CN106962204B - A kind of tissue culture method for breeding of money Pu - Google Patents
A kind of tissue culture method for breeding of money Pu Download PDFInfo
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- CN106962204B CN106962204B CN201710373879.8A CN201710373879A CN106962204B CN 106962204 B CN106962204 B CN 106962204B CN 201710373879 A CN201710373879 A CN 201710373879A CN 106962204 B CN106962204 B CN 106962204B
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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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- 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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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
A kind of method for tissue culture of money Pu, including the step of explant disinfection, induction and Multiplying culture and squamous subculture, culture of rootage and hardening and transplanting.This method reproduction speed is fast; offspring is healthy and strong; cost and production time is greatly saved; the seedling parental source for also solving conventional breeding of method production is complicated; the unstable problem of seedling quality; appropriate criteria, industrial massive production, provide the good seed of unified standard for market, overcome the difficult point that money Pu seedling can not be produced in the anniversary.
Description
Technical field
The invention belongs to field of tissue culture, and in particular to a kind of tissue culture method for breeding of money Pu.
Background technique
Money Pu (AcorusgramineusSoland) is Acorus of Araceae, perennial very water herbaceous plant, branch
It grows thickly.Produce Jiangsu, Zhejiang, Jiangxi, Hubei, Hunan, Guangdong, Guangxi, Shaanxi, Gansu, Sichuan, Guizhou, Yunnan.Money Pu is normal
It is green, leafage is emerald green and has gloss, dignified beautiful, there is fragrance, be suitable for water scenery bank and water body greening.Can also potting it is ornamental or make
Setting is used.Leaf, inflorescence can also make floral holding material.Complete stool fragrance, can make fragrance or mosquito dispersing；Rhizome can be used as medicine, and acrid flavour is warm-natured.
Dampness elimination appetizing, slit phlegm of having one's ideas straightened out, inducing resuscitation intelligence development.In addition to making emergent aquactic plant and watching, it can also make submerged plant or ground cover plant is ornamental, be
A kind of new excellent sight leaf flowering marsh plants.Ornamental and economic value with higher.With the development of decorative indoor plant, product
Supply is constantly in situation in short supply.
Money Pu propagation method is mainly rhizome division propagation, but division propagation quantity is few, plant type easy to damage, is restored very slow
And be subject to seasonal restrictions, a large amount of seedlings cannot be obtained in a short time；It is difficult to carry out pushing away for scale, standardized production and large area
Extensively.Therefore the mating system for exploring a kind of fast and effective money Pu is imperative.
Summary of the invention
In view of this, causing it is an object of the invention to overcome the method for the tradition breeding seedling of existing Propagation of Rhizomes
Reproduction speed is slow, and breeding coefficient is low, and material requirements is high, dosage is big, is difficult to the defects of large-scale production and deficiency, to provide one
Kind can be suitable for the method and special culture media of money Pu industrialized tissue culture and rapid propagation, to meet the extensive life of money Pu high quality seedling
It produces and needs.
I.e. the first object of the present invention is to provide a kind of method for tissue culture of money Pu, comprising the following steps:
1) explant sterilizes: carrying out disinfection to money Pu explant；
2) induction and Multiplying culture: the money Pu explant of disinfection is linked into induced medium, and culture is to growing up to stem
Sharp seedling continues the differentiation for cultivating proliferative induction Multiple Buds；
3) squamous subculture: the explant of induction is transferred in proliferated culture medium, after 25~30d of squamous subculture, is turned
Enter subculture 1 time~2 times under identical proliferated culture medium the same terms, the seedling for turning out induction differentiation is transferred to step (4) culture；Not
The Multiple Buds of seedling are transferred in proliferated culture medium, and with continue to cultivate under identical condition of culture described in squamous subculture, to
The seedling for turning out induction differentiation is transferred to step (4) culture；
4) culture of rootage: the seedling of acquisition is inoculated into root media, and culture to plant base portion sends out roots；
5) hardening and transplanting: the seedling that plant base portion has root is subjected to conventional hardening, transplanting is into dress Seedling bag after disinfection
Growth is to get money Pu transplanted seedling.
Preferably, in the method for tissue culture of the money Pu of the invention, the induced medium and Multiplying culture
Base phase is same, and the Fiber differentiation, Multiplying culture are identical as the condition of culture of squamous subculture, culture of rootage.
Preferably, in the method for tissue culture of the money Pu of the invention, the money Pu explant of the step 1)
The stem apex sprouted for money flagroot stem two sides.
Preferably, in the method for tissue culture of the money Pu of the invention, the sterilization method of the step 1) is to use
Washing powder cleans up, and the tap water of flowing rinses 1 hour, then is impregnated 3 hours with 72% streptomycin sulphate.It is used in ultra-clean ring again
After impregnating 30-40s with the alcohol that mass fraction is 75% under border, it is sequentially placed into the mercuric chloride solution that mass fraction is 0.1%
20min is sterilized, tween two drips, 15min is sterilized in sodium hypochlorite mixed liquor, then with aseptic water washing 4~5 times, every time 1~
2min；Wherein the sodium hypochlorite mixed liquor is that polysorbas20 is added by liquor natrii hypochloritis to mix, liquor natrii hypochloritis
Proportion with polysorbas20 is: 2 drop polysorbas20s being added in the liquor natrii hypochloritis 100ml that mass fraction is 2%.
Preferably, in the method for tissue culture of the money Pu of the invention, the induced medium, proliferated culture medium
Formula are as follows:
MS basic culture solution
Preferably, in the method for tissue culture of the money Pu of the invention, the Fiber differentiation, Multiplying culture with after
Feeding, culture of rootage condition of culture of being commissioned to train be in temperature be 25 DEG C ± 2 DEG C, intensity of illumination is 1800lx~2000lx, when illumination
Between be 10~12h/d；The Fiber differentiation, culture of rootage condition be temperature be 25 DEG C ± 2 DEG C, intensity of illumination 2000lx,
Light application time is 10~12h/d.
Preferably, in the method for tissue culture of the money Pu of the invention, root media in the step 4)
Formula is
MS basic culture solution
Therefore, the present invention also provides the culture mediums of the tissue cultures for money Pu, including state induced medium, proliferation
Culture medium and root media.
Preferably, in the culture medium of the tissue cultures for money Pu of the invention, the induced medium, Multiplying culture
The formula of base are as follows:
MS basic culture solution
The formula of the root media is
MS basic culture solution
Compared with prior art, the present invention the invention has the following advantages that
1, the mating system of the special culture media of money Pu breeding of the present invention and money Pu, is configured with particularly suitable
The special culture media and condition of culture and method of the tissue culture breeding of money Pu, using the special culture media and method reproduction speed
Fastly, 25~30 days are a cycle, and a cycle breeding rate is up to 4~5 times；Culture of rootage 25~30 days or so, every plant of plant
Strain can grow 5~7 roots, Miao Jianzhuan, well developed root system, conducive to growing after bottle outlet.
2, present invention firstly provides the technical solutions to key link in entire money Pu seeling industry techniqueflow, together
Step carries out bud induction and Multiplying culture, and subculture medium is induced with bud and proliferated culture medium is identical, and condition of culture is essentially identical, both
The good culture program in turn simplifying tissue culture technology is achieved the effect that, cost and production time is greatly saved, has also solved often
The seedling parental source for advising breeding of method production is complicated, and the unstable problem of seedling quality, the method for the present invention can make seed and seedling traits
Stablize, source of seedling is single, and appropriate criteria, industrial massive production provide the good seed of unified standard for market.
3, the sterile rootage seedling direct transplantation of the method for the present invention production, which enters in Seedling bag, manages, and eliminates tissue-cultured seedling nutrition
The link of alms bowl transplanting, the seedling of transplant survival are readily transported, and transplanted seedling is adaptable, can directly plant after plucking Seedling bag.
4, whole year production can be realized in mating system of the present invention in culturing room, has not only saved land resource, but also improve
Economic benefit overcomes the difficult point that money Pu seedling can not be produced in the anniversary.
Figure of description
Fig. 1 is the disinfection of money Pu explant and induction outside drawing in one embodiment of the present of invention；
Fig. 2 is the money Pu squamous subculture figure in one embodiment of the present of invention；
Fig. 3 is the money Pu culture of rootage figure in one embodiment of the present of invention；
Fig. 4 is money Pu hardening and transplanting effect picture in one embodiment of the present of invention.
Specific embodiment
Below in conjunction with the embodiment in the present invention, the technical solution in the present invention is clearly and completely described.It is aobvious
So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
Embodiment 1
(1) as shown in Figure 1, the stem apex for taking the money flagroot stem two sides of health to sprout is cleaned with washing powder for explant and done
Only, the tap water of flowing rinses 1 hour, then is impregnated 3 hours with 72% streptomycin sulphate.It is used under super-clean environment again with quality point
After number impregnates 40s for 75% alcohol, it is sequentially placed into the mercuric chloride solution that mass fraction is 0.1% and sterilizes 20min, tween two
It drips, 15min is sterilized in sodium hypochlorite mixed liquor, tween two drips, then with aseptic water washing 4~5 times, every time 1~2min；Wherein institute
The sodium hypochlorite mixed liquor stated is that polysorbas20 is added by liquor natrii hypochloritis to mix, and liquor natrii hypochloritis and polysorbas20 are matched
Than being: 2 drop polysorbas20s being added in the liquor natrii hypochloritis 100ml that mass fraction is 2%.
(2) induction and Multiplying culture: as shown in Fig. 2, the nothing of the sprouting of the side strain for the money flagroot stem that step (1) is obtained
The access of bacterium stem apex fills in the culture bottle of following proliferative induction culture medium:
MS basic culture solution
It is cultivated 20 days, intensity of illumination 2000lx under the conditions of temperature is 25 DEG C, under conditions of light application time is 12h/d,
Culture continues the differentiation for cultivating proliferative induction Multiple Buds to stem sharp is grown up to；
(3) squamous subculture: the bud of step (2) induction is transferred to fill and is trained with proliferative induction described in step (2)
It is 2000lx in intensity of illumination, temperature is 25 DEG C, illumination in the culture bottle for supporting the identical new proliferative induction culture medium of based formulas
Under conditions of time is 12h/d, after squamous subculture 30 days, then it is transferred to and fills and proliferative induction culture medium described in step (2)
Be formulated in the culture bottle of identical new proliferative induction culture medium, and under identical condition of culture described in squamous subculture after
It is commissioned to train feeding 30 days, repeatedly, when being proliferated the quantity that radix reaches required, the seedling for turning out induction differentiation is transferred to step
(4) it cultivates；Depending on the quantity for the tissue-cultured seedling that the number of squamous subculture can according to need, can squamous subculture it is primary, can also
With squamous subculture 2 times or more.
The Multiple Buds of non-seedling, which are transferred to, fills identical with proliferative induction culture medium prescription described in step (2) new lure
Lead in the culture bottle of proliferated culture medium, and with continue to cultivate under identical condition of culture described in squamous subculture, after seedling,
The seedling turned out is transferred to step (4) culture；
(4) root media: the seedling that step (3) obtain is inoculated into the culture bottle for filling following root media:
MS basic culture solution
As shown in figure 3, intensity of illumination be 2000lx, temperature be 25 DEG C, light application time be 12h/d under conditions of cultivate to
Plant base portion sends out roots；
(5) as shown in figure 4, the culture bottle with root is placed in progress in greenhouse conventional hardening 5 days by step (4) plant,
The seedling in culture bottle is taken out again, routinely cleans the culture medium on seedling, is put into the carbendazim solution that mass fraction is 0.1%
After sterilizing 2min, into the Seedling bag equipped with following matrix, the matrix is mixed by peat, perlite and vermiculite for transplanting,
The mass ratio of peat, perlite and vermiculite are as follows: peat: perlite: vermiculite=7:2:1；In sprinkling irrigation water spray, shading and conventional fertilizer application
Under management condition, to get money Pu transplanted seedling after growth 35 days.
Embodiment 2
(1) remaining measure in addition to following measures are different of embodiment 2 is same as Example 1, repeats no more.
The stem apex for taking the money flagroot stem two sides of health to sprout is explant, and it is 75% that mass fraction is used under super-clean environment
Alcohol impregnate 30s after, be sequentially placed into the mercuric chloride solution that mass fraction is 0.1% and sterilize 18min, tween two drips, secondary chlorine
16min is sterilized in sour sodium mixed liquor, tween two drips.
(2) induction and Multiplying culture: the sterile stem apex of the sprouting of the side strain for the money flagroot stem that step (1) is obtained accesses
In the culture bottle for filling following proliferative induction culture medium:
MS basic culture solution
It is cultivated 15 days, intensity of illumination 1800lx under the conditions of temperature is 23 DEG C, under conditions of light application time is 10h/d,
Culture continues the differentiation for cultivating proliferative induction Multiple Buds to stem sharp is grown up to；
(3) squamous subculture: the bud of step (2) induction is transferred to fill and is trained with proliferative induction described in step (2)
It is 1800lx in intensity of illumination, temperature is 23 DEG C, illumination in the culture bottle for supporting the identical new proliferative induction culture medium of based formulas
Under conditions of time is 10h/d, after squamous subculture 25 days, then it is transferred to and fills and proliferative induction culture medium described in step (2)
Be formulated in the culture bottle of identical new proliferative induction culture medium, and under identical condition of culture described in squamous subculture after
It is commissioned to train feeding 25 days, repeatedly, when being proliferated the quantity that radix reaches required, the seedling for turning out induction differentiation is transferred to step
(4) it cultivates；The Multiple Buds of non-seedling, which are transferred to, fills identical with proliferative induction culture medium prescription described in step (2) new lure
Lead in the culture bottle of proliferated culture medium, and with continue to cultivate under identical condition of culture described in squamous subculture, after seedling,
The seedling turned out is transferred to step (4) culture；
(4) root media: the seedling that step (3) obtain is inoculated into the culture bottle for filling following root media:
MS basic culture solution
It is 1800lx in intensity of illumination, temperature is 23 DEG C, and culture is long to plant base portion under conditions of light application time is 10h/d
Root out；
(5) by step (4) plant, the culture bottle with root is placed in greenhouse and carries out conventional hardening 3 days, then by culture bottle
Interior seedling takes out, and routinely cleans the culture medium on seedling, is put into the carbendazim solution that mass fraction is 0.2% and sterilizes 1min
Afterwards, it transplants into the Seedling bag equipped with following matrix, the matrix is mixed by peat and perlite, peat, detritus soil, treasure
The mass ratio of Zhu Yan and vermiculite are as follows: peat: perlite=7:3:1:0.5, in sprinkling irrigation water spray, shading and conventional fertilizer application management condition
Under, to get money Pu transplanted seedling after growth 40 days.
The above various embodiments reproduction speed is fast, is within 25~30 days a breeding cycle, a cycle breeding rate up to 4~
5 times；Culture of rootage 25~30 days or so, every plant of plant can grow 5~7 roots, Miao Jianzhuan, and well developed root system is conducive to
It is grown after bottle
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (6)
1. a kind of method for tissue culture of money Pu, comprising the following steps:
1) explant sterilizes: carrying out disinfection to money Pu explant；
2) induction and Multiplying culture: the money Pu explant of disinfection is linked into induced medium, culture to growing up to stem sharp,
Continue the differentiation of culture proliferative induction Multiple Buds；
3) squamous subculture: the explant of induction is transferred in proliferated culture medium, after 25~30d of squamous subculture, is transferred to phase
With subculture 1 time~2 times under proliferated culture medium the same terms, the seedling for turning out induction differentiation is transferred to step (4) culture；Non- seedling
Multiple Buds be transferred in proliferated culture medium, and with continue to cultivate under identical condition of culture described in squamous subculture, wait cultivate
The seedling of differentiation is induced to be transferred to step (4) culture out；
4) culture of rootage: the seedling of acquisition is inoculated into root media, and culture to plant base portion sends out roots；
5) hardening and transplanting: the seedling that plant base portion has root is subjected to conventional hardening, transplanting is grown into Seedling bag after disinfection, i.e.,
Obtain money Pu transplanted seedling；
The money Pu explant of the step 1) is the stem apex that money flagroot stem two sides are sprouted；
The formula of the induced medium, proliferated culture medium are as follows:
MS basic culture solution
The formula of root media is in the step 4)
MS basic culture solution
2. the method for tissue culture of money Pu according to claim 1, which is characterized in that the induced medium and proliferation
Culture medium is identical, and the Fiber differentiation, Multiplying culture are identical as the condition of culture of squamous subculture, culture of rootage.
3. the method for tissue culture of money Pu according to claim 1, which is characterized in that the sterilization method of the step 1)
To be cleaned up with washing powder, the tap water of flowing is rinsed 1 hour, then is impregnated 3 hours with 72% streptomycin sulphate；Again ultra-clean
After impregnating 30-40s with the alcohol that mass fraction is 75% under environment, it is sequentially placed into the mercuric chloride solution that mass fraction is 0.1%
Middle disinfection 20min is added in the sodium hypochlorite mixed liquor of two drop tweens and sterilizes 15min, then with aseptic water washing 4~5 times, every time
1~2min；Wherein the sodium hypochlorite mixed liquor is that polysorbas20 is added by liquor natrii hypochloritis to mix, and sodium hypochlorite is molten
The proportion of liquid and polysorbas20 is: 2 drop polysorbas20s being added in the liquor natrii hypochloritis 100ml that mass fraction is 2%.
4. the method for tissue culture of money Pu according to claim 1, which is characterized in that the Fiber differentiation, proliferation training
It supports with the condition of culture of squamous subculture, culture of rootage to be 25 DEG C ± 2 DEG C in temperature, intensity of illumination is 1800lx~2000lx,
Light application time is 10~12h/d.
5. the method for tissue culture of money Pu according to claim 4, which is characterized in that the Fiber differentiation, training of taking root
Feeding condition is that temperature is 25 DEG C ± 2 DEG C, intensity of illumination 2000lx, and light application time is 10~12h/d.
6. a kind of culture medium of the tissue cultures for money Pu, including induced medium, proliferated culture medium and root media；
The formula of the induced medium, proliferated culture medium are as follows:
MS basic culture solution
The formula of the root media is
MS basic culture solution
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108849446A (en) * | 2018-05-04 | 2018-11-23 | 南通大学 | A kind of water planting mating system of money Pu |
| CN112106656A (en) * | 2020-09-24 | 2020-12-22 | 安徽农业大学 | Direct organ generation type regeneration method of rhizoma acori graminei |
| CN117158315A (en) * | 2023-05-19 | 2023-12-05 | 淮南师范学院 | Tissue culture method of gardenia lobule |
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| CN104221877A (en) * | 2014-10-14 | 2014-12-24 | 南京帝道农业科技有限公司 | Rapid propagation method for culturing tissues of rhizoma acori graminei |
| CN105706937A (en) * | 2016-03-23 | 2016-06-29 | 宁夏皇达生物科技股份有限公司 | Tissue culture formula and culture method for lonicera tatarica |
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| CN104221877A (en) * | 2014-10-14 | 2014-12-24 | 南京帝道农业科技有限公司 | Rapid propagation method for culturing tissues of rhizoma acori graminei |
| CN105706937A (en) * | 2016-03-23 | 2016-06-29 | 宁夏皇达生物科技股份有限公司 | Tissue culture formula and culture method for lonicera tatarica |
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