CN105230492B - A kind of scythian lamb rhizome spore tissue culture mating system - Google Patents
A kind of scythian lamb rhizome spore tissue culture mating system Download PDFInfo
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Abstract
The present invention relates to field of plant growing technology, in particular to a kind of scythian lamb rhizome spore tissue culture mating system, comprises the following steps：1) sporangium ripe on scythian lamb rhizome sporophyll and sterilization, are gathered；2), sporangium is inoculated into scythian lamb rhizome germination medium and cultivated to spore germination；3), the tissue that will be sprouted it is transferred in scythian lamb rhizome proliferated culture medium and cultivate, induction is grown thickly the generation of original foliage；4), original foliage is transferred in new scythian lamb rhizome differential medium and cultivated, induce differentiation into juvenile sporophyte；5), juvenile sporophyte is transferred in scythian lamb rhizome root media and is trained sporinite.The cultural method can be used for the fast breeding of scythian lamb rhizome, can obtain large-scale neat seedling, and breeding coefficient is high, solves the problems, such as that scythian lamb rhizome can not propagation in scale.
Description
Technical field
The present invention relates to field of plant growing technology, in particular to a kind of scythian lamb rhizome spore tissue culture mating system.
Background technology
Scythian lamb rhizome (Cibotium barometz (L.) J.Sm.) is fern Dicksoniaceae plant, main product in Guangxi,
Guangdong, Guizhou, Jiangxi, the band of Southeast Asia one are also distributed, and plant is protected for national II levels.
Rhizoma cibotii is the dry rhizome of scythian lamb rhizome, has the effect such as wind-damp dispelling, filling liver kidney, strong waist and knee, version over the years《Middle traditional Chinese medicines
Allusion quotation》Record.Strong doctor, Yao nationality medicine are often used it for treating the diseases such as arthralgia pain due to rheumatism, soreness and weakness of waist and knees, weakness of the lower extremities；Zhuang, the Yao nationality
The people are often equipped with meat, bone based food Baoshang is drunk, brewed tea and drunk with rhizome, are eaten with tender leaf cooking, nourishing diseases prevention, are enjoyed
It is delicious.The golden yellow pubescence of growth is used for trauma hemostasis outside its rhizome.Modern study is found：Scythian lamb rhizome have anti-osteocarcinoma,
Osteoporosis, hemostasis, analgesia, antibacterial, anti-inflammatory and other effects are prevented and treated, is that domestic more famous pharmacy corporations of family are used to produce loins-strengthening and kidney-invigorating
Ball, blood quickening pain relieving medicinal ball, zhuanggu guanjie pill, golden pheasant tiger mend the primary raw material of more or less a hundred Chinese patent drugs such as ball, scythian lamb rhizome wine.
Because processing enterprise is continuously increased to the demand of medicinal material, causing mad excavation among the people, wild resource is day by day rare,
China has prohibited excavation wild resource, and increases punishment, and the state such as Vietnam also keeps under strict control the outlet of scythian lamb rhizome, medicinal material
Imbalance between supply and demand is very prominent.Therefore, the artificial growth of scythian lamb rhizome is carried out, into the matter of great urgency.
In the links of artificial growth scythian lamb rhizome, most prominent contradiction is how to breed out the seedling of high performance-price ratio.
Forefathers are studied scythian lamb rhizome cultural method, using root-like stock cutting section propagation seedling, obtain seedling breeding method, but
A large amount of medicinal materials are wasted, and collection wild resource is illegal, and seedling cost is high, is not promoted；There is experiment to show Cibotium barometz
Ridge spore is low in artificial environment breeding coefficient；As can be seen here, the successful reproduction of current conventional art means scythian lamb rhizome is all difficult
To ensure, leisure opinion solves the problems, such as scythian lamb rhizome seedling propagation in scale and cost control.
The content of the invention
It is an object of the invention to provide a kind of scythian lamb rhizome spore tissue culture mating system, described cultural method can be used for
The fast breeding of scythian lamb rhizome, solve the problems, such as that scythian lamb rhizome can not propagation in scale.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
A kind of scythian lamb rhizome spore tissue culture mating system, comprises the following steps：
1) sporangium ripe on scythian lamb rhizome sporophyll and sterilization, are gathered；
2), sporangium is inoculated into scythian lamb rhizome germination medium and cultivated to spore germination；
3), the tissue that will be sprouted it is transferred in scythian lamb rhizome proliferated culture medium and cultivate, induction is grown thickly the generation of original foliage；
4), original foliage is transferred in new scythian lamb rhizome differential medium and cultivated, induce differentiation into juvenile sporophyte；
5), juvenile sporophyte is transferred in scythian lamb rhizome root media and is trained sporinite.
Pteridophyte has the obvious alternation of generations, since haploid spore, to gametophyte on produce sperm and
Ovum, this stage is haploid gametophytic generation (also known as sexual generation), since embryonated egg, to sporinite on caused spore
For ascus miospore mother cell before meiosis, this stage is the sporophyte generation (also known as asexual generation) of diploid.This
Two eposides are regularly alternately accomplished its history of life.Scythian lamb rhizome spore tissue culture mating system described herein is exactly to adapt to
Designed by this heterogamous characteristic of pteridophyte.
Wherein, in step 1), because the conidia powder of scythian lamb rhizome is very tiny, can not carry out disinfection sterilizing after collection.
Therefore can only and sporangium ripe in spore gathered together with sporangium when being not turned on, carry out disinfection sterilizing.Sterilization method
To be carried out disinfection 8~10min with 0.1% mercuric chloride solution or 5% liquor natrii hypochloritis；
In step 2), the purpose that sporangium is inoculated into scythian lamb rhizome germination medium is induction rudiment.Clip can be used
Sporangium is caught broken, conidia powder is scattered in scythian lamb rhizome germination medium and is cultivated；Or by the good spore of sterilization
Ascus is placed on superclean bench, good with aseptic filter paper pad, is opened sporangium, conidia powder is dropped on filter paper, then by filter paper
On conidia powder be inoculated into scythian lamb rhizome germination medium and cultivated；
In step 3), the tissue that will be sprouted from sporangium and is transferred to the master that is cultivated in scythian lamb rhizome proliferated culture medium
Syllabus be by spore cultivate be original foliage；
It by original foliage Fiber differentiation is juvenile sporophyte that the purpose of step 4), which is,；
Step 5) is that induction juvenile sporophyte is further grown up and taken root, and transplants and prepares from now on for it.
The operation of above step is carried out preferably on superclean bench.
The tissue culture mating system is simple and easy, easy to operation, the fast breeding available for scythian lamb rhizome.The present invention uses spore
Son carries out tissue culture and bred, it is only necessary to which gathering sporangium ripe on scythian lamb rhizome sporophyll can be bred, and not had to parent
Injury, does not influence the growth of whole plant.In addition, the present invention has the intrinsic spy for cultivating virus-free seedling of tissue culture method
Point, the scythian lamb rhizome seedling bred out is healthy and strong, and survival rate is high, and the seedling physiological age of acquisition is consistent, and growth is neat, is adapted to batch production
Seedling management and large-scale planting.
Scythian lamb rhizome spore tissue culture mating system as described above, in step 2), the scythian lamb rhizome germination medium
Including modified MS medium, N6Culture medium, Pragre culture mediums.
Preferably, the following raw material component is also included in the scythian lamb rhizome germination medium：
0~0.3mg/L of 6-BA, 0~0.2mg/L of NAA.
Preferably, scythian lamb rhizome spore tissue culture mating system as described above, in step 3)~5) in, the scythian lamb rhizome
Proliferated culture medium, scythian lamb rhizome differential medium and scythian lamb rhizome root media are improvement 1/2MS culture mediums；As described above
Three kinds of culture mediums in also include the following raw material component：0~1.0mg/L of 6-BA, 0~1.0mg/L of NAA.
The modified MS medium mother liquor example (unit of table 1：mg)
In step 2), the effect of preferable modified MS medium is induction scythian lamb rhizome spore rudiment；Step 3)~
5) in, the effect of culture medium be induce rudiment sporogenesis grow thickly original foliage, induction grow thickly original foliage be divided into juvenile sporophyte,
Juvenile sporophyte is trained the sporinite that can be transplanted.In addition, later stage culture can also effectively be reduced using improvement 1/2MS culture mediums
Cost.
Wherein, MS culture mediums are that Murashige and Skoog in 1962 were tobacco cell Training Design, are characterized in
Inorganic salts and ion concentration are higher, are more stable ionic equilibrium solution, and its nitrate content is high, the quantity and ratio of its nutrient
Example is suitable, and still, high salt ionic concentration is unfavorable for scythian lamb rhizome original foliage and is divided into sporinite on the contrary.Therefore, the application is to it
KNO in middle a great number of elements3And NH4NO3Content is improved, and its concrete content is as shown in table 1.
The compound method of modified MS medium is summarized as follows：
One, 1000mL beakers are taken, first add 600mL distilled water, sequentially add a great number of elements mother liquor 100mL, it is micro
Element mother liquor 10mL, mother liquid of iron salt 10mL, Calcisolution 100mL, organic substance mother liquor 20mL, sucrose is some, treats that sucrose is abundant
After dissolving, add agar powder and activated carbon is some.
The beaker for filling culture medium is placed on electromagnetic oven and heated, agar is melted completely (can add at this moment 6-BA and
NAA, and adjust pH), last constant volume to 1000mL.Dispensed after constant volume, wrap up simultaneously high-temperature sterilization；Sterilize wild Oryza species cooling, coagulates
Gu it could use.
Wherein, 6-BA (6-Benzylaminopurine), i.e. 6- benzyls aminoadenine, broad spectrum activity plant growth regulating is belonged to
Agent, have and promote plant cell growth and division, suppress the degraded of plant chlorophyll, improve the content of amino acid, delay blade
The functions such as aging.
NAA (1-Naphthaleneacetic acid), i.e. methyl α-naphthyl acetate, belong to broad spectrum type plant growth regulator, have and promote
Enter cell division with expanding, the function such as induced synthesis adventitious root.
Preferably, by KNO in the modified MS medium3It is adjusted to 800~900mg/L, NH4NO3It is adjusted to 700~
800mg/L；
The improvement 1/2MS culture mediums are by the way that a great number of elements of modified MS medium to be halved to the content and other components
Constant and preparation culture medium.It is adjusted referring specifically to the composition and compound method of above modified MS medium.
Preferably, the modified MS medium and improvement 1/2MS culture mediums include the following raw material component：
4~5g/L of agar, 20~35g/L of sucrose, 0~0.3g/L of activated carbon.
Agar is the polysaccharide body by being extracted in marine alga, has coagulability, stability, can form complex compound etc. with some materials
Physicochemical properties.In plant culture, the effect of solidification is primarily served.
Sucrose plays a part of energy substance and Osmolyte regulator in plant tissue culture media.
Activated carbon has very strong adsorption capacity, can reducing some harmful substances (such as aldehydes matter and peroxidase)
Influence, so as to effectively prevent brown stain；In addition, inhibitory substance caused by medium component degraded when activated carbon is to autoclaving
(such as HMF) also has suction-operated；Activated carbon also produces suction-operated to the BA and NAA of high concentration；Activated carbon makes culture medium
Blackening, so as to provide dark situation, promote the accumulation of light sensitivity auxin in culture medium, be advantageous to plant establishment.
Preferably, the pH of the MS improved culture mediums and 1/2MS improved culture mediums is 5~6.
In step 2), the incubation time of the culture is 20~35 days；
In step 3), the incubation time of the culture is 30~40 days；
In step 4), the incubation time of the culture is 20~30 days；
In step 5), the incubation time of the culture is 30~40 days.
Preferably, in the step 3) and step 4) of scythian lamb rhizome spore tissue culture mating system as described above, specific bag
Include：
The original foliage cultivated is bred, proliferation times are 5~10 times；The original foliage bred is transferred to gold again
Cultivated in hair rhizoma cibotii differential medium, induce differentiation into juvenile sporophyte.
Under field conditions (factors), the propagation method of scythian lamb rhizome generally uses root division method, can also use sporogenesis method, but
It is sporogenesis requirement condition height, it is difficult to differentiation and seedling emergence.And aseptically, after Multiplying culture forms original foliage of growing thickly
Differentiation culture is carried out, differentiation rate is high, can obtain large-scale neat bottle seedling.
Preferably, in step 2)~5 as described above) culture during, condition of culture is：
1000~1500lx of intensity of illumination, hour/day of light application time 7~9,23~25 DEG C of cultivation temperature, cultivate humidity 65
~70%.
Scythian lamb rhizome is pteridophyte, to prevent that illumination is excessively too strong, at the same also should not light application time it is too short, otherwise plant
Strain jaundice, it appears weak or wilting.
Compared with prior art, beneficial effects of the present invention are：
1), the invention provides a kind of efficient scythian lamb rhizome spore tissue culture mating system, technological process is simple, with prophyll
Body form is bred, and can obtain substantial amounts of original foliage, is bred by spore germination, original foliage, sporinite is induced, sporinite is given birth to
The processes such as root, the seedling that largely can be used for production cultivation is obtained, and the seedling physiological age obtained is consistent, growth is neat, is adapted to
Industrial seedling rearing and large-scale planting.
2), the present invention is bred using spore progress tissue culture, it is only necessary to gathers sporangium ripe on scythian lamb rhizome sporophyll
It can be bred, parent is not injured, not influence the growth of whole plant, explant dosage is few, and it is low to breed cost.
3), the scythian lamb rhizome seedling bred with this law, seedling is virus-free or other diseases infect, and plant strain growth is healthy and strong, into
Motility rate is high.
4), scythian lamb rhizome is national second class protection pteridophyte, forbids excavating.It can be bred to obtain a large amount of Cibotium barometzs with this law
Ridge seedling, beneficial to the protection of wild scythian lamb rhizome.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described.
Fig. 1 is photo when step 2) the miospore powder of embodiment 3 cultivates sprouting in 20 days in modified MS medium.
Fig. 2 is in the step 3) of embodiment 3, original foliage after being cultivated 35 days in improveing 1/2MS culture mediums (breed,
Also non-plant division).
Fig. 3 is the sporinite seedling after being cultivated 25 days in improveing 1/2MS culture mediums in the step 4) of embodiment 3.
Fig. 4 is the sporinite after being cultivated 35 days in improveing 1/2MS culture mediums in the step 5) of embodiment 3.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition suggested according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, it is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
Scythian lamb rhizome spore tissue culture mating system, comprises the following steps：
1) sporangium ripe on scythian lamb rhizome sporophyll and sterilization, are gathered；
2) sporangium, is inoculated into N6Cultivated in culture medium to spore germination；Condition of culture is：Intensity of illumination 1000~
1500lx, hour/day of light application time 7~9,23~25 DEG C of cultivation temperature, cultivate humidity 65~70%；Culture in following steps
Condition is identical with this stage condition of culture；
3), the tissue that will be sprouted it is transferred in Pragre culture mediums and cultivate, induces the generation of original foliage；
4), original foliage is transferred in new Pragre culture mediums and cultivated, induce differentiation into juvenile sporophyte；
5), juvenile sporophyte is transferred in new Pragre culture mediums and is trained sporinite.
Embodiment 2
Scythian lamb rhizome spore tissue culture mating system, comprises the following steps：
1) sporangium ripe on scythian lamb rhizome sporophyll, is gathered, is sterilized on superclean bench with 0.1% mercuric chloride solution
8~10min；
2), the sporangium disinfected is connected in modified MS medium, sporangium is caught broken with clip, is attached to conidia powder
20~35 days are cultivated in modified MS medium to spore germination；Condition of culture is：1000~1500lx of intensity of illumination, light application time
23~25 DEG C of 7~9 hours/day, cultivation temperature, cultivate humidity 65~70%；Condition of culture in following steps is trained with this stage
The condition of supporting is identical；
3) tissue that, will be sprouted from sporangium it is transferred in improvement 1/2MS culture mediums and cultivate 30~40 days, induces it
Original foliage is formed, while original foliage is bred, proliferation times are 5~10 times；
4), original foliage is transferred in new improvement 1/2MS culture mediums and cultivated 20~30 days, induce differentiation into young spore
Body；
5), juvenile sporophyte is transferred in new improvement 1/2MS culture mediums and cultivated 30~40 days, obtain sporinite.
The operation of above step is carried out on superclean bench.
Wherein, the modified MS medium and improvement 1/2MS culture mediums include the following raw material component：4~5g/L of agar,
20~35g/L of sucrose, 0~0.3g/L of activated carbon.
In addition, also include the following raw material component in modified MS medium：0~0.3mg/L of 6-BA, 0~0.2mg/L of NAA.
Also include the following raw material component in improvement 1/2MS culture mediums：0~1.0mg/L of 6-BA, 0~1.0mg/L of NAA.
Embodiment 3
Scythian lamb rhizome spore tissue culture mating system, comprises the following steps：
1) sporangium ripe on Cibotium barometz sporophyll, is gathered, is sterilized on superclean bench with 5% liquor natrii hypochloritis
8~10min；
2), the good sporangium of sterilization is placed on superclean bench, it is good with aseptic filter paper pad, allow sporangium to open,
Conidia powder is dropped on filter paper, and the conidia powder on filter paper is shaken off to be attached to improvement MS+6-BA 0.2mg/L+NAA 0.1mg/L+ fine jades
20~35 days are cultivated to spore germination on fat 4.5g/L+ sucrose 30g/L+ activated carbon 0.2g/L culture mediums；Condition of culture is：Illumination
1000~1500lx of intensity, hour/day of light application time 7~9,23~25 DEG C of cultivation temperature, cultivate humidity 65~70%；Walk below
Condition of culture in rapid is identical with this stage condition of culture；
3) tissue that, will be sprouted from sporangium and is transferred to improvement 1/2MS+6-BA 0.8mg/L+NAA 0.35mg/L+
Agar 4.5g/L+ sucrose 30g/L+ activated carbons 0.3g/L；
Cultivated 30~40 days in culture medium, induce it to form original foliage, while original foliage is bred, proliferation times are 5~8
Times；
4), original foliage is transferred to the improvement 1/2MS+6-BA 0.1mg/L+NAA 0.75mg/L+ agar 4g/L newly prepared
+ sucrose 25g/L+ activated carbons 0.3g/L；
Cultivated 20~30 days in culture medium, induce differentiation into juvenile sporophyte；
5), juvenile sporophyte is transferred to the improvement 1/2MS+NAA 0.6mg/L+ agar 4g/L+ sucrose 25g/L+ newly prepared
Cultivated 30~40 days in activated carbon 0.3g/L culture mediums, obtain sporinite.
The operation of above step is carried out on superclean bench.
In modified MS medium described above, KNO3For 800~900mg/L, NH4NO3For 700~800mg/L；Above institute
State in improvement 1/2MS culture mediums, KNO3For 400~450mg/L, NH4NO3For 350~400mg/L；Remaining component is (in embodiment
Unselected a large amount of components, Trace Components, molysite, calcium salt, organic substance) it is customary amount.
It is mostly mountain region the main product of scythian lamb rhizome, height above sea level belongs to subtropical monsoon climate between -100~1000 meters,
16~22 DEG C of average temperature of the whole year, more than 300 days frost-free period of year, 1600~2000 millimeters of average annual precipitation, the winter is without the severe cold summer without cruel
Heat, it is especially suitable for the growth of scythian lamb rhizome.Local farmers are gone up a hill for a long time excavates the sale of rhizoma cibotii medicinal material, and the growth to scythian lamb rhizome is special
Property is very familiar.Major pharmacy corporation set up long-term purchasing site in each main product.
In addition, each main product be mostly forest land, scythian lamb rhizome grows the most vigorous in forest land, cheuch.It is dog using forest land
The good ecological environment that ridge is built, carries out artificial growth, and small investment is quick.Rhizoma cibotii growth enhances forest land water conservation again
Ability, the two is mutually promoted, solve medicinal material plantation with forest development strive ground conspicuous contradiction.
The scythian lamb rhizome spore tissue culture mating system that the application provides can be used for the fast breeding of scythian lamb rhizome, can obtain
Large-scale neat seedling, and breeding coefficient is high, solves the problems, such as that scythian lamb rhizome can not propagation in scale.If widely popularize this
The method provided in application, so that it may solve the problems, such as that seedling is difficult to obtain in a large amount of artificial breedings of scythian lamb rhizome, it is near so as to play
Phase obtain medicine, long-term woods, it is far and near combine, mend the industrialization effect of long coordinated development with short, and then realize ecological protected, agriculture
The people obtain economical win-win situation.
Although illustrate and describing the present invention with specific embodiment, but will be appreciated that without departing substantially from the present invention's
Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (4)
1. a kind of scythian lamb rhizome spore tissue culture mating system, it is characterised in that comprise the following steps：
1）, ripe sporangium and sterilization on collection scythian lamb rhizome sporophyll；
2）, sporangium is inoculated into in scythian lamb rhizome germination medium culture to spore germination；
The scythian lamb rhizome germination medium is modified MS medium+6-BA 0~0.3mg/L+NAA, 0~0.2mg/L+ agar 4
0~0.3g/L of~5g/L+ sucrose 20~35g/L+ activated carbons；
3）, the tissue that will be sprouted be transferred in scythian lamb rhizome proliferated culture medium and cultivate, induction is grown thickly the generation of original foliage；
4）, original foliage is transferred in new scythian lamb rhizome differential medium and cultivated, induce differentiation into juvenile sporophyte；
5）, juvenile sporophyte be transferred in scythian lamb rhizome root media be trained sporinite；
In step 3）~5）In, the scythian lamb rhizome proliferated culture medium, scythian lamb rhizome differential medium and scythian lamb rhizome culture of rootage
Base be MS culture medium+6-BA 0~1.0mg/L+NAA, 0~1.0mg/L+, 4~5g/L+ of the agar sucrose 20 of improvement 1/2~
0~0.3g/L of 35g/L+ activated carbons；
Improved component in the modified MS medium is：KNO3It is adjusted to 800 ~ 900mg/L, NH4NO3It is adjusted to 700 ~
800mg/L；
1/2 MS culture mediums of the improvement are by the way that a great number of elements of modified MS medium is halved and the content of other components not
The culture medium for becoming and preparing.
2. scythian lamb rhizome spore tissue culture mating system according to claim 1, it is characterised in that：
In step 2）In, the incubation time of the culture is 20 ~ 35 days；
In step 3）In, the incubation time of the culture is 30 ~ 40 days；
In step 4）In, the incubation time of the culture is 20 ~ 30 days；
In step 5）In, the incubation time of the culture is 30 ~ 40 days.
3. scythian lamb rhizome spore tissue culture mating system according to claim 1, it is characterised in that step 3）With step 4）
In, specifically include：
The original foliage cultivated is bred, proliferation times are 5 ~ 10 times；The original foliage bred is transferred to Cibotium barometz again
Cultivated in ridge differential medium, induce differentiation into juvenile sporophyte.
4. scythian lamb rhizome spore tissue culture mating system according to claim 1, it is characterised in that condition of culture is：
1000~1500lx of intensity of illumination, hour/day of light application time 7 ~ 9,23~25 DEG C of cultivation temperature, cultivate humidity 65~70%.
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| 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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| CN101911914A (en) * | 2010-09-01 | 2010-12-15 | 中国地质大学（武汉） | Culture medium for spore germination and seedling culturing of pteridophyte |
| CN102860262B (en) * | 2012-10-25 | 2013-07-10 | 南京林业大学 | Propagation method of cyrtomium fortunei |
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