CN109006477A - A kind of sporogenesis method of thick stalk floating fern - Google Patents
A kind of sporogenesis method of thick stalk floating fern Download PDFInfo
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- CN109006477A CN109006477A CN201810784915.4A CN201810784915A CN109006477A CN 109006477 A CN109006477 A CN 109006477A CN 201810784915 A CN201810784915 A CN 201810784915A CN 109006477 A CN109006477 A CN 109006477A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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Abstract
The invention discloses a kind of sporogenesis methods of thick stalk floating fern, the following steps are included: the spore of thick stalk floating fern is sterilized, it is configured to spore suspension with sterile water after disinfection, spore suspension is inoculated in the MS culture medium added with 6- benzyl aminoadenine and methyl α-naphthyl acetate and is cultivated；In MS culture medium, the concentration of 6- benzyl aminoadenine is 0.05-0.15mg/L, and the concentration of methyl α-naphthyl acetate is 0.02-0.1mg/L；The Spore cultivation of thick stalk floating fern is transferred in the MS culture medium added with indolebutyric acid and is cultivated to after sprouting original foliage out, and the concentration of indolebutyric acid is 0.1-0.3mg/L in MS culture medium, cultivates to 2-4 piece blade is grown, obtains test tube seedling；Test tube seedling is taken out, is cultivated after transplanting, obtains slightly obstructing floating fern plant.It is seeded in the MS culture medium for being added to 6- benzyl aminoadenine and methyl α-naphthyl acetate, spore germination rate is high, has reached 60% or more, prothallus development is also higher at the probability of juvenile sporophyte, improves growth coefficient and a large amount of reproductive speeds.
Description
Technical field
The present invention relates to a kind of sporogenesis methods of thick stalk floating fern, belong to water plant and cultivate field.
Background technique
Parkeriaceae (Ceratopteridaceae) all plants are second class protection plant, two type of leaf, metamorphosis compared with
Greatly, while it is edible and medicinal, have unique landscape value and ecological significance.But since floating fern class is to the water of institute's living environment
Matter requirement is relatively high, can not grow in the waters for polluting more, therefore Wild plant is more and more rare.
Floating fern (Ceratopteris thalictroides (L.) Brongn.) has the report of quick-breeding method, Shen at present
Number one kind please be disclosed pass through water for a patent of invention " mating system of Endangered Fern floating fern " of CN201210075022.5
The acquisition of fern spore is inoculated in culture dish, and culture obtains the artificial breeding method of seedling in growth cabinet, application No. is
A kind of patent of invention " method of quick breeding floating fern seedling " of CN201410734844.9 discloses a kind of spore by floating fern
It is inoculated into germination medium to be trained seedling, will be inserted into culture medium after seedling dissection and turn out young shoot, then young shoot is cut into chunks
Squamous subculture is carried out, young shoot is inoculated into root media culture to the method for obtaining floating fern seedling of taking root.
Floating fern has a small amount of artificial breeding nursery in market circulation at present.Thick stalk floating fern (Ceratopteris
Pteridoides (Hook.) Hieron.) opposite floating fern is more rare, and is still in wild state, not yet opened by artificial breeding
Hair.Thick stalk floating fern sporophyll can produce a large amount of spores, but spore maturation time is indefinite, drifts rapidly after mature, it is difficult to acquire, spore
The sub- service life is short, and germination rate is low under natural environment.Moreover, slightly stalk floating fern seedling winter cold resistance is poor, easily freeze to death, planting percent is low；
Seedling is also more sensitive to pollutant, pesticide etc., the more difficult seedling under the environment more than human interference.Current serious environmental pollution
Thick stalk floating fern wild stocks amount is more allowed to gradually decrease, it is endangered, it needs to carry out in situ conservation and artificial breeding.
Summary of the invention
Present invention solves the technical problem that being, the thick floating fern sporophyll that obstructs can produce a large amount of spores, drift rapidly after mature, difficult
With acquisition, the spore service life is short, and germination rate is low under natural environment.Therefore, more slightly obstruct floating fern seedling to expand propagating and cultivating, for thick stalk
Floating fern carry out tissue culture experiments research, filter out be suitable for the plant fast breeding culture medium and cultural method.
The technical scheme is that providing a kind of sporogenesis method of thick stalk floating fern, comprising the following steps:
(1) by after the spore disinfection of thick stalk floating fern, it is configured to spore suspension with sterile water, spore suspension is inoculated in
It is cultivated in MS culture medium added with 6- benzyl aminoadenine and methyl α-naphthyl acetate；In MS culture medium, 6- benzyl aminoadenine
Concentration is 0.05-0.15mg/L, and the concentration of methyl α-naphthyl acetate is 0.02-0.1mg/L；
(2) slightly the Spore cultivation of stalk floating fern is transferred to after sprouting original foliage, juvenile sporophyte out added with indolebutyric acid
It is cultivated in MS culture medium, cultivates to 2-4 piece blade is grown, obtain test tube seedling, the concentration of indolebutyric acid is in MS culture medium
0.8-1.2mg/L；
(3) test tube seedling is taken out, is cultivated after transplanting, obtain slightly obstructing floating fern plant.
Preferably, in step (1), in MS culture medium, the concentration of 6- benzyl aminoadenine is 0.1mg/L, methyl α-naphthyl acetate it is dense
Degree is 0.05mg/L.
Preferably, in step (3), by test tube transplantation of seedlings into the compost by disinfection, the compost is by with the following group
It is grouped as: 65-75wt% peat, 15-25wt% rice hull carbon, 8-12wt% vermiculite.
Preferably, the mode for taking leaching basin water supply, controls the humidity of compost 75%~95%.
Preferably, in the incubation of step (3), foliage-spray is carried out using the nanometer siliceous fertilizer solution of 8-12mg/L, often
It sprays in week once.
Preferably, in step (1) and (2), condition of culture is 27.5-28.5 DEG C, 2000~2500lx of illuminance, illumination 12
~14 hours/day.
Preferably, in step (1), the actual conditions of disinfection are as follows: by the spore of thick stalk floating fern volume fraction 70-75%'s
8-15s is impregnated in ethyl alcohol, then impregnates 8-12min in the sodium hypochlorite of mass fraction 1.5-2.5%.Thick stalk floating fern spore volume
It is small, it selects the stronger disinfectant of toxicity or disinfecting time is too long will affect its activity.Using ethyl alcohol 10s+ sodium hypochlorite 10min
Processing method can effectively sterilize.
Preferably, the density of spore suspension miospore are as follows: 1 × 106A/ml.It takes and is seen under spore suspension to microscope
It examines, sterile water is added to adjust spore density, until there is 50 spores to be advisable in 40 times of visuals field.
Preferably, slightly obstruct the acquisition mode of the spore of floating fern are as follows: have the blade of mature spore under picking, in 3-5 DEG C do
It saves under the conditions of dry and is gradually scattered to spore.
Preferably, in step (1), methyl α-naphthyl acetate is replaced with indolebutyric acid, and in MS culture medium, the concentration of indolebutyric acid is 0.8-
1.2mg/L。
MS culture medium: MS culture medium is made of a great number of elements, microelement, molysite and organic principle, and composition is known
's.Carbon source and agar are added in MS culture medium.Carbon source uses sucrose, concentration 8-12g/L, preferably such as 10g/L；The concentration of agar
5-9g/L, preferably such as 7g/L.
The present invention is further explained below:
1, the acquisition of spore needs lasting observation, gradually deepens when sporangium color, the reduction of sporophyll moisture when, under picking
There is the blade of mature spore, place it in kept dry in envelope or the paper bag of doubling and be gradually scattered to spore, 4 DEG C of low temperature are protected
It deposits, the holding time was no more than 6 months.
2, it sterilizes.Spore is wrapped up with filter paper, filter paper packet is handled using 75% alcohol treatment 10s and 2% sodium hypochlorite
The mode of 10min sterilizes, aseptic water washing, and the conidia powder on filter paper is rinsed into sterile centrifugation tube, takes spore suspension extremely
Microscopically observation adds sterile water to adjust spore density, until there is 50 spores to be advisable in 40 times of visuals field.
3, aseptic inoculation.The MS culture medium of configuration addition 0.1mg/L 6- benzyl aminoadenine+0.05mg/L methyl α-naphthyl acetate, connects
Kind sterilizing spore liquid, induction generate original foliage；Condition of culture is set as 28 DEG C, 2000~2500lx of intensity of illumination, and illumination 12~
14 hours/day.
4, rooting induction.After original foliage grows juvenile sporophyte, taking-up is separated into fritter, prepares switching.It is seeded to addition
The MS culture medium of 1mg/L indolebutyric acid, is trained test tube seedling.Condition of culture is set as 28 DEG C, 2000~2500lx of illuminance, light
According to 12~14 hours/day.
5, bottle outlet hardening.2-4 piece true leaf to be grown, test tube seedling is taken out, and root is cleaned, and plants in by disinfection treatment
In specific compost (peat+rice hull carbon+vermiculite is sterilized with 1% carbendazim).Transplanting early period suitably to shade, take leaching basin to
The mode of water controls soil moisture 75%~95%.Wherein, product of the rice hull carbon, that is, rice husk after charing.
6, fertilizing management.Using foliage-spray nanometer siliceous fertilizer, primary, raising resistance and survival rate are sprayed weekly.
0.1mg/L 6- benzyl aminoadenine+0.05mg/L methyl α-naphthyl acetate is added to the invention has the advantages that being seeded in
MS culture medium in, spore germination rate is high, has reached 65% or more；Prothallus development is also higher at the probability of juvenile sporophyte, reaches
It to 85% or more, can be sprouted within 1 week or so after spore aseptic inoculation, can grow within 2 weeks or so original foliage, 4 weeks or so
Grow juvenile sporophyte seedling, original foliage after cutting can proliferative induction again, can achieve the effect that largely quickly to breed in a short time.
Detailed description of the invention
Fig. 1 spore germination photo early period.
Fig. 2 spore germination grows original foliage photo.
Fig. 3 test tube seedling photo.
Fig. 4 bottle outlet hardening photo.
Fig. 5 bottle outlet hardening enlarged photograph.
Specific embodiment
Below with reference to embodiment, the invention will be further described.
Sucrose, agar are added in MS culture medium.Adding agar is to solidify culture medium, and additive amount can not coagulate less
Gu addition excessively can make culture medium really up to the mark, it is unfavorable for the later period and takes root, the appropriate concentration range of 6-9g/L is conducive to explant culture
Induction and later period take root.Sucrose is as common carbon source, although concentration has certain influence to culture, it is too low may nutrition not
Foot, excessively high to cause reagent waste, 8-12g/L range is suitable concentration.By preliminary test discovery sucrose, agar concentration and hormone
Concentration variation is compared, negligible to inductivity influence degree.So in the culture medium of embodiment by sucrose, agar it is dense
Degree is fixed as 10g/L and 7g/L.
Embodiment
The present embodiment provides a kind of sporogenesis methods of thick stalk floating fern, comprising the following specific steps
When lasting observation to thick stalk floating fern sporangium color gradually deepens, tends to maturation, there is the leaf of mature spore under picking
Piece places it in kept dry in envelope and is gradually scattered to spore, 4 DEG C of cryo-conservations.
Spore is wrapped up with filter paper, 75% ethyl alcohol 10s+2% sodium hypochlorite 10min aseptic water washing 6 times, will be on filter paper
Conidia powder rinse into sterile centrifugation tube, take spore suspension to microscopically observation, add sterile water to adjust spore density, directly
Extremely there are 50 spores to be advisable in 40 times of visuals field.
Spore liquid is inoculated in the MS culture medium of addition 6- benzyl aminoadenine 0.1mg/L+ methyl α-naphthyl acetate 0.1mg/L, setting
Condition of culture is 28 DEG C, 2000~2500lx of illuminance, 12~14 hour/day of illumination.The average germination rate of spore is 58.1%.
After spore germination goes out original foliage, grows juvenile sporophyte, it is forwarded to the MS culture medium of addition 1mg/L indolebutyric acid,
Condition of culture is same as above.
3-4 piece blade to be grown, test tube seedling is taken out, and root is cleaned, and plants in the specific compost by disinfection treatment
In (+10% vermiculite of+20% rice hull carbon of 70% peat, the disinfection of 1% carbendazim).Transplanting early period suitably to shade, take leaching basin to
The mode of water controls soil moisture 75%~95%.Foliage-spray is carried out using 10mg/L nano-silicon solution, sprays one weekly
It is secondary, chlorophyll content can be improved, enhance resistance.
Different sterilization methods have a significant impact to the pollution rate and spore activity of aseptic inoculation, using 75% alcohol treatment
When the mode of 10s and 2% sodium hypochlorite processing 10min sterilize, pollution rate is relatively low, and does not influence spore activity, such as following table
It is shown.
| Serial number | Sterilization method | Pollution rate/spore activity |
| 1 | 0.1% mercuric chloride impregnates 4min | 15% (partial inactivation) |
| 2 | 0.1% mercuric chloride impregnates 6min | 0 (most of inactivation) |
| 3 | 0.1% mercuric chloride impregnates 8min | 0 (most of inactivation) |
| 4 | + 2% sodium hypochlorite (5min) of 75% ethyl alcohol (10s) | 18.5% (not influencing activity) |
| 5 | + 2% sodium hypochlorite (10min) of 75% ethyl alcohol (10s) | 10% (not influencing activity) |
| 6 | + 2% sodium hypochlorite (15min) of 75% ethyl alcohol (10s) | 10% (not influencing activity) |
In preseed stage, after changing condition of culture (hormone of culture medium and addition), spore germination rate has a greater change,
The germination rate highest in the MS culture medium of addition 0.1mg/L 6- benzyl aminoadenine and 0.05mg/L methyl α-naphthyl acetate, up to 65.3 ±
2.4%, as shown in the table.
| Serial number | Culture medium | The hormone added in culture medium | Spore germination rate (%) |
| 1 | MS | 6- benzyl aminoadenine 0.1mg/L+ methyl α-naphthyl acetate 0.1mg/L | 58.1±3.2 |
| 2 | MS | 6- benzyl aminoadenine 0.1mg/L+ methyl α-naphthyl acetate 0.05mg/L | 65.3±2.4 |
| 3 | MS | 6- benzyl aminoadenine 0.1mg/L+ indolebutyric acid 1mg/L | 57.1±2.5 |
| 4 | MS | 6- benzyl aminoadenine 0.1mg/L+ indolebutyric acid 0.05mg/L | 56.2±3.3 |
| 5 | MS | N6-furfuryladenine 2mg/L | 50.5±4.2 |
| 6 | 1/2MS | 6- benzyl aminoadenine 0.1mg/L+ methyl α-naphthyl acetate 0.1mg/L | 43.2±5.5 |
| 7 | 1/2MS | 6- benzyl aminoadenine 0.1mg/L+ methyl α-naphthyl acetate 0.05mg/L | 45.7±2.2 |
| 8 | 1/2MS | 6- benzyl aminoadenine 0.1mg/L+ indolebutyric acid 1mg/L | 42.5±1.6 |
| 9 | 1/2MS | 6- benzyl aminoadenine 0.1mg/L+ indolebutyric acid 0.05mg/L | 46.2±3.5 |
| 10 | 1/2MS | N6-furfuryladenine 2mg/L | 38.6±2.4 |
| 11 | MS | It does not add | 40.3±1.4 |
| 12 | 1/2MS | It does not add | 36.5±1.1 |
In later period rooting induction, change hormone concentration, root length has bigger difference, in addition 1mg/L indolebutyric acid
MS culture medium in average root long longest, up to 2.8 ± 0.5cm, as shown in the table.
| Serial number | Culture medium | The hormone added in culture medium | When bottle outlet average root long (cm) |
| 1 | MS | 0.5mg/L indolebutyric acid | 1.7±0.3 |
| 2 | MS | 1mg/L indolebutyric acid | 2.8±0.5 |
| 3 | MS | 1.5mg/L indolebutyric acid | 2.4±0.4 |
| 4 | MS | 0.5mg/L methyl α-naphthyl acetate | 1.5±0.4 |
| 5 | MS | 1mg/L methyl α-naphthyl acetate | 2.3±0.5 |
| 6 | MS | 1.5mg/L methyl α-naphthyl acetate | 2.1±0.4 |
| 7 | MS | 0 | 1.2±0.3 |
When bottle outlet hardening, the nano-silicon dispersing agent for applying various concentration has larger impact to chlorophyll content in leaf blades, with
10mg/L concentration treatment effect is best, is more than the concentration without significant difference, as shown in the table.
| Serial number | Siliceous fertilizer concentration (mg/L) | Chlorophyll content (SPAD) |
| 1 | 0 | 28.32±3.41 |
| 2 | 2 | 28.45±2.25 |
| 3 | 4 | 30.23±2.63 |
| 4 | 6 | 31.67±2.14 |
| 5 | 8 | 33.74±1.29 |
| 6 | 10 | 35.82±1.17 |
| 7 | 12 | 35.35±1.75 |
Claims (10)
1. a kind of sporogenesis method of thick stalk floating fern, which comprises the following steps:
(1) by after the spore disinfection of thick stalk floating fern, it is configured to spore suspension with sterile water, spore suspension is inoculated in addition
It is cultivated in the MS culture medium for having 6- benzyl aminoadenine and methyl α-naphthyl acetate；In MS culture medium, the concentration of 6- benzyl aminoadenine
For 0.05-0.15mg/L, the concentration of methyl α-naphthyl acetate is 0.02-0.1mg/L；
(2) slightly stalk floating fern Spore cultivation to after sprouting original foliage out, be transferred in the MS culture medium added with indolebutyric acid into
Row is cultivated, and the concentration of indolebutyric acid is 1-1.5mg/L in MS culture medium, is cultivated to 2-4 piece blade is grown, is obtained test tube seedling；
(3) test tube seedling is taken out, is cultivated after transplanting, obtain slightly obstructing floating fern plant.
2. sporogenesis method as described in claim 1, which is characterized in that in step (1), in MS culture medium, 6- benzyl amino
The concentration of adenine is 0.1mg/L, and the concentration of methyl α-naphthyl acetate is 0.05mg/L.
3. sporogenesis method as described in claim 1, which is characterized in that in step (3), by test tube transplantation of seedlings to by disappearing
In the compost of poison, the compost is composed of the following components: 65-75wt% peat, 15-25wt% rice hull carbon, 8-12wt%
Vermiculite.
4. sporogenesis method as claimed in claim 3, which is characterized in that take the mode of leaching basin water supply, control compost
Humidity 75%~95%.
5. sporogenesis method as described in claim 1, which is characterized in that in the incubation of step (3), using 8-
The nanometer siliceous fertilizer solution of 12mg/L carries out foliage-spray, sprays weekly primary.
6. sporogenesis method as described in claim 1, which is characterized in that in step (1) and (2), condition of culture 27.5-
28.5 DEG C, 2000~2500lx of illuminance, 12~14 hour/day of illumination.
7. sporogenesis method as described in claim 1, which is characterized in that in step (1), the actual conditions of disinfection are as follows: will
The spore of thick stalk floating fern impregnates 8-15s, then the secondary chlorine in mass fraction 1.5-2.5% in the ethyl alcohol of volume fraction 70-75%
8-12min is impregnated in sour sodium.
8. sporogenesis method as described in claim 1, which is characterized in that the density of spore suspension miospore are as follows: 1 × 106
A/mL.
9. sporogenesis method as described in claim 1, which is characterized in that the acquisition mode of the spore of thick stalk floating fern are as follows: adopt
The blade for taking mature spore is saved under 3-5 DEG C of drying condition and is gradually scattered to spore.
10. such as the described in any item sporogenesis methods of claim 1-9, which is characterized in that in step (1), use indolebutyric acid
Instead of 6- benzyl aminoadenine and methyl α-naphthyl acetate, in MS culture medium, the concentration of indolebutyric acid is 0.8-1.2mg/L.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110226385A (en) * | 2019-06-11 | 2019-09-13 | 深圳市铁汉生态环境股份有限公司 | A kind of type of seeding of pteridophyte |
| CN112997728A (en) * | 2021-03-19 | 2021-06-22 | 苏州工业园区园林绿化工程有限公司 | Application of multi-walled carbon nanotube in regulation and control of high-temperature stress of paeonia ostii |
-
2018
- 2018-07-17 CN CN201810784915.4A patent/CN109006477A/en active Pending
Non-Patent Citations (2)
| Title |
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| 孙锐等: "粗梗水蕨孢子无菌繁殖的研究", 《华中师范大学学报(自然科学版)》 * |
| 蔡汉权等: "水蕨孢子叶离体培养试验", 《湖北农业科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110226385A (en) * | 2019-06-11 | 2019-09-13 | 深圳市铁汉生态环境股份有限公司 | A kind of type of seeding of pteridophyte |
| CN112997728A (en) * | 2021-03-19 | 2021-06-22 | 苏州工业园区园林绿化工程有限公司 | Application of multi-walled carbon nanotube in regulation and control of high-temperature stress of paeonia ostii |
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Application publication date: 20181218 |