A method for cultivating aseptic seedling by using dried pepper fruits
By using pepper stored seeds as explants, combined with alcohol and hypochlorite sterilization and water agar medium, the time and cost issues of pepper tissue culture were solved, achieving low-cost and high-efficiency sterile seedling cultivation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SPICE & BEVERAGE RES INST CHINESE ACAD OF TROPICAL AGRI SCI
- Filing Date
- 2024-02-07
- Publication Date
- 2026-05-29
AI Technical Summary
In existing technologies, pepper tissue culture is difficult, the selection and sterilization methods for explants are limited, and the harvesting of fresh pepper fruits is highly seasonal, making it difficult to obtain raw materials for explants, resulting in high culture costs and strong time constraints.
Pepper seeds were used as explants and sterilized with 75% alcohol and 10% H2O2 or 20% NaClO. The culture medium was water agar, and the plants were cultured under light conditions with a photoperiod of L:D = 10h:14h, a temperature of 26–30℃, and a culture time of 40–50 days.
It achieves low-cost, environmentally friendly, and efficient cultivation of sterile seedlings, reduces the pollution rate, increases the germination rate, and solves the time-limited problem, making it suitable for pepper breeding and biotechnology research.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of tissue culture technology and relates to a method for cultivating sterile seedlings using dried pepper berries. Background Technology
[0002] Pepper (Piper nigrum L.) is a perennial woody vine belonging to the Piperaceae family and the Piper genus. Known as the "King of Spices," it is an important economic crop in tropical regions worldwide, playing a significant role in both medicinal and industrial applications. Pepper contains volatile oils and piperine, making it widely popular. As early as the Compendium of Materia Medica, it was recorded that pepper has pain-relieving, stomach-warming, and dampness-removing effects. In the medical and industrial fields, pepper is used as a stomachic, antipyretic, and diuretic, and can treat indigestion, colds, and rheumatism. Furthermore, it is used as a preservative in the food industry.
[0003] Plant tissue culture is a routine method for crop genetic breeding and seed propagation, but pepper tissue culture is particularly challenging due to limitations in explant selection, sterilization methods, and germination media. Current research primarily uses fresh pepper fruits as explants to cultivate sterile seedlings. However, the harvest season for fresh pepper fruits is from May to July each year. After the season, seeds become inactive after one month at room temperature, making it difficult to obtain new explant materials, resulting in significant time constraints. Therefore, providing a method for cultivating sterile pepper seedlings that is not limited by time, has low culture costs, and a high germination rate is a primary problem that urgently needs to be solved for pepper tissue culture and genetic transformation research. Summary of the Invention
[0004] In view of this, the present invention provides a simple, environmentally friendly, low-cost method for cultivating sterile pepper seedlings with a high germination rate, using pepper stored seeds as explants.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0006] The method for cultivating aseptic pepper seedlings involves sterilizing pepper seeds and then inoculating them into a culture medium for cultivation.
[0007] The sterilization process involves sterilizing with 75% alcohol, followed by sterilization with 10% H2O2 or 20% NaClO.
[0008] The culture medium consists of water and agar.
[0009] In this invention, the pepper seeds are seeds that have been stored at -20°C for 1-2 years.
[0010] Furthermore, the method for obtaining the pepper seeds is as follows: remove the pericarp from fresh pepper fruits, remove surface moisture from the seeds, and store them at -20℃ for 1-2 years. The method for removing surface moisture from the seeds is to air dry or blot them dry with sterile filter paper.
[0011] In this invention, the sterilization includes: sterilizing with 75% alcohol for 30 seconds, rinsing once with sterile water, sterilizing with 10% H2O2 for 30 minutes or 20% NaClO for 10 minutes, rinsing with sterile water 4 to 5 times to remove surface moisture.
[0012] In this invention, the concentration of agar in the culture medium is 3 to 7 g / L, specifically 3 g / L, 4 g / L, 5 g / L, 6 g / L or 7 g / L.
[0013] In this invention, the cultivation is carried out under light at 26-30℃ for 40-50 days, with a photoperiod L:D = 10h:14h. The cultivation temperature can be 26℃, 27℃, 28℃, 29℃ or 30℃, and the cultivation days can be 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 days.
[0014] In some specific embodiments, the cultivation method provided by the present invention includes the following steps:
[0015] (1) Seed acquisition: After rinsing the mature fresh pepper fruits under running water containing detergent for 24 hours, remove the pericarp, dry the surface moisture of the pepper seeds, and store the seeds in a refrigerator at -20℃ for 1-2 years.
[0016] (2) Culture medium preparation: water agar (4g / L agar powder + 1L distilled water) was sterilized in an autoclave at 121℃ for 20min;
[0017] (3) Seed sterilization: The seeds obtained in step (1) are sterilized in a clean bench with 75% alcohol for 30 seconds, rinsed with sterile water once, and then sterilized with 10% H2O2 for 30 minutes or 20% NaClO for 10 minutes respectively. They are rinsed with sterile water 4 to 5 times and the surface moisture is absorbed with sterile filter paper for later use.
[0018] (4) Inoculation: Under aseptic conditions, inoculate the sterilized seeds from step (3) into the culture medium from step (2);
[0019] (5) Cultivation: Place the Erlenmeyer flask from step (4) under light at 28°C for 45 days, with a photoperiod L:D = 10h:14h.
[0020] In step (1) of this invention, mature fresh pepper fruits are rinsed with running water containing detergent to sterilize the fresh pepper fruits obtained from the field instead of directly removing the peel, thereby reducing the impact of pollutants on the peel surface on the seeds. In addition, rinsing with running water for 24 hours is beneficial for pepper seeds to absorb water and promotes seed germination. After peeling, the seeds are dried and stored in a -20℃ refrigerator to prevent seed inactivation for 1-2 years, providing sufficient raw materials for tissue culture.
[0021] In step (2) of this invention, water agar is selected as the culture medium to replace MS culture medium, which significantly reduces the price and can save a lot of costs for factory seedling production.
[0022] The present invention has found that in step (3), 10% H2O2 and 20% NaClO not only have a sterilizing effect on seeds, but also promote seed germination, and are more environmentally friendly than mercuric chloride.
[0023] This invention uses pepper stored seeds as explants for the cultivation of sterile seedlings, which is not limited by time, economical and environmentally friendly, and of great significance for research on pepper breeding and biotechnology, as well as subsequent efficient industrialized seedling production. Attached Figure Description
[0024] Figure 1 These are the mature pepper fruits and stored seeds harvested in step (1) of this invention;
[0025] Figure 2 This invention utilizes 20% NaClO to sterilize dried pepper seeds to obtain sterile seedlings. Detailed Implementation
[0026] This invention provides a method for cultivating sterile seedlings using dried pepper berries. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method and application of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.
[0027] The test materials used in this invention are all common commercial products and can be purchased on the market.
[0028] In this invention, "stored seeds" and "dried fruit" refer to dried pepper seeds stored in a -20℃ refrigerator for one year. The specific method of obtaining them is as follows: after rinsing mature fresh pepper fruits under running water containing detergent for 24 hours, remove the peel, absorb the surface moisture of the pepper seeds, and store the dried pepper seeds in a -20℃ refrigerator for one year.
[0029] The present invention will be further illustrated below with reference to the embodiments:
[0030] Example 1
[0031] (1) Seed acquisition: Dried pepper seeds stored at -20℃ for one year;
[0032] (2) Culture medium preparation: The culture medium formula is 4 g / L agar powder + 1 L distilled water, and sterilized in an autoclave at 121℃ for 20 min;
[0033] (3) Seed sterilization: The seeds obtained in step (1) are sterilized in a clean bench with 75% alcohol for 30 seconds, rinsed with sterile water once, sterilized with 10% H2O2 for 30 minutes, rinsed with sterile water 4 to 5 times, and the surface moisture is absorbed by sterile filter paper for later use.
[0034] (4) Inoculation: Under aseptic conditions, inoculate the sterilized seeds from step (3) into the culture medium from step (2);
[0035] (5) Cultivation: Place the Erlenmeyer flask from step (4) under light at 28°C for 45 days, with a photoperiod L:D = 10h:14h.
[0036] The seed contamination rate and germination rate were calculated as follows: contamination rate = number of contaminated seeds / total number of inoculated seeds × 100%; germination rate = number of uncontaminated seeds whose radicles broke through the seed coat / total number of inoculated seeds × 100%.
[0037] Example 2
[0038] (1) Seed acquisition: Dried pepper seeds stored at -20℃ for one year;
[0039] (2) Culture medium preparation: The culture medium formula is 4 g / L agar powder + 1 L distilled water, and sterilized in an autoclave at 121℃ for 20 min;
[0040] (3) Seed sterilization: The seeds obtained in step (1) are sterilized in a clean bench with 75% alcohol for 30 seconds, rinsed with sterile water once, sterilized with 20% NaClO for 10 minutes, rinsed with sterile water 4 to 5 times, and the surface moisture is absorbed by sterile filter paper for later use.
[0041] (4) Inoculation: Under aseptic conditions, inoculate the sterilized seeds from step (3) into the culture medium from step (2);
[0042] (5) Cultivation: Place the Erlenmeyer flask from step (4) under light at 28°C for 45 days, with a photoperiod L:D = 10h:14h.
[0043] The seed contamination rate and germination rate were calculated as follows: contamination rate = number of contaminated seeds / total number of inoculated seeds × 100%; germination rate = number of uncontaminated seeds whose radicles broke through the seed coat / total number of inoculated seeds × 100%.
[0044] Comparative Example 1
[0045] (1) Seed acquisition: After rinsing the mature pepper fruits under running water containing detergent for 24 hours, remove the peel and dry the surface moisture of the pepper seeds.
[0046] (2) Culture medium preparation: The culture medium formula is 4.44 g / L MS + 30 g / L sucrose + 2.5 g / L plant gel, pH 5.8~6.0, and sterilized in an autoclave at 121℃ for 20 min;
[0047] (3) Seed sterilization: The seeds obtained in step (1) are sterilized in a clean bench with 75% alcohol for 30 seconds, rinsed with sterile water once, sterilized with 0.1% mercuric chloride for 10 minutes, rinsed with sterile water 4 to 5 times, and the surface moisture is absorbed by sterile filter paper for later use.
[0048] (4) Inoculation: Under aseptic conditions, inoculate the sterilized seeds from step (3) into the culture medium from step (2);
[0049] (5) Cultivation: Place the Erlenmeyer flask from step (4) under light at 28°C for 45 days, with a photoperiod L:D = 10h:14h.
[0050] The seed contamination rate and germination rate were calculated as follows: contamination rate = number of contaminated seeds / total number of inoculated seeds × 100%; germination rate = number of uncontaminated seeds whose radicles broke through the seed coat / total number of inoculated seeds × 100%.
[0051] Comparative Example 2
[0052] (1) Seed acquisition: Dried pepper seeds stored at -20℃ for one year;
[0053] (2) Culture medium preparation: The culture medium formula is 4.44 g / L MS + 30 g / L sucrose + 2.5 g / L plant gel, pH 5.8~6.0, and sterilized in an autoclave at 121℃ for 20 min;
[0054] (3) Seed sterilization: The seeds obtained in step (1) are sterilized in a clean bench with 75% alcohol for 30 seconds, rinsed with sterile water once, sterilized with 0.1% mercuric chloride for 10 minutes, rinsed with sterile water 4 to 5 times, and the surface moisture is absorbed by sterile filter paper for later use.
[0055] (4) Inoculation: Under aseptic conditions, inoculate the sterilized seeds from step (3) into the culture medium from step (2);
[0056] (5) Cultivation: Place the Erlenmeyer flask from step (4) under light at 28°C for 45 days, with a photoperiod L:D = 10h:14h.
[0057] The seed contamination rate and germination rate were calculated as follows: contamination rate = number of contaminated seeds / total number of inoculated seeds × 100%; germination rate = number of uncontaminated seeds whose radicles broke through the seed coat / total number of inoculated seeds × 100%.
[0058] Comparative Example 3
[0059] (1) Seed acquisition: Dried pepper seeds stored at -20℃ for one year;
[0060] (2) Culture medium preparation: The culture medium formula is 4 g / L agar powder + 1 L distilled water, and sterilized in an autoclave at 121℃ for 20 min;
[0061] (3) Seed sterilization: The seeds obtained in step (1) are sterilized in a clean bench with 75% alcohol for 30 seconds, rinsed with sterile water once, sterilized with 0.1% mercuric chloride for 10 minutes, rinsed with sterile water 4 to 5 times, and the surface moisture is absorbed by sterile filter paper for later use.
[0062] (4) Inoculation: Under aseptic conditions, inoculate the sterilized seeds from step (3) into the culture medium from step (2);
[0063] (5) Cultivation: Place the Erlenmeyer flask from step (4) under light at 28°C for 45 days, with a photoperiod L:D = 10h:14h.
[0064] The seed contamination rate and germination rate were calculated as follows: contamination rate = number of contaminated seeds / total number of inoculated seeds × 100%; germination rate = number of uncontaminated seeds whose radicles broke through the seed coat / total number of inoculated seeds × 100%.
[0065] Comparative Example 4
[0066] (1) Seed acquisition: Dried pepper seeds stored at -20℃ for one year;
[0067] (2) Culture medium preparation: The culture medium formula is 4.44 g / L MS + 30 g / L sucrose + 2.5 g / L plant gel, pH 5.8~6.0, and sterilized in an autoclave at 121℃ for 20 min;
[0068] (3) Seed sterilization: The seeds obtained in step (1) are sterilized in a clean bench with 75% alcohol for 30 seconds, rinsed with sterile water once, sterilized with 10% H2O2 for 30 minutes, rinsed with sterile water 4 to 5 times, and the surface moisture is absorbed by sterile filter paper for later use.
[0069] (4) Inoculation: Under aseptic conditions, inoculate the sterilized seeds from step (3) into the culture medium from step (2);
[0070] (5) Cultivation: Place the Erlenmeyer flask from step (4) under light at 28°C for 45 days, with a photoperiod L:D = 10h:14h.
[0071] The seed contamination rate and germination rate were calculated as follows: contamination rate = number of contaminated seeds / total number of inoculated seeds × 100%; germination rate = number of uncontaminated seeds whose radicles broke through the seed coat / total number of inoculated seeds × 100%.
[0072] Comparative Example 5
[0073] (1) Seed acquisition: Dried pepper seeds stored at -20℃ for one year;
[0074] (2) Culture medium preparation: The culture medium formula is 4.44 g / L MS + 30 g / L sucrose + 2.5 g / L plant gel, pH 5.8~6.0, and sterilized in an autoclave at 121℃ for 20 min;
[0075] (3) Seed sterilization: The seeds obtained in step (1) are sterilized in a clean bench with 75% alcohol for 30 seconds, rinsed with sterile water once, sterilized with 20% NaClO for 10 minutes, rinsed with sterile water 4 to 5 times, and the surface moisture is absorbed by sterile filter paper for later use.
[0076] (4) Inoculation: Under aseptic conditions, inoculate the sterilized seeds from step (3) into the culture medium from step (2);
[0077] (5) Cultivation: Place the Erlenmeyer flask from step (4) under light at 28°C for 45 days, with a photoperiod L:D = 10h:14h.
[0078] The seed contamination rate and germination rate were calculated as follows: contamination rate = number of contaminated seeds / total number of inoculated seeds × 100%; germination rate = number of uncontaminated seeds whose radicles broke through the seed coat / total number of inoculated seeds × 100%.
[0079] Table 1. Effects of different sterilization reagents and sterilization times on the contamination rate and germination rate of pepper seeds.
[0080]
[0081]
[0082] As shown in Table 1, when using dried pepper seeds as explants, this invention sterilizes the explants with 10% H2O2 or 20% NaClO, and finally successfully obtains sterile seedlings. Compared with comparative examples 1-5, the contamination rate is significantly reduced, and the germination rate remains at a high level.
[0083] The results of Comparative Examples 4-5 and Examples 1-2 show that the use of water agar instead of traditional MS medium for seed culture in this invention can effectively reduce the contamination rate and achieve a higher germination rate. Compared with the traditional mercuric chloride disinfection method, it has a lower contamination rate and lower cost.
[0084] In summary, this invention successfully obtained sterile seedlings using dried pepper seeds as explants, achieving low-toxicity, low-cost, and efficient year-round acquisition of sterile seedlings.
[0085] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for cultivating sterile pepper seedlings, characterized in that, After sterilizing the pepper seeds, they were inoculated into the culture medium for cultivation. The sterilization process is as follows: sterilize with 75% alcohol for 30 seconds, rinse once with sterile water, then sterilize with 10% H2O2 for 30 minutes or 20% NaClO for 10 minutes, and rinse 4-5 times with sterile water to remove surface moisture; the culture medium consists of water and agar. The pepper seeds mentioned are those stored at -20℃ for 1-2 years.
2. The cultivation method according to claim 1, characterized in that, The method for obtaining pepper seeds is as follows: remove the pericarp from fresh pepper fruits, remove moisture from the surface of the seeds, and store them at -20℃ for 1-2 years.
3. The cultivation method according to claim 2, characterized in that, The method for removing moisture from the seed surface is to air dry or blot dry with sterile filter paper.
4. The cultivation method according to claim 1, characterized in that, The concentration of agar in the culture medium is 3-5 g / L.
5. The cultivation method according to claim 4, characterized in that, The concentration of agar in the culture medium is 4 g / L.
6. The cultivation method according to claim 1, characterized in that, The culture was carried out under light at 26-30℃ for 40-50 days, with a photoperiod L:D = 10 h:14 h.