAILED DESCRIPTION

[0032] The present invention provides the use of glutathione solution in improving the yield of highland barley microspore callus, wherein the concentration of the glutathione solution is 80-100 mg / L. In the present invention, the concentration of the glutathione is preferably 85-95 mg / L.

[0033] In the present invention, young highland barley ears are placed in a glutathione solution and soaked at low temperature. Specifically, the process comprises the following steps:

[0034] 1) Select young highland barley spikelets with microspore development in the middle of the spikelet at the middle to late stages of mononuclear development, pre-treat them at low temperature, and then soak them in a glutathione solution at low temperature;

[0035] 2) taking anthers on young spikelets of highland barley soaked at low temperature in step 1), adding an extract and rotary cutting to obtain a suspension, filtering and centrifuging the suspension to obtain free microspores;

[0036] 3) The free microspores are pretreated with an extract, and then subjected to purification culture and induction culture in sequence.

[0037] In the present invention, young barley spikelets with microspores in the middle of the spikelet in the middle-late mononuclear stage are selected, and after low-temperature pretreatment, they are placed in a glutathione solution for low-temperature soaking. The young barley spikelets retain 1.0-1.5 cm of the base of the two upper leaves, and the leaves are cut off; the low-temperature pretreatment is preferably carried out in a fresh-keeping bag, and the temperature of the low-temperature pretreatment is preferably 3-7°C, more preferably 4-6°C; the time of the low-temperature pretreatment is preferably 5-9d, more preferably 6-8d; the temperature of the low-temperature soaking is preferably 3-7°C, more preferably 4-6°C; the time of the low-temperature soaking is preferably 5-9d, more preferably 6-8d.

[0038] In the present invention, the anthers on the young barley spikelets soaked at low temperature in step 1) are taken, and the extract is added and peeled to obtain a suspension, and the suspension is filtered and centrifuged to obtain free microspores. The young barley spikelets soaked at low temperature are preferably first disinfected, and the disinfection is preferably carried out using disinfectant, and the concentration of the disinfectant is preferably 8-10%, and more preferably 9-11%; the disinfection time is preferably 8-12 minutes, and more preferably 9-11 minutes; after the disinfection, it is washed with sterile water for 4-5 times; the anther collection method is preferably: manually using sterile tweezers on a sterile clean bench to take out the anthers from the flower buds on the young barley spikelets soaked at low temperature after disinfection, and the number of anthers is preferably 350-450 pieces / tube, and more preferably 380-420 pieces / tube. The extract includes the following concentrations of components: 50-70g / L mannitol, 0.8-1.5g / L CaCl2, 0.8-1.2g / L N-morphine ethane sulfonic acid and 8-12 mg / L colchicine; the concentration of mannitol is preferably 55-65 g / L; the concentration of CaCl2 is preferably 0.9-1.4 g / L; the concentration of N-morphine ethane sulfonic acid is preferably 0.9-1.1 g / L; the concentration of colchicine is preferably 9-11 mg / L; the pH of the extract is preferably 5.5-6.2, more preferably 5.6-6.1; the rotary cutting preferably adopts a homogenizer, and the rotary cutting speed is preferably 2500-3500 rpm, more preferably 2800-3200 rpm; the number of the peeling is preferably 2 to 4 times, more preferably 3 times; the peeling time is preferably 8 to 12 seconds / time, more preferably 9 to 11 seconds / time; the filtration is preferably performed using a sieve, the mesh size of the sieve is preferably 100 to 200 meshes, more preferably 150 meshes; the centrifugal speed is preferably 600 to 800 rpm, more preferably 650 to 750 rpm; the number of the centrifugation is preferably 2 to 4 times, more preferably 3 times; the centrifugal time is preferably 3 to 8 min / time, more preferably 4 to 7 min / time.

[0039] In the present invention, the free microspores are pretreated with an extract, and then purified and induced. The pretreatment is preferably carried out in the dark, the pretreatment temperature is preferably 23 to 28°C, more preferably 24 to 27°C; the pretreatment time is preferably 1 to 3 days, more preferably 2 days; the density of the free microspores is preferably 0.5×10 5 ~1.5×10 5 / mL, more preferably 1.0×10 5The induction culture uses an induction medium, which uses N6 medium as the basic medium and includes the following components at the following concentrations: 80-100 g / L maltose, 0.3-0.8 mg / L kinetin, 0.5-1.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.2-0.6 g / L hydrolyzed casein, 0.8-1.2 g / L MES and 1-2 g / L glutamine; the concentration of maltose is preferably 85-95 g / L; the concentration of kinetin is preferably 0.4-0.7 mg / L; the concentration of 2,4-dichlorophenoxyacetic acid is preferably 0.8-1.2 mg / L; the concentration of hydrolyzed casein is preferably 0.3-0.5 g / L; the concentration of MES is preferably 0.9-1.1 g / L; the concentration of glutamine is preferably 1.2-1.8 g / L; the pH of the induction medium is preferably 5.5-6.2, and more preferably 5.6-6.1; the microspores are washed with the induction medium before the induction culture; the induction culture is dark culture, and the temperature of the induction culture is preferably 23-28°C, and more preferably 24-27°C; the time of the induction culture is preferably 17-21 days, and more preferably 18-20 days.

[0040] The invention also provides a highland barley microspore callus induction culture medium, which uses N6 culture medium as a basic culture medium and comprises the following components in concentrations: 80-100 mg / L glutathione, 80-100 g / L maltose, 0.3-0.8 mg / L kinetin, 0.5-1.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.2-0.6 g / L hydrolyzed casein, 0.8-1.2 g / L MES and 1-2 g / L glutamine. In the present invention, the concentration of glutathione is preferably 85-95 mg / L; the concentration of maltose is preferably 85-95 g / L; the concentration of kinetin is preferably 0.4-0.7 mg / L; the concentration of 2,4-dichlorophenoxyacetic acid is preferably 0.8-1.2 mg / L; the concentration of hydrolyzed casein is preferably 0.3-0.5 g / L; the concentration of MES is preferably 0.9-1.1 g / L; the concentration of glutamine is preferably 1.2-1.8 g / L; the pH of the highland barley microspore callus induction medium is preferably 5.5-6.2, and more preferably 5.6-6.1;.

[0041] In the present invention, glutathione is added to the highland barley microspore callus induction medium, which specifically includes the following steps:

[0042] 1) Select young highland barley ears whose microspore development in the middle of the ear is in the middle to late mononuclear stage, and perform low temperature pretreatment;

[0043] 2) taking anthers on young spikelets of highland barley pretreated at low temperature in step 1), adding the extract and rotary cutting to obtain a suspension, filtering and centrifuging the suspension to obtain free microspores;

[0044] 3) pre-treating the free microspores with an extract, purifying and culturing them, and then inducing them with the highland barley microspore callus induction medium.

[0045] In the present invention, young barley spikelets with microspores in the middle of the spikelet in the middle-late mononuclear stage are selected for low-temperature pretreatment. The young barley spikelets retain 1.0-1.5 cm of the base of the two upper leaves and cut off the leaves; the low-temperature pretreatment is preferably carried out in a fresh-keeping bag, the temperature of the low-temperature pretreatment is preferably 3-7°C, more preferably 4-6°C; the time of the low-temperature pretreatment is preferably 5-9 days, more preferably 6-8 days.

[0046] In the present invention, the anthers on the young barley spikelets after low-temperature pretreatment in step 1) are taken, and the extract is added and peeled to obtain a suspension, and the suspension is filtered and centrifuged to obtain free microspores. The young barley spikelets after low-temperature pretreatment are preferably first disinfected, and the disinfection is preferably carried out using disinfectant, and the concentration of the disinfectant is preferably 8-10%, and more preferably 9-11%; the disinfection time is preferably 8-12 minutes, and more preferably 9-11 minutes; after the disinfection, it is washed with sterile water 4-5 times; the anther collection method is preferably: manually using sterile tweezers on a sterile clean bench to take out the anther clips from the flower buds on the young barley spikelets after low-temperature soaking after disinfection, and the number of the anthers is preferably 350-450 pieces / tube, and more preferably 380-420 pieces / tube. The extract includes the following concentrations of components: 50-70g / L mannitol, 0.8-1.5g / L CaCl2, 0.8-1.2g / L N-morphine ethane sulfonic acid and 8-12 mg / L colchicine; the concentration of mannitol is preferably 55-65 g / L; the concentration of CaCl2 is preferably 0.9-1.4 g / L; the concentration of N-morphine ethane sulfonic acid is preferably 0.9-1.1 g / L; the concentration of colchicine is preferably 9-11 mg / L; the pH of the extract is preferably 5.5-6.2, more preferably 5.6-6.1; the rotary cutting preferably adopts a homogenizer, and the rotary cutting speed is preferably 2500-3500 rpm, more preferably 2800-3200 rpm; the number of the peeling is preferably 2 to 4 times, more preferably 3 times; the peeling time is preferably 8 to 12 seconds / time, more preferably 9 to 11 seconds / time; the filtration is preferably performed using a sieve, the mesh size of the sieve is preferably 100 to 200 meshes, more preferably 150 meshes; the centrifugal speed is preferably 600 to 800 rpm, more preferably 650 to 750 rpm; the number of the centrifugation is preferably 2 to 4 times, more preferably 3 times; the centrifugal time is preferably 3 to 8 min / time, more preferably 4 to 7 min / time.

[0047] In the present invention, the free microspores are pretreated with an extract, purified and cultured, and then induced with the highland barley microspore callus induction medium. The pretreatment is preferably carried out in the dark, the pretreatment temperature is preferably 23 to 28°C, more preferably 24 to 27°C; the pretreatment time is preferably 1 to 3 days, more preferably 2 days; the density of the free microspores is preferably 0.5×10 5 ~1.5×10 5 / mL, more preferably 1.0×10 5 / mL; before the induction culture, the microspores are washed using a highland barley microspore callus induction medium; the induction culture is dark culture, and the temperature of the induction culture is preferably 23-28°C, and more preferably 24-27°C; the time of the induction culture is preferably 17-21d, and more preferably 18-20d.

[0048] 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.

[0049] The highland barley variety materials 'Dongqing 19', 'Xila 22', and 'Kunlun 14' were planted in the large fields at the south gate of Shanghai Academy of Agricultural Sciences at the end of November 2022, and the materials began to be harvested in early April 2023.

[0050] Example 1

[0051] Select young highland barley spikelets with middle microspore development in the middle to late mononuclear stage from the field, retain the 1.3 cm base of the two upper leaves, cut off the leaves, put them in a fresh-keeping bag, pre-treat them at 5°C for 7 days, cut open the lower part of the fresh-keeping bag and take out the young spikelets. The young spikelets are immersed in a 90 mg / L GSH aqueous solution at 5°C for 7 days.

[0052] On a sterile clean bench, the young spikelets after low-temperature immersion were disinfected with 10% disinfectant for 10 min, rinsed with sterile water 4 times, and the anther clips were taken out from the flower buds on the young spikelets of highland barley after low-temperature immersion of the disinfection process using sterile tweezers. 400 anthers were accessed into a 50 ml test tube, and 12 ml of extract (60 g / L mannitol, 1.1 g / L CaCl2, 0.976 g / L N-morpholine ethane sulfonic acid (MES) and 10 mg / L colchicine, pH 5.8) were added. A homogenizer was used to perform high-speed rotary cutting to obtain a suspension, and the rotating speed was 3000 rpm. The rotary cutting was performed at room temperature, and each tube was rotary cut 3 times, each time for 10 seconds. The suspension was filtered with a 150 mesh screen, and the filtrate was centrifuged at a low speed of 700 rpm for 5 min at room temperature, repeated 3 times, and free microspores were collected.

[0053] The collected free microspores were pretreated with the extract at 25°C in the dark for 2 days. The pretreated free microspores were purified and cultured. Before culture, they were washed once with induction medium (90 g / L maltose, 0.5 mg / L kinetin (KT), 1.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.4 g / L hydrolyzed casein, 0.976 g / L MES and 1.6 g / L glutamine, pH 5.8), and then the microspore density was adjusted to 1.0×10 5 / mL, take 1.0mL of microspore suspension and inoculate it into a culture dish (35mm×12mm), culture it with induction medium, seal it with sealing film, and culture it at 25℃ in the dark for 19 days. Both the extract and induction medium are sterilized by 0.22μm membrane filtration and stored in a refrigerator at 4℃.

[0054] Example 2

[0055] Select young spikelets of highland barley with middle microspore development in the middle to late mononuclear stage from the field, retain the 1.0 cm base of the two upper leaves, cut off the leaves, put them in a fresh-keeping bag, pre-treat them at 3°C ​​for 5 days, cut open the lower part of the fresh-keeping bag and take out the young spikelets. The young spikelets are immersed in 80 mg / L GSH aqueous solution at 3°C ​​for 5 days.

[0056] On a sterile clean bench, the young spikelets after low-temperature immersion were disinfected with 10% disinfectant for 10 min, rinsed with sterile water 4 times, and the anther clips were taken out from the flower buds on the young spikelets of highland barley after low-temperature immersion of the disinfection process using sterile tweezers. 350 anthers were accessed in a 50 ml test tube, and 12 ml of extract (50 g / L mannitol, 0.8 g / L CaCl2, 0.8 g / L N-morphine ethane sulfonic acid and 8 mg / L colchicine, pH 5.5) was added. A homogenizer was used to perform high-speed rotary cutting to obtain a suspension, and the rotating speed was 2500 rpm. The rotary cutting was performed at room temperature, and each tube was rotary cut 2 times, each time lasting 8 seconds. The suspension was filtered with a 100 mesh screen, and the filtrate was centrifuged at a low speed of 600 rpm for 3 min at room temperature, repeated 2 times, and free microspores were collected.

[0057] The collected free microspores were pretreated with the extract at 23°C in the dark for 1 day. The pretreated free microspores were purified and cultured. Before culture, they were washed once with induction medium (80 g / L maltose, 0.3 mg / L kinetin, 0.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.2 g / L hydrolyzed casein, 0.8 g / L MES and 1 g / L glutamine, pH 5.5), and then the microspore density was adjusted to 0.5×10 5 / mL, take 1.0mL of microspore suspension and inoculate it into a culture dish (35mm×12mm), culture it with induction medium, seal it with sealing film, and culture it in the dark at 23℃ for 17 days. Both the extract and induction medium are sterilized by filtration with a 0.22μm membrane and stored in a refrigerator at 4℃.

[0058] Example 3

[0059] Select young spikelets of highland barley with middle microspore development in the middle to late mononuclear stage from the field, retain the 1.5 cm base of the two upper leaves, cut off the leaves, put them in a fresh-keeping bag, pre-treat them at 7°C for 9 days, cut open the lower part of the fresh-keeping bag and take out the young spikelets. The young spikelets are immersed in a 100 mg / L GSH aqueous solution at 7°C for 9 days.

[0060] On a sterile clean bench, the young spikelets after low-temperature immersion were disinfected with 10% disinfectant for 10 min, rinsed with sterile water 5 times, and the anther clips were taken out from the flower buds on the young spikelets of highland barley after low-temperature immersion of the disinfection process using sterile tweezers. 450 anthers were accessed in a 50 ml test tube, 12 ml of extract (70 g / L mannitol, 1.5 g / L CaCl2, 1.2 g / L N-morphine ethane sulfonic acid and 12 mg / L colchicine, pH 6.2) were added, and a homogenizer was used to high-speed rotary cutting to obtain a suspension, the rotating speed was 3500 rpm, the rotary cutting was normal temperature rotary cutting, and each tube was rotary cutting 4 times, each time lasting 12 seconds, and the suspension was filtered with a 200 mesh screen, and the filtrate was centrifuged at a low speed of 800 rpm for 8 min at normal temperature, repeated 4 times, and free microspores were collected.

[0061] The collected free microspores were pretreated with the extract at 28°C in the dark for 3 days. The pretreated free microspores were purified and cultured. Before culture, they were washed once with induction medium (100 g / L maltose, 0.8 mg / L kinetin, 1.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.6 g / L hydrolyzed casein, 1.2 g / L MES and 2 g / L glutamine, pH 6.2), and then the microspore density was adjusted to 1.5×10 5 / mL, take 1.0mL of microspore suspension and inoculate it into a culture dish (35mm×12mm), culture it with induction medium, seal it with sealing film, and culture it at 28℃ in the dark for 21 days. Both the extract and induction medium are sterilized by 0.22μm membrane filtration and stored in a refrigerator at 4℃.

[0062] Example 4

[0063] Select young highland barley ears with middle microspore development in the middle to late mononuclear stage from the field, retain 1.3 cm from the base of the two upper leaves, cut off the leaves, put them in a fresh-keeping bag, pre-treat them at 5°C for 7 days, cut the lower part of the fresh-keeping bag and take out the young ears.

[0064] On a sterile clean bench, the young spikelets after low temperature pretreatment were disinfected with 10% disinfectant for 10 min, rinsed with sterile water 4 times, and the anther clips were taken out from the flower buds on the young spikelets of highland barley after low temperature immersion of the disinfection treatment by artificial sterile tweezers, 400 anthers were accessed in a 50 ml test tube, 12 ml of extract (60 g / L mannitol, 1.1 g / L CaCl2, 0.976 g / L N-morpholine ethane sulfonic acid (MES) and 10 mg / L colchicine, pH 5.8) were added, and a suspension was obtained by high-speed rotary cutting using a homogenizer, the rotating speed was 3000 rpm, the rotary cutting was normal temperature rotary cutting, and each tube was rotary cut 3 times, each time lasting 10 seconds, and the suspension was filtered with a 150 mesh screen, and the filtrate was centrifuged at a low speed of 700 rpm for 5 min at normal temperature, repeated 3 times, and free microspores were collected.

[0065] The collected free microspores were pretreated with the extract at 25°C in the dark for 2 days. The pretreated free microspores were purified and cultured. Before culture, they were washed once with highland barley microspore callus induction medium (90 mg / L glutathione, 90 g / L maltose, 0.5 mg / L kinetin (KT), 1.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.4 g / L hydrolyzed casein, 0.976 g / L MES and 1.6 g / L glutamine, pH 5.8), and then the microspore density was adjusted to 1.0×10 5 / mL, take 1.0mL of microspore suspension and inoculate it into a culture dish (35mm×12mm), culture it with highland barley microspore callus induction medium, seal it with sealing film, and culture it in the dark at 25℃ for 19 days. Both the extract and highland barley microspore callus induction medium were sterilized by filtration with a 0.22μm membrane and stored in a refrigerator at 4℃.

[0066] Example 5

[0067] Select young highland barley ears with middle microspore development in the middle to late mononuclear stage from the field, retain 1.0 cm from the base of the two upper leaves, cut off the leaves, put them in a fresh-keeping bag, pre-treat them at 3°C ​​for 5 days, cut the lower part of the fresh-keeping bag and take out the young ears.

[0068] On a sterile clean bench, the young spikelets after low temperature pretreatment were disinfected with 10% disinfectant for 10 min, rinsed with sterile water 4 times, and the anther clips were taken out from the flower buds on the young spikelets of highland barley after low temperature immersion of the disinfection treatment by artificial sterile tweezers, 350 anthers were accessed in a 50 ml test tube, 12 ml of extract (50 g / L mannitol, 0.8 g / L CaCl2, 0.8 g / L N-morphine ethane sulfonic acid and 8 mg / L colchicine, pH 5.5) were added, and a homogenizer was used to high-speed rotary cutting to obtain a suspension, the rotating speed was 2500 rpm, the rotary cutting was normal temperature rotary cutting, and each tube was rotary cutting 2 times, each time lasting 8 seconds, and the suspension was filtered with a 100 mesh screen, and the filtrate was centrifuged at a low speed of 600 rpm for 3 min at normal temperature, repeated 2 times, and free microspores were collected.

[0069] The collected free microspores were pretreated with the extract at 23°C in the dark for 1 day. The pretreated free microspores were purified and cultured. Before culture, they were washed once with highland barley microspore callus induction medium (80 mg / L glutathione, 80 g / L maltose, 0.3 mg / L kinetin, 0.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.2 g / L hydrolyzed casein, 0.8 g / L MES and 1 g / L glutamine, pH 5.5), and then the microspore density was adjusted to 0.5×10 5 / mL, take 1.0mL of microspore suspension and inoculate it into a culture dish (35mm×12mm), culture it with highland barley microspore callus induction medium, seal it with sealing film, and culture it in the dark at 23℃ for 17 days. Both the extract and highland barley microspore callus induction medium were sterilized by filtration with a 0.22μm membrane and stored in a refrigerator at 4℃.

[0070] Example 6

[0071] Select young highland barley ears with middle microspore development in the middle to late mononuclear stage from the field, retain 1.5 cm from the base of the two upper leaves, cut off the leaves, put them in a fresh-keeping bag, pre-treat them at 7°C for 9 days, cut the lower part of the fresh-keeping bag and take out the young ears.

[0072] On a sterile clean bench, the young spikelets after low-temperature immersion were disinfected with 10% disinfectant for 10 min, rinsed with sterile water 5 times, and the anther clips were taken out from the flower buds on the young spikelets of highland barley after low-temperature immersion of the disinfection process using sterile tweezers. 450 anthers were accessed in a 50 ml test tube, 12 ml of extract (70 g / L mannitol, 1.5 g / L CaCl2, 1.2 g / L N-morphine ethane sulfonic acid and 12 mg / L colchicine, pH 6.2) were added, and a homogenizer was used to high-speed rotary cutting to obtain a suspension, the rotating speed was 3500 rpm, the rotary cutting was normal temperature rotary cutting, and each tube was rotary cutting 4 times, each time lasting 12 seconds, and the suspension was filtered with a 200 mesh screen, and the filtrate was centrifuged at a low speed of 800 rpm for 8 min at normal temperature, repeated 4 times, and free microspores were collected.

[0073] The collected free microspores were pretreated with the extract at 28°C in the dark for 3 days. The pretreated free microspores were purified and cultured. Before culture, they were washed once with highland barley microspore callus induction medium (100 mg / L glutathione, 100 g / L maltose, 0.8 mg / L kinetin, 1.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.6 g / L hydrolyzed casein, 1.2 g / L MES and 2 g / L glutamine, pH 6.2), and then the microspore density was adjusted to 1.5×10 5 / mL, take 1.0mL of microspore suspension and inoculate it into a culture dish (35mm×12mm), culture it with highland barley microspore callus induction medium, seal it with sealing film, and culture it in the dark at 28℃ for 21 days. Both the extract and highland barley microspore callus induction medium were sterilized by filtration with a 0.22μm membrane and stored in a refrigerator at 4℃.

[0074] Experimental Example 1

[0075] The method of Example 1 was used to culture the callus of 'Dongqing 19' highland barley, and the young highland barley spikelets pretreated by low temperature soaking in water were used as the control. The callus mass produced by each dish of microspore culture for 19 days was counted and expressed in "mg". All test results were analyzed using Microsoft Excel 2010, DPS v7.05 version data processing system.

[0076] Experimental results: Figure 1 As shown. Figure 1 It can be seen that treating the detached spikelets with GSH aqueous solution can significantly increase the microspore culture callus yield of the highland barley variety 'Dongqing 19'. The average microspore culture callus yield of the control group was 98.73 mg per dish, while the average microspore culture callus yield of the detached spikelets treated with 90 mg / L aqueous solution was 126.83 mg per dish, which is 1.28 times that of the control group.

[0077] Experimental Example 2

[0078] The method of Example 4 was used to culture highland barley callus of 'Dongqing 19', 'Xi La 22' and 'Kun Lun 14', and the highland barley microspore callus induction medium without adding 90 mg / L glutathione (GSH) was used as a control. The mass of callus produced by each dish of microspore culture for 19 days was counted and expressed in "mg". All test results were analyzed using Microsoft Excel 2010, DPSv7.05 version data processing system.

[0079] Experimental results: Figure 2 As shown. Figure 2 It can be seen that adding 90 mg / L glutathione (GSH) to the callus induction medium can significantly increase the microspore culture callus yield of highland barley varieties 'Dongqing 19' and 'Kunlun 14', and can also increase the microspore culture callus yield of highland barley variety 'Xila 22'.

[0080] For 'Dongqing 19', the average callus yield of microspore culture in the control group was 134.95 mg per dish. Adding 90 mg / L GSH to the callus induction medium can significantly increase the callus yield of microspore culture in the highland barley variety 'Dongqing 19', which is 180.40 mg per dish on average, which is 1.34 times that of the control group.

[0081] For 'XiLa22', the average callus yield of the control group was 42.87 mg per dish, while the average callus yield of the microspore culture after adding 90 mg / L glutathione (GSH) to the callus induction medium was 48.87 mg per dish.

[0082] For 'Kunlun 14', the average callus yield of the control group was 80.83 mg per dish, while the average callus yield of the microspore culture after adding 90 mg / L glutathione (GSH) to the callus induction medium was 104.33 mg per dish.

[0083] It can be seen from the above embodiments and experimental examples that the present invention can significantly increase the yield of highland barley microspore callus by soaking highland barley spikelets in a glutathione (GSH) aqueous solution or adding glutathione (GSH) to the highland barley microspore callus induction medium.

[0084] 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. The application of glutathione solution in improving the yield of highland barley microspore callus is characterized in that: The concentration of the glutathione solution is 80-100 mg / L.

2. The use according to claim 1, characterized in that: The young highland barley ears are soaked in glutathione solution at low temperature.

3. The use according to claim 2, characterized in that: The specific steps include: 1) Select young highland barley spikelets with microspore development in the middle of the spikelet at the middle to late stages of mononuclear development, pre-treat them at low temperature, and then soak them in a glutathione solution at low temperature; 2) taking anthers on young spikelets of highland barley soaked at low temperature in step 1), adding an extract and rotary cutting to obtain a suspension, filtering and centrifuging the suspension to obtain free microspores; 3) The free microspores are pretreated with an extract, and then subjected to purification culture and induction culture in sequence.

4. The use according to claim 3, characterized in that: Step 1) The temperature of the low temperature pretreatment and the low temperature soaking are both 3-7°C, and the time of the low temperature pretreatment and the low temperature soaking are both 5-9 days; Step 2) The extract comprises the following components in concentrations: 50-70 g / L mannitol, 0.8-1.5 g / L CaCl2, 0.8-1.2 g / L N-morphine ethane sulfonic acid and 8-12 mg / L colchicine; The rotation speed of the peeling is 2500-3500 rpm, the number of the peeling is 2-4 times, and the peeling time is 8-12 seconds / time; The centrifugal speed is 600-800 rpm, the number of centrifugation is 2-4 times, and the centrifugal time is 3-8 min / time.

5. The use according to claim 3, characterized in that: Step 3) The induction culture uses an induction medium, which uses N6 medium as a basic medium and includes the following components in concentrations: 80-100 g / L maltose, 0.3-0.8 mg / L kinetin, 0.5-1.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.2-0.6 g / L hydrolyzed casein, 0.8-1.2 g / L MES and 1-2 g / L glutamine; The induction culture is dark culture, the temperature of the induction culture is 23-28° C., and the time of the induction culture is 17-21 days.

6. The use according to claim 1, characterized in that: Glutathione was added to the highland barley microspore callus induction medium.

7. A highland barley microspore callus induction medium, characterized in that: N6 medium is used as the basic medium, including the following components in concentrations: 80-100 mg / L glutathione, 80-100 g / L maltose, 0.3-0.8 mg / L kinetin, 0.5-1.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.2-0.6 g / L hydrolyzed casein, 0.8-1.2 g / L MES and 1-2 g / L glutamine.

8. The use according to claim 6, characterized in that: The specific steps include: 1) Select young highland barley ears whose microspore development in the middle of the ear is in the middle to late mononuclear stage, and perform low temperature pretreatment; 2) taking anthers on young spikelets of highland barley pretreated at low temperature in step 1), adding the extract and rotary cutting to obtain a suspension, filtering and centrifuging the suspension to obtain free microspores; 3) pre-treating the free microspores with an extract, purifying and culturing them, and then inducing them with the highland barley microspore callus induction medium according to claim 7.

9. The use according to claim 8, characterized in that: Step 1) The temperature of the low temperature pretreatment is 3 to 7°C, and the time of the low temperature pretreatment is 5 to 9 days; Step 3) The induction culture is dark culture, the temperature of the induction culture is 23-28° C., and the time of the induction culture is 17-21 days.

10. The use according to claim 8, characterized in that: Step 2) The extract comprises the following components in concentrations: 50-70 g / L mannitol, 0.8-1.5 g / L CaCl2, 0.8-1.2 g / L N-morphine ethane sulfonic acid and 8-12 mg / L colchicine; The rotation speed of the peeling is 2500-3500 rpm, the number of the peeling is 2-4 times, and the peeling time is 8-12 seconds / time; The centrifugal speed is 600-800 rpm, the number of centrifugation is 2-4 times, and the centrifugal time is 3-8 min / time.

Images