CN101911914A - Culture medium for spore germination and seedling culturing of pteridophyte - Google Patents

Culture medium for spore germination and seedling culturing of pteridophyte Download PDF

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CN101911914A
CN101911914A CN 201010269053 CN201010269053A CN101911914A CN 101911914 A CN101911914 A CN 101911914A CN 201010269053 CN201010269053 CN 201010269053 CN 201010269053 A CN201010269053 A CN 201010269053A CN 101911914 A CN101911914 A CN 101911914A
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moll
medium
acid
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ethylenediamine
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葛台明
郑敏
李继红
王焰新
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China University of Geosciences
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China University of Geosciences
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Abstract

The invention belongs to the technical field of biological engineering, and relates to a culture medium for the spore germination and seedling culturing of pteridophyte, which is characterized by consisting of the following nutritional components: 2 to 60 mmol.L<-1> nitrate ions, 1 to 30 mmol.L<-1> ammonium ions, 0.3 to 8 mmol.L<-1> phosphate radical ions, 2.7 to 66 mmol.L<-1> potassium ions, 0.37 to 12.0 mmol.L<-1> calcium ions, 0.18 to 1.8 mmol.L<-1> magnesium ions, 0.173 to 1.73 mmol.L<-1> sulfate ions, 10 to 100 mumol.L<-1> chelated ferric salt or chelated ferrous salt, 10 to 200 mumol.L<-1> boric acid, 10 to 100 mumol.L<-1> divalent manganese ions, 3 to 30 mumol.L<-1> zinc ions, 0.5 to 8 mumol.L<-1> divalent copper ions or complexes thereof, 0.1 to 1 mumol.L<-1> molybdate ions, 0.01 to 0.1 mumol.L<-1> divalent cobalt ions, 0.05 to 12 mumol.L<-1> chloride ions, 0.3 to 30 mumol.L<-1> thiamine hydrochloride and the balance of water, wherein the pH value of the culture medium is between 4.0 and 9.0. The culture medium can improve the germination rate of spores.

Description

A kind of pteridophyta spore that is suitable for is sprouted and the medium of growing seedlings
Technical field
The invention belongs to technical field of bioengineering, be specifically related to the medium in the factorial seedling growth process of a kind of pteridophyte.
Background technology
Pteridophyte has another name called pteridophyte, gametophyte that tool is lived on one's own life (prothallium) and sporophyte, and its sporophyte tool dimension pipe waits vascular plant for hanging down.China has abundant pteridophyte resource, is described as " fern kingdom ".Pteridophyte is in important meanings of all many-sided tools such as the biological restoration of the ecosystem, contaminated site, social economy, scientific researches.Therefore, the present invention is with a wide range of applications, and now illustrates as follows:
Many pteridophytes have edibility, some is famous mountain delicacy, as common vetch dish (comprising osmund Osmunda japonicThunb. and plant division osmund Osmunda cinnamomea L.), fiddlehead, Matteuccia strthiopteris [Matteuccia struthiopteris (L.) Todaro, another name garden burnet, Guangdong dish], monkey leg (formal name used at school monkey leg female dragon Athyrium multidentatum) etc. is China's important export hill dish of earning foreign exchange.Only the common vetch dish is one, and more than one hundred million dollars of China annual foreign exchange earning E Gaoda is described as " king of mountain delicacy ".Because above-mentioned hill dish is nutritious, tasty, unique flavor, international level rises steadily, and causes the excessive harvesting of wild resource, and wild resource is destroyed, and high-intensity gathering also causes the serious decline of quality.
Many pteridophytes are famous afforestation and ornamental plantss, sit lotus, Cibotium barometz, ciliate desert-grass (not only comprise the Pteris vittata L. of Pteridaceae as " ciliate desert-grass " of flower sale, also comprise the pteridophyte of other many leaf similar centipedes) or the like as Platycerium bifurcatum, brake fern (curb limit grass), chain fern, Osmunda Vachellii Hook, tuber fern, Matteuccia strthiopteris, kwan-yin.At present the pteridophyte sum that success is cultivated in the world wide is approximately 700 kinds, and wherein the overwhelming majority is used for gardens, hotel, home decoration and beautifies as ornamental plants, has become the important component part in the flowers market.Data shows, only American-European market, and annual sales volume has just reached 5,000 ten thousand basins.
Arsenic has very strong toxicity, and human health and environment are caused great harm, but multiple serious health problem such as cause cancer.Discovering in recent years, ciliate desert-grass pteridophytes such as (Pteris vittata L.) has super accumulation ability to arsenic, and the enriching quantity of arsenic is up to 3000mgkg in its blade -1, can be used as the reparation that the biological restoration species are used for the arsenic contamination place.Simultaneously, pteridophytes such as curb limit grass can also enriched lead etc. the heavy metal noxious material, for the biological restoration tool important value of contaminated by heavy metals soil.
Many pteridophyte biologically active materials can be used as important pharmaceutical raw material.As containing a kind of alkaloid that is called huperzine in the Huperzia serrata (LycopodiumserratumThunb.),, be the new drug of treatment senile dementia and juvenile's learning disorder for a kind of efficient, reversible, acetyl cholesterase inhibitor that selectivity is strong.But the huperzine complex structure is difficult to synthesize with the means of chemistry, can only extract from Huperzia serrata.
Also having some pteridophytes is rare species of national protection, and as spinulose tree fern, quillwort, Adiantum reniforme L var sinense Y. X. Lin, Platycerium bifurcatum etc., they have great importance in scientific research.
Because pteridophyte is the low vascular plant of waiting, and mainly relies on sporogenesis.Under the natural situation, spore germination rate is extremely low, thereby has limited development and use and the scientific research of pteridophyte.The present invention proposes in order to address the above problem just.
Summary of the invention
The purpose of this invention is to provide a kind of pteridophyta spore that is suitable for and sprout and the medium of growing seedlings, this medium is used for the pteridophyta spore sprouting and grows seedlings, and can improve the germination rate of spore.
To achieve these goals, the technical solution used in the present invention is: a kind of pteridophyta spore that is suitable for is sprouted and the medium of growing seedlings, and it is characterized in that: medium is formed by comprising following nutrition component: nitrate ion (nitrate nitrogen) 2～60mmolL -1, ammonium ion (ammonium nitrogen) 1～30mmolL -1, phosphate anion 0.3～8mmolL -1, potassium ion 2.7～66mmolL -1, calcium ion 0.37～12.0mmolL -1, magnesium ion 0.18～1.8mmolL -1, sulfate ion 0.173～1.73mmolL -1, chelating molysite (or chelating ferrous salt) 10～100 μ molL -1, boric acid 10～200 μ molL -1, divalent manganesetion 10～100 μ molL -1, zinc ion 3～30 μ molL -1, bivalent cupric ion (or complex compound of bivalent cupric ion) 0.5～8 μ molL -1, molybdenum acid ion 0.1～1 μ molL -1, divalent cobalt ion 0.01～0.1 μ molL -1, bivalent nickel ion 0～0.1 μ molL -1, iodide ion 0～1.2 μ molL -1, chlorion 0.05～12mmolL -1, thiamine hydrochloride 0.3～30 μ molL -1, all the other compositions are water, the medium pH value is 4.0～9.0.
The nutrition component of described medium consists of: NH 4NO 35.15mmolL -1, KNO 34.70mmolL -1, CaCl 22H 2O 0.75mmolL -1, MgSO 47H 2O 0.375mmolL -1, KH 2PO 40.735～1.47mmolL -1, chelating molysite or chelating ferrous salt 25 μ molL -1, H 3BO 325 μ molL -1, MnSO 4H 2O 25 μ molL -1, ZnSO 47H 2O 7.5 μ molL -1, Na 2MoO 42H 2O0.25 μ molL -1, CuSO 45H 2O 0.5～4 μ molL -1, CoCl 26H 2O 0.025 μ molL -1, NiCl 26H 2O 0.025 μ molL -1, KI0～0.3 μ molL -1, thiamine hydrochloride 1.48 μ molL -1, all the other compositions are water, the medium pH value is 6.0 ± 0.5 (promptly 5.5～6.5).
The nutrition component of described medium consists of: NH 4NO 33.50mmolL -1, KNO 311.5mmolL -1, Ca (NO 3) 24H 2O1.25mmolL -1, MgSO 47H 2O 0.50mmolL -1, KH 2PO 40.735～1.47mmolL -1, KCl 0.27mmolL -1, chelated iron or chelating ferrous 25 μ molL -1, H 3BO 325 μ molL -1, MnSO 4H 2O 25 μ molL -1, ZnSO 47H 2O 7.5 μ molL -1, Na 2MoO 42H 2O 0.25 μ molL -1, CuSO 45H 2O 0.5～4 μ molL -1, CoCl 26H 2O 0.025 μ molL -1, NiCl 26H 2O0.025 μ molL -1, KI0～0.3 μ molL -1, thiamine hydrochloride 1.48 μ molL -1, all the other compositions are water, the medium pH value is 6.0 ± 0.5 (promptly 5.5～6.5).
Described chelating molysite (or chelating ferrous salt) is meant with following a kind of or two or more arbitrarily chelating agent (when being two or more is any proportioning), with trivalent or the formed chelate of the divalent iron salt (composite salt that comprises these chelates, as EDTA ferrisodium salt, EDTA iron ammonia salt, DTPA ferrisodium salt, DTPA iron ammonia salt etc.): ethylenediamine tetra-acetic acid (EDTA), diethylene-triamine pentaacetic acid (DTPA), N-ethoxy ethamine triacetic acid (HEDTA), CDTA (DCTA), anti-form-1,2-1,2-diaminocyclohexane tetraacetic acid (CyDTA), ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) (EGTA), 1,3-trimethylen-edinitrilo-tetraacetic acid (PDTA), aminotriacetic acid (NTA), Diethylenetriamine (DETA), triethyl group tetramine six acetate (TTHA), ethylenediamine tetrapropionic acid (EDTP), EDDHA[ethylenediamine-N, N '-bis (2-hydroxyphenylacetic acid), ethylenediamine-N, N '-two hydroxyl phenylacetic acids that face], EDDHMA[ethylendiamine di (2-hydroxy-4-methylphenylacetic) acid, the two hydroxy-4-methyl phenylacetic acids that face of ethylenediamine], EDDHSA[ethylenediamine di (2-hydroxy-5-sulfophenylacetic) acid, the two hydroxyl-5-sulfo group phenylacetic acids that face of ethylenediamine] etc.
The complex compound of described bivalent cupric ion is meant with following a kind of or two or more arbitrarily ionic complexing agent (when being two or more is any proportioning), with the formed complex compound of cupric salt, the composite salt that comprises these complex compounds: ethylenediamine tetra-acetic acid (EDTA), diethylene-triamine pentaacetic acid (DTPA), N-ethoxy ethamine triacetic acid (HEDTA), CDTA (DCTA), anti-form-1,2-1,2-diaminocyclohexane tetraacetic acid (CyDTA), ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) (EGTA), 1,3-trimethylen-edinitrilo-tetraacetic acid (PDTA), aminotriacetic acid (NTA), Diethylenetriamine (DETA), triethyl group tetramine six acetate (TTHA), ethylenediamine tetrapropionic acid (EDTP), EDDHA[ethylenediamine-N, N '-two hydroxyl phenylacetic acids that face, ethylenediamine-N, N '-bis (2-hydroxyphenylacetic acid)], EDDHMA[ethylendiaminedi (2-hydroxy-4-methylphenylacetic) acid, the two hydroxy-4-methyl phenylacetic acids that face of ethylenediamine], EDDHSA[ethylenediamine di (2-hydroxy-5-sulfophenylacetic) acid, the two hydroxyl-5-sulfo group phenylacetic acids that face of ethylenediamine], amino acid and protolysate etc.
Described amino acid is meant: any one in glycine, alanine, valine, leucine, isoleucine, phenyl alanine, tryptophan, tyrosine, aspartic acid, asparagine, glutamic acid, lysine, hydroxylysine, glutamine, methionine, serine, threonine, cystine, cysteine, proline, hydroxyproline, histidine, arginine, the theanine or any mixing more than two kinds, any is any proportioning when mixing more than two kinds.
Described protein hydrolysate is meant: caseinhydrolysate, lactoalbumin hydrolysate, liquid fish, hydrolytic soya bean protein, the hydrolysis peanut protein, the acidolysis casein, the acidolysis milk protein, the acidolysis fish protein, the acidolysis soybean protein, the acidolysis peanut protein, casein hydrolysis, the enzymolysis milk protein, enzymolysis of fish proteins, enzymatic hydrolysis of soybean albumen, the enzymolysis peanut protein, tryptone, casein peptone, the milk protein peptone, beef peptone, fish peptone, the bone protein peptone, soy peptone, in the peanut protein peptone any one or any mixing more than two kinds, any is any proportioning when mixing more than two kinds.
Though only used a certain water of crystallization state of some materials in the described medium, used other water of crystallization state of these materials instead and can realize the present invention equally, as use CaCl 2Or CaCl 26H 2Alternative CaCl such as O 22H 2O is because be the same in solution with a kind of salt of different water of crystallization states of compound.
H in the described medium 3BO 3Can use Na 2B 4O 710H 2O substitutes, Na 2MoO 42H 2O can replace with other molybdate (as ammonium molybdate), and manganese, zinc, copper, cobalt, nickel etc. can replace with the corresponding salt that the acid group (or part) of other kind is formed, and do not influence whole medium effect.
Also can add plant growth regulating substance (or hormone) in the described medium, inositol, nicotinic acid (being also referred to as vitamin B3 or nicotinic acid), puridoxine hydrochloride (being also referred to as vitamin B6), vitamin h (being also referred to as biotin), the common component of medium such as glycine is not [though above-mentioned prescription comprises plant growth regulating substance (or hormone), inositol, nicotinic acid (being also referred to as vitamin B3 or nicotinic acid), puridoxine hydrochloride (being also referred to as vitamin B6), vitamin h (being also referred to as biotin), the component that medium such as glycine are common, institute is essential because above-mentioned substance is not spore germination].
Also can add the culture medium solidifying agent in the described medium (though above-mentioned prescription does not comprise the culture medium solidifying agent, but and do not mean that because of adding the culture medium solidifying agent, after changing the physical behavior of medium, and can not realize the present invention, because solid culture or liquid culture etc. be the problem of cultural method just, but not prescription problem).
Also can add carbon source in the described medium (though above-mentioned prescription does not add carbon sources such as sucrose, but but the present invention's supplementary carbon source, because carbon source can solve by the photosynthesis of culture self, also can change) as replace sucrose etc. with glucose because of different research purposes.
Among the present invention, the total ionic strength adjustment buffer degree of medium approximately is 1/2～1/4 of a standard MS medium, but has improved KH significantly 2PO 4And CuSO 45H 2The concentration of O has been replenished micro-NiCl 26H 2O has reduced the concentration of the KI in the original formulation significantly, and has removed organic substances such as the little inositol of spore germination influence, nicotinic acid, puridoxine hydrochloride, glycine, replaces 1.48 μ molL -1(be equivalent to 0.5mgL -1) thiamine hydrochloride, finally formed optimum formula of the present invention.
The medium that the present invention proposes not only can be used for the sprouting of carrying disease germs of pteridophyta spore, also can be used for axenic germination.There is the problem of dormancy at the part pteridophyta spore, can in medium, adds certain density gibberellin (as GA3 etc.), with breaking dormancy.The GA3 concentration of breaking dormancy can be determined by the test of single-factor concentration gradient is set; Also can carry out preliminary treatment to spore with the certain density GA3 aqueous solution, to break its dormancy, pretreated intensity can be confirmed by simple comparative trial equally.Simultaneously, the best inoculum density that the spore of different pteridophytes needs may be not quite similar, and need grope in advance.
Carry out carrying disease germs of spore when sprouting,, should not add carbon source in the medium, should utilize the photosynthesis of spore germination thing self to satisfy the demand of its growth in order to suppress varied bacteria growing.In order to satisfy photosynthetic requirement, the intensity of illumination of cultivating, periodicity of illumination, light quality etc. there are certain requirement, best illumination condition can be with being confirmed by simple series of comparisons test is set.
If carry out axenic germination, then both can add certain density carbon source, as 30gL -1Sucrose etc., to promote the quick growth of spore germination thing, also can not add carbon source, sprout best illumination condition and cultivate according to above-mentioned carrying disease germs, but will note the ventilative state of culture vessel, satisfy the demand of photosynthesis to carbonic acid gas.
Simultaneously, the present invention also can be used for the pteridophyte demand of breeding fast.When breeding fast, certain density carbon source can be added, in medium as 30gL -1Sucrose etc., promoting the quick growth of spore germination thing, and add certain density auxin substance and cytokinin-like substance, to improve the efficient of breeding.
The present invention does not limit training methods such as adopting solid culture, liquid culture, paper bridge cultivation, sand culture, and application person can correctly select according to different purposes and condition.
Experiment shows, the spore of pteridophyte germination rate height not only on medium proposed by the invention, and also the growth rate of the sporophyte of prothallium of being sprouted and regeneration thereof, growing way all obviously are better than contrast.This prescription be used for the factorial seedling growth of pteridophyte, fast the breeding and breeding work, satisfy the demand of people to the pteridophyte seedling.
The invention has the beneficial effects as follows: this medium is used for the pteridophyta spore sprouting and grows seedlings, and can improve the germination rate of spore, obtains the healthy and strong prothallium and the sporophyte of regeneration.
Common medium with present report, compare as MS medium, KnopShi liquid etc., pteridophyta spore germination rate height not only on basal culture medium, and growth rate, the growing way of the sporophyte of prothallium of being sprouted (gametophyte) and regeneration thereof all obviously are better than contrast.Spore inoculating can be sprouted in the fastest 2～3 days, and germination rate can be up to 95%, and the sporophyte inductivity can be up to 30%, and prothallium, sporophyte color and luster are dark green, robust growth.This medium is used for growing seedlings, breeding fast and breeding work of pteridophyte, satisfies the demand of factorial seedling growth.
Embodiment
In order to understand the present invention better, further illustrate content of the present invention below in conjunction with embodiment, but content of the present invention not only is confined to the following examples.
Embodiment 1～embodiment 7:
A kind of pteridophyta spore that is suitable for is sprouted and the medium of growing seedlings, and forms by comprising following nutrition component, sees Table 1.
Table 1:(is except that the pH value, and the unit of all the other compositions is mmolL -1)
Composition Embodiment 1 Embodiment 2 Embodiment 3 Embodiment 4 Embodiment 5 Embodiment 6 Embodiment 7
NH 4NO 3 5.15 10.30 3.50 7.0 2.0～4.0 20 2.0
KNO 3 4.70 9.40 11.50 23.0 15 40 2.0
CaCl 2·2H 2O 0.75 1.50 0 0 1.36～ 3.7 0.3
3.0
?CaNO 3·4H 2O 0 0 1.25 2.5 0～6.0 0 0
?MgSO 4·7H 2O 0.375 0.75 0.50 1.0 0.75 3.0 0.15
?KH 2PO 4 0.735 1.47 0.75 1.5 1.5 5.9 0.3
?KCl 0 0 0.27 0.54 0 0 0
The EDTA chelated iron 25×10 3 50×10 -3 25×10 -3 50×10 -3 0 100×10 -3 10×10 -3
The EDDHA chelated iron 0 0 0 0 25×10 -3 0 0
?H 3BO 3 25×10 3 50×10 -3 25×10 -3 50×10 -3 25×10 -3 100×10 -3 10×10 -3
?MnSO 4·H 2O 25×10 3 50×10 -3 25×10 -3 50×10 -3 25×10 -3 100×10 -3 10×10 -3
?ZnSO 4·7H 2O 7.5×10 -3 15×10 -3 7.5×10 -3 15×10 -3 7.5×10 -3 30×10 -3 3×10 -3
?Na 2MoO 4·2H 2O 0.25×10 -3 0.50×10 -3 0.25×10 -3 0.50×10 -3 0.25×10 -3 1.0×10 -3 0.1×10 -3
?CuSO 4·5H 2O 1.0×10 -3 2.0×10 -3 0.5×10 -3 1.0×10 -3 0 4×10 -3 0.5×10 -3
The EDTA chelated copper 0 0 0 0 0.5×10 -3 0 0
?CoCl 2·6H 2O 25×10 -6 50×10 -6 25×10 -6 50×10 -6 25×10 -6 0.1×10 -3 0.01×10 -3
?NiCl 2·6H 2O 25×10 -6 50×10 -6 25×10 -6 50×10 -6 25×10 -6 0.1×10 -3 0.01×10 -3
?KI 0.15×10 -3 0.3×10 -3 0.15×10 -3 0.3×10 -3 0.15×10 -3 0.3×10 -3 0
Thiamine hydrochloride 1.48×10 -3 1.48×10 -3 1.48×10 -3 1.48×10 -3 1.48×10 -3 1.48×10 -3 1.48×10 -3
The pH value 6.0±0.5 6.0±0.5 6.0±0.5 6.0±0.5 6.8～7.5 5.5～5.8 6.0±0.5
Concrete configuration method: in 1L water, add above-mentioned composition, adjust the pH value.Can add sucrose or agar etc. according to research purpose.If do axenic germination, then need prepared culture medium moist heat sterilization 20 minutes under 121 ℃ of conditions.In the foregoing description, embodiment 1～4 is fit to the sprouting of most of pteridophyta spores.5 sproutings that are suitable for the fern seed of some basophilia environment of embodiment.6 sproutings that are fit to the spore of some salt tolerant ferns of embodiment.Embodiment 7 is suitable for some sproutings to the more responsive fern seed of salinity.
Application example 1: the sprouting of carrying disease germs of common vetch dish spore
1) medium is prepared: by the requirement preparation medium of embodiment 1～4, and add 6gL -1Agar solidify.Medium need not autoclaving, and direct packaging is in the conical flask of 250mL, and every bottle of about packing 50mL treats after the culture medium solidifying standby.The corresponding medium of formulated of pressing MS medium and KnopShi liquid simultaneously in contrast.All medium all do not add sucrose.
2) spore inoculating: get the spore of a little common vetch dish, be sprinkling upon the surface of medium equably, the conical flask bottleneck is sealed with the plastic closures film of the ventilative filter core of band, prevent the water evaporates in the incubation.
3) condition of culture: postvaccinal conical flask is placed 20～25 ℃, and light intensity is 2000～4000lx, and periodicity of illumination is to cultivate under the condition of 16h illumination/8h dark.
4) spore germination effect: the spore that is seeded on the above-mentioned medium all can be sprouted, wherein on medium of the present invention, spore inoculating promptly can be observed sprouting in 3 days, entered after 4～5 days to sprout the peak period, and the germination rate that utilizes stereomicroscope to add up at the 10th day can be up to 95%.The sprout time of spore on the MS medium one than late 3～5 days on the medium of the present invention, statistics germination rate only about 60%, germination rate of some batch even less than 30%.The sprouting speed of spore on KnopShi liquid is similar to medium of the present invention to germination rate.Show that medium of the present invention is suitable for the sprouting of common vetch dish spore, effect is better than common MS medium.
5) prothallium (gametophyte) growing state: be seeded in the cultivation of spore through about 20 days on the medium of the present invention, gametophytic mean size can reach about 0.5mm, can reach 5mm in 50 days, and the prothallium color and luster is dark green, it is vigorous to grow, most prothallium in addition with 40～80 ° angle of inclination to space growth the surface of medium (rather than be affixed on).After being seeded in the spore germination on the MS medium, similar in growth rate and the present invention.It is slower to be seeded in after the spore germination on the KnopShi liquid growth, growing way also a little less than, growth retardation appears in many prothallium after 30 days, grows slowly, the color and luster of most prothallium is lighter.Show that medium of the present invention extremely is suitable for the growth of common vetch dish prothallium, effect is better than KnopShi liquid.
6) sporophyte growing state: in the cultivation through about 50～60 days on the medium of the present invention, prothallium can be paved with the surface of whole medium, and big I reaches more than the 5mm, and this moment, prothallium was reached maturity.In conical flask, add liquid nutrient medium of the present invention (perhaps running water), make media surface moistening, but do not flood prothallium, carefully siphon away liquid with suction pipe after 30 minutes, repeat above-mentioned processing procedure after 1 week.After inoculation 3～4 months, visible whole media surface is all covered by first leaf of sporophyte, and color and luster is dark green, and it is vigorous to grow.Statistics shows, about 20～30% the equal eruption of gametophyte first sporophyte leaf.Through about 1 month, can grow second sporophyte leaf after the eruption of first sporophyte leaf again as the clover shape, and visible a large amount of fist leaf roll eruption.From the medium beneath, visible a large amount of brown root shape things.And sporophyte significantly is less than of the present inventionly on the control medium, and the sporophyte leaf can not cover media surface fully.Statistics shows, in the control group only about 10% gamete physical efficiency form sporophyte.Especially not only growing way is thin and weak for the sporophyte on the KnopShi liquid, and the color and luster jaundice, even is milk yellow, through 4～5 months cultivation, and most sporophyte growth retardations.Show that medium of the present invention is very suitable for inducing the formation and the growth of common vetch dish sporophyte, and the sporophyte growing way is vigorous, blastoprolepsis, effect is better than MS medium and KnopShi liquid.
Application example 2: the axenic germination of brake fern (curb limit grass) spore
1) prepare medium by the requirement of embodiment 1～4, and additional 30gL -1Sucrose as carbon source, add 6gL -1Agar as curing agent.Medium is sub-packed in the conical flask of 250mL, 121 ℃ of moist heat sterilizations 20 minutes, and the cooling back is standby.
2) when the sporangiorus of brake fern was sepia and sorus and does not ftracture as yet, the clip sporophyll was cut into the leaf section about 1cm.
3) on superclean bench, the leaf section is made surface sterilization 30s with 70% ethanol, with 1 ‰ mercuric chloride solution, the 6～8min that sterilizes, sterile water washes (one is 4～5 times) repeatedly then.
4) the sporophyll section after the surface sterilization being placed one aseptic, diameter is the culture dish of 7.5cm, add a small amount of sterile water,, carefully sporangium is scraped with Small type operating knife by the sterile working standard, and with glass bar or pincet it is smashed to pieces, make spore suspension.
5) draw spore suspension with an aseptic dropper, it is seeded in the surface of medium.If excess moisture can be gone redundant moisture with careful suction of dropper, be not advisable there to be obvious ponding.
6) the conical flask bottleneck is sealed with the plastic closures film of the ventilative filter core of band, placed 20 ℃, light intensity is 4000lx, and periodicity of illumination is to cultivate under the condition of 16h illumination/8h dark.
7) can sprout a large amount of prothallium, the sporophyte that can be regenerated in a large number after 3 months in about about 1 month
Each raw material that the present invention is cited, and the bound of each raw material of the present invention, interval value can both be realized the present invention, do not enumerate embodiment one by one at this.

Claims (7)

1. one kind is suitable for the medium that pteridophyta spore is sprouted and grown seedlings, and it is characterized in that: medium is formed by comprising following nutrition component: nitrate ion 2～60mmolL -1, ammonium ion 1～30mmolL -1, phosphate anion 0.3～8mmolL -1, potassium ion 2.7～66mmolL -1, calcium ion 0.37～12.0mmolL -1, magnesium ion 0.18～1.8mmolL -1, sulfate ion 0.173～1.73mmolL -1, chelating molysite or chelating ferrous salt 10～100 μ molL -1, boric acid 10～200 μ molL -1, divalent manganesetion 10～100 μ molL -1, zinc ion 3～30 μ molL -1, complex compound 0.5～8 μ molL of bivalent cupric ion or bivalent cupric ion -1, molybdenum acid ion 0.1～1 μ molL -1, divalent cobalt ion 0.01～0.1 μ molL -1, bivalent nickel ion 0～0.1 μ molL -1, iodide ion 0～1.2 μ molL -1, chlorion 0.05～12mmolL -1, thiamine hydrochloride 0.3～30 μ molL -1, all the other compositions are water, the medium pH value is 4.0～9.0.
2. a kind of pteridophyta spore that is suitable for according to claim 1 is sprouted and the medium of growing seedlings, and it is characterized in that: the nutrition component of described medium consists of: NH 4NO 35.15mmolL -1, KNO 34.70mmolL -1, CaCl 22H 2O 0.75mmolL -1, MgSO 47H 2O 0.375mmolL -1, KH 2PO 40.735～1.47mmolL -1, chelating molysite or chelating ferrous salt 25 μ molL -1, H 3BO 325 μ molL -1, MnSO 4H 2O 25 μ molL -1, ZnSO 47H 2O 7.5 μ molL -1, Na 2MoO 42H 2O 0.25 μ molL -1, CuSO 45H 2O 0.5～4 μ molL -1, CoCl 26H 2O 0.025 μ molL -1, NiCl 26H 2O 0.025 μ molL -1, KI 0～0.3 μ molL -1, thiamine hydrochloride 1.48 μ molL -1, all the other compositions are water, the medium pH value is 6.0 ± 0.5.
3. a kind of pteridophyta spore that is suitable for according to claim 1 is sprouted and the medium of growing seedlings, and it is characterized in that: the nutrition component of described medium consists of: NH 4NO 33.50mmolL -1, KNO 311.5mmolL -1, Ca (NO 3) 24H 2O 1.25mmolL -1, MgSO 47H 2O 0.50mmolL -1, KH 2PO 40.735～1.47mmolL -1, KCl 0.27mmolL -1, chelated iron or chelating ferrous 25 μ molL -1, H 3BO 325 μ molL -1, MnSO 4H 2O 25 μ molL -1, ZnSO 47H 2O 7.5 μ molL -1, Na 2MoO 42H 2O 0.25 μ molL -1, CuSO 45H 2O 0.5～4 μ molL -1, CoCl 26H 2O 0.025 μ molL -1, NiCl 26H 2O0.025 μ molL -1, KI 0～0.3 μ molL -1, thiamine hydrochloride 1.48 μ molL -1, all the other compositions are water, the medium pH value is 6.0 ± 0.5.
4. according to claim 1,2 or 3 described a kind of pteridophyta spores that are suitable for are sprouted and the medium of growing seedlings, it is characterized in that: described chelating molysite or chelating ferrous salt are meant with following one or more chelating agent, with trivalent or the formed chelate of divalent iron salt: ethylenediamine tetra-acetic acid, diethylene-triamine pentaacetic acid, N-ethoxy ethamine triacetic acid, CDTA, anti-form-1, the 2-1,2-diaminocyclohexane tetraacetic acid, the ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA), 1, the 3-trimethylen-edinitrilo-tetraacetic acid, aminotriacetic acid, Diethylenetriamine, triethyl group tetramine six acetate, ethylenediamine tetrapropionic acid, ethylenediamine-N, N '-two hydroxyl phenylacetic acids that face, the two hydroxy-4-methyl phenylacetic acids that face of ethylenediamine, two hydroxyl-5-sulfo group the phenylacetic acids that face of ethylenediamine.
5. according to claim 1,2 or 3 described a kind of pteridophyta spores that are suitable for are sprouted and the medium of growing seedlings, it is characterized in that: the complex compound of described bivalent cupric ion is meant with following one or more ionic complexing agent, with the formed complex compound of cupric salt, the composite salt that comprises these complex compounds: ethylenediamine tetra-acetic acid, diethylene-triamine pentaacetic acid, N-ethoxy ethamine triacetic acid, CDTA, anti-form-1, the 2-1,2-diaminocyclohexane tetraacetic acid, the ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA), 1, the 3-trimethylen-edinitrilo-tetraacetic acid, aminotriacetic acid, Diethylenetriamine, triethyl group tetramine six acetate, ethylenediamine tetrapropionic acid, ethylenediamine-N, N '-two hydroxyl phenylacetic acids that face, the two hydroxy-4-methyl phenylacetic acids that face of ethylenediamine, two hydroxyl-5-sulfo group the phenylacetic acids that face of ethylenediamine, amino acid, protolysate.
6. a kind of pteridophyta spore that is suitable for according to claim 5 is sprouted and the medium of growing seedlings, it is characterized in that: described amino acid is meant: glycine, alanine, valine, leucine, isoleucine, phenyl alanine, tryptophan, tyrosine, aspartic acid, asparagine, glutamic acid, lysine, hydroxylysine, glutamine, methionine, serine, threonine, cystine, cysteine, proline, hydroxyproline, histidine, arginine, in the theanine any one or any mixing more than two kinds, any is any proportioning when mixing more than two kinds.
7. a kind of pteridophyta spore that is suitable for according to claim 5 is sprouted and the medium of growing seedlings, it is characterized in that: described protein hydrolysate is meant: caseinhydrolysate, lactoalbumin hydrolysate, liquid fish, hydrolytic soya bean protein, the hydrolysis peanut protein, the acidolysis casein, the acidolysis milk protein, the acidolysis fish protein, the acidolysis soybean protein, the acidolysis peanut protein, casein hydrolysis, the enzymolysis milk protein, enzymolysis of fish proteins, enzymatic hydrolysis of soybean albumen, the enzymolysis peanut protein, tryptone, casein peptone, the milk protein peptone, beef peptone, fish peptone, the bone protein peptone, soy peptone, in the peanut protein peptone any one or any mixing more than two kinds, any is any proportioning when mixing more than two kinds.
CN 201010269053 2010-09-01 2010-09-01 Culture medium for spore germination and seedling culturing of pteridophyte Pending CN101911914A (en)

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CN102626033A (en) * 2012-05-04 2012-08-08 天津师范大学 Method utilizing nitrilotriacetic acid (NTA) to improve planting effects of ryegrass turf in garbage compost base material
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CN102613075A (en) * 2012-03-21 2012-08-01 江汉大学 Method for breeding floating ferns of endangered ferns
CN102626033A (en) * 2012-05-04 2012-08-08 天津师范大学 Method utilizing nitrilotriacetic acid (NTA) to improve planting effects of ryegrass turf in garbage compost base material
CN105230492A (en) * 2015-11-04 2016-01-13 广西相成生物科技有限公司 Tissue culture propagation method of cibotium barometz spores
CN105191657A (en) * 2015-11-06 2015-12-30 长沙慧日生物技术有限公司 Rapid breeding method of aseptic huperzia serrata seedling
CN107135949A (en) * 2017-06-16 2017-09-08 黔东南民族职业技术学院 Strawberry adventitious bud induction culture base and preparation method and application
CN109699495A (en) * 2019-02-12 2019-05-03 云南农业大学 A method of improving scythian lamb rhizome spore germination rate
CN109699495B (en) * 2019-02-12 2022-04-08 云南农业大学 Method for improving germination rate of cibotium barometz spores
CN110663548A (en) * 2019-09-24 2020-01-10 福建省农业科学院作物研究所 Aseptic germination seedling raising method for platycerium wallichii spores
CN116584385A (en) * 2022-09-01 2023-08-15 毕节市中药研究所 Culture medium and method for inducing germination of pteris animalis spores and effectively culturing protophylls
CN116584385B (en) * 2022-09-01 2024-06-21 毕节市中药研究所 Culture medium and method for inducing germination of pteris animalis spores and effectively culturing protophylls
CN117089510A (en) * 2023-08-30 2023-11-21 毕节市中药研究所 Culture medium combination for inducing germination and proliferation of Cibotium barometz spores, and method and application for inducing protophylls

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