CN110402816B - System for increasing transplanting seedling emergence amount of test-tube plantlets of grapes and application method thereof - Google Patents

System for increasing transplanting seedling emergence amount of test-tube plantlets of grapes and application method thereof Download PDF

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CN110402816B
CN110402816B CN201910678009.0A CN201910678009A CN110402816B CN 110402816 B CN110402816 B CN 110402816B CN 201910678009 A CN201910678009 A CN 201910678009A CN 110402816 B CN110402816 B CN 110402816B
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transplanting
king
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CN110402816A (en
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师校欣
齐向丽
杜国强
赵跃欣
王莉
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Hebei Agricultural University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
    • A01G17/02—Cultivation of hops or vines
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G2/00—Vegetative propagation
    • A01G2/10—Vegetative propagation by means of cuttings
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G9/00—Cultivation in receptacles, forcing-frames or greenhouses; Edging for beds, lawn or the like
    • A01G9/02—Receptacles, e.g. flower-pots or boxes; Glasses for cultivating flowers
    • A01G9/029—Receptacles for seedlings
    • A01G9/0299—Handling or transporting of soil blocks or seedlings
    • 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
    • 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
    • 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
    • G—PHYSICS
    • G05—CONTROLLING; REGULATING
    • G05D—SYSTEMS FOR CONTROLLING OR REGULATING NON-ELECTRIC VARIABLES
    • G05D27/00—Simultaneous control of variables covered by two or more of main groups G05D1/00 - G05D25/00
    • G05D27/02—Simultaneous control of variables covered by two or more of main groups G05D1/00 - G05D25/00 characterised by the use of electric means
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/25—Greenhouse technology, e.g. cooling systems therefor

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  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
  • Botany (AREA)
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Abstract

The invention relates to the technical field of plant tissue culture application, in particular to a system for increasing the transplanting seedling emergence amount of test-tube grape seedlings of violet king and an application method thereof. Cutting young and tender young shoots of grape plants, removing leaves, cutting into single-bud stem sections, sterilizing, inoculating to a culture medium, subculturing to obtain test-tube seedlings suitable for in vitro culture, hardening the seedlings, cutting into an upper section and a lower section, transplanting at the lower section, cutting at the upper section, changing the test-tube seedlings from one to two, controlling the temperature, humidity and illumination in a small plastic shed by a system for controlling the transplanting seedling emergence of the test-tube seedlings of the grapes, not only increasing the acclimatizing and transplanting seedling emergence of the test-tube seedlings of the grapes, improving the survival rate of the test-tube seedlings of the grapes, reducing the production cost, but also reducing the height of tissue culture seedlings, and overcoming the defect of inconvenient creeping growth management after transplanting.

Description

System for increasing transplanting seedling emergence amount of test-tube plantlets of grapes and application method thereof
Technical Field
The invention relates to the technical field of plant tissue culture application, in particular to a system for increasing the transplanting seedling emergence amount of test-tube grape seedlings of violet king and an application method thereof.
Background
China is a world large grape producing country, but the problems of single main cultivated variety, centralized mature period and the like exist at present, and with the development of market economy and the improvement of the quality requirements of people on grapes, the demand on new varieties is increasingly urgent. The violet king (also called as red king) is a new grape variety cultivated in Japan, and is bred by hybridizing widou-sun rose (or named Wenke-sun rose), the single fruit weight is 25-30g, the color of the fruit peel is dark red, the coloring is easy, the sugar content reaches 23%, the fruit is strong fragrant, sweet, crisp, juicy and not easy to crack, the fruit is storage and transportation resistant, disease resistant, high in yield and stable in yield, and the development potential is very high, and the supply of seedlings in production is not in line.
The grapes are mostly bred by cutting, but the introduction quantity of the new variety branches is limited, so that a large number of seedlings are bred in a short time, and the culture area is very difficult to expand. At present, tissue culture technology is utilized for rapid propagation. The fast propagation of plant tissue culture generally comprises 4 stages of initial culture of explants, subculture propagation, adventitious root induction and test-tube seedling out-of-bottle transplantation. The plant tissue culture technology for propagating the seedlings has the advantages of short propagation period, high propagation coefficient, good quality of the produced seedlings, high uniformity and the like. However, the plant tissue culture is highly specialized, the requirements on production places are high, instruments, equipment and reagent supplies are complicated, and technicians with professional experience are required to culture and manage the plant tissue culture, which are factors causing higher tissue culture cost. In addition, the height of a test-tube seedling is 1.5-3cm to meet the transplanting requirement, but when the tissue culture technology is used for carrying out 'violet king' rapid propagation and bottle-out transplanting on a new grape variety, the height of the test-tube seedling can reach 6-8cm, and grapes are vine fruit trees and cannot grow vertically. When the greenhouse is acclimated and transplanted, in order to improve the utilization rate of facilities, the bottle-out transplanting container is small and densely arranged, and grape seedlings are crawled and grow, so that the seedling management is inconvenient. The technical problem to be solved by the invention is to improve the seedling emergence amount of the domestication and transplantation of the test-tube grape seedlings, improve the survival rate of the test-tube grape seedlings, reduce the cost, reduce the height of the transplanted seedlings and relieve the problem of inconvenient management caused by creeping growth of the survival seedlings of the transplanted grapes to a certain extent.
Disclosure of Invention
The invention provides a system for increasing the transplanting seedling emergence amount of 'violet king' grape test-tube seedlings and an application method thereof, which are used for improving the seedling emergence amount of domestication and transplantation of the grape test-tube seedlings, improving the survival rate of the grape test-tube seedlings, reducing the cost, simultaneously reducing the height of transplanted seedlings and relieving the problem of inconvenient management caused by creeping growth of the survival seedlings of the transplanted grapes to a certain extent.
In order to solve the technical problems, the invention adopts the technical scheme that:
a system for increasing the transplanting seedling emergence amount of test-tube plantlets of 'Violet King',
it is characterized by comprising:
the temperature monitoring module is used for monitoring the temperature in the small plastic shed to obtain a temperature value in the shed;
the humidity monitoring module is used for monitoring the humidity in the small plastic shed to obtain a shed humidity value and a shed soil humidity value;
the illumination monitoring module is used for monitoring the illumination intensity in the small plastic shed to obtain an illumination value in the shed;
control module for
When the temperature value in the small plastic shed obtained by the temperature monitoring module is larger than the maximum value of the preset temperature range, controlling the refrigeration device to start, cooling the interior of the small plastic shed, and when the temperature value in the shed obtained by the temperature monitoring module is smaller than the minimum value of the preset temperature range, controlling the floor heating device to start, and heating the interior of the small plastic shed;
when the humidity value in the greenhouse obtained by the humidity monitoring module is smaller than the minimum value of the preset humidity range, controlling the humidifying device to start, and humidifying the interior of the small plastic greenhouse; when the obtained humidity value in the shed is larger than the maximum value of the preset humidity range, controlling a dehumidifying device to start, and dehumidifying the interior of the small plastic shed;
when the humidity value of the soil in the shed obtained by the humidity monitoring module is smaller than the minimum value of the preset soil humidity range, controlling the irrigation device to start, and irrigating the soil in the small plastic shed;
when the illumination value in the greenhouse obtained by the illumination monitoring module is larger than the maximum value of the preset illumination range, controlling the shading device to start, and shading the illumination supplied to the grape tissue culture seedlings in the small plastic greenhouse; and when the obtained illumination value in the shed is smaller than the minimum value of the preset illumination range, controlling the illumination device to start, and providing illumination for the grape tissue culture seedlings in the small plastic shed.
The method for increasing the transplanting seedling emergence amount of the test-tube grape seedlings of the violet king is characterized by comprising the following steps of:
(1) selecting healthy grape plants without diseases and insect pests in 5-6 months, shearing young and tender young shoots, removing leaves, washing with running water, shearing into single-bud stem segments, sterilizing with sterilizing agent composed of 0.1% mercuric chloride and 0.1% tween-20 in a super clean bench for 6-8min, washing with sterile water for 4-5 times, and cutting off the cut in contact with the sterilizing agent for later use;
(2) inoculating the single-bud stem section treated in the step (1) into a culture medium for initial culture, maintaining the temperature at 23-27 ℃ for illumination culture, cutting off the grown young shoots after the buds germinate, inducing the young shoots into seedlings, replacing the fresh culture medium every 35-40 days, and cutting the test-tube seedlings for subculture propagation;
(3) transferring the test-tube plantlet in the step (2) to a greenhouse, keeping the temperature at 22-35 ℃, the illumination intensity at 18000Lx-30000Lx, and closing the bottle to harden the plantlet for 2 weeks;
(4) taking out the test-tube seedlings after hardening off the seedlings, cutting off the test-tube seedlings which are strong, 5-8cm high and have more than 4 leaves from the middle, dividing the test-tube seedlings into two parts, transplanting the test-tube seedlings with roots at the lower section, and cutting the test-tube seedlings with young shoots at the upper section;
(5) spreading the root system of the seedling with root at the middle lower section in the step (4) in a substrate, filling the substrate to 2/3 parts of a nutrition pot, covering the substrate with vermiculite sterilized by 0.1% carbendazim, and spraying 0.1% carbendazim liquid medicine after transplanting to prevent diseases;
(6) dipping the tender shoots at the upper section in the step (4) with 800-1000mg/L indolebutyric acid (IBA) solution, cutting the tender shoots in a matrix, filling the mixed matrix to 2/3 parts of a nutrition pot, covering the mixed matrix with vermiculite sterilized by 0.1% carbendazim, and spraying 0.1% carbendazim liquid medicine after cutting to prevent diseases;
(7) and (3) placing the tissue culture seedlings subjected to the lower-stage transplanting in the step (5) and the upper-stage young shoot cutting in the step (6) into a built small plastic shed, and gradually opening a plastic film for ventilation after 10-20 days to reduce the humidity until the tissue culture seedlings are completely alive, removing the small plastic shed, appropriately spraying water to the tissue culture seedlings in the period, spraying 0.1% carbendazim liquid every 1-2 weeks to prevent diseases, and pouring Hoagland nutrient solution after 30 days.
Preferably, the first and second liquid crystal materials are,
and (2) inoculating single-bud stem segments with one leaf in the step (1).
Preferably, the first and second liquid crystal materials are,
selection of the medium used in said step (2) B5Or GS as a cultureAnd (5) nutrient base.
Preferably, the first and second liquid crystal materials are,
0.4-0.6mg/LIAA is also added into the culture medium used in the step (2).
Preferably, the first and second liquid crystal materials are,
25-30g/L of sucrose and 6g/L of agar are also added into the culture medium used in the step (2).
Preferably, the first and second liquid crystal materials are,
during the culture process in the step (2),
the conditions for light culture of the test-tube plantlets were light/dark =16 hours/8 hours, and red: blue: white =3:1:1 or red: the LED lamp with blue =4:1 has composite light quality and illumination intensity of 1500Lx-3000 Lx.
Preferably, the first and second liquid crystal materials are,
in the step (5), the substrate is nutrient soil.
Preferably, the first and second liquid crystal materials are,
in step (6), the matrix is vermiculite: nutrient soil =1: 2-2.5.
Preferably, the first and second liquid crystal materials are,
in the step (7), the tissue culture seedling of the upper-section young shoot cuttage in the step (6) is placed into a built small plastic shed, the temperature in the shed is controlled to be 25-30 ℃, and the relative humidity is controlled to be 92-95%.
In the process of the invention, the pollution rate of initial culture is greatly influenced by the sampling period, and the growth and differentiation of the test tube material in the subculture stage are also influenced to a certain extent. The difference of the sampling period is mainly reflected in two aspects, namely, the physiological states of plants are different in different growth and development periods; secondly, the cleanliness of plants growing in different seasons is very different. Generally, selection of explants should avoid the dormant period and avoid the high incidence period of diseases and insect pests. Spring is the most suitable season, the bud enters a natural dormant state in autumn, and the germination rate and the cluster bud induction rate are both low.
The sterilization time and the sterilization mode can affect the pollution rate, the germination rate, the survival rate, the browning degree and the like of the explant, the sterilization time is too long, cell toxicity can be caused, the browning and even death of the explant can be caused, the sterilization time is too short, the explant is not sterilized thoroughly, and the pollution rate is increased.
The culture medium is a nutrient substrate for survival of isolated cells or tissues and organs, provides proper nutrient conditions for isolated culture materials, and meets the requirements of normal growth and maintenance of the complete structure and function of the isolated culture materials.
The subculture multiplication stage is the key to successful and practical application of plant tissue culture, and the culture is rapidly multiplied in a certain multiplication coefficient at the stage and reaches a certain reproduction population at a certain time. When most plant test tube materials are subjected to subculture proliferation, a culture medium contains a high proportion of cytokinin, and the cytokinin 6-BA, namely 6-benzyladenine is an artificially synthesized cytokinin substance, has the main physiological action of promoting cell division and lateral bud germination and growth and is the most commonly used cytokinin for plant tissue culture. In the subculture multiplication stage of tissue culture seedlings of most plant species, 6-BA is added to the culture medium, so that the top end advantage is removed, axillary buds germinate, a large number of clumpy buds and young shoots grow, and the propagation coefficient is high. However, the research of the invention finds that the grapes of the violet king react strongly to the 6-BA, when the 6-BA is added into the culture medium, the roots of the shoot base grow a lot of calluses, the number of the rootuses is reduced, even the rootuses do not grow, the leaves are yellow, the leaves and the stems are deformed, the plants grow weakly, and the plants die after 2-3 subcultures even in the culture medium containing the 6-BA. Therefore, the proliferation culture medium of the test-tube plantlet of the 'violet king' grape only adds auxin and does not add cytokinin, the proliferation is enlarged by adopting the modes of shoot cutting and segmented cuttage, and a root system is grown simultaneously in the proliferation process and can grow into a complete plant once.
Plant tissue culture mainly depends on an electric light source, and a traditional fluorescent light source has low biological energy efficiency and large heat productivity for plants, and is one of higher non-human costs in plant tissue culture. The invention adopts a novel LED light source to regulate and control the light quality in the plant tissue culture, can regulate and control the growth, development and morphogenesis of test-tube plantlets, reduces energy consumption and reduces cost.
The bottle-out transplantation of the test-tube plantlets is the last link of the rapid propagation of plant tissue culture, and is very important, the survival rate of the test-tube plantlets is low, and the previous work can be abandoned, which is one of the biggest problems restricting the wide application of the rapid propagation and the industrialized plantlet culture of plants. Because the test-tube plantlets grow under the special conditions of constant temperature, high humidity, weak light and sterility, the growth conditions of the tissue culture plantlets are directly influenced by the moisture condition, the ventilation condition, the nutrient content, the pH value and the like provided by the matrix in the transplanting process. The bottle-out transplantation firstly needs domestication transition transplantation in a greenhouse or a greenhouse, and the improvement of the survival rate of domestication and transplantation of test-tube seedlings is the key of rapid propagation of plant tissue culture.
The cutting propagation is a propagation method for forming independent plants by cutting roots, stems and leaves of vegetative organs of plants, and has the advantages of easy propagation, quick seedling formation, capability of keeping the excellent characteristics of original varieties and the like. Most grape species and varieties are easy to generate adventitious roots on branches and tendrils, so cutting seedling is mainly used in production practice. On the basis of tissue culture and cutting propagation, the invention researches that the test-tube plantlet is cut off from the middle and is divided into two parts, the lower section of the test-tube plantlet is transplanted with root plantlet, and the upper section of the test-tube plantlet is cut, so that the seedling emergence amount of domestication and transplantation of the test-tube plantlet of the grape is increased.
The nutrient solution is prepared by dissolving compounds containing various nutrient elements necessary for plant growth and development and a small amount of auxiliary materials for making the effectiveness of certain nutrient elements more durable in water according to a certain amount and proportion.
The rapid propagation is carried out by utilizing the tissue culture technology, and the method has the advantages of no land occupation, no season limitation, short propagation period, high propagation coefficient and the like, can shorten the propagation period of the nursery stock, and quickens the popularization and application of the excellent new variety. In the process of tissue culture and subculture proliferation of most plants, a cluster bud proliferation mode is mostly adopted, a culture medium contains a high proportion of cytokinin, the formation of a root system is inhibited, in addition, a specific rooting stage is needed, the cytokinin is removed from the culture medium, and only the auxin is added to induce rooting. The invention discovers that the low-concentration cytokinin is not added or only added to the grape tissue culture propagation culture medium, otherwise the stem leaf deformity is easily caused. The grape has the characteristic of easy rooting in cuttage, so the test-tube plantlet is cut into single-bud stem segments to be inserted into a culture medium for inoculation, after about 10 days, adventitious roots grow out at the base of the stem segments, axillary buds germinate and grow into tender tips, that is, subculture proliferation and rooting are combined to complete the growth of a complete plant once, and the plant is high to the bottle mouth.
Advantageous effects
1. Through monitoring temperature, humidity and illumination intensity in the little plastic shed, can in time adjust the processing to temperature, humidity and illumination intensity in the little plastic shed, provide good living environment for grape tissue culture seedling, further improve the survival rate of grape tissue culture seedling.
2. According to the method, the grape test-tube plantlets are cut when taken out of bottles for transplanting, namely the test-tube plantlets are cut from the middle and are divided into two parts, the upper-section tender tips are dipped with 1000mg/LIBA quickly and then are subjected to cuttage, the survival rate is higher than 60%, and the grape test-tube plantlets grow well; the transplanting survival rate of the lower-section test-tube seedlings is more than 90%, the seedling forming amount of the grape tissue culture seedlings of the violet king is increased by the method combining cuttage and transplanting, and is 1.5 times of the seedling forming amount of the traditional whole plant transplanting, so that the seedling raising cost is reduced by 21%, the height of the transplanted seedlings is reduced, and the problem of inconvenient management caused by creeping growth of the survival seedlings of the grape transplanting is relieved to a certain extent.
3. The production of tissue culture seedlings has short propagation period, high propagation coefficient and environment condition manual control without season limitation, and a certain commodity quantity can be reached in a short period by propagating hundreds of thousands of plants from one stem tip, which is ten thousand times to hundreds of thousands of times faster than that of the conventional method; the produced seedlings have the advantages of good quality, high uniformity, capability of removing part of plant viruses and the like, and are often used as stock seeds, and the seedlings which are collected from the stock seeds and propagated by propagation materials are used for production.
4. Cutting the grape test-tube plantlet into single-bud stem segments with one leaf, about 1-1.5cm, inoculating into culture medium, after more than ten days, making the base portion of stem segment grow adventitious root, at the same time making axillary bud germinate and make tender tip grow gradually, after 35 days, making the stem segment grow into test-tube plant with plant height above 5-8cm, at this time, cutting the test-tube plantlet into 4-6 single-bud stem segments with 1 leaf, inoculating into new culture medium, making subculture and seedling. The method can proliferate 4-6 times every 35 days, and can grow seedlings once when the roots are proliferated, the hereditary character is stable, the culture process is simple, and the transplanting is easy to survive.
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification. In the drawings:
FIG. 1 is a schematic structural diagram of a system for increasing the transplanting seedling emergence amount of test-tube grape seedlings in Violet King according to the present invention;
FIG. 2 is a schematic diagram showing the influence of the test-tube plantlet of Violet King with different subculture times on the plant height;
FIG. 3 is a schematic diagram showing the influence of the test-tube plantlet of Violet King of different subculture times on the propagation coefficient;
FIG. 4 is a schematic diagram showing the influence of the propagation of the test-tube plantlet of Violet King on the rooting rate in different subculture times of the present invention;
FIG. 5 is a schematic diagram showing the effect of different shearing treatments on the cuttage and transplantation survival rate of the test-tube seedlings of the violet king;
FIG. 6 is a schematic diagram showing the effect of different shearing treatments on the height of a test-tube seedling plant of Violet King in the present invention;
FIG. 7 is a schematic diagram showing the effect of different shearing treatments on the leaf number of a test-tube seedling of Violet King according to the present invention;
FIG. 8 is a schematic diagram of the test-tube plantlet before and after shearing of the "Violet King" test-tube plantlet of the present invention;
FIG. 9 is a schematic view showing the growth of the upper cutting after the shearing of the test-tube plantlet of the violet King of the present invention;
FIG. 10 is a schematic view showing the growth of the lower-stage transplanted seedling after shearing the test-tube seedling of the violet King of the present invention;
FIG. 11 is a schematic diagram showing the growth of the whole test-tube plantlet transplanting of the violet King of the present invention;
FIG. 12 is a schematic diagram of the seedlings propagated after cuttage and transplantation of the test-tube seedlings of the violet king test-tube of the invention;
FIG. 13 is a schematic diagram showing the effect of light quality on the subculture proliferation and rooting of a test-tube plantlet of "Violet King" grape in accordance with the present invention;
FIG. 14 is a schematic diagram showing the effect of the light-dark treatment of the present invention on the proliferation and rooting of "Violet King" grape test-tube plantlet.
Detailed Description
A system for increasing the transplanting emergence amount of test-tube grape seedlings of the 'Violet King' grape, as shown in figure 1, comprises:
the temperature monitoring module is used for monitoring the temperature in the small plastic shed to obtain a temperature value in the shed;
the humidity monitoring module is used for monitoring the humidity in the small plastic shed to obtain a shed humidity value and a shed soil humidity value;
the illumination monitoring module is used for monitoring the illumination intensity in the small plastic shed to obtain an illumination value in the shed;
control module for
When the temperature value in the small plastic shed obtained by the temperature monitoring module is larger than the maximum value of the preset temperature range, controlling the refrigeration device to start, cooling the interior of the small plastic shed, and when the temperature value in the shed obtained by the temperature monitoring module is smaller than the minimum value of the preset temperature range, controlling the floor heating device to start, and heating the interior of the small plastic shed;
when the humidity value in the greenhouse obtained by the humidity monitoring module is smaller than the minimum value of the preset humidity range, controlling the humidifying device to start, and humidifying the interior of the small plastic greenhouse; when the obtained humidity value in the shed is larger than the maximum value of the preset humidity range, controlling a dehumidifying device to start, and dehumidifying the interior of the small plastic shed;
when the humidity value of the soil in the shed obtained by the humidity monitoring module is smaller than the minimum value of the preset soil humidity range, controlling the irrigation device to start, and irrigating the soil in the small plastic shed;
when the illumination value in the greenhouse obtained by the illumination monitoring module is larger than the maximum value of the preset illumination range, controlling the shading device to start, and shading the illumination supplied to the grape tissue culture seedlings in the small plastic greenhouse; and when the obtained illumination value in the shed is smaller than the minimum value of the preset illumination range, controlling the illumination device to start, and providing illumination for the grape tissue culture seedlings in the small plastic shed.
The beneficial effects of the above technical scheme are: through monitoring temperature, humidity and illumination intensity in the little plastic shed, can in time adjust the processing to temperature, humidity and illumination intensity in the little plastic shed, for the grape tissue culture seedling provides good living environment, further improve the survival rate of grape tissue culture seedling.
1. Proper collection time of explants
The explants of grape shoots of "violet king" were harvested at 5 and 9 months, respectively, and sterilized for initial culture, and the results are shown in table 1. The shoot explants are collected in 5 months, even if mercury bichloride (mercuric chloride) sterilization treatment is carried out for a short time, the pollution rate and the death rate are both very low, the germination rate is high, and the violet king is suitable for collecting shoot explants to inoculate in spring. Explants collected in the field at the bottom of 9 months are treated with mercuric chloride for 10 min, 15min and 20min, the pollution rate is generally high, the germination rate is very low or even 0min, at the moment, grapes are about to enter a dormancy stage, the vitality of branches is poor, dust, microorganisms and the like on the surfaces of the explants are more, the pollution rate is higher due to incomplete sterilization, the time of the grape explants bearing mercuric chloride sterilization is limited, and especially some young parts are easy to brown and die if the sterilization time is too long. When the grapes are going to enter the dormancy stage in autumn, the grapes are not suitable for explant collection and inoculation.
TABLE 1 initial culture effect of shoot explants of grape harvested in different seasons
Figure 646179DEST_PATH_IMAGE001
2. Influence of the type of inoculation material on the proliferation and rooting of the test-tube plantlet "violet king of kingdom":
selecting the subculture seedlings of the grapes to be tested with consistent growth conditions, inoculating single-bud stem sections of about 1-1.5cm on a subculture medium, and cutting the test tube seedlings of the grapes into single-leaf, half-leaf and non-leaf inoculation treatments. Complete randomized trial design, inoculation of 4 strains per vial per 30 vials treated, was repeated 3 times (the same complete randomized trial design was adopted for the following experiments). After more than ten days, adventitious roots grow out from the base of the stem segment, axillary buds germinate, tender tips grow gradually, and after 35 days, the stem segment grows into a test tube plant with the plant height of more than 4-7 cm. The influence of the existence of leaves and the size of the leaves on the proliferation and rooting in the process of transferring the test-tube plantlets of the violet king is shown in table 2, the plant height, the number of the rooting strips and the propagation coefficient of the transfer material are obviously reduced when the transfer material does not have leaves, the average number of the rooting strips and the propagation coefficient of the single-bud stem segment with one leaf are higher, the numerical value of the rooting rate is slightly lower than that of the single-bud stem segment without leaves and half leaves, but the statistical analysis difference is not obvious. Comprehensively considering, in the cutting propagation stage, a single-bud stem section with one leaf is selected.
TABLE 2 Effect of inoculation Material types on proliferation and rooting of "Violet King" test-tube plantlets
Figure 143019DEST_PATH_IMAGE002
3. Influence of basic culture medium on proliferation and rooting of test-tube plantlet of violet king
Basic culture medium is provided with B 54 culture mediums including GS, MS and 1/2MS are treated, IAA0.5mg/L, cane sugar 25g/L and agar 6g/L are added, the tested grape with consistent growth condition is selected for subculture, and the stem section with leaf and single bud of about 1.5cm is inoculated. The plant height, rooting rate, number of roots and propagation coefficient were investigated after 40 days. The results are shown in Table 3, at B5Or the height and the propagation coefficient of the test-tube plantlet inoculated in the GS culture medium are obviously higher than those of other treatments, in the aspect of rooting condition, the rooting rate of the test-tube plantlet inoculated in the MS culture medium is lower, the rooting number of the test-tube plantlet on the 1/2MS culture medium is more, and then the GS culture medium is used. Because in B5The survival rate of the test-tube plantlets grown in the culture medium can reach more than 90 percent after the test-tube plantlets are taken out of bottles and transplanted, and the height of the test-tube plantlets in the 1/2MS culture medium is shorter, so GS or B can be selected in the stages of subculture proliferation and rooting culture5As a minimal medium. In addition, many components of the GS medium were found to be equivalent to 1/2B5The culture medium, the GS culture medium can be used for lower cost.
TABLE 3 Effect of basic Medium on proliferation and rooting of "Violet King" test-tube plantlets
Figure 918077DEST_PATH_IMAGE003
4. Influence of auxin species and concentration on proliferation and rooting of violet king test-tube plantlet
With B5+ agar 6g/L + sucrose 25g/L as basic cultureThe culture medium was inoculated with IAA (0.2, 0.5, 0.8, 1.0 mg/L), IBA (0.1, 0.2, 0.3, 0.5 mg/L), NAA (0.1, 0.2, 0.3 mg/L) at different concentrations, and the culture was examined after 40 days. The influence of different types and concentrations of auxin on the proliferation and rooting of the test-tube plantlet of the violet king is shown in table 4, the height of the test-tube plantlet in the culture medium added with IAA and IBA is basically not obviously changed, the height of the test-tube plantlet in the culture medium added with NAA is obviously reduced, and the propagation coefficient of the test-tube plantlet of the grape is obviously higher than that of other treatments when the concentration of auxin is 0.5 mg/LIAA; the three auxins have no obvious influence on the rooting rate of the test-tube plantlet of the grape, but the rooting number of NAA treatment is obviously increased, the root system is thick and strong, the callus is few, the effect of NAA induced rooting is superior to IAA and IBA, and 0.5mg/LIAA is added in the subculture multiplication stage of the 'Violan kingdom' in consideration of shearing treatment during the transplantation of the test-tube plantlet.
TABLE 4 influence of auxin type and concentration on proliferation and rooting of "Violet King" test-tube plantlets
Figure 311012DEST_PATH_IMAGE004
5. Influence of sucrose concentration on proliferation and rooting of test-tube plantlet of violet king
With B5+ IAA0.5mg/L + agar 6g/L as the minimal medium, adding sucrose at concentrations of 20g/L, 25g/L, 30g/L, and 35g/L, respectively, for inoculation and culture, and examining after 40 days. The test-tube plantlet of violet king was inoculated in media with different sucrose concentrations, and the results are shown in table 5, where the sucrose content was 25-30g/L, the plants grew vigorously with many roots. The concentration of sucrose reaches 35g/L, and the rooting rate and the propagation coefficient are obviously reduced. Therefore, the concentration of the sucrose suitable for the subculture proliferation and rooting of the test-tube plantlet of the violet king is 25-30 g/L.
TABLE 5 influence of sucrose concentration on proliferation and rooting of "Violet King" test-tube plantlets
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6. Influence of light on proliferation and rooting of test-tube plantlet of violet king
Placing the inoculated test-tube plantlets in a daylight color fluorescent lamp, an LED lamp, a white light lamp and a red lamp respectively: blue: white =3:1:1 LED lamp composite light, red: blue =7:1 LED lamp composite light, red: blue =4:1 LED lamp composite light, red: and (3) performing 6 light qualities of LED lamp composite light with blue =2:3, and investigating after 40 days. FIG. 13 shows the effect of light on the subculture proliferation and rooting of "Violet King" grape test-tube plantlets. As shown in table 6, the results are shown in the daylight fluorescent lamp and LED lamp red: blue: white =3:1:1 and LED lamp red: the height of the grape test-tube seedling strain under 3 kinds of light quality, namely blue =4:1, is higher, and the propagation coefficient is obviously higher than that of other treatments; from the rooting rate, the white light of the LED lamp is not suitable for rooting of the test-tube plantlet of the violet king; the number of roots of the violet king has no influence on the light quality. Daylight color fluorescent lamp, LED lamp red: blue: white =3:1:1 and LED lamp red: blue =4:1, and because the LED lamp has the characteristics of pure light quality, energy saving, environmental protection and the like, red can be selected during the tissue culture and rapid propagation of the violet king: blue: white =3:1:1 or red: blue =4:1 LED lamp composite light quality.
TABLE 6 influence of photoperiod on the subculture proliferation and rooting of "Violet King" test-tube plantlets
Figure 589995DEST_PATH_IMAGE006
7. Influence of dark culture on proliferation and rooting of test-tube plantlet of violet king
The test-tube plantlet is inoculated and then respectively cultured under illumination for 16 hours/day, and cultured in dark for 1-10 days, 11-20 days, 21-30 days, 31-40 days and 24 hours/day after inoculation, and investigation is carried out after 40 days. FIG. 14 shows the effect of light and dark treatment on the proliferation and rooting of "Violet King" grape test-tube plantlets. The results show that: test-tube seedlings which are subjected to dark culture 31-40 days after inoculation have longer internodes, the plant height is obviously higher than that of other treatments, illumination is carried out for 16 hours/day, dark culture and full-dark culture are carried out for 1-10 days after inoculation for 24 hours/day, however, the full-dark culture for 24 hours/day is overgrown seedlings, the seedlings are thin and weak, the internodes are long, and the propagation coefficient is not high; illumination culture for 16 hours/day and dark culture for 1-10 days after inoculation, wherein the propagation coefficient is highest; the rooting rate of the test-tube seedlings of violet king under different light and dark treatments can reach 100 percent; dark culture is beneficial to root development, the number of roots is the most when the culture is performed for 24 hours/day, and dark culture is performed for 1-10 days after inoculation (Table 8). In conclusion, the test-tube plantlet with stronger growth vigor and high propagation coefficient can be obtained by dark culture for 16 hours/day in illumination culture and for 1-10 days after inoculation, compared with the two, the root system development of dark culture for 1-10 days after inoculation is better, and considering that the dark culture for 16 hours/day in illumination culture is more convenient at ordinary times, the test-tube plantlet of the violet king is subjected to illumination culture for 16 hours/day in a subculture multiplication stage, and the dark culture is performed for 1-10 days after inoculation when the last culture is performed before bottle-out transplantation.
TABLE 7 Effect of light and dark treatment on proliferation and rooting of "Violet King" test-tube plantlets
Figure 485139DEST_PATH_IMAGE007
8. Research on propagation characteristics of test-tube plantlets with different subculture times
Selecting test grape test-tube plantlets with consistent growth conditions, subculturing for 1 time every 40 days, and investigating the plant height, rooting rate, rooting number, propagation coefficient and internode length of each generation of the test grape test-tube plantlets during transfer. The growth conditions of each generation are shown in figures 2,3 and 4, and the internode length tends to be stable in the 6 th generation; after 9 times of subculture, the plant height and the propagation coefficient tend to be stable; after 7 subcultures, the rooting rate can reach 100%, and the number of roots is gradually stable. Therefore, the grapes of violet king are completely adapted to the in vitro culture conditions after 9 subcultures, and the propagation efficiency is improved. As shown in fig. 2,3 and 4.
Figure 314554DEST_PATH_IMAGE008
And the influence of shearing treatment on the cuttage and transplantation of the test-tube seedlings of the violet king
Selecting strong test-tube seedlings with more than 4 leaves and consistent growth conditions, closing the bottles in a greenhouse with strong light of more than 18000Lx for 2 weeks, taking out the test-tube seedlings, shearing the test-tube seedlings, cutting the tissue culture seedlings from the middle into two parts, directly planting the lower sections with root systems into a substrate, dipping the base parts of the young tips of the upper sections with 1000mg/LIBA, and then cutting the young tips into the substrate. The whole plant without shearing was used as control. Randomized block trial design was repeated 3 times per 30 treatments. The survival rate, plant height and leaf number were investigated every 30 days, and the survival rate is shown in fig. 5: the upper-section young shoot cuttage survival rate can reach 60%, the lower-section test-tube seedling transplanting survival rate is more than 90%, the whole-plant test-tube seedling transplanting survival rate is 100%, and the seedling number of the tissue culture seedlings after shearing treatment is 1.5 times of the seedling number of the conventional whole-plant test-tube seedlings; the plant height and the leaf number of the tissue culture seedling obtained by the transplantation of the lower section of the test-tube seedling are not different from those of the tissue culture seedling transplanted by the whole plant when 2 months; the tissue culture seedling obtained by the upper-section young shoot cuttage needs a certain time for rooting, so the plant height is lower than that of the tissue culture seedling with root transplantation, and the number of leaves is about the same as that of the tissue culture seedling with the lower-section whole plant transplantation when the number of leaves is 3 months. In conclusion, the cutting and transplanting are simultaneously carried out after the test-tube seedlings are taken out of the bottles and are subjected to the cutting treatment, the seedling forming amount of the tissue culture seedlings of the King of the violet kingdom can be increased, the growth condition of the tissue culture seedlings in the later period is not obviously influenced, the influence of the cutting on the plant height and the leaf number is shown in figures 6-7, the influence of the cutting on the plant height and the leaf number is shown in figure 8 before and after the cutting, and the growth condition is shown in figures 9-11.
The upper-section young shoot cutting seedling is difficult to absorb water because no root exists in the initial stage, so that the high humidity needs to be kept for a long time, and the seedling is easy to infect diseases and die. When the temperature of the greenhouse is low, the system for transplanting the seedling emergence amount of the test-tube grape seedlings of the violet king controls the floor heating device to be started, so that the young shoots of the cuttage can be promoted to root as early as possible, and the cuttage survival rate is improved.
Figure DEST_PATH_IMAGE010A
Influence of substrate on cuttage and transplantation of test-tube seedlings of violet king
Selecting grape test-tube seedlings which are finished in exercise and have consistent growth conditions, dipping 1000mg/LIBA on the cut upper section of the test-tube seedlings, directly transplanting the test-tube seedlings on the lower section, and respectively planting vermiculite and nutrient soil into 5 different matrixes of 1:1 of vermiculite and 1:2 of nutrient soil of vermiculite and 1:1 of nutrient soil of vermiculite and 1:2 of whole non-cut seedlings as a control, and surveying after 40 days.
Figure DEST_PATH_IMAGE012A
Influence of substrate on cutting of tender tips at upper section of violet king
The cut new shoots at the upper section of the test-tube plantlet of the violet king are inserted into different substrates, the investigation result of 40 days is shown in table 8, and the nutrient soil and vermiculite: nutrient soil =2:1 and vermiculite: the survival rate of the tissue culture seedlings in the nutrient soil =1:2 is higher, and all the survival tissue culture seedlings root during investigation. The number of roots and the plant height increase along with the increase of the proportion of the nutrient soil, the tissue culture seedlings in the nutrient soil and the mixed matrix of vermiculite and nutrient soil (the volume ratio is 1:1 and 1: 2) have more roots, and the ratio of the nutrient soil to the vermiculite: the nutrient soil =1:2, the tissue culture seedlings are higher. Vermiculite: the leaf area of the tissue culture seedling in the nutrient soil =1:2 is obviously larger than that of other treatments, and the different substrate proportions have no obvious influence on the leaf number. In conclusion, the substrate suitable for the cutting of the upper young shoots of the grapes of the violet king is vermiculite: nutrient soil =1: 2.
TABLE 8 influence of the substrate on the ex vitro rooting and cutting of the superior seedlings of the "Violet King
Figure 577040DEST_PATH_IMAGE013
10.2 Effect of the substrate on transplanting of tissue culture seedlings with roots of Violet King
The "violet king" rooted tissue culture seedlings were transplanted into different matrices with the results shown in table 9: nutrient soil and vermiculite: the nutrient soil =1:2, and the height of the transplanted tissue culture seedlings at the middle and lower sections is higher. The lower-stage transplanted tissue culture seedlings planted in the vermiculite have fewer leaves. The leaf area of the lower-stage transplanted tissue culture seedling is increased along with the increase of the proportion of the nutrient soil, the maximum in the nutrient soil is, and the vermiculite: nutrient soil =1:2, smallest of vermiculite. The substrate proportion has no obvious influence on the survival rate of the whole transplanted tissue culture seedling; the leaf area of the whole transplanted tissue culture seedling in the nutrient soil is large, and the leaf color is dark green. The tissue culture seedling planted in the vermiculite is short and small, the stem is thin and weak, the leaf area is small, the number of leaves is small, and the method is not suitable for transplanting the tissue culture seedling with roots of the violet king. The substrate suitable for transplanting the tissue culture seedlings with roots of the violet king is nutrient soil.
TABLE 9 influence of the substrate on transplanting of tissue culture seedlings with roots of "Violet King
Figure 212420DEST_PATH_IMAGE014
11. Influence of auxin type and concentration on tender shoot cuttage of upper section of violet king
The young shoots of the upper section of the cut test-tube plantlet are respectively dipped with IAA (1000 mg/L, 2000mg/L, 2500 mg/L), IBA (500 mg/L, 1000mg/L, 2000 mg/L), NAA (500 mg/L, 800mg/L, 1000 mg/L), inserted into nutrient soil and covered with vermiculite. Randomized block trial design was repeated 3 times per 30 treatments. The plant height, leaf number, leaf area, rooting rate and rooting number were investigated after 40 days. The survived tissue culture seedlings all take roots, the growth performance is shown in table 5, 1000mg/LIAA, 2000mg/LIAA, 500mg/LIBA, 1000mg/LIBA and 800mg/LNAA are adopted to dip the upper tender shoots for cuttage, and the survival rate is higher; the shoots dipped with 2000mg/LIAA, 2500mg/LIAA and 1000mg/LIBA have more roots, and when the IBA concentration is 1000mg/L, the plant height, the leaf number and the leaf area of the tissue culture seedling are higher. On the contrary, the tender shoots dipped by NAA of 500mg/L and 1000mg/L have lower survival rate and less rooting number, and are not suitable for the ex-bottle rooting of the tender shoots at the upper section of the 'violet king'. In conclusion, 1000mg/LIBA is suitable for ex-bottle rooting and cutting seedling growth of tender shoots at the upper section of the King of the violet.
TABLE 10 Effect of auxin on rooting and cutting outside the top shoot of the "Violet King
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12. Influence of nutrient solution on strong seedlings of test-tube seedlings of violet king
The cut lower test-tube plantlet is planted in nutrient soil and covered with vermiculite sterilized by 0.1% carbendazim liquid medicine. Performing conventional tube after plantingAfter 40 days, selecting the lower stage transplanted tissue culture seedlings with consistent growth conditions for nutrient solution test, and selecting MS nutrient solution and B nutrient solution5The nutrient solution and Hoagland nutrient solution are 3, and are irrigated for 1 time in 5 days by taking clear water as a control, 30 plants are irrigated for 1L each time, and the plant height, the leaf area, the leaf number and the like are investigated after 70 days. As a result, as shown in Table 11, the growth promoting effect of the tissue culture seedlings by irrigating the nutrient solution was significant, in which the plant height of the tissue culture seedlings by irrigating the Hoagland nutrient solution was significantly higher than that of the other treatments, followed by B5A nutrient solution; the number of leaves of tissue culture seedlings for irrigating the Hoagland nutrient solution is the largest, B5And MS nutrient solution has no obvious influence on the number of leaves; leaf area to water B5And Hoagland nutrient solution, and the color of the leaves is dark green. In conclusion, the Hoagland nutrient solution has the best effect on strengthening the transplanted tissue culture seedlings of the violet king.
TABLE 11 influence of nutrient solution on the strong seedlings after the test-tube seedlings of Violet King
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13. Influence of air temperature and humidity on survival and growth of test-tube plantlet
In the transitional transplanting process, the air humidity has important influence on the survival and growth of the test-tube plantlet, and under a high-humidity environment, as long as the disease can be effectively controlled, the seedling reviving time can be shortened, and the growth of the test-tube plantlet is accelerated. Generally, the humidity can be gradually reduced 10-15 days after the transplantation, and table 12 compares the influence of various environmental conditions on the survival and growth of the test-tube plantlet after the transplantation. According to the test results, a system (an intelligent system for short in table 12) for intelligently controlling the transplanting and seedling emergence amount of the test-tube grape seedlings of the violet king grapes is adopted in the small plastic shed, so that the temperature can be kept at 25-30 ℃ and the humidity can be kept at 92-95% in the small plastic shed, and the growth of tissue culture seedlings is facilitated.
TABLE 12 influence of environmental conditions on test-tube plantlet transplant survival and growth
Figure 915431DEST_PATH_IMAGE017
14. Influence of indoor culture days on cutting of tender tips at upper section of violet king
Culturing the violet king test-tube seedlings of different days indoors, transferring the test-tube seedlings to a greenhouse for hardening for 2 weeks, shearing the test-tube seedlings during transplanting, wherein the growth conditions of the upper-section young shoots after being cut for 40 days are shown in a table 13, the indoor culturing days have no obvious influence on the survival rate of the upper-section young shoots, and the survival tissue culture seedlings completely take roots during investigation; the tissue culture seedlings cultured in the room for 30 and 35 days have more rooting strips and larger leaf area; the height of the tissue culture seedlings cultured indoors for 20 days and 25 days is lower; the number of leaves of the tissue culture seedlings cultured indoors for 25-35d is large; the tissue culture seedling cultured in room for 20d has poor conditions of root number, plant height, leaf number and leaf area growth, and is not suitable for shearing treatment. In conclusion, the tissue culture seedlings obtained by the upper-section young shoot cuttage are developed in root systems and strong in growth vigor after indoor culture for 30-35 days and seedling hardening and shearing treatment.
Watch (A)
Figure 292186DEST_PATH_IMAGE018
Influence of indoor culture days on rooting and cutting outside upper-section young shoot bottle of violet king
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15. Influence of indoor culture days on transplanting of test-tube seedlings with roots of violet king
The effect of the number of days of indoor culture on the transplanting of the test-tube plantlet with roots of "violet king" is shown in table 14: the indoor culture days have no influence on the survival rate of the test-tube plantlets transplanted with the roots. After 40 days of transplanting, the plant height, the leaf number and the leaf area of the lower-stage transplanted tissue culture seedlings with different indoor culture days have no significant difference. The growth condition of the tissue culture seedlings obtained by the upper-stage cuttage is considered, and the tissue culture seedlings are suitable for seedling hardening and shearing treatment after indoor culture for 30-35 d; the plant height and leaf number of the whole transplanted tissue culture seedlings cultured in the indoor culture room for 20, 25, 30 and 35d are not different. The leaf area of the whole transplanted tissue culture seedling is small after indoor culture for 20 days, so that the whole transplanted tissue culture seedling cultured by the violet king in the indoor culture for 25-35 days has strong growth vigor.
TABLE 14 influence of indoor culture days on transplanting of rooted test-tube plantlets of Violet King
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16. Cost accounting for seedling raising
The test is carried out for 18 months after 4 months in 2019, 5720 test-tube plantlets are produced, and the cost of a single test-tube plantlet is 1.134 yuan. (Table 15) the cost of the single plant tissue culture seedling obtained by respectively adopting the conventional transplanting method and the method of combining the shearing, transplanting and cuttage of the test-tube seedling of 500 plants is respectively calculated to be 1.83 yuan and 1.448 yuan, and the cost of the single plant tissue culture seedling is reduced by 21 percent after the method is adopted. (see tables 15, 16 and 17)
TABLE 15 cost constitution of test-tube plantlet (indoor)
Figure DEST_PATH_IMAGE021
TABLE 16 cost composition of tissue culture plantlets (outdoor) (in 500 test-tube plantlets)
Figure DEST_PATH_IMAGE022
TABLE 17 comparison of conventional transplanting methods with the cost of the present invention (in terms of 500 test-tube plantlets)
Figure DEST_PATH_IMAGE023
Example 1:
(1) selecting healthy grape plants without diseases and insect pests in 5-6 months, collecting young and tender young shoots, removing leaves, washing with running water, sterilizing with sterilizing agent composed of 0.1% mercuric chloride and 0.1% tween-20 in a super clean bench for 6min, washing with sterile water for 5 times, and cutting off the cut in contact with the sterilizing agent;
(2) inoculating the single-bud stem section treated in the step (1) into a culture medium for tissue culture, cutting off tender tips after axillary buds germinate and grow into tender tips, inoculating into a new culture medium, and culturing for a period of time to obtain the rooting test-tube plantlet. GS is used as a culture medium, 0.6mg/LIAA, 30g/L sucrose and 6g/L agar are added, a fresh culture medium is replaced every 40 days, the test-tube plantlet is cut into single-bud stem segments with one leaf, the single-bud stem segments are inoculated into the new culture medium, and subculture propagation is carried out. Keeping the temperature at 25 ℃, the relative humidity of air at 60%, selecting red: blue: white =3:1:1 or red: blue =4:1, illumination intensity of 2000Lx, illumination culture for 16 hours/day, 9 subcultures, and dark culture for 10d after inoculation in 9 th subculture rooting culture;
(3) transferring the test-tube plantlet obtained in the step (2) to a greenhouse, keeping the temperature at 28 ℃, the relative humidity of air at 65-70%, the illumination intensity of 20000Lx, and closing the bottle to harden the plantlet for 2 weeks;
(4) taking out the test-tube seedlings after hardening off the bottles, shearing, selecting strong test-tube seedlings with the height of 8cm and more than 5 leaves, cutting off the test-tube seedlings from the middle, dividing the test-tube seedlings into two parts, transplanting the test-tube seedlings with roots at the lower section, and cutting the test-tube seedlings with tender tips at the upper section;
(5) spreading the root system with the root seedlings at the middle and lower sections in the step (4) in a nutrient soil matrix, filling the matrix to 2/3 parts of a nutrition pot, covering the matrix with vermiculite sterilized by 0.1% carbendazim, and spraying 0.1% carbendazim liquid medicine after transplanting to prevent diseases;
(6) dipping the tender shoots at the upper section in the step (4) with 1000mg/L indolebutyric acid (IBA) solution, and cutting the tender shoots in vermiculite: in a substrate with nutrient soil =1:2, the mixed substrate is filled to 2/3 parts of a nutrition pot, then is covered by vermiculite sterilized by 0.1 percent carbendazim, and is sprayed with 0.1 percent carbendazim liquid medicine after cuttage to prevent diseases;
(7) and (3) putting the tissue culture seedlings transplanted in the step (5) and subjected to cuttage in the step (6) into a built plastic shed, wherein the relative air humidity is 95%, the plastic film is gradually opened for ventilation after 10 days of transplanting the tissue culture seedlings in the plastic shed, the humidity is reduced, the plastic film is gradually opened for ventilation after 20 days of cuttage of the tissue culture seedlings, the humidity is reduced, the small plastic shed is removed until the tissue culture seedlings are completely survived, water is properly sprayed on the tissue culture seedlings in the period, 0.1% carbendazim liquid is sprayed every 2 weeks for disease prevention, and the Hoagland nutrient solution is poured after 30 days. The final product is shown in fig. 12.
Comparative example 1
(1) Selecting healthy grape plants without diseases and insect pests in 9 months, collecting young shoots, removing leaves, cutting into single-bud stem segments after washing with running water, sterilizing for 8min and 15min respectively with sterilizing agent composed of 0.1% mercuric chloride and 0.5% tween-20 in a super clean bench, washing with sterile water for 5 times, and cutting off the cut in contact with the sterilizing agent for later use;
(2) inoculating the single-bud stem section treated in the step (1) into a culture medium for tissue culture, cutting off tender tips after axillary buds grow into tender tips, inoculating the tender tips into a new culture medium, inoculating the single-bud stem section in 9 months, wherein the axillary buds slowly germinate, the germinated tender tips grow badly, the stem tips gradually die, and finally discarding. Rooting test-tube plantlets were obtained after culturing for a period of time the sterile propagation lines successfully obtained by inoculating explants according to example 1.
(3) Subculturing the test-tube plantlets obtained in the step (2). Taking GS as a culture medium, adding 0.6mg/LIAA, 30g/L sucrose and 6g/L agar, replacing the fresh culture medium once every 60 days, cutting the test-tube plantlet into single-bud stem segments with one leaf, inoculating the single-bud stem segments into a new culture medium, and carrying out subculture propagation for 5 times. At this time, the multiplication factor was 1.67, while the multiplication factor after 9 subcultures in example 1 was 5.27.
(4) And (4) transferring the test-tube plantlets obtained in the step (3) to a greenhouse, keeping the temperature at 28 ℃, the relative humidity of air at 65% -70%, the illumination intensity of 20000Lx, closing the bottle, hardening seedlings for 2 weeks, and then taking out the bottles for transplantation.
(5) Spreading the root system of the test-tube plantlet in the step (4) in a nutrient soil matrix, filling the matrix to 2/3 parts of a nutrition pot, covering the matrix with vermiculite sterilized by 0.1% carbendazim, and spraying 0.1% carbendazim liquid medicine after transplanting to prevent diseases;
(6) placing the tissue culture seedling in a built plastic shed, gradually opening the plastic film for ventilation after 10 days, reducing the humidity until the tissue culture seedling is completely alive, removing the small plastic shed, properly spraying water to the tissue culture seedling during the period, and spraying 0.1% carbendazim solution every 2 weeks to prevent diseases.
According to the calculation of bottle-out transplantation of 500 test-tube seedlings, the transplantation survival rate is 95 percent in the example, 475 tissue culture seedlings are obtained, and the cost of a single seedling is 1.83 yuan.
Although the present invention has been described in detail in the foregoing embodiments, the description is only intended to serve as a brief description of the design of the invention, rather than as a limitation on the design of the invention, and any combination, addition or modification that does not depart from the scope of the design of the invention is intended to be within the scope of the invention.

Claims (6)

1. A method for increasing the transplanting emergence amount of test-tube grape seedlings of Violet King is characterized in that the method uses a system for increasing the transplanting emergence amount of the test-tube grape seedlings of Violet King, and comprises the following steps:
the temperature monitoring module is used for monitoring the temperature in the small plastic shed to obtain a temperature value in the shed;
the humidity monitoring module is used for monitoring the humidity in the small plastic shed to obtain a shed humidity value and a shed soil humidity value;
the illumination monitoring module is used for monitoring the illumination intensity in the small plastic shed to obtain an illumination value in the shed;
the control module is used for controlling the refrigeration device to start to cool the interior of the small plastic shed when the temperature value in the small plastic shed obtained by the temperature monitoring module is larger than the maximum value of the preset temperature range, and controlling the floor heating device to start to heat the interior of the small plastic shed when the temperature value in the shed obtained by the temperature monitoring module is smaller than the minimum value of the preset temperature range; when the humidity value in the greenhouse obtained by the humidity monitoring module is smaller than the minimum value of the preset humidity range, controlling the humidifying device to start, and humidifying the interior of the small plastic greenhouse; when the obtained humidity value in the shed is larger than the maximum value of the preset humidity range, controlling a dehumidifying device to start, and dehumidifying the interior of the small plastic shed;
when the humidity value of the soil in the shed obtained by the humidity monitoring module is smaller than the minimum value of the preset soil humidity range, controlling the irrigation device to start, and irrigating the matrix in the small plastic shed;
when the illumination value in the greenhouse obtained by the illumination monitoring module is larger than the maximum value of the preset illumination range, controlling the shading device to start, and shading the illumination supplied to the grape tissue culture seedlings in the small plastic greenhouse; when the obtained illumination value in the shed is smaller than the minimum value of the preset illumination range, controlling an illumination device to start and providing illumination for the grape tissue culture seedlings in the small plastic shed;
the method for transplanting seedling emergence amount of test-tube grape seedlings of violet king comprises the following steps:
(1) explant collection: selecting healthy grape plants without diseases and insect pests in 5-6 months, shearing young and tender young shoots, removing leaves, washing with running water, shearing into single-bud stem segments, sterilizing with sterilizing agent composed of 0.1% mercuric chloride and 0.1% tween-20 in a super clean bench for 6-8min, washing with sterile water for 4-5 times, and cutting off the cut in contact with the sterilizing agent for later use;
(2) inoculating the single-bud stem section treated in the step (1) into a culture medium for initial culture, keeping the temperature at 23-27 ℃ for illumination culture, cutting off the grown young shoots after the buds germinate, inducing the young shoots into seedlings, replacing the fresh culture medium every 35-40 days, cutting off the test-tube seedlings for successive propagation;
(3) transferring the test-tube plantlet in the step (2) to a greenhouse, keeping the temperature at 22-35 ℃, the illumination intensity at 18000Lx-30000Lx, closing the bottle, and hardening the plantlet for 2 weeks;
(4) taking out the test-tube seedlings after hardening off the seedlings, cutting off the test-tube seedlings which are strong, 5-8cm high and have more than 4 leaves from the middle, dividing the test-tube seedlings into two parts, transplanting the test-tube seedlings with roots at the lower section, and cutting the test-tube seedlings with young shoots at the upper section;
(5) spreading the root system of the seedling with root at the middle lower section in the step (4) in a substrate, filling the substrate to 2/3 parts of a nutrition pot, covering the substrate with vermiculite sterilized by 0.1% carbendazim, and spraying 0.1% carbendazim liquid medicine after transplanting to prevent diseases;
(6) dipping the tender tips of the upper section in the step (4) with 800-1000mg/L indolebutyric acid (IBA) solution, cutting the tender tips into a matrix, filling the mixed matrix into 2/3 parts of a nutrition pot, covering the mixed matrix with 0.1% carbendazim-disinfected vermiculite, and spraying 0.1% carbendazim liquid medicine after cutting to prevent diseases;
(7) placing the tissue culture seedlings subjected to the lower-stage transplanting in the step (5) and the upper-stage young shoot cuttage in the step (6) into a built small plastic shed, and gradually opening a plastic film for ventilation after 10-20 days to reduce the humidity until the tissue culture seedlings are completely survived, removing the small plastic shed, spraying water properly to the tissue culture seedlings in the period, spraying 0.1% carbendazim solution every 1-2 weeks to prevent diseases, and pouring Hoagland nutrient solution after 30 days;
selection of the medium used in said step (2) B5Or GS as a culture medium;
0.4-0.6mg/L IAA is also added into the culture medium used in the step (2);
25-30g/L of sucrose and 6g/L of agar are also added into the culture medium used in the step (2).
2. The method for increasing the transplanting emergence amount of the test-tube plantlets of the grapes called Violet King in claim 1,
and (3) inoculating single-bud stem segments with one leaf in the step (2).
3. The method for increasing the transplanting emergence amount of the test-tube plantlets of the grapes called Violet King in claim 1,
during the culture process in the step (2),
the conditions for light culture of the test-tube plantlets were light/dark =16 hours/8 hours, and red: blue: white =3:1:1 or red: the LED lamp with blue =4:1 has composite light quality and illumination intensity of 1500Lx-3000 Lx.
4. The method for increasing the transplanting emergence amount of the test-tube plantlets of the grapes called Violet King in claim 1,
in the step (5), the substrate is nutrient soil.
5. The method for increasing the transplanting emergence amount of the test-tube plantlets of the grapes called Violet King in claim 1,
in step (6), the matrix is vermiculite: nutrient soil =1: 2-2.5.
6. The method for increasing the transplanting emergence amount of the test-tube plantlets of the grapes called Violet King in claim 1,
in the step (7), the temperature in the small plastic shed is controlled to be 25-30 ℃, and the relative humidity is controlled to be 92-95%.
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CN101485261B (en) * 2009-03-10 2011-04-27 郭恩才 Minitype cuttage and propagation method in day-light greenhouse for test-tube plantlet of grape
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