CN114073226A - Inoculation method for tissue culture and rapid propagation of raspberry petioles - Google Patents
Inoculation method for tissue culture and rapid propagation of raspberry petioles Download PDFInfo
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- 238000011081 inoculation Methods 0.000 title claims abstract description 49
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- 238000000034 method Methods 0.000 title claims abstract description 43
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 40
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 36
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 32
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000008223 sterile water Substances 0.000 claims abstract description 19
- 230000006698 induction Effects 0.000 claims abstract description 12
- 239000001963 growth medium Substances 0.000 claims abstract description 11
- 230000000249 desinfective effect Effects 0.000 claims abstract description 7
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- 238000011109 contamination Methods 0.000 claims description 10
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 claims description 3
- 229920001817 Agar Polymers 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 238000004364 calculation method Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 abstract description 15
- 239000005708 Sodium hypochlorite Substances 0.000 abstract description 14
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 abstract description 14
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 abstract description 12
- 229960002523 mercuric chloride Drugs 0.000 abstract description 9
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- 230000008929 regeneration Effects 0.000 abstract description 7
- 238000011069 regeneration method Methods 0.000 abstract description 7
- 230000010474 transient expression Effects 0.000 abstract description 6
- 230000001590 oxidative effect Effects 0.000 abstract description 3
- 238000004161 plant tissue culture Methods 0.000 abstract description 3
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- 238000010008 shearing Methods 0.000 abstract 1
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- 238000004519 manufacturing process Methods 0.000 description 3
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 2
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- 235000021013 raspberries Nutrition 0.000 description 2
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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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Abstract
The invention discloses an inoculation method for tissue culture and rapid propagation of raspberry petioles, belonging to the technical field of plant tissue culture. Shearing off leaf stalks of healthy tender leaves on raspberry stems, disinfecting the leaves by using alcohol with the mass concentration of 75% in an ultra-clean workbench, rinsing the leaves by using sterile water, inoculating the leaves into a culture medium for dark culture, wherein the pollution rate of the inoculation at 30d is 5%, the browning rate and the browning rate are both 0, the expansion rate and the callus induction rate are both 95%, and the leaf stalks and the callus thereof grow well. Pollution is reduced by using petioles as explants and adopting a method of not washing before disinfection; the use of 75% alcohol for disinfection avoids the use of highly toxic mercuric chloride and sodium hypochlorite with irritation, corrosivity and strong oxidizing property; the method is simple to operate, time-saving, labor-saving, water-saving, space-saving, reagent-saving, cost-saving, safe and environment-friendly, improves the inoculation quality and efficiency, lays a foundation for establishing a raspberry petiole regeneration system, performing transient expression identification on gene functions by using callus, performing transgenosis and editing genes to culture new germplasm, and is suitable for the fields of scientific research and industrial tissue culture.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to an inoculation method for tissue culture and rapid propagation of raspberry petioles.
Background
The plant tissue culture technology is widely applied to the fields of plant rapid propagation, secondary metabolite production, germplasm preservation, genetic improvement and the like, and is particularly important in scientific research and production application. In the tissue culture process, explant inoculation is the first necessary and critical step for plant to enter the tissue culture process from the natural environment. Tissue culture of many plants takes leaves as explants and the inoculation procedure is usually water washing → disinfection with mercuric chloride solution or sodium hypochlorite solution → sterile water rinsing → disinfection with alcohol solution → sterile water rinsing → inoculation onto culture medium → culture.
For the raspberry, if the leaf is used as the explant, the following problems exist according to the traditional inoculation method: (1) the veins on the leaves of the raspberry are rich and fine and latticed, so that the surface of the tender leaves is uneven and looks like a skirt pleat, and the tender leaves are not easy to clean and disinfect by a disinfectant to cause pollution; (2) the raspberry leaves are very thin, and the raspberry leaves are easily broken to form wounds when the raspberry leaves are wrinkled by hands, so that the risk of pollution caused by the fact that outside bacteria enter the leaves from the wounds is increased; (3) the raspberry leaves are very thin and rich in phenolic substances, and are easy to brown after being disinfected; (4) the leaves of the raspberry are very thin, the phenomenon of adhesion among the leaves can occur during disinfection, pollution is easy to cause, and meanwhile, the inoculation difficulty is increased; (5) observation under a microscope shows that the back surfaces of the raspberry leaves have abundant skin hairs and are interwoven together to form a blanket shape, so that the disinfection difficulty is increased; (6) the technicians in the field generally wash for several hours with detergent and running water before disinfection to reduce the subsequent pollution rate, which is water and time consuming, and in addition, because raspberry leaves are thin and the back surface has skin fur interwoven together, the water washing has the risk of increasing the pollution caused by external bacteria entering the leaves from wounds or rushing into the skin fur areas, and people can be misled to use antibiotics for inhibiting bacteria due to the pollution caused by the endogenous bacteria. (7) Those skilled in the art usually need to disinfect with strong alcohol solution plus mercuric chloride solution or alcohol solution plus sodium hypochlorite solution in order to achieve the purpose of thorough disinfection, however, mercuric chloride is extremely toxic to plants, animals and environment, and sodium hypochlorite has a certain pungent odor, strong oxidizing property and is easy to corrode leaves to brown and die. In conclusion, the traditional method for inoculating raspberry leaves is difficult to operate, time-consuming, labor-consuming, space-consuming, water-consuming, reagent-consuming, high in cost, environment-friendly, safe for operators, prone to causing leaf pollution or browning or death, and further material and time loss causes the problem that the inoculation target cannot be efficiently and excellently completed, subsequent links of tissue culture and rapid propagation are affected, scientific research and production plans are affected, and economic losses are caused.
The leaves and stems of the raspberry are thicker and stick-shaped than the leaves, and the leaves and stems are not close to each other like the leaves during disinfection, so the operation is easy; the petiole has no skirt pleat structure like tender leaf, has less surface skin hair and thorn than leaf, and is easy to be fully contacted with the disinfection reagent to be beneficial to disinfection compared with leaf.
Therefore, the explant inoculation method for tissue culture and rapid propagation of the raspberry, which does not need to replace leaf stalks with leaf stalks as explants, does not need washing before disinfection, does not need mercury bichloride and sodium hypochlorite for disinfection, saves time, labor, water and reagents, is simple to operate, is safe and environment-friendly, and has high quality and high efficiency, is a problem to be solved by technical personnel in the field.
Disclosure of Invention
In view of the above, the invention provides an inoculation method for tissue culture and rapid propagation of raspberry petioles, which is a method for effectively completing the steps of explant disinfection and inoculation without inoculating leaves, washing before disinfection, mercury bichloride and sodium hypochlorite disinfection and closed disinfection; and the petiole has low pollution rate after inoculation and grows callus normally; the method achieves the aims of saving time, labor, space, water and reagent, saving cost, being simple to operate, safe, environment-friendly, high-quality and high-efficiency in completing the raspberry inoculation link, lays a solid material foundation for establishing a raspberry petiole regeneration system, identifying gene functions by using callus for transient expression, transgenosis and cultivating new germplasm through gene editing, and is suitable for the fields of scientific research and industrial tissue culture.
In order to achieve the purpose, the invention adopts the following technical scheme:
an inoculation method for tissue culture and rapid propagation of raspberry petioles comprises the following specific steps:
(1) cutting off the upper petiole of the stem of the raspberry;
(2) disinfecting the petioles by using alcohol with the mass concentration of 75% in a superclean workbench, and rinsing the petioles by using sterile water;
(3) the sterilized and rinsed petioles with sterile water are inoculated into a culture medium for dark culture.
Generally, the criteria for success of inoculation are the presence of contaminating petioles, browning, enlargement and callus growth. Firstly, the theoretical basis of the technical scheme of the invention is as follows: the interior of the healthy petiole without mechanical damage is free from bacteria, so that the surface of the petiole can be disinfected without pollution. Therefore, the technical scheme of the invention only needs 75% alcohol solution to disinfect the petiole, and does not need to use a disinfection method of adding mercuric chloride solution or adding sodium hypochlorite solution into alcohol solution with higher strength like the traditional method, thereby saving time, labor, water, reagent, cost, safety and environmental protection. Secondly, the invention directly takes the leaf stalks as explants, skillfully avoids the factors which are easy to pollute, such as uneven surfaces of tender leaves of the raspberry, much skin hair, thinness, easy breaking and browning, easy approaching of the leaves during disinfection, and the like, the difficulty and complexity of disinfection and inoculation of the leaf stalks are far lower than those of the leaves, the operation is simpler and more convenient, the inoculation effect is excellent, and the time, labor, water, reagent, space and cost are saved. Again, water washing is generally employed by those skilled in the art to reduce the subsequent contamination rate, and it is generally accepted that the longer the wash time, the lower the post-inoculation contamination rate. The invention considers that the water washing method increases the risk of pollution caused by bringing external bacteria into the explant by water, and even misleads people to think the pollution is caused by endophytes. The invention abandons the traditional method of sterilizing by mercuric chloride and alcohol solution or sodium hypochlorite and alcohol solution and washing before sterilization in the prior tissue culture technology, breaks through the limitation of the traditional thought, only uses alcohol solution to sterilize the petiole in a sealing way, and does not need washing before sterilization, thereby achieving better inoculation effect than the flow of washing, sterilizing by mercuric chloride or sodium hypochlorite solution, rinsing by sterile water, sterilizing by alcohol, rinsing by sterile water and inoculating.
Further, the petioles in the step (1) are 0.5-3.0 cm away from the stem tip and are healthy and tender leaves.
Further, the thickness of the petiole in the step (1) is 1.5-4.0 mm, and the petiole is cut into small sections with the length of 0.5-2.0 cm.
Further, the specific operation of the step (2) is as follows: and (3) at an inlet of a super clean bench, filling the petioles into a bottle which is arranged in the super clean bench and is subjected to high-pressure sterilization, then sealing and disinfecting the bottle for 50-70 s by using an alcohol solution with the mass concentration of 75%, and rinsing the bottle by using sterile water.
Further, the rinsing with sterile water in the step (2) is performed for 3-5 times.
Further, the culture medium in the step (3) is prepared by adding the following components in mass concentration into an MS culture medium: 0.1-0.5 mg/L of 6-BA-1、2,4-D 0.5～1.5mg·L-1、NAA 0.2～0.8mg·L-120-30 g.L of sucrose-1And agar 5.5-6.0 g.L-1And the pH value is 5.8-6.2.
Further, the culture temperature in the dark culture in the step (3) is 25. + -. 2 ℃.
Further, during the culture period, the pollution rate, browning rate, enlargement rate and callus induction rate of the petioles are counted, and the growth conditions of the petioles and the callus thereof are observed.
Further, the calculation formulas of the pollution rate, the browning rate, the enlargement rate and the callus induction rate are as follows:
according to the technical scheme, compared with the prior art, the invention discloses and provides the inoculation method for tissue culture and rapid propagation of the raspberry petiole, and the inoculation method has the following beneficial effects: (1) according to the invention, the tender leaf petiole which is 0.5-3.0 cm away from the stem tip and is free from mechanical damage and healthy is taken as the explant, so that the thin leaves with a lot of surface skin hairs and uneven surfaces are replaced, and the disinfection difficulty of the explant is reduced; (2) the method saves the step of washing before disinfection, is simple to operate, saves time, labor and water, and avoids the false phenomena that the water brings outside bacteria into the explant to cause pollution and even causes the pollution of endophyte in tissue culture; (3) based on the theory that 'no bacteria exist in the healthy petioles', the inoculation task can be completed only by disinfecting the surface of the petioles with alcohol solution with the mass concentration of 75%; (4) the use of alcohol solution with the mass concentration of 75% for closed disinfection avoids the use of highly toxic reagent mercuric chloride and the use of sodium hypochlorite with irritation, corrosivity and strong oxidizing property for disinfection; (5) the inoculation method is simple to operate, time-saving, labor-saving, water-saving, reagent-saving, space-saving, cost-saving, safe, environment-friendly, high-quality and efficient, completes the inoculation link in the tissue culture rapid propagation technology of the raspberry petioles, greatly reduces the technical difficulty and complexity, improves the inoculation quality and efficiency, lays a solid material foundation for establishing a raspberry petiole regeneration system, performing transient expression by using callus to identify gene functions, transgenosis and breeding new germplasm by gene editing, and is suitable for the fields of scientific research and industrial tissue culture.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a drawing showing the growth of petioles treated in example 1 of the present invention;
wherein, A is inoculated front petiole, B is inoculated 0d petiole; c is a contaminated petiole; d is an enlarged petiole; e is the petiole of the long callus; f is a petiole with more callus;
FIG. 2 is a graph showing the contamination rate, browning rate, enlargement rate and callus induction rate of raspberry petioles treated 1 in example 1 of the present invention at day 3-30;
FIG. 3 is a graph showing the growth of petioles of comparative example 1 treatment 2 according to the present invention;
wherein, A is inoculated front petiole, B is inoculated 0d petiole; c is a contaminated petiole; d is browned petiole; e is dead petiole; f is an enlarged petiole; g is the petiole with long callus;
FIG. 4 is a graph showing the contamination rate, browning rate, enlargement rate and callus induction rate of raspberry petioles treated in comparative example 1 and 2 cultured for 3-30 days;
FIG. 5 is a drawing showing the growth of the base of the vane of comparative example 2 treatment 3 according to the present invention;
wherein A is the base of the inoculated 0d leaf; b is a browned leaf base; c is the base of the leaf dead from brown; d is an enlarged blade base; e is the leaf base of the long callus.
FIG. 6 is a graph showing the contamination rate, browning rate, enlargement rate and callus induction rate of raspberry leaf culture of comparative example 2 treatment 3 of the present invention at day 3-30.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
(1) Selecting healthy and tender petioles of which the distances from the stem tips are 0.5-3.0 cm to the stem tips of the Berca raspberry, wherein the thickness is 1.5-4.0 mm, and cutting the petioles into small sections of which the lengths are 0.5-2.0 cm.
(2) At the inlet of the clean bench, the petioles were filled into autoclaved bottles in the clean bench, hermetically sterilized in the bottles with an alcohol solution of 75% by mass for 60s (treatment 1), shaken upside down during this time, and then rinsed 3 times with sterile water. 60 petioles were used.
(3) Inoculating sterilized and sterile water rinsed petiole to culture medium (the culture medium is MS culture medium added with 6-BA 0.2 mg. L)-1、2,4-D 1.2mg·L-1、NAA 0.4mg·L-1Sucrose (30 g. L)-1And agar 5.5 g.L-1) The dark culture is carried out at the temperature of 25 +/-2 ℃.
And (3) respectively culturing 3d, 6d, 9d, 12d, 15d, 18d, 21d, 24d, 27d and 30d, counting the pollution rate, browning rate, expansion rate and callus induction rate of the petioles, and observing the growth conditions of the petioles and the callus thereof. Wherein:
the results are shown in FIGS. 1 and 2. The results showed that the contamination rate of treatment 1 at the 6 th day of culture was 5.0%, which did not increase thereafter; the browning rate and the browning rate of treatment 1 were always 0 during the culture; the expansion rate of the treatment 1 on the 9 th day of culture is 95.0%, and the expansion rate is not increased, namely, the uncontaminated petioles are expanded; the callus induction rate of treatment 1 at the 24 th day of culture was 95.0%, which did not increase thereafter, i.e., the enlarged petioles all grew callus and grew well. By the inoculation method, 95.0% of petioles expand after inoculation, callus grows out, and the method can enter links of subculture multiplication, establishment of a petiole regeneration system, verification of gene functions through transient expression, transgenic breeding, gene editing breeding and the like.
The method effectively completes the inoculation link of the leaf stalks in the tissue culture and rapid propagation of the raspberries on the premise of not inoculating leaves, not needing washing agent and running water for flushing before disinfection, and not using mercury bichloride and sodium hypochlorite, has simple operation, time saving, labor saving, water saving, reagent saving, space saving, cost saving, high efficiency, safety and environmental protection, ensures that 95.0 percent of the leaf stalks are expanded and grow callus, lays a foundation for subsequent successive multiplication of the raspberries, establishment of a leaf stalk regeneration system, verification of gene functions through an instant expression technology, transgenic breeding, gene editing breeding and the like, and is suitable for scientific research and industrial seedling culture.
Comparative example 1
(1) Selecting healthy and tender petioles of which the distances from the tips of the stems are 0.5-3.0 cm to the tips of the stems of the Berca rashiki, wherein the thickness is 1.5-4.0 mm, and cutting the petioles into small sections with the length of 0.5-2.0 cm.
(2) Flushing with running water for 2h, sucking water on the surface of petiole with filter paper, placing the petiole into a bottle in a super clean bench and subjected to high-pressure sterilization at an inlet of the super clean bench, performing closed sterilization for 60s by using sodium hypochlorite solution with the effective chlorine concentration of 1%, performing reverse shaking in the period, rinsing with sterile water for 3 times, performing closed sterilization for 60s by using alcohol solution with the mass concentration of 75% (treatment 2), performing reverse shaking in the period, and rinsing with sterile water for 3 times. 60 petioles were used.
The treatment method in the step (3) is the same as that in example 1, i.e., treatment 1.
The results are shown in FIGS. 3 and 4. The results showed that the contamination rate of petioles at the 3 rd day of culture was 1.7% in comparative example 1, treatment 2, and did not increase thereafter; treatment 2 had a browning rate of 10.0% at day 6 of culture, and did not increase thereafter; treatment 2 had a brown mortality of 10.0% at day 15 of culture, and did not increase thereafter; the expansion rate of the treatment 2 at the 18 th day of culture is 88.3 percent, and the expansion rate is not increased later, namely, the expansion rate of the leaf stalks which are not polluted and not dead due to brown is increased; the callus induction rate of treatment 2 at the 27 th day of culture was 88.3%, which did not increase thereafter, i.e., the enlarged petioles all grew callus and grew well. By the inoculation method, 88.3 percent of petioles are expanded after inoculation, the grown callus can enter links of subculture multiplication, petiole regeneration, transient expression verification of gene function, transgenic breeding, gene editing and the like, and the inoculation loss is larger than that of the treatment 1 in the example 1.
Comparative example 2
(1) Selecting healthy tender leaves without mechanical damage and with the leaf area of 2-5 cm near the stem tip of the Berca raspberry2。
(2) Washing for 2 hours by running water, sucking water on the surface of the blade by filter paper, taking the base part of the blade to be 0.5-2 cm2At the inlet of the clean bench, the base of the leaf is placed in an autoclaved bottle in the clean bench, and is sterilized by sodium hypochlorite solution with an effective chlorine concentration of 1% for 60s in a sealed manner, and is shaken upside down during the period, and is rinsed with sterile water for 3 times, and is sterilized by alcohol solution with a mass concentration of 75% for 60s in a sealed manner (treatment 3), and is shaken upside down during the period, and is rinsed with sterile water for 3 times. 60 blades were used.
The treatment method in the step (3) is the same as that in example 1, i.e., treatment 1.
The results are shown in FIGS. 5 and 6. The results showed that the leaf contamination rate of comparative example 2, treatment 3, was 6.7% at day 6 of culture, and did not increase thereafter; treatment 3 had a browning rate of 13.3% at day 6 of culture, which did not increase thereafter; treatment 3 had a brown mortality of 13.3% at day 21 of culture, which did not increase thereafter; the expansion rate of the treatment 3 at the 18 th day of culture is 80.0 percent, and the expansion rate is not increased, namely, the expansion rate of the leaves which are not polluted and not browned is increased; the callus induction rate of the treatment 3 at the 30 th day of culture was 80.0%, that is, the enlarged leaves all grew callus and the growth condition was good. 80.0% of leaves expand after inoculation by the inoculation method, callus grows out, the links of subculture multiplication, establishment of a leaf regeneration system, verification of gene function through transient expression, transgenic breeding, gene editing breeding and the like can be entered, and the inoculation loss is larger than that of the treatment 1 in example 1 and that of the treatment 2 in comparative example 1.
The technical scheme of the invention is also suitable for the inoculation of the petioles of other plants with more epidermal hairs, fine and dense leaf veins, uneven leaf surfaces and thin leaves, avoids the trouble of pollution caused by washing the leaves with water before disinfection and attaching the leaves during disinfection, is also suitable for the inoculation of explants of other plants with healthy and tender petioles, and can be referred by technical personnel in the field according to actual conditions. It should be noted that the sterilization and inoculation steps in the clean bench of the present invention should be performed strictly aseptically, and also strictly when researchers try to use this method in tissue culture of other plant petioles. The invention is helpful to take the method of washing before disinfection and using mercuric chloride or sodium hypochlorite to disinfect the explant, so that the method for disinfecting the explant is simpler, saves water, labor and reagent, saves cost, has high quality and high efficiency, and is safe and environment-friendly.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (9)
1. An inoculation method for tissue culture and rapid propagation of raspberry petioles is characterized by comprising the following specific steps:
(1) cutting off the upper petiole of the stem of the raspberry;
(2) disinfecting the petioles by using alcohol with the mass concentration of 75% in a superclean workbench, and rinsing the petioles by using sterile water;
(3) the sterilized and rinsed petioles with sterile water are inoculated into a culture medium for dark culture.
2. The inoculation method for tissue culture and rapid propagation of raspberry petioles according to claim 1, wherein the petioles in step (1) are healthy and tender leaves 0.5-3.0 cm from the stem tip.
3. The inoculation method for tissue culture and rapid propagation of the raspberry petiole according to claim 1, wherein the thickness of the petiole in the step (1) is 1.5-4.0 mm, and the petiole is cut into small sections with the length of 0.5-2.0 cm.
4. The inoculation method for tissue culture and rapid propagation of raspberry petioles according to claim 1, wherein the specific operation of the step (2) is as follows: and (3) at an inlet of a super clean bench, filling the petioles into a bottle which is arranged in the super clean bench and is subjected to high-pressure sterilization, then sealing and disinfecting the bottle for 50-70 s by using an alcohol solution with the mass concentration of 75%, and rinsing the bottle by using sterile water.
5. The inoculation method for tissue culture and rapid propagation of raspberry petioles according to claim 1, wherein the rinsing with sterile water in step (2) is 3-5 times.
6. The inoculation method for tissue culture and rapid propagation of raspberry petioles according to claim 1, wherein the culture medium in step (3) is MS culture medium added with the following components by mass concentration: 0.1-0.5 mg/L of 6-BA-1、2,4-D 0.5～1.5mg·L-1、NAA 0.2～0.8mg·L-120-30 g.L of sucrose-1And agar 5.5-6.0 g.L-1And the pH value is 5.8-6.2.
7. The inoculation method for tissue culture and rapid propagation of raspberry petiole according to claim 1, wherein the culture temperature of dark culture in step (3) is 25 ± 2 ℃.
8. The inoculation method for tissue culture and rapid propagation of raspberry petioles according to claim 1, wherein during the culture period, the contamination rate, browning rate, enlargement rate and callus induction rate of the petioles are counted, and the growth conditions of the petioles and the callus thereof are observed.
9. The inoculation method for tissue culture and rapid propagation of raspberry petioles according to claim 8, wherein the calculation formulas of the contamination rate, browning rate, enlargement rate and callus induction rate are as follows:
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