CN112335508A - Application method of moss sporophyte suspension containing chitosan/glucan in bare land greening - Google Patents
Application method of moss sporophyte suspension containing chitosan/glucan in bare land greening Download PDFInfo
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Abstract
The invention discloses an application method of moss sporophyte suspension containing chitosan/glucan in bare land greening, which comprises the following steps: collecting bryophyte spores, and adding a hydrogen peroxide solution into the spores to obtain a mixed solution of disinfected spores and hydrogen peroxide; inoculating a mixed solution of sterilized spores and hydrogen peroxide into a spore germination induction culture medium for spore germination induction culture, adding the cultured mixture into a solid subculture multiplication culture medium for subculture multiplication, mixing the cultured mixture with a liquid culture medium, and performing shake culture to obtain a dark green suspension; adding chitosan, glucan, ammonium ferric citrate, phenoxyacetic acid, zinc sulfate and potassium nitrate into the dark green suspension to obtain moss sporophyte suspension; the moss adhesive is uniformly smeared on bare ground, then the moss sporophyte suspension is uniformly sprayed on the surface of the moss adhesive, a sunshade net is used for covering, and spraying irrigation is carried out in time. The moss sporophyte suspension is used for greening bare land, and the greening coverage after growth is high.
Description
Technical Field
The invention relates to the technical field of bare ground greening, in particular to an application method of a moss sporophyte suspension containing chitosan/glucan in bare ground greening.
Background
At present, the bryophyte is a lower-grade higher plant and is widely distributed in the nature, and the bryophyte has the habit of fasciculation and dormancy, strong water absorption capacity, high growth speed and strong cold resistance, is not easy to get ill and cause insect damage. Is a pioneer plant which can rapidly divide and reproduce in a proper environment. The moss can absorb and accumulate substances such as dust particles in the air to form soil, and meanwhile, the moss can change indexes such as the humidity, the pH and the like of the surrounding environment to form soil suitable for the survival of other higher plants. The environment where vegetation cannot grow is improved.
With the development of economy, the greening and beautification of the homeland are the pursuits of people on higher lives. The vast bare land, including bare rock, bare land, bare desertification land, bare engineering section, heavy metal polluted bare land, pesticide polluted bare land, radioactive source polluted bare land and the like, is a greening object which is mainly concerned by people, because the bare land is not covered by soil, the environment is extremely severe, and if no manual intervention is carried out on the bare land, the bare land can not be changed for years or even decades. The method aims to solve the problems that at present, due to the fact that a large number of bare areas exist in the natural environment, ore mining, urban construction and the like, urban landscapes are affected, water and soil loss is prone to occurring, regional development is limited and the like. In the prior art, measures such as net hanging, soil dressing spray seeding, hanging plant planting, climbing plant planting and the like are adopted for carrying out rock greening, but most greening is difficult to be carried out for a long time or the greening effect is not ideal, the moss can cover green on rocks, and the greening problem of rock slopes and the like can be solved. The moss plants are short and small in individuals, simple in morphological structure, free of a real root system and limited in regulation and control capacity on water diversion, but can grow and propagate in extreme environments where other terrestrial plants are difficult to live, such as high temperature, drought, high cold, freezing and weak light, due to the fact that the moss plants have special structures and physiological adaptation mechanisms, many varieties of the small plants are pioneer species in the primary succession stage, the moss plants are usually clustered in open, barren and mostly non-survival places of vascular plants, particularly in large rock bare areas, other plants cannot grow, and the moss plants have unique advantages. With the development of moss greening engineering, the cultivation and processing technology of moss materials is very important, particularly in the aspects of cultivation of moss sporophytes and the like, because the moss sporophytes are bonded on bare land, greening failure is caused due to insufficient nutrition, meanwhile, the selected solid glue has limitations, part of the glue also has pungent smell or contains toxic and harmful components, and the glue cannot resist external wind blowing outdoors, so that the adhesion effect on moss is poor.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages described hereinafter.
To achieve these objects and other advantages in accordance with the present invention, there is provided a method for applying a chitosan/dextran-containing bryozoatum suspension to nude green comprising the steps of:
step one, collecting capsule of moss, cutting the capsule when the capsule cap of the capsule begins to fall off, collecting spores, and washing out the remaining spores of the capsule by using distilled water; adding 3-4% w/w hydrogen peroxide solution into the spores, and shaking for 6-10 minutes to obtain a mixed solution of the sterilized spores and the hydrogen peroxide; the volume ratio of the spores to 3-4% w/w hydrogen peroxide solution is 1: 10;
inoculating the mixed solution of the sterilized spores and hydrogen peroxide into a spore germination induction culture medium, and performing spore germination induction culture for 30-50 days to obtain a suspension containing spores and protonema;
step three, adding the suspension containing the spores and the protonema in the step two into a solid subculture multiplication medium, and performing subculture multiplication for 28-32 days to obtain a suspension containing green colony-like spores and protonema;
step four, mixing the suspension containing the green colony-like spores and protonema in the step four with a liquid culture medium to obtain a mixed solution, and then performing shake culture on the mixed solution for 25-30 days to obtain a dark green suspension;
step five, adding chitosan, glucan, ferric ammonium citrate, phenoxyacetic acid, zinc sulfate and potassium nitrate into the dark green suspension in the step four, and stirring to obtain a moss sporophyte suspension;
and step six, uniformly coating the moss adhesive on the polluted bare land, uniformly spraying the moss sporophyte suspension on the surface of the moss adhesive, covering by using a sunshade net, and spraying and irrigating in time to keep the humidity of the surface layer of the polluted bare land at 20-25%.
Preferably, in the second step, 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 day 6; the moss spore germination induction culture medium comprises: 0.5-1.0 mg/L of MS + KT + 30.2mg/L of GA30.2mg/L + 20g/L of white sugar, and the pH value is 6.0; the volume ratio of the mixed liquid of the sterilized spores and the hydrogen peroxide to the spore germination induction culture medium is 1: 5.
preferably, in the third step, the subculture proliferation conditions are: the temperature is 22-28 ℃, the illumination intensity is 2500-3000 lx, and the illumination time is 8 hours/day; the solid subculture multiplication medium comprises: MS + BA 0.5-1.0 mg/L +2, 4-D0.2 mg/L + IAA 0.3mg/L + white sugar 20g/L, and the pH value is 6.0.
Preferably, in the fourth step, shake culture is performed on a shaking table at 100 r/min; 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/day, 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: MS, 0.1mg/L of BA, 0.2-0.5 mg/L of NAA and 20g/L of white sugar, and the pH value is 6.0.
Preferably, in the fifth step, 100 parts by weight of the dark green suspension in the fourth step is added with 0.5-1 part of chitosan, 0.3-0.5 part of glucan, 0.2-0.4 part of ferric ammonium citrate, 0.1-0.3 part of phenoxyacetic acid, 0.1-0.2 part of zinc sulfate and 0.1-0.3 part of potassium nitrate, and the mixture is stirred to obtain the moss sporophyte suspension.
Preferably, the bare land is any one of bare rock, bare land, bare desertification land, bare engineering section, heavy metal polluted bare land, pesticide polluted bare land and radioactive source polluted soil.
Preferably, the preparation method of the moss adhesive comprises the following steps: adding 10-30 parts by weight of melamine, 10-20 parts by weight of urea, 60-90 parts by weight of attapulgite and 50-80 parts by weight of sepiolite powder into a ball milling tank, adding ball milling balls into the ball milling tank, introducing liquid nitrogen into the ball milling tank to immerse the materials in the liquid nitrogen at the temperature of-160 ℃ to-175 ℃, and keeping the balance between the volatilization amount and the introduction amount of the liquid nitrogen to stabilize the liquid level; carrying out ball milling for 30-45 min after keeping the temperature for 15 min; after ball milling is finished, transferring the ball milling tank into a vacuum glove box, standing for 3-5 hours, and collecting ball milling materials;
and secondly, adding 50-100 parts by weight of konjac glucomannan, 50-100 parts by weight of chitosan and 50-100 parts by weight of collagen into 200-300 parts by weight of water, stirring for 30-60 min at the speed of 1000-1200 r/min, adding half of the ball-milled material at the speed of 10-30 parts/min in the stirring and mixing process, then adding 40-60 parts by weight of latex powder, 10-30 parts by weight of stearic acid and 5-15 parts by weight of ammonium zirconium carbonate, continuously mixing and stirring for 30-45 min at the rotating speed of 1000-1200 r/min, and simultaneously adding the other half of the ball-milled material at the speed of 10-30 parts/min in the stirring and mixing process to obtain the special moss adhesive.
Preferably, the latex powder is one or more than two of ethylene-vinyl acetate copolymer latex powder, acrylate and styrene copolymerized rubber powder and vinyl acetate, acrylate and higher fatty acid vinyl ester ternary copolymerized rubber powder.
Preferably, the collagen is replaced by modified collagen, and the preparation method comprises the following steps: adding 50-100 parts by weight of collagen into 1000-1200 parts by weight of water, carrying out ultrasonic treatment for 30-45 min, adding 5-8 parts by weight of theanine, continuing to carry out ultrasonic treatment for 15-30 min, then adding 15-25 parts by weight of poly-L-glutamic acid, heating to 45-60 ℃, placing under the irradiation of ultraviolet lamp light, carrying out stirring reaction for 15-30 min, and carrying out centrifugal separation to obtain the modified collagen.
Preferably, the power of the ultrasonic wave is 80-120W, and the frequency is 30-50 kHz; the power of ultraviolet lamp light irradiation is 50-500W, and the wavelength of ultraviolet light is 220-400 nm.
The invention at least comprises the following beneficial effects: the invention collects the capsule of the moss, collects the spores, sequentially carries out spore germination induction culture, subculture proliferation culture and liquid medium culture, finally adopts nutrient substances to mix with the obtained suspension to obtain the moss sporophyte suspension with nutrient components, sprays the moss sporophyte suspension on the surface of the moss adhesive, the green covering is carried out on the bare land, the green covering coverage is high after the growth, the adopted moss adhesive has good adhesion effect on moss, is environment-friendly and cannot cause damage to moss plants, meanwhile, the contained konjac glucomannan, chitosan, collagen, attapulgite and sepiolite powder can provide nutrition for moss on one hand, and have the functions of thickening, thixotropic property and water retention on the other hand, can improve the adhesive force and the air permeability of the adhesive and promote the rapid planting of the moss in the severe environments of bare rock, broken stone, fallen wood or bare ground and the like.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
The specific implementation mode is as follows:
the present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The special adhesive for moss prepared in the embodiment 1-7 is subjected to performance detection, wherein the tensile shear strength is determined according to the standard GB/T7124-2008; the peel strength was determined according to GB/T2791-1995.
Example 1:
a method for applying a moss sporophyte suspension containing chitosan/glucan to bare green comprises the following steps:
step one, collecting a capsule of the sphagnum hybridum, shearing the capsule when a capsule cap of the capsule begins to fall off, collecting spores, and washing out the remaining spores of the capsule by using distilled water; adding a 3% w/w hydrogen peroxide solution into the spores, and shaking for 6 minutes to obtain a mixed solution of the sterilized spores and the hydrogen peroxide; the volume ratio of the spores to the 3% w/w hydrogen peroxide solution is 1: 10;
step two, inoculating 2mL of mixed solution of sterilized spores and hydrogen peroxide into 10mL of spore germination induction culture medium, and performing spore germination induction culture for 30 days to obtain suspension containing spores and protonema; the spore germination induction 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: MS culture medium + KT (kinetin) 0.5mg/L + GA3 (gibberellin) 0.2mg/L + white sugar 20g/L, pH 6.0;
step three, adding 2mL of the suspension containing spores and protonema obtained in the step two into 10mL of solid subculture multiplication medium, and performing subculture multiplication for 28 days to obtain a suspension containing green colony-like spores and protonema; the subculture conditions for proliferation are: the temperature is 22 ℃, the illumination intensity is 2500lx, and the illumination time is 8 hours/day; the solid subculture multiplication medium comprises: MS culture medium + BA (benzylamino adenine) 0.5mg/L +2, 4-D (2, 4-dichlorophenoxyacetic acid) 0.2mg/L + IAA (auxin) 0.3mg/L + white sugar 20g/L, pH value is 6.0;
step four, mixing 2mL of the suspension containing the green colony-like spores and protonema in the step four with 10mL of liquid culture medium to obtain a mixed solution, and then performing shake culture on the mixed solution on a shaking table at the rotating speed of 100r/min for 25 days to obtain a dark green suspension; 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/day, 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: MS culture medium + BA 0.1mg/L + NAA (naphthalene acetic acid) 0.2mg/L + white sugar 20g/L, pH value is 6.0;
step five, adding 0.5g of chitosan, 0.3g of glucan, 0.2g of ferric ammonium citrate, 0.1g of phenoxyacetic acid, 0.1g of zinc sulfate and 0.1g of potassium nitrate into 10g of the dark green suspension in the step four, and stirring to obtain a moss sporophyte suspension;
step six, uniformly coating the moss adhesive on the bare rock, uniformly spraying the moss sporophyte suspension on the surface of the moss adhesive, covering by using a sunshade net, and spraying and irrigating in time to keep the humidity of the surface layer of the polluted bare ground at 20%, and culturing for 30 days until the coverage of moss reaches 95%; after 60 days of cultivation, no detached rocks of the moss layer are found;
the preparation method of the moss adhesive comprises the following steps: adding 18g of melamine, 15g of urea, 75g of attapulgite and 72g of sepiolite powder into a ball milling tank, adding ball milling balls into the ball milling tank, introducing liquid nitrogen into the ball milling tank to immerse the materials in the liquid nitrogen at the temperature of-170 ℃, and keeping the volatilization amount and the introduction amount of the liquid nitrogen balanced to stabilize the liquid level; ball milling is started after the constant temperature is kept for 15min, and the ball milling is carried out for 40 min; after ball milling is finished, transferring the ball milling tank into a vacuum glove box, standing for 3 hours, and collecting ball milling materials;
step two, adding 80g of konjac glucomannan, 78g of chitosan and 75g of collagen into 245g of water, stirring for 60min at the speed of 1200r/min, adding half of ball-milled materials at the speed of 20g/min in the stirring and mixing process, then adding 55g of ethylene-vinyl acetate copolymer latex powder, 25g of stearic acid and 12g of ammonium zirconium carbonate, continuously mixing and stirring for 30min at the rotating speed of 1200r/min, and simultaneously adding the other half of ball-milled materials at the speed of 20g/min in the stirring and mixing process to obtain the moss adhesive, wherein the tensile shear strength of the moss adhesive is 10.6 MPa; the peel strength was 11.8N/mm.
Example 2:
a method for applying a moss sporophyte suspension containing chitosan/glucan to bare green comprises the following steps:
step one, collecting a capsule of the sphagnum hybridum, shearing the capsule when a capsule cap of the capsule begins to fall off, collecting spores, and washing out the remaining spores of the capsule by using distilled water; adding a 4% w/w hydrogen peroxide solution into the spores, and shaking for 10 minutes to obtain a mixed solution of the sterilized spores and the hydrogen peroxide; the volume ratio of the spores to the 4% w/w hydrogen peroxide solution is 1: 10;
step two, inoculating 2mL of mixed solution of sterilized spores and hydrogen peroxide into 10mL of spore germination induction culture medium, and performing spore germination induction culture for 50 days to obtain suspension containing spores and protonema; the spore germination induction culture conditions are as follows: the temperature is 28 ℃, the illumination intensity is 3000lx, the illumination time of 1-5 days is 24 hours/day at first, and the illumination time is 8 hours/day after day 6; the moss spore germination induction culture medium comprises: MS culture medium + KT (kinetin) 1.0mg/L + GA3 (gibberellin) 0.2mg/L + white sugar 20g/L, pH 6.0;
step three, adding 2mL of the suspension containing spores and protonema obtained in the step two into 10mL of solid subculture multiplication medium, and performing subculture multiplication for 32 days to obtain a suspension containing green colony-like spores and protonema; the subculture conditions for proliferation are: the temperature is 28 ℃, the illumination intensity is 3000lx, and the illumination time is 8 hours/day; the solid subculture multiplication medium comprises: MS culture medium + BA (benzylamino adenine) 1.0mg/L +2, 4-D (2, 4-dichlorophenoxyacetic acid) 0.2mg/L + IAA (auxin) 0.3mg/L + white sugar 20g/L, pH value is 6.0;
step four, mixing 2mL of the suspension containing the green colony-like spores and protonema in the step four with 10mL of liquid culture medium to obtain a mixed solution, and then performing shake culture on the mixed solution on a shaking table at the rotating speed of 100r/min for 25 days to obtain a dark green suspension; the shake culture conditions on the shaking table are as follows: the temperature is 28 ℃, the illumination intensity is 3000lx, the illumination time is 8 hours/day, 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: MS culture medium + BA 0.1mg/L + NAA (naphthalene acetic acid) 0.5mg/L + white sugar 20g/L, pH value is 6.0;
step five, adding 1g of chitosan, 0.5g of glucan, 0.4g of ferric ammonium citrate, 0.3g of phenoxyacetic acid, 0.2g of zinc sulfate and 0.3g of potassium nitrate into 10g of the dark green suspension in the step four, and stirring to obtain a moss sporophyte suspension;
step six, uniformly coating the moss adhesive on the bare rock, uniformly spraying the moss sporophyte suspension on the surface of the moss adhesive, covering by using a sunshade net, and spraying and irrigating in time to keep the humidity of the surface layer of the polluted bare ground at 25%; the moss is cultivated for 30 days, and the coverage of the moss reaches 94 percent; after 60 days of cultivation, no detached rocks of the moss layer are found;
the preparation method of the moss adhesive comprises the following steps: adding 18g of melamine, 15g of urea, 75g of attapulgite and 72g of sepiolite powder into a ball milling tank, adding ball milling balls into the ball milling tank, introducing liquid nitrogen into the ball milling tank to immerse the materials in the liquid nitrogen at the temperature of-170 ℃, and keeping the volatilization amount and the introduction amount of the liquid nitrogen balanced to stabilize the liquid level; ball milling is started after the constant temperature is kept for 15min, and the ball milling is carried out for 40 min; after ball milling is finished, transferring the ball milling tank into a vacuum glove box, standing for 3 hours, and collecting ball milling materials;
step two, adding 80g of konjac glucomannan, 78g of chitosan and 75g of collagen into 245g of water, stirring for 60min at the speed of 1200r/min, adding half of ball-milled materials at the speed of 20g/min in the stirring and mixing process, then adding 55g of ethylene-vinyl acetate copolymer latex powder, 25g of stearic acid and 12g of ammonium zirconium carbonate, continuously mixing and stirring for 30min at the rotating speed of 1200r/min, and simultaneously adding the other half of ball-milled materials at the speed of 20g/min in the stirring and mixing process to obtain the moss adhesive, wherein the tensile shear strength of the moss adhesive is 10.6 MPa; the peel strength was 11.8N/mm.
Example 3:
a method for applying a moss sporophyte suspension containing chitosan/glucan to bare green comprises the following steps:
step one, collecting a capsule of the sphagnum hybridum, shearing the capsule when a capsule cap of the capsule begins to fall off, collecting spores, and washing out the remaining spores of the capsule by using distilled water; adding a 3.5% w/w hydrogen peroxide solution into the spores, and shaking for 8 minutes to obtain a mixed solution of the sterilized spores and the hydrogen peroxide; the volume ratio of the spores to 3.5% w/w hydrogen peroxide solution is 1: 10;
step two, inoculating 2mL of mixed solution of sterilized spores and hydrogen peroxide into 10mL of spore germination induction culture medium, and performing spore germination induction culture for 40 days to obtain suspension containing spores and protonema; the spore germination induction culture conditions are as follows: the temperature is 25 ℃, the illumination intensity is 2800lx, the illumination time of 1-5 days is 22 hours/day at first, and the illumination time is 8 hours/day after day 6; the moss spore germination induction culture medium comprises: MS culture medium + KT (kinetin) 0.8mg/L + GA3 (gibberellin) 0.2mg/L + white sugar 20g/L, pH 6.0;
step three, adding 2mL of the suspension containing spores and protonema in the step two into 10mL of solid subculture multiplication medium, and performing subculture multiplication for 30 days to obtain a suspension containing green colony-like spores and protonema; the subculture conditions for proliferation are: the temperature is 28 ℃, the illumination intensity is 2800lx, and the illumination time is 8 hours/day; the solid subculture multiplication medium comprises: MS culture medium + BA (benzylamino adenine) 0.8mg/L +2, 4-D (2, 4-dichlorophenoxyacetic acid) 0.2mg/L + IAA (auxin) 0.3mg/L + white sugar 20g/L, pH value is 6.0;
step four, mixing 2mL of the suspension containing the green colony-like spores and protonema in the step four with 10mL of liquid culture medium to obtain a mixed solution, and then performing shake culture on the mixed solution on a shaking table at the rotating speed of 100r/min for 25 days to obtain a dark green suspension; the shake culture conditions on the shaking table are as follows: the temperature is 25 ℃, the illumination intensity is 2800lx, the illumination time is 8 hours/day, 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: MS culture medium + BA 0.1mg/L + NAA (naphthalene acetic acid) 0.4mg/L + white sugar 20g/L, pH value is 6.0;
step five, adding 0.8g of chitosan, 0.4g of glucan, 0.3g of ferric ammonium citrate, 0.2g of phenoxyacetic acid, 0.15g of zinc sulfate and 0.2g of potassium nitrate into 10g of the dark green suspension in the step four, and stirring to obtain a moss sporophyte suspension;
step six, uniformly coating the moss adhesive on the bare rock, uniformly spraying the moss sporophyte suspension on the surface of the moss adhesive, covering by using a sunshade net, and spraying and irrigating in time to keep the humidity of the surface layer of the polluted bare ground at 23%; the moss is cultivated for 30 days, and the coverage of the moss reaches 95 percent; after 60 days of cultivation, no detached rocks of the moss layer are found;
the preparation method of the moss adhesive comprises the following steps: adding 18g of melamine, 15g of urea, 75g of attapulgite and 72g of sepiolite powder into a ball milling tank, adding ball milling balls into the ball milling tank, introducing liquid nitrogen into the ball milling tank to immerse the materials in the liquid nitrogen at the temperature of-170 ℃, and keeping the volatilization amount and the introduction amount of the liquid nitrogen balanced to stabilize the liquid level; ball milling is started after the constant temperature is kept for 15min, and the ball milling is carried out for 40 min; after ball milling is finished, transferring the ball milling tank into a vacuum glove box, standing for 3 hours, and collecting ball milling materials;
step two, adding 80g of konjac glucomannan, 78g of chitosan and 75g of collagen into 245g of water, stirring for 60min at the speed of 1200r/min, adding half of ball-milled materials at the speed of 20g/min in the stirring and mixing process, then adding 55g of ethylene-vinyl acetate copolymer latex powder, 25g of stearic acid and 12g of ammonium zirconium carbonate, continuously mixing and stirring for 30min at the rotating speed of 1200r/min, and simultaneously adding the other half of ball-milled materials at the speed of 20g/min in the stirring and mixing process to obtain the moss adhesive, wherein the tensile shear strength of the moss adhesive is 10.6 MPa; the peel strength was 11.8N/mm.
Example 4:
a method for applying a moss sporophyte suspension containing chitosan/glucan to bare green comprises the following steps:
step one, collecting a capsule of the sphagnum hybridum, shearing the capsule when a capsule cap of the capsule begins to fall off, collecting spores, and washing out the remaining spores of the capsule by using distilled water; adding a 3.5% w/w hydrogen peroxide solution into the spores, and shaking for 8 minutes to obtain a mixed solution of the sterilized spores and the hydrogen peroxide; the volume ratio of the spores to 3.5% w/w hydrogen peroxide solution is 1: 10;
step two, inoculating 2mL of mixed solution of sterilized spores and hydrogen peroxide into 10mL of spore germination induction culture medium, and performing spore germination induction culture for 40 days to obtain suspension containing spores and protonema; the spore germination induction culture conditions are as follows: the temperature is 25 ℃, the illumination intensity is 2800lx, the illumination time of 1-5 days is 22 hours/day at first, and the illumination time is 8 hours/day after day 6; the moss spore germination induction culture medium comprises: MS culture medium + KT (kinetin) 0.8mg/L + GA3 (gibberellin) 0.2mg/L + white sugar 20g/L, pH 6.0;
step three, adding 2mL of the suspension containing spores and protonema in the step two into 10mL of solid subculture multiplication medium, and performing subculture multiplication for 30 days to obtain a suspension containing green colony-like spores and protonema; the subculture conditions for proliferation are: the temperature is 28 ℃, the illumination intensity is 2800lx, and the illumination time is 8 hours/day; the solid subculture multiplication medium comprises: MS culture medium + BA (benzylamino adenine) 0.8mg/L +2, 4-D (2, 4-dichlorophenoxyacetic acid) 0.2mg/L + IAA (auxin) 0.3mg/L + white sugar 20g/L, pH value is 6.0;
step four, mixing 2mL of the suspension containing the green colony-like spores and protonema in the step four with 10mL of liquid culture medium to obtain a mixed solution, and then performing shake culture on the mixed solution on a shaking table at the rotating speed of 100r/min for 25 days to obtain a dark green suspension; the shake culture conditions on the shaking table are as follows: the temperature is 25 ℃, the illumination intensity is 2800lx, the illumination time is 8 hours/day, 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: MS culture medium + BA 0.1mg/L + NAA (naphthalene acetic acid) 0.4mg/L + white sugar 20g/L, pH value is 6.0;
step five, adding 0.8g of chitosan, 0.4g of glucan, 0.3g of ferric ammonium citrate, 0.2g of phenoxyacetic acid, 0.15g of zinc sulfate and 0.2g of potassium nitrate into 10g of the dark green suspension in the step four, and stirring to obtain a moss sporophyte suspension;
step six, uniformly coating the moss adhesive on the bare rock, uniformly spraying the moss sporophyte suspension on the surface of the moss adhesive, covering by using a sunshade net, and spraying and irrigating in time to keep the humidity of the surface layer of the polluted bare ground at 23%; the moss is cultivated for 30 days, and the coverage of the moss reaches 99 percent; after 60 days of cultivation, no detached rocks of the moss layer are found;
the preparation method of the moss adhesive comprises the following steps: adding 18g of melamine, 15g of urea, 75g of attapulgite and 72g of sepiolite powder into a ball milling tank, adding ball milling balls into the ball milling tank, introducing liquid nitrogen into the ball milling tank to immerse the materials in the liquid nitrogen at the temperature of-170 ℃, and keeping the volatilization amount and the introduction amount of the liquid nitrogen balanced to stabilize the liquid level; ball milling is started after the constant temperature is kept for 15min, and the ball milling is carried out for 40 min; after ball milling is finished, transferring the ball milling tank into a vacuum glove box, standing for 3 hours, and collecting ball milling materials;
step two, adding 80g of konjac glucomannan, 78g of chitosan and 75g of modified collagen into 245g of water, stirring for 60min at the speed of 1200r/min, adding half of ball-milled materials at the speed of 20g/min in the stirring and mixing process, then adding 55g of ethylene-vinyl acetate copolymer latex powder, 25g of stearic acid and 12g of ammonium zirconium carbonate, continuously mixing and stirring for 30min at the rotating speed of 1200r/min, and simultaneously adding the other half of ball-milled materials at the speed of 20g/min in the stirring and mixing process to obtain the moss adhesive;
the preparation method of the modified collagen comprises the following steps: adding 80g of collagen into 1000g of water, performing ultrasonic treatment for 45min, adding 8g of catechin, performing continuous ultrasonic treatment for 30min, adding 20g of poly-L-glutamic acid, heating to 60 ℃, placing under the irradiation of an ultraviolet lamp, performing stirring reaction for 30min, and performing centrifugal separation to obtain modified collagen; the power of the ultrasonic wave is 120W, and the frequency is 50 kHz; the power of ultraviolet lamp light irradiation is 300W, the wavelength of ultraviolet light is 254nm, collagen is modified, catechin, poly-L-glutamic acid and collagen are adopted to react to form intramolecular and intermolecular cross-linking, cross-linking bonds are intertwined to form a dense net-shaped structure, the molecular cohesion is greatly improved, and the strength of the adhesive can be improved. The tensile shear strength of the special moss adhesive prepared in the embodiment is 12.7 Mpa; the peel strength was 13.5N/mm.
Example 5:
a method for applying a moss sporophyte suspension containing chitosan/glucan to bare green comprises the following steps:
step one, collecting a capsule of the sphagnum hybridum, shearing the capsule when a capsule cap of the capsule begins to fall off, collecting spores, and washing out the remaining spores of the capsule by using distilled water; adding a 3.5% w/w hydrogen peroxide solution into the spores, and shaking for 8 minutes to obtain a mixed solution of the sterilized spores and the hydrogen peroxide; the volume ratio of the spores to 3.5% w/w hydrogen peroxide solution is 1: 10;
step two, inoculating 2mL of mixed solution of sterilized spores and hydrogen peroxide into 10mL of spore germination induction culture medium, and performing spore germination induction culture for 40 days to obtain suspension containing spores and protonema; the spore germination induction culture conditions are as follows: the temperature is 25 ℃, the illumination intensity is 2800lx, the illumination time of 1-5 days is 22 hours/day at first, and the illumination time is 8 hours/day after day 6; the moss spore germination induction culture medium comprises: MS culture medium + KT (kinetin) 0.8mg/L + GA3 (gibberellin) 0.2mg/L + white sugar 20g/L, pH 6.0;
step three, adding 2mL of the suspension containing spores and protonema in the step two into 10mL of solid subculture multiplication medium, and performing subculture multiplication for 30 days to obtain a suspension containing green colony-like spores and protonema; the subculture conditions for proliferation are: the temperature is 28 ℃, the illumination intensity is 2800lx, and the illumination time is 8 hours/day; the solid subculture multiplication medium comprises: MS culture medium + BA (benzylamino adenine) 0.8mg/L +2, 4-D (2, 4-dichlorophenoxyacetic acid) 0.2mg/L + IAA (auxin) 0.3mg/L + white sugar 20g/L, pH value is 6.0;
step four, mixing 2mL of the suspension containing the green colony-like spores and protonema in the step four with 10mL of liquid culture medium to obtain a mixed solution, and then performing shake culture on the mixed solution on a shaking table at the rotating speed of 100r/min for 25 days to obtain a dark green suspension; the shake culture conditions on the shaking table are as follows: the temperature is 25 ℃, the illumination intensity is 2800lx, the illumination time is 8 hours/day, 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: MS culture medium + BA 0.1mg/L + NAA (naphthalene acetic acid) 0.4mg/L + white sugar 20g/L, pH value is 6.0;
step five, adding 0.8g of chitosan, 0.4g of glucan, 0.3g of ferric ammonium citrate, 0.2g of phenoxyacetic acid, 0.15g of zinc sulfate and 0.2g of potassium nitrate into 10g of the dark green suspension in the step four, and stirring to obtain a moss sporophyte suspension;
uniformly coating the moss adhesive on bare soil polluted by heavy metal lead (removing impurities from the soil, drying, grinding, wet ashing, filtering, drying filtrate, adding distilled water for dissolving, and testing according to the atomic absorption spectrometry process, wherein the lead content in the soil is 45.455mg/kg), then uniformly spraying the moss sporophyte suspension on the surface of the moss adhesive, covering by using a sunshade net, and spraying and irrigating in time to keep the humidity of the surface layer of the polluted bare soil at 23%; the moss is cultivated for 30 days, and the coverage of the moss reaches 92 percent; collecting moss cultured for 90 days, cleaning, oven drying, pulverizing, wet ashing, filtering, oven drying the filtrate, dissolving with distilled water, and testing by atomic absorption spectrometry to obtain moss with lead content of 11.625 mg/kg;
the preparation method of the moss adhesive comprises the following steps: adding 18g of melamine, 15g of urea, 75g of attapulgite and 72g of sepiolite powder into a ball milling tank, adding ball milling balls into the ball milling tank, introducing liquid nitrogen into the ball milling tank to immerse the materials in the liquid nitrogen at the temperature of-170 ℃, and keeping the volatilization amount and the introduction amount of the liquid nitrogen balanced to stabilize the liquid level; ball milling is started after the constant temperature is kept for 15min, and the ball milling is carried out for 40 min; after ball milling is finished, transferring the ball milling tank into a vacuum glove box, standing for 3 hours, and collecting ball milling materials;
step two, adding 80g of konjac glucomannan, 78g of chitosan and 75g of collagen into 245g of water, stirring for 60min at the speed of 1200r/min, adding half of ball-milled materials at the speed of 20g/min in the stirring and mixing process, then adding 55g of ethylene-vinyl acetate copolymer latex powder, 25g of stearic acid and 12g of ammonium zirconium carbonate, continuously mixing and stirring for 30min at the rotating speed of 1200r/min, and simultaneously adding the other half of ball-milled materials at the speed of 20g/min in the stirring and mixing process to obtain the moss adhesive, wherein the tensile shear strength of the moss adhesive is 10.6 MPa; the peel strength was 11.8N/mm.
Example 6:
a method for applying a moss sporophyte suspension containing chitosan/glucan to bare green comprises the following steps:
step one, collecting a capsule of the sphagnum hybridum, shearing the capsule when a capsule cap of the capsule begins to fall off, collecting spores, and washing out the remaining spores of the capsule by using distilled water; adding a 3.5% w/w hydrogen peroxide solution into the spores, and shaking for 8 minutes to obtain a mixed solution of the sterilized spores and the hydrogen peroxide; the volume ratio of the spores to 3.5% w/w hydrogen peroxide solution is 1: 10;
step two, inoculating 2mL of mixed solution of sterilized spores and hydrogen peroxide into 10mL of spore germination induction culture medium, and performing spore germination induction culture for 40 days to obtain suspension containing spores and protonema; the spore germination induction culture conditions are as follows: the temperature is 25 ℃, the illumination intensity is 2800lx, the illumination time of 1-5 days is 22 hours/day at first, and the illumination time is 8 hours/day after day 6; the moss spore germination induction culture medium comprises: MS culture medium + KT (kinetin) 0.8mg/L + GA3 (gibberellin) 0.2mg/L + white sugar 20g/L, pH 6.0;
step three, adding 2mL of the suspension containing spores and protonema in the step two into 10mL of solid subculture multiplication medium, and performing subculture multiplication for 30 days to obtain a suspension containing green colony-like spores and protonema; the subculture conditions for proliferation are: the temperature is 28 ℃, the illumination intensity is 2800lx, and the illumination time is 8 hours/day; the solid subculture multiplication medium comprises: MS culture medium + BA (benzylamino adenine) 0.8mg/L +2, 4-D (2, 4-dichlorophenoxyacetic acid) 0.2mg/L + IAA (auxin) 0.3mg/L + white sugar 20g/L, pH value is 6.0;
step four, mixing 2mL of the suspension containing the green colony-like spores and protonema in the step four with 10mL of liquid culture medium to obtain a mixed solution, and then performing shake culture on the mixed solution on a shaking table at the rotating speed of 100r/min for 25 days to obtain a dark green suspension; the shake culture conditions on the shaking table are as follows: the temperature is 25 ℃, the illumination intensity is 2800lx, the illumination time is 8 hours/day, 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: MS culture medium + BA 0.1mg/L + NAA (naphthalene acetic acid) 0.4mg/L + white sugar 20g/L, pH value is 6.0;
step five, adding 0.8g of chitosan, 0.4g of glucan, 0.3g of ferric ammonium citrate, 0.2g of phenoxyacetic acid, 0.15g of zinc sulfate and 0.2g of potassium nitrate into 10g of the dark green suspension in the step four, and stirring to obtain a moss sporophyte suspension;
uniformly coating the moss adhesive on bare soil polluted by heavy metal lead (removing impurities from the soil, drying, grinding, wet ashing, filtering, drying filtrate, adding distilled water for dissolving, and testing according to the atomic absorption spectrometry process, wherein the lead content in the soil is 45.455mg/kg), then uniformly spraying the moss sporophyte suspension on the surface of the moss adhesive, covering by using a sunshade net, and spraying and irrigating in time to keep the humidity of the surface layer of the polluted bare soil at 23%; the moss is cultivated for 30 days, and the coverage of the moss reaches 96 percent; collecting moss cultured for 90 days, cleaning, oven drying, grinding, wet ashing, filtering, oven drying the filtrate, dissolving with distilled water, and testing by atomic absorption spectrometry to obtain moss with lead content of 15.854 mg/kg;
the preparation method of the moss adhesive comprises the following steps: adding 18g of melamine, 15g of urea, 75g of attapulgite and 72g of sepiolite powder into a ball milling tank, adding ball milling balls into the ball milling tank, introducing liquid nitrogen into the ball milling tank to immerse the materials in the liquid nitrogen at the temperature of-170 ℃, and keeping the volatilization amount and the introduction amount of the liquid nitrogen balanced to stabilize the liquid level; ball milling is started after the constant temperature is kept for 15min, and the ball milling is carried out for 40 min; after ball milling is finished, transferring the ball milling tank into a vacuum glove box, standing for 3 hours, and collecting ball milling materials;
step two, adding 80g of konjac glucomannan, 78g of chitosan and 75g of modified collagen into 245g of water, stirring for 60min at the speed of 1200r/min, adding half of ball-milled materials at the speed of 20g/min in the stirring and mixing process, then adding 55g of ethylene-vinyl acetate copolymer latex powder, 25g of stearic acid and 12g of ammonium zirconium carbonate, continuously mixing and stirring for 30min at the rotating speed of 1200r/min, and simultaneously adding the other half of ball-milled materials at the speed of 20g/min in the stirring and mixing process to obtain the moss adhesive;
the preparation method of the modified collagen comprises the following steps: adding 80g of collagen into 1000g of water, performing ultrasonic treatment for 45min, adding 8g of catechin, performing continuous ultrasonic treatment for 30min, adding 20g of poly-L-glutamic acid, heating to 60 ℃, placing under the irradiation of an ultraviolet lamp, performing stirring reaction for 30min, and performing centrifugal separation to obtain modified collagen; the power of the ultrasonic wave is 120W, and the frequency is 50 kHz; the power of ultraviolet lamp light irradiation is 300W, the wavelength of ultraviolet light is 254nm, collagen is modified, catechin, poly-L-glutamic acid and collagen are adopted to react to form intramolecular and intermolecular cross-linking, cross-linking bonds are intertwined to form a dense net-shaped structure, the molecular cohesion is greatly improved, and the strength of the adhesive can be improved. The tensile shear strength of the special moss adhesive prepared in the embodiment is 12.7 Mpa; the peel strength was 13.5N/mm.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention may be embodied with additional modifications as would be readily apparent to those skilled in the art, and the invention is therefore not limited to the details given herein and to the examples shown and described without departing from the generic concept as defined by the claims and their equivalents.
Claims (10)
1. A method for applying a moss sporophyte suspension containing chitosan/glucan to bare green is characterized by comprising the following steps:
step one, collecting capsule of moss, cutting the capsule when the capsule cap of the capsule begins to fall off, collecting spores, and washing out the remaining spores of the capsule by using distilled water; adding 3-4% w/w hydrogen peroxide solution into the spores, and shaking for 6-10 minutes to obtain a mixed solution of the sterilized spores and the hydrogen peroxide; the volume ratio of the spores to 3-4% w/w hydrogen peroxide solution is 1: 10;
inoculating the mixed solution of the sterilized spores and hydrogen peroxide into a spore germination induction culture medium, and performing spore germination induction culture for 30-50 days to obtain a suspension containing spores and protonema;
step three, adding the suspension containing the spores and the protonema in the step two into a solid subculture multiplication medium, and performing subculture multiplication for 28-32 days to obtain a suspension containing green colony-like spores and protonema;
step four, mixing the suspension containing the green colony-like spores and protonema in the step four with a liquid culture medium to obtain a mixed solution, and then performing shake culture on the mixed solution for 25-30 days to obtain a dark green suspension;
step five, adding chitosan, glucan, ferric ammonium citrate, phenoxyacetic acid, zinc sulfate and potassium nitrate into the dark green suspension in the step four, and stirring to obtain a moss sporophyte suspension;
and step six, uniformly coating the moss adhesive on bare ground, uniformly spraying the moss sporophyte suspension on the surface of the moss adhesive, covering by using a sunshade net, and spraying and irrigating in time to keep the humidity of the surface layer of the polluted bare ground at 20-25%.
2. The method of applying chitosan/dextran-containing bryozoatum suspension in nude green as claimed in claim 1, wherein in the second step, the spore germination inducing culture conditions are: 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 day 6; the moss spore germination induction culture medium comprises: 0.5-1.0 mg/L of MS + KT + 30.2mg/L of GA30.2mg/L + 20g/L of white sugar, and the pH value is 6.0; the volume ratio of the mixed liquid of the sterilized spores and the hydrogen peroxide to the spore germination induction culture medium is 1: 5.
3. the method for applying chitosan/dextran-containing bryozoatum suspension to nude green according to claim 1, wherein in step three, the subculture proliferation conditions are: the temperature is 22-28 ℃, the illumination intensity is 2500-3000 lx, and the illumination time is 8 hours/day; the solid subculture multiplication medium comprises: MS + BA 0.5-1.0 mg/L +2, 4-D0.2 mg/L + IAA 0.3mg/L + white sugar 20g/L, and the pH value is 6.0.
4. The method for applying chitosan/dextran-containing bryozoatum suspension to nude green according to claim 1, wherein the fourth step is shake cultivation on a shaker of 100 r/min; 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/day, 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: MS, 0.1mg/L of BA, 0.2-0.5 mg/L of NAA and 20g/L of white sugar, and the pH value is 6.0.
5. The method of claim 1, wherein in the fifth step, the moss sporophyte suspension containing chitosan/glucan is added with 0.5-1 part of chitosan, 0.3-0.5 part of glucan, 0.2-0.4 part of ferric ammonium citrate, 0.1-0.3 part of phenoxyacetic acid, 0.1-0.2 part of zinc sulfate and 0.1-0.3 part of potassium nitrate into 100 parts of the dark green suspension in the fourth step by weight, and the moss sporophyte suspension is obtained after stirring.
6. The method of claim 1, wherein the open land is any one of bare rock, bare land, bare sandy land, bare engineering section, heavy metal contaminated open land, pesticide contaminated open land, and radioactive source contaminated soil.
7. The method of applying a chitosan/dextran-containing bryoid suspension to bare green according to claim 1, wherein the bryoid adhesive is prepared by: adding 10-30 parts by weight of melamine, 10-20 parts by weight of urea, 60-90 parts by weight of attapulgite and 50-80 parts by weight of sepiolite powder into a ball milling tank, adding ball milling balls into the ball milling tank, introducing liquid nitrogen into the ball milling tank to immerse the materials in the liquid nitrogen at the temperature of-160 ℃ to-175 ℃, and keeping the balance between the volatilization amount and the introduction amount of the liquid nitrogen to stabilize the liquid level; carrying out ball milling for 30-45 min after keeping the temperature for 15 min; after ball milling is finished, transferring the ball milling tank into a vacuum glove box, standing for 3-5 hours, and collecting ball milling materials;
and secondly, adding 50-100 parts by weight of konjac glucomannan, 50-100 parts by weight of chitosan and 50-100 parts by weight of collagen into 200-300 parts by weight of water, stirring for 30-60 min at the speed of 1000-1200 r/min, adding half of the ball-milled material at the speed of 10-30 parts/min in the stirring and mixing process, then adding 40-60 parts by weight of latex powder, 10-30 parts by weight of stearic acid and 5-15 parts by weight of ammonium zirconium carbonate, continuously mixing and stirring for 30-45 min at the rotating speed of 1000-1200 r/min, and simultaneously adding the other half of the ball-milled material at the speed of 10-30 parts/min in the stirring and mixing process to obtain the special moss adhesive.
8. The method of claim 7, wherein the latex powder is one or more of ethylene-vinyl acetate copolymer latex powder, acrylate-styrene copolymer latex powder, and vinyl acetate-acrylate-higher fatty acid vinyl ester terpolymer latex powder.
9. The method of using a chitosan/dextran-containing bryozoatum suspension in nude green according to claim 7, wherein the collagen is replaced with a modified collagen prepared by: adding 50-100 parts by weight of collagen into 1000-1200 parts by weight of water, carrying out ultrasonic treatment for 30-45 min, adding 5-8 parts by weight of theanine, continuing to carry out ultrasonic treatment for 15-30 min, then adding 15-25 parts by weight of poly-L-glutamic acid, heating to 45-60 ℃, placing under the irradiation of ultraviolet lamp light, carrying out stirring reaction for 15-30 min, and carrying out centrifugal separation to obtain the modified collagen.
10. The method for applying the moss sporophyte suspension containing chitosan/glucan to bare ground greening according to claim 7, wherein the power of the ultrasound is 80-120W, and the frequency is 30-50 kHz; the power of ultraviolet lamp light irradiation is 50-500W, and the wavelength of ultraviolet light is 220-400 nm.
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| CN116584381A (en) * | 2023-05-10 | 2023-08-15 | 江西农业大学 | Litsea cubeba embryogenic callus induction medium and induction method |
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| GB2597602A (en) | 2022-02-02 |
| GB202109405D0 (en) | 2021-08-11 |
| CN112335508B (en) | 2021-05-18 |
| GB2597602B (en) | 2022-07-20 |
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