CN107996398A - A kind of method for tissue culture of matrimony vine - Google Patents
A kind of method for tissue culture of matrimony vine Download PDFInfo
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- CN107996398A CN107996398A CN201610972990.4A CN201610972990A CN107996398A CN 107996398 A CN107996398 A CN 107996398A CN 201610972990 A CN201610972990 A CN 201610972990A CN 107996398 A CN107996398 A CN 107996398A
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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)
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Abstract
The present invention relates to technical field of tissue culture, more particularly to a kind of method for tissue culture of matrimony vine.The method for tissue culture includes：Matrimony vine tissue is inoculated in inducing culture, first light culture 7~9 days, then illumination cultivation 30~40 days, then the matrimony vine seedling induced is transferred and carries out culture of rootage into root media, obtains tissue culture of Lycium barbarum seedling；Inducing culture includes 6 BA (6 benzyl aminoadenine), NAA (methyl α-naphthyl acetate) and MS culture mediums.Matrimony vine method for tissue culture using the present invention is substantially shorter the seedling time, can significantly improve the growth coefficient of matrimony vine tissue.Meanwhile using the method for the present invention evoked callus, culture medium is same culture medium used by evoked callus is cultivated with differentiation, directly break up Germination And Seedling after inducing few callus, that is forming seedling through one step culture, simplifies operation, cost and manpower is greatly saved.
Description
Technical field
The present invention relates to technical field of tissue culture, more particularly to a kind of method for tissue culture of matrimony vine.
Background technology
Matrimony vine is the multi-branched shrub plant of Solanaceae Lycium, and matrimony vine whole body is precious, can not only be used for medicinal plant, can also make
For food plant, it may also be used for gardens, its is widely used, and economic value is higher.
The medical value of matrimony vine：Fruit (Chinese medicine claims the fruit of Chinese wolfberry), nature and flavor are sweet, flat, have effects that nourishing the liver, nourshing kidney, moistening lung；
Root skin (Chinese medicine claims the root bark of Chinese wolf-berry), there is the effectiveness of antipyretic cough-relieving；It is wolfberry leaf bitter, sweet, it is cool in nature, there is qi-restoratives strengthening the essence, heat-clearing improving eyesight
The effect of.
The forestry value of matrimony vine：Since matrimony vine is drought-resistant, sand ground can be grown in, thus can as the shrub of water and soil conservation,
And due to its saline-alkali tolerant, become salt-soda soil and open tree pioneer.
The ornamental value of matrimony vine：Lycium barbarum is tree-like graceful, and leaf is emerald green, spends pale purple, fruit ruby, is that good potted landscape is seen
Plant is appreciated, there is now part matrimony vine appreciation and cultivation, but due to its drought-enduring intolerant to waterlogging that resists cold, so in the rainy more flood areas in Jiangnan very
Hardly possible plantation lycium barbarum.
The matrimony vine edible value of matrimony vine：Tender leaf can make vegetables, and on Guangdong, Guangxi and other places, Lycium chinense is very popular,
Lycium chinense can be bought in food market, but south is essentially Chinese matrimony vine, without lycium barbarum.In the Northwests such as Ningxia, make
It is less to make vegetables with matrimony vine tender leaf.The fruit of Chinese wolfberry is classified as " medicine-food two-purpose " kind by the Ministry of Public Health, and the fruit of Chinese wolfberry can be processed into various foods
Product, beverage, health liquor, health products etc..Matrimony vine is also frequently added in Baoshang or cook congee when.Seed oil can lubricating oil processed
Or edible oil, also it is processed into health products, Chinese wolfberry fruit oil.
Due to the various health-care efficacies that matrimony vine has, pursued year by year for people, its fancy price makes many people carry out
Destructive mad picking, wild resource is by considerable degree of destruction.Therefore, it is necessary to breed substantial amounts of matrimony vine kind
Seedling.But matrimony vine conventional seedbed system percentage of seedgermination is very low.Tissue cultures are by the in vitro of life under the aseptic condition artificially created
Organ (such as root, stem, leaf, stem section, protoplast), tissue or cell are placed in culture medium, and are placed in suitable environment, are carried out
It is continuous to cultivate to obtain cell, tissue or the technology of individual.This technology can obtain substantial amounts of regeneration plant in a short time,
It is widely used in agricultural and biology, the research of medicine.
Traditional matrimony vine tissue cultures mode is divided into two steps：1. evoked callus；2. callus is forwarded to point again
Change culture medium seedling differentiation.The tradition training method light culture 8 days, optical culture form callus in 25 days, and cultivation cycle is longer；
It is only 4.68 and the growth coefficient of traditional tissue cultures mode is relatively low.Accordingly, it is desirable to provide when one kind can shorten seedling
Between, improve growth coefficient matrimony vine method for tissue culture.
The content of the invention
In view of this, the present invention provides a kind of method for tissue culture of matrimony vine.The method for tissue culture is substantially shorter
The seedling time, improves growth coefficient.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The present invention provides a kind of method for tissue culture of matrimony vine, including：Matrimony vine tissue is inoculated in inducing culture,
First light culture 7~9 days, then illumination cultivation 30~40 days, then the matrimony vine seedling induced is transferred given birth into root media
Root culture, obtains tissue culture of Lycium barbarum seedling；Inducing culture includes 6-BA (6- benzyls aminoadenine), NAA (methyl α-naphthyl acetate), agar, sugarcane
Sugar and MS culture mediums.
Matrimony vine method for tissue culture using the present invention is substantially shorter the seedling time, can significantly improve the increasing of matrimony vine tissue
Grow coefficient.Meanwhile using the method for the present invention evoked callus, culture medium is used by evoked callus is cultivated with differentiation
Same culture medium, directly breaks up Germination And Seedling, i.e. forming seedling through one step culture after inducing few callus, simplifies operation, save significantly
Cost and manpower are saved.
Preferably, concentration of the 6-BA in inducing culture is 1.0~2.0mg/L.
Preferably, concentration of the 6-BA in inducing culture is 1.5mg/L.
Preferably, concentration of the NAA in inducing culture is 0.2~0.4mg/L.
Preferably, concentration of the NAA in inducing culture is 0.3mg/L.
Preferably, concentration of the agar in inducing culture is 4.5~6g/L.
In embodiment provided by the invention, concentration of the agar in inducing culture is 5.5g/L.
Preferably, concentration of the sucrose in inducing culture is 30g/L.
In embodiment provided by the invention, during Fiber differentiation, first light culture 8 days, then illumination cultivation 30 days.
Preferably, the pH value of inducing culture is 5.8~6.2.
Preferably, the pH value of inducing culture is 5.8.
Preferably, the periodicity of illumination of illumination cultivation is 14~16h/ days, intensity of illumination 1500Lx, temperature is 24~26
℃。
Preferably, the periodicity of illumination of illumination cultivation is 16h/ days, intensity of illumination 1500Lx, and temperature is 24 DEG C.
Preferably, root media includes IBA, agar, sucrose and 1/2MS culture mediums.
Preferably, concentration of the IBA in root media is 0.4~0.6mg/L.
Preferably, concentration of the IBA in root media is 0.5mg/L.
Preferably, concentration of the agar in root media is 5.5g/L.
Preferably, concentration of the sucrose in root media is 30g/L.
Preferably, the pH value of root media is 5.8~6.5.
Preferably, the pH value of root media is 6.0.
Preferably, the periodicity of illumination of culture of rootage is 14~16h/ days, intensity of illumination 1500Lx, temperature is 24~26
℃。
Preferably, the periodicity of illumination of culture of rootage is 16h/ days, intensity of illumination 1500Lx, and temperature is 24 DEG C.
In embodiment provided by the invention, the time of culture of rootage is 18~22 days.
Preferably, the step of hardening is further included after culture of rootage.
In the present invention, matrimony vine is organized as matrimony vine blade or matrimony vine petiole.
In the present invention, the place that carries out disinfection to matrimony vine tissue is further included before matrimony vine tissue is inoculated in inducing culture
The step of reason.
The present invention provides a kind of method for tissue culture of matrimony vine.The method for tissue culture includes：Matrimony vine is organized to be inoculated with
In inducing culture, first light culture 7~9 days, then illumination cultivation 30~40 days, then the matrimony vine seedling induced is transferred into life
Root culture medium carries out culture of rootage, obtains tissue culture of Lycium barbarum seedling；Inducing culture includes 6-BA (6- benzyls aminoadenine), NAA (naphthalenes
Acetic acid) and MS culture mediums.The present invention at least has one of following advantage：
1st, matrimony vine method for tissue culture using the present invention is substantially shorter the seedling time：Light culture 8 days, optical culture 10 days
After can form a small amount of callus；And traditional matrimony vine method for tissue culture needs light culture 8 days, optical culture ability shape after 25 days
Into callus；
2nd, matrimony vine method for tissue culture using the present invention can significantly improve the growth coefficient of matrimony vine tissue, and growth coefficient exists
Between 5.5~6, the growth coefficient of tradition tissue training method is only 4.68；
3rd, using the method for the present invention evoked callus, culture medium is same used by evoked callus is cultivated with differentiation
One culture medium, directly breaks up Germination And Seedling, i.e. forming seedling through one step culture after inducing few callus, simplifies operation, greatly save
Cost and manpower.
Brief description of the drawings
Fig. 1, which shows, is inoculated with the petiole growing state that 3 days are induced in culture medium Y1；
Fig. 2, which shows, is inoculated with the leaf growth situation that 3 days are induced in culture medium Y1；
Fig. 3 shows light culture illumination cultivation leaf growth situation of 30 days after 8 days；
Fig. 4 shows that the seedling for inducing inducer blade is forwarded to the illumination cultivation bud growing state of 18 days in culture medium H1.
Embodiment
The invention discloses a kind of method for tissue culture of matrimony vine, those skilled in the art can use for reference present disclosure, fit
When modified technique parameter is realized.In particular, all similar substitutions and modifications are for a person skilled in the art
It is it will be apparent that they are considered as being included in the present invention.The present invention method and application by preferred embodiment into
Gone description, related personnel substantially can not depart from present invention, in spirit and scope to method described herein and application
It is modified or suitably changes with combining, realizes and using the technology of the present invention.
Term is explained：
6-BA is 6- benzyl aminoadenines, alias：6-benzyl aminopurine, the basic element of cell division, English common name：6-
Benzylaminopurine, molecular formula：C12H11N5.6-BA has the characteristics that efficient, stable, cheap and easy to use, thus quilt
It is widely used, and the favorite basic element of cell division of person that is tissue cultures.The main function of BA is the formation for promoting bud, can also
Evoked callus occurs.Available for improve tealeaves, tobacco quality and yield, the fresh-keeping and rootless bean sprouts of veterinary antibiotics
Cultivate, hence it is evident that improve the quality of fruit and blade.
Methyl α-naphthyl acetate (1-Naphthylacetic acid), abbreviation NAA, is a kind of organic compound, is that one kind has been soluble in
The colorless solid of solvent.Its structure is substituted for No. 1 position of naphthalene with carboxymethyl.It is the growth in plant growth regulator
Plain analog, is usually used in the root of hair powder or rooting agent of commercialization, is used when plant is using cuttage breeding.It can also be used for planting
Thing tissue cultures.
In embodiments of the present invention, the periodicity of illumination of illumination cultivation and culture of rootage is 16h/ days, and intensity of illumination is
1500Lx, temperature are 24 DEG C.
MS culture mediums and 1/2MS culture mediums are conventional commercial product in the embodiment of the present invention.
Used medium component can be bought by market in the method for tissue culture of matrimony vine provided by the invention.
With reference to embodiment, the present invention is further explained：
Embodiment 1
The sterile tissue-cultured seedling of matrimony vine is chosen, blade is cut into 0.5cm × 0.5cm fritters, faces up and is connected to culture medium Y1 (MS
+ 1.5mg/L 6-BA+0.3mg/L NAA+5.5g/L agar+30g/L sucrose, pH value 5.8) in, petiole is cut into the small of 0.5cm
Section, is inoculated in culture medium Y1 (the MS+1.5mg/L 6-BA+0.3mg/L NAA+5.5g/L agar+30g/L of evoked callus
Sucrose, pH value 5.8) in, first light culture 8 days, then illumination cultivation, illumination cultivation 10 days or so, forms a small amount of callus, and in callus
On have black dot occur, illumination cultivation 30 days or so, the dot of black grows up to the seedling of 2cm or so, then will induce
Given birth in seedling access root media H1 (1/2MS+0.5mg/L IBA+5.5g/L agar+30g/L sucrose, pH value 6.0)
Root culture, is transferred to illumination cultivation 20 days or so in H1, obtains complete tissue-cultured seedling.Have recorded during Fiber differentiation petiole and
The growing state of blade, is shown in Fig. 1~Fig. 4.Wherein, Fig. 1, which shows, is inoculated with the petiole growing state that 3 days are induced in culture medium Y1；Fig. 2
Show and be inoculated with the leaf growth situation that 3 days are induced in culture medium Y1；Fig. 3 shows the light culture illumination cultivation blade of 30 days life after 8 days
Long situation；Fig. 4 shows that the seedling for inducing inducer blade is forwarded to the illumination cultivation bud growing state of 18 days in culture medium H1.
The growth coefficient of matrimony vine tissue cultures is calculated after Fiber differentiation, computational methods are：Growth coefficient=Tip density/inoculation
Number * 100%.
From experimental result, illumination cultivation can form a small amount of callus in 10 days again for first light culture 8 days, and have black
Color bud point occurs.Using the present embodiment method, the growth coefficient of matrimony vine tissue cultures is 6.
Embodiment 2
The sterile tissue-cultured seedling of matrimony vine is chosen, blade is cut into 0.5cm × 0.5cm fritters, faces up and is connected to culture medium Y1 (MS
+ 1.0mg/L 6-BA+0.4mg/L NAA+5.5g/L agar+30g/L sucrose, pH value 5.8) in, petiole is cut into the small of 0.5cm
Section, is inoculated in culture medium Y1 (the MS+1.0mg/L 6-BA+0.4mg/L NAA+5.5g/L agar+30g/L of evoked callus
Sucrose, pH value 5.8) in, first light culture 8 days, then illumination cultivation, illumination cultivation 10 days or so, forms a small amount of callus, and in callus
On have black dot occur, illumination cultivation 30 days or so, the dot of black grows up to the seedling of 2cm or so, then will induce
Given birth in seedling access root media H1 (1/2MS+0.4mg/L IBA+5.5g/L agar+30g/L sucrose, pH value 6.0)
Root culture, is transferred to illumination cultivation 20 days or so in H1, obtains complete tissue-cultured seedling.Have recorded during Fiber differentiation petiole and
The growing state of blade, calculates the growth coefficient of matrimony vine tissue cultures after Fiber differentiation.
From experimental result, illumination cultivation can form a small amount of callus in 10 days again for first light culture 8 days, and have black
Color bud point occurs.Using the present embodiment method, the growth coefficient of matrimony vine tissue cultures is 5.5.
Embodiment 3
The sterile tissue-cultured seedling of matrimony vine is chosen, blade is cut into 0.5cm × 0.5cm fritters, faces up and is connected to culture medium Y1 (MS
+ 2.0mg/L 6-BA+0.2mg/L NAA+5.5g/L agar+30g/L sucrose, pH value 5.8) in, petiole is cut into the small of 0.5cm
Section, is inoculated in culture medium Y1 (the MS+2.0mg/L 6-BA+0.2mg/L NAA+5.5g/L agar+30g/L of evoked callus
Sucrose, pH value 5.8) in, first light culture 8 days, then illumination cultivation, illumination cultivation 10 days or so, forms a small amount of callus, and in callus
On have black dot occur, illumination cultivation 30 days or so, the dot of black grows up to the seedling of 2cm or so, then will induce
Given birth in seedling access root media H1 (1/2MS+0.6mg/L IBA+5.5g/L agar+30g/L sucrose, pH value 5.8)
Root culture, is transferred to illumination cultivation 20 days or so in H1, obtains complete tissue-cultured seedling.Have recorded during Fiber differentiation petiole and
The growing state of blade, calculates the growth coefficient of matrimony vine tissue cultures after Fiber differentiation.
From experimental result, illumination cultivation can form a small amount of callus in 10 days again for first light culture 8 days, and have black
Color bud point occurs.Using the present embodiment method, the growth coefficient of matrimony vine tissue cultures is 5.8.
Comparative example 1
Traditional tissue culture method concrete operations are as follows：
The sterile tissue-cultured seedling of matrimony vine is chosen, blade is cut into 0.5cm × 0.5cm fritters, facing up is connected to callus and lures
Lead in culture medium, first light culture 8 days, then illumination cultivation, illumination cultivation 28 days or so, starts to generate a small amount of callus and starts to break up
Go out bud point, illumination cultivation 35-45 days, bud point starts a large amount of differential growths.
From experimental result, illumination cultivation forms a small amount of callus for 25 days again for first light culture 8 days, and 40 days or so
A large amount of differentiation bud points.Using this comparative example method, the growth coefficient of matrimony vine tissue cultures is 4.68.
According to the experimental result of this comparative example, it can be contracted significantly using the matrimony vine method for tissue culture of the present embodiment 1 to 3
The short seedling time, can significantly improve the growth coefficient of matrimony vine tissue.Meanwhile using the method for the present invention evoked callus, induction
Culture medium be same culture medium used by callus and differentiation culture, only induces directly to break up after few callus and sprouts
Send out seedling, can forming seedling through one step culture, simplify operation, cost and manpower be greatly saved.
The above is only the 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 also should
It is considered as protection scope of the present invention.
Claims (10)
- A kind of 1. method for tissue culture of matrimony vine, it is characterised in that including：Matrimony vine tissue is inoculated in inducing culture, first Light culture 7~9 days, then illumination cultivation 30~40 days, then the matrimony vine seedling induced is transferred take root into root media Culture, obtains tissue culture of Lycium barbarum seedling；The inducing culture includes 6-BA, NAA, agar, sucrose and MS culture mediums.
- 2. method for tissue culture according to claim 1, it is characterised in that concentration of the 6-BA in inducing culture For 1.0~2.0mg/L, concentration of the NAA in inducing culture is 0.2~0.4mg/L.
- 3. method for tissue culture according to claim 1 or 2, it is characterised in that the agar is in inducing culture Concentration is 4.5~6g/L, and concentration of the sucrose in inducing culture is 30g/L.
- 4. method for tissue culture according to any one of claim 1 to 3, it is characterised in that the inducing culture PH value is 5.8~6.2.
- 5. method for tissue culture according to any one of claim 1 to 4, it is characterised in that the light of the illumination cultivation It it is 14~16h/ days, intensity of illumination 1500Lx according to the cycle, temperature is 24~26 DEG C.
- 6. method for tissue culture according to any one of claim 1 to 5, it is characterised in that the root media bag Include IBA, agar, sucrose and 1/2MS culture mediums.
- 7. method for tissue culture according to claim 6, it is characterised in that concentration of the IBA in root media For 0.4~0.6mg/L.
- 8. method for tissue culture according to any one of claim 1 to 7, it is characterised in that the light of the culture of rootage It it is 14~16h/ days, intensity of illumination 1500Lx according to the cycle, temperature is 24~26 DEG C.
- 9. method for tissue culture according to any one of claim 1 to 8, it is characterised in that the culture of rootage when Between be 18~22 days.
- 10. method for tissue culture according to any one of claim 1 to 9, it is characterised in that after the culture of rootage also The step of including hardening.
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| CN201610972990.4A CN107996398A (en) | 2016-10-28 | 2016-10-28 | A kind of method for tissue culture of matrimony vine |
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| CN114766357A (en) * | 2022-04-14 | 2022-07-22 | 中国科学院华南植物园 | Culture medium combination and method for tissue culture and propagation of lycium ruthenicum murr |
| CN116762700A (en) * | 2023-07-17 | 2023-09-19 | 新疆中亚果树产业研究院有限公司 | Rapid propagation method for synchronously developing rooting and hardening of Chinese wolfberry tissue culture seedlings |
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Application publication date: 20180508 |