CN105981636B - A kind of bog moss protonema rapid propagation method - Google Patents
A kind of bog moss protonema rapid propagation method Download PDFInfo
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- CN105981636B CN105981636B CN201510070439.6A CN201510070439A CN105981636B CN 105981636 B CN105981636 B CN 105981636B CN 201510070439 A CN201510070439 A CN 201510070439A CN 105981636 B CN105981636 B CN 105981636B
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Abstract
The present invention relates to organic synthesis field, discloses a kind of bog moss protonema rapid propagation method and include the following steps, prepare culture medium, prepare suspension, culture, continuous culture proliferation number generation.Culture of the present invention can be bred at any time, not influenced by external factor such as season, environment, and cost is relatively low, and occupied space resource is less；Necessary growth elements and environment are provided for the growth of bog moss protonema.Meanwhile, it is capable to quickly breed bog moss protonema, solve that the speed of growth is slow, big technical problem affected by environment.The pH value and bog moss natural habitat meta-acid soil that the present invention selects are coincide.The sucrose that the present invention is added provides carbon source, greatly improves bog moss protonema yield.The present invention selects 6- benzyl aminoadenine (6-BA) to be used as the basic element of cell division, is greater than other auxin to the effect of bog moss protonema.The optium concentration that the present invention filters out hormone 6-BA is 0.05-0.2 μm of ol/L, inhibits to grow instead more than or less than this concentration.
Description
Technical field
The present invention relates to horticultural gardening technical field more particularly to a kind of bog moss protonema rapid propagation methods.
Background technique
Peat is rich in organic matter and humic acid, can be directly as cultivation medium, it is also possible to make the fertilizer of improvement soil, in recent years
Come, the high speed development of horticulture industry has driven the formation of peat industry.However, the market demand of peat is increasing, peat
Wetland is also constantly reduced by exhaustive exploitation, area, and China is gradually converted into importer from peat exported country.Peat wetland
It destroys and not only vegetation distribution, ecological diversity is caused to seriously threaten, also result in the reduction of nature ability to pool carbon, human living ring
A series of consequences such as border deterioration.To realize the sustainable use of mud and charcoal, while the development of agricultural and horticulture industry is not influenced,
The substitute for finding peat is a problem to be solved.
Bog moss (Sphagnum.sp.) is also known as sphagna, be Genus Sphagnum plant general designation and formtion of peat it is main
Source.America and Europe's gardening developed country such as Germany has been studied using bog moss substitution peat as growth substrate, and is successfully applied to
The cultivations of plants such as cuckoo, brier.Therefore, the exploitation of bog moss and application can promote the development of horticulture industry.
Summary of the invention
The present invention is directed to the disadvantage in existing, discloses a kind of bog moss protonema rapid propagation method.
In order to solve the above-mentioned technical problem, the present invention is addressed by following technical proposals.
A kind of bog moss protonema rapid propagation method, includes the following steps,
A. the phosphoric acid of the ammonium nitrate of 0.1-0.3g, the calcium chloride of 0.05-0.15g, 0.05-0.15g are added in every 1 liter of water
Potassium dihydrogen, the magnesium sulfate of 0.05-0.15g, 0.02-0.035g iron chloride；PH value is adjusted to 5-6, then adds the sugarcane of 5-10g
The 6- benzyl aminoadenine of sugar and 0.05-0.2 μm of ol, obtains culture medium；High pressure sterilization handles culture medium；
B. bog moss protonema is crushed, the bog moss protonema crushed is moved into sterile water, suspension is made by filtering
Liquid；
C. suspension prepared by step B is added in the culture medium prepared to step A, wherein being added in every 10ml culture medium
0.5-1.5ml suspension is put into shaking table, revolving speed 130-150r/min, illumination 2800-3500lux, and the photoperiod is day night 10
h/ 14 h；The condition of culture solves that the bog moss protonema speed of growth is slow, technical problem easily affected by environment.
D. after cultivating 20 days, bog moss protonema is crushed again, is dispensed in the culture medium prepared to step A after filtering,
In 0.5-1.5ml suspension is added in every 10ml culture medium, be put into shaking table, revolving speed 130-150r/min, illumination 2800-
3500lux, photoperiod are 10 h/ of day night, 14 h；After culture 20 days, culture medium nutrient depletion in former bottle, by the precursor of proliferation
Body crushes again.The condition of culture solves the slow technical problem of the bog moss protonema speed of growth, reaches the mesh of fast breeding
's.
E. step D, continuous culture proliferation number generation are repeated.
The culture medium that the present invention uses is Beneck culture medium.Culture medium provides necessary for the growth of bog moss protonema
Growth elements and environment, so the screening of culture medium occupies an important position in entire quickly reproductive process.In research process, into
The screening of minimal medium is gone, test medium is respectively as follows: Beneck, Knop, BCD, MS, 1/2MS, water.Pass through different trainings
The culture experiment discovery of base is supported, Beneck the and Knop culture medium of Low-salinity is relatively suitble to the growth of bog moss；And equally it is moss
The BCD culture medium of common culture medium is then not suitable for bog moss growth；The various concentration MS of high salinity is less useful for the life of bog moss
It is long, or even with the extension of incubation time, protonema can gradually turn yellow.
In research process, the screening of pH value is carried out, test pH gradient is respectively 4,5,6,7,8.Result of study shows, mud
Charcoal moss protonema is also proliferated comparatively fast on the lower culture medium of pH, and pH5.0-6.0 is its optimum growth pH value, with
The raising of pH, bog moss protonema increment is reduced, or even is stopped growing.This coincide with bog moss natural habitat meta-acid soil.
The sucrose of addition provides carbon source, greatly improves bog moss protonema yield.In research process, carbon source has been carried out
And its screening of concentration, test carbon source is respectively sucrose and glucose, and concentration is respectively 5 g/L, 10 g/L, 20 g/L, 40g/
L.Result of study shows that the addition of carbon source greatly improves bog moss protonema yield: sucrose and glucose have it significantly
Effect, but it is higher to the utilization efficiency of sucrose, and the sucrose (5-10g/L) of low concentration is more advantageous to the proliferation of bog moss, proliferation effect
Rate is 2.7-4.3 times more than high concentration processing.
The present invention selects 6- benzyl aminoadenine (6-BA) to be used as the basic element of cell division, is greater than to the effect of bog moss protonema
Other auxin, such as 6-BA are greater than auxin indolebutyric acid (IBA) to the effect of bog moss protonema, and IBA increases protonema
Grow no remarkable effect.The optium concentration that the present invention filters out hormone 6-BA is 0.05-0.2 μm of ol/L, is more than or less than this concentration
Inhibit growth instead.In research process, the screening of hormone and its concentration is carried out, test hormone is respectively IBA and 6-BA, concentration
Respectively 0.1 μm of ol/L, 1 μm of ol/L, 10 μm of ol/L.Result of study shows that basic element of cell division 6-BA is to bog moss protonema
Effect is greater than auxin IBA.IBA is smaller to bog moss protonema proliferation function, and high concentration (10 μm of ol/L) can also inhibit its life
It is long.Low concentration (0.05-0.2 μm of ol/L) 6-BA significantly improves bog moss yield, weakens as concentration increases facilitation.
Preferably, the di(2-ethylhexyl)phosphate of the ammonium nitrate of 0.2g, the calcium chloride of 0.1g, 0.1g are added in every 1 liter of water in step A
Hydrogen potassium, the magnesium sulfate of 0.1g, 0.028g iron chloride；PH value adjusted to 5-6, then adds the sucrose and 0.1 μm of ol of 10g
6- benzyl aminoadenine, obtains culture medium.The formula for the optimal medium that the present invention filters out: it is added 0.2g's in every 1 liter of water
Ammonium nitrate, the calcium chloride of 0.1g, the potassium dihydrogen phosphate of 0.1g, the magnesium sulfate of 0.1g, 0.028g iron chloride.Culture medium is peat
The growth of moss protonema, which provides necessary growth elements and environment, the culture medium of the proportion, can quickly breed bog moss precursor
Body.
Preferably, in step A, magnesium sulfate MgSO4·7H2O。
Preferably, in step A, iron chloride FeCl3 ·6 H2O。
Preferably, culture medium is fitted into 500ml triangular flask in step A, per bottled about 200ml, high pressure sterilization is waited for
With.
Preferably, bog moss protonema crushes 1min, specific breakage rate 18000r/ in superclean bench in step B
Min, the filtering of 200 mesh cell sieves；The bog moss protonema crushed is moved into sterile water, suspension is made.
Preferably, 1ml suspension is added in every 10ml culture medium in step C.
Preferably, being put into shaking table, revolving speed 140r/min, illumination 3000lux in step C, the photoperiod is day night 10
h/ 14 h。
Preferably, being put into shaking table, revolving speed 140r/min, illumination 3000lux in step D, the photoperiod is day night 10
h/ 14 h。
Preferably, 1ml suspension is added in every 10ml culture medium in step D.
Compared with prior art, the invention has the benefit that
Culture of the present invention can be bred at any time, not influenced by external factor such as season, environment, and cost compared with
Low, occupied space resource is less；Necessary growth elements and environment are provided for the growth of bog moss protonema.Meanwhile, it is capable to fast
Speed breeding bog moss protonema, solves that the speed of growth is slow, big technical problem affected by environment.
The optimal pH that the present invention selects is 5-6, is coincide with bog moss natural habitat meta-acid soil.The sugarcane that the present invention is added
Sugar provides carbon source, greatly improves bog moss protonema yield.The optimum carbon source that the present invention selects is sucrose, and utilization efficiency is most
Height, and the yield of bog moss protonema reaches highest.The present invention selects 6- benzyl aminoadenine (6-BA) to be used as the basic element of cell division,
Other auxin are greater than to the effect of bog moss protonema, for example 6-BA is greater than auxin indoles to the effect of bog moss protonema
Butyric acid (IBA), IBA are proliferated without remarkable effect protonema.The optium concentration that the present invention filters out hormone 6-BA is 0.05-0.2 μ
Mol/L inhibits to grow instead more than or less than this concentration.
Specific embodiment
Embodiment 1
A kind of bog moss protonema rapid propagation method, includes the following steps,
A. the sulfuric acid of the ammonium nitrate of 0.2g, the calcium chloride of 0.1g, the potassium dihydrogen phosphate of 0.1g, 0.1g are added in every 1 liter of water
The iron chloride of magnesium, 0.028g；PH value is adjusted to 5, then adds the sucrose of 10g and the 6- benzyl aminoadenine of 0.1 μm of ol, is obtained
To culture medium；High pressure sterilization handles culture medium；
B. bog moss protonema is crushed, the bog moss protonema crushed is moved into sterile water, suspension is made by filtering
Liquid；
C. suspension prepared by step B is added in the culture medium prepared to step A, wherein being added in every 10ml culture medium
1ml suspension is put into shaking table, revolving speed 140r/min, illumination 3000lux, and the photoperiod is 10 h/ of day night, 14 h；
D. after cultivating 20 days, bog moss protonema is crushed again, is dispensed in the culture medium prepared to step A after filtering,
In 1ml suspension is added in every 10ml culture medium, be put into shaking table, revolving speed 140r/min, illumination 3000lux, the photoperiod be daytime/
10 h/ of night, 14 h；
E. step D, continuous culture proliferation number generation are repeated.
Embodiment 2
A kind of bog moss protonema rapid propagation method, includes the following steps,
A. the ammonium nitrate of 0.1g, the calcium chloride of 0.05g, the potassium dihydrogen phosphate of 0.05g, 0.05g are added in every 1 liter of water
The iron chloride of magnesium sulfate, 0.02g；PH value is adjusted to 5, then adds the sucrose of 5g and the 6- benzyl aminoadenine of 0.05 μm of ol,
Obtain culture medium；High pressure sterilization handles culture medium；
B. bog moss protonema crushes 1min, specific breakage rate 18000r/min, 200 mesh cell sieves in superclean bench
Filtering；The bog moss protonema crushed is moved into sterile water, suspension is made；
C. suspension prepared by step B is added in the culture medium prepared to step A, wherein being added in every 10ml culture medium
0.5ml suspension is put into shaking table, revolving speed 130r/min, illumination 2800lux, and the photoperiod is 10 h/ of day night, 14 h；
D. after cultivating 20 days, bog moss protonema is crushed again, is dispensed in the culture medium prepared to step A after filtering,
In 0.5ml suspension is added in every 10ml culture medium, be put into shaking table, revolving speed 130r/min, illumination 2800lux, the photoperiod is
10 h/ of day night, 14 h；
E. step D, continuous culture proliferation number generation are repeated.
Embodiment 3
A kind of bog moss protonema rapid propagation method, includes the following steps,
A. the ammonium nitrate of 0.3g, the calcium chloride of 0.15g, the potassium dihydrogen phosphate of 0.15g, 0.15g are added in every 1 liter of water
The iron chloride of magnesium sulfate, 0.035g；By pH value adjust to 6, then add 10g sucrose and 0.2 μm of ol 6- benzyl amino gland it is fast
Purine obtains culture medium；Culture medium is fitted into 500ml triangular flask, per bottled about 200ml, high pressure sterilization is stand-by.
B. bog moss protonema is crushed, the bog moss protonema crushed is moved into sterile water, suspension is made by filtering
Liquid；
C. suspension prepared by step B is added in the culture medium prepared to step A, wherein being added in every 10ml culture medium
1.5ml suspension is put into shaking table, revolving speed 150r/min, illumination 3500lux, and the photoperiod is 10 h/ of day night, 14 h；
D. after cultivating 20 days, bog moss protonema is crushed again, is dispensed in the culture medium prepared to step A after filtering,
In 1.5ml suspension is added in every 10ml culture medium, be put into shaking table, revolving speed 150r/min, illumination 3500lux, the photoperiod is
10 h/ of day night, 14 h；
E. step D, continuous culture proliferation number generation are repeated.
Embodiment 4
A kind of bog moss protonema rapid propagation method, includes the following steps,
A. the ammonium nitrate of 0.15g, the calcium chloride of 0.08g, the potassium dihydrogen phosphate of 0.08g, 0.08g are added in every 1 liter of water
MgSO4·7H2O, the FeCl of 0.025g3 ·6 H2O；PH value adjusted to 5.5, then adds the sucrose and 0.08 μm of ol of 6g
6- benzyl aminoadenine, obtains culture medium；High pressure sterilization handles culture medium；
B. bog moss protonema is crushed, the bog moss protonema crushed is moved into sterile water, suspension is made by filtering
Liquid；
C. suspension prepared by step B is added in the culture medium prepared to step A, wherein being added in every 10ml culture medium
0.8ml suspension is put into shaking table, revolving speed 135r/min, illumination 2900lux, and the photoperiod is 10 h/ of day night, 14 h；
D. after cultivating 20 days, bog moss protonema is crushed again, is dispensed in the culture medium prepared to step A after filtering,
In 0.8ml suspension is added in every 10ml culture medium, be put into shaking table, revolving speed 135r/min, illumination 2900lux, the photoperiod is
10 h/ of day night, 14 h；
E. step D, continuous culture proliferation number generation are repeated.
Embodiment 5
A kind of bog moss protonema rapid propagation method, includes the following steps,
A. the ammonium nitrate of 0.28g, the calcium chloride of 0.12g, the potassium dihydrogen phosphate of 0.12g, 0.14g are added in every 1 liter of water
The iron chloride of magnesium sulfate, 0.032g；By pH value adjust to 6, then add 9g sucrose and 0.18 μm of ol 6- benzyl amino gland it is fast
Purine obtains culture medium；High pressure sterilization handles culture medium；
B. bog moss protonema is crushed, the bog moss protonema crushed is moved into sterile water, suspension is made by filtering
Liquid；
C. suspension prepared by step B is added in the culture medium prepared to step A, wherein being added in every 10ml culture medium
1.2ml suspension is put into shaking table, revolving speed 140r/min, illumination 3400lux, and the photoperiod is 10 h/ of day night, 14 h；
D. after cultivating 20 days, bog moss protonema is crushed again, is dispensed in the culture medium prepared to step A after filtering,
In 1.2ml suspension is added in every 10ml culture medium, be put into shaking table, revolving speed 140r/min, illumination 3400lux, the photoperiod is
10 h/ of day night, 14 h；
E. step D, continuous culture proliferation number generation are repeated.
Embodiment 6
A kind of bog moss protonema rapid propagation method, includes the following steps,
A. the sulfuric acid of the ammonium nitrate of 0.2g, the calcium chloride of 0.1g, the potassium dihydrogen phosphate of 0.1g, 0.1g are added in every 1 liter of water
The iron chloride of magnesium, 0.028g；PH value is adjusted to 5, then adds the sucrose of 10g and the 6- benzyl aminoadenine of 0.1 μm of ol, is obtained
To culture medium；High pressure sterilization handles culture medium；
B. bog moss protonema crushes 1min, specific breakage rate 18000r/min, 200 mesh cell sieves in superclean bench
Filtering；The bog moss protonema crushed is moved into sterile water, suspension is made；
C. suspension prepared by step B is added in the culture medium prepared to step A, wherein being added in every 10ml culture medium
1ml suspension is put into shaking table, revolving speed 140r/min, illumination 3000lux, and the photoperiod is 10 h/ of day night, 14 h；
D. after cultivating 20 days, bog moss protonema is crushed again, is dispensed in the culture medium prepared to step A after filtering,
In 1ml suspension is added in every 10ml culture medium, be put into shaking table, revolving speed 140r/min, illumination 3000lux, the photoperiod be daytime/
10 h/ of night, 14 h；
E. step D, continuous culture proliferation number generation are repeated.
Embodiment 7
A kind of bog moss protonema rapid propagation method, includes the following steps,
A. the ammonium nitrate of 0.2g, the calcium chloride of 0.1g, the potassium dihydrogen phosphate of 0.1g, 0.1g are added in every 1 liter of water
MgSO4·7H2O, the FeCl of 0.028g3 ·6 H2O；PH value is adjusted to 5, then adds the sucrose of 10g and the 6- of 0.1 μm of ol
Benzyl aminoadenine, obtains culture medium；High pressure sterilization handles culture medium；
B. bog moss protonema is crushed, the bog moss protonema crushed is moved into sterile water, suspension is made by filtering
Liquid；
C. suspension prepared by step B is added in the culture medium prepared to step A, wherein being added in every 10ml culture medium
1ml suspension is put into shaking table, revolving speed 140r/min, illumination 3000lux, and the photoperiod is 10 h/ of day night, 14 h；
D. after cultivating 20 days, bog moss protonema is crushed again, is dispensed in the culture medium prepared to step A after filtering,
In 1ml suspension is added in every 10ml culture medium, be put into shaking table, revolving speed 140r/min, illumination 3000lux, the photoperiod be daytime/
10 h/ of night, 14 h；
E. step D, continuous culture proliferation number generation are repeated.
In short, the foregoing is merely presently preferred embodiments of the present invention, it is all according to equalization made by scope of the present invention patent
Variation and modification, shall all be covered by the patent of the invention.
Claims (8)
1. a kind of bog moss protonema rapid propagation method, it is characterised in that: include the following steps,
A. in every 1 liter of water be added the ammonium nitrate of 0.2g, the calcium chloride of 0.1g, the potassium dihydrogen phosphate of 0.1g, 0.1g magnesium sulfate,
The iron chloride of 0.028g；PH value is adjusted to 5-6, then adds the sucrose of 10g and the 6- benzyl aminoadenine of 0.1 μm of ol, is obtained
Culture medium；High pressure sterilization handles culture medium；
B. bog moss protonema is crushed into 1min, specific breakage rate 18000r/min in superclean bench, 200 mesh cells are sieved through
Filter；The bog moss protonema crushed is moved into sterile water, suspension is made；
C. suspension prepared by step B is added in the culture medium prepared to step A, wherein 0.5- is added in every 10ml culture medium
1.5ml suspension is put into shaking table, revolving speed 130-150r/min, illumination 2800-3500lux, and the photoperiod is day night 10h/
14h；
D. after cultivating 20 days, bog moss protonema is crushed again, is dispensed in the culture medium prepared to step A after filtering, wherein often
0.5-1.5ml suspension is added in 10ml culture medium, is put into shaking table, revolving speed 130-150r/min, illumination 2800-3500lux,
Photoperiod is day night 10h/14h；
E. step D, continuous culture proliferation number generation are repeated.
2. bog moss protonema rapid propagation method according to claim 1, it is characterised in that: in step A, magnesium sulfate
For MgSO4·7H2O。
3. bog moss protonema rapid propagation method according to claim 1, it is characterised in that: in step A, iron chloride
For FeCl3·6H2O。
4. bog moss protonema rapid propagation method according to claim 1, it is characterised in that: in step A, will cultivate
Base is fitted into 500ml triangular flask, and per bottled about 200ml, high pressure sterilization is stand-by.
5. bog moss protonema rapid propagation method according to claim 1, it is characterised in that: in step C, every 10ml
1ml suspension is added in culture medium.
6. bog moss protonema rapid propagation method according to claim 1, it is characterised in that: in step C, be put into and shake
Bed, revolving speed 140r/min, illumination 3000lux, photoperiod are day night 10h/14h.
7. bog moss protonema rapid propagation method according to claim 1, it is characterised in that: in step D, be put into and shake
Bed, revolving speed 140r/min, illumination 3000lux, photoperiod are day night 10h/14h.
8. bog moss protonema rapid propagation method according to claim 1, it is characterised in that: in step D, every 10ml
1ml suspension is added in culture medium.
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| CN109089882B (en) * | 2018-08-30 | 2022-02-08 | 云南省农业科学院花卉研究所 | Moss tissue culture and seedling culture method directly induced by spores |
| CN109169021B (en) * | 2018-10-18 | 2020-11-24 | 贵州工程应用技术学院 | Method for quickly laying and planting sphagnum on wet land of river bank of plateau city |
| CN109588315B (en) * | 2018-12-31 | 2021-05-28 | 西南民族大学 | Method for disinfecting bryophyte explants |
| CN113265369B (en) * | 2021-05-25 | 2024-01-16 | 内蒙古大学 | Liquid culture method for rapidly improving biomass of short leaf para-dentate protonema |
| 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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