CN110810240A - Stem tissue culture and rapid propagation method for Lanzhou lily plants - Google Patents
Stem tissue culture and rapid propagation method for Lanzhou lily plants Download PDFInfo
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- CN110810240A CN110810240A CN201911089083.5A CN201911089083A CN110810240A CN 110810240 A CN110810240 A CN 110810240A CN 201911089083 A CN201911089083 A CN 201911089083A CN 110810240 A CN110810240 A CN 110810240A
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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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention discloses a method for tissue culture and rapid propagation of stem segments of Lanzhou lily plants, which comprises the following steps: s1 selecting a stock plant; s2 stem segment treatment; culturing the stem segment of S3; s4 cutting the adventitious bud for subculture; s5 culturing aseptic tissue culture seedlings; s6 culturing the stem tip of the tissue culture seedling; s7 stem section detoxification culture; s8 stem tip bulb induction culture; s9 bulblet proliferation culture; s10 rooting culture of the value-added bulb; s11 dormant for cold storage. By the process means, the bulb bulbs can be cultured by using the dried lily stems, so that the consumption of high-quality lily bulbs in the virus-free tissue culture of lily is greatly reduced, and the culture cost is reduced; the cultivation is to select high-quality lily plants as explants, can ensure that bulb bulbs obtained by tissue culture propagation inherit excellent genes, and the yield and the quality rate of lily can be improved by using the bulb bulbs for planting, thereby improving the income of growers.
Description
Technical Field
The invention belongs to the technical field of lily production, and particularly relates to a tissue culture and rapid propagation method for lily plant stems.
Background
The Lanzhou lily is suitable for dry land cultivation and has high requirement on the condition of ecological environment. Generally, the elevation is about 1800-2200 m, the soil layer is deep, and the texture is soft; the gradient is required to be more than 15 degrees, so that the drainage is convenient, and the organic matter is rich; the climate requires cold, cool and moist, the temperature difference between day and night is large, and the growth period is as long as 6-9 years. Since the lily of Lanzhou is planted by asexual propagation for a long time, the lily bulbs are in the original disc seed state of self-conservation and seed transmission for many years. The long-term planting leads the virus infection of the plants to be very common, causes the quality degradation and the yield reduction of the Lanzhou lily, influences the income increase and seriously restricts the development of the Lanzhou lily, so that the research on virus-free plants has very important value.
Through virus detection research, the viruses harmful to Lanzhou lily are determined to mainly comprise Cucumber Mosaic Virus (CMV), lily mottle virus (LMoV) and Lily Symptomless Virus (LSV), the infection rate reaches 100 percent, and the virus is 3 virus compound infection. After virus infection, lily shows symptoms of short plant bodies and light yellow leaf color in different degrees, which causes quality reduction and yield reduction. The method solves the problem of the degeneration of Lanzhou lily variety and quality, and the approaches for obtaining virus-free plants include natural selection, physical methods, chemical agent treatment and biological methods, wherein the most effective method is a biological detoxification method.
The traditional biological detoxification mainly uses lily scales as explants, and because the scales need to be selected from the middle layer with large and thick scales, the scales on the outer layer and the inner layer are generally discarded completely, so that the consumption of high-quality seed balls is huge, and the breeding cost is high.
Disclosure of Invention
The invention provides a stem tissue culture and rapid propagation method of a Lanzhou lily plant, and aims to solve the problem of high culture cost caused by huge consumption of high-quality seed balls in the conventional lily virus-free tissue culture and propagation process.
Therefore, the invention adopts the following technical scheme:
a method for tissue culture and rapid propagation of stem segments of Lanzhou lily plants comprises the following steps:
s1: selecting a stock plant: selecting a good-growing, no-pest and no-damage Lanzhou lily original plant seedling with a growth period of 55-65 days, shearing a dry stem from more than 10-14 leaves to the top as an explant, wherein the dry stem contains at least four leaves or at least two plant bud points;
s2: treating stem segments: cleaning and sterilizing the stem cut in the step S1 to obtain a sterilized stem section;
s3: stem section culture: cutting the stem sections obtained in the step S2 into 1-1.5 cm long sections, baking cut sections at two ends and plant bud points for 3-5S, and inserting the cut sections and the plant bud points into a tissue culture seedling culture medium; controlling the temperature to be 20-26 ℃, the illumination intensity to be 2000-2500 lx, and the illumination time to be 10-12 h per day for culturing;
after 25-35 days of culture, adventitious buds can grow on the plant bud points of the stem segments, water and water-soluble chitosan oligosaccharide are diluted according to the proportion of 2-5: 1, and chitosan oligosaccharide diluent is injected on each adventitious bud;
s4: subculturing: cutting the adventitious buds 12-16 days after chitosan oligosaccharide diluent is injected, putting the adventitious buds into a subculture medium added with EDTA diluent, and carrying out subculture for 18-25 days to obtain a sterile tissue culture seedling;
s5: and (3) culturing sterile tissue culture seedlings: culturing the sterile tissue culture seedlings obtained in the step S4 for 25-30 days under the conditions that the temperature is 39 +/-1 ℃, the illumination intensity is 2000-3000 lx and the illumination is 12-14 h every day; then culturing for 18-22 days under the conditions that the temperature is 25 +/-1 ℃, the illumination intensity is 2000-2500 lx and the daily illumination is 7-9 h, thus obtaining the heat-treated tissue culture seedling;
s6: and (3) culturing the stem tip of the tissue culture seedling: taking the heat-treated tissue culture seedlings obtained in the step S5 as a detoxification material, stripping stem tips with the diameter of 0.2-0.5 mm, then putting the stem tips into a stem tip culture medium, culturing under the conditions that the temperature is controlled to be 20-26 ℃, the illumination intensity is 2000-25001 x and the illumination time is 10-12 h per day, and obtaining the detoxification stem tips after 29-42 days;
s7: stem section detoxification culture: shearing 1-1.5 cm of the detoxified stem tip, soaking in common washing powder water with the mass concentration of 0.01-0.1 g/ml for 5-12 min, taking out, cleaning and sterilizing.
Then cutting stem tip growing points with different sizes according to the length of 1.0-1.5 mm, 0.8-1.0 mm, 0.5-0.8 mm, 0.3-0.5 mm and less than 0.3mm, and putting the stem tip growing points into a detoxification culture medium; culturing under the conditions that the temperature is controlled to be 22-28 ℃, the illumination intensity is 2000-25001 x and the illumination time is 12-15 h every day, and obtaining the detoxified stem tip after culturing for 25-35 days;
s8: and (3) stem tip bulb induction culture: putting the detoxified stem tip obtained in the step S7 into a bulb induction culture medium, and differentiating small bulbs after culturing for 30-40 days;
s9: and (3) proliferation culture of bulblets: placing the bulblets obtained in the step S8 into a proliferation culture medium, culturing under the conditions that the temperature is controlled to be 22-25 ℃, the illumination intensity is 2000-25001 x, and the daily illumination time is 15-22 h, and obtaining the proliferation bulbs after 25-30 days;
s10: and (3) rooting culture of the multiplication bulb: placing the proliferated bulbs obtained in the step S9 in a rooting medium; culturing under the conditions that the temperature is controlled to be 20-25 ℃, the illumination intensity is 2000-2500 lx and the illumination time is 8-10 h per day, and obtaining bulb balls with the diameter of 0.5-1 cm after 25-35 days;
s11: and (4) dormancy refrigeration: and (5) placing the bulb balls obtained in the step (S10) into a refrigeration house, controlling the temperature to be 5 +/-0.5 ℃, carrying out low-temperature treatment for 4-6 weeks, and then carrying out refrigeration in the refrigeration house at the temperature of-2-0 ℃.
Further, the neutral detergent described in step S2 refers to: diluting ordinary washing powder according to the mass ratio of 1:75 to obtain an aqueous solution.
Further, the tissue culture seedling culture medium in step S3 is a culture medium with pH value of 5.8 obtained by adding 6-benzylpurine at a concentration of 1.0-2.0 mg/L and naphthylacetic acid at a concentration of 0.1-0.5 mg/L to MS culture medium.
Further, the shoot tip medium described in step S6 is a medium having a pH of 5.8 obtained by adding ribavirin, 6-benzylpurine, and naphthylacetic acid to an MS medium at a concentration of 6mg/L, 0.3-0.5 mg/L, and 0.1-0.3 mg/L, respectively.
Further, the detoxified medium described in step S7 is a medium having a pH of 4.9 obtained by adding 6-benzylpurine at a concentration of 0.05mg/L and naphthaleneacetic acid at a concentration of 0.05mg/L to MS medium.
Further, the bulb induction medium described in step S8 is a medium having a pH of 5.8 obtained by adding 6-benzylpurine at a concentration of 0.2 to 0.5mg/L and naphthylacetic acid at a concentration of 0.5 to 1.0mg/L to an MS medium.
Further, the growth medium described in step S9 is a medium having a pH of 4.9 obtained by adding 6-benzylpurine at 0.2 to 0.5mg/L, naphthylacetic acid at 0.5 to 1.0mg/L, and sucrose at 40 to 60g/L to an MS medium.
Further, the rooting medium described in step S10 is a medium obtained by adding 0.2 to 0.5mg/L of naphthylacetic acid (NAA) and 0.5mg/L of sucrose to 1/2 of MS medium, and has a pH of 5.8.
The invention has the beneficial effects that: by the process means, the bulb bulbs can be cultured by using the dried lily stems, so that the consumption of high-quality lily bulbs in the virus-free tissue culture of lily is greatly reduced, and the culture cost is reduced; the cultivation is to select high-quality lily plants as explants, can ensure that bulb bulbs obtained by tissue culture propagation inherit excellent genes, and the yield and the quality rate of lily can be improved by using the bulb bulbs for planting, thereby improving the income of growers.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments.
Example 1:
a method for tissue culture and rapid propagation of stem segments of Lanzhou lily plants comprises the following steps:
s1: selecting a stock plant: selecting a good-growing, no-pest and no-damage Lanzhou lily original plant seedling with a growth period of 55 days, shearing a stem from more than the 12 th leaf to the top end as an explant, wherein the stem contains four leaves or has two plant bud points;
s2: treating stem segments: washing the stem cut in the step S1 with neutral detergent to remove dust and impurities on the surface, and then washing with tap water for 15 min; soaking with 10% sodium hypochlorite solution for 5min, soaking with 84 disinfectant for 2min, washing with sterile water for 1 time (10 min for 1 time);
then placing the cut surfaces and the plant bud points at the two ends on an alcohol lamp for baking for 5 s; soaking in 0.1% mercuric chloride solution for 10 min; washing with sterile water for 1 time;
then transferring the mixture into alcohol with the volume concentration of 75% to be soaked for 30s, and then washing the mixture for 3 times by using sterile water; soaking in 0.2% mercuric chloride solution for 8min, and washing with sterile water for 6 times; finally, absorbing water on the surface of the stem by using sterilized filter paper to obtain a sterilized stem section;
s3: stem section culture: cutting the sterilized stem segments obtained in the step S2 into 1cm in length, then placing cut surfaces at two ends and plant bud points on an alcohol lamp for baking for 3S, and inserting the stem segments into a tissue culture seedling culture medium according to the quantity of 20 segments of inoculated seeds per 150ml wide-mouth bottle; controlling the temperature to be 20 ℃, the illumination intensity to be 2500lx, and culturing for 12h per day;
after 25-35 days, adventitious buds can grow on the plant bud points of the stem segments, water and water-soluble chitosan oligosaccharide are diluted according to the proportion of 3:1, and the diluted water and water-soluble chitosan oligosaccharide are injected into each adventitious bud by a sterilized needle tube;
s4: subculturing: cutting the adventitious bud after injecting chitosan oligosaccharide for 12 days, then placing the cut adventitious bud into a subculture medium added with EDTA diluent, and carrying out subculture for 22 days to obtain the lily aseptic tissue culture seedling;
s5: and (3) culturing sterile tissue culture seedlings: culturing the lily aseptic tissue culture seedling obtained in the step S4 for 25 days under the conditions that the temperature is 39 +/-1 ℃, the illumination intensity is 3000lx and the daily illumination is 14 h; culturing at 25 + -1 deg.C under illumination intensity of 2500lx for 20 days under illumination for 9h per day to obtain heat-treated tissue culture seedling;
s6: and (3) culturing the stem tip of the tissue culture seedling: taking the heat-treated tissue culture seedlings obtained in the step S5 as a detoxification material, stripping 0.2mm stem tips, putting the stem tips into a stem tip culture medium according to the number of 20 inoculated segments per 150ml wide-mouth bottle, and culturing under the conditions that the temperature is controlled to be 26 ℃, the illumination intensity is 20001x and the daily illumination time is 12 hours, wherein the detoxification stem tips are obtained after 29-42 days;
s7: stem section detoxification culture: cutting 1cm of the detoxified stem tip, soaking in 0.01g/ml common washing powder water for 8min, and washing with tap water for 60 s; soaking in 70% ethanol for 30s, soaking in 0.1% mercuric chloride solution for 8min for sterilization, and washing with sterile water for 6 times;
then cutting stem tip growing points with different sizes according to the length of 1.0mm, 0.8mm, 0.5mm, 0.3mm and less than 0.3mm under a dissecting mirror, and inoculating 35 inoculation quantities of the stem tip growing points into a detoxification culture medium according to the inoculation quantity of each 150ml wide-mouth bottle; culturing under the conditions that the temperature is controlled to be 22 ℃, the illumination intensity is 2500lx and the illumination time is 15h every day, and obtaining the detoxified stem tip after 25-35 days;
s8: and (3) stem tip bulb induction culture: transferring the detoxified stem tip obtained in the step S7 into a bulb induction culture medium, and differentiating bulblet after 30-40 days;
s9: and (3) proliferation culture of bulblets: transferring the bulblets obtained in the step S8 into a proliferation culture medium according to the inoculation amount of 18 bulblets in each 150ml wide-mouth bottle, and culturing under the conditions that the temperature is controlled to be 25 ℃, the illumination intensity is 20001x and the illumination time is 22h every day, so that the proliferation bulblets are obtained after 25-30 days;
s10: and (3) rooting culture of the multiplication bulb: transferring the proliferated bulbs obtained in the step S9 into a rooting culture medium; culturing under the conditions that the temperature is controlled to be 20 ℃, the illumination intensity is 2000lx and the daily illumination time is 10h, and obtaining bulb balls with the diameter of 0.5-1 cm after 25-35 days;
s11: and (4) dormancy refrigeration: and (5) placing the bulb balls obtained in the step (S10) into a refrigeration house, controlling the temperature to be 5 +/-0.5 ℃, carrying out low-temperature treatment for 4-6 weeks, and then carrying out refrigeration in the refrigeration house at the temperature of-2-0 ℃.
Example 2:
a method for tissue culture and rapid propagation of stem segments of Lanzhou lily plants comprises the following steps:
s1: selecting a stock plant: selecting a good-growing, no-pest and no-damage Lanzhou lily original plant seedling with a growth period of 60 days, shearing a stem from more than 10 th leaf to the top as an explant, wherein the stem contains four leaves or has two plant bud points;
s2: treating stem segments: washing the stem cut in the step S1 with neutral detergent to remove dust and impurities on the surface, and then washing with tap water for 10 min; soaking with 10% sodium hypochlorite solution for 6min, soaking with 84 disinfectant for 1.5min, washing with sterile water for 1 time (10 min for 1 time);
then placing the cut surfaces and the plant bud points at the two ends on an alcohol lamp for baking for 4 s; soaking in 0.1% mercuric chloride solution for 10 min; washing with sterile water for 1 time;
then transferring the mixture into alcohol with the volume concentration of 75% to be soaked for 40s, and then washing the mixture for 3 times by using sterile water; soaking in 0.2% mercuric chloride solution for 6min, and washing with sterile water for 6 times; finally, absorbing water on the surface of the stem by using sterilized filter paper to obtain a sterilized stem section;
s3: stem section culture: cutting the sterilized stem segments obtained in the step S2 into 1.2cm in length, then placing cut surfaces at two ends and plant bud points on an alcohol lamp for baking for 3S, and inserting the stem segments into a tissue culture seedling culture medium according to the quantity of 20 segments inoculated in each 150ml wide-mouth bottle; controlling the temperature to be 20 ℃, the illumination intensity to be 2500lx, and culturing the cells in the illumination time of 11h every day;
after 25-35 days, adventitious buds can grow on the plant bud points of the stem segments, water and water-soluble chitosan oligosaccharide are diluted according to the proportion of 3:1, and the diluted water and water-soluble chitosan oligosaccharide are injected into each adventitious bud by a sterilized needle tube;
s4: subculturing: cutting the adventitious bud after injecting chitosan oligosaccharide for 12 days, then placing the cut adventitious bud into a subculture medium added with EDTA diluent, and carrying out subculture for 25 days to obtain a lily aseptic tissue culture seedling;
s5: and (3) culturing sterile tissue culture seedlings: culturing the lily aseptic tissue culture seedling obtained in the step S4 for 25 days under the conditions that the temperature is 39 +/-1 ℃, the illumination intensity is 3000lx and the daily illumination is 14 h; culturing at 25 + -1 deg.C under illumination intensity of 2500lx for 20 days under illumination for 9h per day to obtain heat-treated tissue culture seedling;
s6: and (3) culturing the stem tip of the tissue culture seedling: taking the heat-treated tissue culture seedlings obtained in the step S5 as a detoxification material, stripping 0.5mm stem tips, putting the stem tips into a stem tip culture medium according to the number of 15 inoculated segments per 150ml wide-mouth bottle, and culturing under the conditions that the temperature is controlled to be 26 ℃, the illumination intensity is 20001x and the daily illumination time is 10 hours, wherein the detoxification stem tips are obtained after 29-42 days;
s7: stem section detoxification culture: cutting 1.5cm of the detoxified stem tip, soaking in 0.01g/ml common washing powder water for 10min, and washing with tap water for 60 s; soaking in 70% ethanol for 30s, soaking in 0.1% mercuric chloride solution for 8min for sterilization, and washing with sterile water for 6 times;
then cutting stem tip growing points with different sizes according to the length of 1.4mm, 0.9mm, 0.7mm, 0.4mm and less than 0.4mm under a dissecting mirror, and inoculating 25 inoculation amounts of the stem tip growing points into a detoxification culture medium according to the inoculation amount of each 150ml wide-mouth bottle; culturing under the conditions that the temperature is controlled to be 25 ℃, the illumination intensity is 2500lx and the daily illumination time is 14h, and obtaining the detoxified stem tip after 25-35 days;
s8: and (3) stem tip bulb induction culture: transferring the detoxified stem tip obtained in the step S7 into a bulb induction culture medium, and differentiating bulblet after 30-40 days;
s9: and (3) proliferation culture of bulblets: transferring the bulblets obtained in the step S8 into a proliferation culture medium according to the inoculation amount of 20 bulblets in each 150ml wide-mouth bottle, and culturing under the conditions that the temperature is controlled to be 22 ℃, the illumination intensity is 20001x and the illumination time is 20h per day, so as to obtain the proliferation bulbs after 25-30 days;
s10: and (3) rooting culture of the multiplication bulb: transferring the proliferated bulbs obtained in the step S9 into a rooting culture medium; culturing under the conditions that the temperature is controlled to be 22 ℃, the illumination intensity is 2000lx and the daily illumination time is 9h, and obtaining bulb bulbs with the diameter of 0.5-1 cm after 25-35 days;
s11: and (4) dormancy refrigeration: and (5) placing the bulb balls obtained in the step (S10) into a refrigeration house, controlling the temperature to be 5 +/-0.5 ℃, carrying out low-temperature treatment for 4-6 weeks, and then carrying out refrigeration in the refrigeration house at the temperature of-2-0 ℃.
Example 3:
a method for tissue culture and rapid propagation of stem segments of Lanzhou lily plants comprises the following steps:
s1: selecting a stock plant: selecting a good-growing, no-pest and no-damage Lanzhou lily stock plant seedling with a growth period of 65 days, shearing a stem from more than the 12 th leaf to the top as an explant, wherein the stem contains four leaves or has two plant bud points;
s2: treating stem segments: the stem cut in step S1 was subjected to cleaning and sterilization in the same manner as in example 1.
S3: stem section culture: cutting the sterilized stem segments obtained in the step S2 into 1.5cm in length, then placing cut surfaces at two ends and plant bud points on an alcohol lamp for baking for 5S, and inserting the stem segments into a tissue culture seedling culture medium according to the number of 15 segments inoculated in each 150ml wide-mouth bottle; controlling the temperature to be 25 ℃, the illumination intensity to be 2500lx, and culturing for 12h per day;
after 25-35 days, adventitious buds can grow on the plant bud points of the stem segments, water and water-soluble chitosan oligosaccharide are diluted according to the proportion of 3:1, and the diluted water and water-soluble chitosan oligosaccharide are injected into each adventitious bud by a sterilized needle tube;
s4: subculturing: cutting the adventitious bud 16 days after chitosan oligosaccharide injection, then placing the cut adventitious bud into a subculture medium added with EDTA diluent, and carrying out subculture for 22 days to obtain the lily aseptic tissue culture seedling;
s5: and (3) culturing sterile tissue culture seedlings: culturing the lily aseptic tissue culture seedling obtained in the step S4 for 25 days under the conditions that the temperature is 39 +/-1 ℃, the illumination intensity is 3000lx and the illumination is 12h every day; culturing at 25 + -1 deg.C under illumination intensity of 2500lx for 20 days under illumination of 8h per day to obtain heat-treated tissue culture seedling;
s6: and (3) culturing the stem tip of the tissue culture seedling: taking the heat-treated tissue culture seedlings obtained in the step S5 as a detoxification material, stripping 0.3mm stem tips, putting the stem tips into a stem tip culture medium according to the number of 18 inoculated segments per 150ml wide-mouth bottle, and culturing under the conditions that the temperature is controlled to be 22 ℃, the illumination intensity is 20001x and the daily illumination time is 11h, wherein the detoxification stem tips are obtained after 29-42 days;
s7: stem section detoxification culture: cutting 1.2cm of the detoxified stem tip, soaking in 0.01g/ml common washing powder water for 10min, and washing with tap water for 60 s; soaking in 70% ethanol for 30s, soaking in 0.1% mercuric chloride solution for 8min for sterilization, and washing with sterile water for 6 times;
then cutting stem tip growing points with different sizes according to the length of 1.2mm, 0.7mm, 0.5mm and less than 0.5mm under a dissecting mirror, and inoculating the stem tip growing points into a detoxification culture medium according to the inoculation amount of 28 seeds in each 150ml wide-mouth bottle; culturing under the conditions that the temperature is controlled to be 24 ℃, the illumination intensity is 2500lx and the illumination time is 12h per day, and obtaining the detoxified stem tip after 25-35 days;
s8: and (3) stem tip bulb induction culture: transferring the detoxified stem tip obtained in the step S7 into a bulb induction culture medium, and differentiating bulblet after 30-40 days;
s9: and (3) proliferation culture of bulblets: transferring the bulblets obtained in the step S8 into a proliferation culture medium according to the inoculum size of 22 bulbs per 150ml wide-mouth bottle, and culturing under the conditions that the temperature is controlled to be 22 ℃, the illumination intensity is 20001x and the illumination time is 19 hours per day, so as to obtain the proliferation bulbs after 25-30 days;
s10: and (3) rooting culture of the multiplication bulb: transferring the proliferated bulbs obtained in the step S9 into a rooting culture medium; culturing under the conditions that the temperature is controlled to be 25 ℃, the illumination intensity is 2000lx and the daily illumination time is 10h, and obtaining bulb balls with the diameter of 0.5-1 cm after 25-35 days;
s11: and (4) dormancy refrigeration: and (5) placing the bulb balls obtained in the step (S10) into a refrigeration house, controlling the temperature to be 5 +/-0.5 ℃, carrying out low-temperature treatment for 4-6 weeks, and then carrying out refrigeration in the refrigeration house at the temperature of-2-0 ℃.
It should be noted that the above are only some embodiments of the present invention, and it should be noted that, for those skilled in the art, many modifications and substitutions can be made without departing from the technical principle of the present invention, and these modifications and substitutions should also be regarded as the protection scope of the present invention.
Claims (8)
1. A method for tissue culture and rapid propagation of a stem segment of a Lanzhou lily plant is characterized by comprising the following steps:
s1: selecting a stock plant: selecting a good-growing, no-pest and no-damage Lanzhou lily original plant seedling with a growth period of 55-65 days, shearing a dry stem from more than 10-14 leaves to the top as an explant, wherein the dry stem contains at least four leaves or at least two plant bud points;
s2: treating stem segments: cleaning and sterilizing the stem cut in the step S1 to obtain a sterilized stem section;
s3: stem section culture: cutting the stem sections obtained in the step S2 into 1-1.5 cm long sections, baking cut sections at two ends and plant bud points for 3-5S, and inserting the cut sections and the plant bud points into a tissue culture seedling culture medium; controlling the temperature to be 20-26 ℃, the illumination intensity to be 2000-2500 lx, and the illumination time to be 10-12 h per day for culturing;
after 25-35 days of culture, adventitious buds can grow on the plant bud points of the stem segments, water and water-soluble chitosan oligosaccharide are diluted according to the proportion of 5-2: 1, and chitosan oligosaccharide diluent is injected on each adventitious bud;
s4: subculturing: cutting the adventitious buds 12-16 days after chitosan oligosaccharide diluent is injected, putting the adventitious buds into a subculture medium added with EDTA diluent, and carrying out subculture for 18-25 days to obtain a sterile tissue culture seedling;
s5: and (3) culturing sterile tissue culture seedlings: culturing the sterile tissue culture seedlings obtained in the step S4 for 25-30 days under the conditions that the temperature is 39 +/-1 ℃, the illumination intensity is 2000-3000 lx and the illumination is 12-14 h every day; then culturing for 18-22 days under the conditions that the temperature is 25 +/-1 ℃, the illumination intensity is 2000-2500 lx and the daily illumination is 7-9 h, thus obtaining the heat-treated tissue culture seedling;
s6: and (3) culturing the stem tip of the tissue culture seedling: taking the heat-treated tissue culture seedlings obtained in the step S5 as a detoxification material, stripping stem tips with the diameter of 0.2-0.5 mm, then putting the stem tips into a stem tip culture medium, culturing under the conditions that the temperature is controlled to be 20-26 ℃, the illumination intensity is 2000-25001 x and the illumination time is 10-12 h per day, and obtaining the detoxification stem tips after 29-42 days;
s7: stem section detoxification culture: shearing 1-1.5 cm of the detoxified stem tip, soaking in common washing powder water with the mass concentration of 0.01-0.1 g/ml for 5-12 min, taking out, cleaning and sterilizing;
then cutting stem tip growing points with different sizes according to the length of 1.0-1.5 mm, 0.8-1.0 mm, 0.5-0.8 mm, 0.3-0.5 mm and less than 0.3mm, and putting the stem tip growing points into a detoxification culture medium; culturing under the conditions that the temperature is controlled to be 22-28 ℃, the illumination intensity is 2000-25001 x and the illumination time is 12-15 h every day, and obtaining the detoxified stem tip after culturing for 25-35 days;
s8: and (3) stem tip bulb induction culture: putting the detoxified stem tip obtained in the step S7 into a bulb induction culture medium, and differentiating small bulbs after culturing for 30-40 days;
s9: and (3) proliferation culture of bulblets: placing the bulblets obtained in the step S8 into a proliferation culture medium, culturing under the conditions that the temperature is controlled to be 22-25 ℃, the illumination intensity is 2000-25001 x, and the daily illumination time is 15-22 h, and obtaining the proliferation bulbs after 25-30 days;
s10: and (3) rooting culture of the multiplication bulb: placing the proliferated bulbs obtained in the step S9 in a rooting medium; culturing under the conditions that the temperature is controlled to be 20-25 ℃, the illumination intensity is 2000-2500 lx and the illumination time is 8-10 h per day, and obtaining bulb balls with the diameter of 0.5-1 cm after 25-35 days;
s11: and (4) dormancy refrigeration: and (5) placing the bulb balls obtained in the step (S10) into a refrigeration house, controlling the temperature to be 5 +/-0.5 ℃, carrying out low-temperature treatment for 4-6 weeks, and then carrying out refrigeration in the refrigeration house at the temperature of-2-0 ℃.
2. The method for tissue culture and rapid propagation of stem segments of lilium davidii var unicolor plants as claimed in claim 1, wherein the neutral detergent in step S2 is: diluting ordinary washing powder according to the mass ratio of 1:75 to obtain an aqueous solution.
3. The method for tissue culture and rapid propagation of stem segments of Lilium Lanzhou Hei et al plants according to claim 1, wherein the tissue culture seedling culture medium in step S3 is a culture medium with pH of 5.8 obtained by adding 6-benzylpurine at a concentration of 1.0-2.0 mg/L and naphthylacetic acid at a concentration of 0.1-0.5 mg/L to MS culture medium.
4. The method for tissue culture and rapid propagation of stem segments of Lilium Lanzhou Hei L.var.Lanzhou in claim 1, wherein the stem tip culture medium in step S6 is a culture medium with pH of 5.8 obtained by adding ribavirin, 6-benzylpurine, and naphthylacetic acid at a ratio of 6mg/L, 0.3-0.5 mg/L, and 0.1-0.3 mg/L, to an MS culture medium.
5. The method for tissue culture and rapid propagation of stem segments of Lilium lanzhou Hei et al plants according to claim 1, wherein the detoxified medium in step S7 is a medium with pH 4.9 obtained by adding 6-benzylpurine at a concentration of 0.05mg/L and naphthaleneacetic acid at a concentration of 0.05mg/L to MS medium.
6. The method for tissue culture and rapid propagation of stem segments of Lilium Lanzhou Hei L.var.Lanzhou in claim 1, wherein the bulb induction medium in step S8 is a medium with pH of 5.8 obtained by adding 6-benzylpurine at a concentration of 0.2-0.5 mg/L and naphthylacetic acid at a concentration of 0.5-1.0 mg/L to MS medium.
7. The method for tissue culture and rapid propagation of stem segments of Lilium Lanzhou Hei et al plants according to claim 1, wherein the propagation medium in step S9 is a medium with pH of 4.9 obtained by adding 6-benzylpurine at a concentration of 0.2-0.5 mg/L, naphthylacetic acid at a concentration of 0.5-1.0 mg/L, and sucrose at a concentration of 40-60 g/L to an MS medium.
8. The method for tissue culture and rapid propagation of stem segments of Lilium lanzhou Hei et al plants according to claim 1, wherein the rooting medium in step S10 is a medium with pH of 5.8 obtained by adding 0.2-0.5 mg/L naphthylacetic acid (NAA) and 0.5mg/L sucrose to 1/2 MS medium.
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| CN113207689A (en) * | 2021-05-21 | 2021-08-06 | 中国农业科学院蔬菜花卉研究所 | Rapid propagation method for directly inducing bulblet by lily stem segment |
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| CN113207689A (en) * | 2021-05-21 | 2021-08-06 | 中国农业科学院蔬菜花卉研究所 | Rapid propagation method for directly inducing bulblet by lily stem segment |
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Inventor after: Shang Yongqiang Inventor after: Lv Feibin Inventor before: Shang Yongqiang Inventor before: Lv Feibin Inventor before: Wang Xianling Inventor before: Sun Kepei Inventor before: Yang Hua |