CN116439135A - Quick cultivation method for capsicum by using molecular markers - Google Patents
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- CN116439135A CN116439135A CN202310511998.0A CN202310511998A CN116439135A CN 116439135 A CN116439135 A CN 116439135A CN 202310511998 A CN202310511998 A CN 202310511998A CN 116439135 A CN116439135 A CN 116439135A
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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
- A01H4/008—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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
- A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
-
- 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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
- A01H1/08—Methods for producing changes in chromosome number
-
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Botany (AREA)
- Biotechnology (AREA)
- Environmental Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Microbiology (AREA)
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- Biophysics (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a quick cultivation method of capsicum by using molecular markers, which comprises the steps of seed collection and treatment, induction cultivation, proliferation cultivation, rooting cultivation, seedling hardening, chromosome doubling and transplanting, ploidy identification and virus detoxification rate detection by using molecular markers. The cultivation method is free from the influence of seasons, temperature and regions, a large number of aseptic seedlings are cultivated, the growth and development speed of the peppers is effectively improved, the yield of the peppers is improved, the planting and cultivation benefits are improved, the market competitiveness is very high, meanwhile, a large number of seedlings are propagated in a short period, a molecular marker is utilized to screen out homozygous plants meeting target requirements, data support is provided for the creation of pepper germplasm resources, in addition, a theoretical basis and technical support are provided for the subsequent study of analysis and identification of main pepper flavor substances, genetic analysis of pepper flavor and molecular breeding of pepper flavor improvement through the establishment of a pepper aseptic propagation system, and the cultivation method has very important significance.
Description
Technical Field
The invention relates to the technical field of crop tissue culture, in particular to a quick cultivation method of capsicum by using molecular markers.
Background
Molecular markers are genetic markers based on nucleotide sequence variations within genetic material between individuals, and are a direct reflection of genetic polymorphisms at the DNA level. Compared with other genetic markers, namely morphological markers, biochemical markers and cytological markers, the DNA molecular markers have the following advantages: most of the molecular markers are co-dominant, so that the selection of recessive characters is very convenient; genomic variation is extremely abundant, and the number of molecular markers is almost unlimited; at different stages of biological development, DNA from different tissues can be used for marker analysis; molecular markers reveal variations from DNA; the expression is neutral, the expression of the target character is not influenced, and the linkage with the bad character is avoided; the detection means is simple and rapid. With the development of molecular biology technology, the DNA molecular marker technology has tens of types, and is widely applied to genetic breeding, genome mapping, gene positioning, species genetic relationship identification, gene library construction, gene cloning and other aspects.
The capsicum is an important vegetable crop, is a annual or limited perennial herb plant, has spicy taste and strong irritation, has a good appetizing function, contains rich vitamin C, vitamin B, carotene, calcium, iron and other mineral substances, has the functions of sterilization, corrosion prevention, seasoning, nutrition, cold dispelling and the like, and plays a positive role in preventing and treating diseases and the like for people. According to statistics of a large number of vegetable systems in the agricultural department, the annual seeding area of the capsicum in China accounts for 8-10% of the total seeding area of the vegetables in China in recent years, and the capsicum occupies the first place of the vegetables. The capsicum is one of the most important special agricultural industries in Guizhou province, and mainly focuses on the city and county of Zunyi, pichia and the like.
At present, the planting scale, the processing industry scale, the product distribution scale and the like of Guizhou peppers are first nationwide, the peppers are important vegetable types affecting the adjustment of the Guizhou agricultural production structure and the income of farmers, and the peppers are planted as one of main cash crops by agricultural farmers. The amount of capsicum planted is increasing each year due to the greater demand for capsicum. The planting method of the peppers mainly comprises sowing and seedling culturing, and after transplanting and culturing, phenomena of dead seedlings, yellowing of leaves, long seedling-recovering time, no growth and the like are easy to occur, so that the overall survival rate of the peppers is not high. The final yield and quality of the capsicum can not be well improved according to the conventional method, and the conventional planting method needs to be improved in order to promote the planting popularization range of the capsicum, so that cultivation of the capsicum virus-free seedlings is particularly important.
Disclosure of Invention
The invention aims at solving the technical problems in the background technology, adopts pepper fruits as explants, and directly cultures into seedlings by tissue culture technology under aseptic condition, thereby establishing a plant aseptic seedling system, greatly shortening the cultivation time, obtaining a large number of aseptic seedlings in a short period, reducing the death phenomenon, improving the overall survival rate, thereby improving the yield and meeting the market demand, and in particular relates to a rapid cultivation method of peppers by using molecular markers.
In order to solve the technical problems, the invention adopts the following technical scheme: the quick pepper cultivation method by using molecular markers comprises the steps of seed collection and treatment, induction cultivation, proliferation cultivation, rooting cultivation, seedling hardening, chromosome doubling and transplanting, ploidy identification and virus detoxification rate detection by using molecular markers, and specifically comprises the following steps:
s1, collecting and processing seeds: taking out the seeds after the pepper fruits are collected, soaking the seeds in clear water, sterilizing, soaking in ethanol, and then washing with sterile water for later use;
s2, induction culture: the seeds obtained in the step S1 are dried by using sterile filter paper, inoculated into a sterile induction culture medium, and cultivated in an illumination incubator under the conditions that the temperature is 25+/-1 ℃ and the illumination time is 8-12 h/d and the illumination intensity is 2800-3500 lx, so as to induce adventitious buds;
s3, proliferation cultivation: cutting the adventitious bud obtained in the step S2 into single plants by using a sterile scalpel, and reserving 2-3 leaves for each plant; inoculating the seeds into a proliferation culture medium in a sterile environment, culturing the seeds in an illumination incubator under the conditions that the temperature is 25+/-1 ℃ and the illumination time is 8-12 h/d and the illumination intensity is 2800-3500 lx, and then cutting out the grown buds and transferring the buds into the proliferation culture medium for secondary proliferation culture;
s4, rooting cultivation: cutting tender buds grown by proliferation and cultivation by using a sterile scalpel, soaking in IBA solution, reserving 3-4 leaves for each plant, selecting single plant seedlings with better growth vigor, inoculating the single plant seedlings into a rooting culture medium to induce rooting in a sterile environment, culturing the single plant seedlings in an illumination incubator at the temperature of 25+/-1 ℃ for 8-12 h/d under the illumination intensity of 2800-3500 lx, and obtaining robust rooting seedlings after culturing for one week;
s5, hardening seedlings, doubling chromosomes and transplanting: removing the rooting seedling obtained in the step (4) together with a culture container from an illumination incubator, placing the rooted pepper tissue culture seedling in a normal room temperature, standing at room temperature for hardening off for 5-10 days, taking out plants from the culture medium, removing the culture medium at the root of the seedling, soaking the root system with colchicine solution for chromosome doubling, and finally transplanting the seedling in the incubator with soil;
s6, ploidy identification: culturing the cultivated seedlings obtained in the step S5 for 20 days, and taking new leaves for ploidy identification to obtain homozygous diploid;
s7, detecting virus detoxification rate by using molecular markers: extracting genome DNA of tissue cultured pepper seedlings, taking the genome DNA as a template, carrying out PCR amplification reaction by using forward and reverse primers of molecular markers to obtain PCR amplification products, and screening out homozygous plants meeting target requirements through PCR amplification.
Further, in the step S1, the pepper fruits are collected and then the seeds are taken out, the seeds are soaked in clear water at the temperature of 40-45 ℃ for 2-3 days, then the seeds are sterilized with 0.1-0.2% potassium permanganate solution for 5-8 min, then the seeds are soaked in 75-80% ethanol for 8-12S, and finally the seeds are washed with sterile water for 3-5 times.
Further, in the S2 step, the induction culture medium is selected from an MS culture medium added with 1-3 mg/L triacontanol, 0.05-0.1 mg/L LKT (kinetin) and 0.05-0.1 mg/L NAA (naphthylacetic acid), wherein the MS culture medium comprises sucrose with the mass concentration of 3% and agar with the mass concentration of 6 g/L.
Further, the rapid cultivation method of capsicum by using molecular markers is characterized in that: in the step S3, the proliferation culture medium is selected from MS culture medium added with 2-4 mg/L2, 4-D (2, 4-dichlorophenoxyacetic acid), 0.5-1 mg/L6-BA (6-benzylaminopurine) and 0.05-0.1 mg/L NAA (naphthalene acetic acid), wherein the MS culture medium comprises sucrose with the mass concentration of 3 percent and agar with the mass concentration of 6 g/L.
Further, in the step S3, the method of subculture is to cut and transfer the grown buds into a proliferation culture medium, and to perform subculture for 2-4 times in an illumination incubator under the conditions of the temperature of 25+/-1 ℃ and the illumination time length of 8-12 h/d and the illumination intensity of 2800-3500 lx until a subcultured tissue is obtained, wherein the time of each subculture is 7-10 days.
Further, in the S4 step, the rooting medium is 1/2MS medium added with 0.5-1 mg/L IBA (indolebutyric acid) and 1.0-1.5 mg/LIAA (indoleacetic acid), wherein the 1/2MS medium contains sucrose with the mass concentration of 1.5% and agar with the mass concentration of 6 g/L.
Further, in the rapid cultivation method of capsicum using molecular markers of the present invention, in the step S5, the chromosome doubling treatment is to take out the young seedling after hardening the seedling for 5 to 7 days, wash the culture medium of the root, soak the root system with colchicine with concentration of 0.2 to 0.4% for 36 hours, and perform chromosome doubling.
Further, in the rapid pepper cultivation method using molecular markers, in the step S6, the ploidy identification adopts a flow cytometer, and haploid and polyploid plants are removed by taking normal diploid peppers as a control.
Further, in the method for rapidly culturing capsicum using molecular markers according to the present invention, in the step S7, when a PCR amplification reaction is performed using a forward primer and a reverse primer using molecular markers, the nucleotide sequence of the forward primer is: 5'-ctgtgatatggaaggggaacc-3', and the nucleotide sequence of the reverse primer is 5'-aactcgctgggaaaagaatg-3'.
Further, according to the rapid cultivation method of capsicum by using molecular markers, the induction medium, the proliferation medium and the rooting medium are sterilized at 121 ℃ for 30 minutes, and the pH value of the capsicum is adjusted to 5.6-5.8 before sterilization.
The MS culture medium in the culture medium is a basic culture medium formula commonly used in plant tissue culture technology, has higher inorganic salt concentration, can ensure mineral nutrition required by tissue growth, can accelerate the growth of callus, is a stable ion balance solution, has high nitrate content, and has proper nutrient quantity and proportion, can meet the nutrition and physiological needs of plant cells, thus the application range is wider, and most plant tissue culture rapid propagation uses the MS culture medium as the basic culture medium of the culture medium.
Triacontanol is a natural biological product that enhances amylase, polyoxidase, peroxidase activity; and the plant callus can be greened by adding triacontanol, and the induction rate of the plant callus can be improved.
The Chinese name of KT is kinetin/6-furfuryl amino purine, one of plant cytokinins, is a non-natural cytokinin, and has a chemical name of 6-glycosylamino purine (or N6-furfuryl methyl adenine) and a molecular formula of C10H9N5O. Is insoluble in water and soluble in strong acid, alkali and glacial acetic acid; besides the function of promoting cell division, the plant growth regulator also has the functions of delaying the aging of in vitro leaves and cut flowers, inducing bud differentiation and development and increasing the opening degree of stomata.
NAA is named as naphthalene acetic acid and one of auxins, and can promote cell division and expansion, induce adventitious root formation to increase fruit setting, prevent fruit drop, change female and male flower ratio, etc. and may be introduced into plant via tender skin and seed of leaf and branch for transmission along with nutrient flow.
The Chinese name indolebutyric acid of IBA is one of auxins, is mainly used for rooting cuttings, can induce the formation of root protoplasm, promotes cell differentiation and division, is favorable for the generation of new roots and the differentiation of vascular bundle systems, promotes the formation of adventitious roots of cuttings, and is widely applied to the cuttage rooting of trees and flowers.
The Chinese name indoleacetic acid of IAA is one of auxins, is used as a hormone reagent for stimulating plant growth, and is widely applied to agricultural production.
The Chinese name benzyl purine/6-benzyl amino purine of 6-BA is one kind of plant cytokinin, and has the functions of inhibiting the decomposition of chlorophyll, nucleic acid and protein in plant leaf, protecting green and resisting aging, and regulating amino acid, auxin, inorganic salt, etc. to the treated part.
2, 4-D Chinese name 2, 4-dichlorophenoxyacetic acid is a plant growth regulator, and can promote rooting of cuttings and premature fruit, prevent flower and fruit from falling, etc.
Compared with the prior art, the quick pepper cultivation method using the molecular marker has the beneficial effects that: the method can be used for rapidly cultivating a large number of pepper aseptic seedlings without being influenced by seasons, temperatures and regions, can effectively promote the growth and development speed of the peppers, promotes the yield of the peppers, further improves the planting and cultivating benefits, has market competitiveness, is suitable for large-scale cultivation, simultaneously, can provide data support for the creation of Guizhou pepper germplasm resources by breeding a large number of seedlings in a short period and utilizing molecular markers to screen homozygous plants meeting target requirements, and has important significance for the subsequent research of analysis and identification of main flavor substances of the Guizhou peppers, genetic analysis of the Guizhou peppers and molecular breeding of pepper flavor improvement.
Description of the embodiments
In order to more fully explain the practice of the invention, the invention will be further illustrated by the following examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.
The reagents used in the examples of the present invention, if not specifically described, may be commercially available to achieve the present invention, and the technical schemes involved, if not specifically described, may be conventional techniques in the art.
Examples
The quick pepper cultivation method by using molecular markers comprises the steps of seed collection and treatment, induction cultivation, proliferation cultivation, rooting cultivation, seedling hardening, chromosome doubling and transplanting, ploidy identification and virus detoxification rate detection by using molecular markers, and specifically comprises the following steps:
s1, collecting and processing seeds: taking out the seeds after the pepper fruits are collected, soaking the seeds in clear water at 40 ℃ for 2 days, then sterilizing with 0.1% potassium permanganate solution for 5min, soaking in 75% ethanol for 8s, and finally flushing with sterile water for 3 times for later use;
s2, induction culture: the seeds obtained in the step S1 are dried by using sterile filter paper, inoculated into a sterile induction culture medium, and cultivated in an illumination incubator under the conditions that the temperature is 25+/-1 ℃ and the illumination time is 8h/d and the illumination intensity is 2800-3000 lx, so as to induce adventitious buds; the induction culture medium is an MS culture medium added with 1mg/L triacontanol, 0.05mg/L kinetin and 0.05mg/L NAA (naphthylacetic acid), wherein the MS culture medium comprises sucrose with the mass concentration of 3 percent and agar with the mass concentration of 6g/L, and triacontanol, KT (kinetin) and NAA (naphthylacetic acid) are added into the MS culture medium, and the triacontanol, the KT (kinetin) and the NAA (naphthylacetic acid) can effectively promote the growth and differentiation of capsicum cells through the combination of the triacontanol, so that the capsicum cells have high growth speed, vigorous tissue metabolism and strong stress resistance;
s3, proliferation cultivation: cutting the adventitious bud obtained in the step S2 into single plants by using a sterile scalpel, and reserving 2-3 leaves for each plant; inoculating the seedlings to a proliferation culture medium in a sterile environment, culturing in an illumination incubator under the conditions that the temperature is 25+/-1 ℃ and the illumination time is 8h/d and the illumination intensity is 2800-3000 lx, then cutting out the grown buds, transferring the seedlings to the proliferation culture medium for secondary proliferation culture, and culturing the seedlings in the proliferation culture medium for 2-4 times in the illumination incubator under the conditions that the temperature is 25+/-1 ℃ and the illumination time is 8h/d and the illumination intensity is 2800-3000 lx, wherein the time of each secondary culture is 7-10 days; the proliferation culture medium is selected to be added with 2mg/L of 2, 4-D (2, 4-dichlorophenoxyacetic acid), 0.5mg/L of 6-BA (6-benzylaminopurine) and 0.05mg/L of NAA (naphthylacetic acid), wherein the MS culture medium comprises sucrose with the mass concentration of 3 percent and agar with the mass concentration of 6 g/L;
s4, rooting cultivation: cutting tender buds grown by proliferation and cultivation by using a sterile scalpel, soaking in IBA solution, reserving 3-4 leaves for each plant, selecting single plant seedlings with better growth vigor, inoculating the single plant seedlings into a rooting culture medium to induce rooting in a sterile environment, culturing the single plant seedlings in an illumination incubator at the temperature of 25+/-1 ℃ for 8-12 h/d under the illumination intensity of 2800-3500 lx, and obtaining robust rooting seedlings after culturing for one week; the rooting culture medium is 1/2MS culture medium added with 0.5mg/L IBA (indolebutyric acid) and 1.0mg/LIAA (indoleacetic acid), wherein the 1/2MS culture medium comprises sucrose with the mass concentration of 1.5% and agar with the mass concentration of 6 g/L;
s5, hardening seedlings, doubling chromosomes and transplanting: removing the rooting seedling obtained in the step (4) together with a culture container into an illumination incubator, placing the rooted chilli tissue culture seedling in a normal room temperature, standing and hardening the seedling for 5 days at room temperature, taking out the plant from the culture medium, removing the culture medium at the root of the seedling, cleaning the culture medium at the root, soaking the root system for 36 hours by using colchicine with the concentration of 0.2%, doubling the chromosome, and finally transplanting the seedling in the cultivation incubator with soil;
s6, ploidy identification: culturing the cultivated seedlings obtained in the step S5 for 20 days, and taking new leaves for ploidy identification to obtain homozygous diploid; the ploidy identification adopts a flow cytometer, and a haploid plant and a polyploid plant are removed by taking normal diploid capsicum as a control;
s7, detecting virus detoxification rate by using molecular markers: extracting genome DNA of tissue cultured pepper seedlings, taking the genome DNA as a template, carrying out PCR amplification reaction by using forward and reverse primers of molecular markers to obtain PCR amplification products, and screening out homozygous plants meeting target requirements through PCR amplification; wherein, when the forward primer and the reverse primer of the molecular marker are used for PCR amplification reaction, the forward primer and the reverse primer are both existing primers, and the nucleotide sequence of the forward primer is as follows: 5'-ctgtgatatggaaggggaacc-3', and the nucleotide sequence of the reverse primer is 5'-aactcgctgggaaaagaatg-3'.
Examples
The quick pepper cultivation method by using molecular markers comprises the steps of seed collection and treatment, induction cultivation, proliferation cultivation, rooting cultivation, seedling hardening, chromosome doubling and transplanting, ploidy identification and virus detoxification rate detection by using molecular markers, and specifically comprises the following steps:
s1, collecting and processing seeds: taking out the seeds after the pepper fruits are collected, soaking the seeds in clear water at 42 ℃ for 3 days, then sterilizing the seeds in 0.15% potassium permanganate solution for 6min, soaking the seeds in 78% ethanol for 10s, and finally flushing the seeds with sterile water for 4 times for later use;
s2, induction culture: the seeds obtained in the step S1 are dried by using sterile filter paper, inoculated into a sterile induction culture medium, and cultivated in an illumination incubator under the conditions that the temperature is 25+/-1 ℃ and the illumination time is 12h/d and the illumination intensity is 3000-3300 lx, so as to induce adventitious buds; the induction culture medium is an MS culture medium added with 2mg/L triacontanol, 0.08mg/L kinetin and 0.08mg/L NAA (naphthylacetic acid), wherein the MS culture medium comprises sucrose with the mass concentration of 3 percent and agar with the mass concentration of 6g/L, and triacontanol, KT (kinetin) and NAA (naphthylacetic acid) are added into the MS culture medium, and the triacontanol, the KT (kinetin) and the NAA (naphthylacetic acid) can effectively promote the growth and differentiation of capsicum cells through the combination of the triacontanol, so that the capsicum cells have high growth speed, vigorous tissue metabolism and strong stress resistance;
s3, proliferation cultivation: cutting the adventitious bud obtained in the step S2 into single plants by using a sterile scalpel, and reserving 2-3 leaves for each plant; inoculating the culture medium into a proliferation culture medium in a sterile environment, culturing in an illumination incubator under the conditions that the temperature is 25+/-1 ℃ and the illumination time is 12h/d and the illumination intensity is 3000-3300 lx, then cutting out the grown tender buds, transferring the tender buds into the proliferation culture medium for secondary proliferation culture, and performing secondary culture for 2-4 times in the illumination incubator under the conditions that the temperature is 25+/-1 ℃ and the illumination time is 12h/d and the illumination intensity is 3000-3300 lx, wherein the time of each secondary culture is 7-10 days; the proliferation culture medium is selected to be added with 3mg/L of 2, 4-D (2, 4-dichlorophenoxyacetic acid), 0.8mg/L of 6-BA (6-benzylaminopurine) and 0.08mg/L of NAA (naphthylacetic acid), wherein the MS culture medium comprises 3% of sucrose and 6g/L of agar;
s4, rooting cultivation: cutting tender buds grown by proliferation culture by using a sterile scalpel, soaking in IBA solution, reserving 3-4 leaves for each plant, selecting single plant seedlings with better growth vigor, inoculating the single plant seedlings into a rooting culture medium to induce rooting in a sterile environment, culturing in an illumination incubator at the temperature of 25+/-1 ℃ for 12h/d under the illumination time of 3000-3300 lx, and culturing for one week to obtain robust rooting seedlings; the rooting culture medium is 1/2MS culture medium added with 0.8mg/L IBA (indolebutyric acid) and 1.2mg/LIAA (indoleacetic acid), wherein the 1/2MS culture medium comprises sucrose with the mass concentration of 1.5% and agar with the mass concentration of 6 g/L;
s5, hardening seedlings, doubling chromosomes and transplanting: removing the rooting seedling obtained in the step (4) together with a culture container into an illumination incubator, placing the rooted pepper tissue culture seedling in a normal room at room temperature, standing and hardening the seedling for 7 days at room temperature, taking out the plant from the culture medium, removing the culture medium at the root of the seedling, cleaning the culture medium at the root, soaking the root system for 36 hours by using colchicine with the concentration of 0.3%, doubling the chromosome, and finally transplanting the seedling into the cultivation incubator with soil;
s6, ploidy identification: culturing the cultivated seedlings obtained in the step S5 for 20 days, and taking new leaves for ploidy identification to obtain homozygous diploid; the ploidy identification adopts a flow cytometer, and a haploid plant and a polyploid plant are removed by taking normal diploid capsicum as a control;
s7, detecting virus detoxification rate by using molecular markers: extracting genome DNA of tissue cultured pepper seedlings, taking the genome DNA as a template, carrying out PCR amplification reaction by using forward and reverse primers of molecular markers to obtain PCR amplification products, and screening out homozygous plants meeting target requirements through PCR amplification; wherein, when the forward primer and the reverse primer of the molecular marker are used for PCR amplification reaction, the forward primer and the reverse primer are both existing primers, and the nucleotide sequence of the forward primer is as follows: 5'-ctgtgatatggaaggggaacc-3', and the nucleotide sequence of the reverse primer is 5'-aactcgctgggaaaagaatg-3'.
Examples
The quick pepper cultivation method by using molecular markers comprises the steps of seed collection and treatment, induction cultivation, proliferation cultivation, rooting cultivation, seedling hardening, chromosome doubling and transplanting, ploidy identification and virus detoxification rate detection by using molecular markers, and specifically comprises the following steps:
s1, collecting and processing seeds: taking out the seeds after the capsicum fruits are collected, soaking the seeds in clear water at 42 ℃ for 2 days, then sterilizing with 0.2% potassium permanganate solution for 8min, soaking in 80% ethanol for 12s, and finally washing with sterile water for 5 times for later use;
s2, induction culture: the seeds obtained in the step S1 are dried by using sterile filter paper, inoculated into a sterile induction culture medium, and cultivated in an illumination incubator under the conditions that the temperature is 25+/-1 ℃ and the illumination time is 10h/d and the illumination intensity is 3200-3500 lx, so as to induce adventitious buds; the induction culture medium is an MS culture medium added with 3mg/L triacontanol, 0.1mg/L LKT (kinetin) and 0.1mg/L NAA (naphthylacetic acid), wherein the MS culture medium comprises sucrose with the mass concentration of 3 percent and agar with the mass concentration of 6g/L, and triacontanol, KT (kinetin) and NAA (naphthylacetic acid) are added into the MS culture medium, and the triacontanol, the KT (kinetin) and the NAA (naphthylacetic acid) can effectively promote the growth and differentiation of capsicum cells through the combination of the triacontanol, so that the capsicum cells have high growth speed, vigorous tissue metabolism and strong stress resistance;
s3, proliferation cultivation: cutting the adventitious bud obtained in the step S2 into single plants by using a sterile scalpel, and reserving 2-3 leaves for each plant; inoculating the culture medium into a proliferation culture medium in a sterile environment, culturing in an illumination incubator under the conditions that the temperature is 25+/-1 ℃ and the illumination time is 10h/d and the illumination intensity is 3200-3500 lx, then cutting out the grown tender buds, transferring the tender buds into the proliferation culture medium for secondary proliferation culture, cutting out the grown tender buds, transferring the tender buds into the proliferation culture medium, and performing secondary culture for 2-4 times in the illumination incubator under the conditions that the temperature is 25+/-1 ℃ and the illumination time is 10h/d and the illumination intensity is 3200-3500 lx until a secondary culture tissue is obtained, wherein the time of each secondary culture is 7-10 days; the proliferation culture medium is selected from MS culture medium added with 4mg/L2, 4-D (2, 4-dichlorophenoxyacetic acid), 1.0 mg/L6-BA (6-benzylaminopurine) and 0.1mg/L NAA (naphthylacetic acid), wherein the MS culture medium comprises sucrose with the mass concentration of 3 percent and agar with the mass concentration of 6 g/L;
s4, rooting cultivation: cutting tender buds grown by proliferation culture by using a sterile scalpel, soaking in IBA solution, reserving 3-4 leaves for each plant, selecting single plant seedlings with better growth vigor, inoculating the single plant seedlings into a rooting culture medium to induce rooting in a sterile environment, culturing in an illumination incubator at the temperature of 25+/-1 ℃ for 10h/d under the illumination time length of 3200-3500 lx, and culturing for one week to obtain robust rooting seedlings; the rooting culture medium is 1/2MS culture medium added with 1.0mg/L IBA (indolebutyric acid) and 1.5mg/LIAA (indoleacetic acid), wherein the 1/2MS culture medium comprises sucrose with the mass concentration of 1.5% and agar with the mass concentration of 6 g/L;
s5, hardening seedlings, doubling chromosomes and transplanting: removing the rooting seedling obtained in the step (4) together with a culture container into an illumination incubator, placing the rooted pepper tissue culture seedling in a normal room at room temperature, standing and hardening the seedling for 7 days at room temperature, taking out the plant from the culture medium, removing the culture medium at the root of the seedling, cleaning the culture medium at the root, soaking the root system for 36 hours by using colchicine with the concentration of 0.4%, doubling the chromosome, and finally transplanting the seedling into the cultivation incubator with soil;
s6, ploidy identification: culturing the cultivated seedlings obtained in the step S5 for 20 days, and taking new leaves for ploidy identification to obtain homozygous diploid; the ploidy identification adopts a flow cytometer, and a haploid plant and a polyploid plant are removed by taking normal diploid capsicum as a control;
s7, detecting virus detoxification rate by using molecular markers: extracting genome DNA of tissue cultured pepper seedlings, taking the genome DNA as a template, carrying out PCR amplification reaction by using forward and reverse primers of molecular markers to obtain PCR amplification products, and screening out homozygous plants meeting target requirements through PCR amplification; wherein, when the forward primer and the reverse primer of the molecular marker are used for PCR amplification reaction, the forward primer and the reverse primer are both existing primers, and the nucleotide sequence of the forward primer is as follows: 5'-ctgtgatatggaaggggaacc-3', and the nucleotide sequence of the reverse primer is 5'-aactcgctgggaaaagaatg-3'.
The cultivation method of the present invention was adopted, wherein the induction medium, proliferation medium and rooting medium in examples 1 to 3 were sterilized at 121℃for 25 minutes and the pH was adjusted to 5.6 to 5.8 before sterilization.
In order to explain the cultivation condition of the cultivation method, the Zunyi pod pepper is adopted as an explant, homozygous plants meeting the target requirements are screened out through a tissue culture technology under the aseptic condition, and the final transplanting survival rate reaches more than 98% through outdoor cultivation.
The above description is not intended to limit the invention to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. A method for rapidly cultivating capsicum by using molecular markers is characterized in that: the cultivation method comprises the steps of seed collection and treatment, induction culture, proliferation culture, rooting culture, seedling hardening, chromosome doubling and transplanting, ploidy identification and virus detoxification rate detection by using a molecular marker, and specifically comprises the following steps:
s1, collecting and processing seeds: taking out the seeds after the pepper fruits are collected, soaking the seeds in clear water, sterilizing, soaking in ethanol, and then washing with sterile water for later use;
s2, induction culture: the seeds obtained in the step S1 are dried by using sterile filter paper, inoculated into a sterile induction culture medium, and cultivated in an illumination incubator under the conditions that the temperature is 25+/-1 ℃ and the illumination time is 8-12 h/d and the illumination intensity is 2800-3500 lx, so as to induce adventitious buds;
s3, proliferation cultivation: cutting the adventitious bud obtained in the step S2 into single plants by using a sterile scalpel, and reserving 2-3 leaves for each plant; inoculating the young buds into a proliferation culture medium in a sterile environment, culturing the young buds in an illumination incubator under the conditions that the temperature is 25+/-1 ℃, the illumination time is 8-12 h/d and the illumination intensity is 2800-3500 lx, and then cutting off the young buds and transferring the young buds into the proliferation culture medium for secondary proliferation culture;
s4, rooting cultivation: cutting tender buds grown by proliferation and cultivation by using a sterile scalpel, soaking in IBA solution, reserving 3-4 leaves for each plant, selecting single plant seedlings with better growth vigor, inoculating the single plant seedlings into a rooting culture medium to induce rooting in a sterile environment, culturing the single plant seedlings in an illumination incubator at the temperature of 25+/-1 ℃ for 8-12 h/d under the illumination intensity of 2800-3500 lx, and obtaining robust rooting seedlings after culturing for one week;
s5, hardening seedlings, doubling chromosomes and transplanting: removing the rooting seedling obtained in the step (4) together with a culture container from an illumination incubator, placing the rooted pepper tissue culture seedling in a normal room temperature, standing at room temperature for hardening off for 5-10 days, taking out plants from the culture medium, removing the culture medium at the root of the seedling, soaking the root system with colchicine solution for chromosome doubling, and finally transplanting the seedling in the incubator with soil;
s6, ploidy identification: culturing the cultivated seedlings obtained in the step S5 for 20 days, and taking new leaves for ploidy identification to obtain homozygous diploid;
s7, detecting virus detoxification rate by using molecular markers: extracting genome DNA of tissue cultured pepper seedlings, taking the genome DNA as a template, carrying out PCR amplification reaction by using forward and reverse primers of molecular markers to obtain PCR amplification products, and screening out homozygous plants meeting target requirements through PCR amplification.
2. The method for rapid cultivation of capsicum using molecular markers according to claim 1, wherein: in the step S1, after the capsicum fruits are collected, the seeds are taken out, the seeds are soaked in clear water at the temperature of 40-45 ℃ for 2-4 days, then sterilized with 0.1-0.2% potassium permanganate solution for 5-8 min, soaked in 75-80% ethanol for 8-12S, and finally washed with sterile water for 3-5 times.
3. The method for rapid cultivation of capsicum using molecular markers according to claim 1, wherein: in the step S2, the induction culture medium is selected from an MS culture medium added with 1-3 mg/L triacontanol, 0.05-0.1 mg/L LKT (kinetin) and 0.05-0.1 mg/L NAA (naphthalene acetic acid), wherein the MS culture medium comprises sucrose with the mass concentration of 3% and agar with the mass concentration of 6 g/L.
4. The method for rapid cultivation of capsicum using molecular markers according to claim 1, wherein: in the step S3, the proliferation culture medium is selected from MS culture medium added with 2-4 mg/L2, 4-D (2, 4-dichlorophenoxyacetic acid), 0.5-1 mg/L6-BA (6-benzylaminopurine) and 0.05-0.1 mg/L NAA (naphthalene acetic acid), wherein the MS culture medium comprises sucrose with the mass concentration of 3 percent and agar with the mass concentration of 6 g/L.
5. The method for rapid cultivation of capsicum using molecular markers according to claim 1, wherein: in the step S3, the method for subculture is to cut out the grown buds and transfer the cut buds into a proliferation culture medium, and the subculture is carried out for 2 to 4 times in an illumination incubator under the conditions that the temperature is 25+/-1 ℃, the illumination time is 8 to 12 hours/d and the illumination intensity is 2800 to 3500lx until a subculture tissue is obtained, wherein the time of each subculture is 7 to 10 days.
6. The method for rapid cultivation of capsicum using molecular markers according to claim 1, wherein: in the step S4, the rooting culture medium is 1/2MS culture medium added with 0.5-1 mg/LIBA (indolebutyric acid) and 1.0-1.5 mg/LIAA (indoleacetic acid), wherein the 1/2MS culture medium contains sucrose with the mass concentration of 1.5% and agar with the mass concentration of 6 g/L.
7. The method for rapid cultivation of capsicum using molecular markers according to claim 1, wherein: in the step S5, the chromosome doubling treatment is to take out the seedlings after hardening seedlings for 5-7 days, wash the culture medium of the roots, soak the root system for 36 hours with colchicine with the concentration of 0.2-0.4%, and carry out chromosome doubling.
8. The method for rapid cultivation of capsicum using molecular markers according to claim 1, wherein: in the step S6, the ploidy identification adopts a flow cytometer, and haploid and polyploid plants are removed by taking normal diploid peppers as a control.
9. The method for rapid cultivation of capsicum using molecular markers according to claim 1, wherein: in the step S7, when a PCR amplification reaction is performed using a forward primer and a reverse primer, which are molecular markers, the nucleotide sequence of the forward primer is: 5'-ctgtgatatggaaggggaacc-3', and the nucleotide sequence of the reverse primer is 5'-aactcgctgggaaaagaatg-3'.
10. The method for rapid cultivation of capsicum using molecular markers according to claim 1, wherein: wherein the induction culture medium, the proliferation culture medium and the rooting culture medium are sterilized at the temperature of 121 ℃ for 30 minutes, and the pH value of the induction culture medium, the proliferation culture medium and the rooting culture medium is adjusted to be 5.6-5.8 before sterilization.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19990031523A (en) * | 1997-10-13 | 1999-05-06 | 임용표 | Mass production method of redied plants of red pepper (Caｐｓｉｃｕｍ ｎｎｕｕｍ L．) using tissue culture technology |
| CN1970790A (en) * | 2006-11-29 | 2007-05-30 | 广东省农业科学院蔬菜研究所 | Molecular marker-assisted selection method for capsicum blight-resistant breeding |
| KR20090042100A (en) * | 2007-10-25 | 2009-04-29 | 경상대학교산학협력단 | Micropropagation method of paprika |
| CN108077072A (en) * | 2016-11-21 | 2018-05-29 | 巴中精致现代农业开发有限公司 | Windbell green pepper tissue cultures culture medium and rapid propagation method |
| CN115486372A (en) * | 2022-11-08 | 2022-12-20 | 海南大学三亚南繁研究院 | Method for constructing in-vitro regeneration system of drooping hot pepper |
-
2023
- 2023-05-09 CN CN202310511998.0A patent/CN116439135A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19990031523A (en) * | 1997-10-13 | 1999-05-06 | 임용표 | Mass production method of redied plants of red pepper (Caｐｓｉｃｕｍ ｎｎｕｕｍ L．) using tissue culture technology |
| CN1970790A (en) * | 2006-11-29 | 2007-05-30 | 广东省农业科学院蔬菜研究所 | Molecular marker-assisted selection method for capsicum blight-resistant breeding |
| KR20090042100A (en) * | 2007-10-25 | 2009-04-29 | 경상대학교산학협력단 | Micropropagation method of paprika |
| CN108077072A (en) * | 2016-11-21 | 2018-05-29 | 巴中精致现代农业开发有限公司 | Windbell green pepper tissue cultures culture medium and rapid propagation method |
| CN115486372A (en) * | 2022-11-08 | 2022-12-20 | 海南大学三亚南繁研究院 | Method for constructing in-vitro regeneration system of drooping hot pepper |
Non-Patent Citations (4)
| Title |
|---|
| 宋志荣: "几种药剂对辣椒种子发芽的影响研究", 中国种业, no. 06, pages 26 - 27 * |
| 张爱民等: "贵州地方辣椒材料疫病抗性鉴定的分子标记引物筛选", 贵州农业科学, vol. 45, no. 11, pages 129 - 14 * |
| 苏荣存等: "辣椒砧木"骄珍108"的组培快繁技术研究", 德州学院学报, vol. 29, no. 06, pages 83 - 85 * |
| 蒋向辉等: "观赏辣椒一步成苗诱导条件的筛选", 植物生理学通讯, vol. 43, no. 04, pages 705 - 717 * |
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