CN105265064B - Fern protonema Liquid Culture method for culturing seedlings - Google Patents

Fern protonema Liquid Culture method for culturing seedlings Download PDF

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Publication number
CN105265064B
CN105265064B CN201510789978.5A CN201510789978A CN105265064B CN 105265064 B CN105265064 B CN 105265064B CN 201510789978 A CN201510789978 A CN 201510789978A CN 105265064 B CN105265064 B CN 105265064B
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spore
protonema
culture
fern
water
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CN105265064A (en
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曹皓普
袁明
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Sichuan Agricultural University
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Sichuan Agricultural University
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Abstract

The invention discloses a kind of fern protonema Liquid Culture method for culturing seedlings, including spore germination step:The spore of harvesting is added in clean clear water after soaking to remove the floating object of the water surface and moisture, the scheduled time is optionally frozen to promote spore germination, add appropriate clean clear water and handled with gibberellin to promote spore germination, spore after processing, which is soaked in water, to be cultivated, in the medicine that Initial stage of culture is optionally grown using killing or suppressing exogenous microbial;There is bubble to produce in spore settling zone in incubation and then show that spore has been sprouted, protonema starts to grow, if having spore agglomerate buoyance lift in incubation in the water surface, should use predetermined means so that spore agglomerate settles.The method of the invention is simple and easy, and can effectively avoid not sprouting spore and go mouldy, and protonema Liquid Culture system controllability is strong, breeds efficiency high, available for fern scale breeding.

Description

Fern protonema Liquid Culture method for culturing seedlings
Technical field
The invention belongs to the breeding method of pteridophyte seedling, in particular it relates to which a kind of fern protonema Liquid Culture is educated Seedling-growing method.
Background technology
Fern is the vascular plant that earliest extant occurs on the earth.Many ferns great ornamental value because of plant shape grace; It is deep to be liked by consumers in general meanwhile substantial amounts of fern has medicinal or edibility.However, due to long-term, substantial amounts of nothing The excavation of predation formula is limited, the deterioration of the ecological environment in addition, wild fern resource is seriously damaged, even in original producton location, Hen Duoye Raw distribution types are also drastically reduced, or even extinction, the pteridophyte of natural propagation can not meet the needs of people.Therefore, it is raw Start to carry out artificial breeding and cultivation to the pteridophyte being worth with Important Economic in production.
At present, pteridophyte artificial breeding mainly uses the methods of vegetative reproduction, sporogenesis and tissue cultures.
Vegetative reproduction is to produce asexual gemma using positions such as the roots, root-like stock, leaf of fern, or passes through apical meristem group Knit and produce new plant.This method is simple, easy, and technical requirements are not high, but proliferation rates are small, need to take seeding-growth strain, right The destruction Of resources are very big, in the long run consider, and this method has larger limitation, and the fern seedling breeding method of non-optimal.
Tissue culture technique is a kind of fast and effectively fern mating system, and explant dosage is few, and natural resources is not made Into destruction, growth coefficient is high, and advantage is notable in Germ-plasma resources protection, extensive modernization commercial seedling production.It is but logical at present Cross that the fern that tissue cultures are successfully bred is less, every kind of fern needs suitable culture medium and condition of culture, and screening operation amount is big, Culture medium versatility is not strong, and breeding cycle is long, seedling transplanting survival rate is not high, need to establish tissue culture system, equipment, place into This is higher, and operating technology requirement is strict, and general production unit is difficult to carry out related work.Therefore, fern tissue cultivating and seedling skill Art is also by a definite limitation.
Sporogenesis is that sporinite is formed after being sprouted in solid matrix using spore caused by fern, then passes through sporinite Caused gametophyte obtains seedling.It is excellent that this method has that cost is low, technical requirements are not high, easy to operation, breeding amount is big etc. Point, used particularly suitable for general planting household, but this mode of reproduction is easily by spore germination rate, sowing media, thickness of sowing, culture The factors such as condition influence, do not sprout spore and easily go mouldy, gametophyte is formed it is totally unfavorable, and sporogenous tissue cultivate it is relatively multiple It is miscellaneous, cumbersome.
The content of the invention
The technical problems to be solved by the invention, which are to provide one kind, to be had simple and easy and can effectively avoid not sprouting spore The method that the sporogenesis easily to go mouldy cultivates pteridophyte seedling.
Technical scheme is used by the present invention solves the above problems:
Fern protonema Liquid Culture method for culturing seedlings, including
Spore germination step:The spore of harvesting is added in clean clear water and soaked, after the floating object and moisture that remove the water surface, The scheduled time is optionally frozen to promote spore germination, appropriate clean clear water is added and is handled with gibberellin to promote spore to sprout Hair, the spore after processing, which is soaked in water, to be cultivated, and optionally using killing or suppresses exogenous microbial life in Initial stage of culture Long medicine(Such as, optionally using antiseptic to handle the situation of bacteria breed, or optionally using primary dynamic Thing agent for killing with handle protozoan growth situation);There is bubble to produce then table in spore settling zone in incubation Bright spore has been sprouted, and protonema starts to grow, if having spore agglomerate buoyance lift in incubation in the water surface, should use predetermined means with Settle spore agglomerate.
The gibberellin used in the present invention is broad spectrum activity plant growth regulator, widely distributed, vdiverse in function, can be released not Sleep, promote seed to sprout, the gibberellin of debita spissitudo can improve pteridophyta spore germination rate.In the prior art generally will harvesting Spore afterwards is seeded in solid matrix(Soil after usually handling)Middle culture, sprouting, and the mould in upper soll layer is preferably Oxygen, heterotroph.Oxygen content in liquid internal environment is much lower compared with soil surface;The present invention is using Aquaponic ring simultaneously Border, the content of organic matter is extremely low (it is seldom not to be sprouted the organic matter of spores release, can easily and fast be dropped in incubation by changing water The low content of organic matter), it can not meet needed for heterotroph fungus growth.So it can prevent that fern seed from sprouting and protonema grows During go mouldy.
Wherein, in the spore germination step, the temperature frozen is 0 to -20 DEG C, and the time frozen is a Zhou Zhiyi Individual month.
Wherein, in the spore germination step, if need frozen and preserved spore be according to fern grown area whether Belong to winter outdoor temperature in area below freezing to carry out selection, be specially:When the area that fern is grown belongs to the winter Season, outdoor temperature was area below freezing, then should carry out freezing operation;If the area that fern is grown belongs to winter outdoor temp Spend for area above freezing, the then optional operation without freezing.
Wherein, in the spore germination step, the floating object generally refers to float on the spore of the water surface, spore Capsule, and/or other debris.
Wherein, in the spore germination step, final concentration of 100~300ppm of the gibberellin, the gibberellin Processing time is 15 minutes to 24 hours.
Wherein, in the spore germination step, the condition that the spore after the processing is soaked in when being cultivated in water is:15 ~20 DEG C, natural lighting, and culture vessel, which have, can avoid exogenous microbial or other species spores from entering culture vessel So as to cause the covering protection thing of pollution.
Wherein, in the spore germination step, the spore is soaked in water during culture, it is necessary to periodically or not Water is regularly added, to keep the volume of culture vessel reclaimed water to be basically unchanged.
Wherein, in the spore germination step, the preferred agricultural streptomycin of antiseptic, the end of the agricultural streptomycin Concentration is 0.01 ~ 0.1%, the preferred Tinidazole of protozoan agent for killing, final concentration of 0.05 ~ 0.5mg/ of the Tinidazole mL。
Wherein, in the spore germination step, the predetermined means can make what spore agglomerate settled to be any Means, specifically, such as it can stir by stirring rod or rock culture vessel mode and realize.
Wherein, because the present invention is a kind of breeding method of pteridophyte seedling, therefore, the water used should be met necessarily Cell culture water or plant culture water requirement, all to avoid exogenous microbial from polluting to require spore, Specifically, the water used in the present invention can be free of contamination clean river, well water or running water, wherein, phreatic water can be direct Use;River needs to boil more than 30 minutes, to make contained microorganism and spore inactivation;Running water needs exposure more than 3 days, Need also exist for boiling more than 30 minutes.And the clean clear water being particularly limited to the application, most preferably aqua sterilisa or through boil place Water after reason, to avoid living contaminants.
Wherein, the method for culturing seedlings can also include:
Protonema incubation step:When spore occurs by the spore germination step culture to protonema in natural lighting Under the conditions of continue to cultivate, add urea and potassium dihydrogen phosphate by foliar fertilizer dosage in culture, to promote the growth of protonema, Predetermined means should be used in incubation(As it was noted above, the means such as stirring can be used)To avoid protonema aggregation, winding shape Into agglomerate so as to reduce appreciation rate, when protonema content is higher than predetermined value in culture vessel, using expand cultivate by the way of after Continuous culture.
Fern protonema can carry out photosynthesis, and Liquid Culture improves intensity of illumination in appropriate degree, promotes original Mycelial growth, culture can be expanded in incubation in good time in addition, therefore improve the propagation efficiency of protonema.Due to protonema be Cultivated in the glass container of printing opacity, water, fertilising can be changed at any time, once occur going mouldy, microbiological contamination or grow protozoan without During method processing, you can reject pollution group, while culture can be expanded in good time according to protonema growing state.Therefore, incubation Middle controllability is directly seeded in much better than in soil compared with spore.
Wherein, it is described expand culture operation be specifically:After stir culture thing, by stoste: new liquid is(1~5): 10 body Product continues to expand culture than accessing new culture vessel.
Wherein, in the protonema incubation step, judgement of the protonema content higher than predetermined value can pass through the shape of protonema State is carried out, and such as when protonema produces lumps, can determine that protonema too high levels, higher than predetermined value, for raising culture efficiency, It can continue to cultivate by the way of culture is expanded.
Wherein, the method for culturing seedlings can also include:
Gametophyte incubation step, sporinite incubation step, sporinite (seedling) transplant step.
Wherein, the gametophyte incubation step is:Choose free of contamination fertile soil, pH6.0~7.0, exposure under sunlight, do Thoroughly, crush, go the removal of impurity, take it is appropriate as matrix and fill basin to the 2/3 ~ 4/5 of volume, separately take the fertile soil mistake after above-mentioned processing Culture basin soil table, 1 ~ 5cm of thickness are overlying on after 50 eye mesh screens, and soil in basin is infiltrated by predetermined way, will be through the precursor The protonema sprinkle that body incubation step obtains ensures protonema more than 80% in flakes in Culture basin soil surface, is placed in illumination Incubated at room temperature in environment, ground moistening and holding should be kept to be unfavorable for the temperature of heterotroph fungi growth during culture (That is, low temperature should be avoided), cultivate to gametophyte and expose soil surface.
Wherein, in the gametophyte incubation step, allow the predetermined way that soil infiltrates in basin that basin is placed in pallet, The water filling into pallet, make the purpose that soil infiltrates in basin so as to realize.
Wherein, in the gametophyte incubation step, for increase seedling yield, by protonema sprinkle after Culture basin, if Soil surface still has many places blank --- and without protonema, it can again be inoculated with without protonema blank space in soil surface, also may be used in good time The mode being repeatedly inoculated with directly is taken, to ensure protonema more than 80% in flakes.
Wherein, the sporinite incubation step is:After gametophyte exposes soil surface, keep ground moistening so that sperm with Ovum combines to form embryonated egg, continues culture until there is sporinite seedling.
If protonema is inoculated with the gametophyte incubation step, the protonema 100% in Culture basin continues to cultivate in flakes About 20-50 strains/cm can then be generated2Sporinite seedling.
Wherein, sporinite (seedling) transplant step is:By 4cm × 4cm plants after sporinite grows 3~4 leaves Away from first time transplanting is carried out, using conventional soil as matrix, indoor culture, after 7~15 days seedlings of transplanting survive for the first time, press Seeding row spacing 6cm × 10cm is transplanted on outdoor seedbed, continues to cultivate, and is grown after seedling to the height (10~15cm) that meets the requirements, In autumn then or spring in next year production Tanaka is colonized in by appropriate seeding row spacing.
Wherein, the method for culturing seedlings can also include:
Field daily management operation:Seedling transplant planting routinely requires to be operated after Tanaka is produced.Pay attention to avoiding Strong light direct beam, fertilising in good time, watering, weeding, loosens the soil and removes sick seedling, dead seedling, prevent pest and disease damage in time.
Wherein, the method for culturing seedlings can also include
Spore acquisition step:In spore maturation (different ferns different regions mature period different), treat sporophyll by When green is changed into yellowish-brown (before spore distributes), sporophyll is cut, is dried, room temperature storage or low temperature (being less than 4 DEG C) storage are to prolong Long spore activity, crushes sporophyll, crosses 100-200 eye mesh screens and sifts out spore and sporangium.
Wherein, in the spore acquisition step, after the sporophyll is dried, whether the area grown according to fern is category Choose whether to carry out to freeze operation in area below freezing in winter outdoor temperature, be specially:The area that fern is grown Belonging to winter temperature need to freeze one to three month or so in area below freezing, sporophyll after drying(Cryogenic temperature about- 10 DEG C to -20 DEG C)To improve spore germination rate, the area that fern is grown belongs to winter temperature in area above freezing, optional Select without the above-mentioned operation frozen.
To sum up, the beneficial effects of the invention are as follows:
1st, the present invention using liquid environment culture spore and promotes it to sprout into protonema, and solid matrix can be avoided to go mouldy and made Into adverse effect, by freezing and the various ways such as gibberellin improve the germination rate of spore.
2nd, protonema Liquid Culture system controllability is strong, breed efficiency high, while avoid solid matrix go mouldy caused by not Profit influences, and can obtain a large amount of protonemas by expanding culture, meaning is very big in scale breeding.
Embodiment
With reference to embodiment, the present invention is described in further detail, but the implementation of the present invention is not limited to this.
Breeding method of the present invention may be adapted to each pteridophyte, act including but not limited to set forth below it is several specific Pteridophyte, mainly by using Liquid Culture, can effectively prevent from occurring in spore germination and protonema growth course Go mouldy, improve the propagation efficiency of protonema, strengthen the controllability of protonema cultivating system, following examples give different ferns What plant seedling was cultivated schematically illustrates.
Embodiment 1
1. spore acquisition step:
After August part, during Matteuccia strthiopteris spore maturation, when sporophyll is changed into yellowish-brown from green (before spore distributes), The afternoon (taken outdoors need to be after dew disperses) of continuous sunny is selected, sporophyll is cut with clean scissors, is placed in clean light In sliding paper bag, drying is air-dried, room temperature storage or low temperature (being less than 4 DEG C) storage are to extend spore activity, by above-mentioned obtained spore Leaf fully pulverizes, and 100-200 eye mesh screens sift out spore (and sporangium), goes the removal of impurity, obtains spore(Contain sporangium).Spore Leaf need to be one month or so in refrigerating chamber freezing processing after drying(Cryogenic temperature is about -20 DEG C), to improve spore germination rate.
2. spore germination step:
By the spore of acquisition pollution-free clean clear water (preferably aqua sterilisa or the water boiled, to avoid miscellaneous bacteria in right amount Pollution) fully mix and soak after, removal floats on spore, sporangium, other debris and the moisture of the water surface, is frozen in -20 DEG C One week to promote spore germination.Appropriate clean clear water (it is required that with foregoing) is added, it is small with the processing 24 of final concentration 100ppm gibberellin When or so, to promote spore germination.
Spore after processing is soaked in water (water quality requirement:Free of contamination clean river, well water, running water.Processing:It is deep Well water can be used directly;River needs to boil more than 30 minutes, to make contained microorganism, spore inactivation;Running water needs to expose Shine more than 3 days, need to equally boil processing), spore suspension is placed in glass container, under the conditions of 15~20 DEG C, natural lighting (covering such as culture vessel available paper covers, and avoids exogenous microbial or other species spores from falling into, causes dirt for culture Dye).The clean water after frequent addition processing is paid attention in incubation, keeps water volume in culture vessel to be basically unchanged.
If Initial stage of culture has bacterium largely to grow, the agricultural streptomycin processing of final concentration 0.02% can be added;If big Amount protozoan grows the Tinidazole processing that can add final concentration 0.1mg/mL.If there is gas in spore settling zone in incubation Bubble, which produces, then shows that spore has been sprouted, and protonema starts to grow, it is a large amount of when producing oxygen bubbles can by spore agglomerate buoyance lift in water meter, Suitably stirred or other optional predetermined means make spore agglomerate be settled down to the bottom.
3. protonema incubation step:
About one week or so spore of culture starts to sprout, and starts green protonema occur after 2 weeks.It can now be pressed in culture Foliar fertilizer dosage adds urea and potassium dihydrogen phosphate, to promote the growth of protonema.
Often stirred in incubation, protonema aggregation can be avoided, agglomerate is wound and reduces appreciation rate.
(that is, there is agglomerate) when protonema too high levels in culture vessel, culture can be expanded, method is:By culture Stir, by stoste: new liquid is(1~5): 10 volume ratio accesses new culture vessel, continues to expand culture, breeds efficiency Up to more than 5 times.
4. gametophyte is cultivated:
Choose free of contamination fertile soil (pH6.0~7.0), thorough exposure under sunlight, parch, crush, go the removal of impurity, take suitable Amount is as matrix and fills basin to the 4/5 of volume, separately takes the fertile soil after above-mentioned processing to cross 50 eye mesh screens, by the tiny fertile soil of gained Granule is overlying on Culture basin soil table, thickness about 5cm, is subsequently placed in pallet, the water filling into pallet, soil in basin is fully infiltrated.
Culture is obtained into protonema to mix, sprinkle is uniformly distributed protonema, be placed in photoenvironment in Culture basin soil table Incubated at room temperature, pay attention to the often water filling into pallet, keep ground moistening.Low temperature is avoided, to prevent the growth of heterotroph fungi.
Culture about 20 days or so, the visible diameter of soil surface about 1cm green gametophyte.For increase seedling yield, with It in daughter cultivating process, can be again inoculated with without protonema blank space in soil surface in good time, also can directly take what is be repeatedly inoculated with Mode, to ensure protonema final more than 80% in flakes.
5. sporinite incubation step:
After gametophyte occurs, soil is kept to be sufficiently humidified so as to, so that sperm combines to form embryonated egg with ovum.It is straight to continue culture To starting sporinite seedling occur.In the present embodiment, protonema 100% in flakes when can generate about 20-50 strains/cm2Seedling.
Sporinite 6. (seedling) transplant step:
First time transplanting is carried out by 4cm × 4cm seeding row spacings after sporinite grows 3~4 leaves, using conventional soil conduct Matrix, indoor culture.
After 7~15 days seedlings of transplanting survive for the first time, it is transplanted to by seeding row spacing 6cm × 10cm on outdoor seedbed, continues to train Support.(10~15cm) is grown to the height that meets the requirements after seedling, is colonized in autumn then or spring in next year by appropriate seeding row spacing Produce Tanaka.
7. field daily management operates:
Routinely require to carry out.Pay attention to avoiding strong light direct beam, fertilising in good time, watering, weeding, loosen the soil in time and remove disease Seedling, dead seedling, prevent pest and disease damage.
Embodiment 2
1. spore acquisition step:
After August part, during female dragon spore maturation, when sporophyll is changed into yellowish-brown from green (before spore distributes), The afternoon (taken outdoors need to be after dew disperses) of continuous sunny is selected, sporophyll is cut with clean scissors, is placed in clean light In sliding paper bag, drying is air-dried, room temperature storage or low temperature (being less than 4 DEG C) storage are to extend spore activity, by above-mentioned obtained spore Leaf fully pulverizes, and 100-200 eye mesh screens sift out spore (and sporangium), goes the removal of impurity, obtains spore(Contain sporangium).Spore Leaf need to be three months or so in refrigerating chamber freezing processing after drying(Cryogenic temperature is about -10 DEG C), to improve spore germination rate.
2. spore germination step:
By the spore of acquisition pollution-free clean clear water (preferably aqua sterilisa or the water boiled, to avoid miscellaneous bacteria in right amount Pollution) fully mix and soak after, removal floats on spore, sporangium, other debris and the moisture of the water surface, is frozen in -5 DEG C One month to promote spore germination.Appropriate clean clear water (it is required that with foregoing) is added, it is small with the processing 5 of final concentration 300ppm gibberellin When, to promote spore germination.
Spore after processing is soaked in water (water quality requirement:Free of contamination clean river, well water, running water.Processing:It is deep Well water can be used directly;River needs to boil more than 30 minutes, to make contained microorganism, spore inactivation;Running water needs to expose Shine more than 3 days, need to equally boil processing), spore suspension is placed in glass container, under the conditions of 15~20 DEG C, natural lighting (covering such as culture vessel available paper covers, and avoids exogenous microbial or other species spores from falling into, causes dirt for culture Dye).The clean water after frequent addition processing is paid attention in incubation, keeps water volume in culture vessel to be basically unchanged.
If Initial stage of culture has bacterium largely to grow, the agricultural streptomycin processing of final concentration 0.01% can be added;If big Amount protozoan grows the Tinidazole processing that can add final concentration 0.05mg/mL.If have in incubation in spore settling zone Bubble, which produces, then shows that spore has been sprouted, and protonema starts to grow, largely can be by spore agglomerate buoyance lift in water during generation oxygen bubbles Table, is suitably stirred or other optional predetermined means make spore agglomerate be settled down to the bottom.
3. protonema incubation step:
About one week or so spore of culture starts to sprout, and starts green protonema occur after 2 weeks.Now can be in culture Urea and potassium dihydrogen phosphate are added by foliar fertilizer dosage, to promote the growth of protonema.
Often stirred in incubation, protonema aggregation can be avoided, agglomerate is wound and reduces appreciation rate.
(that is, there is agglomerate) when protonema too high levels in culture vessel, culture can be expanded, method is:By culture Stir, by stoste: new liquid is(1~5): 10 volume ratio accesses new culture vessel, continues to expand culture, breeds efficiency Up to more than 5 times.
4. gametophyte is cultivated:
Choose free of contamination fertile soil (pH6.0~7.0), thorough exposure under sunlight, parch, crush, go the removal of impurity, take suitable Amount is as matrix and fills basin to the 2/3 of volume, separately takes the fertile soil after above-mentioned processing to cross 50 eye mesh screens, by the tiny fertile soil of gained Granule is overlying on Culture basin soil table, thickness about 1cm, is subsequently placed in pallet, the water filling into pallet, soil in basin is fully infiltrated.
Culture is obtained into protonema to mix, sprinkle is uniformly distributed protonema, be placed in photoenvironment in Culture basin soil table Incubated at room temperature, pay attention to the often water filling into pallet, keep ground moistening.Low temperature is avoided, to prevent the growth of heterotroph fungi.
Culture about 20 days or so, the visible diameter of soil surface about 1cm green gametophyte.For increase seedling yield, with It in daughter cultivating process, can be again inoculated with without protonema blank space in soil surface in good time, also can directly take what is be repeatedly inoculated with Mode, to ensure protonema final more than 80% in flakes.
5. sporinite incubation step:
After gametophyte occurs, soil is kept to be sufficiently humidified so as to, so that sperm combines to form embryonated egg with ovum.It is straight to continue culture To starting sporinite seedling occur.In the present embodiment, protonema 100% in flakes when can generate about 20-50 strains/cm2Seedling.
Sporinite 6. (seedling) transplant step:
First time transplanting is carried out by 4cm × 4cm seeding row spacings after sporinite grows 3~4 leaves, using conventional soil conduct Matrix, indoor culture.
After 7~15 days seedlings of transplanting survive for the first time, it is transplanted to by seeding row spacing 6cm × 10cm on outdoor seedbed, continues to train Support.(10~15cm) is grown to the height that meets the requirements after seedling, is colonized in autumn then or spring in next year by appropriate seeding row spacing Produce Tanaka.
7. field daily management operates:
Routinely require to carry out.Pay attention to avoiding strong light direct beam, fertilising in good time, watering, weeding, loosen the soil in time and remove disease Seedling, dead seedling, prevent pest and disease damage.
Embodiment 3
1. spore acquisition step:
After June, during potted plant venushair fern spore maturation, (spore distributes when sporangium is changed into yellowish-brown from green Before), the afternoon (taken outdoors need to be after dew disperses) of continuous sunny is selected, sporophyll is cut with clean scissors, is placed in dry In net smooth paper bag, drying is air-dried, room temperature storage or low temperature (being less than 4 DEG C) storage to extend spore activity, are obtained above-mentioned Sporophyll fully pulverizes, and 100-200 eye mesh screens sift out spore (and sporangium), goes the removal of impurity, obtains spore(Contain sporangium).
2. spore germination step:
By the spore of acquisition pollution-free clean clear water (preferably aqua sterilisa or the water boiled, to avoid miscellaneous bacteria in right amount Pollution) fully mix and soak after, removal floats on spore, sporangium, other debris and the moisture of the water surface.Add appropriate clean Clear water (it is required that with foregoing), handled 12 hours with final concentration 200ppm gibberellin, to promote spore germination.
Spore after processing is soaked in water (water quality requirement:Free of contamination clean river, well water, running water.Processing:It is deep Well water can be used directly;River needs to boil more than 30 minutes, to make contained microorganism, spore inactivation;Running water needs to expose Shine more than 3 days, need to equally boil processing), spore suspension is placed in glass container, under the conditions of 15~20 DEG C, natural lighting (covering such as culture vessel available paper covers, and avoids exogenous microbial or other species spores from falling into, causes dirt for culture Dye).The clean water after frequent addition processing is paid attention in incubation, keeps water volume in culture vessel to be basically unchanged.
If Initial stage of culture has bacterium largely to grow, the agricultural streptomycin processing of final concentration 0.1% can be added;If a large amount of Protozoan grows the Tinidazole processing that can add final concentration 0.5mg/mL.If there is bubble in spore settling zone in incubation Generation then shows that spore has been sprouted, and protonema starts to grow, and can be fitted when largely producing oxygen bubbles by spore agglomerate buoyance lift in water meter When being stirred or other optional predetermined means make spore agglomerate be settled down to the bottom.
3. protonema incubation step:
About one week or so spore of culture starts to sprout, and starts green protonema occur after 2 weeks.Now can be in culture Urea and potassium dihydrogen phosphate are added by foliar fertilizer dosage, to promote the growth of protonema.
Often stirred in incubation, protonema aggregation can be avoided, agglomerate is wound and reduces appreciation rate.
(that is, there is agglomerate) when protonema too high levels in culture vessel, culture can be expanded, method is:By culture Stir, by stoste: new liquid is(1~5): 10 volume ratio accesses new culture vessel, continues to expand culture, efficiency of rising in value Up to more than 10 times.
4. gametophyte is cultivated:
Choose free of contamination fertile soil (pH6.0~7.0), thorough exposure under sunlight, parch, crush, go the removal of impurity, take suitable Amount is as matrix and fills basin to the 3/4 of volume, separately takes the fertile soil after above-mentioned processing to cross 50 eye mesh screens, by the tiny fertile soil of gained Granule is overlying on Culture basin soil table, thickness about 3cm, is subsequently placed in pallet, the water filling into pallet, soil in basin is fully infiltrated.
Culture is obtained into protonema to mix, sprinkle is uniformly distributed protonema, be placed in photoenvironment in Culture basin soil table Incubated at room temperature, pay attention to the often water filling into pallet, keep ground moistening.Low temperature is avoided, to prevent the growth of heterotroph fungi.
Culture about 20 days or so, the visible diameter of soil surface about 1cm green gametophyte.For increase seedling yield, with It in daughter cultivating process, can be again inoculated with without protonema blank space in soil surface in good time, also can directly take what is be repeatedly inoculated with Mode, to ensure protonema final more than 80% in flakes.
5. sporinite incubation step:
After gametophyte occurs, soil is kept to be sufficiently humidified so as to, so that sperm combines to form embryonated egg with ovum.It is straight to continue culture To starting sporinite seedling occur.In the present embodiment, protonema 100% in flakes when can generate about 20-50 strains/cm2Seedling.
Sporinite 6. (seedling) transplant step:
First time transplanting is carried out by 4cm × 4cm seeding row spacings after sporinite grows 3~4 leaves, using conventional soil conduct Matrix, indoor culture.
After 7~15 days seedlings of transplanting survive for the first time, it is transplanted to by seeding row spacing 6cm × 10cm on outdoor seedbed, continues to train Support.(10~15cm) is grown to the height that meets the requirements after seedling, is colonized in autumn then or spring in next year by appropriate seeding row spacing Produce Tanaka.
7. field daily management operates:
Routinely require to carry out.Pay attention to avoiding strong light direct beam, fertilising in good time, watering, weeding, loosen the soil in time and remove disease Seedling, dead seedling, prevent pest and disease damage.
As described above, it can preferably realize the present invention.
The above described is only a preferred embodiment of the present invention, any formal limitation not is made to the present invention, according to According to the technical spirit of the present invention, within the spirit and principles in the present invention, any simple modification for being made to above example, Equivalent substitution and improvement etc., still fall within the protection domain of technical solution of the present invention.

Claims (8)

1. fern protonema Liquid Culture method for culturing seedlings, it is characterised in that including
Spore germination step:The spore of harvesting is added in clean clear water and soaked, after the floating object and moisture that remove the water surface, selection Property freeze the scheduled time to promote spore germination, the temperature frozen be 0 to -20 DEG C, the time frozen be one week to one Month, add appropriate clean clear water and handled with gibberellin to promote spore germination, the gibberellin final concentration of 100~ 300ppm, the gibberellin processing time are 15 minutes to 24 hours, and the spore after processing, which is soaked in water, to be cultivated, in culture just The medicine that phase is optionally grown using killing or suppressing exogenous microbial;There is bubble production in spore settling zone in incubation Raw then show that spore has been sprouted, protonema starts to grow, if having spore agglomerate buoyance lift in incubation in the water surface, should use predetermined Means are so that spore agglomerate settles.
2. fern protonema Liquid Culture method for culturing seedlings according to claim 1, it is characterised in that in the spore germination In step, if need frozen and preserved spore to be whether the area grown according to fern belongs to winter outdoor temperature below freezing Area carries out selection, is specially:It is area below freezing when the area that fern is grown belongs to winter outdoor temperature, then It should carry out freezing operation;If it is area above freezing that the area that fern is grown, which belongs to winter outdoor temperature, select to enter The operation that row freezes.
3. fern protonema Liquid Culture method for culturing seedlings according to claim 1, it is characterised in that in the spore germination In step, the condition that the spore after the processing is soaked in when being cultivated in water is:15~20 DEG C, natural lighting, and culture vessel With the covering protection thing that exogenous microbial can be avoided to enter culture vessel.
4. fern protonema Liquid Culture method for culturing seedlings according to claim 1, it is characterised in that the breeding method is also Including
Protonema incubation step:When spore occurs by the spore germination step culture to protonema, pressed in culture Foliar fertilizer dosage adds urea and potassium dihydrogen phosphate, to promote the growth of protonema, uses predetermined means to keep away in incubation Exempt from protonema and form agglomerate, when protonema content is higher than predetermined value in culture vessel, continue to train by the way of culture is expanded Support.
5. fern protonema Liquid Culture method for culturing seedlings according to claim 4, it is characterised in that the breeding method is also Including:
Gametophyte incubation step, sporinite incubation step and seedling replanting step.
6. fern protonema Liquid Culture method for culturing seedlings according to claim 5, it is characterised in that the gametophyte is cultivated Step is:Choose the fertile soil of pH6.0~7.0, exposure under sunlight, parch, crush, go the removal of impurity, take in right amount as matrix simultaneously Basin is filled to the 2/3~4/5 of volume, separately takes the fertile soil after above-mentioned processing to cross after 50 eye mesh screens and is overlying on Culture basin soil table, thickness 1~ 5cm, and infiltrate soil in basin by predetermined way, by the protonema sprinkle obtained through the protonema incubation step in culture Basin soil surface, and ensure protonema more than 80% in flakes, incubated at room temperature in photoenvironment is placed in, should be kept during culture Ground moistening and holding are unfavorable for the temperature of heterotroph fungi growth, cultivate to gametophyte and expose soil surface.
7. fern protonema Liquid Culture method for culturing seedlings according to claim 6, it is characterised in that the sporinite is cultivated Step is:After gametophyte exposes soil surface, ground moistening is kept so that sperm and ovum combine to form embryonated egg, continues to cultivate Until there is sporinite seedling;
The seedling replanting step is:First time transplanting is carried out by 4cm × 4cm seeding row spacings after sporinite grows 3~4 leaves, Using conventional soil as matrix, indoor culture, after 7~15 days seedlings of transplanting survive for the first time, moved by seeding row spacing 6cm × 10cm Plant on outdoor seedbed, continue to cultivate, grown after seedling to the height that meets the requirements, appropriate strain is pressed in autumn then or spring in next year Line-spacing is colonized in production Tanaka.
8. fern protonema Liquid Culture method for culturing seedlings according to claim 1, it is characterised in that the breeding method is also Including
Spore acquisition step:In spore maturation, filemot sporophyll is cut, is dried, room temperature storage or cryopreservation are to prolong Long spore activity, crushes sporophyll, crosses 100-200 eye mesh screens and sifts out spore and sporangium.
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