CN112616662A - Method for separating and purifying polyploid from chimeric polyploid by using pear regeneration system - Google Patents
Method for separating and purifying polyploid from chimeric polyploid by using pear regeneration system Download PDFInfo
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- CN112616662A CN112616662A CN202011491016.9A CN202011491016A CN112616662A CN 112616662 A CN112616662 A CN 112616662A CN 202011491016 A CN202011491016 A CN 202011491016A CN 112616662 A CN112616662 A CN 112616662A
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- pear
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- polyploid
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- polyploids
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- 208000020584 Polyploidy Diseases 0.000 title claims abstract description 43
- 235000014443 Pyrus communis Nutrition 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 20
- 230000008929 regeneration Effects 0.000 title claims abstract description 17
- 238000011069 regeneration method Methods 0.000 title claims abstract description 17
- 239000001963 growth medium Substances 0.000 claims abstract description 19
- 210000001672 ovary Anatomy 0.000 claims abstract description 17
- 230000001939 inductive effect Effects 0.000 claims abstract description 11
- 230000000392 somatic effect Effects 0.000 claims abstract description 11
- 210000002257 embryonic structure Anatomy 0.000 claims abstract description 9
- 238000010494 dissociation reaction Methods 0.000 claims abstract description 4
- 230000005593 dissociations Effects 0.000 claims abstract description 4
- 210000000056 organ Anatomy 0.000 claims abstract description 4
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 4
- 239000002609 medium Substances 0.000 claims description 15
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 12
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 7
- 229940088597 hormone Drugs 0.000 claims description 7
- 239000005556 hormone Substances 0.000 claims description 7
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- 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 6
- 229930006000 Sucrose Natural products 0.000 claims description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 6
- 238000005286 illumination Methods 0.000 claims description 6
- 229910052742 iron Inorganic materials 0.000 claims description 6
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 6
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 235000010333 potassium nitrate Nutrition 0.000 claims description 6
- 239000004323 potassium nitrate Substances 0.000 claims description 6
- 239000008223 sterile water Substances 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 3
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 210000001161 mammalian embryo Anatomy 0.000 claims description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims 2
- 241000220324 Pyrus Species 0.000 description 16
- 241000196324 Embryophyta Species 0.000 description 13
- 230000035772 mutation Effects 0.000 description 5
- 235000013399 edible fruits Nutrition 0.000 description 3
- 208000035199 Tetraploidy Diseases 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000002269 spontaneous effect Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 235000021017 pears Nutrition 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
Classifications
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to a method for separating and purifying polyploids from chimeric polyploids by using a pear regeneration system, which comprises the following steps: 1) establishing an explant; 1.1) obtaining of explants: taking pear flower organs as explant sources; 1.2) disinfection of explants; 1.3) preculture of explants: inoculating the pear ovaries on a PIM culture medium for pre-culture; 2) inducing somatic embryos into seedlings: transferring the pre-cultured ovaries to an IM (instant Messaging) culture medium under the aseptic condition for culture, and inducing somatic embryos to differentiate into seedlings; 3) ploidy identification of seedlings: selecting healthy leaves, dissociating cells by using a dissociation solution, and identifying ploidy by using a cell flow instrument; 4) and (5) propagation of seedlings. The method can simply, quickly and efficiently separate and purify the polyploid plant from the pear mosaic polyploid.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for separating and purifying polyploids from chimeric polyploids by using a pear regeneration system.
Background
Polyploid breeding is one of the important means for germplasm innovation of horticultural plants. For pear varieties, the fruits of polyploid varieties are generally larger than those of diploids, and the fruits have excellent quality and strong stress resistance. The spontaneous bud mutation of fruit trees is an important source of the existing polyploid variety, but the bud mutation breeding still has a plurality of defects. For example, when spontaneous bud mutation occurs, the mutant cells and wild cells are associated with each other, so that chimera is easily formed and the chimeric type is difficult to control, thereby causing unstable mutant characters and easy loss of mutant characters. The instability not only restricts the development of bud mutation and seed selection, but also causes great loss of excellent mutation traits and special germplasm resources.
Disclosure of Invention
The invention provides a method for separating and purifying polyploidy from chimeric polyploidy by using a pear regeneration system, which can separate and purify polyploidy plants from the chimeric polyploidy simply, in a short time and efficiently.
The technical solution of the invention is as follows: the invention relates to a method for separating and purifying polyploids from chimeric polyploids by using a pear regeneration system, which is characterized by comprising the following steps: the method comprises the following steps:
1) establishing an explant;
1.1) obtaining of explants: taking pear flower organs as explant sources;
1.2) disinfection of explants;
1.3) preculture of explants: inoculating the pear ovaries on a PIM culture medium for pre-culture;
2) inducing somatic embryos into seedlings: transferring the pre-cultured ovaries to an IM (instant Messaging) culture medium under the aseptic condition for culture, and inducing somatic embryos to differentiate into seedlings;
3) ploidy identification of seedlings: selecting healthy leaves, dissociating cells by using a dissociation solution, and identifying ploidy by using a cell flow instrument;
4) and (5) propagation of seedlings.
Preferably, the chimeric polyploid pear 9712 is selected as a material in the step 1.1); selecting the pear 7 days before the full-bloom stage (the specific date depends on the climatic conditions of the current year, and using a non-flowering bud and an ovary as materials for establishing the explant.
Preferably, the specific steps of step 1.2) are as follows: washing the picked pear ovaries at 25 ℃ for 6 hours by clear water; sterilizing with 70% alcohol for 30 s; rinsing with sterile water for 30 seconds, and repeating for 4 times; sterilizing with 15% sodium hypochlorite for 20 min, and shaking for 4 times; finally, rinsing with sterile water for 30 seconds, and repeating for 4 times.
Preferably, the specific formula of the PIM medium in step 1.3) is: basic elements: 1/2QL medium (current commercial medium) supplemented with 24mg/LEDTA chelated iron, 250mg/L potassium nitrate, 97mg/L magnesium sulfate heptahydrate, 407mg/L ammonium nitrate; hormones: 0.2 mg/LIBA; carbon source: 30g/L of sucrose.
Preferably, the pre-culture conditions in step 1.3) are specifically: dark culture was carried out at 25 ℃ for 4 days.
Preferably, the inoculation mode in the step 1.3) is specifically as follows: cutting the sterile bud and the ovary at the lower part into two parts along the radial direction in a sterile super clean bench, exposing anther and ovule, cutting off redundant pedicel, and sticking the cut surface downwards on a culture medium.
Preferably, the specific formula of the IM medium in the step 2) is as follows: basic elements: 1/2QL medium (current commercial medium) supplemented with 24mg/LEDTA chelated iron, 250mg/L potassium nitrate, 97mg/L magnesium sulfate heptahydrate, 407mg/L ammonium nitrate, 2.2 mg/LTDZ; hormones: 0.2 mg/LIBA; carbon source: 30g/L of sucrose.
Preferably, the culture conditions for inducing the somatic embryos to differentiate into seedlings in the step 2) are as follows: culturing at 25 deg.C in dark for 60 days, and changing to new IM medium after culturing for 30 days.
Preferably, the specific steps of step 4) are as follows: after 4 weeks of seedling culture, selecting a tissue culture seedling with strong growth vigor, cutting off callus and redundant leaves on the lower part of the plant, keeping about 2-3 cm of the upper half part of the plant, transferring the plant into an MS culture medium for subculture and culture, wherein the culture temperature is 25 +/-2 ℃, the illumination culture is carried out, the light source is a fluorescent lamp, the illumination intensity is 2000lx, and the light cycle is 16 h.
The invention has the following advantages:
1. adopts a brand new induction formula: the invention reselects the element proportion and hormone concentration combination of the culture medium, adjusts the dark culture time, and leads the immature ovule and anther which are not pollinated to directly develop into a new plant.
2. Separating and purifying polyploid by adopting a brand new explant: the invention utilizes the cell totipotency and the gene type source unicity of the plant ovule and the anther to directly utilize the ovule and the anther to obtain a regeneration plant with a single gene type. The regenerated plants are all diploid or tetraploid through detection of a cell flow instrument, and no chimera appears.
Detailed Description
The method of the embodiment of the invention comprises the following steps:
1) establishing an explant;
1.1) obtaining of explants: taking pear flower organs as explant sources; selecting 9712 as a material for embedding polyploid pears; selecting the bud and ovary which are not bloomed as the materials for establishing the explant 7 days before the full-bloom period of the pear (the specific date depends on the climatic conditions of the current year).
1.2) disinfection of explants: washing the picked pear ovaries at 25 ℃ for 6 hours by clear water; sterilizing with 70% alcohol for 30 s; rinsing with sterile water for 30 seconds, and repeating for 4 times; sterilizing with 15% sodium hypochlorite for 20 min, and shaking for 4 times; finally, rinsing with sterile water for 30 seconds, and repeating for 4 times.
1.3) preculture of explants: inoculating the pear ovaries on a PIM culture medium for pre-culture; the specific formula of the PIM culture medium is as follows: basic elements: 1/2QL medium (current commercial medium) supplemented with 24mg/LEDTA chelated iron, 250mg/L potassium nitrate, 97mg/L magnesium sulfate heptahydrate, 407mg/L ammonium nitrate; hormones: 0.2 mg/LIBA; carbon source: 30g/L sucrose; the pre-culture conditions are specifically as follows: culturing at 25 deg.C in dark for 4 days; the inoculation mode is specifically as follows: cutting the sterile bud and the ovary at the lower part into two parts along the radial direction in a sterile super clean bench, exposing anther and ovule, cutting off redundant pedicel, and sticking the cut surface downwards on a culture medium.
2) Inducing somatic embryos into seedlings; transferring the pre-cultured ovaries to an IM (instant Messaging) culture medium under the aseptic condition for culture, and inducing somatic embryos to differentiate into seedlings; the specific formula of the IM culture medium is as follows: basic elements: 1/2QL medium (current commercial medium) supplemented with 24mg/LEDTA chelated iron, 250mg/L potassium nitrate, 97mg/L magnesium sulfate heptahydrate, 407mg/L ammonium nitrate, 2.2 mg/LTDZ; hormones: 0.2 mg/LIBA; carbon source: 30g/L sucrose; the culture conditions for inducing somatic embryo differentiation into seedlings are as follows: culturing at 25 deg.C in dark for 60 days, and changing to new IM medium after culturing for 30 days.
3) Ploidy identification of seedlings: healthy leaves are selected, and after cells are dissociated by using a dissociation solution, the ploidy is identified by using a cell flow meter.
4) And (3) propagation of seedlings: after 4 weeks of seedling culture, selecting a tissue culture seedling with strong growth vigor, cutting off callus and redundant leaves on the lower part of the plant, keeping about 2-3 cm of the upper half part of the plant, transferring the plant into an MS culture medium for subculture and culture, wherein the culture temperature is 25 +/-2 ℃, the illumination culture is carried out, the light source is a fluorescent lamp, the illumination intensity is 2000lx, and the light cycle is 16 h.
The regenerated plants obtained by the method for separating and purifying the polyploids from the chimeric polyploids by utilizing the pear regeneration system are detected by a cell flow instrument, and the regenerated plants are all diploid or tetraploid without chimeras.
The present invention and the technical contents not specifically described in the above embodiments are the same as the prior art.
The present invention is not limited to the above-described embodiments, and the present invention can be implemented with the above-described advantageous effects.
The above embodiments are only specific embodiments disclosed in the present invention, but the scope of the present invention is not limited thereto, and the scope of the present invention disclosed in the present invention should be subject to the scope of the claims.
Claims (9)
1. A method for separating and purifying polyploids from chimeric polyploids by using a pear regeneration system is characterized by comprising the following steps: the method comprises the following steps:
1) establishing an explant;
1.1) obtaining of explants: taking pear flower organs as explant sources;
1.2) disinfection of explants;
1.3) preculture of explants: inoculating the pear ovaries on a PIM culture medium for pre-culture;
2) inducing somatic embryos into seedlings: transferring the pre-cultured ovaries to an IM (instant Messaging) culture medium under the aseptic condition for culture, and inducing somatic embryos to differentiate into seedlings;
3) ploidy identification of seedlings: selecting healthy leaves, dissociating cells by using a dissociation solution, and identifying ploidy by using a cell flow instrument;
4) and (5) propagation of seedlings.
2. The method for separating and purifying polyploids from chimeric polyploids using the pear regeneration system according to claim 1, wherein: selecting 9712 of the chimeric polyploid pear as a material in the step 1.1); the bud and ovary which do not bloom are selected as the materials for establishing the explant 7 days before the full bloom stage of the pear.
3. The method for separating and purifying polyploids from chimeric polyploids using the pear regeneration system according to claim 2, wherein: the specific steps of step 1.2) are as follows: washing the picked pear ovaries at 25 ℃ for 6 hours by clear water; sterilizing with 70% alcohol for 30 s; rinsing with sterile water for 30 seconds, and repeating for 4 times; sterilizing with 15% sodium hypochlorite for 20 min, and shaking for 4 times; finally, rinsing with sterile water for 30 seconds, and repeating for 4 times.
4. The method for separating and purifying polyploids from chimeric polyploids using the pear regeneration system according to claim 3, wherein: the specific formula of the PIM culture medium in the step 1.3) is as follows: basic elements: 1/2QL medium supplemented with 24mg/L EDTA chelated iron, 250mg/L potassium nitrate, 97mg/L magnesium sulfate heptahydrate, 407mg/L ammonium nitrate; hormones: 0.2mg/L IBA; carbon source: 30g/L of sucrose.
5. The method of claim 4, wherein the polyploid is isolated and purified from the chimeric polyploid by using pear regeneration system, wherein: the pre-culture conditions in the step 1.3) are specifically as follows: dark culture was carried out at 25 ℃ for 4 days.
6. The method for separating and purifying polyploids from chimeric polyploids using the pear regeneration system according to claim 5, wherein: the inoculation mode in the step 1.3) is specifically as follows: cutting the sterile bud and the ovary at the lower part into two parts along the radial direction in a sterile super clean bench, exposing anther and ovule, cutting off redundant pedicel, and sticking the cut surface downwards on a culture medium.
7. The method of claim 6, wherein the polyploid is isolated and purified from the chimeric polyploid by using pear regeneration system, wherein: the specific formula of the IM culture medium in the step 2) is as follows: basic elements: 1/2QL medium supplemented with 24mg/L EDTA chelated iron, 250mg/L potassium nitrate, 97mg/L magnesium sulfate heptahydrate, 407mg/L ammonium nitrate, 2.2mg/L TDZ; hormones: 0.2mg/L IBA; carbon source: 30g/L of sucrose.
8. The method of claim 7, wherein the polyploid is isolated and purified from the chimeric polyploid by using pear regeneration system, wherein: the culture conditions for inducing the somatic embryo to differentiate into seedlings in the step 2) are as follows: culturing at 25 deg.C in dark for 60 days, and changing to new IM medium after culturing for 30 days.
9. The method of claim 8, wherein the polyploid is isolated and purified from the chimeric polyploid using pear regeneration system, wherein: the specific steps of the step 4) are as follows: after 4 weeks of seedling culture, selecting a tissue culture seedling with strong growth vigor, cutting off callus and redundant leaves on the lower part of the plant, keeping about 2-3 cm of the upper half part of the plant, transferring the plant into an MS culture medium for subculture and culture, wherein the culture temperature is 25 +/-2 ℃, the illumination culture is carried out, the light source is a fluorescent lamp, the illumination intensity is 2000lx, and the light cycle is 16 h.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011491016.9A CN112616662A (en) | 2020-12-17 | 2020-12-17 | Method for separating and purifying polyploid from chimeric polyploid by using pear regeneration system |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011491016.9A CN112616662A (en) | 2020-12-17 | 2020-12-17 | Method for separating and purifying polyploid from chimeric polyploid by using pear regeneration system |
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| CN202011491016.9A Pending CN112616662A (en) | 2020-12-17 | 2020-12-17 | Method for separating and purifying polyploid from chimeric polyploid by using pear regeneration system |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113897329A (en) * | 2021-10-11 | 2022-01-07 | 河北农业大学 | Method for inducing and culturing pear anther callus |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB8714160D0 (en) * | 1986-06-26 | 1987-07-22 | Oji Paper Co | Mass propagation through shoot primordia |
| CN105557522A (en) * | 2015-12-16 | 2016-05-11 | 青岛百瑞吉生物工程有限公司 | Detached leaf somatic embryo induction-based rapid-propagation cultivation method for pear trees |
| CN106688881A (en) * | 2016-12-16 | 2017-05-24 | 北京林业大学 | Method for separating homozygotes from ploidy chimera plant |
| CN112042543A (en) * | 2020-10-22 | 2020-12-08 | 郭爱英 | Method for obtaining haploid plant through in vitro culture of pear unfertilized ovule |
-
2020
- 2020-12-17 CN CN202011491016.9A patent/CN112616662A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB8714160D0 (en) * | 1986-06-26 | 1987-07-22 | Oji Paper Co | Mass propagation through shoot primordia |
| CN105557522A (en) * | 2015-12-16 | 2016-05-11 | 青岛百瑞吉生物工程有限公司 | Detached leaf somatic embryo induction-based rapid-propagation cultivation method for pear trees |
| CN106688881A (en) * | 2016-12-16 | 2017-05-24 | 北京林业大学 | Method for separating homozygotes from ploidy chimera plant |
| CN112042543A (en) * | 2020-10-22 | 2020-12-08 | 郭爱英 | Method for obtaining haploid plant through in vitro culture of pear unfertilized ovule |
Non-Patent Citations (4)
| Title |
|---|
| MASANORI KADOTA ET AL.: "Production of triploid plants of Japanese pear (Pyrus pyrifolia Nakai) by anther culture", 《EUPHYTICA》, vol. 138, 31 December 2004 (2004-12-31), pages 141 - 147 * |
| 何子顺 等: "梨多倍体及其利用研究进展", 《山西果树》, no. 3, 31 December 2016 (2016-12-31), pages 15 - 18 * |
| 刘淑芳 等: "七月酥梨花粉植株的诱导研究", 《江苏农业科学》, no. 1, 31 December 2010 (2010-12-31), pages 180 - 181 * |
| 张绿萍: "刺梨花药和未受精胚珠离体培养", 《中国优秀博硕士学位论文全文数据库(硕士) 农业科技辑》, no. 1, 15 December 2011 (2011-12-15), pages 048 - 130 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113897329A (en) * | 2021-10-11 | 2022-01-07 | 河北农业大学 | Method for inducing and culturing pear anther callus |
| CN113897329B (en) * | 2021-10-11 | 2023-09-15 | 河北农业大学 | Method for inducing and culturing pear anther callus |
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Application publication date: 20210409 |