CN115486372A - Method for constructing in-vitro regeneration system of drooping hot pepper - Google Patents
Method for constructing in-vitro regeneration system of drooping hot pepper Download PDFInfo
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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
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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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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
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- Y02A40/22—Improving land use; Improving water use or availability; Controlling erosion
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
The invention discloses a method for constructing a pendulous chili in-vitro regeneration system, and belongs to the technical field of plant tissue culture. The construction method comprises the steps of obtaining aseptic seedlings, induction culture, subculture, rooting culture and hardening seedling transplantation. The invention establishes an efficient regeneration system of the drooping pepper obtained by the induction of sterile seedling stemmed cotyledon, and improves the regeneration rate of the drooping pepper. Based on an organogenesis approach, the invention takes the cotyledon of the drooping pepper with the stalk as an explant, and adds the plant hormones 6-BA and IAA, thereby simplifying the construction of the regeneration system of the drooping pepper in vitro, establishing a stable and efficient regeneration system, and providing technical support for the functional genome research and the molecular assisted breeding of the drooping pepper.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a construction method of a pendulous pepper in-vitro regeneration system.
Background
Hot pepper (A)Capsicumspp.) isThe annual or perennial plants of the capsicum genus of the solanaceae family, originating in the central and south america region, are one of the earliest domesticated vegetable crops in the world. Because the pepper has the functions of vegetables and seasonings, the pepper has extremely high economic value worldwide. The pepper has the functions of eating, viewing, medicine and the like, and has extremely high economic and research values. Currently, 35 species have been identified in the capsicum genus, including 5 pepper cultivars, each of which is: annual pepper (Zhao)C. aunuumL., chinese pepper (Chili pepper)C. chinenseJacq.), frutex pepper (capsicum frutescens (r) ((r))C. frutescensL. and drooping pepper (L.), (C. baccatumL.) and paprika (C. pubesensRuiz & Pavon). Drooping peppers are produced in south america in monogamy and the corolla has yellow or green spots, and there are significant morphological and genetic differences with other pepper cultivars and reproductive isolation.
The in vitro regeneration research is the key of the plant biotechnology and the plant biology basic research, and the in vitro regeneration approaches taking plant part organs as explants have two ways: somatic embryogenesis pathways and organogenesis pathways. The somatic embryogenesis way is subjected to stages of embryogenic callus, early proembryo, spherical embryo, heart-shaped embryo, torpedo embryo and the like to form mature cotyledon embryo, and finally the mature cotyledon embryo grows into a complete plant; the organ generation way directly generates bud primordium of the adventitious bud when the explant generates callus, then develops the adventitious bud, induces the adventitious root and finally grows a complete plant. The time for the organogenic pathway to form a whole plant is generally shorter than the somatic embryogenesis pathway.
Compared with other species in Solanaceae, the construction and development of the hot pepper in-vitro regeneration system are slow, and the research and development of hot pepper functional genes are severely limited. The regeneration system of pepper is mainly focused on annual pepper (capsicum annuum)C. annuum) And Chinese Chili pepper (C)C. chinense) Part of the varieties and strains in (1). The pepper is a recalcitrant plant for tissue culture, different genotypes (different germplasm and varieties) in the same cultivar have obvious difference, the reference property between different cultivars is weak, and a great deal of effort is required to explore the type, the hormone concentration, the regeneration way and the like of an explant, so that various cultivars or varieties can be obtainedAn in vitro regeneration system with ideal quality. The same explant and hormone formula are utilized by the team to test a plurality of drooping pepper varieties or germplasm, the complete plant is not regenerated in vitro, and the fact that the drooping pepper needs to be continuously explored for the regeneration in vitro is prompted.
Disclosure of Invention
Aiming at the defects of the existing in-vitro regeneration system of the drooping hot pepper, the invention provides a construction method of the stable, high-efficiency and high-inductivity in-vitro regeneration system of the drooping hot pepper.
The purpose of the invention is realized by the following technical scheme: a construction method of a prolapsed pepper in-vitro regeneration system comprises the following steps:
s1, obtaining aseptic seedlings: soaking drooping pepper seeds in ultrapure water, sterilizing, drying by using sterile filter paper, and inoculating the drooping pepper seeds to an MS solid culture medium to obtain sterile seedlings;
s2, induced culture: cutting the aseptic seedling growing for 15-25 days to take the cotyledon with the stalk as an explant, inoculating the explant to an induction culture medium for culturing for 20-25 days, and generating callus and adventitious buds at two ends of the cotyledon with the stalk; wherein the induction culture medium is MS +3 mg/L6-BA +0.5mg/L IAA;
s3, subculturing: transferring the callus and the adventitious bud generated in the step S2 into a subculture medium for culturing for 15-20 days, wherein the adventitious bud grows to 4-6 cm; wherein the subculture medium is the same as the induction medium;
s4, rooting culture: cutting the adventitious bud in the step S2 or S3, inoculating the cut adventitious bud into a rooting culture medium, and culturing for 2-3 weeks to grow 5-8 root systems; wherein the rooting medium is MS +0.2 mg/L IAA;
s5, hardening and transplanting seedlings: and (5) after the height of the rooting seedling plant in the step (S4) is 7-10 cm, removing the bottle cap, hardening the seedling for 1-2 d, taking the regenerated seedling out of the culture bottle, washing off the root culture medium, planting the regenerated seedling into the peat of the Pingshi, culturing for 20-25 d, and transplanting to the outdoors.
Further, the sterilization method in step S1 is: soaking in 75% alcohol for 1-2 min, washing with sterile water for 2 times, shaking with 10% (v/v) sodium hypochlorite solution for 15-20 min, and washing with sterile water for 5-8 times.
Further, the length of the cotyledon with stalk in the step S2 is 0.5-1 cm.
Further, the light conditions for the culture in steps S1, S2, S3 and S4 are: the illumination culture time is 16-18 h, and the illumination culture intensity is 3000-4000 lux.
The invention has the following advantages:
(1) Based on an organogenesis approach, the cotyledon with the handle of the drooping pepper is used as an explant, and the plant hormones 6-BA and IAA are added, so that the steps of the in-vitro regeneration system of the drooping pepper are simplified, a stable and efficient regeneration system is constructed, the highest induction rate reaches more than 72%, and technical support is provided for functional genome research and molecular assisted breeding of the drooping pepper;
(2) The invention establishes an efficient regeneration system for obtaining the drooping hot pepper through the induction of sterile seedling stemmed cotyledons, improves the regeneration rate of the drooping hot pepper, and provides technical support for the rapid propagation and genetic transformation of the hot pepper.
(3) The invention can quickly obtain the regeneration plant of the drooping pepper in a short time, is not limited by regions, seasons and climate, has relatively stable hereditary characters, provides germplasm resource materials for the research of the resistance of the drooping pepper and lays a foundation for the research of the stress resistance gene.
(4) The method constructs and optimizes the in-vitro regeneration system of the drooping hot pepper, solves the problem of low efficiency of the in-vitro regeneration system of the drooping hot pepper, provides great driving force for functional genomics research and molecular assisted breeding development of the drooping hot pepper, and can accelerate the breeding process and shorten the breeding time.
Drawings
FIG. 1 is a comparison of the tissue culture regeneration process of the present invention: in the figure, A is an explant of a drooping pepper cotyledon with a stalk; b is explant induced callus; c, differentiating the callus to form adventitious buds; D-E is the adventitious bud subculture for inducing rooting of the pepper explant; F-G is a regenerated seedling for inducing rooting; h, transplanting the regenerated seedlings in a phytotron; i is the regenerated plant transplanted to the base.
Detailed Description
The invention is further described below with reference to the following figures and examples, without limiting the scope of the invention to the following:
example 1: a construction method of a prolapsed pepper in-vitro regeneration system comprises the following steps:
s1, obtaining aseptic seedlings: soaking drooping pepper seeds in ultrapure water, then soaking in 75% ethanol for 1min, washing with sterile water for 2 times, then shaking with 10% (v/v) sodium hypochlorite solution for 15min, washing with sterile water for 5 times, sucking dry with sterile filter paper, and inoculating to MS solid culture medium for culture to obtain sterile seedlings; the illumination conditions of the culture are as follows: the illumination culture time is 16h, and the illumination culture intensity is 3000lux;
s2, induced culture: cutting sterile seedlings growing for 15 days to take cotyledons with stalks and with the length of 0.5-1 cm as explants, inoculating the cotyledons with stalks to an induction culture medium for culturing for 20 days, and generating calluses and adventitious buds at two ends of the cotyledons with stalks; wherein the induction culture medium is MS +3 mg/L6-BA +0.5mg/L IAA; the illumination conditions of the culture are as follows: the illumination culture time is 16h, and the illumination culture intensity is 3000lux;
s3, subculturing: transferring the callus and the adventitious buds generated in the step S2 into a subculture medium for culturing for 15d, wherein the culture conditions are as follows: the illumination culture time is 16h, and the illumination culture intensity is 3000lux; the adventitious bud grows to 4-6 cm; wherein the subculture medium is the same as the induction medium;
s4, rooting culture: and (3) cutting the adventitious bud in the step S2 or S3, inoculating the cut adventitious bud into a rooting culture medium for culture, wherein the culture illumination condition is as follows: the illumination culture time is 16h, the illumination culture intensity is 3000lux, 5-8 root systems grow after 2 weeks of culture; wherein the rooting medium is MS +0.2 mg/L IAA;
s5, hardening and transplanting seedlings: and (5) after the height of the rooting seedling plant in the step (S4) is 7-10 cm, removing the bottle cap, hardening the seedling for 1d, taking the regenerated seedling out of the culture bottle, washing off the root culture medium, planting the regenerated seedling into the peat moss, culturing for 20d, and transplanting to the outdoors.
Example 2: a construction method of a prolapsed pepper in-vitro regeneration system comprises the following steps:
s1, obtaining aseptic seedlings: soaking drooping pepper seeds in ultrapure water, soaking in 75% ethanol for 2min, washing with sterile water for 2 times, shaking with 10% (v/v) sodium hypochlorite solution for 20min, washing with sterile water for 8 times, drying with sterile filter paper, and inoculating to MS solid culture medium to obtain sterile seedlings; the lighting conditions of the culture are as follows: the illumination culture time is 18h, and the illumination culture intensity is 4000lux;
s2, induced culture: cutting sterile seedlings growing for 25d, taking cotyledons with stalks and with the length of 0.5-1 cm as explants, inoculating the cotyledons with stalks to an induction culture medium for culturing for 25d, and generating calluses and adventitious buds at two ends of the cotyledons with stalks; wherein the induction culture medium is MS +3 mg/L6-BA +0.5mg/L IAA; the illumination conditions of the culture are as follows: the illumination culture time is 18h, and the illumination culture intensity is 4000lux;
s3, subculturing: transferring the callus and the adventitious bud generated in the step S2 to a subculture medium for culturing for 20d, wherein the culturing illumination condition is as follows: the illumination culture time is 18h, and the illumination culture intensity is 4000lux; the adventitious bud grows to 4-6 cm; wherein the subculture medium is the same as the induction medium;
s4, rooting culture: and (3) cutting the adventitious bud in the step S2 or S3, inoculating the cut adventitious bud into a rooting culture medium for culture, wherein the culture illumination condition is as follows: the illumination culture time is 18h, the illumination culture intensity is 4000lux, and 5-8 root systems grow after 3 weeks of culture; wherein the rooting medium is MS +0.2 mg/L IAA;
s5, hardening and transplanting seedlings: and (5) after the height of the rooted seedling plant in the step (S4) is 7-10 cm, removing the bottle cap, hardening off the seedling for 2d, taking the regenerated seedling out of the culture bottle, washing off the root culture medium, planting the regenerated seedling into the peat, culturing for 25d, and transplanting to the outdoor.
Example 3: a construction method of a prolapsed pepper in-vitro regeneration system comprises the following steps:
s1, obtaining aseptic seedlings: soaking drooping pepper seeds in ultrapure water, then soaking in 75% ethanol for 1min, washing with sterile water for 2 times, shaking with 10% (v/v) sodium hypochlorite solution for 18min, washing with sterile water for 7 times, drying with sterile filter paper, and inoculating to MS solid culture medium to obtain sterile seedlings; the lighting conditions of the culture are as follows: the illumination culture time is 17h, and the illumination culture intensity is 3500lux;
s2, induced culture: cutting sterile seedlings which grow for 20d, taking stalk-carrying cotyledons with the length of 0.5-1 cm as explants, inoculating the stalk-carrying cotyledons to an induction culture medium for culture for 23d, and generating calluses and adventitious buds at two ends of the stalk-carrying cotyledons; wherein the induction culture medium is MS +3 mg/L6-BA +0.5mg/L IAA; the illumination conditions of the culture are as follows: the illumination culture time is 17h, and the illumination culture intensity is 3000-4000 lux;
s3, subculturing: transferring the callus and the adventitious bud generated in the step S2 to a subculture medium for 18d, wherein the illumination condition for culturing is as follows: the illumination culture time is 17h, and the illumination culture intensity is 3500lux; the adventitious bud grows to 4-6 cm; wherein the subculture medium is the same as the induction medium;
s4, rooting culture: and (3) cutting the adventitious bud in the step S2 or S3, inoculating the cut adventitious bud into a rooting culture medium for culture, wherein the culture illumination condition is as follows: the illumination culture time is 17h, the illumination culture intensity is 3500lux, 5-8 root systems grow after 2-3 weeks of culture; wherein the rooting medium is MS +0.2 mg/L IAA;
s5, hardening and transplanting seedlings: and (5) after the height of the rooted seedling plant in the step (S4) is 7-10 cm, removing the bottle cap, hardening the seedling for 1.5d, taking the regenerated seedling out of the culture bottle, washing off the root culture medium, planting the regenerated seedling into the peat, culturing for 23d, and transplanting the regenerated seedling to the outdoor.
The following experiments illustrate the beneficial effects of the present invention:
1. explant
Soaking drooping pepper seeds in ultrapure water for 2h, soaking in 75% ethanol for 1min, washing with sterile water for 2 times, shaking with 10% (v/v) sodium hypochlorite solution for 15min, washing with sterile water for 8 times, drying with sterile filter paper, and inoculating to MS solid culture medium to obtain sterile seedlings;
respectively cutting sterile seedlings growing for 20 days into cotyledons with stalks by 0.5-1 cm, cotyledons and hypocotyls serving as explants, inoculating the explants to an induction culture medium for culture for 25 days, and generating calluses and adventitious buds at two ends of the cotyledons with stalks; wherein the induction culture medium is MS +3 mg/L6-BA +0.5mg/L IAA;
the average differentiation rate of different explants was counted and calculated, and the experimental results are shown in table 1:
TABLE 1 Effect of different explants on adventitious bud Induction
As is clear from Table 1, the average differentiation rate was the highest when the cotyledon with stalk was used as the explant.
2. Induction of adventitious buds by different phytohormone concentrations
TABLE 2 Effect of different phytohormone concentrations on adventitious bud Induction
As is clear from Table 2, 3mg/L of 6-BA +0.5mg/L of IAA had the best effect on the induction of adventitious buds.
3. Root induction by different phytohormone concentrations
As shown in table 3, the effect of different phytohormone concentrations on root induction results:
TABLE 3 Effect of different phytohormone concentrations on root Induction
As is clear from Table 3, 0.2mg/L of IAA had the best effect on root induction.
4. Conclusion
The sterilized drooping pepper seeds are inoculated in MS without phytohormone, and 15-20d seedling-age cotyledons with stalks, cotyledons and hypocotyls are taken as explants. Experiments show that the cotyledon can generate a small amount of callus as an explant, the induction rate is about 20%, hypocotyls have the problem of browning, only a small amount of callus can be generated, and the induction rate of adventitious buds is below 3%. The cotyledon with the stalk is taken as the explant and can expand at two ends to generate callus and adventitious buds, and the induced differentiation rate is more than 40 percent, so the cotyledon with the stalk is taken as the explant to induce the adventitious buds of the hot pepper to be the optimal choice.
Using the 6-BA +2,4-D formula studied by predecessors, through several germplasm experiments of drooping pepper, it was found that the explants were easy to brown and intact plants were not regenerated in vitro. The hormones commonly used in the tissue culture of hot pepper include 6-BA, IAA, ZT, etc. The adventitious buds can be differentiated by the combination of ZT and IAA through experiments, but the invention finds that the 6-BA + IAA formula is superior to ZT + IAA. Then, through different hormone concentration gradient design experiments, the optimal culture medium formula is obtained; MS +6-BA3.0mg/L + IAA0.5mg/L, and the highest regeneration rate of the adventitious bud reaches 72 percent. Compared with the reported research, the induction rate of the adventitious bud of the drooping pepper is found to be remarkably improved. According to the invention, the regeneration rate of the drooping hot pepper is greatly improved through subculture in the induction medium, so that a regeneration system is optimized, and the method has remarkable progress.
The auxin is beneficial to the induction of adventitious roots, commonly used hormones for inducing rooting comprise IAA, NAA and IBA, the IAA rooting induction rate is higher through the test of different hormones, and the optimal rooting induction culture medium formula is found through the test: MS + IAA 0.2mg/L.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution of the present invention and the inventive concept within the technical scope of the present invention.
Claims (3)
1. A method for constructing a prolapsed pepper in-vitro regeneration system is characterized by comprising the following steps:
s1, obtaining aseptic seedlings: taking drooping pepper seeds, soaking the drooping pepper seeds in ultrapure water and then sterilizing, wherein the sterilizing method comprises the following steps: soaking in 75% alcohol for 1-2 min, washing with sterile water for 2 times, shaking with 10% (v/v) sodium hypochlorite solution for 15-20 min, washing with sterile water for 5-8 times, sucking with sterile filter paper, and inoculating to MS solid culture medium to obtain sterile seedling;
s2, induced culture: cutting the sterile seedling growing for 15-25 days to take stalk cotyledon as explant, inoculating to inducing culture medium for culturing for 20-25 days to generate callus and adventitious bud; wherein the induction culture medium is MS +3 mg/L6-BA +0.5mg/L IAA;
s3, subculturing: transferring the callus and the adventitious bud generated in the step S2 into a subculture medium for culturing for 15-20 days, wherein the adventitious bud grows to 4-6 cm; wherein the subculture medium is the same as the induction medium;
s4, rooting culture: cutting the adventitious bud in the step S2 or S3, inoculating the cut adventitious bud into a rooting culture medium, and culturing for 2-3 weeks to grow 5-8 root systems; wherein the rooting medium is MS +0.2 mg/L IAA;
s5, hardening and transplanting seedlings: and (5) after the height of the rooting seedling plant in the step (S4) is 7-10 cm, removing the bottle cap, hardening the seedling for 1-2 d, taking the regenerated seedling out of the culture bottle, washing off the root culture medium, planting the regenerated seedling into the peat of the Pingshi, culturing for 20-25 d, and transplanting to the outdoors.
2. The method for constructing the in-vitro regeneration system of the drooping pepper as claimed in claim 1, wherein the cotyledon with stalk in the step S2 has a length of 0.5-1 cm.
3. The method for constructing the in-vitro regeneration system of the drooping pepper as claimed in claim 1, wherein the light conditions for the culture of the steps S1, S2, S3 and S4 are as follows: the illumination culture time is 16-18 h, and the illumination culture intensity is 3000-4000 lux.
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| CN202211390003.1A CN115486372B (en) | 2022-11-08 | 2022-11-08 | Method for constructing in-vitro regeneration system of drooping hot pepper |
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| CN116439135A (en) * | 2023-05-09 | 2023-07-18 | 遵义职业技术学院 | Quick cultivation method for capsicum by using molecular markers |
| CN117561981A (en) * | 2024-01-17 | 2024-02-20 | 浙江大学海南研究院 | Method for establishing callus regeneration system of capsicum |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117561981A (en) * | 2024-01-17 | 2024-02-20 | 浙江大学海南研究院 | Method for establishing callus regeneration system of capsicum |
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