CN112293255A - Tissue culture rapid propagation method for large cherry rootstock Gisela No. 6 - Google Patents
Tissue culture rapid propagation method for large cherry rootstock Gisela No. 6 Download PDFInfo
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- CN112293255A CN112293255A CN202011224241.6A CN202011224241A CN112293255A CN 112293255 A CN112293255 A CN 112293255A CN 202011224241 A CN202011224241 A CN 202011224241A CN 112293255 A CN112293255 A CN 112293255A
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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
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Abstract
The invention provides a tissue culture and rapid propagation method for large cherry rootstock Gisela No. 6, which comprises the steps of explant disinfection establishment, primary culture, subculture, rooting culture and transplantation domestication. The invention simplifies the transplanting steps, improves the rooting rate and the transplanting survival rate, reduces the production cost of the nursery stock to 70 percent, further improves the quality of the nursery stock to meet the market demand, and simultaneously provides theoretical basis for the propagation of virus-free nursery stock of the future cherries, the preservation of germplasm resources and the genetic improvement of the cherries by utilizing the gene technology.
Description
Technical Field
The invention belongs to the technical field of artificial propagation of plants, and particularly relates to a tissue culture rapid propagation method for large cherry rootstock Gisela No. 6.
Background
The large cherry is a fruit tree of Prunus of Rosaceae, which is sour and sweet, rich in multiple vitamins, microelements, etc., and has high edible value, and is popular in recent years and has short supply in the market. The plant tissue culture technology has the advantages of high propagation speed, no influence of seasons and the like, can obviously shorten the seedling culture period, and can obtain a large number of high-quality seedlings in a short time. In the prior art, the method of dwarf stock close planting can realize early bearing and early high yield of large cherry fruit trees, and the stock is selected for popularizing the core technology of dwarf stock close planting of large cherry. Through regional and production test observation for many years, the Jisaila No. 6 has better grafting affinity with most of large cherries, also has the advantages of obvious early bearing property, early high yield, disease resistance, waterlogging resistance, wide soil application range and the like, and is an excellent rootstock which is rare for popularizing the thick planting of the short rootstocks of the large cherries. The number 6 of the Geisera is triploid clone stock, which can not be propagated by seeds, and the traditional propagation modes are layering, root breeding and cuttage, but have the problems of low propagation speed and inconsistent growth, can not obtain a large amount of stock seedlings with high quality in time, and can not meet the production requirement. At present, the problems of bottle seedling vitrification, low rooting rate, low transplanting survival rate and the like in the production process of the Gisela No. 6 in the tissue culture and propagation process due to unreasonable culture medium formula are solved.
Disclosure of Invention
Aiming at the explanation of the background technology, the invention provides a tissue culture rapid propagation method for large cherry rootstock Gisela 6, and solves the technical problems of vitrification, plant stem tip necrosis, growth emaciation, low rooting rate and low transplanting survival rate of the Gisela 6 in the tissue culture rapid propagation process.
In order to achieve the purpose, the invention provides the following technical scheme:
a tissue culture rapid propagation method for large cherry rootstock Gisela No. 6 comprises the following steps:
step one, explant disinfection establishment: selecting strong and healthy annual Gesela No. 6 stock branches without diseases and insect pests in the middle ten days of 4 months to 5 months in spring, cutting the strong and healthy Gesela No. 6 stock branches into single-bud stem sections of about 2-3cm, cleaning the stem sections for 2min by using detergent, and washing for 2h by running water; then operating in a clean bench, and sterilizing the used utensils and tools at high temperature and high pressure; transferring the stem segments into a sterile bottle by using a forceps, pouring 75% alcohol, disinfecting for 10-15s by using the alcohol, quickly pouring out, cleaning for 3 times by using sterile water, then disinfecting and disinfecting for 6-8min by using mercuric chloride with the concentration of 1g/L, adding 1-2 drops of Tween 20, pouring out the mercuric chloride, cleaning for 6 times by using the sterile water, and continuously shaking each time to ensure that the cleaning is thorough to obtain an explant;
step two, primary culture: cutting off wounds at the upper end and the lower end of a stem section, which are in contact with mercuric chloride, of the explant, peeling off a leaf stalk, vertically inoculating the cut explant into a primary culture medium, placing 2 explants in each bottle, and then placing the bottles in a culture room for culture under the conditions that the temperature is 24 +/-2 ℃, the illumination intensity is 4000Lux, the photoperiod is 16h/8h in daytime/darkness, and the growth period is 4 weeks;
step three, subculture, namely, cutting off a new bud of about 1.5-2cm, which is germinated from a stem section, of the explant after 4 weeks of primary culture, transferring the bud to the subculture for multiplication culture, and expanding the amount of tissue culture seedlings, wherein the culture conditions are the same as those of the primary culture, the growth period is 4 weeks, and the multiplication coefficient is 3-4;
step four, rooting culture: selecting strong plants with the height of 2-2.5cm from the proliferated seedlings, cutting off base leaves, and vertically inoculating the plants into a rooting culture medium for rooting culture; the culture conditions are that the temperature is 22 +/-2 ℃, the illumination intensity is 2000-;
step five, transplanting and domesticating: after rooting culture is carried out for 2 weeks, transplanting and domesticating after the root system of the tissue culture seedling reaches 1cm, wherein the transplanting and domesticating medium is peat: coconut husk: perlite is 12:5: 3; placing the prepared substrate into a 50-hole forest wood plug for standby, taking the rooted plants out of a bottle, washing the culture medium from clear water, soaking the whole plants in 1000 times of carbendazim for 5s, vertically planting the plants into the prepared substrate, placing the plants into a pre-prepared small arched shed for acclimation, paying attention to proper ventilation during the acclimation, ensuring that the air humidity is over 90 percent in 1 week before the acclimation, ensuring that the temperature is between 24 and 26 ℃, sending out new roots after 1 week, gradually reducing the temperature and humidity requirements, removing a plastic film after 3 weeks, and transferring the plants into a hardening chamber for rapid growth and lignification exercise.
In the technical scheme, the bactericide comprises etoposide, removazide, carbendazim and tetramycin, and the insecticide comprises imidacloprid, deltamethrin, cypermethrin and pyridaben.
In the technical scheme, the primary culture medium comprises an improved MS culture medium, 0.5-0.8 mg/L6-benzylamino adenine and 0.06-0.1mg/L indolebutaneAcid, 30g/L sucrose and 6g/L agar, the hydrogen ion concentration index is 5.8-6.0, and the agar strength is 1300kg/cm2The primary medium was sterilized at 121 ℃ for 20 min.
In the technical scheme, the subculture medium comprises an improved MS culture medium, 0.8-1 mg/L6-benzylamino adenine, 0.02-0.06mg/L indolebutyric acid, 30g/L sucrose and 6g/L agar, the hydrogen ion concentration index value is 5.8-6.0, the agar strength is 1300kg/cm2, and the subculture medium is sterilized for 20min at 121 ℃.
In the technical scheme, the improved MS culture medium comprises macroelements, microelements and organic matters, wherein the macroelements comprise 1650mg/L ammonium nitrate, 950mg/L potassium nitrate, 370mg/L magnesium sulfate heptahydrate, 170mg/L potassium dihydrogen phosphate, 980mg/L calcium nitrate tetrahydrate, 780mg/L potassium sulfate and 440mg/L calcium chloride dihydrate; the microelements comprise 16.9mg/L of manganese sulfate monohydrate, 8.6mg/L of zinc sulfate heptahydrate, 6.2mg/L of boric acid, 0.83mg/L of potassium iodide, 0.025mg/L of copper sulfate pentahydrate, 0.025mg/L, EDDHA mg/L of cobalt chloride hexahydrate, 100mg/L of chelated iron and 0.25mg/L of sodium molybdate dihydrate, and the organic substances comprise 2mg/L of glycine, 0.5mg/L of pyridoxine, 0.5mg/L of nicotinic acid, 0.1mg/L of thiamine and 100mg/L of inositol.
In the technical scheme, the rooting medium is an improved MS medium, wherein macroelements are reduced by half, microelements and organic matters are kept unchanged, the rooting medium also comprises 0.2-0.5mg/L of indolebutyric acid and 0.5-1mg/L of naphthylacetic acid, the content of sucrose is 20g/L, the content of agar is 6g/L, the hydrogen ion concentration index value is 5.8-6.0, the strength of the agar is 1300kg/cm2, and the rooting medium is sterilized for 20min at 121 ℃.
The method improves the commonly used MS culture medium, reduces the content of potassium nitrate, simultaneously increases the calcium nitrate tetrahydrate and the potassium sulfate, directly solves the problems of vitrification and stem tip necrosis of the cherry tissue culture seedlings in the production process, does not need a strong seedling culture stage, does not need to close and open the bottle for hardening seedlings in the transplanting and domesticating process, directly transplants the rooted seedlings to a substrate, and then puts the matrix into a small arched shed for domestication, and only needs 3 weeks in the whole transplanting and domesticating process, thereby greatly saving the labor cost, shortening the production period of the seedlings, and improving the rooting rate and the transplanting survival rate of the bottle seedlings. The technology simplifies the transplanting step, reduces the production cost of the nursery stock to 70%, improves the quality of the nursery stock to meet the market demand, and provides a theoretical basis for the propagation of virus-free nursery stock of the cherry in the future, the preservation of germplasm resources and the genetic improvement of the cherry by utilizing a gene technology.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A tissue culture rapid propagation method for large cherry rootstock Gisela No. 6 comprises the following steps:
step one, explant disinfection establishment: selecting strong and healthy annual Gesela No. 6 stock branches without diseases and insect pests in the middle ten days of 4 months to 5 months in spring, cutting the strong and healthy Gesela No. 6 stock branches into single-bud stem sections of about 2-3cm, cleaning the stem sections for 2min by using detergent, and washing for 2h by running water; then operating in a clean bench, and sterilizing the used utensils and tools at high temperature and high pressure; transferring the stem segments into a sterile bottle by using a forceps, pouring 75% alcohol, disinfecting for 10-15s by using the alcohol, quickly pouring out, cleaning for 3 times by using sterile water, then disinfecting and disinfecting for 6-8min by using mercuric chloride with the concentration of 1g/L, adding 1-2 drops of Tween 20, then pouring out the mercuric chloride, cleaning for 6 times by using the sterile water, and continuously shaking each time to ensure that the cleaning is thorough to obtain the explant.
Step two, primary culture: cutting off wounds at the upper end and the lower end of a stem section, which are in contact with mercuric chloride, of the explant, peeling off a leaf stalk, vertically inoculating the cut explant into a primary culture medium, placing 2 explants in each bottle, and then placing the bottles in a culture room for culture under the conditions that the temperature is 24 +/-2 ℃, the illumination intensity is 4000Lux, the photoperiod is 16h/8h in daytime/darkness, and the growth period is 4 weeks;
step three, subculturing: germinating explant buds after 4 weeks of primary culture, cutting new buds of about 1.5-2cm from the buds of stem segments, transferring to subculture for proliferation culture, and expanding the amount of tissue culture seedlings, wherein the culture conditions are the same as those of the primary culture, the growth cycle is 4 weeks, and the proliferation coefficient is 3-4; 6-benzylaminopurine and indolebutyric acid are added into a culture medium in a propagation culture stage, an optimal hormone proportion is selected, and the problems of vitrification and plant growth emaciation are solved under the combined action of the optimal hormone proportion and the improved MS.
Step four, rooting culture: selecting strong plants with the height of 2-2.5cm from the proliferated seedlings, cutting off base leaves, and vertically inoculating the plants into a rooting culture medium for rooting culture; the culture conditions are that the temperature is 22 +/-2 ℃, the illumination intensity is 2000-.
Step five, transplanting and domesticating: after rooting culture is carried out for 2 weeks, transplanting and domesticating after the root system of the tissue culture seedling reaches 1cm, wherein the transplanting and domesticating medium is peat: coconut husk: perlite is 12:5: 3; placing the prepared substrate into a 50-hole forest tree plug for standby use, taking the rooted plants out of a bottle, washing the culture medium from clear water, soaking the whole plants in carbendazim for 5s, vertically planting the plants into the prepared substrate, placing the plants into a small pre-prepared arched shed for acclimation, paying attention to proper ventilation during the acclimation, ensuring that the air humidity is over 90 percent and the temperature is between 24 and 26 ℃ in 1 week before the acclimation, and gradually reducing the requirements on the temperature and the humidity after new roots are sent out after 1 week. During the domestication period, diluted bactericide and pesticide are sprayed to prevent diseases and pests, the bactericide and pesticide are used according to the use instructions, after 3 weeks, the plastic film can be removed, and the plastic film is moved into a hardening room for rapid growth and lignification exercise.
The improved MS culture medium contains macroelements, microelements and organic matters, the types of compounds contained in the macroelements and the concentrations of working solutions of the compounds are 1650mg/L of ammonium nitrate, 950mg/L of potassium nitrate, 370mg/L of magnesium sulfate heptahydrate, 170mg/L of monopotassium phosphate, 980mg/L of calcium nitrate tetrahydrate, 780mg/L of potassium sulfate and 440mg/L of calcium chloride dihydrate respectively; the compound types and working solution concentrations of the trace elements are respectively 16.9mg/L of manganese sulfate monohydrate, 8.6mg/L of zinc sulfate heptahydrate, 6.2mg/L of boric acid, 0.83mg/L of potassium iodide, 0.025mg/L of copper sulfate pentahydrate, 150mg/L of cobalt chloride hexahydrate, and 0.25mg/L of sodium molybdate dihydrate, and the compound types and working solution concentrations of the organic matters are respectively 2mg/L of glycine, 0.5mg/L of pyridoxine, 0.5mg/L of nicotinic acid, 0.1mg/L of thiamine and 100mg/L of inositol.
Primary culture medium: the primary culture medium comprises the components in an improved MS culture medium, the content of the components is the same as that of the working solution raw materials, the content of 6-benzylaminopurine is 0.5-0.8mg/L, the content of indolebutyric acid is 0.02-0.6mg/L, the content of sucrose is 30g/L, the content of agar is 6g/L, the hydrogen ion concentration index is 5.8-6.0, the agar strength is 1300kg/cm2, and the culture medium is sterilized for 20min at 121 ℃.
Subculture medium: the subculture medium comprises the components in an improved MS culture medium, the content of the components is the same as that of the working solution raw materials, the content of 6-benzylaminopurine is 0.8-1mg/L, the content of indolebutyric acid is 0.06-0.1mg/L, the content of sucrose is 30g/L, the content of agar is 6g/L, the hydrogen ion concentration index value is 5.8-6.0, the agar strength is 1300kg/cm2, and the culture medium is sterilized for 20min at 121 ℃.
Rooting culture medium: the rooting culture medium is 1/2 modified MS culture medium, namely the content of macroelements in 1L modified MS culture medium is halved, the content of trace elements and organic matters is kept unchanged, the types of hormones and the concentrations of working solution are 0.2-0.5mg/L of indolebutyric acid, 0.5-1mg/L of naphthylacetic acid, 20g/L of sucrose, 6g/L of agar, 5.8-6.0 of hydrogen ion concentration index value and 1300kg/cm2 of agar, and the culture medium is sterilized for 20min at the temperature of 121 ℃.
The first embodiment is as follows: the preparation method of the macroelement solution, the trace element solution and the organic matter solution in the MS culture medium comprises the steps of preparing 40 times of 1L of mother liquor from macroelements for standby, preparing 2 kinds of mother liquor from the macroelements due to the fact that calcium ions and sulfate ions in the macroelements can be combined to generate calcium sulfate precipitation, preparing calcium chloride dihydrate and calcium nitrate tetrahydrate for convenient distinguishing, and mixing and preparing the mother liquor 1 from calcium chloride dihydrate and the calcium nitrate tetrahydrate, and mixing and preparing the rest 5 kinds of compounds 2; preparing 200 times of 1L of mother liquor with microelements for later use, and naming the mother liquor as mother liquor 3; the organic matter is prepared into mother liquor of 200 times 1L for standby, and the mother liquor is named as mother liquor 4.
Preparing a mother solution 1: weighing 17.6g of calcium chloride dihydrate and 39.2g of calcium nitrate tetrahydrate, fully dissolving and mixing uniformly in turn, and transferring into a volumetric flask to fix the volume to 1L.
Preparing a mother solution 2: weighing 66g of ammonium nitrate, 38g of potassium nitrate, 14.8g of magnesium sulfate heptahydrate, 6.8g of monopotassium phosphate and 31.2g of potassium sulfate, fully dissolving and uniformly mixing the materials in turn, and transferring the mixture into a volumetric flask to achieve a constant volume of 1L.
Preparation of mother liquor 3: weighing 3.38g of manganese sulfate monohydrate, 1.72g of zinc sulfate heptahydrate, 1.24g of boric acid, 0.166g of potassium iodide, 0.005g of copper sulfate pentahydrate, 0.005g of cobalt chloride hexahydrate and 0.05g of sodium molybdate dihydrate, fully dissolving and mixing uniformly in turn, and transferring into a volumetric flask to keep the volume to 1L.
Preparing a mother solution 4: weighing 0.4g of glycine, 0.1g of pyridoxine, 0.1g of nicotinic acid, 0.02g of thiamine and 20g of inositol, fully dissolving and mixing uniformly in turn, and transferring into a volumetric flask to fix the volume to 1L.
Example two: 100mL of each solution of 1mg/L plant growth regulating hormone 6-benzylamino adenine, indolebutyric acid and naphthylacetic acid is prepared.
6-benzylamino adenine is one of cytokinins, promotes cell division and bud formation, promotes differentiation of non-differentiated tissues, and induces formation of callus; indolebutyric acid is one of auxin, promotes the growth of stem and branch, and can promote the growth of root, especially adventitious root; naphthylacetic acid is one of the growth factors, and can promote the growth of stems and branches and promote rooting.
Preparing a 6-benzylamino adenine solution: weighing 100mg of 6-benzylaminopurine powder, dissolving the 6-benzylaminopurine powder by using 1mL of sodium hydroxide solution with the concentration of 1moL/L, adding pure water, transferring the solution to a 100mL volumetric flask, fixing the volume, uniformly mixing, and putting the solution into a refrigerator at 4 ℃ for later use;
preparing an indolebutyric acid solution: weighing 100mg of indolebutyric acid powder, dissolving the indolebutyric acid powder by using 1mL of sodium hydroxide solution with the concentration of 1moL/L, adding pure water, transferring the solution to a 100mL volumetric flask, fixing the volume, uniformly mixing, and putting the solution into a refrigerator at 4 ℃ for later use;
preparing a naphthylacetic acid solution: weighing 100mg of naphthylacetic acid powder, dissolving with 1mL of 1moL/L sodium hydroxide solution, adding pure water, transferring to a 100mL volumetric flask, fixing the volume, uniformly mixing, and placing in a refrigerator at 4 ℃ for later use;
example three: preparing a primary culture medium, a multiplication culture medium and a rooting culture medium.
Preparing a primary culture medium: 25mL of the mother solution 1 solution, 25mL of the mother solution 2 solution, 5mL of the mother solution 3 solution and 5mL of the mother solution 4 solution in the first example are weighed, and 100mg of EDDHA-chelated iron, 30g of sucrose and 6g of agar powder are weighed. Mixing and dissolving the solution and the powder by using pure water, then adding 800 mu L of the 6-benzylamino adenine solution and 50-100 mu L of the indolebutyric acid solution in the embodiment 2, fixing the volume to 1L, adjusting the pH value to 5.8, heating and boiling, subpackaging into 300mL wide-mouth bottles, placing into a high-temperature and high-pressure sterilizing pot for sterilization for 20min, and placing the culture medium in a condensing chamber for cooling after the sterilization is finished for standby.
Preparing a proliferation culture medium: in the first example, 25mL of a 1-mother solution, 25mL of a 2-mother solution, 5mL of a 3-mother solution and 5mL of a 4-mother solution were weighed, and 100mg of chelated iron, 30g of sucrose and 6g of agar powder were weighed. Mixing and dissolving the above solution and powder with pure water, adding 800-1000 μ L of 6-benzylamino adenine solution and 60-100 μ L of indolebutyric acid solution in example II, diluting to 1L, and adjusting pH to 5.8. Heating to boil, subpackaging into 300mL wide-mouth bottles, sterilizing in a high-temperature high-pressure sterilizing pot for 20min, and cooling the culture medium in a condensing chamber after sterilization.
Preparing a rooting culture medium: in the first example, 12.5mL of the mother liquor 1 solution, 12.5mL of the mother liquor 2 solution, 5mL of the mother liquor 3 solution and 5mL of the mother liquor 4 solution were weighed, and 100mg of chelated iron, 20g of sucrose and 6g of agar powder were weighed. And mixing and dissolving the solution and the powder by using pure water, adding 500 mu L of the indolebutyric acid solution 200-.
Under the condition of the prior art, the problems of vitrification, stem tip necrosis, plant growth emaciation and the like can occur in the propagation culture process of the bottle seedlings. The withering and death of plants are easy to occur in the rooting process, the rooting rate is low, most of the plants are 80-90%, and the rooting time is long and is more than 20 days. By utilizing the traditional technology, the rooting bottle seedling needs to be hardened by closing and opening the bottle, the time of 7-10 days is needed, and the survival rate of the plant after transplanting is not high and is between 80 and 90 percent. By utilizing the technology of the invention, the bottle seedling growth of the Gisela No. 6 in the process of subculture is strong, the vitrification problem is avoided, the height and thickness of the plant are obviously increased, the color of the leaf is deepened, the adventitious buds are increased, and the growth vigor is good. The rooting time of the plants is short, partial plants can emerge root tips after 10 days, the rooting rate after 15 days can reach 95 percent, the number of roots is large and the roots are thick and strong, the rooting time is shortened, and the rooting rate is improved. The technology simplifies the transplanting and domesticating process, bottle closing and bottle opening are not needed to be carried out in the transplanting and domesticating process of the bottle seedlings, the bottle seedlings are not needed to be moved to a domesticating room, the root-taking plants which can be transplanted can be taken out of the bottles in a laboratory, the culture medium is washed off, the plants are kept moist, and the plants are taken into the domesticating room to be planted into the matrix after a certain amount of the plants are accumulated. In the industrialized production process, the technology effectively improves the production efficiency, simplifies the production link, shortens the production period and can reduce the production cost to 70 percent of the original production cost.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (6)
1. A tissue culture rapid propagation method for large cherry rootstock Gisela No. 6 comprises the following steps:
step one, explant disinfection establishment: selecting strong and healthy annual Gesela No. 6 stock branches without diseases and insect pests in the middle ten days of 4 months to 5 months in spring, cutting the strong and healthy Gesela No. 6 stock branches into single-bud stem sections of about 2-3cm, cleaning the stem sections for 2min by using detergent, and washing for 2h by running water; operating in a clean bench, and sterilizing the used utensils and tools at high temperature and high pressure; transferring the stem segment into a sterile bottle by using a forceps, pouring 75% alcohol, disinfecting for 10-15s by using the alcohol, quickly pouring out, cleaning for 3 times by using sterile water, then disinfecting and disinfecting for 6-8min by using mercuric chloride with the concentration of 1g/L, adding 1-2 drops of Tween 20, pouring out the mercuric chloride, cleaning for 6 times by using the sterile water, and continuously shaking each time to ensure that the cleaning is thorough to obtain a disinfected explant;
step two, primary culture: cutting off wounds at the upper end and the lower end of a stem section, which are in contact with mercuric chloride, of the explant, peeling off a leaf stalk, vertically inoculating the cut explant into a primary culture medium, placing 2 explants in each bottle, and then placing the bottles in a culture room for culture under the conditions that the temperature is 24 +/-2 ℃, the illumination intensity is 4000Lux, the photoperiod is 16h/8h in daytime/darkness, and the growth period is 4 weeks;
step three, subculturing: the explant after 4 weeks of primary culture is cut to obtain a new bud with the stem length of about 1.5-2cm, and the new bud is transferred to subculture for multiplication culture, so that the tissue culture seedling quantity is enlarged, the culture condition is the same as that of the primary culture, the growth period is 4 weeks, and the multiplication coefficient is 3-4;
step four, rooting culture: selecting strong plants with the height of 2-2.5cm from the proliferated seedlings, cutting off base leaves, and vertically inoculating the plants into a rooting culture medium for rooting culture; the culture conditions are that the temperature is 22 +/-2 ℃, the illumination intensity is 2000-;
step five, transplanting and domesticating: after rooting culture is carried out for 2 weeks, transplanting and domesticating after the root system of the tissue culture seedling reaches 1cm, wherein the transplanting and domesticating medium is peat: coconut husk: perlite is 12:5: 3; placing the prepared substrate into a 50-hole forest wood plug for standby, taking the rooted plants out of a bottle, washing the culture medium from clear water, soaking the whole plants in 1000 times of carbendazim for 5s, vertically planting the plants into the prepared substrate, placing the plants into a pre-prepared small arched shed for acclimation, paying attention to proper ventilation during the acclimation, ensuring that the air humidity is over 90 percent in 1 week before the acclimation, ensuring that the temperature is between 24 and 26 ℃, sending out new roots after 1 week, gradually reducing the temperature and the humidity, removing a plastic film after 3 weeks, and moving the plants into a hardening chamber for rapid growth and lignification exercise.
2. The tissue culture rapid propagation method for large cherry rootstock Gisela No. 6 according to claim 1, characterized in that: the bactericide comprises methidathion, removazide, carbendazim and tetramycin, and the insecticide comprises imidacloprid, deltamethrin, cypermethrin and pyridaben.
3. The tissue culture rapid propagation method for large cherry rootstock Gisela No. 6 according to claim 1, characterized in that: the primary culture medium comprises an improved MS culture medium, 0.5-0.8 mg/L6-benzylamino adenine, 0.06-0.1mg/L indolebutyric acid, 30g/L sucrose and 6g/L agar, the hydrogen ion concentration index is 5.8-6.0, and the agar strength is 1300kg/cm2The primary medium was sterilized at 121 ℃ for 20 min.
4. The tissue culture rapid propagation method for large cherry rootstock Gisela No. 6 according to claim 1, characterized in that: the subculture medium comprises an improved MS culture medium, 0.8-1 mg/L6-benzylamino adenine, 0.02-0.06mg/L indolebutyric acid, 30g/L sucrose and 6g/L agar, wherein the hydrogen ion concentration index value is 5.8-6.0, the agar strength is 1300kg/cm2, and the subculture medium is sterilized for 20min at 121 ℃.
5. The tissue culture rapid propagation method for large cherry rootstock Gisela No. 6 according to claim 1, characterized in that: the improved MS culture medium comprises macroelements, microelements and organic matters, wherein the macroelements comprise 1650mg/L of ammonium nitrate, 950mg/L of potassium nitrate, 370mg/L of magnesium sulfate heptahydrate, 170mg/L of monopotassium phosphate, 980mg/L of calcium nitrate tetrahydrate, 780mg/L of potassium sulfate and 440mg/L of calcium chloride dihydrate; the microelements comprise 16.9mg/L of manganese sulfate monohydrate, 8.6mg/L of zinc sulfate heptahydrate, 6.2mg/L of boric acid, 0.83mg/L of potassium iodide, 0.025mg/L of copper sulfate pentahydrate, 0.025mg/L, EDDHA mg/L of cobalt chloride hexahydrate, 100mg/L of chelated iron and 0.25mg/L of sodium molybdate dihydrate, and the organic substances comprise 2mg/L of glycine, 0.5mg/L of pyridoxine, 0.5mg/L of nicotinic acid, 0.1mg/L of thiamine and 100mg/L of inositol.
6. The tissue culture rapid propagation method for large cherry rootstock Gisela No. 6 according to claim 1, characterized in that: the rooting medium is an improved MS medium, wherein macroelements are reduced by half, microelements and organic matters are kept unchanged, the rooting medium also comprises 0.2-0.5mg/L of indolebutyric acid and 0.5-1mg/L of naphthylacetic acid, the content of sucrose is 20g/L, the content of agar is 6g/L, the hydrogen ion concentration index value is 5.8-6.0, the strength of agar is 1300kg/cm2, and the rooting medium is sterilized for 20min at 121 ℃.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114097616A (en) * | 2021-11-19 | 2022-03-01 | 灵宝市鼎宏农业科技开发有限公司 | Large cherry breeding method |
| CN116034876A (en) * | 2023-01-10 | 2023-05-02 | 重庆市铜梁区果之王园艺研究院 | GF677 peach stock culture medium and cultivation method thereof |
| CN116034875A (en) * | 2023-01-10 | 2023-05-02 | 重庆市铜梁区果之王园艺研究院 | Cherry stock tissue culture seedling culture medium and tissue culture seedling culture method thereof |
| CN116210592A (en) * | 2023-04-07 | 2023-06-06 | 上海交通大学 | Large cherry stock Creams Ke 5 tissue culture rapid propagation method |
| CN116439130A (en) * | 2023-01-18 | 2023-07-18 | 重庆市铜梁区果之王园艺研究院 | Culture medium for autumn tissue culture seedlings and culture method for tissue culture seedlings |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102090328A (en) * | 2010-11-03 | 2011-06-15 | 天津樱桃谷农业科技发展有限公司 | Cherry rootstock tissue culture medium and improvement method of culture medium |
| CN103155867A (en) * | 2011-12-10 | 2013-06-19 | 天水市果树研究所 | Large cherry rootstock G-7 rapid propagation method |
| CN105325299A (en) * | 2015-11-23 | 2016-02-17 | 枣庄市农业科学研究院 | Tissue culture method and culture media for large cherry rootstock Gisela |
-
2020
- 2020-11-05 CN CN202011224241.6A patent/CN112293255B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102090328A (en) * | 2010-11-03 | 2011-06-15 | 天津樱桃谷农业科技发展有限公司 | Cherry rootstock tissue culture medium and improvement method of culture medium |
| CN103155867A (en) * | 2011-12-10 | 2013-06-19 | 天水市果树研究所 | Large cherry rootstock G-7 rapid propagation method |
| CN105325299A (en) * | 2015-11-23 | 2016-02-17 | 枣庄市农业科学研究院 | Tissue culture method and culture media for large cherry rootstock Gisela |
Non-Patent Citations (1)
| Title |
|---|
| 沙玉芬等: "樱桃砧木吉塞拉6号组培快繁技术研究", 《山东农业科学》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114097616A (en) * | 2021-11-19 | 2022-03-01 | 灵宝市鼎宏农业科技开发有限公司 | Large cherry breeding method |
| CN116034876A (en) * | 2023-01-10 | 2023-05-02 | 重庆市铜梁区果之王园艺研究院 | GF677 peach stock culture medium and cultivation method thereof |
| CN116034875A (en) * | 2023-01-10 | 2023-05-02 | 重庆市铜梁区果之王园艺研究院 | Cherry stock tissue culture seedling culture medium and tissue culture seedling culture method thereof |
| CN116439130A (en) * | 2023-01-18 | 2023-07-18 | 重庆市铜梁区果之王园艺研究院 | Culture medium for autumn tissue culture seedlings and culture method for tissue culture seedlings |
| CN116210592A (en) * | 2023-04-07 | 2023-06-06 | 上海交通大学 | Large cherry stock Creams Ke 5 tissue culture rapid propagation method |
| CN116210592B (en) * | 2023-04-07 | 2024-05-28 | 上海交通大学 | Large cherry stock Creams Ke 5 tissue culture rapid propagation method |
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