CN106171999B - The fertile Spore cultivation of Guangdong osmund and its propagation method - Google Patents
The fertile Spore cultivation of Guangdong osmund and its propagation method Download PDFInfo
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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
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The present invention provides a kind of fertile Spore cultivation of Guangdong osmund and its propagation method, comprise the following steps：Be seeded in after sporangium fritter or conidia powder are disinfected on MS minimal mediums and add 6 0.5~1.5 ㎎ of benzyl purine/L, 0.01~0.1 ㎎ of methyl α-naphthyl acetate/L does culture processing, gradually grow up to original foliage by filamentous, plates after spore germination；Then it is transferred to culture in 1/2MS, 1/4MS culture medium to handle, when original foliage volume significantly increases, then is transferred in identical solid medium and cultivates, form sporinite seedling；Sporinite seedling inoculation is done into culture processing in the culture medium containing 0.5~1.0 ㎎ of indolebutyric acid/L again, 20d or so grows young root, after root is formed, continues to cultivate 1 month or so in former bottle, moves into basin and cultivates.The present invention solves the problems, such as that current Guangdong osmund breeding is difficult, can quickly breed Guangdong osmund seedling in a short time, effectively protect the scarce resource of Guangdong osmund.
Description
Technical field
The invention belongs to technical field of pteridophyte tissue culture, more particularly to a kind of fertile Spore cultivation of Guangdong osmund and its
Propagation method.
Background technology
Pteridophyte plays an important role as species resource and aspect of keeping ecological balance；Secondly, in research plant
The Origin of Species, also great scientific value in terms of evolution and classification；3rd, it is also medicinal, the use that eats, view and admire etc.
On the way.Therefore tissue culture technique is widely used in the research and production of pteridophyte.At present in the tissue of fern
In culture studies, explant can use underground stem section or stem apex, tender leaf or phyllopodium, spore etc..The underground stem section of usual fern
Or stem apex, tender leaf or phyllopodium are all coated with scale, disinfection is more difficult, and rare species individual amount is inherently seldom, and material comes
Source is very precious in itself, therefore is bred more using spore as explant.
Guangdong osmund (Osmunda mildei C.Chr.) is perennial medium-sized draft pteridophyte, distribution be detected in Hong Kong,
Shenzhen and the The Qiyun Mountain of Jiangxi, are distinctive herbaceous plant, rare, and its precious.But Guangdong osmund is a heterozygosis
Diploid kind, spore full maturity and natural propagation are close to 100% abortion.The Guangdong osmund sum reported is no more than 10 plants, is
Expand the individual amount of the species, achieve the purpose that this kind of child care, work out a kind of sprouting of the suitable fertile spore of Guangdong osmund
It is very necessary with tissue culture and rapid propagation method.
Patent document 201010594485.3 disclosed in Patent Office of the People's Republic of China《A kind of method of Guangdong osmund tissue cultures》Using
Be young tender stem point induction and breeding culture, since Guangdong osmund sum is very rare, this pattern it is more or less meeting injury
Former strain, causes the death of former strain, causes the individual reduction naturally of Guangdong osmund, is unfavorable for wild individual protection.
The content of the invention
It is an object of the invention to overcome the problem of breeding of Guangdong osmund plant spore is difficult in the prior art, there is provided one kind is adapted to
The fertile spore germination of heterozygosis species Guangdong osmund and tissue culture and rapid propagation method.
The fertile Spore cultivation of Guangdong osmund provided by the invention and its propagation method, comprise the steps of：
S1 spores are disinfected：
3~May Guangdong osmund sporangium by it is dark green just switch to pale green when, spore blade is taken, conidia powder is collected, with going out
Bacterium water soaks 5-10min, then removes supernatant, adds 0.1% HgCl2Solution disinfection handles 2-6min, and aqua sterilisa rinses
Afterwards plus appropriate aqua sterilisa mixes and suspension is made；
Alternatively, 3~May Guangdong osmund sporangium by it is dark green just switch to pale green when, each spore leaflet is taken, use
0.1% HgCl2Solution disinfection handles 2-6min, and aqua sterilisa blots surface moisture after rinsing, and is cut into containing 2~3 sporangiums
Fritter；
The acquisition of S2 original foliages：
By the step S1 sporangium fritters disinfected or conidia powder, it is seeded in MS minimal mediums and to add 6- benzyls fast
0.5~1.5 ㎎ of purine/L, 0.01~0.1 ㎎ of methyl α-naphthyl acetate/L, do culture processing, gradual by filamentous, plates after spore germination
Grow up to original foliage；
The formation of juvenile sporophyte in S3 original foliages：
The step S2 original foliages obtained are transferred in 1/2MS, 1/4MS culture medium, do culture processing, treat original foliage volume
When significantly increasing, then it is transferred in identical solid medium and cultivates, forms sporinite seedling；
S4 culture of rootage and transplanting：
The sporinite seedling inoculation that step S3 is produced is cultivated in the culture medium containing 0.5~1.0 ㎎ of indolebutyric acid/L
Processing, 20d grows young root, after root is formed, continues to cultivate 1 month in former bottle, moves into basin and cultivates.
The fertile Spore cultivation of Guangdong osmund provided by the invention and its propagation method, have drawn from Guangdong osmund plant spore, utilizes
Plant tissue culture technique obtains a large amount of clones, and the original foliage and juvenile sporophyte cultivated, will not both injure plant original
Strain, and solve the problems, such as that current Guangdong osmund breeding is difficult, Guangdong osmund seedling can be quickly bred in a short time, be disclosure satisfy that and studied it
A large amount of materials of endangered mechanism and species Forming Mechanism need, and contribute for rescue endangered plants Guangdong osmund；Simultaneously because
Pteridophyte has the function that important in urban green space system bio-diversity, and the especially exploitation of wild ferns, works as greening
It can largely be produced in the short time when needing, will there is vast potential for future development in Greenland Application.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The present invention provides the fertile Spore cultivation of Guangdong osmund and its propagation method, mainly includes：(1) spore is disinfected；
(2) acquisition of original foliage；(3) in original foliage juvenile sporophyte formation；(4) sporinite seedling culture of rootage and transplanting.It is above-mentioned
Step cultivates original foliage and juvenile sporophyte by choosing Guangdong osmund plant spore, solves the problems, such as that current Guangdong osmund breeding is difficult,
Guangdong osmund seedling can be quickly bred in a short time, effectively protect the scarce resource of Guangdong osmund.
The present invention is further described with reference to embodiment.
Embodiment one：
The present embodiment comprises the steps of：
S1 spores are disinfected：
3~May, Guangdong osmund sporophyll was fully deployed, sporangium start from bottle green to pale green change when, by sporophyll
Piece is taken, and washes away surface floating dust with clear water immediately, after the moisture of spore leaf surface dries, collects conidia powder；Alternatively, by spore
Leaf is placed in the culture dish for being covered with template, is placed in drier and conidia powder is collected after drying process.Then in superclean bench
On, put it into small test tube, soak 5-10min with aqua sterilisa, then with removing supernatant after 3500 revs/min of centrifuge
Liquid, adds 0.1% HgCl2Solution disinfection 2-6min, aqua sterilisa rinse 5-6 times, finally add appropriate aqua sterilisa mixing to be made outstanding
Supernatant liquid.
In the step, with disinfection, 3 minutes are optimal, the pure Multiplying culture not for the purpose of observing Development of Gametophytes,
Convenient operation is directly disinfected with sporophyll.
The acquisition of S2 original foliages：
The conidia powder that step S1 is disinfected, is seeded on MS minimal mediums, and addition hormone 6- benzyl purines 0.5~
1.5 ㎎/L, 0.01~0.1 ㎎ of methyl α-naphthyl acetate/L, in temperature (25 ± 2) DEG C, illumination 12~16h/ days, 1500~2000lx of illuminance
Under conditions of do culture processing, gradually grow up to original foliage by filamentous, plates after spore germination.
In this step, MS minimal mediums are this area conventional medium, by combining 6- benzyls on MS minimal mediums
Base purine and methyl α-naphthyl acetate, can promote spore division, growth and development, be conducive to the sprouting of spore and the formation of original foliage.
This step also further can carry out increment culture to above-mentioned original foliage using following S21 steps.
The original foliage of acquisition is seeded in MS minimal mediums and containing 1.5~2.0 ㎎ of 6- benzyl purines/L, naphthalene second by S21
In the proliferated culture medium of acid 0.1~0.5 ㎎/L, carry out Multiplying culture, 45d or so and transfer 1 time.Its culture processing procedure of rising in value
In, add 6- benzyl purines and methyl α-naphthyl acetate can further improve the growth rate of original foliage, form a large amount of original foliages in a short time, reach
To the purpose quickly bred.
The formation of juvenile sporophyte in S3, original foliage：
The original foliage that step S2 is obtained, because propagation quantity is more, original foliage individual is small, is unfavorable for the formation of sexual organ, very
Difficult formation sporinite.Needing to be transferred to 1/2MS, 1/4MS at this time, (described 1/2MS, 1/4MS are in MS minimal medium components
A great number of elements is reduced to 1/2 or 1/4, remaining components unchanged) in culture medium, in temperature (25 ± 2) DEG C, illumination 12~16h/ days,
Cultivated under conditions of 1500~2000lx of illuminance, treat that original foliage volume significantly increases, then be transferred to identical solid medium (such as
Agar medium) in culture, the aqua sterilisa of 1mm or so is added during inoculation in solid culture primary surface, to increase the movement of sperm,
Combined easy to sperm with egg cell, form sporinite seedling.By the culture of 1 month or so, there is more than the 30% prophyll bodily form
Into sporinite seedling.
In this step, sporinite seedling can directly be transferred to follow-up transplant step or be transferred to following step S31 juvenile sporophytes
Multiplying culture is carried out in proliferated culture medium, does not form the original foliage of juvenile sporophyte, can continue to be transferred to S2 steps original foliage propagation
Multiplying culture is carried out in culture medium, can also continue to cultivate in 1/2,1/4MS, to produce more sporinite seedling.
The Multiplying culture of S31 juvenile sporophytes：
The juvenile sporophyte that step S3 is produced, is seeded in containing 0.5~1.0 ㎎ of 6- furfuryl group adenines/L, methyl α-naphthyl acetate 0.1
In the sporinite proliferated culture medium of ㎎/L, in temperature (25 ± 2) DEG C, 12~16h/d of illumination, the bar of 1500~2000lx of illuminance
Culture processing, 45d or so are under part can induce new juvenile sporophyte in its base portion.
In this step, combined using the 6- furfuryl groups adenine and methyl α-naphthyl acetate of high concentration, juvenile sporophyte can be promoted to breed, into one
Step accelerates the breeding of sporinite seedling.
S4 culture of rootage：
Step S3 or the S31 juvenile sporophyte produced are seeded in the culture medium containing 0.5~1.0 ㎎ of indolebutyric acid/L,
In temperature (25 ± 2) DEG C, under conditions of illumination 12~16h/ days, 1500~2000lx of illuminance culture handle, 20d or so can
To grow young root, after root is formed, continuing to cultivate 1 month or so in former bottle to transplant.
In this step, add indolebutyric acid, can induce the formation of sporinite seedling root restriction, promote root restriction division and
Differentiation, is conducive to new root generation and the differentiation of fibrovascular system, the quantity of young root is improved, beneficial to juvenile sporophyte transplant survival.
S5 is transplanted：
Juvenile sporophyte continues to cultivate 45d or so in same medium, you can is taken out from blake bottle, wash clean culture
Base, moves into basin and cultivates.
Cultivation can use following matrix after the juvenile sporophyte is taken root：
(1) loess：Humus, its ratio be 1：1；
(2) river sand：Loess：Humus, its ratio be 1：1：3；
(3) perlite：Loess：Humus, its ratio be 1：1：3.
After cultivation, start that moisturizing can be covered with plastic film, open 1 hour ventilation of plastic film daily, increase after a week
Add ventilation time, 20d can remove plastic film completely later.
Seedling percent in above-mentioned three kinds of cultivation matrixes can reach more than 90%, wherein with (2) river sand：Loess：
Seedling growth in the cultivation matrix of humus proportioning is best.
Embodiment two：
S1 3~May Guangdong osmund sporophyll be fully deployed, sporangium start from bottle green to pale green change when, by spore
Blades are taken, and wash away surface floating dust with clear water immediately, and each spore leaflet is taken, on superclean bench, with 0.1%
HgCl2Solution disinfection 2-6min, aqua sterilisa rinse 5-6 times, and sterilized filter paper blots surface moisture, are cut into containing 2~3 sporangiums
Fritter.
In the step, with disinfection, 5 minutes are optimal, the pure Multiplying culture not for the purpose of observing Development of Gametophytes,
Convenient operation is directly disinfected with sporophyll.
The acquisition of S2 original foliages：
The sporangium fritter that step S1 is disinfected is seeded on MS minimal mediums, adds hormone 6- benzyl purines
0.5~1.5 ㎎/L, 0.01~0.1 ㎎ of methyl α-naphthyl acetate/L, in temperature (25 ± 2) DEG C, illumination 12~16h/ days, illuminance 1500~
Culture processing is done under conditions of 2000lx, gradually grows up to original foliage by filamentous, plates after spore germination.
In this step, MS minimal mediums are this area conventional medium, by combining 6- benzyls on MS minimal mediums
Base purine and methyl α-naphthyl acetate, can promote the spore in sporangium to divide, growth and development, be conducive to sprouting and the original foliage of spore
Formed.
This step also further can carry out increment culture to above-mentioned original foliage, be properly increased in its culture processing procedure of rising in value
The concentration of 6- benzyl purines and methyl α-naphthyl acetate, is specially：The original foliage of acquisition is seeded in MS minimal mediums and contains 6- benzyls
1.5~2.0 ㎎ of purine/L, 0.1~0.5 ㎎ of methyl α-naphthyl acetate/L proliferated culture medium in, carry out Multiplying culture, 45d or so switching 1
It is secondary.It rises in value in culture processing procedure, improves 6- benzyl purines and methyl α-naphthyl acetate can further improve the growth rate of original foliage, short
A large amount of original foliages are formed in phase, achieve the purpose that quickly to breed.
The formation of juvenile sporophyte in S3, original foliage：
The original foliage that step S2 is obtained, because propagation quantity is more, original foliage individual is small, is unfavorable for the formation of sexual organ, very
Difficult formation sporinite.Needing to be transferred to 1/2MS, 1/4MS, (described 1/2MS, 1/4MS are a large amount of in MS minimal medium components
Element is reduced to 1/2 or 1/4, remaining components unchanged) in culture medium, in temperature (25 ± 2) DEG C, illumination 12~16h/ days, illumination
Cultivated under conditions of 1500~2000lx of degree, treat that original foliage volume significantly increases, then be transferred to identical solid medium (such as agar
Culture medium) in culture, the aqua sterilisa of 1mm or so is added during inoculation in solid culture primary surface, to increase the movement of sperm, is easy to
Sperm is combined with egg cell, forms sporinite seedling.By the culture of 1 month or so, the original foliage for having more than 30% formed spore
Daughter seedling.
In this step, sporinite seedling can directly be transferred to follow-up transplant step or be transferred in juvenile sporophyte proliferated culture medium
Multiplying culture is carried out, does not form the original foliage of juvenile sporophyte, can continue to be transferred in original foliage proliferated culture medium and carry out propagation training
Support, can also continue to cultivate in 1/2,1/4MS, to produce more sporinite seedling.
The Multiplying culture of S31 juvenile sporophytes：
The juvenile sporophyte that step S3 is produced, is seeded in containing 0.5~1.0 ㎎ of 6- furfuryl group adenines/L, methyl α-naphthyl acetate 0.1
In the sporinite proliferated culture medium of ㎎/L, in temperature (25 ± 2) DEG C, 12~16h/d of illumination, the bar of 1500~2000lx of illuminance
Culture processing, 45d or so are under part can induce new juvenile sporophyte in its base portion.
In this step, combined using the 6- furfuryl groups adenine and methyl α-naphthyl acetate of high concentration, juvenile sporophyte can be promoted to breed, into one
Step accelerates the breeding of sporinite seedling.
S4 culture of rootage：
The sporinite seedling inoculation that step S3 or S31 are produced is in the culture medium containing 0.5~1.0 ㎎ of indolebutyric acid/L
In, in temperature (25 ± 2) DEG C, under conditions of illumination 12~16h/ days, 1500~2000lx of illuminance culture handle, 20d or so
Young root can be grown, after root is formed, continuing to cultivate 1 month or so in former bottle to transplant.
In this step, add indolebutyric acid, can induce the formation of sporinite seedling root restriction, promote root restriction division and
Differentiation, is conducive to new root generation and the differentiation of fibrovascular system, the quantity of young root is improved, beneficial to juvenile sporophyte transplant survival.
S5 is transplanted：
Juvenile sporophyte continues to cultivate 45d or so in same medium, you can is taken out from blake bottle, wash clean culture
Base, moves into basin and cultivates.
Cultivation can use following matrix after the juvenile sporophyte is taken root：
(1) loess：Humus, its ratio be 1：1；
(2) river sand：Loess：Humus, its ratio be 1：1：3；
(3) perlite：Loess：Humus, its ratio be 1：1：3.
After cultivation, start that moisturizing can be covered with plastic film, open 1 hour ventilation of plastic film daily, increase after a week
Add ventilation time, 20d can remove plastic film completely later.
Seedling percent in above-mentioned three kinds of cultivation matrixes can reach more than 90%, wherein with (2) river sand：Loess：
Seedling growth in the cultivation matrix of humus proportioning is best.
In conclusion be only the part of present pre-ferred embodiments shown in the above embodiment of the present invention, can not be with this office
The limit present invention, under conditions of marrow of the present invention is not departed from, any modification that those skilled in the art are made, equivalent substitution and changes
Into etc., all belong to protection scope of the present invention.
Claims (6)
1. the fertile Spore cultivation of Guangdong osmund and its propagation method, it is characterised in that comprise the steps of：
S1 spores are disinfected：
3~May Guangdong osmund sporangium by it is dark green just switch to pale green when, spore blade is taken, collect conidia powder, use aqua sterilisa
5-10min is soaked, supernatant is then removed, adds 0.1% HgCl2Solution disinfection handles 2-6min, and aqua sterilisa adds after rinsing
Appropriate aqua sterilisa, which mixes, is made suspension；
Alternatively, 3~May Guangdong osmund sporangium by it is dark green just switch to pale green when, each spore leaflet is taken, with 0.1%
HgCl2Solution disinfection handles 2-6min, and aqua sterilisa blots surface moisture after rinsing, is cut into the fritter containing 2~3 sporangiums；
The acquisition of S2 original foliages：
By the step S1 sporangium fritters disinfected or conidia powder, it is seeded in MS minimal mediums and adds 6- benzyl purines
0.5~1.5 ㎎/L, 0.01~0.1 ㎎ of methyl α-naphthyl acetate/L, do culture processing, are gradually grown by filamentous, plates after spore germination
Into original foliage；
The formation of juvenile sporophyte in S3 original foliages：
The step S2 original foliages obtained are transferred in 1/2MS, 1/4MS culture medium, do culture processing, treat that original foliage volume is obvious
During increase, then it is transferred in identical solid medium and cultivates, forms sporinite seedling；
S4 culture of rootage and transplanting：
The sporinite seedling inoculation that step S3 is produced is done at culture in the culture medium containing 0.5~1.0 ㎎ of indolebutyric acid/L
Reason, 20d grows young root, after root is formed, continues to cultivate 1 month in former bottle, moves into basin and cultivates.
2. the fertile Spore cultivation of Guangdong osmund as claimed in claim 1 and its propagation method, it is characterised in that the step S1
In, sporangium just by it is dark green switch to pale green when, sporophyll is taken, conidia powder is collected after drying process.
3. the fertile Spore cultivation of Guangdong osmund as claimed in claim 1 and its propagation method, it is characterised in that the step S2 it
After original foliage obtains, the original foliage of acquisition can be also seeded in MS minimal mediums and 1.5~2.0 ㎎ of benzyl purine containing 6-/L,
In the proliferated culture medium of 0.1~0.5 ㎎ of methyl α-naphthyl acetate/L, Multiplying culture is carried out, 45d transfers 1 time.
4. the fertile Spore cultivation of Guangdong osmund as claimed in claim 1 and its propagation method, it is characterised in that the step S3 it
After juvenile sporophyte is formed in original foliage, the Multiplying culture step of juvenile sporophyte is further included：The juvenile sporophyte that step S3 is produced, connects
Kind 0.5~1.0 ㎎ of the adenine of furfuryl group containing 6-/L, 0.1 ㎎ of methyl α-naphthyl acetate/L sporinite proliferated culture medium in, culture processing after,
45d induces new juvenile sporophyte in its base portion.
5. the fertile Spore cultivation of Guangdong osmund as claimed in claim 1 and its propagation method, it is characterised in that the juvenile sporophyte
Move into basin and use following cultivation matrixes：
Loess：Humus, ratio 1：1；
Alternatively, river sand：Loess：Humus, ratio 1：1：3；
Alternatively, perlite：Loess：Humus, ratio 1：1：3.
6. such as the fertile Spore cultivation of claim 1-5 any one of them Guangdong osmund and its propagation method, it is characterised in that described
Cultivating treatment conditions is：Temperature (25 ± 2) DEG C, illumination 12~16h/ days, 1500~2000lx of illuminance.
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| CN106718899A (en) * | 2016-12-08 | 2017-05-31 | 大连民族大学 | A kind of method of mountain region high-yield culturing common vetch dish |
| CN109601387B (en) * | 2019-01-25 | 2021-08-24 | 徐州生物工程职业技术学院 | Tissue culture propagation method of osmunda vachellii with GGB route induced by juvenile sporocyst group |
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