CN113068619A - Pepper living body regeneration method - Google Patents
Pepper living body regeneration method Download PDFInfo
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- CN113068619A CN113068619A CN202110564494.6A CN202110564494A CN113068619A CN 113068619 A CN113068619 A CN 113068619A CN 202110564494 A CN202110564494 A CN 202110564494A CN 113068619 A CN113068619 A CN 113068619A
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- 238000011069 regeneration method Methods 0.000 title claims abstract description 75
- 235000002566 Capsicum Nutrition 0.000 title claims abstract description 44
- 239000006002 Pepper Substances 0.000 title claims abstract description 41
- 235000016761 Piper aduncum Nutrition 0.000 title claims abstract description 41
- 235000017804 Piper guineense Nutrition 0.000 title claims abstract description 41
- 235000008184 Piper nigrum Nutrition 0.000 title claims abstract description 41
- 244000203593 Piper nigrum Species 0.000 title 1
- 230000008929 regeneration Effects 0.000 claims abstract description 60
- 241000722363 Piper Species 0.000 claims abstract description 40
- 241000196324 Embryophyta Species 0.000 claims abstract description 33
- 229920000742 Cotton Polymers 0.000 claims abstract description 21
- 239000001963 growth medium Substances 0.000 claims abstract description 16
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 12
- 238000012258 culturing Methods 0.000 claims abstract description 5
- 238000009630 liquid culture Methods 0.000 claims abstract description 5
- 230000003203 everyday effect Effects 0.000 claims abstract description 4
- 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 7
- 229930006000 Sucrose Natural products 0.000 claims description 7
- 239000005720 sucrose Substances 0.000 claims description 7
- 239000011782 vitamin Substances 0.000 claims description 7
- 229940088594 vitamin Drugs 0.000 claims description 7
- 229930003231 vitamin Natural products 0.000 claims description 7
- 235000013343 vitamin Nutrition 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 6
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 230000001172 regenerating effect Effects 0.000 claims description 4
- 238000009331 sowing Methods 0.000 claims description 4
- 239000000758 substrate Substances 0.000 claims description 4
- 238000002791 soaking Methods 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 206010021033 Hypomenorrhoea Diseases 0.000 claims description 2
- 230000000249 desinfective effect Effects 0.000 claims description 2
- 230000001737 promoting effect Effects 0.000 claims description 2
- 238000000338 in vitro Methods 0.000 abstract description 6
- 238000005286 illumination Methods 0.000 abstract 1
- 240000008574 Capsicum frutescens Species 0.000 description 11
- 235000002568 Capsicum frutescens Nutrition 0.000 description 8
- 230000017423 tissue regeneration Effects 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 238000010362 genome editing Methods 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 241000227653 Lycopersicon Species 0.000 description 3
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 3
- 239000001390 capsicum minimum Substances 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 230000009261 transgenic effect Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 210000003370 receptor cell Anatomy 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000207199 Citrus Species 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000028446 budding cell bud growth Effects 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Images
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
- A01H4/008—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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- 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 discloses a regeneration method of a pepper living body. The cotyledon is completely unfolded, the pepper seedling grown from the first new leaf is used as an explant, the seedling growing point and the lateral bud growing point are completely removed by a sharp scalpel, and the cotyledon is kept intact. Wrapping the cut with sterile cotton, and dripping the improved MS liquid culture medium on the sterile cotton by a micropipette every day to keep the sterile cotton moist and promote the regeneration of the callus. And (5) culturing the treated plants in an illumination incubator, and carrying out normal management. After about 30 days, bud points appear on the callus, sterile cotton is removed, 45 days of regeneration buds are elongated, and 60 days of regeneration buds grow into new plants. The method has the advantages that the optimal culture medium does not need to be screened, and the genotype dependence problem is solved; the problems of difficult root regeneration and long time in-vitro regeneration are solved by utilizing the root system of the plant; no sterile environment is needed, and the pollution problem in vitro tissue culture is avoided; high efficiency, and 2-3 new regenerated buds can be regenerated for each single plant.
Description
Technical Field
The invention relates to a regeneration method of a hot pepper living body, which has great promotion effect on hot pepper tissue regeneration, transgenosis and gene editing technology and belongs to the field of plant genetic engineering.
Background
The plant tissue regeneration is the precondition for the smooth implementation of plant transgenosis and gene editing technology, because exogenous genes need to be inserted into the DNA of receptor cells in the transgenosis process, the receptor cells carrying the exogenous genes are divided, differentiated and regenerated again, and finally, new transgenic plants are formed. Although many plants can be successfully subjected to isolated tissue culture to obtain regenerated plants, the plants with large specific gravity can not or hardly obtain the regenerated plants through isolated tissue culture, and the implementation of a transgenic technology and a gene editing technology is greatly limited.
The pepper belongs to a regenerated recalcitrant plant, has serious genotype dependence, and cannot establish a universal isolated tissue regeneration system. In the isolated culture, the induction and differentiation of pepper regeneration buds are key factors influencing pepper regeneration, different genotypes, different regeneration explants and different hormone ratios are adopted in different researches, the obtained results are different, and a unified conclusion cannot be obtained. The elongation of the regeneration bud in the isolated culture of the pepper is another difficulty of pepper regeneration, and a large amount of experiments are needed to screen an optimal induced growth culture medium. The regeneration of roots also needs to screen a culture medium for inducing root regeneration, the regeneration time is long, and some materials can grow a regenerated root system after 2-3 months.
Living regeneration techniques have been reported successfully on other plants, the most successful being tomato and citrus. The regeneration technology of living body on tomato uses 5-6 true leaf plants as explant. Because the lignification degree of the pepper stems is far higher than that of tomatoes, growth points are removed when 5-6 main leaves are cut, the pepper cannot form wound healing and cannot regenerate new plants. The explant used in the living orange regeneration technology is hypocotyl from which growth points and cotyledons are removed, and because orange is a woody plant and pepper is a herbaceous plant, the regeneration mechanisms of the orange and the capsicum are different. When the hypocotyl of growing points and cotyledons is removed from hot peppers, the wounds will wither and dry, and new plants cannot be regenerated.
At present, regeneration plants are mainly obtained on pepper through isolated tissue culture, and living body regeneration is not reported yet. Due to the lack of a universal in-vitro regeneration system, different laboratories need to start from the beginning in the related research aspect, a great deal of manpower and material resources are spent on the related research, and most research results show that the regeneration efficiency is low. Aiming at the problems in regeneration of isolated tissues of hot pepper, a new hot pepper living body regeneration method is created to improve the regeneration efficiency.
Disclosure of Invention
The invention aims to: aiming at the problems of difficult regeneration of isolated tissues of the hot pepper, large workload, low efficiency and the like, a set of new living body regeneration method is established, the problem of difficult regeneration of the hot pepper is effectively solved, and the regeneration efficiency is improved.
The purpose of the invention can be realized by the following technical scheme:
a regeneration method of living pepper comprises the following steps:
1) sowing the disinfected seeds, completely unfolding cotyledons, and taking the seedling with the first new leaf as an explant for regeneration of a pepper living body;
(2) completely cutting off the seedling growing point and the lateral bud growing point, and keeping the cotyledon intact; wrapping the cut with sterile cotton, and dripping 380-;
(3) the treated plants were placed in a light incubator with light of 10000-12000LX and light cycle of day/night: culturing for 14-16h/8-10h at day and night temperature of 24-26 deg.C/19-21 deg.C and relative humidity of 70% -75%, and normally managing; and after 28-32 days, bud points appear on the callus, sterile cotton is removed, the regeneration bud is elongated after 40-48 days, and a new plant containing the regeneration bud is grown after 58-65 days.
As a preferred aspect of the present invention, the formula of the modified MS liquid medium is: vitamin-containing MS culture medium 4-5g/L, sucrose 28-30g/L, TDZ 2.0-2.5mg/L, NAA 0.1-0.2mg/L and Ph 5.8.
As a further preferred aspect of the present invention, the formula of the modified MS liquid medium is: vitamin-containing MS culture medium 4.43g/L, sucrose 30g/L, TDZ 2.0mg/L, NAA 0.1mg/L and Ph 5.8.
Preferably, the pepper seeds are soaked in warm water at 55 ℃ for 30 minutes, sterilized and disinfected, and the disinfected seeds are sowed in 50-hole plug trays filled with pepper special seedling raising substrates, wherein 1 seed is planted in each hole.
Preferably, the treated plant is placed in a light incubator, the light is 12000LX, the light cycle is day/night: culturing for 16h/8h at 26 deg.C/20 deg.C day and night and 75% relative humidity, and normally managing. After about 30 days, bud points appear on the callus, sterile cotton is removed, 45 days of regeneration buds are elongated, 60 days of regeneration buds grow into new plants, and the new plants have 2-3 regeneration buds.
As a further preferred aspect of the present invention, the method for regenerating a living body of capsicum comprises the steps of:
1) soaking pepper seeds in warm water at 55 ℃ for 30 minutes, sterilizing and disinfecting, sowing the disinfected seeds in a 50-hole plug filled with a pepper special seedling substrate, wherein 1 seed is planted in each hole, cotyledon is completely unfolded, and the seedling with the first new leaf growing out is used as an explant for pepper living body regeneration;
(2) completely cutting off the seedling growing point and the lateral bud growing point by using a sharp scalpel, keeping cotyledon intact, wrapping the incision by using sterile cotton, dripping 400 mu L of improved MS liquid culture medium on the sterile cotton by using a micropipette every day, keeping the sterile cotton moist and promoting the regeneration of callus; the formula of the improved MS liquid culture medium is as follows: vitamin-containing MS culture medium 4.43g/L, sucrose 30g/L, TDZ 2.0mg/L, NAA 0.1mg/L and Ph 5.8;
(3) the treated plant is placed in a light incubator, the light is 12000LX, the light period is 16h/8h day/night, the day and night temperature is 26 ℃/20 ℃, the relative humidity is 75%, the plant is cultured under the condition of normal management, after 30d, the bud points appear on the callus, the sterile cotton is removed, the regeneration buds grow for 45d, the new plant is grown for 60d, and generally the new plant has 2-3 regeneration buds.
Has the advantages that:
the invention aims to create a regeneration method of a pepper living body. Compared with an in vitro tissue regeneration method, the method disclosed by the invention does not need to be operated in an aseptic environment, is simpler to operate and has loose environmental requirements; the method mainly utilizes endogenous hormones of the plant to promote the regeneration of the buds, does not need to spend a large amount of time to optimize the hormone proportion in a culture medium, and overcomes the genotype dependence problem of in vitro tissue regeneration; the root system of the plant is utilized, so that the problems of difficult regeneration and long time of the root system in the in vitro regeneration are solved. According to the invention, the regeneration verification is carried out on the pepper material CM334, the regeneration plants can be successfully obtained within 2 months, and each plant has 2-3 regeneration buds, so that the efficiency is higher. The results show that the method can be used for the tissue regeneration of the pepper, and lays a foundation for pepper transgenic technology and gene editing.
Drawings
FIG. 1 shows a method for treating a regeneration plant of a living pepper, a: the size of the plant to be treated; b: removing a growing point by using a scalpel; c: the incision was wrapped with sterile cotton soaked with MS liquid medium.
FIG. 2. regeneration process of living body of Capsici fructus, a: bud points appear at the callus; b: extending the regenerated bud; c: the regenerated bud is elongated to form a new plant.
Detailed Description
Soaking the CM334 capsicum seeds in warm water at 55 ℃ for 30 minutes, and sterilizing. The disinfected seeds are sown in 50-hole trays filled with seedling culture medium special for hot pepper, and each hole contains 1 seed. The cotyledons were fully expanded and the seedling from which the first new leaf emerged served as the explant for pepper regeneration in vivo (FIG. 1 a).
The seedling growth point and lateral bud growth point were completely excised with a sharp scalpel, leaving the cotyledons intact (FIG. 1 b). The incision was wrapped with sterile cotton, and 400. mu.L of MS liquid medium (MS basal media with vitamins (PhytoTech)4.43g/L + sucrose 30g/L + TDZ 2.0mg/L + NAA 0.1mg/L, Ph 5.8) was dropped onto the sterile cotton by a micropipette every day, keeping it moist (FIG. 1c), to promote regeneration of callus.
The treated plants are placed in a light incubator, the light is 12000LX, the light cycle is 16h/8h (day/night), the day and night temperature is 26 ℃/20 ℃, the relative humidity is 75%, and the normal management is carried out.
In the test, a pepper material CM334 was selected as the test material, and the transformation effect is shown in FIG. 2. The living regeneration method is used for generating bud points in the callus at about 30d (figure 2a), the regeneration buds grow at about 40d (figure 2b) and grow into new plants at about 50d, and the new plants generally have 2-3 regeneration buds (figure 2 c). The above results show that the regeneration method of living pepper can effectively form regenerated plants, improve the regeneration efficiency and shorten the regeneration time.
Claims (6)
1. A regeneration method of living pepper is characterized by comprising the following steps:
(1) sowing the disinfected seeds, completely unfolding cotyledons, and taking the seedling with the first new leaf as an explant for regeneration of a pepper living body;
(2) completely cutting off the seedling growing point and the lateral bud growing point, and keeping the cotyledon intact; wrapping the cut with sterile cotton, and dripping 380-;
(3) the treated plants were placed in a light incubator with light of 10000-12000LX and light cycle of day/night: culturing for 14-16h/8-10h at day and night temperature of 24-26 deg.C/19-21 deg.C and relative humidity of 70% -75%, and normally managing; and after 28-32 days, bud points appear on the callus, sterile cotton is removed, the regeneration bud is elongated after 40-48 days, and a new plant containing the regeneration bud is grown after 58-65 days.
2. The regeneration method of living pepper as claimed in claim 1, wherein the modified MS liquid medium formula is: vitamin-containing MS culture medium 4-5g/L, sucrose 28-30g/L, TDZ 2.0-2.5mg/L, NAA 0.1-0.2mg/L and Ph 5.8.
3. The regeneration method of living pepper as claimed in claim 2, wherein the modified MS liquid medium formula is: vitamin-containing MS culture medium 4.43g/L, sucrose 30g/L, TDZ 2.0mg/L, NAA 0.1mg/L and Ph 5.8.
4. The regeneration method of living bodies of pepper as claimed in claim 2, wherein pepper seeds are soaked in warm water at 55 ℃ for 30 minutes, sterilized and disinfected, and the disinfected seeds are sown in 50-hole trays filled with seedling substrate dedicated to pepper, 1 seed per hole.
5. The method for regenerating living pepper bodies according to claim 2, wherein the treated plants are placed in a light incubator with a light intensity of 12000LX and a light cycle of day/night: culturing for 16h/8h at 26 deg.C/20 deg.C day and night and 75% relative humidity, and normally managing. After about 30 days, bud points appear on the callus, sterile cotton is removed, 45 days of regeneration buds are elongated, 60 days of regeneration buds grow into new plants, and the new plants have 2-3 regeneration buds.
6. The method for regenerating living pepper as claimed in any one of claims 1-5, wherein the method for regenerating living pepper comprises the steps of:
1) soaking pepper seeds in warm water at 55 ℃ for 30 minutes, sterilizing and disinfecting, sowing the disinfected seeds in a 50-hole plug filled with a pepper special seedling substrate, wherein 1 seed is planted in each hole, cotyledon is completely unfolded, and the seedling with the first new leaf growing out is used as an explant for pepper living body regeneration;
(2) completely cutting off the seedling growing point and the lateral bud growing point by using a sharp scalpel, keeping cotyledon intact, wrapping the incision by using sterile cotton, dripping 400 mu L of improved MS liquid culture medium on the sterile cotton by using a micropipette every day, keeping the sterile cotton moist and promoting the regeneration of callus; the formula of the improved MS liquid culture medium is as follows: vitamin-containing MS culture medium 4.43g/L, sucrose 30g/L, TDZ 2.0mg/L, NAA 0.1mg/L and Ph 5.8;
(3) the treated plant is placed in a light incubator, the light is 12000LX, the light period is 16h/8h day/night, the day and night temperature is 26 ℃/20 ℃, the relative humidity is 75%, the plant is cultured under the condition of normal management, after 30d, the bud points appear on the callus, the sterile cotton is removed, the regeneration buds grow for 45d, the new plant is grown for 60d, and generally the new plant has 2-3 regeneration buds.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110564494.6A CN113068619A (en) | 2021-05-24 | 2021-05-24 | Pepper living body regeneration method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110564494.6A CN113068619A (en) | 2021-05-24 | 2021-05-24 | Pepper living body regeneration method |
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| CN113068619A true CN113068619A (en) | 2021-07-06 |
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| CN202110564494.6A Pending CN113068619A (en) | 2021-05-24 | 2021-05-24 | Pepper living body regeneration method |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429521A (en) * | 2007-11-08 | 2009-05-13 | 河南农业大学 | Cucumber Flamingo-bill explant transgenic method |
| CN101449658A (en) * | 2007-12-06 | 2009-06-10 | 河南农业大学 | Green cucumber high-frequency regeneration method |
| CN105039353A (en) * | 2015-08-31 | 2015-11-11 | 华南农业大学 | Gene CaMS1 related to chili pollen development and application thereof |
| CN105039398A (en) * | 2015-08-31 | 2015-11-11 | 华南农业大学 | Method for preparing male sterile peppers |
| CN106106149A (en) * | 2016-06-23 | 2016-11-16 | 四川农业大学 | Efficient hot pepper genetic transformation and the method for plant regeneration |
-
2021
- 2021-05-24 CN CN202110564494.6A patent/CN113068619A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429521A (en) * | 2007-11-08 | 2009-05-13 | 河南农业大学 | Cucumber Flamingo-bill explant transgenic method |
| CN101449658A (en) * | 2007-12-06 | 2009-06-10 | 河南农业大学 | Green cucumber high-frequency regeneration method |
| CN105039353A (en) * | 2015-08-31 | 2015-11-11 | 华南农业大学 | Gene CaMS1 related to chili pollen development and application thereof |
| CN105039398A (en) * | 2015-08-31 | 2015-11-11 | 华南农业大学 | Method for preparing male sterile peppers |
| CN106106149A (en) * | 2016-06-23 | 2016-11-16 | 四川农业大学 | Efficient hot pepper genetic transformation and the method for plant regeneration |
Non-Patent Citations (2)
| Title |
|---|
| 何道一等: ""农杆菌介导的小麦活体转化"", 《中国农业科学》 * |
| 曹慧颖等: ""番茄micro-TOM活体再生体系的建立"", 《中国蔬菜》 * |
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Application publication date: 20210706 |