CN114073226B - Inoculation method for tissue culture and rapid propagation of raspberry leaf stalks - Google Patents
Inoculation method for tissue culture and rapid propagation of raspberry leaf stalks Download PDFInfo
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- 240000007651 Rubus glaucus Species 0.000 title claims abstract description 44
- 238000011081 inoculation Methods 0.000 title claims abstract description 44
- 235000011034 Rubus glaucus Nutrition 0.000 title claims abstract description 35
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- 238000000034 method Methods 0.000 title claims abstract description 33
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- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 31
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 20
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
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- 238000004659 sterilization and disinfection Methods 0.000 abstract description 27
- 239000005708 Sodium hypochlorite Substances 0.000 abstract description 13
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 abstract description 13
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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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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a tissue culture and rapid propagation inoculation method of raspberry leaf stalks, and belongs to the technical field of plant tissue culture. Cutting off leaf stalks of healthy tender leaves on raspberry stems, sterilizing the healthy tender leaves in an ultra-clean workbench by using alcohol with the mass concentration of 75%, rinsing the healthy tender leaves by using sterile water, inoculating the healthy tender leaves into a culture medium for dark culture, wherein the 30d pollution rate of the inoculated raspberry stems is 5%, the browning rate and the browning rate are 0, the swelling rate and the callus induction rate are 95%, and the leaf stalks and the callus grow well. Pollution is reduced by taking the petioles as explants and not washing before disinfection; the disinfection with 75% alcohol avoids the use of highly toxic mercury lift and sodium hypochlorite which is irritant, corrosive and highly oxidative; the method has the advantages of simple operation, time saving, labor saving, water saving, space saving, reagent saving, safety, environmental protection, improvement of inoculation quality and efficiency, laying a foundation for establishing a raspberry leaf stalk regeneration system, carrying out transient expression and identification of gene functions by using callus, and carrying out transgene and gene editing and cultivation of new germplasm, and is suitable for the field of scientific research and industrialized tissue culture.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture and rapid propagation inoculation method of raspberry leaf stalks.
Background
The plant tissue culture technology is widely applied to the fields of plant rapid propagation, production of secondary metabolites, germplasm preservation, genetic improvement and the like, and the plant tissue culture technology is particularly important in scientific research and production application. During tissue culture, explant inoculation is the first necessary and critical step in the process of plant entry into tissue culture from the natural environment. Tissue culture of many plants uses leaves as explants, and the inoculation process is usually water washing, sterilization with mercuric chloride solution or sodium hypochlorite solution, sterile water rinsing, sterilization with alcohol solution, sterile water rinsing, inoculation onto culture medium, and culture.
However, if leaves are used as explants for raspberry, the following problems exist according to the conventional inoculation method: (1) The veins on the raspberry leaves are rich, fine and compact, so that the surfaces of the tender leaves are uneven like skirt folds, are not easy to clean and are not easy to be disinfected by disinfectants, thereby causing pollution; (2) The raspberry leaves are thin, and the raspberry leaves are easy to break and have wounds when the raspberry leaves are washed by hands, so that the risk of pollution caused by external bacteria entering the raspberry leaves from the wounds is increased; (3) The raspberry leaves are thin and rich in phenolic substances, and are easy to brown after disinfection; (4) The raspberry leaves are thin, the phenomenon of close contact between the leaves can occur during disinfection, pollution is easy to cause, and meanwhile, the inoculation difficulty is increased; (5) Microscopic observation shows that the back of the raspberry leaf has abundant epidermis hair and interweaves to form a blanket shape, thereby increasing the disinfection difficulty; (6) The person skilled in the art generally reduces the subsequent contamination rate by flushing with detergent and running water for several hours before disinfection, which is water-consuming and time-consuming, and furthermore, since raspberry leaves are thin and have surface hairs interwoven together on the back, water-flushing has the risk of increasing contamination of the external bacteria from the wound into the leaves or into the surface hair area, which misleads the person to use antibiotics for bacteriostasis for contamination caused by endophytes. (7) Those skilled in the art generally need to use a strong alcohol solution plus a mercuric chloride solution or an alcohol solution plus a sodium hypochlorite solution to disinfect in order to achieve the purpose of thorough disinfection, however, mercuric chloride is extremely toxic to plants, animals and the environment, and sodium hypochlorite has a certain pungent smell, has strong oxidizing property and is easy to corrode leaves to brown and die. In summary, the traditional method for grafting raspberry leaves has the disadvantages of high operation difficulty, time and labor waste, space and water and reagent consumption, high cost, environmental pollution, safety risk to operators, easy initiation of leaf pollution or browning or death, and further material and time loss, so that the inoculation target cannot be efficiently and excellently finished, the follow-up links of tissue culture and rapid propagation are affected, scientific research and production planning are affected, and economic loss is caused.
The stems of raspberries are thicker than the leaves and are in a stick shape, and the stems are not close to each other like the leaves during disinfection, so that the raspberries are easy to operate; the petioles have no skirt pleat structure like tender leaves, have fewer surface fur and thorns than the leaves, are easier to fully contact with the disinfectant than the leaves, and are beneficial to disinfection.
Therefore, the method for inoculating the explant for tissue culture and rapid propagation of raspberries, which is simple to operate, safe, environment-friendly, high-quality and efficient, is provided, and is a problem to be solved by a person skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a tissue culture and rapid propagation inoculation method of raspberry leaf stalks, which is a method capable of effectively completing the disinfection and inoculation links of explants without inoculating leaves, washing before disinfection, disinfecting with mercuric chloride and sodium hypochlorite and sealing; the pollution rate of the petioles after inoculation is low, and the petioles normally expand to grow callus; the method achieves the purposes of saving time, labor, space, water, reagent and cost, being simple to operate, being safe and environment-friendly, high-quality and high-efficiency and completing the raspberry inoculation link, lays a solid material foundation for establishing a raspberry stem regeneration system, carrying out transient expression and identification gene function by using callus, transferring genes and culturing new germplasm by gene editing, and is suitable for the field of scientific research and industrialized tissue culture.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the inoculation method for tissue culture and rapid propagation of raspberry leaf stalks comprises the following specific steps:
(1) Cutting off leaf stalks on the stems of the raspberries;
(2) Sterilizing the petioles in an ultra-clean workbench by using alcohol with the mass concentration of 75%, and rinsing by using sterile water;
(3) The sterilized, sterile water rinsed petioles were inoculated into medium for dark culture.
In general, criteria for successful inoculation are seen in the cases of petiole contamination, browning, death, swelling and callus growth. First, the theoretical basis of the technical scheme of the invention is as follows: the interior of the healthy petiole without mechanical damage is bacteria-free, so that the surface of the petiole can be disinfected without pollution. Therefore, the technical scheme of the invention only needs to disinfect the petioles by using the alcohol solution with the mass concentration of 75%, and the disinfection method of adding the mercuric chloride solution or the sodium hypochlorite solution into the alcohol solution with higher strength is not needed as the traditional method, so that the time, the labor, the water, the reagent and the cost are saved, and the method is safe and environment-friendly. In addition, the invention directly takes the petioles as explants, skillfully avoids the factors that the surface of the tender leaves of raspberries is uneven, the surface of the tender leaves is thin, the surface is thin, the raspberries are easy to break and brown, the leaves are easy to be close to each other during disinfection, and the like, and is easy to cause pollution, the difficulty and the complexity of the petioles disinfection and inoculation are far lower than those of the leaves, the operation is simpler and more convenient, the inoculation effect is excellent, the time, the labor, the water, the reagent and the space are saved, and the cost is saved. Again, those skilled in the art generally employ water washes to reduce the subsequent contamination rate, and generally consider that the longer the rinse time, the lower the contamination rate after inoculation. The present invention considers that the use of water washing increases the risk of contamination by bringing outside bacteria into the interior of the explant and even misleading people to contamination by endophytes. The invention abandons the traditional method of sterilizing by adding alcohol solution with mercuric chloride or adding alcohol solution with sodium hypochlorite in the prior tissue culture technology and using water before sterilizing, breaks through the limitation of the traditional thought, only uses alcohol solution to seal and sterilize the petiole, and does not need water before sterilizing, thereby achieving better inoculation effect than the flow of adopting water washing, mercuric chloride or sodium hypochlorite solution sterilization, sterile water rinsing, alcohol sterilization, sterile water rinsing and inoculation.
Further, the petioles in the step (1) are petioles of healthy tender leaves, wherein the distance between the petioles and the stem tip is 0.5-3.0 cm.
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 described in the step (2) is as follows: at the inlet of the super clean bench, the petiole is put into a bottle which is in the super clean bench and is sterilized by high pressure, and then the bottle is sterilized by an alcohol solution with the mass concentration of 75% in a sealing way for 50-70 s, and the bottle is rinsed by sterile water.
Further, the step (2) of sterile water rinsing is to rinse 3-5 times with sterile water.
Further, the culture medium in the step (3) is prepared by adding the following components in mass concentration into an MS culture medium: 6-BA 0.1-0.5 mg.L -1 、2,4-D 0.5～1.5mg·L -1 、NAA 0.2～0.8mg·L -1 Sucrose 20-30 g.L -1 And agar 5.5-6.0g.L -1 The pH value is 5.8-6.2.
Further, the culturing temperature of the dark culture in the step (3) was 25.+ -. 2 ℃.
Further, during the culture period, the pollution rate, browning rate, brown death rate, swelling rate and callus induction rate of the petioles were counted, and the growth of the petioles and the callus thereof was observed.
Further, the pollution rate, browning rate, brown death rate, swelling rate and callus induction rate are calculated as follows:
compared with the prior art, the invention discloses an inoculation method for tissue culture and rapid propagation of raspberry leaf stalks, which has the following beneficial effects: (1) According to the invention, the tender leaf stems which are 0.5-3.0 cm away from the stem tip and have no mechanical damage and are healthy are used as explants, so that thin and uneven-surface leaves with a plurality of surface fur are replaced, and the disinfection difficulty of the explants is reduced; (2) The method omits the step of washing before disinfection, is simple to operate, saves time, labor and water, and avoids the false image of pollution caused by bringing external bacteria into the explant and even pollution caused by endophytes in tissue culture; (3) Based on the theory that no bacteria exist in the healthy petioles, the invention can finish the inoculation task by only sterilizing the surfaces of the petioles by using an alcohol solution with the mass concentration of 75%; (4) The alcohol solution with the mass concentration of 75% is used for closed disinfection, so that the use of highly toxic reagent mercury lift and the disinfection of sodium hypochlorite with irritation, corrosiveness and strong oxidization are avoided; (5) The method has the advantages of simple operation, time saving, labor saving, water saving, reagent saving, space saving, cost saving, safety, environmental protection, high quality and high efficiency, and is used for completing the inoculation link in the tissue culture rapid propagation technology of the raspberry leaf stalks, greatly reducing the technical difficulty and complexity, improving the inoculation quality and efficiency, laying a solid material foundation for establishing a raspberry leaf stalk regeneration system, carrying out transient expression and identification gene function by using callus, transferring genes and culturing new germplasm by gene editing, and being suitable for the field of scientific research and industrialized 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 that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a drawing showing the growth of the petiole of treatment 1 of example 1 of the present invention;
wherein A is the petiole before inoculation, B is the petiole after inoculation 0 d; c is a polluted petiole; d is an enlarged petiole; e is the petiole of the long callus; f is the petiole with more long calli;
FIG. 2 is a graph showing the pollution rate, browning rate, brown death rate, swelling rate and callus induction rate of the raspberry stem culture of example 1 of the present invention on days 3 to 30;
FIG. 3 is a graph showing the growth of the petioles of treatment 2 of comparative example 1 of the present invention;
wherein A is the petiole before inoculation, B is the petiole after inoculation 0 d; c is a polluted petiole; d is a brown petiole; e is a brown dead petiole; f is an enlarged petiole; g is the petiole of the long callus;
FIG. 4 is a graph showing the pollution rate, browning rate, brown death rate, swelling rate and callus induction rate of the raspberry stem of comparative example 1, treatment 2, cultured for 3 to 30 days;
FIG. 5 is a drawing showing the growth of the blade base of comparative example 2 treatment 3 of the present invention;
wherein A is inoculating 0d blade base; b is the brown leaf base; c is the base of the brown dead leaf; 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, brown death rate, swelling rate, callus induction rate of the raspberry leaves of comparative example 2, treatment 3, culture for 3 to 30 days of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
(1) Selecting a healthy tender leaf stalk of the Boerca raspberry with the thickness of 1.5-4.0 mm and no mechanical damage from the stem tip by 0.5-3.0 cm, and cutting the leaf stalk into small sections with the length of 0.5-2.0 cm.
(2) At the entrance of the super-clean bench, the petiole was put into an autoclaved bottle in which it was sterilized hermetically with an alcohol solution of 75% by mass concentration for 60s (treatment 1), and the shaking was reversed during this period, followed by rinsing 3 times with sterile water. 60 petioles were used.
(3) Inoculating sterilized and sterile water rinsed petiole into culture medium (the culture medium comprises MS culture medium containing 6-BA 0.2mg.L -1 、2,4-D 1.2mg·L -1 、NAA 0.4mg·L -1 Sucrose 30 g.L -1 And agar 5.5 g.L -1 ) Dark culture was performed at 25.+ -. 2 ℃.
Culturing 3d, 6d, 9d, 12d, 15d, 18d, 21d, 24d, 27d and 30d, counting pollution rate, browning rate, brown death rate, swelling rate and callus induction rate of petiole, and observing growth condition of petiole and callus. Wherein:
the results are shown in FIGS. 1 and 2. The results showed that the contamination rate of treatment 1 at day 6 of cultivation was 5.0% and did not increase thereafter; the browning and browning rates 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 leaf stalks which are not increased after that, namely the uncontaminated leaf stalks are expanded; the callus induction rate of treatment 1 at 24 days of culture was 95.0%, and after that, no increase was observed, i.e., the expanded petioles had grown callus and had good growth conditions. The inoculation method has 95.0% of petioles which are expanded after inoculation and grow callus, and can enter the links of subculture multiplication, petiole regeneration system establishment, gene function verification through transient expression, transgenic breeding, gene editing breeding and the like.
The invention effectively completes the inoculation link of the petioles in the tissue culture and rapid propagation of raspberries without inoculating leaves, washing with washing agent and running water and using mercuric chloride and sodium hypochlorite before disinfection, has simple operation, time saving, labor saving, water saving, reagent saving, space saving, cost saving, high efficiency, safety and environmental protection, ensures 95.0 percent of petioles to expand and grow callus, lays a foundation for subsequent subculture propagation of raspberries, establishment of a petiole regeneration system, verification of gene functions through transient expression technology, transgenic breeding, gene editing breeding and the like, and is suitable for scientific research and industrialized seedling cultivation.
Comparative example 1
(1) Selecting a healthy tender leaf stalk of the Boerca raspberry with the thickness of 1.5-4.0 mm and no mechanical damage near 0.5-3.0 cm from the stem tip, and cutting the leaf stalk into small sections with the length of 0.5-2.0 cm.
(2) Washing with flowing water for 2h, sucking the water on the surface of the petiole by filter paper, placing the petiole into an autoclave bottle in the super clean bench at the inlet of the super clean bench, sealing and sterilizing with sodium hypochlorite solution with the effective chlorine concentration of 1% for 60s, reversely shaking during the process, rinsing with sterile water for 3 times, sealing and sterilizing with alcohol solution with the mass concentration of 75% for 60s (treatment 2), reversely shaking during the process, and rinsing with sterile water for 3 times. 60 petioles were used.
The treatment method of step (3) is the same as that of example 1, i.e., treatment 1.
The results are shown in FIGS. 3 and 4. The results showed that the pollution rate of the petiole at the 3 rd day of cultivation was 1.7% in comparative example 1, treatment 2, and was not increased after that; treatment 2 showed a browning rate of 10.0% at day 6 of culture, after which it was not increased; treatment 2 had a brown death rate of 10.0% at 15 days of culture, after which no increase was observed; the expansion rate of the treatment 2 at the 18 th day of culture is 88.3%, and the leaf stalks which are not increased after that, namely, the leaf stalks which are not polluted and not brown dead are expanded; treatment 2 the callus induction rate was 88.3% at day 27 of culture, after which no increase was observed, i.e. the expanded petioles had grown callus and had good growth. By the inoculation method, 88.3% of petioles are expanded after inoculation, callus can grow into links such as subculture multiplication, petiole regeneration, transient expression verification gene function, transgenic breeding and gene editing, and the inoculation loss is larger than that of the treatment 1 in the embodiment 1.
Comparative example 2
(1) Selecting healthy tender leaves of Boerca raspberry with no mechanical damage and leaf area of 2-5 cm and near 0.5-3.0 cm from the stem tip 2 。
(2) Washing with flowing water for 2h, sucking water on the surface of the leaf with filter paper, taking 0.5-2 cm of the base of the leaf 2 At the entrance of the ultra-clean bench, the blade base is put into an autoclave bottle in the ultra-clean bench, sterilized by sealing with a sodium hypochlorite solution having an effective chlorine concentration of 1% for 60s, and then by shaking upside down, rinsing with sterile water for 3 times, and then by sealing with an alcohol solution having a mass concentration of 75% for 60s (treatment 3), and then by rinsing with sterile water for 3 times. 60 blades were used.
The treatment method of step (3) is the same as that of 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 cultivation, and was not increased thereafter; treatment 3 showed 13.3% browning at day 6 of culture, after which no increase was observed; treatment 3 had a brown death rate of 13.3% at day 21 of culture, after which no increase was observed; the expansion rate of the treatment 3 at the 18 th day of culture is 80.0%, and the expansion rate is not increased after that, namely, the uncontaminated and non-browned leaves are expanded; the callus induction rate of treatment 3 at day 30 of culture was 80.0%, i.e. the expanded leaves had grown callus and had good growth conditions. According to the inoculation method, 80.0% of leaves are expanded after inoculation, callus grows, the leaves can enter the links of subculture multiplication, establishment of a leaf regeneration system, verification of gene functions through transient expression, transgenic breeding, gene editing breeding and the like, and the inoculation loss is larger than that of the example 1, namely the treatment 1, and the comparative example 1, namely the treatment 2.
The technical scheme of the invention is also suitable for inoculation of other plant petioles with more leaf surface fur, fine veins, uneven leaf surfaces and thin leaf surfaces, avoids the trouble of washing the leaf before disinfection and pollution caused by the close approach of the leaf during disinfection, is also suitable for explant inoculation of other plants with healthy tender petioles, and can be referred by the person skilled in the art according to actual conditions. It should be noted that the sterilization and inoculation steps in the ultra-clean bench of the present invention should be strictly sterile, and that scientific researchers should also be strictly working when attempting to use this method in tissue culture of other plant petioles. The invention is helpful for washing before sterilization, and the method for sterilizing the explant by using mercuric chloride or sodium hypochlorite, so that the method for sterilizing the explant is simpler, saves water, labor and reagent, saves cost, is 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 (2)
1. The inoculation method for tissue culture and rapid propagation of raspberry leaf stalks is characterized by comprising the following specific steps:
(1) Cutting off leaf stalks on the stems of the raspberries;
the petioles are petioles of healthy tender leaves with a distance of 0.5-3.0 cm from the stem tip;
the thickness of the petiole is 1.5-4.0 mm, and the petiole is cut into small sections with the length of 0.5-2.0 cm;
(2) In an ultra-clean workbench, sterilizing the petioles by using alcohol with the mass concentration of 75%, and rinsing by using sterile water, wherein the specific operation is as follows: at the inlet of the super clean bench, the petiole is put into a bottle which is in the super clean bench and is sterilized under high pressure, and then the bottle is sterilized in a sealed way by alcohol solution with the mass concentration of 75% for 50-70 s, and the bottle is swayed upside down and rinsed by sterile water;
(3) Inoculating the sterilized and sterile water rinsed petioles into a culture medium for dark culture;
the culture medium is prepared by adding the following components in mass concentration into an MS culture medium: 6-BA 0.2 mg.L -1 、2,4-D 1.2mg·L -1 、NAA 0.4mg·L -1 Sucrose 20-30 g.L -1 And agar 5.5-6.0g.L -1 The pH value is 5.8-6.2;
during the culture period, counting the pollution rate, browning rate, brown death rate, swelling rate and callus induction rate of the petioles, and observing the growth condition of the petioles and the callus;
the pollution rate, the browning rate, the brown death rate, the swelling rate and the callus induction rate are calculated according to the following formulas:
the raspberry is a Boer raspberry;
and (3) rinsing with sterile water for 3-5 times.
2. The inoculation method for tissue culture and rapid propagation of raspberry leaf stalks according to claim 1, wherein the culture temperature of the dark culture in the step (3) is 25±2℃.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2080059C1 (en) * | 1994-02-17 | 1997-05-27 | Всероссийский селекционно-технологический институт садоводства и питомниководства | Method of plant preparing - raspberry regenerants in vitro |
| CN101946706A (en) * | 2010-08-24 | 2011-01-19 | 中国农业科学院生物技术研究所 | Method for regeneration system establishment of sweet sorghum young ear callus |
| CN102893862A (en) * | 2012-07-16 | 2013-01-30 | 中国热带农业科学院橡胶研究所 | Oil palm embryonic callus induction method |
| CN103238516A (en) * | 2012-02-02 | 2013-08-14 | 黄涛 | Factorial seedling culturing method of blueberries |
| CN107079816A (en) * | 2017-05-26 | 2017-08-22 | 广州甘蔗糖业研究所湛江甘蔗研究中心 | A kind of tissue culture and rapid propagation method of new variety of sugarcane Guangdong sugar 03 373 |
| CN111202005A (en) * | 2020-03-24 | 2020-05-29 | 广西壮族自治区农业科学院 | Sugarcane variety embryonic callus induction method |
| CN111631135A (en) * | 2020-07-02 | 2020-09-08 | 江苏农牧科技职业学院 | Blueberry seedling detoxification breeding method |
| RU2019143343A (en) * | 2019-12-24 | 2021-06-24 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Пущинский государственный естественно-научный институт" (ПущГЕНИ) | METHOD FOR IN VITRO REPRODUCTION OF STAMP VARIETIES OF RASPBERRY |
-
2021
- 2021-11-23 CN CN202111396046.6A patent/CN114073226B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2080059C1 (en) * | 1994-02-17 | 1997-05-27 | Всероссийский селекционно-технологический институт садоводства и питомниководства | Method of plant preparing - raspberry regenerants in vitro |
| CN101946706A (en) * | 2010-08-24 | 2011-01-19 | 中国农业科学院生物技术研究所 | Method for regeneration system establishment of sweet sorghum young ear callus |
| CN103238516A (en) * | 2012-02-02 | 2013-08-14 | 黄涛 | Factorial seedling culturing method of blueberries |
| CN102893862A (en) * | 2012-07-16 | 2013-01-30 | 中国热带农业科学院橡胶研究所 | Oil palm embryonic callus induction method |
| CN107079816A (en) * | 2017-05-26 | 2017-08-22 | 广州甘蔗糖业研究所湛江甘蔗研究中心 | A kind of tissue culture and rapid propagation method of new variety of sugarcane Guangdong sugar 03 373 |
| RU2019143343A (en) * | 2019-12-24 | 2021-06-24 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Пущинский государственный естественно-научный институт" (ПущГЕНИ) | METHOD FOR IN VITRO REPRODUCTION OF STAMP VARIETIES OF RASPBERRY |
| CN111202005A (en) * | 2020-03-24 | 2020-05-29 | 广西壮族自治区农业科学院 | Sugarcane variety embryonic callus induction method |
| CN111631135A (en) * | 2020-07-02 | 2020-09-08 | 江苏农牧科技职业学院 | Blueberry seedling detoxification breeding method |
Non-Patent Citations (3)
| Title |
|---|
| "Arapaho"黑莓叶片与叶柄愈伤组织的诱导;王鲁北等;《中国南方果树》;20110320;第40卷(第02期);第19-23页 * |
| 培养基成分对黑莓Kiowa叶片和叶柄愈伤组织诱导的影响;汤飞云等;《江苏农业科学》;20131025;第41卷(第10期);第42-44页 * |
| 植物生长调节物质及外植体部位对树莓愈伤组织诱导的影响试验初报;陆杨辉;《南方农业》;20180615;第12卷(第17期);第137-138、140页 * |
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