CN112438201A - Tissue culture rapid propagation method of wine grapes - Google Patents
Tissue culture rapid propagation method of wine grapes Download PDFInfo
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- CN112438201A CN112438201A CN201910801972.3A CN201910801972A CN112438201A CN 112438201 A CN112438201 A CN 112438201A CN 201910801972 A CN201910801972 A CN 201910801972A CN 112438201 A CN112438201 A CN 112438201A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- 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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Abstract
The invention relates to the technical field of plant tissue culture seedling culture, in particular to a tissue culture rapid propagation method of wine grapes. The method comprises the following steps: taking dormant branches of wine grapes, cutting the dormant branches into single-bud stem sections, breaking physiological dormancy, carrying out water culture until sprouting, and sampling when the length of a young shoot reaches 10-20 cm to obtain an explant; cutting the top and base wounds of the explant, and inoculating the cut to an initial culture medium for initial culture; when young shoots grow to 5-6 cm, inoculating the young shoots into a proliferation culture medium for proliferation culture, and carrying out subculture for 3-4 generations every 40-50 days; selecting tissue culture seedlings to harden and transplant. The initial culture medium can rapidly induce the stem of the wine grape to grow new shoots or cluster buds. The new shoots or cluster buds of the multiplication culture medium after initial culture can be simultaneously germinated and rooted to induce seedlings in one step through multiplication culture, and only one multiplication culture medium needs to be prepared, so that the propagation efficiency is improved.
Description
Technical Field
The invention relates to the technical field of plant tissue culture seedling culture, in particular to a tissue culture rapid propagation method of wine grapes.
Background
The wine grape can be used for brewing wine, has large market demand and great economic benefit, so the rapid propagation of high-quality seedlings is very important. The rapid propagation technology of tissue culture can obtain a large amount of virus-free seedlings in a short time, and the domestication and transplantation technology can ensure that the tissue culture seedlings meet the requirement of large-scale field seedling culture. Different wine grapes can brew wine with different styles due to different varieties and characteristics. The wine grape 'Weidai' (Vidal blanc) belongs to white grape varieties, has thick skins, is mainly used for brewing noble rot wine or ice wine, and has a certain planting area in northeast China. 'Chardonnay' (Chardonnay) belongs to European Asia grape wine, is originally produced from French Bouguet (Bourggogong), belongs to precocious white grape variety, can be used for brewing white wine and champagne, and is planted in the main grape wine production areas in China. 'Matherland' (Marselan), a red grape variety native to France, a middle-late-maturing variety, is now gradually becoming a distinctive wine grape variety in wine grape producing areas of China.
To rapidly propagate wine grapes by tissue culture techniques, proper explant material is selected, proper medium components and plant growth regulator concentrations are set, and proper temperature and illumination culture conditions are given. In the process of acclimatizing and transplanting the tissue culture seedlings, the tissue culture seedlings are gradually adapted to the field environment through the seedling acclimatization process, and the transplanting survival rate is improved.
At present, the commonly adopted tissue culture technology comprises the following steps: the explants of the wine grape 'Henbinuo', 'Cabernet' and 'Amur grape' are taken from aseptic tissue culture seedlings, stem segments with buds and an integral leaf are cut and cultured on a start culture medium GS +20g/L sucrose +3.5 or 4g/L agar +0.002g/L sodium penicillin +0.1mg/L NAA, the pH value is 5.8, the culture environment is at the temperature of 25 +/-2 ℃, the illumination intensity is 4000-. In the existing tissue culture and rapid propagation technology of wine grapes, sterile seedlings are used as explant materials, so that the materials cannot be obtained in large quantities, a complete solution for domesticating and transplanting tissue culture seedlings is not provided, and the induced tissue culture seedlings cannot be directly applied to field planting.
Disclosure of Invention
In view of the above, the invention provides a tissue culture and rapid propagation method of wine grapes. The method is not limited by time and season, and can greatly improve the survival rate of transplanting the tissue culture seedlings to the field and improve the efficiency of large-scale field seedling culture.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a tissue culture and rapid propagation method of wine grapes, which comprises the following steps:
taking dormant branches of wine grapes, shearing the dormant branches into single-bud stem sections, breaking physiological dormancy, carrying out water culture on the single-bud stem sections until the single-bud stem sections germinate, sampling when the length of a new shoot reaches 10-20 cm, shearing the stem sections into 4-6 cm, and disinfecting to obtain explants;
cutting the top and base wounds of the explant, and inoculating the cut to an initial culture medium for initial culture; the initial culture medium is an MS culture medium containing 0.1-0.3 mg/L IBA, 0.5-1.5 mg/L, KT 0.2.2-0.8 mg/L6-BA, 2.0-6.0 mg/L adenine, 25-35 g/L sucrose and 8-9 g/L agar;
when young shoots grow to 5-6 cm, inoculating the young shoots into a proliferation culture medium for proliferation culture, and carrying out subculture for 3-4 generations every 40-50 days; the multiplication culture medium is a WPM culture medium containing 0.1-0.3 mg/L of IBA, 25-35 g/L of sucrose and 6-7 g/L of agar;
after subculture, selecting tissue culture seedlings with the plant height of more than or equal to 5cm, the root length of more than or equal to 3cm and the adventitious root of more than or equal to 3, hardening off and transplanting.
In the invention, the variety of the wine grape is Vidale, Chardonnay or Matheran.
Preferably, the upper end of the bud of the single-bud stem section is flat and 2-3 cm away from the bud, and the lower end of the bud is an inclined opening and 3-4 cm away from the bud.
Preferably, the conditions for breaking physiological dormancy are: treating the mixture in a water bath at 44-46 ℃ for 85-95 min.
Preferably, the conditions for breaking physiological dormancy are: treating with 45 deg.C water bath for 90 min.
Preferably, the hydroponic culture is: floating the single-bud stem in water by using a foam board for culturing, and changing water once after 3-4 days for culturing for 30-40 days; the light intensity of water culture is 30-40 mu mol.m-2·s-1The illumination time is 14-16 h, and the temperature is 25 +/-2 ℃.
Preferably, the procedure for sterilization is: cleaning the stem segments, soaking the stem segments in 75% ethanol for 30s, and washing the stem segments with sterile water for 2-3 times; and then soaking the stem segments in a sodium hypochlorite solution with the mass volume percentage concentration of 0.3-0.5% for 15-20 min, washing the stem segments for 4-5 times by using sterile water, and removing the water on the surfaces of the stem segments.
Preferably, the mixture is soaked for 20min by sodium hypochlorite solution with the mass volume percentage concentration of 0.4%.
Preferably, the initiation medium is MS medium containing IBA0.2mg/L, 6-BA 1.0mg/L, KT 0.5.5 mg/L, adenine 4.0mg/L, sucrose 30g/L, agar 8.2 g/L.
Preferably, the propagation medium is a WPM medium containing 0.2mg/L of IBA, 30g/L of sucrose and 6.5-7 g/L of agar.
Preferably, the conditions for initiating the culture are light intensity of 30-40 μmol · m-2·s-1The illumination time is 14-16 h, and the temperature is 25 +/-2 ℃.
Preferably, the conditions for the proliferation culture are light intensity of 30-40 μmol/m-2·s-1The illumination time is 14-16 h, and the temperature is 25 +/-2 ℃.
Preferably, the seedling exercising method comprises the following steps: and (3) under the greenhouse condition, placing the tissue culture seedlings in Hoagland semi-nutrient solution, performing film covering culture for 6-8 days, then performing Hoagland total nutrient solution culture for 6-8 days, and then uncovering the film to harden the seedlings for 1-2 weeks.
Preferably, the seedling exercising is as follows: and (3) under the greenhouse condition, placing the tissue culture seedlings in Hoagland semi-nutrient solution, performing film covering culture for 7 days, then performing Hoagland total nutrient solution culture for 7 days, and then removing the film and hardening the seedlings for 1-2 weeks.
Preferably, the transplanting is as follows: after hardening, transplanting the tissue culture seedlings into a nutrient medium covered with a greenhouse film, wherein the nutrient medium consists of garden soil, a peat medium and vermiculite, and the volume ratio of the garden soil to the peat medium to the vermiculite is 1: 1: 1; gradually opening the greenhouse film, and controlling the illumination intensity to be 90-180 mu mol.m-2·s-1Controlling the temperature at 20-28 ℃; and removing the shed film in the 4 th week after transplanting, and planting the tissue culture seedlings in the field.
The invention provides a tissue culture and rapid propagation method of wine grapes. The method comprises the following steps: taking dormant branches of wine grapes, shearing the dormant branches into single-bud stem sections, breaking physiological dormancy, carrying out water culture on the single-bud stem sections until the single-bud stem sections germinate, sampling when the length of a new shoot reaches 10-20 cm, shearing the stem sections into 4-6 cm, and disinfecting to obtain explants; cutting the top and base wounds of the explant, and inoculating the cut to an initial culture medium for initial culture; the initial culture medium is an MS culture medium containing 0.1-0.3 mg/L IBA, 0.5-1.5 mg/L, KT 0.2.2-0.8 mg/L6-BA, 2.0-6.0 mg/L adenine, 25-35 g/L sucrose and 8-9 g/L agar; when young shoots grow to 5-6 cm, inoculating the young shoots into a proliferation culture medium for proliferation culture, and carrying out subculture for 3-4 generations every 40-50 days; the multiplication culture medium is a WPM culture medium containing 0.1-0.3 mg/L of IBA, 25-35 g/L of sucrose and 6-7 g/L of agar; after subculture, selecting tissue culture seedlings with the plant height of more than or equal to 5cm, the root length of more than or equal to 3cm and the adventitious root of more than or equal to 3, hardening off and transplanting. The invention has the following technical effects:
(1) the invention takes the new shoot of the dormant branch after water culture as the explant material, and the water culture is used for promoting the germination after the branch dormancy is broken through the warm water bath, thereby reducing the tissue culture experiment pollution risk caused by directly taking materials from the field, expanding the time range of taking materials and leading the materials to be more convenient.
(2) The invention adopts 75% ethanol and low concentration sodium hypochlorite (0.3% -0.5%) solution as the disinfectant of the explant in the tissue culture experiment, has low toxicity, proper concentration and good disinfection effect, and reduces the difficulty of disinfection operation.
(3) The initial culture medium of the invention is MS + IBA0.2mg/L +6-BA 1.0mg/L + KT 0.5mg/L + adenine 4.0mg/L + sucrose 30g/L + agar 8.2g/L, and induces the stem of the wine grape to grow new shoots or cluster buds.
(4) The multiplication culture medium is WPM + IBA0.2mg/L + sucrose 30g/L + agar 6.5-7 g/L, and the young shoots or cluster buds after initial culture can be simultaneously germinated and rooted through multiplication culture to induce seedling in one step, and only one multiplication culture medium needs to be prepared, so that the propagation efficiency is improved.
(5) The invention needs to make the tissue culture seedling pass through the water culture hardening process of Hoagland nutrient solution before domestication and transplantation, has simple operation and high water culture survival rate of more than 90 percent, makes the tissue culture seedling gradually adapt to the subsequent domestication and transplantation environment, and increases the transplantation survival rate.
(6) The invention transplants the tissue culture seedling after water culture seedling hardening into the nutrition pot during domestication and transplantation, and gradually adapts to the low-humidity and strong-light environment by means of gradually uncovering the simple shed film to reduce the environmental humidity, gradually increasing the illumination and the like, so as to achieve the effect that the transplanting survival rate of the tissue culture seedling is more than 85 percent.
Detailed Description
The invention discloses a tissue culture and rapid propagation method of wine grapes, which can be realized by appropriately improving process parameters by referring to the content of the culture medium. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
Interpretation of terms:
explants (explants): plant tissue culture uses isolated organs (roots, stems, leaves, stem tips, flowers, fruits, etc.), tissues (cambium, epidermis, cortex, myelin cells, endosperm, etc.) or cells of a plant body and protoplasts, called explants.
Contamination (contamination): the culture medium or culture material has a phenomenon that microorganisms such as fungi or bacteria grow.
Sterilization (sterilation): the process of killing all microbes including spore, etc. through physical, chemical and physical and chemical methods.
Explant browning (explant browning): in the isolated culture, the explants are damaged by surface disinfectants and shearing, have brown appearance and die when the appearance is serious.
Initial culture (initiation culture): inoculating the sterilized explant to a sterile culture medium, and inducing callus or adventitious buds under proper culture conditions after the explant is alive.
Proliferation culture (proliferation culture): the process of periodically dividing the culture material (propagule), transferring the divided culture material into a fresh culture medium and continuing the culture is also called subculture.
Proliferation coefficient (proliferation coeffecient): the multiplication multiple of the test-tube plantlet in one culture period is the average multiplication number of 1 propagule after one period of culture.
Tissue culture plantlets (plantlets): plant tissue, organ or cell is used as explant, and plant tissue culture technology is adopted to obtain complete plant with complete root, stem and leaf.
Seedling exercising (plantlet hardening): the sterile tissue culture seedlings are moved from the culture room to the outdoor environment close to the natural environment for culture, and the lignification degree of the seedlings and the adaptability to the external environment (natural illumination and temperature and humidity change) are improved.
Domestication (acclimatization): the tissue culture seedling changes the growth state of the tissue culture seedling to adapt to the external environment (natural illumination and temperature and humidity change) so as to survive the tissue culture seedling.
Transplanting (transplanting): the domesticated tissue culture seedlings are planted in a matrix and gradually adapt to the process under natural conditions in a greenhouse or other protection facility conditions.
MS medium was designed in 1962 by Murashige and Skoog for culturing tobacco cells. It features high concentration of inorganic salt (nitrate) and ions, and wide application in culturing plant organs, anthers, cells and protoplasts.
WPM (wood plant medium) medium is a low-salt medium, designed for stem tip culture of Laurel (Kalmia latifolia) by Lloyd and McCown in 1981, and widely used for woody plant culture. Compared with MS culture medium, the use concentration of WPM macroelement is low, and NH4 is reduced+、NO3-Potassium sulfate was used instead of potassium nitrate, and the ammonium nitrate concentration corresponded to 1/4 MS. The variety of trace elements is reduced.
The plant growth regulating substance is artificial plant hormone substances, such as IAA, IBA, NAA, 2, 4-D of auxin, GA3 of gibberellin, KT, 6-BA, TDZ, ZT of cytokinin, etc.
Hoagland (Hoagland) nutrient solution was developed by Hoagland and Arnon in 1938, and improved in 1950, and is suitable for water culture of most plants. The concentrations of each element used were N210 ppm (Mg/L), K235 ppm, Ca 200ppm, P31 ppm, S64 ppm, Mg 48ppm, B0.5ppm, Fe 1-5 ppm, Mn 0.5ppm, Zn 0.05ppm, Cu 0.02ppm and Mo 0.01 ppm. On the basis, the culture solution formula can be improved for different crops. The following table shows the ingredients of the modified nutritional liquids used in the present invention.
TABLE 1 modified Hoagland Total nutrient solution ingredients and amounts
The culture medium, the material, the matrix and the like used in the tissue culture and rapid propagation method of the wine grapes can be purchased from the market.
The invention is further illustrated by the following examples:
example 1
(1) Explant Collection
Collecting dormant branches of virus-free wine grape 'Weidai', and cutting the dormant branches into single-bud stem sections, wherein the upper ends of the buds are flat, 2-3 cm away from the buds, the lower ends of the buds are oblique, and the distance between the buds is 3-4 cm. And (3) breaking physiological dormancy by treating the stem in a constant-temperature water bath at 45 ℃ for 90min, floating the single-bud stem in clear water by using a foam plate for culturing, changing water once after 3-4 days, and germinating after culturing for 30-40 days. The culture environment is light intensity of 30-40 mu mol.m-2·s-1(i.e. 2000 Lx-3000 Lx), the illumination time is 14-16 h, and the temperature is 25 +/-2 ℃. And sampling when the young shoots reach 10-20 cm in length, and cutting into 5cm stem sections to be used as explant materials.
(2) Explant surface disinfection
Putting the stem segments into a container, soaking the stem segments in detergent for 5-10 min, then washing the stem segments for 2-3 h with running water, finally washing the stem segments with distilled water, and transferring the stem segments into a super-clean workbench. Soaking the mixture in 75% ethanol for 30s in a superclean workbench, and then washing the mixture for 2-3 times by using sterile water; and then disinfecting with a low-concentration sodium hypochlorite (0.4%) solution for a long time (20min), washing with sterile water for 4-5 times, and finally absorbing excess water on the surface of the stem segment by using sterilized filter paper for inoculation for later use.
(3) Inoculation of explants
In a sterile inoculation vessel, cutting the top end and the base part of a young shoot of the explant, reducing the influence of a disinfectant on the wound and the young shoot tip, inoculating the cut and the young shoot tip on an initial culture medium, culturing for 3 days, cutting the explant without pollution and browning into single-bud stem sections again, and transferring the single-bud stem sections on the initial culture medium. And uniformly inoculating 3-4 buds or single buds in each 100ml culture bottle.
(4) Initial culture
The initial culture medium is MS + IBA0.2mg/L +6-BA 1.0mg/L + KT 0.5mg/L + adenine 4.0mg/L + sucrose 30g/L + agar 8.2g/L, and is used after being sterilized by high pressure steam at 121 ℃ for 20 min. The initial culture environment is light intensity of 30-40 μmol/m-2·s-1(or 2000 Lx-3000 Lx), the illumination time is 14-16 h, and the temperature is 25 +/-2 ℃.
And (4) germinating axillary buds of the inoculated stem segments within 10-15 d, and performing subculture multiplication when the young shoots grow to 5-6 cm (about 30 d).
(5) Proliferation culture
And inoculating the new shoots or cluster buds formed by the primary culture induction to a proliferation culture medium for culture, and subculturing every 40-50 days. The multiplication culture medium is WPM + IBA0.2mg/L + sucrose 30g/L + agar 6.5-7 g/L, and is used after being sterilized by high-pressure steam at 121 ℃ for 20min, so that the multiplication culture medium has dual functions of germination and rooting, and can enable young shoots or multiple shoots to be grown into seedlings in one step. The culture environment is the same as the initial culture.
The second generation of the 'wital' has a multiplication coefficient of 3.6, sprouts and roots, and has developed root system and higher density of main roots, and leaves and root systems have no browning condition.
(6) Water culture hardening seedling
Culturing the tissue culture seedlings for 35-40 days per generation, selecting the tissue culture seedlings cultured to the plant height of not less than 5cm, the root system length of not less than 3cm and the adventitious root of not less than 3 after 3-4 generations of subculture, washing with water to remove the root culture medium, and fixing on a foam floating seedling culture tray (the seedling culture tray is soaked in 0.1% potassium permanganate solution for 4 hours before use and then cleaned with clear water for use). Under the condition of natural scattered light of a greenhouse, placing the tissue culture seedling hole tray in a transparent plastic box, covering the film with improved Hoagland semi-nutrient solution (reducing the number of macroelements by half) for culturing for 1 week, then culturing for 1 week with improved Hoagland full-nutrient solution, and then uncovering the film and hardening seedlings for 1-2 weeks. And (5) observing whether the tissue culture seedlings wither or not during membrane removal, and performing membrane removal culture if the tissue culture seedlings do not wither after 2 hours. The nutrient solution needs to be replaced every week. The survival rate of the 'wital' water culture is 94.4 percent.
(7) Domesticated transplantation
After water culture and seedling hardening, transplanting the tissue culture seedlings in a nutrition pot, and standing bamboo sticks and binding the seedlings. Adding soil into a nutrition pot (12X 12): peat substrate: 1, vermiculite: 1: 1, and mixing the culture medium uniformly. Building a simple greenhouse film around the nutrition pot, keeping 100% relative humidity in the early stage of transplanting, and ventilating at irregular time; after 1-2 weeks, lifting the periphery of the greenhouse film, and covering the greenhouse film in a floating manner, so that moisture and air are preserved; after the first new leaf was fully opened, the film was gradually opened to reduce the humidity. During the culture period, proper watering is carried out to keep the soil humidity, and water is controlled in the later period to prevent root rot. The illumination intensity is controlled at 90 mu mol.m-2·s-1～180μmol·m-2·s-1(5000 Lx-10000 Lx), and the temperature is controlled to be 20-28 ℃. And removing the covering on the 4 th week after transplanting, and planting the tissue culture seedlings in the field. The survival rate of the Weidall transplantation is 90.0 percent.
Comparative example 1:
dormant branches of virus-free wine grapes 'wital' are collected, the dormant branches are subjected to water culture treatment, the surface of the explant is disinfected, the explant is inoculated, and the initial culture conditions are the same as those of example 1. Only the formulation of the proliferation medium was changed: WPM + IBA0.2mg/L +6-BA 0.5mg/L + sucrose 30g/L + agar 6.5-7 g/L. The growth coefficient in the growth medium was 4.0, but the number of adventitious roots was large.
Example 2:
the dormant branches of the virus-free wine grape 'Chardonnay' are collected, the dormant branches are subjected to water culture treatment, the surface of the explant is disinfected, the explant is inoculated, and the initial propagation culture conditions are the same as the example I. In the enrichment culture, when the culture medium conditions are WPM + IBA0.2mg/L + sucrose 30g/L + agar 6.5-7 g/L, the second-generation multiplication coefficient of Chardonnay' is 4.5, the germination and rooting are realized, the root system is developed, the density of the main root is high, and the leaves and the root system are not browned; the steps of water culture hardening seedling and domestication transplanting are the same as the example I, the water culture survival rate of Chardonnay is 96.3 percent, and the transplanting survival rate is 85.0 percent.
Comparative example 2:
dormant branches of virus-free wine grape 'Chardonnay' are collected, the dormant branches are subjected to water culture treatment, the surface of the explant is disinfected, and the explant is inoculated and cultured initially under the same conditions as in example 2. Only the formulation of the proliferation medium was changed: WPM + IBA0.2mg/L +6-BA 0.5mg/L + sucrose 30g/L + agar 6.5-7 g/L. The growth coefficient in the growth medium was 2.6.
Example 3:
the dormant branches of virus-free wine grape Matherland are collected, the dormant branches are subjected to water culture, the surface of the explant is disinfected, the explant is inoculated, and the culture conditions for initial proliferation are the same as the example I. In the proliferation culture, when the conditions of a culture medium are WPM, IBA0.2mg/L, sucrose 30g/L and agar 6.5-7 g/L, the second-generation proliferation coefficient of Matherland is 2.9, the germination and rooting are realized, the root system is developed, the density of the main root is high, and the leaves and the root system are not browned; the steps of water culture hardening seedling and domestication transplanting are the same as the example I, the water culture survival rate of the Matheran is 93.6%, and the transplanting survival rate is 85.0%.
Comparative example 3:
dormant branches of virus-free wine grape "mashelan" were collected, the dormant branches were hydroponically treated, the surface of the explant was sterilized, the explant was inoculated, and the initial culture conditions were the same as in example 3. Only the formulation of the proliferation medium was changed: WPM + IBA0.2mg/L +6-BA 0.5mg/L + sucrose 30g/L + agar 6.5-7 g/L. The growth coefficient in the growth medium was 3.3.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A tissue culture and rapid propagation method of wine grapes is characterized by comprising the following steps:
taking dormant branches of wine grapes, shearing the dormant branches into single-bud stem sections, breaking physiological dormancy, carrying out water culture on the single-bud stem sections until the single-bud stem sections germinate, sampling when the length of a new shoot reaches 10-20 cm, shearing the stem sections into 4-6 cm, and disinfecting to obtain explants;
cutting the top and base wounds of the explant, and inoculating the cut to an initial culture medium for initial culture; the initial culture medium is an MS culture medium containing 0.1-0.3 mg/L IBA, 0.5-1.5 mg/L, KT 0.2.2-0.8 mg/L6-BA, 2.0-6.0 mg/L adenine, 25-35 g/L sucrose and 8-9 g/L agar;
when young shoots grow to 5-6 cm, inoculating the young shoots into a proliferation culture medium for proliferation culture, and carrying out subculture for 3-4 generations every 40-50 days; the multiplication culture medium is a WPM culture medium containing 0.1-0.3 mg/L of IBA, 25-35 g/L of sucrose and 6-7 g/L of agar;
after subculture, selecting tissue culture seedlings with the plant height of more than or equal to 5cm, the root length of more than or equal to 3cm and the adventitious root of more than or equal to 3, hardening off and transplanting.
2. The tissue culture rapid propagation method according to claim 1, wherein the upper end of the bud of the single bud stem section is flat and 2-3 cm away from the bud, and the lower end is oblique and 3-4 cm away from the bud.
3. The tissue culture rapid propagation method according to claim 1, wherein the condition for breaking physiological dormancy is as follows: treating the mixture in a water bath at 44-46 ℃ for 85-95 min.
4. The tissue culture rapid propagation method according to claim 1, wherein the water culture is: floating the single-bud stem in water by using a foam board for culturing, and changing water once after 3-4 days for culturing for 30-40 days; the light intensity of water culture is 30-40 mu mol.m-2·s-1The illumination time is 14-16 h, and the temperature is 25 +/-2 ℃.
5. The tissue culture rapid propagation method according to claim 1, wherein the disinfection procedure is as follows: cleaning the stem segments, soaking the stem segments in 75% ethanol for 30s, and washing the stem segments with sterile water for 2-3 times; and then soaking the stem segments in a sodium hypochlorite solution with the mass volume percentage concentration of 0.3-0.5% for 15-20 min, washing the stem segments for 4-5 times by using sterile water, and removing the water on the surfaces of the stem segments.
6. The tissue culture rapid propagation method according to claim 1, wherein the initiation medium is MS medium containing IBA0.2mg/L, 6-BA 1.0mg/L, KT 0.5.5 mg/L, adenine 4.0mg/L, sucrose 30g/L, agar 8.2 g/L.
7. The tissue culture rapid propagation method according to claim 1, wherein the propagation medium is a WPM medium containing IBA0.2mg/L, sucrose 30g/L and agar 6.5-7 g/L.
8. The tissue culture rapid propagation method according to claim 1, wherein the condition for initiating the culture is that the light intensity is 30-40 μmol-m-2·s-1The illumination time is 14-16 h, and the temperature is 25 +/-2 ℃;
the condition of the proliferation culture is that the light intensity is 30-40 mu mol.m-2·s-1The illumination time is 14-16 h, and the temperature is 25 +/-2 ℃.
9. The tissue culture rapid propagation method according to claim 1, characterized in that the seedling exercising is: and (3) under the greenhouse condition, placing the tissue culture seedlings in Hoagland semi-nutrient solution, performing film covering culture for 6-8 days, then performing Hoagland total nutrient solution culture for 6-8 days, and then uncovering the film to harden the seedlings for 1-2 weeks.
10. The tissue culture rapid propagation method according to any one of claims 1 to 9, wherein the transplanting is: after hardening, transplanting the tissue culture seedlings into a nutrient medium covered with a greenhouse film, wherein the nutrient medium consists of garden soil, a peat medium and vermiculite, and the volume ratio of the garden soil to the peat medium to the vermiculite is 1: 1: 1; gradually opening the greenhouse film, and controlling the illumination intensity to be 90-180 mu mol.m-2·s-1Controlling the temperature at 20-28 ℃; and removing the shed film in the 4 th week after transplanting, and planting the tissue culture seedlings in the field.
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| CN114698549A (en) * | 2022-04-18 | 2022-07-05 | 新疆农业大学 | Tissue culture medium and tissue culture method for rapid propagation of grape rootstock stem |
| CN115316271A (en) * | 2022-07-26 | 2022-11-11 | 江苏省农业科学院宿迁农科所 | Tissue culture method of muscadine |
| CN115413581A (en) * | 2022-10-09 | 2022-12-02 | 西北农林科技大学 | Plant proliferation culture medium and method for promoting regeneration of adventitious roots of plant tissue culture seedlings by using same |
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| CN114698549A (en) * | 2022-04-18 | 2022-07-05 | 新疆农业大学 | Tissue culture medium and tissue culture method for rapid propagation of grape rootstock stem |
| CN114698549B (en) * | 2022-04-18 | 2023-05-23 | 新疆农业大学 | Tissue culture medium and tissue culture method for rapid propagation of grape stock stem segments |
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| CN115413581A (en) * | 2022-10-09 | 2022-12-02 | 西北农林科技大学 | Plant proliferation culture medium and method for promoting regeneration of adventitious roots of plant tissue culture seedlings by using same |
| CN115413581B (en) * | 2022-10-09 | 2023-11-10 | 西北农林科技大学 | Plant proliferation culture medium and method for promoting plant tissue culture seedling adventitious root regeneration |
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