CN109089882B - Moss tissue culture and seedling culture method directly induced by spores - Google Patents
Moss tissue culture and seedling culture method directly induced by spores Download PDFInfo
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- CN109089882B CN109089882B CN201810999295.6A CN201810999295A CN109089882B CN 109089882 B CN109089882 B CN 109089882B CN 201810999295 A CN201810999295 A CN 201810999295A CN 109089882 B CN109089882 B CN 109089882B
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
- A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
- A01G24/15—Calcined rock, e.g. perlite, vermiculite or clay aggregates
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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
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
- A01G24/22—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing plant material
- A01G24/23—Wood, e.g. wood chips or sawdust
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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
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
- A01G24/28—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing peat, moss or sphagnum
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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
- A01G31/00—Soilless cultivation, e.g. hydroponics
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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
Abstract
A method for tissue culture and seedling culture of moss by direct induction of spores includes selection and pretreatment of sporophytes, collection of spores, direct sterilization of spores, induction of spore germination, successive multiplication of spores and protonema, oscillation differentiation culture, spray inoculation and seedling culture. The method improves the efficiency of establishing the sterile system and the proliferation efficiency, has low death and pollution rate, and has high effective seed feeding rate, subculture proliferation rate and seedling coverage rate after transplantation. Compared with the prior art, the tissue culture seed-feeding death rate is reduced by 6.5%, the pollution rate is reduced by 70.4%, and the effective seed-feeding rate is improved by 45.4%. The subculture multiplication rate is improved by 2.63 times, and the coverage rate of the moss plants cultured by seedling culture reaches 85.6 percent on average.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture and rapid propagation, and particularly relates to a technique for explant selection and disinfection, spore and protonema induced proliferation and field seedling culture in the tissue culture and rapid propagation of bryophytes.
Background
Bryophytes (bryophytes) are plants of the phylum bryophyte (Musci) and are the most widely distributed, most varied and structurally simplest higher plants in the world. The moss fresh plant has special habits of growing in a low-light and high-humidity environment, being rich in water content, strong in vitality, thin in ridge resistance and the like, and plays an important role in water and soil conservation, water source conservation, nutrient substance circulation and storage in nature. With the development of urban and garden economy, the use and demand in yard beautification, landscaping, bryophyte turf, vertical greening, roof garden, and ecological restoration has increased in recent years. However, at present, more technical problems still exist in the artificial propagation of bryophytes, especially, bottlenecks such as establishment of a tissue culture high-efficiency sterile system and rapid propagation in large-scale production are not completely solved, for example, the death rate is high when gametophytes are used as explants, the effective seed-feeding rate is low when capsules are used as explants due to easy pollution of the shells of the capsules or easy death of spores after the capsules fall off, the tissue culture rapid propagation sterile system is difficult to establish, the propagation rate is not high in the propagation stage, and the bryophytes are difficult to survive after being taken out of bottles and transplanted. Therefore, the mass moss materials required for production and application are mainly collected in the field in large quantities, or the vegetative mass (or gametophyte) and sporophyte are taken as materials to carry out artificial cutting propagation, division propagation and spore sowing propagation. A large amount of field mining and digging can seriously damage the natural and ecological environment, the artificial propagation is greatly influenced by the environment and seasons, the propagation efficiency of the cutting and the plant division propagation is low, the spore sowing propagation has the problems of difficult spore collection, low spore germination rate and the like. Therefore, the research and the solution of the critical technology of the tissue culture propagation of the moss and the establishment of the efficient, economic and ecological moss propagation technology are nearly to be waited.
Disclosure of Invention
In order to solve the technical problems, the invention realizes the artificial large-scale tissue culture propagation and seedling culture of moss by selecting and pretreating sporophytes, collecting spores, directly sterilizing the spores, inducing the germination of the spores, carrying out subculture proliferation of the spores and protonema, carrying out shake differentiation culture, spraying inoculation and seedling culture.
The technical scheme of the invention is as follows:
1. a moss tissue culture and seedling culture method directly induced by spores is characterized by comprising the following steps:
(1) selection and pretreatment of sporozoites
Selecting plump sporophytes of the sporophyte when the capsule and the peduncle of the moss are changed into yellow brown and the capsule cap is not fallen off, shearing the sporophytes, putting the sporophytes into a culture dish, culturing for 28-32 minutes under an ultraviolet lamp, and culturing for 2-4 days under the sunlight light with the illumination intensity of 3000lx at the culture temperature of 22-28 ℃;
(2) spore collection
When the capsule cap of the capsule cultured in the step (1) begins to fall off and spores are scattered, cutting the capsule or picking the capsule cap by using tweezers to scatter the spores, and collecting the bryophyte spores;
(3) direct disinfection of spores
Putting the spores obtained in the step (2) into a centrifugal tube, adding 3-4% w/w hydrogen peroxide solution according to the volume ratio of the spores to the 3-4% w/w hydrogen peroxide solution being 1: 10-20, and shaking for 6-10 minutes to obtain a mixed solution of sterilized spores and hydrogen peroxide;
(4) spore germination induction
32-35ml of moss spore germination induction culture medium is filled in each culture bottle, and the mixed solution of the sterilized spores and the hydrogen peroxide obtained in the step (3) is absorbed by a sterilized pipette or a sterilized dropperThe moss spore germination induction culture medium inoculated into the culture bottle is on the surface, and is gently shaken to make the mixed solution of the disinfected spores and the hydrogen peroxide uniformly distributed on the surface, the spore germination induction culture is carried out for 30-50 days, the spores germinate to form spores and protonema, and the spore germination induction culture conditions are as follows: the temperature is 22-28 ℃, the illumination intensity is 2500-3000 lx, the illumination time of 1-5 days is 20-24 hours/day at first, and the illumination time is 8 hours/day after the 6 th day; the moss spore germination induction culture medium comprises: improved MS + KT 0.5-1.0 mg/L + GA30.2mg/L, 6g/L of agar, 20g/L of white sugar and 6.0 of pH value;
(5) subculture propagation of spores and protonema
Filling a solid successive transfer multiplication culture medium into a new culture bottle, wherein the solid successive transfer multiplication culture medium filled into each culture bottle is 32-35ml, adding 4-6 ml of sterile water into the culture bottle of the spores and the protonema obtained by spore germination induction culture in the step (4), shaking to form a high-concentration spore and protonema suspension, inoculating the high-concentration spore and protonema suspension onto the surface of the solid successive transfer multiplication culture medium by using a sterile inoculating spoon or inoculating loop for successive transfer multiplication culture, forming green colony-like spores and protonema on the surface of the solid successive transfer multiplication culture medium after successive transfer multiplication culture for 28-32 days, and inoculating 10-15 bottles of culture bottles filled with the solid successive transfer multiplication culture medium into each bottle of the high-concentration spore and protonema suspension, wherein the successive transfer multiplication culture conditions are as follows: the temperature is 22-28 ℃, the illumination intensity is 2500-3000 lx, and the illumination time is 8 hours; the solid subculture multiplication medium comprises: modified MS + BA 0.5-1.0 mg/L +2, 4-D0.2 mg/L + IAA0.3mg/L + agar 7g/L + white sugar 20g/L, pH value is 6.0;
(6) shaking differentiation culture
And (3) adding 10-15 ml of sterile water into each culture bottle in which green colony-like spores and protonema are formed after the culture in the step (5), diluting into a diluted suspension, and mixing the diluted suspension with a liquid culture medium according to the volume ratio of the diluted suspension to the liquid culture medium of 1:5 mixing to obtain a mixed solution, and then performing shake culture on the mixed solution on a shaking table with the rotating speed of 100r/min for 25-30 days, wherein the shake culture conditions on the shaking table are as follows: the temperature is 22-28 ℃, the illumination intensity is 2500-3000 lx, the illumination time is 8 hours, and a dark green suspension is obtained when the mixed solution is thickened and the color is dark green; the liquid culture medium is as follows: modified MS + BA0.1mg/L + NAA 0.2-0.5 mg/L + white sugar 20g/L, pH 6.0;
(7) spray inoculation and seedling culture
The moss seedling culture substrate is loaded into a seedling culture plate and prepared by the following method: mixing turf, vermiculite and sawdust according to the volume ratio of turf to vermiculite to sawdust of 6:1: 1;
diluting the dark green suspension obtained by the culture in the step (6) with water to obtain a diluent, wherein the dilution ratio is 1: 15-20 according to the volume ratio of the dark green suspension to the water, the diluent is sprayed or brushed onto the surface of a moss seedling culture substrate in a spraying mode or a brushing mode, a seedling culture tray is placed on a seedbed in a greenhouse for seedling culture, a small arched shed is covered above the seedling culture tray, a colorless plastic film is covered on the small arched shed, a sunshade net with the shading rate of 75% is covered on the colorless plastic film, and the seedling culture conditions are as follows: keeping 90-95% of the relative humidity of air in the small arched shed from the 1 st day to the 15 th day of seedling culture, ventilating colorless plastic films on the small shed for 3-5 times every day at intervals from the 16 th day to the 30 th day, keeping 85-90% of the relative humidity of air in the small arched shed, completing seedling culture from the 31 st day to the 31 st day, ventilating the colorless plastic films on the small shed before 10 am and after 5 pm, keeping 75-85% of the relative humidity of air in the small arched shed, controlling the temperature in the big shed to be 20-28 ℃ in the daytime, controlling the temperature in the big shed to be not less than 15 ℃ at night, keeping the surface of a moss seedling culture substrate moist, and completing seedling culture when moss is formed on the surface of the moss seedling culture substrate during the 1 st day to the seedling culture;
the steps (3) to (6) are operated under the aseptic condition;
the modified MS in each of the media described in step (4), step (5) and step (6) is:
KN03 950mg/L，NH4N03 825mg/L，MgSO4·7H2O 185mg/L,KH2PO4 85mg/L，CaCl2·2H2O220mg/L，MnSO4·4H2O 11.15mg/L，ZnSO4·7H2O 4.3mg/L，H3BO3 3.1mg/L,KI 0.415mg/L,Na2MoO4·2H2O 0.125mg/L，CuSO4·5H2O 0.0125mg/L，CoCl2·6H2O 0.0125mg/L，Na2-EDTA 18.65mg/L，FeSO4·4H2o13.9 mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, vitamin B60.5mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L, Ca (NO)3)2·4H2O 140mg/L。
2. The tissue culture and seedling culture method of moss directly induced by spores according to the technical scheme 1 is characterized in that: and (5) repeating the step (5), and realizing the rapid proliferation of the spores and the protonema through the secondary proliferation and the secondary transfer of the spores and the protonema for more than two times.
3. The tissue culture and seedling culture method for moss directly induced by spores according to the technical scheme 1 or 2 is characterized in that: and (3) adding 100 g of 50% carbendazim wettable powder into the moss seedling culture substrate in the step (7) according to each cubic meter of the moss seedling culture substrate, uniformly mixing, then loading into a seedling culture tray, spraying or brushing the diluent on the surface of the moss seedling culture substrate mixed with the 50% carbendazim wettable powder in the seedling culture tray according to the subsequent operation, and placing the seedling culture tray on a seedling bed in a greenhouse for seedling culture.
Compared with the prior art, the invention has the following innovation points and beneficial effects:
1. the efficiency of sterile system establishment is improved.
Aiming at the problems that the opening time of a capsule cap is not easy to determine when a moss capsule in the field is mature and the difficulty of directly collecting spores is high, the capsule and the capsules with yellow-brown stalks and without shedding of the capsule cap are collected firstly, then the surfaces of the capsules are pretreated by irradiation culture of a fluorescent lamp after ultraviolet irradiation disinfection, and thus mature, high-purity and clean spores can be obtained, and a comprehensive measure of directly disinfecting the spores by using a 3-4% hydrogen peroxide solution is adopted, so that the efficiency of establishing a sterile system is improved. Firstly, the spore in the capsule is not easily damaged by ultraviolet irradiation for disinfecting the capsule, the capsule with clean appearance can be obtained, then the mature spore is obtained by culturing the capsule under the light of sunlight at proper temperature and time, and then the spore is directly disinfected by hydrogen peroxide, so that the damage to the spore is small, the pollution is less, the death rate of the spore is low, a reaction product is water, no harmful residue is generated, no sterile water is needed for cleaning, and a sterile tissue culture system of moss can be conveniently and quickly established. Compared with the existing method for sterilizing mercuric chloride by adopting capsule as an explant, the method has the advantages that the tissue culture death rate is reduced by 6.5%, the pollution rate is reduced by 70.4%, and the effective seed-feeding rate is improved by 45.4%. [ Steps (1) and (2) ]
2. The spore and the protonema are cultured on a solid subculture multiplication medium by adopting suspension, so that the multiplication efficiency is obviously improved.
The moss subculture multiplication material is spore and protonema suspension liquid (step (5)), and is difficult to cut and transfer like the subculture of adventitious buds (seedlings) in the conventional tissue culture. Aiming at the particularity, a proper amount of sterile water is added into the spores and the protonema obtained in the step (4) to form a high-concentration spore and protonema suspension, the high-concentration spore and protonema suspension is inoculated to the surface of a solid successive transfer culture medium for culture, and a bottle of a mother bottle of the high-concentration spore and protonema suspension can be transferred into 10-15 bottles of successive transfer bottles (culture bottles containing the solid successive transfer culture medium) for carrying out average transfer culture by 12.23 times. The high concentration spore and protonema suspension is inoculated on the solid subculture multiplication medium, so that the nutrition, moisture, humidity and oxygen requirements for growth of the spores and protonema can be ensured.
3. Constructing the culture technique suitable for the in-vitro seedling formation of the moss suspension liquid bottle.
The invention can lead the moss suspension liquid after spraying (coating) and transplanting to quickly grow to form moss plants through proper illumination, staged humidity and temperature control and proper moss seedling culture substrate, and the moss plant coverage rate of seedling culture can averagely reach 85.6%. Meanwhile, the transplantation adopts a spraying (coating) method after diluting dark green suspension obtained by oscillation differentiation culture, thus being beneficial to forming moss blocks which are uniformly distributed and grow tidily, being capable of better meeting the requirements of various gardens, greenness and households and having good popularization prospect.
In conclusion, through the synergistic effect of the steps, the invention solves the technical problems of high death rate when gametophyte is taken as an explant during moss tissue culture, low effective seed feeding rate caused by easy pollution of capsule or high death rate of spore falling off of capsule cap when capsule is taken as the explant, difficult establishment of a tissue culture rapid propagation sterile system, low proliferation rate in a proliferation stage, and poor growth and survival after bottle-out and transplantation.
Compared with the prior art that capsules are used as explants and protofilament is differentiated and cultured into plants and then is subjected to segmental proliferation, the method reduces the tissue culture death rate by 6.5 percent, reduces the pollution rate by 70.4 percent, improves the effective seed-feeding rate by 45.4 percent, improves the subculture proliferation rate by 2.63 times, greatly improves the tissue culture and propagation efficiency of moss, ensures that the coverage rate of moss plants cultured by seedling reaches 85.6 percent on average, and establishes an artificial propagation technology suitable for mass and large-scale production of moss.
Detailed Description
The present invention will be further described below by way of examples of tissue culture production of ` Cucurbita moschata `, ` Petasites angustifolia ` and ` Petasites aurantiaca ` without particular reference to the following examples, which are conventional methods.
Example 1 (Process according to the invention)
(1) Selection and pretreatment of sporozoites
Selecting sporophytes with good development and plump capsules when the capsules and the peduncles of the cucurbitacin moss turn yellow brown and the capsules and caps are not fallen off in 10 months, shearing the capsules, putting the capsules into a culture dish, culturing for 30 minutes under a 20W ultraviolet lamp, and culturing for 3 days under the sunlight light with the illumination intensity of 3000lx at the culture temperature of 26 ℃.
(2) Spore collection
When the capsule cap of the capsule cultured in the step (1) begins to fall off and spores are scattered, cutting the capsule by using a surgical blade or slightly picking the capsule cap by using tweezers to scatter the spores, and collecting moss spores for tissue culture after picking other sundries such as peduncles, sporangiums and the like by using the tweezers;
(3) direct disinfection of spores
Putting the spores obtained in the step (2) into a centrifugal tube, and mixing the spores and the 3% w/w hydrogen peroxide solution according to the volume ratio of 1:15, adding a hydrogen peroxide solution with the concentration of 3% w/w, and shaking for 8 minutes to obtain a mixed solution of the sterilized spores and the hydrogen peroxide;
(4) spore germination induction
35ml of moss spore germination inducing culture medium is filled in each culture bottle. And (2) inoculating (dropping) the mixed liquid of the sterilized spores and the hydrogen peroxide obtained in the step (3) to the surface of a moss spore germination induction culture medium in a culture bottle by using a sterilized pipette or a sterilized dropper, slightly shaking to ensure that the mixed liquid of the sterilized spores and the hydrogen peroxide is uniformly distributed on the surface of the mixed liquid, performing spore germination induction culture for 35 days, wherein the color of the spores inoculated to the surface of the moss spore germination induction culture medium is changed from faint yellow to green, namely, the spores germinate to form spores and protonema, and the spore germination induction culture conditions are as follows: the temperature is 25 ℃, the illumination intensity is 3000lx, the illumination time of the first 1-5 days is 24 hours/day, and the illumination time is 8 hours/day after the 6 th day (the illumination intensity is still 3000 lx); the moss spore germination induction culture medium comprises: improved MS + KT 1.0mg/L + GA30.2mg/L, 6g/L of agar, 20g/L of white sugar and 6.0 of pH value.
(5) Subculture propagation of spores and protonema
And (3) filling a solid successive multiplication culture medium into a new culture bottle, wherein the solid successive multiplication culture medium filled into each culture bottle is 35ml, adding 5ml of sterile water into the culture bottle of the spores and the protonema obtained by spore germination induction culture in the step (4), shaking to form a high-concentration spore and protonema suspension, inoculating the high-concentration spore and protonema suspension onto the surface of the solid successive multiplication culture medium by using a sterile inoculating spoon or inoculating loop for successive multiplication culture, inoculating 10-15 bottles of culture bottles filled with the solid successive multiplication culture medium into each bottle of the high-concentration spore and protonema suspension after 30 days of successive multiplication culture, and enabling the successive multiplication rate to be 10.9-13.4 times. The subculture conditions for proliferation are: the temperature is 25 ℃, the illumination intensity is 3000lx, and the illumination time is 8 hours; the solid subculture multiplication medium comprises: modified MS + BA0.5 mg/L +2, 4-D0.2 mg/L + IAA0.3mg/L + agar 7g/L + white sugar 20g/L, and pH value is 6.0.
Repeating the step (5) of subculture proliferation of the spores and the protonema, and obtaining a large number of spore and protonema proliferation bottles through 3-5 times of subculture transfer, wherein the average subculture proliferation rate can reach 12.8 times.
(6) Shaking differentiation culture
In the culture bottle which forms green colony-like spores and protonema after the culture in the step (5), adding 12ml of sterile water into each bottle to dilute into diluted suspension, mixing the diluted suspension and a liquid culture medium according to the volume ratio of the diluted suspension to the liquid culture medium of 1:5 to obtain mixed liquid, and then performing shake culture on the mixed liquid on a shaking table at the rotating speed of 100r/min for 25 days, wherein the shake culture conditions on the shaking table are as follows: the temperature is 25 ℃, the illumination intensity is 3000lx, the illumination time is 8 hours, the color of the mixed solution gradually turns green, and a dark green suspension is obtained when the mixed solution becomes thick and the color is dark green; the liquid culture medium is as follows: modified MS + BA0.1mg/L + NAA 0.2mg/L + white sugar 20g/L, pH 6.0.
(7) Spray inoculation and seedling culture
Preparing a moss seedling culture substrate: mixing grass carbon, vermiculite and sawdust according to the volume ratio of the grass carbon to the vermiculite to the sawdust of 6:1:1 to form the moss seedling culture substrate.
Adding 100 g of 50% carbendazim wettable powder into each cubic meter of the bryophyte seedling culture substrate, adding 50% carbendazim wettable powder into the bryophyte seedling culture substrate, mixing to obtain the bryophyte seedling culture substrate (hereinafter referred to as substrate) mixed with the 50% carbendazim wettable powder, and filling the substrate into a seedling culture tray.
Diluting the dark green suspension obtained by the culture in the step (6) with water to obtain a diluent, wherein the dilution ratio is 1:15 according to the volume ratio of the dark green suspension to the water, the diluent is sprayed or brushed onto the surface of a substrate in a seedling tray in a spraying mode or a brushing mode, the seedling tray is placed on a seedbed in a greenhouse for seedling culture, a small arched shed is covered above the seedling tray, a colorless plastic film is covered on the small arched shed, a sunshade net with the shading rate of 75% is covered on the colorless plastic film to keep humidity and semi-shade conditions, and the seedling culture conditions are as follows: from 1 st to 15 th days of seedling culture on a seedling bed placed in a greenhouse from a seedling culture plate, spraying 3-5 times of water in a small arched shed every day to keep the relative humidity of air in the small arched shed at 90% -95%, from 16 th to 30 th days, ventilating 3-5 times by uncovering a colorless plastic film on a small greenhouse every day to keep the relative humidity of air in the small arched shed at 85% -90%, from 31 st to finish seedling culture, and uncovering the colorless plastic film on the small greenhouse for ventilation before 10 am and after 5 pm every day to keep the relative humidity of air in the small arched shed at 75% -85%, and after 45 th day, the surface of a substrate begins to turn green, and after 80 th day, uniformly and densely distributed moss plants are formed on the surface of the substrate, and the seedling culture is finished when the moss plants are formed on the surface of the substrate, wherein the moss can be used for garden beautification, and the moss culture can be finished, Landscaping, moss turf, vertical greening, roof garden, ecological restoration, and the like. And (3) controlling the temperature in the greenhouse to be 20-28 ℃ in the daytime and controlling the temperature in the greenhouse to be not lower than 15 ℃ at night during the period from 1 day when the seedling plate is placed on a seedbed in the greenhouse for seedling culture to the period when the seedling culture is finished, keeping the surface of the substrate moist, and enabling the coverage rate of the moss plants obtained by seedling culture to reach 87.5%.
The moss plant coverage rate is calculated in the following way:
moss plant coverage (%) - (number of moss × 1cm2) Total area cm2]×100，
Taking the area of the substrate sprayed or brushed with the diluent in the step (7) as the total area, wherein the unit of the total area is cm2The total area is drawn in 1cm2The method is characterized in that the method is a grid of one cell, more than 2/3 areas are full of moss plants in each cell to form a moss plant, the area of each cell smaller than 1/3 area of the moss plants is full of moss plants to form a moss plant, the moss plants are not grown in each cell to form a moss plant, the number of the moss plants is counted, and the moss plant coverage rate can be calculated according to the formula.
The steps (3) to (6) are operated under the aseptic condition;
the modified MS in each of the media described in step (4), step (5) and step (6) is:
KN03 950mg/L，NH4N03 825mg/L，MgSO4·7H2O 185mg/L,KH2PO4 85mg/L，CaCl2·2H2O220mg/L，MnSO4·4H2O 11.15mg/L，ZnSO4·7H2O 4.3mg/L，H3BO3 3.1mg/L,KI 0.415mg/L,Na2MoO4·2H2O 0.125mg/L，CuSO4·5H2O 0.0125mg/L，CoCl2·6H2O 0.0125mg/L，Na2-EDTA 18.65mg/L，FeSO4·4H2o13.9 mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, vitamin B60.5mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L, Ca (NO)3)2·4H2O 140mg/L。
Macroelement, CaCl in the improved MS2·2H2The contents of O, trace elements and iron salt are half of the MS culture medium, the organic elements are the total amount of the MS culture medium, and Ca (NO) is increased3)2.4H2O 140mg/L。
Example 2 (method of the invention)
Example 2 the procedure was the same as in example 1 except that the following procedure was different.
(1) Selection and pretreatment of sporozoites
When the capsule and the stemming of the sphagnum angustifolium become yellow brown and the capsule cap does not fall off in 8 months, a pair of scissors is used to put the well-developed and plump capsule into a culture dish, the well-developed and plump capsule is firstly cultured under a 20W ultraviolet lamp for 30 minutes and then is irradiated by a fluorescent lamp with the temperature of 22 ℃ and the illumination intensity of 2500lx for 4 days.
(4) Spore germination induction
33ml of moss spore germination inducing culture medium is filled in each culture bottle. And (2) inoculating (dropping) the mixed liquid of the sterilized spores and the hydrogen peroxide obtained in the step (3) to the surface of a moss spore germination induction culture medium in a culture bottle by using a sterilized pipette or a sterilized dropper, slightly shaking to ensure that the mixed liquid of the sterilized spores and the hydrogen peroxide is uniformly distributed on the surface of the mixed liquid, performing spore germination induction culture for 48 days, and changing the color of the spores inoculated to the surface of the moss spore germination induction culture medium from faint yellow to green, namely, the spores germinate to form spores and protonema, wherein the spore germination induction culture conditions are as follows: the temperature is 22 ℃, the illumination intensity is 2500lx, the illumination time of the initial 1-5 days is 20 hours/day,the illumination time is 8 hours/day after the 6 th day; the moss spore germination induction culture medium comprises: improved MS + KT 0.6mg/L + GA30.2mg/L, 6g/L of agar, 20g/L of white sugar and 6.0 of pH value.
(5) Subculture propagation of spores and protonema
Filling a solid successive transfer multiplication culture medium into a new culture bottle, wherein the solid successive transfer multiplication culture medium filled into each culture bottle is 35ml, adding 6ml of sterile water into the culture bottle of the spores and protonema obtained by spore germination induction culture in the step (4), shaking to form a high-concentration spore and protonema suspension, inoculating the high-concentration spore and protonema suspension onto the surface of the solid successive transfer multiplication culture medium by using a sterile inoculating spoon or inoculating loop for successive transfer multiplication culture, after successive transfer multiplication culture for 31 days, forming green colony-like spores and protonema on the surface of the solid successive transfer multiplication culture medium, and inoculating 15 culture bottles containing the solid successive transfer multiplication culture medium into each high-concentration spore and protonema suspension; the subculture conditions for proliferation are: the temperature is 22 ℃, the illumination intensity is 2500lx, and the illumination time is 8 hours; the solid subculture multiplication medium comprises: modified MS + BA 0.8mg/L +2, 4-D0.2 mg/L + IAA0.3mg/L + agar 7g/L + white sugar 20g/L, pH value is 6.0.
(6) Shaking differentiation culture
Adding 15ml of sterile water into each culture bottle which is cultured in the step (5) to dilute into a diluted suspension, mixing the diluted suspension and a liquid culture medium according to the volume ratio of the diluted suspension to the liquid culture medium of 1:5 to obtain a mixed solution, and then carrying out shake culture on the mixed solution on a shaking table at the rotating speed of 100r/min for 25 days, wherein the shake culture conditions on the shaking table are as follows: the temperature is 22 ℃, the illumination intensity is 2500lx, the illumination time is 8 hours, the color of the mixed solution gradually turns green, and a dark green suspension is obtained when the mixed solution becomes thick and the color is dark green; the liquid culture medium is as follows: modified MS + BA0.1mg/L + NAA 0.5mg/L + white sugar 20g/L, pH 6.0.
(7) Spray inoculation and seedling culture
The substrate in the seedling culture plate is a moss seedling culture substrate. The moss plant coverage rate reaches 83.6 percent.
And (3) comparison test:
carrying out the following tests by using cucurbit moss, stenotropha angustifolia and trichophyton aurantiaca as materials, wherein 1, the influence of different explants and disinfection methods on the seed feeding effect is carried out; 2. and (3) experimental study on the influence of different subculture methods on the proliferation efficiency. Experiments further prove that the method for establishing the moss sterile system and carrying out subculture can improve the efficiency of moss sterile system establishment and subculture multiplication, obtain a large amount of moss tissue culture seedlings, enable the moss tissue culture seedlings to grow well and has high seedling rate through the methods of sporophyte pretreatment, spore collection, direct spore sterilization by hydrogen peroxide, spore germination induction, spore and protonema subculture multiplication, oscillation differentiation culture, field spraying inoculation and seedling culture.
1. Effect of different explants and Disinfection methods on seed feeding Effect
The 'cucurbit moss', 'sphagnum angustifolium' and 'sphagnum chrysosporium' yellowish brown capsules and spores collected after pretreatment by adopting the method are taken as explants, and the hydrogen peroxide sterilization method of the spores and the mercury rising sterilization method of the conventional capsules are respectively adopted for explant sterilization. The hydrogen peroxide sterilization method is to sterilize the implant directly with a 3% w/w hydrogen peroxide solution for 8 minutes, and the mercuric chloride sterilization method is to sterilize the implant with a 0.1% w/w mercuric chloride solution for 8 minutes.
The spores are explants: hydrogen peroxide disinfection method, in a centrifugal tube containing spores, according to the volume ratio of the spores to 3% w/w hydrogen peroxide solution of 1:15 to a concentration of 3% w/w hydrogen peroxide solution, shaking for 8 minutes to obtain a mixture of sterilized spores and hydrogen peroxide, and inoculating the mixture onto the surface of the bryozoan spore germination induction medium described in example 1.
Capsule is explant: the mercuric chloride disinfection method comprises the steps of disinfecting capsule by 0.1% w/w mercuric chloride solution for 8 minutes, then shearing the capsule, taking out spores, putting the spores into a centrifugal tube, adding sterile water according to the volume ratio of the spores to the sterile water of 1:1.5, uniformly mixing to form suspension, and then inoculating the suspension onto the surface of the moss spore germination induction culture medium described in example 1.
After the inoculation, the moss spores are cultured in the germination induction culture medium for 25 days (the spore germination induction culture conditions are the same as the step (4) in the example 1), and statistics of pollution, death and effective seed feeding rate are carried out. The test results of table 1 show that: compared with the conventional explant mercury-lift disinfection method of the capsule, the method disclosed by the invention has the advantages that the average death rate is reduced by 6.5%, the pollution rate is reduced by 70.4%, and the effective seed-feeding rate is improved by 45.4%.
TABLE 1 Effect of different explants and Disinfection methods on seed feeding
Remarking: the death criterion was that there were no or very few greening spores in the flask after 25 days of culture.
Mortality (%) × 100 (dead bottle/inoculum bottle).
The contamination rate (%) × 100 (contaminated bottle/seed bottle).
Effective seed feeding rate (%) [ (inoculation bottle-dead bottle-contaminated bottle)/inoculation bottle ] × 100.
2. Experimental study on influence of different subculture proliferation modes on proliferation rate
Subjecting spores and protonema obtained by inducing the cucurbit moss, the stenotrophomonas angustifolia and the trichophyton chrysosporium in the step (4) to subculture proliferation according to two different subculture methods, wherein the first method is a stem and leaf proliferation method: transferring spores and protonema formed by spore germination induction into a solid differentiation medium (the formula of the solid differentiation medium is improved MS + BA0.1mg/L + NAA 0.3mg/L + white sugar 20g/L, and the pH value is 6.0) to culture for 42 days to form plants with stem-leaf bodies, cutting the plants with the stem-leaf bodies into plant segments of 0.1-0.3cm, inoculating the plant segments into the solid subculture multiplication medium described in the embodiment 1 to culture (35 ml of the solid subculture multiplication medium is contained in each culture bottle, 12-15 plant segments are inoculated into each culture bottle containing the solid subculture multiplication medium according to the conventional method), and the subculture period of the stem-leaf bodies is 40 days/time; the second method is a subculture method of inoculating the high-concentration spore and protonema suspension formed in step (5) of the present invention onto the solid subculture growth medium described in example 1 (35 ml of the solid subculture growth medium is contained in each culture bottle, the high-concentration spore and protonema suspension is dropped onto the solid subculture growth medium by using a0.5 ml pipette, 6 drops of the high-concentration spore and protonema suspension are dropped into each culture bottle containing the solid subculture growth medium), the subculture cycle is 40 days/time, and after 80 days of culture, the growth rates of the two subculture methods are counted.
TABLE 2 Effect of different subculture proliferation methods on proliferation rates
The multiplication factor (multiple) of subculture is the number of subculture bottles/number of mother bottles.
The test results in table 2 show that: the average multiplication of the method reaches 12.23 times, the multiplication of the stem and leaf bodies is only 3.37 times, and the multiplication rate of the method is improved by 2.63 times compared with the stem and leaf body multiplication method.
The experiments show that the tissue culture method for three mosses can achieve the technical effects of improving the effective seed feeding rate and the subculture proliferation rate, quickly establish a sterile system in a short time and quickly perform artificial tissue culture propagation of the mosses, and is suitable for batch and large-scale tissue culture production of different mosses.
Claims (3)
1. A moss tissue culture and seedling culture method directly induced by spores is characterized by comprising the following steps:
(1) selection and pretreatment of sporozoites
Selecting plump sporophytes of the sporophyte when the capsule and the peduncle of the moss are changed into yellow brown and the capsule cap is not fallen off, shearing the sporophytes, putting the sporophytes into a culture dish, culturing for 28-32 minutes under an ultraviolet lamp, and culturing for 2-4 days under sunlight light with the illumination intensity of 3000lx at the culture temperature of 22-28 ℃;
(2) spore collection
When the capsule cap of the capsule cultured in the step (1) begins to fall off and spores are scattered, cutting the capsule or picking the capsule cap by using tweezers to scatter the spores, and collecting the bryophyte spores;
(3) direct disinfection of spores
Putting the spores obtained in the step (2) into a centrifugal tube, and mixing the following materials according to the ratio of the spores: the volume ratio of the 3% w/w hydrogen peroxide solution is 1: adding a 3% w/w hydrogen peroxide solution in a proportion of 10-20, and shaking for 6-10 minutes to obtain a mixed solution of sterilized spores and hydrogen peroxide;
(4) spore germination induction
The moss spore germination inducing culture medium is filled in 33ml of each culture bottle, the mixed liquid of the disinfected spores obtained in the step (3) and hydrogen peroxide is absorbed by a disinfected pipette or a dropper to be inoculated on the surface of the moss spore germination inducing culture medium in the culture bottle, and the mixed liquid of the disinfected spores and the hydrogen peroxide is gently shaken to be uniformly distributed on the surface of the moss spore germination inducing culture medium, spore germination inducing culture is carried out for 48 days, spores are germinated to form spores and protonema, and the spore germination inducing culture conditions are as follows: the temperature is 22 ℃, the illumination intensity is 2500lx, the illumination time of 1-5 days is 20 hours/day at first, and the illumination time is 8 hours/day after day 6; the moss spore germination induction culture medium comprises: improved MS + KT 0.6mg/L + GA30.2mg/L, 6g/L of agar, 20g/L of white sugar and 6.0 of pH value;
(5) subculture propagation of spores and protonema
Filling a solid successive transfer multiplication culture medium into a new culture bottle, wherein the solid successive transfer multiplication culture medium filled into each culture bottle is 35ml, adding 6ml of sterile water into the culture bottle of the spores and protonema obtained by spore germination induction culture in the step (4), shaking to form a high-concentration spore and protonema suspension, inoculating the high-concentration spore and protonema suspension onto the surface of the solid successive transfer multiplication culture medium by using a sterile inoculating spoon or inoculating loop for successive transfer multiplication culture, forming green colony-like spores and protonema on the surface of the solid successive transfer multiplication culture medium after 31 days of successive transfer multiplication culture, and inoculating 10-15 bottles of culture bottles filled with the solid successive transfer multiplication culture medium into each bottle of the high-concentration spore and protonema suspension, wherein the successive transfer multiplication culture conditions are as follows: the temperature is 22 ℃, the illumination intensity is 2500lx, and the illumination time is 8 hours; the solid subculture multiplication medium comprises: modified MS + BA 0.8mg/L +2, 4-D0.2 mg/L + IAA0.3mg/L + agar 7g/L + white sugar 20g/L, pH value is 6.0;
(6) shaking differentiation culture
In the culture bottles in which green colony-like spores and protonema are formed after the culture in the step (5), 15ml of sterile water is added into each bottle to dilute the mixture into a diluted suspension, and the diluted suspension is mixed with a liquid culture medium according to the dilution suspension: volume ratio of liquid medium 1:5 mixing to obtain a mixed solution, and then performing shake culture on the mixed solution on a shaking table with the rotating speed of 100r/min for 25 days, wherein the shake culture conditions on the shaking table are as follows: the temperature is 22 ℃, the illumination intensity is 2500lx, the illumination time is 8 hours, and a dark green suspension is obtained when the mixed solution is thickened and the color is dark green; the liquid culture medium is as follows: modified MS + BA0.1mg/L + NAA 0.5mg/L + white sugar 20g/L, pH 6.0;
(7) spray inoculation and seedling culture
The moss seedling culture substrate is loaded into a seedling culture plate and prepared by the following method: mixing grass peat, vermiculite and sawdust according to the following ratio: vermiculite: the volume ratio of sawdust is 6:1:1, mixing the raw materials in a ratio;
diluting the dark green suspension obtained by the culture in the step (6) with water to obtain a diluent, wherein the dilution ratio is as follows: the volume ratio of water is 1:15 ~ 20, with the spraying mode or scribble rinse the mode with the diluent spraying or scribble rinse to moss seedling culture substrate surface to will educate seedling tray and put and carry out the seedling culture on the seedbed in the big-arch shelter, cover little arched shed and cover colorless plastic film on little arched shed in the top of educating seedling tray, cover the sunshade net that the shading rate is 75% on colorless plastic film, the condition is cultivateed to the seedling: keeping the relative humidity of air in the small arched shed to be 90-95% from the 1 st day to the 15 th day of seedling culture, opening the colorless plastic film on the small arched shed at intervals every day for ventilation from the 16 th day to the 30 th day, keeping the relative humidity of the air in the small arched shed to be 85-90%, and keeping the relative humidity of the air in the small arched shed to be 85-90% from the 31 st day to the completion of seedling culture, opening the colorless plastic film on the small arched shed for ventilation before 10 am every day and after 5 pm, keeping the relative humidity of the air in the small arched shed to be 75-85%, controlling the temperature in the big shed to be 20-28 ℃ in the daytime and controlling the temperature in the big shed to be not less than 15 ℃ at night during the period from the 1 st day to the completion of seedling culture, keeping the surface of a moss seedling culture substrate to be moist, and completing the seedling culture when the moss is formed on the surface of the moss seedling culture substrate;
the steps (3) to (6) are operated under the aseptic condition;
the modified MS in each of the media described in step (4), step (5) and step (6) is:
KNO3 950mg/L，NH4NO3 825mg/L，MgSO4·7H2O 185mg/L， KH2PO4 85mg/L，CaCl2·2H2O 220mg/L，MnSO4·4H2O 11.15mg/L，ZnSO4·7H2O 4.3mg/L，H3BO3 3.1mg/L， KI 0.415mg/L，Na2MoO4·2H2O 0.125mg/L，CuSO4·5H2O 0.0125mg/L，CoCl2·6H2O 0.0125mg/L，Na2-EDTA 18.65mg/L，FeSO4·4H2o13.9 mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, vitamin B60.5mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L, Ca (NO)3)2·4H2O 140mg/L。
2. The tissue culture and seedling culture method of moss induced directly by spores as claimed in claim 1, wherein: and (5) repeating the step (5) to realize the rapid proliferation of the spores and the protonema through the secondary proliferation and the secondary transfer of the spores and the protonema for more than two times.
3. The tissue culture and seedling culture method of moss induced directly by spores as claimed in claim 1 or 2, wherein: and (3) adding 100 g of 50% carbendazim wettable powder into the moss seedling culture substrate in the step (7) according to each cubic meter of the moss seedling culture substrate, uniformly mixing, then loading into a seedling culture tray, spraying or brushing the diluent on the surface of the moss seedling culture substrate mixed with the 50% carbendazim wettable powder in the seedling culture tray according to the subsequent operation, and placing the seedling culture tray on a seedling bed in a greenhouse for seedling culture.
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| CN111869566A (en) * | 2020-08-01 | 2020-11-03 | 梁江 | Ginseng free microspore induction culture method |
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| CN114600771B (en) * | 2022-03-18 | 2023-03-14 | 湖北大学 | Landscape type moss spore large-scale mutagenesis screening method |
| CN116636463A (en) * | 2023-07-10 | 2023-08-25 | 广西壮族自治区中国科学院广西植物研究所 | Cultivation method of sphagnum moss in warm land in high-altitude area |
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