CN114514879A - Rapid propagation and sugar-free rooting culture method for Nicotiana benthamiana - Google Patents
Rapid propagation and sugar-free rooting culture method for Nicotiana benthamiana Download PDFInfo
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- A—HUMAN NECESSITIES
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- 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
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Abstract
The invention belongs to the technical field of tobacco propagation, and discloses a rapid propagation method and a sugar-free rooting culture method for nicotiana benthamiana.A seed with plump seed grains, no disease, excellent growth vigor and high germination capacity is collected in a field to be used as an explant material, and the explant material is sterilized and then inoculated into a primary culture medium, and then is placed in a light environment for culture; after the seeds germinate and grow into seedlings, inoculating the seedlings into a subculture medium, selecting the seedlings which are strong and have consistent growth vigor after the seedlings grow for 25-30 days, inoculating the seedlings into a sugar-free rooting medium, putting the medium on a special culture shelf for sugar-free culture, introducing carbon dioxide gas, and performing sugar-free rooting culture in an illumination environment for 20-25 days to obtain seedlings. The method improves the rooting rate and the transplanting survival rate of the transplanting of the Nicotiana benthamiana, accelerates the chemical seedling raising of the production of the Nicotiana benthamiana, and can be used as a model plant of scientific research materials to accelerate the rapid test process of scientific research.
Description
Technical Field
The invention belongs to the technical field of tobacco propagation, and particularly relates to a rapid propagation and sugar-free rooting culture method for Nicotiana benthamiana.
Background
At present, the breeding mode of tobacco in China mostly takes seeding or plant division breeding as the main mode, tobacco seeds have small seeds, each gram of the tobacco seeds is 11500, the seeds have small seeds and are difficult to seed, and pelleted seeds are generally adopted for seeding in production. In scientific research and production, the tobacco is taken as a model plant and plays an important role in scientific research because of the short growth cycle and the easy observation of expression and characters. In scientific research, the traditional tissue culture technology is generally adopted to carry out tissue culture seeding on tobacco, culture tissue culture seedlings, and carry out rooting domestication and transplanting. Tissue culture is one of the important ways for tobacco rooting propagation, but the traditional tissue culture technology has a plurality of problems. For example, the culture medium adopted by the traditional tissue culture rooting takes sugar as a plant carbon source, and the culture medium is easy to pollute, so that the breeding of microorganisms such as bacteria, fungi and the like is serious, and the pollution is caused; because of the adoption of agar and other organic substances, the tissue culture seedling has compact texture and poor air permeability, is not beneficial to the root respiration of the tissue culture seedling and causes poor rooting. In order to reduce pollution, only a closed small container can be adopted, the communication with the external gas is less, the internal environment is poor, the growth vigor of the tissue culture seedlings is poor, the production period is long, the links are many, direct transplantation cannot be carried out after rooting, acclimation and seedling hardening are carried out firstly, but in the links of seedling hardening and subsequent transplantation, a large number of tissue culture seedlings die, the survival rate of the tissue culture seedlings is low, and the cost is increased.
Tobacco is an annual or limited perennial herb, with glandular hairs throughout the body, needles, or ovoids, tapering at the tip, strong roots, 0.7-2 meters high stem, and slightly lignified at the base. The tobacco is a temperature-preference crop, and the moisture is very important for the tobacco, namely, the tobacco plants are tall and big, the leaf surface is wide, the leaf area coefficient is large, the transpiration effect is strong, and more moisture is required to be supplied; secondly, the effectiveness of the fertilizer is influenced by moisture; third, moisture affects the extension of the leaves. When the water supply is sufficient, the expansion pressure of the tobacco leaves is large, the tobacco leaves can be fully stretched, the tobacco leaves can be lengthened and widened, and the structure is loose; the whole tobacco plant can also be used as pesticide, or medicinal preparation, and can be used as anesthetic, sweating, tranquilizing and emetic; native to south America, and widely cultivated in the provinces of south and north China.
In the past, pelleted seeds are adopted, when a orchard is built, the uniformity of stocks grown in the breeding mode is poor, the growth vigor of tree bodies is uneven, the appearance of the orchard is uneven, and much inconvenience is brought to subsequent orchard management. The Bunsen tobacco seedlings produced under the tissue culture condition grow neatly, and can be planted in a classified manner according to the sizes of the seedlings, so that the management difficulty is greatly reduced, the labor and the labor are saved, and the management cost is also reduced.
In summary, the problems of the prior art are as follows: the traditional tissue culture technology has unstable rooting performance, unstable hardening-seedling transplanting survival rate, difficult cost control and the like. The sugar-free rooting culture can effectively save time, reduce the transplanting cost, is convenient to operate, has small damage to the root system, and improves the survival rate of tobacco transplanting. The traditional tissue culture technology has the advantages that the culture medium is easy to pollute, the rooting of a common culture medium is not particularly good, the survival rate of later-stage transplantation is low, the domestication time is long, and the cost is high; conditions such as climate and the like have higher requirements and are not suitable for planting in severe areas, so domestication and transplantation are particularly important, sugar-free rooting culture skillfully combines seedling exercising and domestication, time is greatly saved, and the seedling revival of tobacco tissue culture seedlings is improved; the tree body of the traditional benthamiana propagated by seedling growth is not uniform in growth and appearance after garden formation, so that much inconvenience is brought to subsequent management, and the cost is correspondingly increased.
The difficulty of solving the technical problems is as follows: most of culture media used for rooting in traditional tissue culture are sugar-containing organic substances, the organic substances are very easy to pollute, in order to reduce the pollution rate, a culture bottle with a small inner space has to be adopted, so that the environment humidity for the internal growth of the tissue culture seedlings is high, the air exchange with the outside is basically not carried out, the seedlings grow in a weaker way, and the rooting is poor. Therefore, if the problem that the traditional tissue culture rooting culture medium is easy to pollute is not fundamentally solved, the problem that the death rate of the tissue culture seedlings is high in the seedling exercising and transplanting processes cannot be solved, and the aims of reducing the cost, and achieving high quality and high yield are more difficult to achieve.
The significance of solving the technical problems is as follows: if the problem that a tissue culture rooting medium is easy to pollute can be solved from the source, a precondition is provided for using a large-scale incubator, the internal humidity of the incubator can be reduced, so that gas exchange can be carried out between the internal environment and the external environment, the growth environment of tissue culture seedlings is improved, the growth of the tissue culture seedlings is facilitated, the produced tissue culture seedlings grow vigorously and have good rooting, seedling hardening and domestication are not needed, the tissue culture seedlings can be directly transplanted, the survival rate of the transplanted tissue culture seedlings is improved, one step is omitted, the seedling reviving period is short, survival is easy, the time is saved, and the production cost is reduced.
At present, the tobacco commonly used in scientific research comprises Bunsen tobacco and big leaf tobacco, the characters and the phenotypes of the tobacco are excellent, and the tobacco can be used as model plants of scientific research materials, transgenosis, treatment of test materials and the like in scientific research, so that the rapid test process of the scientific research can be added; meanwhile, the problems of garden construction and irregular garden appearance of the traditional seed seedling stock are solved, and the method has a wide application prospect.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a rapid propagation and sugar-free rooting culture method and application of nicotiana benthamiana.
The technical scheme adopted by the invention is as follows:
a rapid propagation and sugar-free rooting culture method for Nicotiana benthamiana comprises the following steps:
(1) selection and inoculation of the nicotiana benthamiana explants: inoculating the sterilized Nicotiana benthamiana seeds into a primary culture medium, and culturing the inoculated explants under the environment with illumination of 16h/d and darkness of 8h/d, wherein the illumination is 2000-4000 lx and the room temperature is 25 +/-2 ℃;
(2) proliferation and propagation culture: taking out the seedlings, cutting off roots and large plant leaves, inoculating the seedlings into a subculture medium, and culturing the inoculated explants under the environment with illumination of 16h/d and 8h/d in darkness at the illumination intensity of 2000-4000 lx and at the room temperature of 25 +/-2 ℃;
(3) sugar-free rooting culture: pouring vermiculite and nutrient solution into a sugar-free culture box, and uniformly mixing to obtain a sugar-free rooting culture medium; selecting Nicotiana benthamiana seedlings, cutting off callus on the base parts, and inoculating the cut callus to a sugar-free rooting culture medium; and (3) carrying out dark culture on the inoculated seedlings at the room temperature of 25 +/-2 ℃ for 3 days, then placing the seedlings on a special sugar-free culture rack, introducing carbon dioxide gas, and carrying out sugar-free rooting culture under the conditions of 16h/d illumination, 8h/d darkness, 2000-4000 lx illumination and 25 +/-2 ℃ at the room temperature, wherein the culture time is 20-25 days, so that seedlings can emerge.
Further, in the step (1), the Nicotiana benthamiana is used as a material, and seeds with plump seed grains, no disease, excellent growth vigor and high germination capacity are collected in a field to be used as explant materials.
Further, in the step (1), the step of disinfecting the seeds comprises: and (3) putting the washed seeds into 75% alcohol for disinfection for 10-15 minutes in a super-clean workbench, shaking during the disinfection, sucking the seeds out by using a liquid transfer gun, and putting the seeds into sterile water for washing for 3-5 times.
Further, in the step (1), the disinfected seeds are inoculated into a primary culture medium; and culturing the inoculated explant for 25-30 days in an environment with illumination of 16h/d and darkness of 8h/d, illumination of 2000-4000 lx and room temperature of 25 +/-2 ℃. In general, the seeds can germinate for 5 days and can be propagated for 20-25 days.
Further, the composition of the primary medium was: 1/2MS +30g/L sucrose +7-8g/L agar, pH5.8-6.2.
Further, in the step (2): after the primary explant is cultured for 25-30 days, carrying out subculture propagation culture; taking out the seedlings in a clean bench, cutting off the base parts and the large leaves of the plants, inoculating the seedlings into a subculture medium, and culturing the inoculated explants for 25d to 30d in an environment with illumination of 16h/d and darkness of 8h/d, illumination of 2000 to 4000lx and room temperature of 25 +/-2 ℃.
Further, the composition of the subculture medium was: 1/2MS +30g/L sucrose +7-8g/L agar, pH5.8-6.2.
Further, in the step (3), the sugar-free rooting medium comprises the following components: mixing a vermiculite matrix and a nutrient solution, wherein the nutrient solution comprises 1/2MS + 0.5-1.0 mg/L IBA, the pH value is 5.8-6.0, the water content of the mixed matrix is 75-80%, and the water content of the preferred matrix is 80%.
Further, the sugar-free rooting medium is prepared by the following method: assembling a sugar-free culture box, putting vermiculite into a high-temperature high-pressure sterilization pot at 121 ℃ for sterilization for 21min, taking out, drying in a drying oven at 65 ℃ for 12h, weighing 400 g/box under aseptic condition, and preparing 1000-1100 ml/box of nutrient solution, wherein the nutrient solution comprises the following components: 1/2MS + 0.5-1.0 mg/L IBA, pH5.8, and the water content of the mixed substrate is 75-80%.
Further, the step (3) is preferably operated as follows: pouring the prepared vermiculite and nutrient solution into a sugar-free culture box in an ultra-clean workbench, and uniformly mixing to obtain a sugar-free rooting culture medium with the water content of 75-80%; selecting the good-growing seedling of the Nicotiana benthamiana which grows for 25-30 days and is strong (the height is 2.5.0-4.0cm), the growth vigor of which is consistent, and has 3-5 or more leaves, cutting off the callus of the base part, and inoculating the seedling to a sugar-free rooting culture medium; and (2) carrying out dark culture on the inoculated seedlings at the room temperature of 25 +/-2 ℃ for 3 days, putting the seedlings on a special sugar-free culture rack for 3 days, introducing carbon dioxide gas with the concentration of 800-1300 ppm, and carrying out culture under the environment of illumination of 16h/d, darkness of 8h/d, illumination of 2000-4000 lx and room temperature of 25 +/-2 ℃, wherein the seedling can emerge after the culture time of 20-25 days.
The culture environment parameters are obtained by comparing experiments, the culture medium parameters are also results obtained by screening for many times, and the culture medium and the cultured seedlings are prepared under the parameters, so that the Nicotiana benthamiana has the advantages of strong growth vigor, no growth stress phenomenon, good rooting effect and developed root system.
The primary culture medium and the secondary culture medium are not added with auxin substances, but have good rooting effect, and the cost can be saved by using the auxin-free culture medium.
The invention also aims to provide the application of the rapid propagation and sugar-free rooting culture method of the Nicotiana benthamiana in tobacco propagation.
In summary, the advantages and positive effects of the invention are: the Sugar-free tissue culture technique (also called photoautotrophic tissue culture technique or photoautotrophic tissue culture technique) of plants refers to the technique of using photosynthesis of plant explants to cultivate CO2Instead of sugar as carbon source of plant body, by introducing CO2Gas and environmental factors such as illumination temperature of 16h/d, humidity and the like which influence the growth and development of the seedlings are controlled, the photosynthesis of the plants is promoted, the photosynthetic rate of the plants is enhanced, the seedlings are changed from the mixotrophic type to the autotrophic type, and a novel plant micropropagation technology for producing high-quality seedlings is provided。
The culture medium of the invention does not contain sugar and uses CO2The carbon source of plant body instead of sugar can reduce microbial contamination caused by microorganism, such as fungus and bacteria; the sugar-free rooting technology is carried out while domesticating the rooting strong seedlings, has convenient operation and short transition period, does not need to clean the roots, can be directly transplanted into a matrix, avoids the damage to the roots and improves the survival rate of the transplantation; the vermiculite is loose and breathable, and provides a good living environment for transplanting the tobacco, so that the rooting rate of the tissue culture seedlings is improved, the environmental factors such as sufficient carbon dioxide, illumination, temperature, humidity and the like in the sugar-free rooting process improve the high-quality tobacco, and the tobacco tissue culture seedlings which are rooted out by the sugar-free rooting process have a large number of roots, are strong and grow vigorously. The transplanting survival rate of the tissue culture seedlings produced by sugar-free tissue culture can reach about 98-99.8%, the autotrophic capacity of the tissue culture seedlings is far higher than that of the tissue culture seedlings of traditional tissue culture rooting, the tissue culture seedlings grow vigorously after being transplanted, the later-stage management is facilitated, the chemical seedling culture of the Nicotiana benthamiana production is accelerated, an efficient rapid propagation mode is provided for industrial agricultural seedling culture, the tissue culture seedling culture method has important guiding significance for asexual propagation of the tissue culture seedlings, and the tissue culture seedling culture method can be used as a model plant of scientific research materials, such as transgenosis, application of treatment of test materials and the like, and can be added with a rapid test process of scientific research.
Drawings
FIG. 1 is a flow chart of a rapid propagation and sugar-free rooting culture method for Nicotiana benthamiana provided in an embodiment of the present invention.
Fig. 2 is a drawing of a sugarless rooting device.
FIG. 3 photo of Nicotiana benthamiana at seedling stage after sugar-free rooting culture for 25 days.
FIG. 4 is a photograph of the full-length period of the sugar-free rooting cultured tissue culture seedlings of Nicotiana benthamiana transplanted into the matrix for 30d regrowth.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
Aiming at the problems in the prior art, the invention provides a rapid propagation and sugar-free rooting culture method for nicotiana benthamiana, and the invention is described in detail below with reference to the accompanying drawings.
As shown in FIG. 1, the rapid propagation and sugar-free rooting culture method for Nicotiana benthamiana provided by the embodiment of the invention comprises the following steps:
s101: inoculating the disinfected seeds into a primary culture medium, and culturing the inoculated explants under the environment with illumination of 16h/d and darkness of 8h/d, wherein the illumination is 2000-4000 lx and the room temperature is 25 +/-2 ℃;
s102: taking out the seedlings, cutting off callus and bottom leaves, inoculating the seedlings into a subculture medium, and culturing the inoculated explants under the environment with illumination of 16h/d and 8h/d in darkness and illumination of 2000-4000 lx at room temperature of 25 +/-2 ℃;
s103: pouring the prepared vermiculite and nutrient solution into a sugar-free culture box, and uniformly mixing; selecting Nicotiana benthamiana seedlings, cutting out callus and bottom leaves, and inoculating the cut leaves into a sugar-free rooting culture medium; and (3) placing the seedlings on a culture rack special for sugar-free culture, introducing carbon dioxide gas, and carrying out sugar-free rooting culture in an environment with illumination of 16h/d, darkness 8h/d, room temperature of 25 +/-2 ℃ and illumination of 2000-4000 lx.
The technical solution of the present invention is further described with reference to the following specific examples.
Example 1
First, establishment of explants
(1) Taking the Nicotiana benthamiana as a material, and collecting seeds with full seed grains, no disease, excellent growth vigor and high germination capacity in the field as explant materials;
(2) and (3) putting the washed seeds into 75% alcohol for disinfection for 10-15 minutes in a super-clean workbench, shaking during the disinfection, sucking the seeds out by using a liquid transfer gun, and putting the seeds into sterile water for washing for 3-5 times.
(3) Inoculating the disinfected seeds into a primary culture medium, wherein the formula of the culture medium is as follows: 1/2MS +30g/L sucrose +8g/L agar, pH5.8. And culturing the inoculated explants for 25d in an environment with illumination of 16h/d and darkness of 8h/d, illumination of 2000-4000 lx and room temperature of 25 +/-2 ℃.
Second, subculture
And after culturing the primary explant for 20-25 days, carrying out subculture propagation. Taking out the seedlings in a clean bench, cutting off callus and bottom leaves, and inoculating the seedlings to a subculture medium, wherein the formula of the subculture medium is as follows: 1/2MS +30g/L sucrose +8g/L agar, pH5.8. And culturing the inoculated tissue culture seedlings in an environment with illumination of 16h/d and darkness of 8h/d, illumination of 2000-4000 lx and room temperature of 25 +/-2 ℃ for 25 d.
Thirdly, sugar-free rooting culture
(1) Assembling a sugar-free culture box, putting vermiculite into a high-temperature high-pressure sterilization pot at 121 ℃ for sterilization for 21min, taking out, drying in a drying oven at 65 ℃ for 12h, and weighing 400 g/box under aseptic conditions; 1100ml of nutrient solution is prepared per box, and the formula of the nutrient solution is as follows: 1/2MS +0.5mg/L IBA, pH5.8 or so.
(2) Pouring the vermiculite and the nutrient solution prepared in the step into a sugar-free culture box in an ultra-clean workbench, and uniformly mixing to obtain a sugar-free rooting culture medium with the water content of 80%; selecting good growing seedling of the Nicotiana benthamiana which grows for 25-30 days and has 5 or more leaves, cutting off callus and bottom leaves, and inoculating the seedling to a sugar-free rooting culture medium. And carrying out dark culture on the inoculated seedlings at the room temperature of 25 +/-2 ℃ for 3 days, putting the seedlings on a special sugar-free culture frame after 3 days, introducing carbon dioxide gas with the concentration of 800-1300 ppm, and carrying out sugar-free rooting culture under the environment of illumination of 16h/d, 8h/d in the dark, the room temperature of 25 +/-2 ℃ and the illumination of 2000-4000 lx, wherein the culture time is 25 days. The photo of the seedlings cultured for 25d without sugar rooting is shown in figure 3, which shows that the seedlings grow strongly and root well. The tissue culture seedlings were transplanted into the substrate, and the photograph of the growth for 30d is shown in FIG. 4.
After sugar-free rooting culture for 25 days, randomly extracting 15 plants to count the plant height and the root number, and obtaining the results shown in the following table 1:
TABLE 1 statistics of sugar-free rooting culture 25d physiological data
Example 2
The procedure was as in example 1, except that the water content of the substrate of the sugarless rooting medium in step (3) was 75%. After culturing for 25d according to the same operation, randomly extracting 15 plants and counting the plant height and the root number, wherein the average plant height is 18.15cm, and the root number is 101.7 per plant.
The growth of the seedlings in the sugar-free rooting medium with the matrix water content of 80 percent is better than that of the seedlings with the matrix water content of 75 percent.
Comparative example 1
The procedure was as in example 1, except that the water content of the substrate of the sugarless rooting medium in step (3) was 70%. After culturing for 25 days according to the same operation, randomly extracting 15 plants to count the plant height and the root number, wherein the average plant height is 16.28cm, and the root number is 77.6 per plant.
Comparative example 2
The procedure was as in example 1, except that the water content of the substrate of the sugarless rooting medium in step (3) was 90%. After culturing for 25 days according to the same operation, randomly extracting 15 plants to count the plant height and the root number, wherein the average plant height is 15.75cm, and the root number is 75.7 per plant.
As can be seen from the comparative examples, the water content of the substrate is too low or too high, which results in slow growth of seedlings and poor rooting.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (8)
1. A rapid propagation and sugar-free rooting culture method for Nicotiana benthamiana is characterized by comprising the following steps of:
(1) selection and inoculation of the nicotiana benthamiana explants: inoculating the sterilized Nicotiana benthamiana seeds into a primary culture medium, and culturing the inoculated explants under the environment with illumination of 16h/d and darkness of 8h/d, wherein the illumination is 2000-4000 lx and the room temperature is 25 +/-2 ℃;
(2) proliferation and propagation culture: taking out the seedlings, cutting off roots and large plant leaves, inoculating the seedlings into a subculture medium, and culturing the inoculated explants under the environment with illumination of 16h/d and 8h/d in darkness at the illumination intensity of 2000-4000 lx and at the room temperature of 25 +/-2 ℃;
(3) sugar-free rooting culture: pouring vermiculite and nutrient solution into a sugar-free culture box, and uniformly mixing to obtain a sugar-free rooting culture medium; selecting Nicotiana benthamiana seedlings, cutting off callus on the base parts, and inoculating the cut callus to a sugar-free rooting culture medium; dark culturing the inoculated seedlings at the room temperature of 25 +/-2 ℃ for 3 days, placing the seedlings on a special culture rack for sugar-free culture, introducing carbon dioxide gas, and performing sugar-free rooting culture at the room temperature of 25 +/-2 ℃ under the conditions of illumination of 16h/d, darkness of 8h/d, illumination of 2000-4000 lx and room temperature of 25 +/-2 ℃, wherein seedlings can emerge after the culture time of 20-25 days; the sugar-free rooting culture medium comprises the following components: mixing a vermiculite matrix and a nutrient solution, wherein the nutrient solution comprises 1/2MS + 0.5-1.0 mg/LIBA, the pH value is 5.8-6.0, and the water content of the mixed matrix is 75-80%.
2. The rapid propagation and sugar-free rooting culture method of nicotiana benthamiana according to claim 1, wherein in the step (1), the step of sterilizing the seeds comprises: and (3) putting the washed seeds into 75% alcohol for disinfection for 10-15 minutes in a super-clean workbench, shaking during the disinfection, sucking the seeds out by using a liquid transfer gun, and putting the seeds into sterile water for washing for 3-5 times.
3. The rapid propagation and sugar-free rooting culture method of nicotiana benthamiana according to claim 1, wherein in step (1), the sterilized seeds are inoculated onto a primary medium; and culturing the planted seeds for 25 d-30 d in an environment with illumination of 16h/d and darkness of 8h/d, illumination of 2000-4000 lx and room temperature of 25 +/-2 ℃.
4. The rapid propagation and sugar-free rooting culture method of nicotiana benthamiana according to claim 1, wherein in the step (1), the composition of the primary culture medium is as follows: 1/2MS +30g/L sucrose +7-8g/L agar, pH5.8-6.2.
5. The rapid propagation and sugar-free rooting culture method for nicotiana benthamiana according to claim 1, wherein in the step (2), the primary explants are cultured for 25-30 days and then subcultured for propagation; taking out the seedlings in a clean bench, cutting off roots and large plant leaves, inoculating the seedlings into a subculture medium, and culturing the inoculated explants under the environment of illumination of 16h/d and 8h/d in darkness at the illumination intensity of 2000-4000 lx and at the room temperature of 25 +/-2 ℃ for 25 d-30 d.
6. The rapid propagation and sugar-free rooting culture method of nicotiana benthamiana according to claim 5, wherein in the step (2), the composition of the subculture medium is as follows: 1/2MS +30g/L sucrose +7-8g/L agar, pH5.8-6.2.
7. The rapid propagation and sugar-free rooting culture method of nicotiana benthamiana according to claim 5, wherein in the step (3), the sugar-free rooting culture medium is prepared by the following method: assembling a sugar-free culture box, putting vermiculite into a high-temperature high-pressure sterilization pot at 121 ℃ for sterilization for 21min, taking out, drying in a drying oven at 65 ℃ for 12h, weighing 400 g/box under aseptic condition, and preparing 1000-1100 ml/box of nutrient solution, wherein the nutrient solution comprises the following components: 1/2MS + 0.5-1.0 mg/L IBA, pH5.8, and the water content of the mixed substrate is 75-80%.
8. The rapid propagation and sugar-free rooting culture method for Nicotiana benthamiana as claimed in claim 5, wherein the prepared vermiculite and nutrient solution are poured into a sugar-free culture box in an ultra-clean bench and mixed uniformly to obtain a sugar-free rooting culture medium with a water content of 75-80%; selecting good growing seedlings of the Nicotiana benthamiana which grow for 25-30 days and have 3-5 leaves or more, cutting off the callus of the base part, and inoculating the seedlings to a sugar-free rooting culture medium; and (2) carrying out dark culture on the inoculated seedlings for 3d at the room temperature of 25 +/-2 ℃, putting the seedlings on a sugar-free culture special culture frame after 3d, introducing carbon dioxide gas with the concentration of 800-1300 ppm, and carrying out culture under the environment with the illumination of 16h/d, the darkness of 8h/d, the illumination of 2000-4000 lx and the room temperature of 25 +/-2 ℃, wherein the seedling can emerge after the culture time of 20-25 d.
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| CN115486360A (en) * | 2022-08-29 | 2022-12-20 | 甘肃勇馨科技农业有限公司 | Novel sugar-free efficient rooting culture and transplanting method for detoxified ginseng fruit seedlings |
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