CN108157180B - Open type factory rapid propagation method for potato virus-free seedlings - Google Patents
Open type factory rapid propagation method for potato virus-free seedlings Download PDFInfo
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- CN108157180B CN108157180B CN201810062415.XA CN201810062415A CN108157180B CN 108157180 B CN108157180 B CN 108157180B CN 201810062415 A CN201810062415 A CN 201810062415A CN 108157180 B CN108157180 B CN 108157180B
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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
- A01H4/001—Culture apparatus for tissue culture
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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
Abstract
The invention discloses an open type factory rapid propagation method of potato virus-free seedlings. The invention does not need a culture medium, and abandons the process of high-pressure sterilization culture medium; no bacteriostatic agent is needed, so that the influence of the bacteriostatic agent on the growth of the virus-free seedlings is avoided; directly propagating the virus-free tissue culture seedlings in an open and bacteria-free environment without an ultra-clean workbench; the speed of shearing stem sections in the average-per-person rapid propagation is more than 10 times that of the traditional mode; the propagation coefficient is 3-4 times; the propagation time is shortened to 14-15 d; culturing in an open and bacteria-bearing greenhouse, so that the potato virus-free seedlings grow strongly; the survival rate of the detoxified seedlings after entering the original seed production can reach 100 percent, the rapid industrial production cost is 1/6-1/7 of the traditional production cost, and the number of the potatoes is obviously higher than that of the detoxified seedlings in the traditional meaning. The method provided by the invention provides an important technical support for effectively reducing the factory rapid propagation production cost of the potato virus-free seedlings, improving the propagation efficiency, producing strong and high-quality virus-free seedlings and planting and producing the original seeds, and producing the original seeds through aeroponics or soilless culture.
Description
Technical Field
The invention belongs to the technical field of rapid propagation of potato virus-free seedlings, and particularly relates to an open type factory rapid propagation method of potato virus-free seedlings.
Background
Potatoes, native to south america peru, are later cultivated in large numbers around the world. The potatoes are introduced into China at the beginning of Ming, Finisheng and Qing, and the cultivation history of the potatoes is nearly 400 years in China. The potato is a globally important crop used as both grain and vegetable, and has the advantages of short growth period, wide adaptability, high yield, wide application, rich nutrition, and storage and transportation resistance. However, due to virus invasion, the potato is generally degraded in the cultivation process, so that the quality and the yield of the potato are linearly reduced. The virus harming the potatoes is very much, the potatoes are asexual propagation crops, the virus is accumulated in potato tubers and handed over by generations, the harm is serious year by year, and the conventional propagation mode has no effect on the virus. In 1955, French plant tissue culture researchers obtained virus-free plants from diseased potato plants by means of stem tip tissue culture, and opened up a brand new field for plant detoxification. After the 60 s in the 20 th century, the development of plant tissue culture technology is rapid, the research of plant detoxification technology is greatly developed, and the excellent seed potatoes are produced by producing the detoxified seedlings through stem tip detoxification, subculturing and expanding propagation and establishing an excellent seed breeding system, which is the main breeding technology for potato production at present. The conventional quick propagation technology of potato virus-free seedlings mainly comprises the following steps: a test-tube seedling (tissue culture seedling) cutting and rapid propagation technology, a cutting seedling rapid propagation technology and a detoxified seed potato bud breaking and rapid propagation technology. The cutting and rapid propagation technology of the virus-free test-tube seedling (tissue culture seedling) of the potato is an important key technology in a virus-free seed potato propagation technology system of the potato and is widely applied to production. However, the rapid propagation technology of potato virus-free seedlings has many problems in the technical link developed to the present. Among them, the problem of eubacterial pollution in tissue culture production is the first difficult problem to solve. The traditional test-tube plantlet (tissue culture plantlet) cutting and rapid propagation modes are all carried out under strict aseptic closed conditions, and the environmental requirements are strict and harsh. The culture medium and culture vessels are sterilized at high temperature in a sterile environment, any vessel used for handling and processing test-tube plantlets (tissue culture plantlets) needs to be sterilized, and the handling process needs to be carried out in a sterile environment such as an ultra-clean workbench to reduce the risk of contamination by eubacteria.
In recent years, a new method of open tissue culture is developed in potato tissue culture production, but the method is not popularized in production, because the open tissue culture needs to add a certain amount of bacteriostatic agent into a culture medium, the culture medium is not well solidified, and the potato tissue culture stem section is difficult to root and is easy to have weak seedlings and albino seedlings. The inhibition effect on some bacteria is not ideal. If a rapid propagation culture technology of virus-free seedlings without sterilization can be established and antibacterial agents are not used, the pollution rate can be reduced to 0, and the cutting and rapid propagation technology of the virus-free potato seedlings can be qualitatively leaped. Therefore, it is very important to invent an open virus-free seedling rapid propagation technology without a sterilization procedure.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide an open type factory rapid propagation method of potato virus-free seedlings.
Aiming at the problems that the environment requirement of a tissue culture room is strict, the tissue culture room needs to be carried out under strict aseptic conditions, the operation procedure is complicated, the efficiency is low, the culture cost is high, the transplanting survival rate is low, the quantity of the potatoes grown in a cutting process is small and the like in the current potato virus-free seedling cutting culture seedling process, systematic research is carried out on the rapid propagation technology of the potato virus-free seedlings, the novel method for industrially and rapidly propagating the open potato virus-free seedlings is invented, the rapid propagation of the potato virus-free seedlings is separated from the strict aseptic operation environment, the operation is carried out in the natural and open aseptic environment, the rapid propagation operation procedure is simplified, the propagation speed is increased by more than 10 times compared with that of the traditional method, the quality and the survival rate of the potato virus-free seedlings are greatly improved, and the production cost is 1/6-1/7 of the traditional method.
It is another object of the present invention to provide the use of the above method.
The purpose of the invention is realized by the following technical scheme:
an open type factory rapid propagation method of potato virus-free seedlings comprises the following steps:
(1) preparation of culture carrier: culturing the detoxified seedling carrier as inorganic vermiculite without carbon source;
(2) uniformly paving the vermiculite obtained in the step (1) on an off-ground seedbed of a greenhouse;
(3) spraying water on the seedbed in the step (2) until vermiculite is in a saturated water state;
(4) taking potato virus-free seedlings with the height of 10-14 cm, removing a culture medium, removing roots and clustering;
(5) directly shearing the cluster potato virus-free seedlings obtained in the step (4) into 1.0-3.0 cm long stem sections with axillary buds by using sterilized medical scissors in an open indoor environment;
(6) putting the stem sections obtained in the step (5) into a sterilized clean and dry container;
(7) uniformly spreading the stem sections contained in the container in the step (6) on vermiculite;
(8) spraying 100-200 ppm indolebutyric acid to the stem segments of the virus-free seedlings in the step (7) for 1 time;
(9) spraying water to the stem segments of the detoxified seedlings in the step (8) for 1 time every 60-120 min;
(10) culturing the potato virus-free seedlings in the step (9) for 5-7 d, observing rooting and raising heads, scattering a small amount of vermiculite on rooted stems, and continuing culturing;
(11) and (3) culturing the potato virus-free seedlings in the step (10) for about 10-15 days until the potato virus-free seedlings grow to 8-10 cm and 3-5 leaves, and then pulling up the potato virus-free seedlings with roots and transferring the potato virus-free seedlings into a miniature potato production field to produce miniature potatoes.
In order to better achieve the aim of the invention, the method further comprises the following steps:
preferably, the vermiculite in the step (1) is fresh vermiculite, namely, unused vermiculite formed by high-temperature firing.
Preferably, the thickness of the vermiculite on the above-ground seedbed in the step (2) is 5-8 cm.
Preferably, the seedbed in the step (2) is isolated from the ground; the seedbed is a culture bed of an iron frame, a plastic net frame or a seedbed made of asbestos tiles.
Preferably, the temperature of the greenhouse in the step (2) is kept at 18-25 ℃, the greenhouse is illuminated by sunlight all day long, and the greenhouse is clean and sanitary;
preferably, the potato virus-free seedlings in the step (4) are cultured in a tissue culture room for 20-25 days, and the height of the seedlings is 10-14 cm;
preferably, after the virus-free seedlings in the step (4) are taken out of the culture bottle, the culture medium at the root is removed, the roots are removed, and then the virus-free seedlings are clustered;
preferably, the sterilization in the step (6) is to wipe and clean the inner surface of the dried container by using 75% alcohol;
preferably, the container in step (6) is a vessel such as a tray, a basin, a box and the like;
the container is made of glass, ceramics, plastics, metal and other materials;
preferably, the density of the stem segments uniformly spread on the vermiculite in the step (7) is 5000-10000 stem segments per square meter.
Preferably, the indoor humidity of the stem section of the detoxified seedling in the rooting stage in the step (9) is kept at 80-90%, and preferably, water drops are visible on the stem section by naked eyes.
Preferably, the water is sprayed for 1 time in the step (9), and each time lasts for 5-15 min.
Preferably, in the rooting stage of the stem segments of the virus-free seedlings in the step (10), the indoor environment should be shaded, a shading net is 60-80 meshes, and the illumination intensity in the daytime is kept at 200-1000 LX.
Preferably, the culture humidity is kept at 80-90% in the rooting stage of the virus-free seedling culture in the step (10).
Preferably, the nutrient solution is sprayed for 1 time every 3-5 days during the culture of the detoxified seedlings in the step (11).
Preferably, the nutrient solution is MS culture solution without organic components, sucrose and inositol.
Preferably, the culture stage of the detoxified seedlings in the step (11) is full sunlight.
The open type factory rapid propagation method of the potato virus-free seedlings can be used for large-scale production and providing the potato virus-free seedling culture of facility planting production stock seeds, aeroponics or soilless culture production stock seeds.
The mechanism of the invention is as follows:
the characteristic that true bacteria of potato pathogens cannot survive in an inorganic environment is utilized, and the problem of rapid propagation of virus-free seedling tissues polluted by the true bacteria is solved. The invention screens and determines the open type virus-free seedling rapid propagation technology which is simple and easy to be operated in factory through systematic research. The method provides an important technical support for effectively reducing the factory rapid propagation production cost of the potato virus-free seedlings, improving the propagation efficiency and producing strong and high-quality virus-free seedlings to plant and produce the original seeds, and for producing the original seeds by aeroponics or soilless culture.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention gets rid of the culture container and adopts the off-ground seedbed as the culture container; a culture medium is not needed, and the process of high-pressure sterilization of the culture medium is abandoned; no bacteriostatic agent is needed, so that the influence of the bacteriostatic agent on the growth of the virus-free seedlings is avoided; directly propagating the virus-free tissue culture seedlings in an open and bacteria-free environment without an ultra-clean workbench; the speed of shearing stem sections in the average-per-person rapid propagation is more than 10 times that of the traditional mode; the propagation coefficient is 3-4 times; the propagation time is shortened to 14-15 d; culturing in an open and bacteria-bearing greenhouse, so that the potato virus-free seedlings grow strongly; the survival rate of the detoxified seedlings after entering the original seed (miniature potato) production can reach 100 percent, which is obviously higher than that of the traditional mode, the rapid industrial production cost is 1/6-1/7 of the traditional mode, and the number of the potatoes is obviously higher than that of the detoxified seedlings in the traditional sense.
(2) The invention has no high requirements on workers and operating environment, and does not need a tissue culture room with higher investment and construction cost.
(3) The cultivated detoxified seedlings have developed roots, roots are not easy to damage during transplanting, and transplanting survival rate is high.
(4) The cost of the potato virus-free seedlings produced by the method is 0.031 yuan per plant, which is 1/7 of the rapid propagation production cost of the traditional virus-free seedlings, and the manpower cost, the production data cost and the electric power cost of the rapid propagation of the potato virus-free seedlings are greatly saved.
(5) The invention makes the quick propagation of the potato virus-free seedlings simple and easy to operate.
(6) The method of the invention is to directly and rapidly propagate potato virus-free seedlings in open and bacteria-containing environment, the produced potato virus-free seedlings are strong and have roots, and can be directly transplanted into a greenhouse or a net shed to produce miniature potatoes, thus being an effective new method which can greatly improve the speed of factory rapid propagation of the potato virus-free seedlings, get rid of the pollution of the virus-free seedlings, improve the quality of the virus-free seedlings, increase the number of the miniature potatoes and greatly reduce the production cost of the virus-free seedlings.
Drawings
FIG. 1 is a process flow diagram of the present invention.
FIG. 2 is a schematic illustration of a tiled stem segment of the method of the invention.
FIG. 3 is a schematic diagram of stem segments of rooting heads in the method of the present invention.
FIG. 4 is a schematic representation of a potato virus-free seedling produced by the method of the present invention.
FIG. 5 is a schematic representation of potato stock produced by the method of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the experimental materials, reagents and the like used are commercially available unless otherwise specified.
The process flow diagram of the invention is shown in figure 1. The stem segments of the method of the invention are laid out schematically as shown in figure 2. The stem segment of the rooting new head of the method is shown in figure 3. The potato virus-free seedlings produced by the method of the invention are shown in figure 4. The potato breeder's seed produced by the method of the present invention is shown in figure 5.
Example 1
Material (one):
2 ten thousand Dutch fifteen-potato virus-free tissue culture seedlings are selected in 2015, 3 months and 24 days, and the height of each seedling is 10-14 cm; preparing a culture carrier, wherein the culture carrier is 20 bags of new vermiculite processed at high temperature; preparing a clean greenhouse 1, wherein the greenhouse is an intelligent greenhouse called Alternate cliff in inner Mongolia, the temperature of the intelligent greenhouse is 20-25 ℃ in the daytime, the temperature of the intelligent greenhouse is 18-22 ℃ at night, and the humidity of the intelligent greenhouse is 80-90%.
(II) method:
(1) a layer of gardening ground fabric is paved on the seedbed;
(2) and then, uniformly paving the culture carrier vermiculite on an off-ground seedbed of a greenhouse, wherein the thickness is 5-8 cm.
(3) Spraying water to the culture carrier vermiculite to form a saturated water state, and pressing with fingers to form water, preferably, the culture carrier vermiculite does not form water with naked eyes.
(4) Taking potato tissue culture virus-free seedlings with the height of 10-14 cm, removing a culture medium, removing roots and clustering;
(5) in an open indoor environment for 3 months and 24 days, directly shearing the clustered potato virus-free seedlings into stem sections with axillary buds, wherein the length of each stem section is 1.0-3.0 cm, and 3-5 stem sections can be obtained from 1 plant of tissue culture seedlings generally;
(6) putting the cut stem segments into a clean and dry container;
(7) putting the container into a greenhouse, and uniformly scattering the stem sections on a culture carrier vermiculite;
(8) spraying 100-200 ppm indolebutyric acid to the stem segments of the detoxified seedlings for 1 time;
(9)3, 24 days after month, the stem sections of the detoxified seedlings are cultured in a greenhouse, water is sprayed for 1 time every 60-120 min in the daytime, 5-10 minutes every 1 time, water is not sprayed at night, dew is preferably on the stem sections visible to naked eyes, and the greenhouse is shaded by a 60-80-mesh shading net with 60 percent;
(10) culturing the stem sections of the virus-free potato seedlings for 5-7 days, after the rooting and raising are observed, scattering a small amount of vermiculite on the rooted stem sections, spraying water once every 2-3 hours for 5-10 minutes, not spraying water at night, removing a sunshade net, and continuously culturing by full-day illumination;
(11) after the stem sections of the detoxified seedlings take roots, spraying the nutrient solution for 1 time every 3-5 days;
(12)4, growing the stem of the potato virus-free seedling to 8-10 cm in 15 days after 4 months, pulling out the virus-free seedling with 3-5 leaves and transplanting the virus-free seedling with the root into a greenhouse for producing original seeds to produce the original seeds;
(14) starting to produce conventional original seeds in a greenhouse at 16 days 4 months, planting 8 million virus-free potato seedlings produced by the method with the planting density of 1.3 mu: 90 strains/m2(ii) a Simultaneously planting 2 ten thousand potatoes cultured by common tissue culture in a greenhouse, wherein the planting density is as follows: 90 strains/m2；
(15) Fifteen stock seeds of the Netherlands are harvested in 30 days after 6 months.
(III) investigation
The production cost of 8 million potato virus-free seedlings produced by the two methods is investigated in 3 months and 30 days, and the production cost comprises labor cost, production data cost, water and electricity cost, per-capita propagation speed and the like.
4, 20 days in 4 months, investigating the transplanting survival number, the root length and the root number of the seedlings cultured by the two methods;
the number of single-plant potatoes of the original seed and the average single-grain weight are investigated after 6 months and 30 days.
Randomly sampling the harvested miniature potato blocks in 9 months and 11 days, and detecting the carrying condition of potato block virus by using an RT-PCR method.
(IV) results
The results of investigating the production cost of 8 ten thousand virus-free potato seedlings within 3 months and 30 days are detailed in the following table:
the growth condition of the detoxified seedlings is investigated in 20 days after 4 months and is shown in the following table:
| survey item | Number of plants | Number of surviving (plant) | Survival rate (%) | Length of main root (cm) | Root number (strips/plants) |
| The method for producing virus-free seedlings | 2000 | 2000 | 100％ | 12.32 | 12.52 |
| Conventional production of virus-free seedlings | 2000 | 1573 | 78.65 | 6.142 | 6.35 |
The details of the potato bearing condition of the detoxified seedlings are investigated in 30 days after 6 months and are shown in the following table:
| survey item | Number of plants | Average number of single potato plants | Average number of potatoes (number/m)2) | Average single particle weight (g) |
| The method for producing virus-free seedlings | 200 | 6.35 | 571.5 | 9.25 |
| Conventional production of virus-free seedlings | 200 | 2.45 | 173.4 | 5.85 |
The results of detecting the virus carried by the miniature potato tuber produced by the method in 9 months and 11 days are detailed in the following table:
| viral species | PVY | PVX | PLRV | PVA | PVS | PVH | PSTVd |
| Whether or not to carry | — | — | — | — | — | — | — |
One is not carrying the virus; +. is carrying the virus)
Example 2
Preparation work:
preparing a clean greenhouse 1 in 2016, 4 months and 3 days, wherein the greenhouse is a thick-wall sunlight greenhouse in Biclicun of a huoerstone hutch and Haote; preparing 10 thousands of virus-free seedling tissue culture seedlings of the kanibek potatoes; preparing a culture carrier, wherein the culture carrier is 100 bags of new vermiculite processed at high temperature; during the culture period, the temperature of the greenhouse is kept at 21-25 ℃ in the daytime, 18-21 ℃ at night and the humidity is 80-90%.
(II) method:
(1) a layer of gardening ground fabric is paved on the off-ground seedbed;
(2) and then, uniformly paving the culture carrier vermiculite on an off-ground seedbed of a greenhouse, wherein the thickness is 5-8 cm.
(3) Spraying water to the culture carrier vermiculite to form a saturated water state, and pressing with fingers to form water, preferably, the culture carrier vermiculite does not form water with naked eyes.
(4) Taking the well-cultured potato tissue culture virus-free seedlings, removing the culture medium, removing the roots and clustering;
(5)4, in a clean and tidy room for 3 days after 4 months, wiping scissors with a 75% alcohol cotton ball, and directly cutting the clustered potato virus-free seedlings into 1.0-3.0 cm long stem sections with axillary buds, wherein 3-5 stem sections can be obtained from each plant;
(6) wiping the container with 75% alcohol cotton ball, air drying, and placing the cut stem into a dry container;
(7) putting the container into a greenhouse, and uniformly scattering the stem sections on a culture carrier vermiculite;
(8) spraying 100-200 ppm indolebutyric acid to the stem segments of the detoxified seedlings for 1 time;
(9)4, 3 days after 4 months, the stem sections of the detoxified seedlings are cultured in a greenhouse, water is sprayed for 1 time every 60-120 min in the daytime, 5-10 minutes are sprayed for 1 time, water is not sprayed at night, dew is preferably on the stem sections visible to naked eyes, and the greenhouse is shaded by a 60-80-mesh shading net with 60 percent;
(10) and 4, month and 11 days, culturing the stem segments of the potato virus-free seedlings for 8 days, observing rooting and raising heads, and removing the sunshade net. Spreading a small amount of vermiculite on the rooted stems, 8: spraying water once every 2-3h after 00 hours, and continuously culturing by illumination all day for 5-10 minutes;
(11) after the stem segments of the virus-free seedlings take roots in 4 months and 15 days, spraying the nutrient solution for 1 time every 4 days;
(12)4, growing the stem of the potato virus-free seedling to 8-10 cm in 25 days after 4 months, pulling out the virus-free seedling with 3-5 leaves and transplanting the virus-free seedling with the root into a greenhouse for producing original seeds to produce the original seeds;
(13) starting to produce conventional stock seeds in a greenhouse at 4 months and 15 days, planting 40 million virus-free potato seedlings produced by the method in 10 greenhouses, wherein the planting density is as follows: 90 strains/m2(ii) a Simultaneously, 2 ten thousand of potato tissue culture seedlings produced by common tissue culture are planted in 1 greenhouse, and the planting density is as follows: 90 strains/m2；
(14) And 7, harvesting the original Connibeck seeds on 8 days in 7 months.
(III) investigation
The production cost of 40 million virus-free potato seedlings produced by the two methods is investigated at 4 months and 15 days, and the production cost comprises labor cost, production data cost, water and electricity cost, per-capita propagation speed and the like.
4, surveying the transplanting survival number, the root length and the root number of the seedlings cultured by the two methods within 30 days of 4 months;
the number of single-plant tubers and the average single-grain weight of the original seed were investigated at harvest time of 7 months and 8 days.
Randomly sampling the harvested miniature potato blocks in 10 months and 21 days, and detecting the virus carrying condition of the potato blocks by adopting an RT-PCR method.
(IV) results
The production cost results of 40 ten thousand virus potato virus-free seedlings investigated in 2016 (4 months and 15 days) are detailed in the following table:
the growth of the detoxified seedlings is investigated in 2016, 4, 30 days and is shown in the following table:
| survey item | Number of plants | Number of surviving (plant) | Survival rate (%) | Length of main root (cm) | Root number (strips/plants) |
| The method for producing virus-free seedlings | 2000 | 2000 | 100％ | 11.75 | 11.65 |
| Conventional production of virus-free seedlings | 2000 | 1649 | 82.45 | 5.85 | 7.50 |
The details of the potato bearing condition of the detoxified seedlings in 2016, 7, 8 days are shown in the following table:
| survey item | Number of plants | Average number of single potato plants | Average number of potatoes (number/m)2) | Average single particle weight (g) |
| The method for producing virus-free seedlings | 200 | 7.55 | 679.50 | 8.65 |
| Conventional production of virus-free seedlings | 200 | 3.70 | 274.55 | 6.03 |
The method for detecting the virus carrying results of the miniature potato tuber produced in 2016, 10, 21 and is detailed in the following table:
| viral species | PVY | PVX | PLRV | PVA | PVS | PVH | PSTVd |
| Whether or not to carry | — | — | — | — | — | — | — |
One is not carrying the virus; +. is carrying the virus)
Example 3
Preparation work:
preparing a clean greenhouse 1 in 3 months in 2017, wherein the greenhouse is a non-wave sunlight greenhouse called inner Mongolia and having a great fresh water river and a great river town; preparing an off-ground seedbed; preparing 20 ten thousand of detoxified seedling tissue culture seedlings of the summer cliff potatoes; preparing a culture carrier, wherein the carrier is 200 bags of new vermiculite processed at high temperature; during the culture period, the temperature of the greenhouse is kept at 21-25 ℃ in the daytime, 18-21 ℃ at night and the humidity is 80-90%.
(II) method:
(1) a layer of gardening ground fabric is paved on the off-ground seedbed;
(2) and then, uniformly paving the culture carrier vermiculite on an off-ground seedbed of a greenhouse, wherein the thickness is 5-8 cm.
(3) Spraying water to the culture carrier vermiculite to form a saturated water state, pressing the culture carrier vermiculite with a finger to form water, and keeping the culture carrier vermiculite from being soaked with water to the naked eye.
(4) Taking the well-cultured potato tissue culture virus-free seedlings, removing the culture medium, removing the roots and clustering;
(5)3 months and 22 days in a clean and tidy room, wiping scissors with a 75% alcohol cotton ball, and then directly cutting the clustered potato virus-free seedlings into 1.0-3.0 cm long stem sections with axillary buds, wherein 3-5 stem sections can be obtained from each plant;
(6) wiping the container with 75% alcohol cotton ball, air drying, and placing the cut stem into a dry container;
(7) putting the container into a greenhouse, and uniformly scattering the stem sections on a culture carrier vermiculite;
(8) spraying 100-200 ppm indolebutyric acid to the stem segments of the detoxified seedlings for 1 time;
(9)3, 23 days after 3 months, the stem segments of the detoxified seedlings are cultured in a greenhouse, water is sprayed for 1 time and 10 minutes every 120min at the speed of 8: 00-17: 30, water is not sprayed at night, dew on the stem segments visible to naked eyes is taken as a standard, and the greenhouse is shaded by adopting a 60-80-mesh shading net with the concentration of 60%;
(10)4, 1 day after 4 months, and after the rooting and raising heads, the sunshade net is removed. Spreading a small amount of vermiculite on the rooted stems, 8: spraying water once every 2-3h after 00 hours for 15 minutes, and continuously culturing by illumination all day long;
(11) after the stem segments of the virus-free seedlings take roots in 4 months and 3 days, spraying the nutrient solution for 1 time every 4 days;
(12)4, growing the stem of the potato virus-free seedling to 8-10 cm in 13 days after 4 months, pulling out the virus-free seedling with 3-5 leaves and transplanting the virus-free seedling with the root into a greenhouse for producing the original stock seed to produce the original stock seed;
(13) starting to produce conventional stock seeds in a greenhouse at 14 days 4 months, planting 80 million virus-free potato seedlings produced by the method in 20 greenhouses, wherein the planting density is as follows: 90 strains/m2(ii) a Simultaneously, 2 ten thousand of potato tissue culture seedlings produced by common tissue culture are planted in 1 greenhouse, and the planting density is as follows: 90 strains/m2；
(14) And (5) harvesting the summer clitora original seeds from 7 and 2 days.
(III) investigation
The production cost of 80 million virus-free potato seedlings produced by the two methods is investigated at 4 months and 13 days, and the production cost comprises labor cost, production data cost, water and electricity cost, per-capita propagation speed and the like.
4, 23 days in 4 months, and the transplanting survival number, the root length and the root number of the two methods for seedling culture are investigated;
the number of single-plant tubers and the average single-grain weight of the original seed were investigated at harvest time of 7 months and 2 days.
Randomly sampling the harvested miniature potato blocks in 23 days in 9 months, and detecting the carrying condition of potato block virus diseases by adopting an RT-PCR method.
(IV) results
The production cost results of 80 ten thousand virus potato virus-free seedlings investigated in 2017, 4 months and 13 days are detailed in the following table:
the growth condition of the detoxified seedlings is investigated in 2017, 4, 23 and the following table:
| survey item | Number of plants | Number of surviving (plant) | Survival rate (%) | Length of main root (cm) | Root number (strips/plants) |
| The method for producing virus-free seedlings | 2000 | 2000 | 100％ | 5.85 | 6.55 |
| Conventional production of virus-free seedlings | 2000 | 1588 | 79.4 | 1.35 | 2.50 |
The details of the potato bearing condition of the detoxified seedlings are investigated in 2017, 7, month and 2 and are shown in the following table:
| survey item | Number of plants | Average number of single potato plants | Average number of potatoes (number/m)2) | Average single particle weight (g) |
| The method for producing virus-free seedlings | 200 | 6.30 | 567.00 | 9.35 |
| Conventional production of virus-free seedlings | 200 | 2.55 | 182.22 | 6.35 |
The results of detecting the miniature potato block virus disease carried by the method in 2017, 9 and 23 days are shown in the following table:
| viral species | PVY | PVX | PLRV | PVA | PVS | PVH | PSTVd |
| Whether or not to carry | — | — | — | — | — | — | — |
One is not carrying the virus; +. is carrying the virus)
According to the results of the embodiments 1 to 3, the method of the invention can completely separate the potato tissue culture seedling detoxification and rapid propagation process from strict sterile operation environment without culture medium under the inorganic environment condition which is not beneficial to the existence of pathogenic bacteria, fungi and viruses of potatoes, especially, the method can completely carry out factory rapid propagation of potato detoxification seedlings in an open bacteria-carrying environment without preparing culture medium and adding medicament to the culture medium to inhibit the growth of true bacteria without high-pressure sterilization, thereby completely simplifying the cultivation link of the potato detoxification seedlings, improving the seedling cultivation efficiency, completely solving the problem of true bacteria pollution, greatly reducing the tissue culture seedling cultivation production cost of the potato detoxification seedlings and the construction cost of the tissue culture workshop of the tissue culture seedling cultivation workshop, simultaneously cultivating the potato detoxification seedlings with the survival rate of 100 percent, greatly improving the number of the grown potatoes in the minitype potato production link, generally, the yield of the miniature potatoes is 4-18 per plant, which is greatly higher than that of the miniature potatoes produced by common tissue culture seedlings and cutting seedlings.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. An open type factory rapid propagation method of potato virus-free seedlings is characterized by comprising the following steps:
(1) preparation of culture carrier: the culture carrier is inorganic vermiculite without carbon source;
(2) uniformly paving the vermiculite obtained in the step (1) on an off-ground seedbed of a greenhouse;
(3) spraying water on the seedbed in the step (2) until vermiculite is in a saturated water state;
(4) taking potato virus-free seedlings with the height of 10-14 cm, removing a culture medium, removing roots and clustering;
(5) directly clustering and shearing the virus-free clustered potato seedlings obtained in the step (4) into 1.0-3.0 cm long stem sections with axillary buds by using sterilized medical scissors in an open indoor environment;
(6) putting the stem sections obtained in the step (5) into a sterilized clean and dry container;
(7) uniformly spreading the stem sections contained in the container in the step (6) on vermiculite; the stem sections are uniformly scattered on the vermiculite, and the density of the stem sections is 5000-10000 per square meter;
(8) spraying 100-200 ppm indolebutyric acid to the stem segments of the virus-free seedlings in the step (7) for 1 time;
(9) spraying water to the stem segments of the detoxified seedlings in the step (8) for 1 time every 60-120 min;
(10) culturing the stem of the detoxified seedling in the step (9) for 5-7 d, observing rooting and raising heads, scattering a small amount of vermiculite on the rooted stem, and continuing culturing; in the rooting stage of the stem segments of the detoxified seedlings, the indoor sun is shaded, a shading net is 60-80 meshes, and the illumination intensity is kept at 200-1000 LX in the daytime;
(11) and (3) carrying out full-day illumination culture on the potato virus-free seedlings in the step (10) for 10-15 days until the potato virus-free seedlings grow to 8-10 cm and 3-5 leaves, pulling out the potato virus-free seedlings with roots, and transplanting the potato virus-free seedlings into a production field for producing miniature potatoes.
2. The open-type factory rapid propagation method of potato virus-free seedlings according to claim 1, characterized in that: the vermiculite in the step (1) is new vermiculite.
3. The open-type factory rapid propagation method of potato virus-free seedlings according to claim 1, characterized in that: and (3) the thickness of the vermiculite on the off-ground seedbed in the step (2) is 5-8 cm.
4. The open-type factory rapid propagation method of potato virus-free seedlings according to claim 1, characterized in that: isolating the seedbed in the step (2) from the land; the seedbed is a culture bed of an iron frame, a plastic net frame or a seedbed made of asbestos tiles.
5. The open-type factory rapid propagation method of potato virus-free seedlings according to claim 1, characterized in that: and (3) keeping the temperature of the greenhouse in the step (2) at 18-25 ℃, illuminating the greenhouse by full sunlight, and cleaning and sanitation in the greenhouse.
6. The open-type factory rapid propagation method of potato virus-free seedlings according to claim 1, characterized in that:
the sterilization in the step (6) is to wipe and clean the inner surface of the dried container by using 75% alcohol;
the container in the step (6) is a tray, a basin or a box;
the container is made of glass, ceramics, plastics and metal materials.
7. The open-type factory rapid propagation method of potato virus-free seedlings according to claim 1, characterized in that:
keeping the indoor humidity at 80-90% in the rooting stage of the stem section of the virus-free seedling in the step (9), and preferably, water drops are visible on the stem section by naked eyes;
and (4) spraying water for 1 time, and each time lasts for 5-15 min.
8. The open-type factory rapid propagation method of potato virus-free seedlings according to claim 1, characterized in that:
and (5) maintaining the culture humidity of the rooting stage of culturing the virus-free seedlings in the step (10) at 80-90%.
9. The open-type factory rapid propagation method of potato virus-free seedlings according to claim 1, characterized in that: spraying the nutrient solution for 1 time every 3-5 days during the culture of the virus-free seedlings in the step (11);
the nutrient solution is MS culture solution without organic components and sucrose.
10. The application of the open type factory rapid propagation method of potato virus-free seedlings as claimed in any one of claims 1 to 9, is characterized in that: the method can be used for large-scale production, and provides the potato virus-free seedlings of facility planting production stock seeds and soilless culture production stock seeds.
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| CN108967193A (en) * | 2018-08-07 | 2018-12-11 | 遵义华富生物科技有限公司 | A kind of Potato Shoot-tips removing fast breeding technique method |
| CN109042198A (en) * | 2018-10-16 | 2018-12-21 | 云南省农业技术推广总站 | A kind of tissue culture seedlings of potatoes Field planting breeding method |
| CN109997641A (en) * | 2019-04-04 | 2019-07-12 | 新疆农业科学院综合试验场 | The method for culturing seedlings for preventing and treating potato soil-borne disease |
| CN110612907B (en) * | 2019-11-12 | 2021-01-19 | 甘肃农业大学 | Potato seed culture system of detoxication of potato |
| CN111528097B (en) * | 2020-06-09 | 2023-03-21 | 吉林省蔬菜花卉科学研究院 | Method for breeding potato mini-potatoes by utilizing stem segments of potato stolons |
| CN111602595A (en) * | 2020-06-28 | 2020-09-01 | 甘肃省农业科学院马铃薯研究所 | Open type rapid propagation method for potato tissue culture seedlings |
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| CN1048141A (en) * | 1990-08-06 | 1991-01-02 | 天津市蔬菜研究所 | Fast propagation technique for potato using depoisoned small seed-potato |
| CN1582624A (en) * | 2004-06-16 | 2005-02-23 | 宜昌市高农科技有限公司 | Quick and open liquid cultivation method and apparatus for detoxicated potato seedlings and miniature potatoes |
| CN101999308A (en) * | 2010-10-27 | 2011-04-06 | 何建栋 | Efficient fast-propagation technology for framework disc type tissue culture seedlings of potato |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1048141A (en) * | 1990-08-06 | 1991-01-02 | 天津市蔬菜研究所 | Fast propagation technique for potato using depoisoned small seed-potato |
| CN1582624A (en) * | 2004-06-16 | 2005-02-23 | 宜昌市高农科技有限公司 | Quick and open liquid cultivation method and apparatus for detoxicated potato seedlings and miniature potatoes |
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