A method for improving the green seedling differentiation rate of barley microspore callus after Agrobacterium infection and its application

Abstract

The present invention relates to the field of barley planting technology, and in particular to a method for improving the green shoot differentiation rate of barley microspore callus after Agrobacterium infection and its application. The method for improving the green shoot differentiation rate of barley microspore callus after Agrobacterium infection of the present invention comprises the following steps: (1) inoculating barley microspores into an induction medium to obtain microspore callus; (2) inoculating the microspore callus into a solid induction medium for subculture to obtain subculture microspore callus; (3) mixing the subculture microspore callus with an Agrobacterium suspension, transferring the subculture to a co-culture medium for co-culture, and obtaining infected callus; (4) treating the infected callus with negative pressure, transferring the infected callus to a differentiation medium for differentiation culture, and obtaining barley microspore callus green shoots. The method of the present invention can significantly improve the green shoot differentiation rate of barley microspore callus after Agrobacterium infection.

Description

Technical Field

[0001] The present invention relates to the field of barley planting, and in particular to a method for improving the green seedling differentiation rate of barley microspore callus after Agrobacterium infection and an application thereof. Background Art

[0002] Plant microspore culture is a single-cell, haploid germ cell culture system. Through chromosome doubling, plant morphology can be rapidly homozygous and stabilized, while also allowing for the generation of high-frequency regeneration plants. Currently, mature, isolated microspore culture systems have been established for a variety of crops, including tobacco, rapeseed, corn, and pepper. Our laboratory has established a microspore culture regeneration system and an Agrobacterium-mediated genetic transformation system for barley. During microspore culture, the green shoot differentiation rate is a key factor in determining the high-frequency regeneration of microspores. Previous studies have shown that the green shoot differentiation capacity of microspore callus after Agrobacterium infection is significantly reduced. Therefore, further improving the green shoot differentiation rate of barley microspore callus after Agrobacterium infection, based on existing research, will enable barley microspore regeneration technology to better serve haploid molecular breeding. Summary of the Invention

[0003] The purpose of the present invention is to provide a method and application thereof which can significantly improve the green shoot differentiation rate of barley microspore callus after Agrobacterium infection.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for improving the green shoot differentiation rate of barley microspore callus after Agrobacterium infection, comprising the following steps:

[0006] (1) Barley microspore induction culture:

[0007] The barley microspores were inoculated into the induction medium and cultured for 25 to 30 days to obtain microspore callus tissue;

[0008] (2) Subculture of barley microspore callus:

[0009] inoculating the microspore callus into a solid induction medium and subculturing for 10 to 15 days to obtain subculture microspore callus;

[0010] (3) Agrobacterium infection of barley microspore callus:

[0011] The subcultured microspore callus tissue is mixed with the Agrobacterium suspension for 25 to 35 minutes, and then transferred to a co-culture medium for co-culture for 2 to 4 days to obtain infected callus tissue;

[0012] (4) Post-infection treatment:

[0013] The infected callus tissue is treated with negative pressure and then transferred to a differentiation medium for differentiation culture for 25 to 30 days to obtain barley microspore callus green shoots.

[0014] Preferably, the induction medium is based on N6 medium and further comprises the following components at the following concentrations:

[0015] 80-100 g / L maltose, 0.4-0.6 mg / L 6-furfurylaminopurine, 0.8-1.2 mg / L dichlorophenoxyacetic acid, 0.960-0.980 g / L 2-morpholineethanesulfonic acid, 300-500 mg / L hydrolyzed casein, and 1400-1800 mg / L glutamine;

[0016] The pH of the induction culture medium is 5.6-6.0.

[0017] Preferably, in step (1), the culture temperature is 20-30° C.; the induction medium is replaced when the barley microspores are cultured for 10-15 days;

[0018] In step (2), the temperature of the subculture is 20-30°C.

[0019] Preferably, the solid induction medium is based on N6 medium and further comprises the following components at the following concentrations:

[0020] 80-100 g / L maltose, 0.4-0.6 mg / L 6-furfurylaminopurine, 0.8-1.2 mg / L dichlorophenoxyacetic acid, 0.960-0.980 g / L 2-morpholineethanesulfonic acid, 300-500 mg / L hydrolyzed casein, 1400-1800 mg / L glutamine, and 6-10 g / L agar powder;

[0021] The pH of the solid induction culture medium is 5.6-6.0.

[0022] Preferably, the method for preparing the Agrobacterium suspension comprises the following steps:

[0023] 1) transferring the vector into an Agrobacterium strain to obtain Agrobacterium containing the vector; inoculating the Agrobacterium containing the vector into a YEB medium for propagation to obtain propagated Agrobacterium;

[0024] 2) centrifuging the expanded Agrobacterium 2 to 4 times to obtain an Agrobacterium precipitate;

[0025] 3) diluting the Agrobacterium precipitate with induction medium to obtain an Agrobacterium suspension;

[0026] In step 1), the vector is pCambia1305.1; the Agrobacterium strain is LBA4404;

[0027] The YEB culture medium uses water as a solvent and includes the following components at the following concentrations: 4-6 g / L beef extract, 0.5-1.5 g / L yeast extract, 4-6 g / L peptone, 4-6 g / L sucrose, and 0.3-0.7 g / LMgSO4·H2O; the pH of the YEB culture medium is 6.5-7.5;

[0028] The OD of the Agrobacterium after the propagation 600 0.6~0.8;

[0029] In step 2), the speed of each centrifugation is 2500-3500 rpm; the time of each centrifugation is 4-6 minutes;

[0030] In step 3), the OD of the Agrobacterium suspension is 600 0.6~0.8;

[0031] The induction medium is based on N6 medium and also includes the following components at the following concentrations:

[0032] 80-100g / L maltose, 0.4-0.6mg / L 6-furfurylaminopurine, 0.8-1.2mg / L dichlorophenoxyacetic acid, 0.960-0.980g / L 2-morpholineethanesulfonic acid, 300-500mg / L hydrolyzed casein, 1400-1800mg / L glutamine;

[0033] The pH of the induction culture medium is 5.6-6.0.

[0034] Preferably, in step (3), the subculture microspore callus and the Agrobacterium suspension are mixed in a volume ratio of 150-250:40-60 mL;

[0035] The co-culture medium is based on N6 medium and also includes the following components at the following concentrations:

[0036] 80-100 g / L maltose, 0.4-0.6 mg / L 6-furfurylaminopurine, 0.8-1.2 mg / L dichlorophenoxyacetic acid, 0.960-0.980 g / L 2-morpholineethanesulfonic acid, 80-120 mg / L acetosyringone, 750-850 mg / L L-cysteine, and 6-10 g / L agar powder;

[0037] The pH of the co-culture medium is 5.6-6.0.

[0038] Preferably, the pressure of the negative pressure treatment is 40 to 80 KPa; the number of negative pressure treatments is 2 to 4 times, and the time of the negative pressure treatment is 30 to 180 s per time.

[0039] Preferably, the differentiation medium is based on 2 / 3 MS medium and further comprises the following components at the following concentrations:

[0040] 20-40 g / L maltose, 0.3-0.7 mg / L 6-benzylaminopurine, 1.0-2.0 mg / L 6-furfurylaminopurine, 0.02-0.08 mg / L α-naphthaleneacetic acid, 6-10 g / L agar powder;

[0041] The pH of the differentiation medium is 5.6-6.0.

[0042] Preferably, in step (3), the co-cultivation temperature is 20-30°C;

[0043] In step (4), the temperature of the differentiation culture is 20-30°C.

[0044] The invention also provides application of the method in barley breeding.

[0045] Beneficial effects of the present invention:

[0046] The present invention addresses the problem of poor differentiation ability of barley microspore callus after infection and provides a method that can significantly improve the green shoot differentiation rate of barley microspore callus after Agrobacterium infection. The method of the present invention can make the green shoot differentiation rate of barley microspore callus reach 26.67-43.33%. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The green seedling differentiation rate of barley microspore callus obtained under different pressure treatments (different letters on the error line indicate p < 0.05). DETAILED DESCRIPTION

[0048] The present invention provides a method for improving the green shoot differentiation rate of barley microspore callus after Agrobacterium infection, comprising the following steps:

[0049] (1) Barley microspore induction culture:

[0050] The barley microspores were inoculated into the induction medium and cultured for 25 to 30 days to obtain microspore callus tissue;

[0051] (2) Subculture of barley microspore callus:

[0052] inoculating the microspore callus into a solid induction medium and subculturing for 10 to 15 days to obtain subculture microspore callus;

[0053] (3) Agrobacterium infection of barley microspore callus:

[0054] The subcultured microspore callus tissue is mixed with the Agrobacterium suspension for 25 to 35 minutes, and then transferred to a co-culture medium for co-culture for 2 to 4 days to obtain infected callus tissue;

[0055] (4) Post-infection treatment:

[0056] The infected callus tissue is treated with negative pressure and then transferred to a differentiation medium for differentiation culture for 25 to 30 days to obtain barley microspore callus green shoots.

[0057] In the present invention, the induction medium is based on N6 medium and also includes the following components at the following concentrations:

[0058] 80-100 g / L maltose, preferably 90 g / L;

[0059] 0.4-0.6 mg / L 6-furfurylaminopurine, preferably 0.5 mg / L

[0060] 0.8-1.2 mg / L dichlorophenoxyacetic acid, preferably 1.0 mg / L,

[0061] 0.960-0.980 g / L 2-morpholineethanesulfonic acid, preferably 0.970 g / L,

[0062] 300-500 mg / L hydrolyzed casein, preferably 400 mg / L,

[0063] 1400-1800 mg / L glutamine, preferably 1600 mg / L;

[0064] The N6 medium includes the following components at the following concentrations:

[0065] 2830mg / L potassium nitrate, 463mg / L ammonium sulfate, 400mg / L potassium dihydrogen phosphate, 185mg / L magnesium sulfate (MgSO4·7H2O), 165mg / L calcium chloride (CaCl2·2H2O), 74.6mg / L disodium edetate, 55.6mg / L ferrous sulfate (FeSO4·7H2O), 4.4mg / L manganese sulfate (MnSO4·H2O), 1.5mg / L zinc sulfate (ZnSO4·7H2O), 1.6mg / L boric acid, 0.8mg / L potassium iodide, 1.0mg / L thiamine hydrochloride (vitamin B1), 0.5mg / L pyridoxine hydrochloride (vitamin B6), 0.5mg / L niacin, 2.0mg / L glycine;

[0066] The pH of the induction medium is 5.6-6.0, preferably 5.8.

[0067] In the present invention, in step (1), the culture time is preferably 27.5 days; the culture temperature is 20-30°C, preferably 25°C; the induction medium is replaced when the barley microspores are cultured for 10-15 days, preferably 12 days;

[0068] In step (2), the subculture time is preferably 12.5 days; the subculture temperature is 20-30°C, preferably 25°C.

[0069] In the present invention, the solid induction medium is based on N6 medium and further includes the following components at the following concentrations:

[0070] 80-100 g / L maltose, preferably 90 g / L;

[0071] 0.4-0.6 mg / L 6-furfurylaminopurine, preferably 0.5 mg / L

[0072] 0.8-1.2 mg / L dichlorophenoxyacetic acid, preferably 1.0 mg / L,

[0073] 0.960-0.980 g / L 2-morpholineethanesulfonic acid, preferably 0.970 g / L,

[0074] 300-500 mg / L hydrolyzed casein, preferably 400 mg / L,

[0075] 1400-1800 mg / L glutamine, preferably 1600 mg / L;

[0076] 6-10 g / L agar powder, preferably 8 g / L;

[0077] The components of the N6 medium are as described above;

[0078] The pH of the solid induction culture medium is 5.6-6.0, preferably 5.8.

[0079] In the present invention, in step (3), the mixing time is preferably 30 minutes, the co-cultivation time is preferably 3 days; and the co-cultivation temperature is 20-30°C, preferably 25°C.

[0080] In the present invention, the method for preparing the Agrobacterium suspension comprises the following steps:

[0081] 1) transferring the vector into an Agrobacterium strain to obtain Agrobacterium containing the vector; inoculating the Agrobacterium containing the vector into a YEB medium for propagation to obtain propagated Agrobacterium;

[0082] 2) centrifuging the expanded Agrobacterium 2 to 4 times to obtain an Agrobacterium precipitate;

[0083] 3) diluting the Agrobacterium precipitate with induction medium to obtain an Agrobacterium suspension;

[0084] In step 1), the vector is pCambia1305.1; the Agrobacterium strain is LBA4404;

[0085] The YEB culture medium uses water as a solvent and includes the following components in the following concentrations:

[0086] 4-6g / L beef extract, preferably 5g / L,

[0087] 0.5-1.5 g / L yeast extract, preferably 1.0 g / L,

[0088] 4-6 g / L peptone, preferably 5 g / L,

[0089] 4-6 g / L sucrose, preferably 5 g / L,

[0090] 0.3-0.7 g / L MgSO4·H2O, preferably 0.5 g / L;

[0091] The pH of the YEB medium is 6.5-7.5, preferably 7.0;

[0092] The OD of the Agrobacterium after the propagation 600 0.6 to 0.8, preferably 0.7;

[0093] In step 2), the speed of each centrifugation is 2500-3500 rpm, preferably 3000 rpm; the time of each centrifugation is 4-6 min, preferably 5 min; the number of centrifugation is preferably 3 times;

[0094] In step 3), the OD of the Agrobacterium suspension is 600 0.6 to 0.8, preferably 0.7;

[0095] The induction medium is based on N6 medium and also includes the following components at the following concentrations:

[0096] 80-100 g / L maltose, preferably 90 g / L;

[0097] 0.4-0.6 mg / L 6-furfurylaminopurine, preferably 0.5 mg / L

[0098] 0.8-1.2 mg / L dichlorophenoxyacetic acid, preferably 1.0 mg / L,

[0099] 0.960-0.980 g / L 2-morpholineethanesulfonic acid, preferably 0.970 g / L,

[0100] 300-500 mg / L hydrolyzed casein, preferably 400 mg / L,

[0101] 1400-1800 mg / L glutamine, preferably 1600 mg / L;

[0102] The components of the N6 medium are as described above;

[0103] The pH of the induction medium is 5.6-6.0, preferably 5.8.

[0104] In the present invention, in step (3), the subculture microspore callus and the Agrobacterium suspension are mixed in a volume ratio of 150 to 250:40 to 60 mL, preferably 200:50 mL;

[0105] The co-culture medium is based on N6 medium and also includes the following components at the following concentrations:

[0106] 80-100 g / L maltose, preferably 90 g / L;

[0107] 0.4-0.6 mg / L 6-furfurylaminopurine, preferably 0.5 mg / L;

[0108] 0.8-1.2 mg / L dichlorophenoxyacetic acid, preferably 1.0 mg / L;

[0109] 0.960-0.980 g / L 2-morpholineethanesulfonic acid, preferably 0.970 g / L;

[0110] 80-120 mg / L acetosyringone, preferably 100 mg / L;

[0111] 750-850 mg / L L-cysteine, preferably 800 mg / L;

[0112] 6-10 g / L agar powder, preferably 8 g / L;

[0113] The components of the N6 medium are as described above;

[0114] The pH of the co-culture medium is 5.6-6.0, preferably 5.8.

[0115] In the present invention, the pressure of the negative pressure treatment is 40-80Kpa, preferably 60Kpa; the number of negative pressure treatments is 2-4 times, preferably 3 times, and the time of the negative pressure treatment is 30s-180s / time, preferably 90-120s / time, and more preferably 105s / time.

[0116] In the present invention, the differentiation medium is based on 2 / 3 MS medium and further includes the following components at the following concentrations:

[0117] 20-40 g / L maltose, preferably 30 g / L;

[0118] 0.3-0.7 mg / L 6-benzylaminopurine, preferably 0.5 mg / L;

[0119] 1.0-2.0 mg / L 6-furfurylaminopurine, preferably 1.5 mg / L;

[0120] 0.02-0.08 mg / L α-naphthylacetic acid, preferably 0.05 mg / L;

[0121] 6-10 g / L agar powder, preferably 8 g / L;

[0122] The 2 / 3MS culture medium includes the following components at the following concentrations:

[0123] 1267mg / L potassium nitrate, 1100mg / L potassium nitrate, 113mg / L ammonium nitrate, 247mg / L potassium dihydrogen phosphate, 293mg / L calcium chloride (CaCl2·2H2O), 0.55mg / L potassium iodide, 4.1mg / L boric acid, 17.9mg / L manganese sulfate (MnSO4·H2O), 5.7mg / L zinc sulfate (ZnSO4·7H2O), 0.17mg / L sodium molybdate (Na2MoO4·2H2O), 0. 0.17 mg / L copper sulfate (CuSO4·5H2O), 0.017 mg / L cobalt chloride (CoCl2·6H2O), 24.83 mg / L disodium edetate, 18.57 mg / L ferrous sulfate (FeSO4·7H2O), 66.6 mg / L inositol, 1.3 mg / L glycine, 0.067 mg / L thiamine hydrochloride (vitamin B1), 0.33 mg / L pyridoxine hydrochloride (vitamin B6), 0.33 mg / L niacin;

[0124] The pH of the differentiation medium is 5.6-6.0, preferably 5.8.

[0125] In the present invention, in step (4), the differentiation culture time is preferably 27.5 days; the differentiation culture temperature is 20-30°C, preferably 25°C.

[0126] The invention also provides application of the method in barley breeding.

[0127] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0128] The barley variety used in the examples and comparative examples of the present invention is "Hua 22", and the seeds are from the Institute of Cell Engineering, Shanghai Academy of Agricultural Sciences.

[0129] The Agrobacterium strain used in the examples and comparative examples of the present invention is LBA4404, which was purchased from Shanghai Weidi Biotechnology Co., Ltd.;

[0130] The culture medium used in the examples and comparative examples of the present invention is as follows:

[0131] Induction medium: weigh 2830 mg potassium nitrate, 463 mg ammonium sulfate, 400 mg potassium dihydrogen phosphate, 185 mg magnesium sulfate (MgSO4·7H2O), 165 mg calcium chloride (CaCl2·2H2O), 74.6 mg disodium edetate, 55.6 mg ferrous sulfate (FeSO4·7H2O), 4.4 mg manganese sulfate (MnSO4·H2O), 1.5 mg zinc sulfate (ZnSO4·7H2O), 1.6 mg boric acid, 0.8 mg potassium iodide, 1.0 mg thiamine hydrochloride (vitamin B1), 0.5 mg pyridoxine hydrochloride (vitamin B6), 0.5 mg niacin, 2.0 mg glycine, 90000 mg maltose, 0.5 mg 6-furfurylaminopurine, 1.0 mg dichlorophenoxyacetic acid, 976 mg 2-Morpholineethanesulfonic acid, 400 mg hydrolyzed casein, and 1600 mg glutamine were mixed with distilled water to make up to 1 L, the pH was adjusted to 5.8, and sterilized.

[0132] The amount of iron salt added in this induction medium is twice that of the conventional N6 medium.

[0133] Solid induction medium: weigh 2830 mg potassium nitrate, 463 mg ammonium sulfate, 400 mg potassium dihydrogen phosphate, 185 mg magnesium sulfate (MgSO4·7H2O), 165 mg calcium chloride (CaCl2·2H2O), 74.6 mg disodium edetate, 55.6 mg ferrous sulfate (FeSO4·7H2O), 4.4 mg manganese sulfate (MnSO4·H2O), 1.5 mg zinc sulfate (ZnSO4·7H2O), 1.6 mg boric acid, 0.8 mg potassium iodide, 1.0 mg thiamine hydrochloride (vitamin B1), 0.5 mg pyridoxine hydrochloride (vitamin B6), 0.5 mg niacin, 2.0 mg glycine, 90000 mg maltose, 0.5 mg 6-furfurylaminopurine, 1.0 mg dichlorophenoxyacetic acid, 976 mg 2-Morpholineethanesulfonic acid, 400 mg hydrolyzed casein, 1600 mg glutamine, 8000 mg agar powder were mixed with distilled water to make up to 1 L, the pH was adjusted to 5.8, and sterilized.

[0134] The amount of iron salt added in this induction medium is twice that of the conventional N6 medium.

[0135] YEB medium: 5 g beef extract, 1 g yeast extract, 5 g peptone, 5 g sucrose, 0.5 g MgSO4·H2O, and distilled water were mixed to 1 L, the pH was adjusted to 7.0, and sterilized.

[0136] Co-culture medium: weigh 2830 mg potassium nitrate, 463 mg ammonium sulfate, 400 mg potassium dihydrogen phosphate, 185 mg magnesium sulfate (MgSO4·7H2O), 165 mg calcium chloride (CaCl2·2H2O), 74.6 mg disodium edetate, 55.6 mg ferrous sulfate (FeSO4·7H2O), 4.4 mg manganese sulfate (MnSO4·H2O), 1.5 mg zinc sulfate (ZnSO4·7H2O), 1.6 mg boric acid, 0.8 mg potassium iodide, 1.0 mg thiamine hydrochloride (vitamin B1), 0.5 mg pyridoxine hydrochloride (vitamin B6), 0.5 mg niacin, 2.0 mg glycine, 90000 mg maltose, 0.5 mg 6-furfurylaminopurine, 1.0 mg dichlorophenoxyacetic acid, 976 mg 2-Morpholineethanesulfonic acid, 100 mg acetosyringone, 800 mg L-cysteine, and 8000 mg agar powder were mixed with distilled water to make up to 1 L, the pH was adjusted to 5.8, and sterilized.

[0137] The amount of iron salt added in this induction medium is twice that of the conventional N6 medium.

[0138] Differentiation medium: 1267 mg potassium nitrate, 1100 mg potassium nitrate, 113 mg ammonium nitrate, 247 mg potassium dihydrogen phosphate, 293 mg calcium chloride (CaCl2·2H2O), 0.55 mg potassium iodide, 4.1 mg boric acid, 17.9 mg manganese sulfate (MnSO4·H2O), 5.7 mg zinc sulfate (ZnSO4·7H2O), 0.17 mg sodium molybdate (Na2MoO4·2H2O), 0.017 mg sulfur Copper sulfate (CuSO4·5H2O), 0.017 mg cobalt chloride (CoCl2·6H2O), 24.83 mg disodium edetate, 18.57 mg ferrous sulfate (FeSO4·7H2O), 66.6 mg inositol, 1.3 mg glycine, 0.067 mg thiamine hydrochloride (vitamin B1), 0.33 mg pyridoxine hydrochloride (vitamin B6), 0.33 mg niacin, 30,000 mg maltose, 0.5 mg 6-benzylaminopurine, 1.5 mg 6-furfurylaminopurine, 0.05 mg α-naphthaleneacetic acid, 8,000 mg agar powder were mixed with distilled water to 1 L, the pH was adjusted to 5.8, and sterilized.

[0139] Example 1 Barley microspore induction culture

[0140] Soak "Hua 22" in a 15 cm culture dish with filter paper until it turns white. Germinate the seeds and transplant them into an artificial climate chamber. The plant culture conditions are: temperature 18°C ​​at night, 20°C during the day; humidity 65%; 12 hours of light and 12 hours of darkness.

[0141] Select the "Hua 22" spikelet with the florets and microspores in the middle of the young spikelet in the early to middle stages of uninucleate development, cut off the leaves (retain the 1.5 cm base of the two upper leaves), put it in a fresh-keeping bag to keep it moist, mark the collection date and quantity, and put it in a 4°C refrigerator for low-temperature pretreatment for 21 days;

[0142] After the low-temperature treatment of "Hua 22", the flower buds of 4 ears of flower buds were taken and 12 ml of extract (mannitol 60 g / L + CaCl2 1.1 g / L + 2-morpholineethanesulfonic acid 0.976 g / L + colchicine 20 mg / L, pH 5.8) was added and ultra-speed rotary cutting was performed using a high-speed disperser; the rotary cutting suspension was filtered through a 300-mesh sieve, and the filtrate was centrifuged at 700 rpm for 5 minutes, repeated 3 times, and the microspores were collected. The collected microspores were pretreated with the extract at 25°C in the dark for 2 days, and then resuspended in the induction medium and adjusted to a density of 1.0 × 10 5 After mixing, take 1.5 ml (1.5×10 5 The microspore suspension was inoculated into a culture dish (30×15 mm), sealed with Parafilm, and cultured at 25°C in the dark for 12 days. The induction medium was replaced and cultured for another 14 days to obtain microspore callus.

[0143] Example 2 Subculture of barley microspore callus

[0144] The microspore callus described in Example 1 was inoculated into a solid induction medium and subcultured at 25° C. for 14 days to obtain subculture microspore callus.

[0145] Example 3 Agrobacterium infection of wheat microspore callus

[0146] (1) The vector pCambia1305.1 was transformed into the Agrobacterium strain LBA4404 and the Agrobacterium was propagated using YEB medium to an OD 600 =0.7, and the expanded Agrobacterium was obtained;

[0147] (2) Centrifuge the expanded Agrobacterium at 3000 rpm for 5 min, remove the supernatant, and repeat three times to obtain the Agrobacterium precipitate;

[0148] (3) Use induction medium to dilute the Agrobacterium precipitate to OD 600 =0.7, and obtain Agrobacterium suspension;

[0149] (4) 200 microspore calli described in Example 2 were inoculated into 50 mL of Agrobacterium suspension and placed on a shaker. After infection at 100 rpm for 30 min, the infected microspore calli were placed on three layers of filter paper to absorb the bacterial solution and transferred to a co-culture medium for 3 days to obtain infected calli.

[0150] Example 4 Post-infection treatment

[0151] The infected callus tissue described in Example 3 was treated with 80 kPa negative pressure for 1 minute three times, then transferred to differentiation medium and cultured at 25°C for 28 days to obtain green shoots of barley microspore callus. The number of green shoots was counted and the green shoot differentiation rate was calculated. The calculation results are shown in Table 1 and Figure 1 shown.

[0152] The calculation formula is as follows:

[0153]

[0154] Example 5

[0155] Referring to Example 4, the pressure was set to 60KPa and the treatment was repeated 3 times, each time for 1 minute. The number of green seedlings was counted and the green seedling differentiation rate was calculated according to the calculation formula of Example 4. The calculation results are shown in Table 1 and Figure 1 shown.

[0156] Example 6

[0157] Referring to Example 4, the pressure was set to 40KPa and the treatment was repeated 3 times, each time for 1 minute. The number of green seedlings was counted and the green seedling differentiation rate was calculated according to the calculation formula of Example 4. The calculation results are shown in Table 1 and Figure 1 shown.

[0158] Comparative Example 1

[0159] Referring to Example 4, the pressure was set to 100KPa and the treatment was performed 3 times, each time for 1 minute. The number of green seedlings was counted and the green seedling differentiation rate was calculated according to the calculation formula of Example 4. The calculation results are shown in Table 1 and Figure 1 shown.

[0160] Comparative Example 2

[0161] Referring to Example 4, the pressure was set to 20KPa and the treatment was repeated 3 times, each time for 1 minute. The number of green seedlings was counted and the green seedling differentiation rate was calculated according to the calculation formula of Example 4. The calculation results are shown in Table 1 and Figure 1 shown.

[0162] Table 1 Green seedling differentiation rate

[0163] Treatment group Green seedling differentiation rate / % Example 4 (80Kpa) 26.67 Example 5 (60Kpa) 43.33 Example 6 (40Kpa) 28.33 Comparative Example 1 (100Kpa) 13.33 Comparative Example 2 (20Kpa) 18.33

[0164] Through Table 1 and Figure 1 It can be seen that the method of the present invention can make the green shoot differentiation rate of barley microspore callus after Agrobacterium infection reach 26.67-43.33%, which is significantly better than the comparative example.

[0165] From the above examples, it can be seen that the present invention provides a method that can significantly improve the green shoot differentiation rate of barley microspore callus after Agrobacterium infection. The method of the present invention can make the green shoot differentiation rate of barley microspore callus after Agrobacterium infection reach 26.67-43.33%.

[0166] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for increasing the green shoot differentiation rate of barley microspore callus after Agrobacterium infection, characterized in that: The steps include: (1) Barley microspore induction culture: The barley microspores were inoculated into the induction medium and cultured for 25-30 days to obtain microspore callus. (2) Subculture of barley microspore callus: inoculating the microspore callus into a solid induction medium and subculturing for 10 to 15 days to obtain subculture microspore callus; (3) Agrobacterium infection of barley microspore callus: The subcultured microspore callus tissue is mixed with the Agrobacterium suspension for 25 to 35 minutes, and then transferred to a co-culture medium for co-culture for 2 to 4 days to obtain infected callus tissue; (4) Post-infection treatment: The infected callus tissue was treated with negative pressure, and then transferred to a differentiation medium for differentiation culture for 25 to 30 days to obtain barley microspore callus green shoots; In step (4), the pressure of the negative pressure treatment is 40~80KPa; the number of times of the negative pressure treatment is 2~4 times, and the time of the negative pressure treatment is 30~180s / time.

2. The method according to claim 1, characterized in that The induction medium is based on N6 medium, and the following components are added: 80-100 g / L maltose, 0.4-0.6 mg / L 6-furfurylaminopurine, 0.8-1.2 mg / L dichlorophenoxyacetic acid, 0.960-0.980 g / L 2-morpholineethanesulfonic acid, 300-500 mg / L hydrolyzed casein, and 1400-1800 mg / L glutamine; The pH of the induction medium is 5.6-6.

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3. The method according to claim 2, characterized in that In step (1), the culture temperature is 20-30°C; the induction medium is replaced when the barley microspores are cultured for 10-15 days; In step (2), the temperature of the subculture is 20-30°C.

4. The method according to claim 3, characterized in that The solid induction medium is based on N6 medium, and the following components are added: 80-100 g / L maltose, 0.4-0.6 mg / L 6-furfurylaminopurine, 0.8-1.2 mg / L dichlorophenoxyacetic acid, 0.960-0.980 g / L 2-morpholineethanesulfonic acid, 300-500 mg / L hydrolyzed casein, 1400-1800 mg / L glutamine, and 6-10 g / L agar powder; The pH of the solid induction culture medium is 5.6-6.

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5. The method according to claim 4, characterized in that The preparation method of the Agrobacterium suspension comprises the following steps: 1) Transforming the vector into an Agrobacterium strain to obtain Agrobacterium containing the vector; inoculating the Agrobacterium containing the vector into YEB medium for propagation to obtain propagated Agrobacterium; 2) Centrifuging the expanded Agrobacterium 2-4 times to obtain an Agrobacterium precipitate; 3) Dilute the Agrobacterium precipitate with induction medium to obtain an Agrobacterium suspension; In step 1), the vector is pCambia1305.1; the Agrobacterium strain is LBA4404; The YEB medium uses water as a solvent and is composed of the following components at the following concentrations: 4-6 g / L beef extract, 0.5-1.5 g / L yeast extract, 4-6 g / L peptone, 4-6 g / L sucrose, and 0.3-0.7 g / L MgSO4·H2O; the pH of the YEB medium is 6.5-7.5; The OD of the agrobacterium after the propagation 600 0.6~0.8; In step 2), the speed of each centrifugation is 2500-3500 rpm; the time of each centrifugation is 4-6 minutes; In step 3), the OD of the Agrobacterium suspension is 600 0.6~0.8; The induction medium is based on N6 medium, and the following components are added: 80-100g / L maltose, 0.4-0.6mg / L 6-furfurylaminopurine, 0.8-1.2mg / L dichlorophenoxyacetic acid, 0.960-0.980g / L 2-morpholineethanesulfonic acid, 300-500mg / L hydrolyzed casein, 1400-1800mg / L glutamine; The pH of the induction medium is 5.6-6.

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6. The method according to claim 5, characterized in that In step (3), the subculture microspore callus and the Agrobacterium suspension are mixed in a volume ratio of 150-250:40-60 mL; The co-culture medium is based on N6 medium, and the following components are added: 80-100 g / L maltose, 0.4-0.6 mg / L 6-furfurylaminopurine, 0.8-1.2 mg / L dichlorophenoxyacetic acid, 0.960-0.980 g / L 2-morpholineethanesulfonic acid, 80-120 mg / L acetosyringone, 750-850 mg / L L-cysteine, and 6-10 g / L agar powder; The pH of the co-culture medium is 5.6-6.

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7. The method according to claim 6, characterized in that The differentiation medium is based on 2 / 3 MS medium, and is further supplemented with the following components at the following concentrations: 20-40 g / L maltose, 0.3-0.7 mg / L 6-benzylaminopurine, 1.0-2.0 mg / L 6-furfurylaminopurine, 0.02-0.08 mg / L α-naphthylacetic acid, and 6-10 g / L agar powder; The pH of the differentiation medium is 5.6-6.

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8. The method according to claim 7, characterized in that In step (3), the co-cultivation temperature is 20-30°C; In step (4), the temperature of the differentiation culture is 20-30°C.

9. Use of the method according to any one of claims 1 to 8 in barley breeding.

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