CN105766646B - A kind of method for tissue culture of vesicle grass - Google Patents
A kind of method for tissue culture of vesicle grass Download PDFInfo
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- CN105766646B CN105766646B CN201610197564.8A CN201610197564A CN105766646B CN 105766646 B CN105766646 B CN 105766646B CN 201610197564 A CN201610197564 A CN 201610197564A CN 105766646 B CN105766646 B CN 105766646B
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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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- Engineering & Computer Science (AREA)
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Abstract
The invention discloses a kind of method for tissue culture of vesicle grass, this method comprises the following steps：Explant sterilizing, the primary Fiber differentiation of vesicle grass bud, shoot proliferation culture, tissue culture seedling rooting and tissue culture transplantation of seedlings.The method of the present invention is used for the tissue cultures of vesicle grass, has the advantages of reproduction speed is fast, inductivity is high and transplanting survival rate is high.The method of the present invention is the modernization development of anaesthetic, and commerial growing, which produces, and utilization vesicle is careless and belongs to other kinds of plant resourceses together provides foundation and reference.
Description
Technical field
The present invention relates to field of plant tissue culture technique, more particularly to a kind of method for tissue culture of vesicle grass.
Background technology
The dry root of vesicle grass system plant of Solanaceae vesicle careless (Physochlaina Physaloides (L.) G.Don), makees
Be anaesthetic " mixed-great day element " first recorded in hide, Mongolia Medicine classical works《Errorless anaesthetic mirror》In,《Recognize medicine Bai Jingjian》Have Deng document
Record, the kind recorded for Drug Standard of Ministry of Public Health of the Peoples Republic of China (anaesthetic fascicle) [cloud unicorn, Song Hongchun, Zhang Hongyu,
Influence [J] National medicine magazine .2008.3 of the different concocting methods of Liu Dongming vesicles grass to acute toxicity and drug effect:57
~58].In existing vesicle grass hyoscyamine assay pertinent literature [Zhang Runxiang .HPLC methods measure mongolian medicine vesicle grass (it is mixed-
It is good Japan-Soviet) in hyoscyamine content China the drug standards [J] .2011.12 (6):419~421], its main component is Tangut Anisodus Radix
[Reinouts the van Haga, R such as alkali, hyoscyamine, hyoscine, new rutin and Cuscohygrine:cuscohygrine,a
normal constituent alkaloid of Atropa belladonna[J].Nature.1954.174:833], Mongolian medicine
Medicine theoretical research shows that " mixed-great day element " has and kill " glutinous ", detumescence, desinsection, analgesia, spasmolysis, establishing-Yang and other effects, for " glutinous " property
Gastritis, Adam's apple, hair disease, worm disease, brain shouting pain, treatment [the herbal anaesthetic volume of State Administration of Traditional Chinese Medicine China of headache, impotence etc.
[M] Science and Technology of Shanghai publishing house .2004.233].Vesicle grass main product in Inner Mongolia Autonomous Region Siklingelei Abaga Banner, exhale Lun Bei
The provinces and regions such as your Ewenki flag and Wulanchabu, Xinjiang in China, Heilungkiang, Hebei are also distributed, and are born in hillside, gully and grass
Ground.Because resource is few, Distribution Area limitation, therefore Tissue culture assays are carried out, for the modernization development of anaesthetic, commerial growing
Produce and utilization vesicle is careless and belongs to other kinds of plant resourceses offer foundations and reference together.
It is many that the material that explant is cultivated is can be used as in tissue cultures, for example, stem segment with axillary bud, root, stem apex etc., flower
The floral organs such as silk, bennet, holder, style, ovary can also be used for explant culture, and [Zhang Yanni influence Plant Tissue Breeding success
Factor [J] north gardening, 2003 (3):132~133；The Tissue culture assays research of the lilies such as Yao Lianfang, Zhou Junguo
[J] Guizhou Agricultural Sciences .1999.27 (3):47~48；The lily genetic transformation embryo callus receptor systems such as Tang Dongqin
Foundation [J] Zhejiang Forestry Colleges .200320 (3):273~276；The tissue cultures of Bai Meifa green hills lily with it is quick numerous
Grow [J] Guizhou Agricultural Sciences .2003.31 (6):25~26；Tissue culture and rapid proliferation [J] of Li Rui Lilium longiflorums is sweet
Respectful forestry science and technology .2003.28 (2):13~14], need to be selected according to the proliferating way and test objective of bud in actual production
Select suitable explant.
Requirement of the different plant tissues to nutritional ingredient is different, the Anther Culture of Cereal, can select the N of high phosphorus6
Culture medium, and the Anther Culture of strawberry is convenient with the White culture mediums ratio of less salt, Verenaceae arbor and teak must be in height
Ability seedling in the MS culture mediums of salt, the Shoot Tip Culture of azalea often [it is economical to apply skill sound with 1/4MS or using 6,7-V culture mediums
Plant extensive quick breeding technology [M] Chemical Industry Press .2009.35].There is presently no carry out tissue to vesicle grass
The report to succeed is cultivated, needs to screen suitable vesicle grass culture medium in actual production.
The content of the invention
In view of this, it is an object of the invention to propose a kind of method for tissue culture of vesicle grass, this method is used for vesicle
The tissue cultures of grass, there is the advantages of reproduction speed is fast, inductivity is high and transplanting survival rate is high.
Based on above-mentioned purpose, a kind of method for tissue culture of vesicle grass provided by the invention, comprise the following steps：
(1) explant sterilizes
It is explant to choose blade of the vesicle grass then on raw, healthy and strong branch, and explant is cut into 5 × 5mm with scalpel
Square, washed with neutral detergent after explant square 10min washes surface blot off, 20min rinsed under running water, filter paper is inhaled
Superclean bench sterilizing is placed in after solid carbon dioxide point, is that 0.1% mercuric chloride carries out disinfection 2~3min of processing with mass fraction, sterilized water punching
Wash 3~5 times；
(2) the primary Fiber differentiation of vesicle grass bud
The explant square to have sterilized is inoculated into primary inducing culture, primary Fiber differentiation 7~15 days, to sprouting
Grow；
(3) shoot proliferation culture
The sprouting of acquisition is inoculated into subculture multiplication medium, shoot proliferation culture 20~26 days, to Elongation of adventitious bud
To 2~3cm；
(4) tissue culture seedling rooting
Adventitious bud is cut from base portion, is inoculated on root media, is cultivated 18~21 days, extend to 2 to young root~
3cm；
(5) tissue culture transplantation of seedlings
Young root is extended into the tissue-cultured seedling to 2~3cm at room temperature after hardening 2~3 days, removes the sealed membrane of blake bottle,
Addition 8mL water continues hardening 3~5 days in blake bottle, then takes out tissue-cultured seedling, the unnecessary culture medium adhered on root is washed away, with dilution
10 times of carbendazim solution soaks 8~10min, is transplanted in the sterile vegetative soil that water content is 68%~71%, is cladded with plastics
Film moisturizing, crop field is transplanted to after 14~15 days, you can.
Preferably, the condition of primary Fiber differentiation is described in step (2)：It is 20~28 DEG C to cultivate room temperature, illumination 12-
14h, intensity of illumination 2000-2500Lx.
Preferably, primary inducing culture described in step (2) is MS minimal mediums；The primary Fiber differentiation when
Between be 7~10 days.
Preferably, the condition of shoot proliferation culture is described in step (3)：It is 20~28 DEG C to cultivate room temperature, illumination 12-
14h, intensity of illumination 2000-2500Lx.
Preferably, subculture multiplication medium described in step (3) is MS minimal mediums；The shoot proliferation culture when
Between be 21~23 days.
Preferably, the condition of tissue culture seedling rooting is described in step (4)：It is 20~28 DEG C to cultivate room temperature, illumination 12-
14h, intensity of illumination 2000-2500Lx.
Preferably, root media described in step (4) is to add 0~0.5mg/L IBA in MS minimal mediums to obtain
Arrive.
Preferably, the root media adds 0.02~0.03mg/L IBA in MS minimal mediums and obtained.
Preferably, the soil of sterile vegetative described in step (5) includes humus and perlite, wherein humus and perlite
Mass ratio is 1~2.
From the above it can be seen that the method for tissue culture of vesicle grass provided by the invention, method for tissue culture choosing
It is explant that is suitable and easily obtaining with vesicle blade of grass piece, its differentiation capability is strong, not tight to culture medium requirement, pollution rate is low, increases
It is fast to grow speed；Explant material is that 0.1% mercuric chloride carries out disinfection 2~3min of processing with mass fraction, inductivity up to 100% and
It is pollution-free；Widely used MS minimal mediums are selected, it need not be added in explant culture and shoot proliferation culture appoints
What hormone, you can obtain preferable cultivation effect, can effectively reduce cost；And when the young root of vesicle grass grows to 2~3cm,
Transplanting survival rate is up to 96.7%.
Brief description of the drawings
Fig. 1 is the tissue cultures figure of vesicle grass；When wherein A figures are shoot proliferation culture 22 days, adventitious buds differentiation situation map；
B figures are the situation map of taking root that adventitious bud is cultivated 18~21 days in the root media containing 0.025mg/L IBA.
Embodiment
For the object, technical solutions and advantages of the present invention are more clearly understood, below in conjunction with specific embodiment, and reference
Accompanying drawing, the present invention is described in more detail.
Embodiment 1
The method for tissue culture of vesicle grass, this method comprise the following steps：
The foundation of 1.1 explants
Material to be tested is that Inner Mongol farming and animal husbandry academy of sciences grass cultivation center is adopted in Inner Mongolia Autonomous Region Xilinguole League Ah bar's loud, high-pitched sound
The vesicle grass plant of flag, plant in seedling alms bowl, the blade in selection life then, healthy and strong branch is as explant.
1.2 test method
1.2.1 explant sterilizes
Blade of the selection then on raw, healthy and strong branch is explant, and 5 × 5mm square is cut into scalpel, is washed with neutrality
Wash agent washing explant square 10min wash surface blot off after, 20min is rinsed under running water, is placed in after filter paper suck dry moisture super
Net workbench sterilizing, it is that 0.1% mercuric chloride carries out disinfection processing with mass fraction, processing time is shown in Table 1, aseptic water washing 3~5
It is secondary, then it is inoculated in MS culture mediums, statistics, the culture effect of more different sterilization times is observed after 20 days.
Table 1 disinfects mode
1.2.2MS prepared by minimal medium
MS minimal mediums preparation method used in the present invention is as follows：MS culture medium powders are purchased from Qingdao GaoKeYuan Hai Bo
Bioisystech Co., Ltd, after the MS culture medium powders 4.74g of purchase is dissolved in into 1000mL sterilized waters, 20g/L sucrose is added,
7g/L agar, regulation pH value are 5.8, standby in 121 DEG C of autoclaving 30min.
1.2.3 condition of culture
It is 25 ± 1 DEG C, illumination 12-14h, intensity of illumination 2000-2500Lx to cultivate room temperature.
1.2.4 the primary Fiber differentiation of vesicle grass bud
The present invention does not add any hormone, the explant square to have sterilized is inoculated in into MS bases using MS as minimal medium
After basal culture medium, according to 1.2.3 condition of culture primary Fiber differentiation 7~15 days, grown to sprouting；Seen in time in incubation
Statistics is examined, records the situation of bud differentiation on culture medium.
1.2.5 shoot proliferation culture
Using MS as minimal medium, the sterile bud that Initial culture obtains is inoculated on MS minimal mediums, according to 1.2.3
Condition of culture shoot proliferation culture, culture 20~26 days after, observe and count adventitious bud proliferation situation.
1.2.6 tissue culture seedling rooting
Adventitious bud length is cut to during 2~3cm from base portion, is inoculated in the generation of inducing adventitious root on root media, is taken root
Culture medium adds plant growth regulator in MS minimal mediums and is prepared, plant growth regulator selection IBA (0,
0.025,0.05,0.1,0.2,0.3,0.4,0.5mg/L) 7 concentration gradients, sucrose concentration is 20g/L in root media, fine jade
Lipid concentration is 7g/L, pH value 5.8, standby in 121 DEG C of autoclaving 30min.Each processing inoculation 10, is repeated 3 times, presses
Condition of culture culture according to 1.2.3 counts rooting rate after 18~21 days, filters out optimal root media.
1.2.7 tissue culture transplantation of seedlings
When the young root of vesicle grass grows to 2~3cm, blake bottle is removed into illumination box, at room temperature hardening 2~3 days
Afterwards, the sealed membrane of blake bottle is removed, 8mL water is added in blake bottle and continues hardening 4~5 days, tissue-cultured seedling is then taken out, carefully washes
The unnecessary culture medium adhered on root is removed, is soaked in 8~10min in the carbendazim solution of 10 times of dilution, is transferred to water content as 68%
In~71% sterile vegetative native (humus/perlite (m/m)=1~2), plastic sheeting moisturizing is cladded with, is moved after 14~15 days
Plant to crop field, transplanting survival rate is counted after 30 days.
2 results are with discussing
Influence of the 2.1 different sterilization times to vesicle grass explant
Most basic in Plant Tissue Breeding, most important link is the culture of aseptic explant, is largely successfully cultivated sterile
Explant can effectively accelerate test process.The present invention have studied influence of the different sterilization times to vesicle grass cultured in vitro effect,
2 are the results are shown in Table, is that 0.1% mercuric chloride carries out disinfection processing 2~3min best results with mass fraction as can be seen from Table 2,7~
It can sprout within 10 days, and it is pollution-free.With the extension of processing time, bud induction rate declines, time lengthening of sprouting, bud induction rate with
Inverse correlation relation is presented in the budding time.
The difference of table 2 disinfects influence of the mode to vesicle grass explant
Need to select suitable explant according to the proliferating way and test objective of bud in actual production, with regard to vesicle grass
For, its spring turns green morning, and the middle ten days and the last ten days in April can bloom, and blade is explant that is suitable and easily obtaining, and its differentiation capability is strong,
It is not tight to culture medium requirement, pollution rate is low, growth rate is fast.
The sterilization of explant material is to influence the important step of tissue cultures process below, and disinfecting time is long, material
Material be poisoned effect it is larger, inductivity is influenceed after inoculation, easily causes lopsided seedling；Disinfecting time is too short, and sterilization is not thorough, easily causes
Pollution；Suitable disinfecting time is screened, is advantageous to the recovery of material after sterilization, improves the bud induction rate of explant after inoculation.Bubble
Capsule grass is that 0.1% mercuric chloride carries out disinfection processing 2~3min best results with mass fraction, and inductivity is up to 100% and pollution-free.
2.2 shoot proliferation cultures
The propagation that squamous subculture is used for material is fast numerous and preserve, and is the critical process that Plant Tissue Breeding is quickly bred.Root
According to vesicle grass explant culture experiment result, in the case where not adding any hormone, explant is on MS minimal mediums
Can quick differentiation and proliferation, therefore, on the premise of cost is not increased, squamous subculture still selects MS minimal mediums, and inductivity can
Up to 100%, incubation time 22 days is optimal, sees Figure 1A, and adventitious bud growing way is preferable.More than 22 days, there is contamination phenomenon in portion of material,
Pollution rate is up to 6.67%, and leaf color turns yellow, and growing way is deteriorated.
For the tissue cultures of medicinal economic plant, it may be considered that selection MS minimal mediums, vesicle grass is as illiteracy
Medicine plant, widely used MS minimal mediums are selected effectively to reduce cost, it is in explant culture and shoot proliferation culture
In need not add any hormone, you can obtain preferable cultivation effect.
2.3 tissue-cultured seedling is taken root
Committed step before tissue culture transplantation of seedlings is to take root, the speed of rootage duration and the quality of root gesture, determines entering for transplanting
Degree and survival rate.The influence that hormon concentration is taken root to vesicle grass in the present invention is shown in Table 3.IBA concentration is using 0.025mg/L as most
Good root media, showing as taking root, quantity is more, and robust growth, rooting rate is shown in Figure 1B up to 100%.With IBA concentration liters
Height, quantity of taking root gradually decrease, and when IBA concentration reaches 0.5mg/L, tissue-cultured seedling is without phenomenon of taking root, and plant leaf turns white, and
Budlet growth is slower.
The influence that the different culture media of table 3 is induced vesicle grass roots
2.4 hardenings and transplanting
Whether the quality and function of tissue-cultured seedling root long gesture perfect, and determine transplanting survival rate.According to test statistics, when vesicle grass
Young root when growing to 2~3cm, transplanting survival rate highest, up to 96.7%, easy adhesion, fracture, shadow when long coring is transplanted
Absorption of the root system to nutrition is rung, reduces transplanting survival rate, when root long is shorter than 1cm, fibrous root quantity is few, and root function is unsound, shadow
Nutrient absorption is rung, is unfavorable for surviving.
Those of ordinary skills in the art should understand that：The discussion of any of the above embodiment is exemplary only, not
It is intended to imply that the scope of the present disclosure (including claim) is limited to these examples；Under the thinking of the present invention, above example
Or can also be combined between the technical characteristic in different embodiments, and the different aspect of the present invention as described above be present
Many other changes, in order to it is concise they do not provided in details.Therefore, within the spirit and principles of the invention,
Any omission for being made, modification, equivalent substitution, improvement etc., should be included in the scope of the protection.
Claims (8)
1. a kind of method for tissue culture of vesicle grass, it is characterised in that comprise the following steps：
(1) explant sterilizes
It is explant to choose blade of the vesicle grass then on raw, healthy and strong branch, and explant is cut into 5 × 5mm with scalpel2 Side
Block, washed with neutral detergent after explant square 10min washes surface blot off, 20min is rinsed under running water, filter paper blots water
Superclean bench sterilizing is placed in after point, is that 0.1% mercuric chloride carries out disinfection 2~3min of processing with mass fraction, aseptic water washing 3~
5 times；
(2) the primary Fiber differentiation of vesicle grass bud
The explant square to have sterilized is inoculated into primary inducing culture, primary Fiber differentiation 7~15 days, grown to sprouting
Go out；The primary inducing culture is MS minimal mediums；
(3) shoot proliferation culture
The sprouting of acquisition is inoculated into subculture multiplication medium, shoot proliferation culture 20~26 days, to Elongation of adventitious bud to 2~
3cm；The subculture multiplication medium is MS minimal mediums；
(4) tissue culture seedling rooting
Adventitious bud is cut from base portion, is inoculated on root media, cultivates 18~21 days, is extended to young root to 2~3cm；Institute
State root media 0~0.5mg/L IBA are added in MS minimal mediums and obtain；
(5) tissue culture transplantation of seedlings
Young root is extended into the tissue-cultured seedling to 2~3cm at room temperature after hardening 2~3 days, the sealed membrane of blake bottle is removed, is cultivating
Addition 8mL water continues hardening 3~5 days in bottle, then takes out tissue-cultured seedling, washes away the unnecessary culture medium adhered on root, with dilution 10
Carbendazim solution again soaks 8~10min, is transplanted in the sterile vegetative soil that water content is 68%~71%, it is thin to be cladded with plastics
Film moisturizing, crop field is transplanted to after 14~15 days, you can.
2. the method for tissue culture of vesicle grass according to claim 1, it is characterised in that primary lures described in step (2)
The condition for leading culture is：It is 20~28 DEG C, illumination 12-14h, intensity of illumination 2000-2500Lx to cultivate room temperature.
3. the method for tissue culture of vesicle grass according to claim 1, it is characterised in that primary lures described in step (2)
The time for leading culture is 7~10 days.
4. the method for tissue culture of vesicle grass according to claim 1, it is characterised in that subculture described in step (3) increases
The condition for growing culture is：It is 20~28 DEG C, illumination 12-14h, intensity of illumination 2000-2500Lx to cultivate room temperature.
5. the method for tissue culture of vesicle grass according to claim 1, it is characterised in that subculture described in step (3) increases
The time for growing culture is 21~23 days.
6. the method for tissue culture of vesicle grass according to claim 1, it is characterised in that tissue-cultured seedling described in step (4)
The condition taken root is：It is 20~28 DEG C, illumination 12-14h, intensity of illumination 2000-2500Lx to cultivate room temperature.
7. the method for tissue culture of vesicle grass according to claim 1, it is characterised in that the root media is in MS
Add what 0.02~0.03mg/L IBA were obtained in minimal medium.
8. the method for tissue culture of vesicle grass according to claim 1, it is characterised in that sterile battalion described in step (5)
Support soil and include humus and perlite, the wherein mass ratio of humus and perlite is 1~2.
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