CN113170734A - Tissue culture, transplantation and domestication method for bird's nest fruit - Google Patents
Tissue culture, transplantation and domestication method for bird's nest fruit Download PDFInfo
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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
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/05—Fruit crops, e.g. strawberries, tomatoes or cucumbers
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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 artificial culture of plant tissues, and provides a tissue culture, transplantation and domestication method of bird's nest fruit, which comprises the following steps: s1, selecting explants; s2, disinfecting explants; s3, induction of sterile buds: cutting the explant into small sections, and culturing to obtain sterile buds; s4, carrying out subculture cyclic multiplication on the sterile buds to obtain the propagated sterile single buds: cutting the aseptic buds into small sections for subculture circulating multiplication, and transferring the intermediate aseptic buds to a multiplication culture medium added with activated carbon in the last multiplication step to obtain fast-growing, upright and robust multiplied aseptic single buds; s5, transplanting and domesticating the proliferated sterile single buds: and sequentially carrying out shearing, rooting and seedling raising treatment on the proliferated sterile single buds to obtain the tissue culture seedlings directly used for seedling hardening planting. The propagation efficiency of the invention can reach more than 5, and the tissue culture solves the detoxification effect of the bird's nest fruit, and the produced bird's nest fruit aseptic seedlings grow fast and are aseptic.
Description
Technical Field
The invention relates to the technical field of artificial culture of plant tissues, in particular to a method for tissue culture, transplantation and domestication of bird's nest fruits.
Background
Pitaya, a succulent shrub of Cactaceae family, belonging to the perennial climbing genus, is native to the north of central to south America.
The bird nest fruit, also known as the kylin fruit, has yellow peel and white pulp, is transparent, is fine and smooth like the bird nest, has the sweetness of more than 18 degrees, has a plurality of small thorns on the surface of the peel, is rich in water-soluble dietary fiber and anthocyanin which are rarely seen in common plants, has natural fragrance, has excellent taste after being eaten, is a new high-grade fruit with low energy and high fiber, but is difficult to produce because seedlings are lacked in China, the yield is low, the import is mainly depended on, the planting management requirement is relatively high, the fruit maturity is relatively long, and therefore, the price is higher and is dozens of times higher than that of the common red pulp dragon fruit.
At present, bird's nest fruit seedlings are mainly bred in a cutting mode, but the breeding coefficient is low, the breeding speed is low, the bird's nest fruit seedlings are easy to rot, and the breeding quantity can not meet the market demand of the bird's nest fruit at all.
The plant tissue culture technology can breed a large amount of high-quality, uniform-growth, stable-character and detoxified healthy seedlings in a short period, can preserve excellent bird's nest fruit germplasm resources, has less plant culture material consumption, small damage to parent plant materials, high propagation coefficient and high propagation speed, and becomes an important way for producing seedlings in an industrial way.
The prior cubilose fruit tissue culture system adopts the following technical route: firstly, selecting healthy plants, collecting tender fleshy stems as explants, cutting the explants into sections, disinfecting the surfaces, carrying out cluster bud induction, then carrying out rooting induction, and finally transplanting and domesticating.
Disclosure of Invention
In order to solve the defects of slow tissue culture and propagation speed and low propagation coefficient of the cubilose fruit, the invention provides a tissue culture, transplantation and domestication method of the cubilose fruit. According to the invention, through selection of explants and screening of proliferation subculture medium, the subculture growth period is generally 35-40 days, the rooting link of the dragon fruit is directly saved, when the proliferation culture medium grows to a healthy single bud of about 3-5 cm, the proliferation culture medium is cut off in a workbench and directly enters a greenhouse for rooting and domestication, and the growth period is greatly shortened.
The invention has the technical scheme that the method for tissue culture, transplantation and domestication of the bird's nest fruits comprises the following steps:
s1, selecting explants;
s2, disinfecting explants;
s3, induction of sterile buds: shearing the sterilized explant into small sections, and culturing to obtain sterile buds;
s4, carrying out subculture cyclic multiplication on the sterile buds to obtain the propagated sterile single buds: cutting the aseptic buds into small segments for cyclic proliferation, transferring the aseptic buds to a proliferation culture medium added with activated carbon when carrying out the last proliferation subculture to obtain the proliferated aseptic single buds;
s5, transplanting and domesticating the proliferated sterile single buds: and sequentially carrying out shearing, rooting and seedling raising treatment on the proliferated sterile single buds to obtain the tissue culture seedlings directly used for seedling hardening planting.
Further, in step S1, the method includes: selecting young stem sections on the robust mother plant from the explant, wherein the length of the young stem sections is about 5 cm.
Further, the method for sterilizing the explant in the step S2 comprises: placing the young stem segments in a container, washing with sterile water for 1 time, and soaking in 75% alcohol for 10 s after 5 min; then placing the mixture in mercuric chloride solution for disinfection for 13 minutes; and finally, washing with sterile water for 5 times, and washing with sterile water for 3 minutes each time, wherein the mercuric chloride solution is a liquid obtained by dissolving 0.1 gram liter of mercury in 1 liter of sterile water.
Further, the method for inducing aseptic buds in the step S3 includes: cutting the sterilized explant into stem sections with the length of 1-1.5 cm, removing medulla parts of the stem sections of the bird's nest fruits along wavy edges, longitudinally cutting the stem sections into edge pieces, horizontally placing the stem sections in a bud induction culture medium, culturing at the temperature of 28 +/-1 ℃, with the illumination intensity of 1500-2000 lx and the light cycle of 16h/8h in daytime/darkness, and culturing for 30-40 days to obtain sterile buds; the bud induction culture medium is as follows: DKW basic culture medium, 2-4 mg/L6-BA, 0.4-0.6 mg/L IAA, 30g/L white granulated sugar, 0.5g/L tryptone and 5g/L agar powder, and adjusting the pH value of the bud induction culture medium to 6.0 by using HCl or NaOH; the cutting of the edge into edge pieces along the edge and horizontal placement in the bud induction culture medium means that the edge pieces are kept with the thorn bases upwards and inoculated in the bud induction culture medium.
Further, the method for the sterile bud subculture cyclic multiplication in the step S4 includes:
the method for the subculture cyclic multiplication of the sterile buds in the step S4 comprises the following steps:
s41, keeping the medullary part of the aseptic bud obtained in the step S3, directly transversely cutting the stem segment into small segments of 1-1.5 cm, wherein the small segments are provided with three wave-shaped edges, and horizontally placing the small segments in an aseptic bud multiplication medium 1 to be subjected to illumination culture for 30-40 days to complete first generation multiplication of the aseptic bud to obtain an intermediate generation aseptic bud;
wherein: the proliferation culture medium 1 is: DKW basic culture medium, 0.4-0.6 mg/L6-BA, 0.4-0.6 mg/L IAA, 30g/L white granulated sugar, 0.5g/L tryptone and 5g/L agar powder, and adjusting the pH value of the culture medium to be 6.0 by using HCl or NaOH; the horizontal placement means that two edges of the small section with three wave-shaped edges are attached to the culture medium in parallel, and one edge is arranged below the culture medium;
s42, keeping the medullary part of the intermediate generation sterile bud obtained in the step S41, directly transversely cutting the stem segment into small segments of 1-1.5 cm, wherein the small segments are provided with three wave-shaped edges, and horizontally placing the small segments in a sterile bud multiplication culture medium 1 to perform illumination culture for 30-40 days to complete the second generation multiplication of the sterile bud to obtain the intermediate generation sterile bud;
s43, taking the intermediate generation sterile bud obtained in the previous step as a parent, and circularly executing the operations of transversely cutting, horizontally placing and culturing in the culture medium 1 in the step S42 until the intermediate generation sterile bud of 8-10 generations is obtained;
s44, keeping the medullary part of the obtained 8-10-generation propagated intermediate generation sterile bud, directly cutting the stem into small segments of 1-1.5 cm, horizontally transferring the small segments to a propagation medium 2 added with activated carbon, and performing light propagation culture for 30-40 days to obtain propagated sterile single buds; the proliferation culture medium 2 is: DKW basic culture medium, 0.4-0.6 mg/L6-BA, 0.4-0.6 mg/L IAA, 30g/L white granulated sugar, 0.5g/L tryptone, 0.5g/L active carbon and 5g/L agar powder, and HCl or NaOH is used for adjusting the pH value of the culture medium to be 6.0.
According to the invention, researches show that the multiplication culture medium 1 can be used for quickly obtaining the intermediate generation aseptic buds with high propagation efficiency, but if the multiplication culture medium 1 is still used in the final generation multiplication, the finally obtained multiplication aseptic single buds have the problems of insufficient robustness, insufficient bending straightness, more aerial roots and the like, and the culture medium 2 obtained through the researches can ensure that the finally obtained multiplication aseptic buds are upright and robust while the multiplication of the intermediate generation aseptic buds is realized, so that the culture medium is convenient for transplanting and survival. Therefore, the combined use of the multiplication medium 1 and the multiplication medium 2 in the subculture is considered to achieve both the multiplication effect and the quality of the finally obtained multiplied sterile single shoots.
Further, the method for transplant acclimatization in step S5 includes: directly cutting the proliferated sterile single buds growing to 3-5 cm in the step S4 from the bottom of the single buds in a workbench, soaking the single buds in a bactericide, placing the single buds in a moist and cool place in a warm shed, placing the single buds for about 4 days, and transplanting the single buds into a seedling cup to grow for about 14 days after the bottom of the single buds is dry and new roots grow out; repeatedly applying fertilizer for 2 times per week in the next continuous 4 weeks; and slowly fertilizing once a week in the next 8 weeks, and directly hardening the seedlings for planting after the seedlings in the seedling cup grow to 12-15 cm long tissue culture seedlings.
Further, in step S5, the method includes: the bactericide is carbendazim and water, and the ratio of the carbendazim to the water is 4 g: dissolving the formed solution by 3 liters of water for 2-5 minutes; the seedling cup comprises the following components of coconut chaff, nutrient soil and cow dung according to the weight ratio of 10: 3: 3 in a mass ratio; the fertilizer used for the service fertilization is water, compound fertilizer and urea according to the ratio of 600: 3: 1, wherein the compound fertilizer is a fertilizer containing 16% of nitrogen, phosphorus and potassium by mass percent; the slow fertilizer is prepared from water, compound fertilizer, urea and potassium chloride according to the weight ratio of 300: 3: 1: 1 to prepare a formed solution; the compound fertilizer is a fertilizer containing 16% of nitrogen, phosphorus and potassium by mass percent respectively.
The invention uses young stem section of the cubilose fruit as the explant, obtains a large amount of cubilose fruit sterile materials by bud induction and sterile bud circulation proliferation, the explant selection and the culture medium optimization lead the propagation efficiency to be more than 5, the production and experimental requirements can be met, the cubilose fruit detoxification effect is solved by tissue culture, the produced cubilose fruit sterile seedlings grow rapidly and are sterile, and excellent materials are provided for the propagation of healthy seedlings of the cubilose fruit.
According to the method, the proliferation culture medium 2 containing the activated carbon is adopted for carrying out the final subculture proliferation of the aseptic buds, the activated carbon is added, an inhibitor in-vitro culture can be adsorbed, the browning phenomenon of an explant is reduced, the grown aseptic buds generally grow fast, upright and robust and are mostly single buds, the transplanting domestication of the aseptic buds is facilitated, the adding of the activated carbon has a promoting effect on the subculture proliferation of the bird's nest fruit, but the proliferation efficiency is slightly lower than that of the proliferation culture medium 1 without the activated carbon; and the sterile buds are proliferated in the proliferation culture medium 1 without adding the active carbon, the proliferation efficiency of the sterile buds is higher than that of the proliferation culture medium 2 with the active carbon, the sterile buds are in a cluster bud shape, but generally grow downwards, are immersed in the culture medium, grow upwards after reaching a culture substrate, have poor erectility, have a plurality of aerial roots and are not beneficial to direct transplantation and domestication. The invention selects the tender stem section of the bird's nest fruit as the explant material, has the advantages of convenient material source, simple process flow, low production cost, strong regeneration plant and great application value.
Detailed Description
The present invention will be described in further detail below with reference to specific embodiments in order to make the present invention better understood by those skilled in the art.
Example 1
A cubilose fruit tissue culture and transplantation domestication method comprises the following steps:
selecting young stem segments of the bird's nest fruit as explants, wherein the length of the young stem segments is about 5 cm, cutting the stem segments into small segments with the length of 1-1.5 cm under an aseptic condition, cutting the small segments into edge pieces along edges, horizontally placing the edge pieces on a bud induction culture medium, and carrying out illumination culture for 30-40 days at the culture temperature of 28 +/-1 ℃, the illumination intensity of 1500-2000 lx and the light cycle of day/dark which is 16h/8 h; the step of horizontally placing the stem segments of the bird's nest fruit in the bud induction culture medium along the edge refers to the step of longitudinally cutting the stem segments of the bird's nest fruit into the edge along the wavy edge after removing the pulp part, and keeping the thorn bases upwards to inoculate in the bud induction culture medium. The explant is germinated in a bud induction culture medium to generate aseptic buds, a stem section is directly cut into small sections of 1-1.5 cm without removing a medulla part, the small sections are horizontally placed in a multiplication culture medium 1 to multiply the aseptic buds, a large number of aseptic buds are obtained within 30-40 days, the multiplication efficiency can reach more than 5, the aseptic buds are robust, and the stem has aerial roots. When the aseptic buds are subjected to the last multiplication subculture, the stem segments are directly cut into small segments of 1-1.5 cm without removing the medulla part, and the small segments are horizontally transferred to a multiplication culture medium 2 added with activated carbon, so that the aseptic multiplication single buds which are upright, strong and fast in growth can be obtained. And (5) transplanting and domesticating when the plant grows to about 3-5 cm. The proliferation efficiency was calculated as: and (3) expanding propagation in a propagation subculture medium, and at least averagely propagating 5 tissue culture bottles with the same length and number by placing 1 tissue culture bottle of 4 explants of 1-1.5 cm.
The transplanting and domesticating process comprises: selecting about 3-5 cm of sterile single buds to proliferate, directly cutting off the bottom of the single buds in a workbench, taking the single buds into a greenhouse, soaking the single buds in a bactericide (4 g of carbendazim dissolved in 3 liters of water) for 2-5 minutes, placing the single buds in a damp and cool place of the greenhouse, placing the single buds for about 4 days, transplanting the single buds into a seedling cup (containing coconut husk, nutrient soil and cow dung according to a mass ratio of 10: 3: 3) to grow for about 14 days after the bottom of the single buds is dry and new roots grow out; repeatedly applying fertilizer for 2 times per week in the next 4 consecutive weeks (water, compound fertilizer and urea in a mass ratio of 600: 3: 1); and slowly fertilizing once a week within 8 weeks (water, compound fertilizer urea and potassium chloride are in a mass ratio of 300: 3: 1: 1), and directly using the seedlings as hardening seedlings for planting after the seedlings in the seedling cup grow to 12-15 cm.
The sterile bud induction culture medium is a DKW basic culture medium, 2-4 mg/L6-BA, 0.4-0.6 mg/L IAA, 30g/L white granulated sugar, 0.5g/L tryptone and 5g/L agar powder. The proliferation culture medium 1 is a DKW basic culture medium, 0.4-0.6 mg/L6-BA, 0.4-0.6 mg/L IAA, 30g/L white granulated sugar, 0.5g/L tryptone and 5g/L agar powder. The multiplication culture medium 2 is a DKW basic culture medium, 0.4-0.6 mg/L6-BA, 0.4-0.6 mg/L IAA, 30g/L white granulated sugar, 0.5g/L tryptone, 0.5g/L active carbon and 5g/L agar powder. HCl or NaOH adjusted the pH of the medium to 6.0.
The horizontal placement means that the stem section of the edible bird nest fruit is not removed of the medulla part and is transversely cut into small sections of 1-1.5 cm, each small section is provided with three wave-shaped edges, two edges are attached to the culture medium in parallel, and one edge is arranged below the culture medium.
Example 2:
a cubilose fruit tissue culture and transplantation domestication method comprises the following steps:
selecting young stem segments of the bird's nest fruit as explants, wherein the length of the young stem segments is about 5 cm, cutting the stem segments into small segments with the length of 1-1.5 cm under an aseptic condition, cutting the small segments into edge pieces along edges, horizontally placing the edge pieces on a bud induction culture medium, and carrying out illumination culture for 30-40 days at the culture temperature of 28 +/-1 ℃, the illumination intensity of 1500-2000 lx and the light cycle of day/dark which is 16h/8 h; the step of horizontally placing the stem segments of the bird's nest fruit in the bud induction culture medium along the edge refers to the step of longitudinally cutting the stem segments of the bird's nest fruit into the edge along the wavy edge after removing the pulp part, and keeping the thorn bases upwards to inoculate in the bud induction culture medium. The explant is germinated in a bud induction culture medium to generate aseptic buds, a stem section is directly cut into small sections of 1-1.5 cm without removing a medulla part, the small sections are horizontally placed in a multiplication culture medium 2 to multiply the aseptic buds, a large number of aseptic buds are obtained within 30-40 days, the multiplication efficiency can reach about 4, the aseptic buds are upright and robust, and fewer aerial roots are generated. And (5) transplanting and domesticating when the plant grows to about 3-5 cm. The proliferation efficiency was calculated as: and (3) expanding propagation in a propagation subculture medium, and uniformly propagating about 4 tissue culture bottles with the same length and number by placing 1 tissue culture bottle of 4 explants of 1-1.5 cm.
The transplanting and domesticating process comprises: selecting sterile multiplication single buds of about 3-5 cm, directly cutting off the bottom of the sterile single buds in a workbench, taking the single buds into a greenhouse, soaking the single buds in a bactericide (4 g of carbendazim dissolved in 3 liters of water) for 2-5 minutes, placing the single buds in a damp and cool place of the greenhouse, placing the single buds for about 4 days, transplanting the single buds into a seedling cup (containing coconut husk, nutrient soil and cow dung according to the mass ratio of 10: 3: 3) to grow for about 14 days after the bottom of the single buds is dry and new roots grow out; repeatedly applying fertilizer for 2 times per week in the next 4 consecutive weeks (water, compound fertilizer and urea in a mass ratio of 600: 3: 1); and slowly fertilizing once a week within 8 weeks (water, compound fertilizer urea and potassium chloride are in a mass ratio of 300: 3: 1: 1), and directly using the seedlings as hardening seedlings for planting after the seedlings in the seedling cup grow to 12-15 cm.
The sterile bud induction culture medium is a DKW basic culture medium, 2-4 mg/L6-BA, 0.4-0.6 mg/L IAA, 30g/L white granulated sugar, 0.5g/L tryptone and 5g/L agar powder. The multiplication culture medium 2 is a DKW basic culture medium, 0.4-0.6 mg/L6-BA, 0.4-0.6 mg/L IAA, 30g/L white granulated sugar, 0.5g/L tryptone, 0.5g/L active carbon and 5g/L agar powder. HCl or NaOH adjusted the pH of the medium to 6.0.
The horizontal placement means that the stem section of the edible bird nest fruit is not removed of the medulla part and is transversely cut into small sections of 1-1.5 cm, each small section is provided with three wave-shaped edges, two edges are attached to the culture medium in parallel, and one edge is arranged below the culture medium.
Under the condition of the prior art, the cubilose fruit tissue culture is subjected to two steps of cluster bud induction and rooting, and the length of a proliferation bud is only 1.82 cm in the proliferation culture process for about 4 weeks; according to the tissue culture and transplantation domestication method, the multiplication buds can reach about 3 cm in about 30 days, the aseptic buds grow well, are upright and robust in a subculture medium, each multiplication subculture period is 30-40 days, and high-quality materials can be provided for transplantation domestication. According to the invention, the cut explants are horizontally placed in the proliferation culture medium, more bud points can be obtained in the proliferation process, the proliferation efficiency is more than 5 and higher than that of other placement methods (the proliferation efficiency is about 3.5 when the explants are vertically placed, and the proliferation efficiency is about 2 when the explants are cut into prismatic sheets along the edges). Compared with an MS basic culture medium, the basic culture medium DKW used in the invention can reduce the formation of red callus at the bottom of the cubilose fruit explant under the same hormone level, and is beneficial to the induction and proliferation of buds.
The method omits a rooting technology, directly transplants and acclimates the 3-5 cm proliferated single buds, simplifies the process, shortens the time, ensures that the survival rate of transplantation reaches more than 90 percent, can grow new roots in about 18 days, can absorb nutrition, fertilizes, can obtain 12-15 cm tissue culture seedlings in about 90 days, and can directly acclimatize the seedlings for field planting.
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (7)
1. A method for tissue culture and transplantation domestication of bird's nest fruit is characterized by comprising the following steps:
s1, selecting explants;
s2, disinfecting explants;
s3, induction of sterile buds: shearing the sterilized explant into small sections, and culturing to obtain sterile buds;
s4, carrying out subculture cyclic multiplication on the sterile buds to obtain the propagated sterile single buds: cutting the aseptic buds into small segments, carrying out subculture cyclic proliferation, and transferring the intermediate aseptic buds to a proliferation culture medium added with activated carbon during the last proliferation to obtain proliferated aseptic single buds;
s5, transplanting and domesticating the proliferated sterile single buds: and sequentially carrying out shearing, rooting and seedling raising treatment on the proliferated sterile single buds to obtain the tissue culture seedlings directly used for seedling hardening planting.
2. The method of tissue culture and transplant acclimatization of bird's nest fruit according to claim 1, characterized in that,
in the step S1: selecting young stem sections on the robust mother plant from the explant, wherein the length of the young stem sections is 5 cm.
3. The method of tissue culture and transplant acclimatization of bird's nest fruit according to claim 1, characterized in that,
the method for disinfecting the explants in the step S2 comprises the following steps: placing young stem segments of the healthy bird's nest fruit stock plant in a container, cleaning the young stem segments with sterile water for 1 time, and soaking the young stem segments in 75% alcohol for 10 seconds; sterilizing in 0.1% mercuric chloride solution for 13 min; finally, washing with sterile water for 5 times, and washing with sterile water for 3 minutes each time; the 0.1% mercuric chloride solution is a solution of 0.1 g mercuric chloride dissolved in 1L of sterile water.
4. The method of tissue culture and transplant acclimatization of bird's nest fruit according to claim 1, characterized in that,
the method for inducing the sterile bud in the step S3 comprises the following steps: cutting the sterilized explant into stem sections with the length of 1-1.5 cm, removing medulla parts of the stem sections along wavy edges, longitudinally cutting the stem sections into edge pieces, horizontally placing the stem sections in a bud induction culture medium, culturing at the temperature of 28 +/-1 ℃, under the illumination intensity of 1500-2000 lx and under the light cycle of 16h/8h in daytime/darkness, and culturing for about 30-40 days to obtain sterile buds; the bud induction culture medium is as follows: DKW basic culture medium, 2-4 mg/L6-BA, 0.4-0.6 mg/L IAA, 30g/L white granulated sugar, 0.5g/L tryptone and 5g/L agar powder, and adjusting the pH value of the bud induction culture medium to 6.0 by using HCl or NaOH; the horizontal placement in the bud induction culture medium means that the prism sheet is kept with the thorn seat upwards and inoculated in the bud induction culture medium.
5. The method of tissue culture and transplant acclimatization of bird's nest fruit according to claim 1, characterized in that,
the method for the subculture cyclic multiplication of the sterile buds in the step S4 comprises the following steps:
s41, keeping the medullary part of the aseptic bud obtained in the step S3, directly transversely cutting the stem segment into small segments of 1-1.5 cm, wherein the small segments are provided with three wave-shaped edges, and horizontally placing the small segments in an aseptic bud multiplication medium 1 to perform illumination culture for 30-40 days to complete first generation multiplication of the aseptic bud and obtain an intermediate generation aseptic bud;
wherein: the proliferation culture medium 1 is: DKW basic culture medium, 0.4-0.6 mg/L6-BA, 0.4-0.6 mg/L IAA, 30g/L white granulated sugar, 0.5g/L tryptone and 5g/L agar powder, and adjusting the pH value of the culture medium to be 6.0 by using HCl or NaOH; the horizontal placement means that two edges of the small section with three wave-shaped edges are attached to the culture medium in parallel, and one edge is arranged below the culture medium;
s42, keeping the medullary part of the intermediate generation sterile bud obtained in the step S41, directly transversely cutting the stem segment into small segments of 1-1.5 cm, wherein the small segments are provided with three wave-shaped edges, and horizontally placing the small segments in a sterile bud multiplication culture medium 1 to perform illumination culture for 30-40 days to complete the second generation multiplication of the sterile bud to obtain the intermediate generation sterile bud;
s43, taking the intermediate generation sterile bud obtained in the previous step as a parent, and circularly executing the operations of transversely cutting, horizontally placing and culturing in the culture medium 1 in the step S42 until the intermediate generation sterile bud of 8-10 generations is obtained;
s44, keeping the medullary part of the obtained 8-10-generation propagated intermediate generation sterile bud, directly cutting the stem into small segments of 1-1.5 cm, horizontally transferring the small segments to a propagation medium 2 added with activated carbon, and performing light propagation culture for 30-40 days to obtain propagated sterile single buds; the proliferation culture medium 2 is: DKW basic culture medium, 0.4-0.6 mg/L6-BA, 0.4-0.6 mg/L IAA, 30g/L white granulated sugar, 0.5g/L tryptone, 0.5g/L active carbon and 5g/L agar powder, and adjusting the pH value of the culture medium to be 6.0 by using HCl or NaOH.
6. The method of tissue culture and transplant acclimatization of bird's nest fruit according to claim 1, characterized in that,
the method for transplanting and domesticating in the step S5 comprises the following steps: directly cutting the proliferated sterile single buds growing to 3-5 cm in the step S4 from the bottom of the single buds in a workbench, soaking the single buds in a bactericide, placing the single buds in a moist and cool place in a warm shed, placing the single buds for about 4 days, and transplanting the single buds into a seedling cup to grow for about 14 days after the bottom of the single buds is dry and new roots grow out; repeatedly applying fertilizer for 2 times per week in the next continuous 4 weeks; and slowly fertilizing once a week in the next 8 weeks, and directly using the seedlings as hardening seedlings for planting after the seedlings in the seedling cup grow to tissue culture seedlings of 12-15 centimeters.
7. The method for tissue culture and transplant acclimatization of bird' S nest fruit of claim 1, wherein in the step S5:
the bactericide is carbendazim and water, and the ratio of the carbendazim to the water is 4 g: dissolving the formed solution by 3 liters of water for 2-5 minutes;
the seedling cup comprises the following components of coconut chaff, nutrient soil and cow dung according to the weight ratio of 10: 3: 3 in a mass ratio;
the fertilizer used for the service fertilization is water, compound fertilizer and urea according to the ratio of 600: 3: 1, wherein the compound fertilizer is a fertilizer containing 16% of nitrogen, phosphorus and potassium by mass percent;
the slow fertilizer is prepared from water, compound fertilizer, urea and potassium chloride according to the weight ratio of 300: 3: 1: 1 to prepare a formed solution; the compound fertilizer is a fertilizer containing 16% of nitrogen, phosphorus and potassium by mass percent respectively.
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| CN116472959A (en) * | 2023-04-11 | 2023-07-25 | 广西硕果农业开发有限公司 | Culture method for bird's nest fruit seedlings |
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| CN115226577A (en) * | 2022-09-21 | 2022-10-25 | 海南省农业科学院三亚研究院(海南省实验动物研究中心) | Method for adjusting and increasing yield of bird's nest fruit in production period |
| CN115226577B (en) * | 2022-09-21 | 2023-02-17 | 海南省农业科学院三亚研究院(海南省实验动物研究中心) | Method for adjusting and increasing yield of bird's nest fruit in production period |
| CN116472959A (en) * | 2023-04-11 | 2023-07-25 | 广西硕果农业开发有限公司 | Culture method for bird's nest fruit seedlings |
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