CN116584391A - Induction method of mature sorghum seed somatic embryo - Google Patents
Induction method of mature sorghum seed somatic embryo Download PDFInfo
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- 230000006698 induction Effects 0.000 title claims abstract description 31
- 230000000392 somatic effect Effects 0.000 title claims abstract description 23
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- 238000000034 method Methods 0.000 title claims abstract description 20
- 240000006394 Sorghum bicolor Species 0.000 title claims description 57
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 57
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- 230000004069 differentiation Effects 0.000 claims abstract description 18
- 238000012258 culturing Methods 0.000 claims abstract description 17
- 239000002609 medium Substances 0.000 claims abstract description 17
- 238000007789 sealing Methods 0.000 claims abstract description 17
- 238000009331 sowing Methods 0.000 claims abstract description 11
- 239000012883 rooting culture medium Substances 0.000 claims abstract description 7
- 239000002689 soil Substances 0.000 claims abstract description 6
- 239000011259 mixed solution Substances 0.000 claims description 35
- 230000001105 regulatory effect Effects 0.000 claims description 28
- 238000005286 illumination Methods 0.000 claims description 27
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 claims description 23
- 239000012452 mother liquor Substances 0.000 claims description 17
- 229920001817 Agar Polymers 0.000 claims description 15
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 15
- 229930006000 Sucrose Natural products 0.000 claims description 15
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- 238000001816 cooling Methods 0.000 claims description 15
- 210000002257 embryonic structure Anatomy 0.000 claims description 15
- 239000000843 powder Substances 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 15
- 239000005720 sucrose Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 230000001939 inductive effect Effects 0.000 claims description 14
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- 239000007788 liquid Substances 0.000 claims description 9
- 235000013339 cereals Nutrition 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000005520 cutting process Methods 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 5
- 238000009928 pasteurization Methods 0.000 claims description 5
- 239000012882 rooting medium Substances 0.000 claims description 5
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 5
- 239000012498 ultrapure water Substances 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 238000011049 filling Methods 0.000 claims description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 229920001214 Polysorbate 60 Polymers 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
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- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 24
- 239000003617 indole-3-acetic acid Substances 0.000 description 12
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- HXKWSTRRCHTUEC-UHFFFAOYSA-N 2,4-Dichlorophenoxyaceticacid Chemical compound OC(=O)C(Cl)OC1=CC=C(Cl)C=C1 HXKWSTRRCHTUEC-UHFFFAOYSA-N 0.000 description 4
- 239000010413 mother solution Substances 0.000 description 4
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- JAWMENYCRQKKJY-UHFFFAOYSA-N [3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-ylmethyl)-1-oxa-2,8-diazaspiro[4.5]dec-2-en-8-yl]-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]methanone Chemical compound N1N=NC=2CN(CCC=21)CC1=NOC2(C1)CCN(CC2)C(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F JAWMENYCRQKKJY-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
- A01H6/4666—Sorghum, e.g. sudangrass
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- Health & Medical Sciences (AREA)
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Abstract
The invention discloses an induction method of mature sorghum seed somatic embryo, which comprises the following steps: (1) sterilizing mature sorghum seeds; (2) preparing a callus induction medium: (3) Uniformly sowing the sorghum seeds treated in the step (1) on the culture dish containing the callus induction culture medium obtained in the step (2); (4) Sealing the culture dish treated in the step (3), placing the culture dish in an incubator at 20-30 ℃, and continuously culturing for 6-8 weeks at 16h/8h to obtain embryogenic callus; (5) preparing a differentiation medium; (6) differentiation culture; (7) preparing a rooting culture medium; (8) rooting culture; (9) Sealing the inoculated culture bottle in the step (8), placing the bottle in an incubator at 20-30 ℃, continuously culturing for 4-6 weeks with a photoperiod of 16h/8h, rooting, and transplanting the root into soil to complete the life history.
Description
Technical Field
The invention belongs to the technical field of crop biology, in particular to the field of molecular plant breeding, and particularly relates to an induction method of mature sorghum seed somatic embryos.
Background
Sorghum is a widely planted grain crop. The difficulty of genetic transformation of sorghum plants limits the industrial application of genome editing. An easily prepared receptor system is a prerequisite for genetic transformation. The regeneration system of sorghum is usually derived from Immature Zygotic Embryos (IZE), but the production of immature zygotic embryos requires a long period, is season dependent, or requires costly climatic chambers. Furthermore, immature zygotic embryos exhibit genotype dependence, and successful reports are mostly focused on inbred lines such as Tx430, tx623, P898102, and are difficult to integrate into existing crossbreeding systems.
Disclosure of Invention
The invention aims to: the invention improves the problems existing in the prior art, namely the invention discloses a method for inducing mature sorghum seed somatic embryos.
The technical scheme is as follows: a method for inducing mature sorghum seed somatic embryo comprises the following steps:
(1) Sterilizing mature sorghum seeds;
(2) Preparing a callus induction culture medium:
(21) Respectively adding 2.3-9.2 g of MS culture medium, 1-5 g of sucrose, 7-15 g of agar powder and 1L of water into a container, uniformly stirring to obtain a mixed solution, and then regulating the pH value to 5.5-6.2;
(22) Sterilizing the mixed solution at 110-130 ℃ for 10-60 minutes by high pressure steam, cooling to RT-75 ℃, then adding a proper amount of 2,4-D (namely 2, 4-dichlorophenoxyacetic acid) mother solution into the mixed solution, and regulating the concentration of 2,4-D to 4-20 mg/L;
(23) Then CuSO is added thereto 4 Mother liquor, adjust CuSO 4 The concentration of the culture medium is 0.5-2 mu mol/L, shaking evenly, pouring the culture medium into a plate, pouring 20-30 ml of the culture medium into a culture dish, and condensing the culture medium on an ultra-clean workbench to obtain the culture dish containing the callus induction culture medium;
(3) Uniformly sowing the sorghum seeds treated in the step (1) onto the culture dish containing the callus induction medium obtained in the step (2), wherein:
the embryo of the sorghum seeds faces downwards, and the sowing density is controlled to be 10-30 grains/culture dish;
(4) Sealing the culture dish treated in the step (3), placing the culture dish in an incubator at 20-30 ℃, and continuously culturing for 6-8 weeks at 16h/8h to obtain embryogenic callus;
(5) Preparation of differentiation medium:
(51) Respectively adding 2.3-9.2 g of MS culture medium, 1-5 g of sucrose, 7-15 g of agar powder and 1L of water into a container, uniformly stirring to obtain a mixed solution, and then regulating the pH value to 5.5-6.2;
(52) Sterilizing the mixed solution at 110-130 ℃ by high-pressure steam for 10-60 minutes, and cooling to RT-75 ℃;
(53) Adding a proper amount of IAA (indoleacetic acid) mother liquor into the mixed liquor, and regulating the concentration of the IAA to 1-5 mg/L;
(54) Adding BAP (6-BA, 6-benzylaminopurine) mother liquor into the mixed liquor, and regulating the concentration of BAP to 1-5 mg/L;
(55) Adding CuSO into the mixed solution 4 Mother liquor, adjust CuSO 4 The concentration of the culture medium is 0.5-2 mu mol/L, the culture medium is obtained after shaking, the plate is poured, every 20-30 ml of the culture medium is poured into a breathable glass culture flask, and the culture flask containing the differentiation culture medium is obtained after condensing on an ultra-clean workbench;
(6) And (3) differentiation culture:
(61) Inoculating the embryogenic callus induced in the step (4) to a culture bottle containing the differentiation medium obtained in the step (5);
(62) Sealing the inoculated culture bottle, placing the bottle in an incubator at 20-30 ℃, continuously culturing for 4-8 weeks at 16h/8h, and allowing embryogenic callus to see complete main stems;
(7) Preparing a rooting culture medium:
(71) Adding 2.3-9.2 g of MS culture medium, 1-5 g of sucrose, 7-15 g of agar powder and 1L of water into a container respectively, stirring uniformly to obtain a mixed solution, and then regulating the pH value to 5.5-6.2;
(72) Sterilizing the mixed solution at 110-130 ℃ by high-pressure steam for 10-60 minutes, and cooling to RT-75 ℃;
(73) Adding NAA (1-naphthylacetic acid) mother liquor into the mixed liquor, and regulating the concentration of NAA to 0.5-2 mg/L;
(74) Adding IAA mother liquor into the mixed liquor, and regulating the concentration of IAA to 0.5-2 mg/L;
(75) Adding IBA (indolebutyric acid) mother liquor into the mixed liquor, and regulating the concentration of IBA to 0.5-2 mg/L;
(76) Adding CuSO into the mixed solution 4 Mother liquor, adjust CuSO 4 The concentration of the culture medium is 0.5-2 mu mol/L, the culture medium is obtained after shaking, the plate is poured, each 20-30 ml of culture medium is poured into a breathable glass culture flask, and the culture flask containing rooting culture medium is obtained after condensing on an ultra-clean workbench;
(8) Rooting culture:
cutting off the main stem of the embryogenic callus induced in the step (6), discarding the residual callus, and inoculating the residual callus to a culture flask containing a rooting medium obtained in the step (7), wherein:
each flask was inoculated with only one main stem;
(9) Sealing the inoculated culture bottle in the step (8), placing the bottle in an incubator at 20-30 ℃, continuously culturing for 4-6 weeks with a photoperiod of 16h/8h, rooting, and transplanting the root into soil to complete the life history.
Further, the step (1) includes the steps of:
(11) Taking 10-20 ml of mature sorghum seeds, placing the mature sorghum seeds into a 50ml centrifuge tube, then dripping 1-3 drops of surfactant into the centrifuge tube, then adding a proper amount of 75% v/v ethanol, filling the centrifuge tube, and covering a cover;
(12) Vibrating and sterilizing the centrifuge tube on an oscillator at a rotating speed of 60-100 rpm for 30-60 minutes, and discarding liquid after completion;
(13) Adding a proper amount of pasteurization liquid with the volume of 2% v/v into the centrifuge tube, vibrating and disinfecting for 2-4 hours at the rotating speed of 60-100 rpm, removing the liquid, and washing at least three times by using high-pressure sterilized ultrapure water to obtain the treated sorghum seeds.
Further, the surfactant in the step (11) is one of tween 20, tween 40, tween 60 and tween 80.
Further, the concentration of 2,4-D is adjusted to 6-10 mg/L in the step (22).
Further, the illumination intensity in the illumination in the step (4) is 50 to 500 mu mol m -2 s -1 。
Further, in step (6), the embryogenic callus to be inoculated needs to be crushed or mashed off prior to inoculation.
Further, the illumination intensity at the time of illumination in the step (62) is 50 to 500. Mu. Mol m -2 s -1 。
Further, the length of the main stem cut in the step (8) is 2cm or more.
Further, the illumination intensity at the time of illumination in the step (9) is 50 to 500. Mu. Mol m -2 s -1 。
The beneficial effects are that: the invention has the following beneficial effects:
somatic embryos are directly induced from mature sorghum seeds, embryogenic calli can be effectively induced from embryogenic axis cells through relatively high concentration of 2,4-D, the somatic embryogenesis rate is about 60% on average, and the induction efficiency of some varieties can be as high as 70%.
Drawings
FIGS. 1a and 1b are histological microscopic images of mature sorghum seed somatic embryogenesis of control example 1.
FIGS. 1c and 1d are histological microscopic images of mature sorghum seed somatic embryogenesis of example 1.
FIG. 2 is a schematic representation of the generation of two calli from control example 1 and example 1.
FIG. 3 is a schematic representation of somatic embryo induction assays for four hybrid varieties.
The specific embodiment is as follows:
the following detailed description of specific embodiments of the invention.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
MS medium was purchased from Duchefa reagent, netherlands.
Example 1
A method for inducing mature sorghum seed somatic embryo comprises the following steps:
(1) Sterilizing mature sorghum seeds;
(2) Preparing a callus induction culture medium:
(21) Respectively adding 4.6g of MS culture medium, 3g of sucrose, 10g of agar powder and 1L of water into a container, uniformly stirring to obtain a mixed solution, and then regulating the pH value to 5.8;
(22) Sterilizing the mixed solution at 120 ℃ for 20 minutes by high-pressure steam, cooling to 70 ℃, and then adding a proper amount of 2,4-D (namely 2, 4-dichlorophenoxyacetic acid) mother solution into the mixed solution, and regulating the concentration of the 2,4-D to 8mg/L;
(23) Then adding CuSO thereto 4 Mother liquor, adjust CuSO 4 The concentration of the culture medium is 1 mu mol/L, shaking evenly, pouring the culture medium into a plate, pouring 25ml of the culture medium into the plate, and condensing the culture medium on an ultra-clean workbench to obtain the plate containing the callus induction culture medium;
(3) Uniformly sowing the sorghum seeds treated in the step (1) onto the culture dish containing the callus induction medium obtained in the step (2), wherein:
the embryo of the sorghum seeds faces downwards, and the sowing density is controlled to be 15 grains/culture dish;
(4) Sealing the culture dish treated in the step (3), placing the culture dish in an incubator at 25 ℃, and continuously culturing for 7 weeks at 16h/8h to obtain embryogenic callus;
(5) Preparation of differentiation medium:
(51) Respectively adding 4.6g of MS culture medium, 3g of sucrose, 10g of agar powder and 1L of water into a container, uniformly stirring to obtain a mixed solution, and then regulating the pH value to 5.8;
(52) Sterilizing the mixed solution at 120 ℃ by high-pressure steam for 20 minutes, and cooling to 70 ℃;
(53) Adding a proper amount of IAA (indoleacetic acid) mother liquor into the mixed liquor, and regulating the concentration of the IAA to 1mg/L;
(54) Adding BAP (6-BA, 6-benzylaminopurine) mother liquor into the mixed liquor, and regulating the concentration of BAP to 1mg/L;
(55) Adding CuSO into the mixed solution 4 Mother liquor, adjust CuSO 4 The concentration of the culture medium is 1 mu mol/L, the culture medium is obtained after shaking, the culture medium is poured into a breathable glass culture flask every 25ml, and the culture flask containing the differentiation culture medium is obtained after condensing on an ultra-clean workbench;
(6) And (3) differentiation culture:
(61) Inoculating the embryogenic callus induced in the step (4) to a culture flask containing the differentiation medium obtained in the step (5);
(62) Sealing the inoculated culture bottle, placing the bottle in an incubator at 25 ℃, continuously culturing for 6 weeks at 16h/8h in a photoperiod, and allowing embryogenic callus to see complete main stems;
(7) Preparing a rooting culture medium:
(71) Respectively adding 4.6g of MS culture medium, 3g of sucrose, 10g of agar powder and 1L of water into a container, uniformly stirring to obtain a mixed solution, and then regulating the pH value to 5.8;
(72) Sterilizing the mixed solution at 120 ℃ for 20 minutes by high-pressure steam, and cooling to 70 ℃;
(73) Adding NAA (1-naphthylacetic acid) mother liquor into the mixed liquor, and regulating the concentration of NAA to 1mg/L;
(74) Adding IAA mother liquor into the mixed liquor, and regulating the concentration of IAA to 1mg/L;
(75) Adding IBA (indolebutyric acid) mother liquor into the mixed liquor, and regulating the concentration of IBA to 1mg/L;
(76) Adding CuSO into the mixed solution 4 Mother liquor, adjust CuSO 4 The concentration of the culture medium is 1 mu mol/L, the culture medium is obtained after shaking, the culture medium is poured into a breathable glass culture flask every 25ml, and the culture flask containing rooting culture medium is obtained after condensation on an ultra-clean workbench;
(8) Rooting culture:
cutting off the main stem of the embryogenic callus induced in the step (6), discarding the residual callus, and inoculating the residual callus to a culture flask containing a rooting medium obtained in the step (7), wherein:
each flask was inoculated with only one main stem;
(9) Sealing the inoculated culture bottle in the step (8), placing the bottle in an incubator at 25 ℃, and culturing for 5 weeks continuously, wherein the photoperiod is 16h/8h, rooting, transplanting the root into soil after strengthening, and completing the life history.
Further, the step (1) includes the steps of:
(11) Taking 10ml of mature sorghum seeds, placing the mature sorghum seeds into a 50ml centrifuge tube, then dripping 1 drop of surfactant into the centrifuge tube, then adding 45ml of 75% v/v ethanol, filling the centrifuge tube, and closing a cover;
(12) Vibrating and sterilizing the centrifuge tube on an oscillator at a rotating speed of 80rpm for 30 minutes, and discarding liquid after completion;
(13) And adding a proper amount of pasteurization solution with the volume of 2% v/v into the centrifuge tube, vibrating and sterilizing for 3 hours at the rotating speed of 80rpm, discarding the solution, and washing with high-pressure sterilized ultrapure water for three times to obtain the treated sorghum seeds. The induction efficiency of the technical scheme of example 1 was calculated to be 70%.
Further, the surfactant in the step (11) is tween 20.
Further, the illumination intensity upon illumination in the step (4) was 400. Mu. Mol m -2 s -1 。
Further, in step (6), the embryogenic callus to be inoculated needs to be crushed prior to inoculation.
Further, the illumination intensity upon illumination in the step (62) was 400. Mu. Mol m -2 s -1 。
Further, the length of the main stem cut in the step (8) is 2cm or more.
Further, the illumination intensity upon illumination in the step (9) was 400. Mu. Mol m -2 s -1 。
Example 2
A method for inducing mature sorghum seed somatic embryo comprises the following steps:
(1) Sterilizing mature sorghum seeds;
(2) Preparing a callus induction culture medium:
(21) Respectively adding 2.3g of MS culture medium, 1g of sucrose, 7g of agar powder and 1L of water into a container, uniformly stirring to obtain a mixed solution, and then regulating the pH value to 5.5;
(22) Sterilizing the mixed solution at 110 ℃ for 60 minutes by high-pressure steam, cooling to RT, adding a proper amount of 2,4-D (namely 2, 4-dichlorophenoxyacetic acid) mother solution, and regulating the concentration of 2,4-D to 4mg/L;
(23) Then adding CuSO thereto 4 Mother liquor, adjust CuSO 4 The concentration of the culture medium is 0.5 mu mol/L, shaking evenly, pouring the culture medium into a plate, pouring 20ml of the culture medium into the culture dish, and condensing the culture medium on an ultra-clean workbench to obtain the culture dish containing the callus induction culture medium;
(3) Uniformly sowing the sorghum seeds treated in the step (1) onto the culture dish containing the callus induction medium obtained in the step (2), wherein:
the embryo of the sorghum seeds faces downwards, and the sowing density is controlled to be 10 grains/culture dish;
(4) Sealing the culture dish treated in the step (3), placing the culture dish in an incubator at 20 ℃, and continuously culturing for 8 weeks at 16h/8h to obtain embryogenic callus;
(5) Preparation of differentiation medium:
(51) Respectively adding 2.3g of MS culture medium, 1g of sucrose, 7g of agar powder and 1L of water into a container, uniformly stirring to obtain a mixed solution, and then regulating the pH value to 5.5;
(52) Sterilizing the mixed solution at 110 ℃ by high-pressure steam for 60 minutes, and cooling to RT ℃;
(53) Adding a proper amount of IAA (indoleacetic acid) mother liquor into the mixed liquor, and regulating the concentration of the IAA to 3mg/L;
(54) Adding BAP (6-BA, 6-benzylaminopurine) mother liquor into the mixed liquor, and regulating the concentration of BAP to 3mg/L;
(55) Adding CuSO into the mixed solution 4 Mother liquor, adjust CuSO 4 The concentration of the culture medium is 0.5 mu mol/L, the culture medium is obtained after shaking, the plate is poured, each 20ml of culture medium is poured into a breathable glass culture flask, and the culture flask containing the differentiation culture medium is obtained after condensing on an ultra-clean workbench;
(6) And (3) differentiation culture:
(61) Inoculating the embryogenic callus induced in the step (4) to a culture flask containing the differentiation medium obtained in the step (5);
(62) Sealing the inoculated culture bottle, placing the bottle in an incubator at 20 ℃, continuously culturing for 8 weeks at 16h/8h in a photoperiod, and enabling embryogenic callus to see complete main stems;
(7) Preparing a rooting culture medium:
(71) Respectively adding 2.3g of MS culture medium, 1g of sucrose, 7g of agar powder and 1L of water into a container, uniformly stirring to obtain a mixed solution, and then regulating the pH value to 5.5;
(72) Sterilizing the mixed solution at 110 ℃ for 60 minutes by high-pressure steam, and cooling to RT ℃;
(73) Adding NAA (1-naphthylacetic acid) mother liquor into the mixed liquor, and regulating the concentration of NAA to 0.5mg/L;
(74) Adding IAA mother liquor into the mixed liquor, and regulating the concentration of IAA to 0.5mg/L;
(75) Adding IBA (indolebutyric acid) mother liquor into the mixed liquor, and regulating the concentration of IBA to 0.5mg/L;
(76) Adding CuSO into the mixed solution 4 Mother liquor, adjust CuSO 4 The concentration of the mixture is 0.5 mu mol/L, the culture solution is obtained after shaking, the plate is poured, and each 20ml of culture solution is poured into the plateIn the gas glass culture flask, condensing on an ultra-clean workbench to obtain a culture flask containing a rooting culture medium;
(8) Rooting culture:
cutting off the main stem of the embryogenic callus induced in the step (6), discarding the residual callus, and inoculating the residual callus to a culture flask containing a rooting medium obtained in the step (7), wherein:
each flask was inoculated with only one main stem;
(9) Sealing the inoculated culture bottle in the step (8), placing the bottle in an incubator at 20 ℃, continuously culturing for 6 weeks, rooting the bottle, and transplanting the bottle into soil to complete the life history, wherein the photoperiod is 16h/8 h. The induction efficiency of the technical scheme of example 2 was calculated to be 48%.
Further, the step (1) includes the steps of:
(11) 15ml of mature sorghum seeds are taken and placed in a 50ml centrifuge tube, 2 drops of surfactant are added into the centrifuge tube, 40ml of 75% v/v ethanol is added into the centrifuge tube, the centrifuge tube is filled, and a cover is closed;
(12) Vibrating and sterilizing the centrifuge tube on an oscillator at a rotating speed of 60rpm for 60 minutes, and discarding liquid after completion;
(13) And adding a proper amount of pasteurization solution with the volume of 2% v/v into the centrifuge tube, vibrating and sterilizing for 4 hours at the rotating speed of 60rpm, discarding the solution, and washing for 4 times by using high-pressure sterilized ultrapure water to obtain the treated sorghum seeds.
Further, the surfactant in the step (11) is tween 40.
Further, the illumination intensity upon illumination in the step (4) was 50. Mu. Mol m -2 s -1 。
Further, in step (6), the embryogenic callus to be inoculated needs to be tamped open prior to inoculation.
Further, the illumination intensity upon illumination in the step (62) was 50. Mu. Mol m -2 s -1 。
Further, the length of the main stem cut in the step (8) is 2cm or more.
Further, the light in the illumination in the step (9)The irradiation intensity was 50. Mu. Mol m -2 s -1 。
Example 3
A method for inducing mature sorghum seed somatic embryo comprises the following steps:
(1) Sterilizing mature sorghum seeds;
(2) Preparing a callus induction culture medium:
(21) Respectively adding 9.2g of MS culture medium, 5g of sucrose, 15g of agar powder and 1L of water into a container, uniformly stirring to obtain a mixed solution, and then regulating the pH value to 6.2;
(22) Sterilizing the mixed solution at 130 ℃ for 10 minutes by high-pressure steam, cooling to 75 ℃, and then adding a proper amount of 2,4-D (namely 2, 4-dichlorophenoxyacetic acid) mother solution into the mixed solution, and regulating the concentration of the 2,4-D to 20mg/L;
(23) Then adding CuSO thereto 4 Mother liquor, adjust CuSO 4 The concentration of the culture medium is 2 mu mol/L, the culture medium is poured into a plate after shaking evenly, 20-30 ml of the culture medium is poured into a culture dish, and the culture dish containing the callus induction culture medium is obtained after condensing on an ultra-clean workbench;
(3) Uniformly sowing the sorghum seeds treated in the step (1) onto the culture dish containing the callus induction medium obtained in the step (2), wherein:
the embryo of the sorghum seeds faces downwards, and the sowing density is controlled to be 30 grains/culture dish;
(4) Sealing the culture dish treated in the step (3), placing the culture dish in an incubator at 20-30 ℃, and continuously culturing for 8 weeks at 16h/8h to obtain embryogenic callus;
(5) Preparation of differentiation medium:
(51) Respectively adding 9.2g of MS culture medium, 5g of sucrose, 15g of agar powder and 1L of water into a container, uniformly stirring to obtain a mixed solution, and then regulating the pH value to 6.2;
(52) Sterilizing the mixed solution at 110-130 ℃ by high-pressure steam for 10-60 minutes, and cooling to 75 ℃;
(53) Adding a proper amount of IAA (indoleacetic acid) mother liquor into the mixed liquor, and regulating the concentration of the IAA to 5mg/L;
(54) Adding BAP (6-BA, 6-benzylaminopurine) mother liquor into the mixed liquor, and regulating the concentration of BAP to 5mg/L;
(55) Adding CuSO into the mixed solution 4 Mother liquor, adjust CuSO 4 The concentration of the culture medium is 2 mu mol/L, the culture medium is obtained after shaking, the culture medium is poured into a breathable glass culture flask every 20 to 30ml, and the culture flask containing the differentiation culture medium is obtained after condensing on an ultra-clean workbench;
(6) And (3) differentiation culture:
(61) Inoculating the embryogenic callus induced in the step (4) to a culture flask containing the differentiation medium obtained in the step (5);
(62) Sealing the inoculated culture bottle, placing the bottle in an incubator at 230 ℃, continuously culturing for 4 weeks at 16h/8h in a photoperiod, and allowing embryogenic callus to see complete main stems;
(7) Preparing a rooting culture medium:
(71) Respectively adding 9.2g of MS culture medium, 5g of sucrose, 15g of agar powder and 1L of water into a container, uniformly stirring to obtain a mixed solution, and then regulating the pH value to 6.2;
(72) Sterilizing the mixed solution at 130 ℃ for 10 minutes by high-pressure steam, and cooling to 75 ℃;
(73) Adding NAA (1-naphthylacetic acid) mother liquor into the mixed liquor, and regulating the concentration of NAA to 2mg/L;
(74) Adding IAA mother liquor into the mixed liquor, and regulating the concentration of IAA to 2mg/L;
(75) Adding IBA (indolebutyric acid) mother liquor into the mixed liquor, and regulating the concentration of IBA to 2mg/L;
(76) Adding CuSO into the mixed solution 4 Mother liquor, adjust CuSO 4 The concentration of the culture medium is 2 mu mol/L, the culture medium is obtained after shaking, the culture medium is poured into a breathable glass culture flask every 30ml, and the culture flask containing rooting culture medium is obtained after condensation on an ultra-clean workbench;
(8) Rooting culture:
cutting off the main stem of the embryogenic callus induced in the step (6), discarding the residual callus, and inoculating the residual callus to a culture flask containing a rooting medium obtained in the step (7), wherein:
each flask was inoculated with only one main stem;
(9) Sealing the inoculated culture bottle in the step (8), placing the bottle in an incubator at 30 ℃, continuously culturing for 6 weeks, rooting the bottle, and transplanting the bottle into soil to complete the life history, wherein the photoperiod is 16h/8 h. The induction efficiency of the technical scheme of example 2 was calculated to be 60%.
Further, the step (1) includes the steps of:
(11) Taking 20ml of mature sorghum seeds, placing the mature sorghum seeds into a 50ml centrifuge tube, then dripping 3 drops of surfactant into the centrifuge tube, then adding 35ml of 75% v/v ethanol, filling the centrifuge tube, and closing a cover;
(12) Vibrating and sterilizing the centrifuge tube on an oscillator at a rotating speed of 100rpm for 30 minutes, and discarding liquid after completion;
(13) And adding a proper amount of pasteurization solution with the volume of 2% v/v into the centrifuge tube, vibrating and sterilizing for 2 hours at the rotating speed of 100rpm, discarding the solution, and washing for 5 times by using high-pressure sterilized ultrapure water to obtain the treated sorghum seeds.
Further, the surfactant in the step (11) is tween 60. In another embodiment, the surfactant in step (11) is tween 80.
Further, the illumination intensity upon illumination in the step (4) was 500. Mu. Mol m -2 s -1 。
Further, in step (6), the embryogenic callus to be inoculated needs to be crushed prior to inoculation.
Further, the illumination intensity upon illumination in the step (62) was 500. Mu. Mol m -2 s -1 。
Further, the length of the main stem cut in the step (8) is 2cm or more.
Further, the illumination intensity upon illumination in the step (9) was 500. Mu. Mol m -2 s -1 。
Verification test
1. Effect of 2,4-D concentration on Induction efficiency
The technical scheme of the embodiment 1 is adopted for verification test, the concentrations of 2,4-D are respectively set to be 0mg/L, 2mg/L, 4mg/L, 6mg/L, 8mg/L, 10mg/L and 20mg/L according to a control variable method, and the specific test results are shown in the following table:
| concentration of 2,4-D (mg/L) | Induction efficiency | |
| Blank control test | 0 | 0 |
| Comparative example 1 | 2 | 20％ |
| Comparative example 2 | 4 | 50％ |
| Comparative example 3 | 6 | 66％ |
| Example 1 | 8 | 70％ |
| Comparative example 4 | 10 | 63％ |
| Control implementationExample 5 | 20 | 40％ |
Wherein: the blank and comparative examples 1-5 are substantially identical to example 1, except that a different concentration setting of 2,4-D is used in step (22).
2. When the control examples 1 and 1 were carried out for 60 days, they were subjected to histological examination, respectively. FIGS. 1a and 1b are histological microscopic images of mature sorghum seed somatic embryogenesis of control example 1.
FIGS. 1c and 1d are histological microscopic images of mature sorghum seed somatic embryogenesis of example 1. Wherein:
mature sorghum seeds induced at low concentration of 2,4-D (2 mg/L) of control example 1, mostly seen as white loose non-embryogenic callus (as shown in FIGS. 1a and 1 b), showed root organogenesis of parenchyma, with no ability to develop into whole plants.
Mature sorghum seeds induced at high concentration of 2,4-D (8 mg/L) of example 1, visible yellow embryogenic callus, SE at globular embryo and pear embryo stage were clearly distinguishable (as shown in FIGS. 1c and 1D), wherein:
rp represents a root primordium; rc represents a root crown cell; mc represents meristematic cells; vb represents the bundle of vascular tubes;
SE means somatic embryo.
3. FIG. 2 is a schematic representation of the generation of two calli from control example 1 and example 1, wherein:
the development process of the non-embryogenic callus of control example 1 corresponds to A-E on the left side of FIG. 2;
the development of the yellow embryogenic callus of example 1 corresponds to F-J on the right side of FIG. 2, wherein:
rp represents a root primordium; sc represents the seed coat; SE means somatic embryo.
4. FIG. 3 is a schematic representation of somatic embryo induction assays for four hybrid varieties. As shown in FIG. 3, the mature seeds of four sorghum hybrid varieties "Liao-za 27 (Liao-za 27)", "Liao-za 36 (Liao-za 36)", 'Liao-za 52 (Liao-za 52)' and 'Liao-tan 3 (Liao-tie 3)' were subjected to embryo induction using the technical scheme of example 1 with a2, 4-D gradient, and the results showed that no obvious genotype dependence was found in the four varieties tested.
In summary, the induction efficiency of the invention can reach 70%, and can replace the traditional IZE (immature zygotic embryo) receptor system; overcomes the defects of long preparation period, high cost and genotype dependence of IZE and solves the problem of annual supply of sorghum DNA-free gene editing receptors.
The embodiments of the present invention have been described in detail. However, the present invention is not limited to the above-described embodiments, and various modifications may be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.
Claims (9)
1. A method for inducing mature sorghum seed somatic embryos, which is characterized by comprising the following steps:
(1) Sterilizing mature sorghum seeds;
(2) Preparing a callus induction culture medium:
(21) Respectively adding 2.3-9.2 g of MS culture medium, 1-5 g of sucrose, 7-15 g of agar powder and 1L of water into a container, uniformly stirring to obtain a mixed solution, and then regulating the pH value to 5.5-6.2;
(22) Sterilizing the mixed solution at 110-130 ℃ for 10-60 minutes by high pressure steam, cooling to RT-75 ℃, adding a proper amount of 2,4-D mother liquor, and regulating the concentration of 2,4-D to 4-20 mg/L;
(23) Then CuSO is added thereto 4 Mother liquor, adjust CuSO 4 The concentration of the culture medium is 0.5-2 mu mol/L, shaking evenly, pouring the culture medium into a plate, pouring 20-30 ml of the culture medium into a culture dish, and condensing the culture medium on an ultra-clean workbench to obtain the culture dish containing the callus induction culture medium;
(3) Uniformly sowing the sorghum seeds treated in the step (1) onto the culture dish containing the callus induction medium obtained in the step (2), wherein:
the embryo of the sorghum seeds faces downwards, and the sowing density is controlled to be 10-30 grains/culture dish;
(4) Sealing the culture dish treated in the step (3), placing the culture dish in an incubator at 20-30 ℃, and continuously culturing for 6-8 weeks at 16h/8h to obtain embryogenic callus;
(5) Preparation of differentiation medium:
(51) Respectively adding 2.3-9.2 g of MS culture medium, 1-5 g of sucrose, 7-15 g of agar powder and 1L of water into a container, uniformly stirring to obtain a mixed solution, and then regulating the pH value to 5.5-6.2;
(52) Sterilizing the mixed solution at 110-130 ℃ by high-pressure steam for 10-60 minutes, and cooling to RT-75 ℃;
(53) Adding a proper amount of IAA mother liquor into the mixed liquor, and regulating the concentration of IAA to 1-5 mg/L;
(54) Adding BAP mother liquor into the mixed liquor, and regulating the concentration of BAP to 1-5 mg/L;
(55) Adding CuSO into the mixed solution 4 Mother liquor, adjust CuSO 4 The concentration of the culture medium is 0.5-2 mu mol/L, the culture medium is obtained after shaking, the plate is poured, every 20-30 ml of the culture medium is poured into a breathable glass culture flask, and the culture flask containing the differentiation culture medium is obtained after condensing on an ultra-clean workbench;
(6) And (3) differentiation culture:
(61) Inoculating the embryogenic callus induced in the step (4) to a culture bottle containing the differentiation medium obtained in the step (5);
(62) Sealing the inoculated culture bottle, placing the bottle in an incubator at 20-30 ℃, continuously culturing for 4-8 weeks at 16h/8h, and allowing embryogenic callus to see complete main stems;
(7) Preparing a rooting culture medium:
(71) Adding 2.3-9.2 g of MS culture medium, 1-5 g of sucrose, 7-15 g of agar powder and 1L of water into a container respectively, stirring uniformly to obtain a mixed solution, and then regulating the pH value to 5.5-6.2;
(72) Sterilizing the mixed solution at 110-130 ℃ by high-pressure steam for 10-60 minutes, and cooling to RT-75 ℃;
(73) Adding NAA mother liquor into the mixed liquor, and regulating the concentration of NAA to 0.5-2 mg/L;
(74) Adding IAA mother liquor into the mixed liquor, and regulating the concentration of IAA to 0.5-2 mg/L;
(75) Adding IBA mother liquor into the mixed liquor, and adjusting the concentration of IBA to 0.5-2 mg/L;
(76) Adding CuSO into the mixed solution 4 Mother liquor, adjust CuSO 4 The concentration of the culture medium is 0.5-2 mu mol/L, the culture medium is obtained after shaking, the plate is poured, each 20-30 ml of culture medium is poured into a breathable glass culture flask, and the culture flask containing rooting culture medium is obtained after condensing on an ultra-clean workbench;
(8) Rooting culture:
cutting off the main stem of the embryogenic callus induced in the step (6), discarding the residual callus, and inoculating the residual callus to a culture flask containing a rooting medium obtained in the step (7), wherein:
each flask was inoculated with only one main stem;
(9) Sealing the inoculated culture bottle in the step (8), placing the bottle in an incubator at 20-30 ℃, continuously culturing for 4-6 weeks with a photoperiod of 16h/8h, rooting, and transplanting the root into soil to complete the life history.
2. The method of inducing somatic embryos in mature sorghum seeds according to claim 1 wherein step (1) comprises the steps of:
(11) Taking 10-20 ml of mature sorghum seeds, placing the mature sorghum seeds into a 50ml centrifuge tube, then dripping 1-3 drops of surfactant into the centrifuge tube, then adding a proper amount of 75% v/v ethanol, filling the centrifuge tube, and covering a cover;
(12) Vibrating and sterilizing the centrifuge tube on an oscillator at a rotating speed of 60-100 rpm for 30-60 minutes, and discarding liquid after completion;
(13) Adding a proper amount of pasteurization liquid with the volume of 2% v/v into the centrifuge tube, vibrating and disinfecting for 2-4 hours at the rotating speed of 60-100 rpm, removing the liquid, and washing at least three times by using high-pressure sterilized ultrapure water to obtain the treated sorghum seeds.
3. The method of inducing somatic embryos of mature sorghum seeds according to claim 2, wherein the surfactant in step (11) is one of tween 20, tween 40, tween 60 and tween 80.
4. The method of inducing somatic embryos in mature sorghum seeds according to claim 1, wherein the concentration of 2,4-D is adjusted to 6-10 mg/L in step (22).
5. The method for inducing somatic embryos of mature sorghum seeds according to claim 1, wherein the illumination intensity in the illumination in the step (4) is 50-500 μmolm -2 s -1 。
6. A method of inducing somatic embryos from mature sorghum seeds according to claim 1 wherein in step (6) the embryogenic callus to be inoculated is crushed or mashed prior to inoculation.
7. The method of inducing somatic embryos of mature sorghum seeds according to claim 1, wherein the light intensity upon illumination in step (62) is 50-500 μmolm -2 s -1 。
8. The method of inducing somatic embryos from mature sorghum seeds according to claim 1, wherein the length of the main stem cut in step (8) is above 2 cm.
9. The method for inducing somatic embryos of mature sorghum seeds according to claim 1, wherein the light intensity upon illumination in the step (9) is 50-500 μmolm -2 s -1 。
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