CN106857239A - A kind of efficient Wheat Anther Culture Callus method - Google Patents
A kind of efficient Wheat Anther Culture Callus method Download PDFInfo
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- CN106857239A CN106857239A CN201510924268.9A CN201510924268A CN106857239A CN 106857239 A CN106857239 A CN 106857239A CN 201510924268 A CN201510924268 A CN 201510924268A CN 106857239 A CN106857239 A CN 106857239A
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- wheat
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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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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
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Abstract
It is as follows the step of the colored culture method the invention discloses a kind of efficient Wheat Anther Culture Callus method：（1）Take fringe and pretreatment；（2）The pretreated wheat head is sterilized；（3）The wheat head is aseptically carried out into mechanical dissection, the wheat head of chopping is poured into centrifuge tube and shaken after sealing；（4）Centrifuge tube after preliminary concussion is put into high speed freezing centrifuge, centrifugation obtains in vitro flower pesticide；（5）In vitro flower pesticide is inoculated with and Fiber differentiation is gone out anther callus；（6）Differentiation, subculture and transplanting, complete the flower training process.The present invention is crushed by way of taking machine cuts to wheat anther, then fragment is separated using centrifugal force, pure wheat anther is extracted, and by optimizing centrifugation medium and using sterile working, get the wheat anther that flushes and further flower train successfully, obtain healthy and strong bloom control；Flower training operating efficiency is improved while improving operational ton.
Description
Technical field
The present invention relates to a kind of efficient Wheat Anther Culture Callus method, belong to technical field of agriculture science.
Background technology
Wheat is a kind of grass in extensive plantation all over the world, is the general designation of wheat platymiscium, earliest origin
In the crescent fertile soil area in the Middle East.Wheat is one of three big cereal, and the overwhelming majority only there are about 1/6th as feeding as edible
Material is used.Wheat is also Chinese second largest cereal crops as the cereal crops of total output second in the world, in compatriots' food
Play key player.According to statistics, in the grain ration total quantity consumed of China, wheat accounts for 43% or so.In recent years, people from world
Mouth increases and people's living standard is improved constantly, grain demand sustainable growth, and at the same time, arable land gradually decreases, water resource
Not enough, wheat diseases take place frequently, the deterioration of the ecological environment, natural calamity take place frequently, and feed-use grain and bio-fuel consumption grain increase etc.
Stern challenge is brought to world food supply, Wheat Production becomes more and more important in the status of grain security.
Anther Culture is to utilize plant tissue culture technique, the flower pesticide of development to certain phase, by sterile working skill
Art, is seeded on synthetic medium to change the development program of pollen grain in flower pesticide, induces it to break up, and has been carried out continuously silk point
Split, form cell mass, and then form a parenchymal tissue-callus without differentiation, or be divided into embryoid, then make callus
Tissue is divided into the process of complete plant, therefore Breeding by anther culture is also haploid breeding.
Compared with traditional breeding method, haploid breeding can shorten breeding time and improve efficiency of selection, what it was produced
Doubled haploid（DH）Plant is also the good material for carrying out genetic transformation and build molecular labeling colony.From 20th century 70
Since age, haploid breeding technology has been widely used in wheat breeding, and many improved seeds have been cultivated so far, is that wheat is educated
Kind one of effective way, and haploid induction can be combined something lost for crop with molecular labeling and Genetic Manipulative Technology
Pass Upgrading.
Although Wheat mitochondria culture has obtained remarkable progress in recent years, anther culture technique is simple, is still to lure at present
Lead haploid main path.Although Wheat midge has carried out excessive quantifier elimination, and establishes Anther culture breeding system,
Still suffer from that inductivity is low, colony is small, efficiency is also than relatively low problem at present, it is impossible to meet the demand of breeding, Anther Culture is current
Still in the application feature in research.Therefore, update with optimization culture method, improve Anther Culture Efficiency to Wheat Anther Culture Callus
Extensive use of the breeding technique in breeding is very necessary.
At this stage Anther culture breeding on anther culture method there are still workload is big, ineffective contradiction, limitation
Wider application of the colored training technology in breeding.How to improve operating efficiency, accelerating selection efficiency, shorten breeding cycle,
Innovation Germplasm material, the application for coordinating other biotechnologys, are quickly sheerly, and make full use of colored training technology to be ground in basic theory
The richness basis with Breeding Application research is studied carefully, as our problems in the urgent need to address.Practical experience discovery for many years, Hua Pei
Ineffective most important reason is the limitation of flower training operating method.It is that current flower pesticide separation is most universal to cut grain husk and tremble medicine method
Operating method, however, cutting grain husk and trembling medicine process had both needed professional tissue culture to operate, need to consume substantial amounts of work again, as flower
Train the rate-limiting step of operation.How quick separation wheat flower pesticide, and wheat anther maintains vigor and pollution-free after ensureing to separate
Suitable cultured in vitro, is to solve the poorly efficient key of flower training operation.
Centrifuge is to utilize centrifugal force, separates the machine of each component in mixture of the liquid with solid particle or liquid with liquid
Tool.Centrifuge is mainly used in separating the solid particle in suspension and liquid, or two kinds of density in emulsion are different and mutual
Immiscible liquid is separated.The characteristics of sinking speed is different in a liquid using the solid particle of different densities or granularity, have
Sedimentation centrifuge can be also classified to solid particle by density or granularity.
The content of the invention
The purpose of the present invention is directed to the costly and time consuming effort of professional operation requirement of existing vitro anther culture operating method
Defect, there is provided a kind of raising operational ton, the efficient Wheat Anther Culture Callus method of high working efficiency.
The purpose of the present invention is solved by the following technical programs：
A kind of efficient Wheat Anther Culture Callus method, it is characterised in that：The step of colored culture method, is as follows：
（1）Take fringe and pretreatment；
（2）The pretreated wheat head is sterilized；
（3）The wheat head is aseptically carried out into mechanical dissection, the wheat head of chopping is poured into centrifuge tube and shaken after sealing；
（4）Centrifuge tube after preliminary concussion is put into high speed freezing centrifuge, centrifugation obtains in vitro flower pesticide；
（5）In vitro flower pesticide is inoculated with and Fiber differentiation is gone out anther callus；
（6）Differentiation, subculture and transplanting, complete the flower training process.
The detailed step of the colored culture method is as follows：
（1）Take fringe and pretreatment：
Taking the fringe time is typically chosen the fine day morning 9:00-11:00, after materials, with 75% ethanol surface sterilization, the yarn for then moistening
Cloth wraps up the whole wheat head, keeps wheat head moistening, is put into refrigerator, Cold pretreatment 2-5 days at 3-5 DEG C；
（2）The pretreated wheat head is sterilized：
After pretreatment, the wheat head is cut, the wheat head is soaked in 2~3min for the treatment of in 75% alcohol, then shift in super the wheat head
In net workbench, with after the gauze wrapped wheat head sterilized with the mercuric chloride that concentration is 0.1% sterilize and between or shake, sterilized water floats
Wash 3~4 times；
（3）The wheat head is aseptically carried out into mechanical dissection, the wheat head of chopping is poured into centrifuge tube and shaken after sealing：
The wheat head is aseptically carried out into mechanical dissection, the wheat head that 20-30g is shredded is poured into and is contained 60-80ml sucrose solutions
100 milliliters of centrifuge tubes in, cover centrifugation and lid and tightly closed centrifuge tube headkerchief position with preservative film, from superclean bench
Middle taking-up centrifuge tube, is placed in 300-400rpm concussions 8-10min on shaking table；
（4）Centrifuge tube after preliminary concussion is put into high speed freezing centrifuge, centrifugation obtains in vitro flower pesticide：
Centrifuge tube is put into high speed freezing centrifuge and is centrifuged, after the completion of then gently take out centrifuge tube, it is possible to find broken grain husk
Shell and branch stalk are deposited to centrifugation bottom of the tube, and flower pesticide swims in liquid level, and centrifuge tube is gently put back in superclean bench, aseptic behaviour
Make, gently tear rupture disk, open centrifugation lid, upper strata flower pesticide is poured into and fills sterilized sucrose solution container；
（5）In vitro flower pesticide is inoculated with and Fiber differentiation is gone out anther callus：
With the mesh screen spoon agitation sucrose solution in the suitable aperture for sterilizing, make flower pesticide dispersed, then scoop out so that flower pesticide is equal
It is even to be distributed on mesh screen spoon, drop is controlled out, flower pesticide is knocked in the blake bottle equipped with inducing culture, 27-29 DEG C of light culture,
Evoked callus；
（6）Differentiation, subculture and transplanting, complete the flower training process：
After after anther callus diameter 1.0-1.5cm, transfer in optical culture on differential medium, differentiate flower training seedling, treat
Bloom control height of seedling to reach transfers after 3cm carry out squamous subculture in root media, long to transplanting to Turnover Box after 8cm after bloom control
Strong sprout, crop field is transplanted into after 15~20 days.
The step（1）In take fringe with microspore development be in mid-late uninucleate stage boot stage wheat children tassel as standard takes
Fringe.
The step（2）In 0.1% mercuric chloride sterilization time be 10-12min.
The step（3）In the wheat head to carry out the distance of mechanical dissection be 4.0-4.4mm/ sections.
The step（4）In centrifuge parameters be set to：Centrifugal rotational speed 12000-14000rpm, centrifugation time 8-10 point
Clock, 10-20 DEG C of centrifuging temperature.
The step（3）With（4）In sucrose solution mass fraction be 18%.
The step（5）In suitable aperture mesh screen spoon aperture be 36-42 mesh.
The step（6）In the condition of optical culture be：27~29 DEG C of temperature, intensity of illumination 1500-2000lx, photoperiod
The dark 12h of 12h/.
The present invention has the following advantages compared to existing technology：
The present invention is crushed by way of creatively taking machine cuts to wheat anther, is then adopted according to specific needs
Fragment is separated with different size of centrifugal force, has successfully extracted pure wheat anther, and be situated between by optimizing centrifugation
Matter and sterile working is used, successfully got the wheat anther that flushes and further flower train successfully, obtained healthy and strong
Bloom control；Flower training operating efficiency is greatly improved while improving operational ton.
Specific embodiment
With reference to embodiment, the present invention is further illustrated.
The detailed technology scheme of efficient Wheat Anther Culture Callus method that the present invention is provided is：
（1）Take fringe and pretreatment：
Take fringe and the boot stage wheat children tassel of mid-late uninucleate stage is in as standard takes fringe with microspore development, taking the fringe time is typically chosen
The fine day morning 9:00-11:00, after materials, with 75% ethanol surface sterilization, the whole wheat head of gauze wrapped for then moistening keeps
The wheat head is moistened, and is put into refrigerator, Cold pretreatment 2-5 days at 3-5 DEG C；
（2）The pretreated wheat head is sterilized：
After pretreatment, the wheat head is cut, the wheat head is soaked in 2~3min for the treatment of in 75% alcohol, then shift in super the wheat head
In net workbench, with after the gauze wrapped wheat head sterilized with the mercuric chloride that concentration is 0.1% sterilize 10-12min and between or shake,
Rinsed with sterile water 3~4 times；
（3）The wheat head is aseptically carried out into mechanical dissection, the wheat head of chopping is poured into centrifuge tube and shaken after sealing：
The wheat head is aseptically carried out into mechanical dissection according to 4.0-4.4mm/ sections of distance, the wheat head that 20-30g is shredded is fallen
Enter in 100 milliliters of centrifuge tubes containing the sucrose solution that 60-80ml mass fractions are 18%, cover centrifugation lid and use preservative film
Centrifuge tube headkerchief position is tightly closed, centrifuge tube is taken out from superclean bench, be placed in 300-400rpm concussions 8- on shaking table
10min；
（4）Centrifuge tube after preliminary concussion is put into high speed freezing centrifuge, centrifugation obtains in vitro flower pesticide：
Centrifuge tube is put into high speed freezing centrifuge and is centrifuged, centrifuge parameters are set to：Centrifugal rotational speed 12000-14000rpm,
Centrifugation time 8-10 minutes, 10-20 DEG C of centrifuging temperature, after the completion of then gently take out centrifuge tube, it is possible to find broken glume and
Branch stalk is deposited to centrifugation bottom of the tube, and flower pesticide swims in liquid level, and centrifuge tube is gently put back in superclean bench, sterile working,
Rupture disk is gently torn, centrifugation lid is opened, upper strata flower pesticide is poured into the sucrose solution for filling that sterilized mass fraction is 18%
Container；
（5）In vitro flower pesticide is inoculated with and Fiber differentiation is gone out anther callus：
With the mesh screen spoon agitation sucrose solution that the aperture for sterilizing is 36-42 mesh, make flower pesticide dispersed, then scoop out so that
Flower pesticide is evenly spread on mesh screen spoon, controls out drop, and flower pesticide is knocked in the blake bottle equipped with inducing culture, and 27-29 DEG C dark
Culture, evoked callus；
（6）Differentiation, subculture and transplanting, complete the flower training process：
After after anther callus diameter 1.0-1.5cm, transferring in optical culture on differential medium, the condition of optical culture is temperature
27~29 DEG C, intensity of illumination 1500-2000lx, the dark 12h of photoperiod 12h/, differentiate flower training seedling, treat that bloom control height of seedling reaches
Transferred after 3cm and carry out squamous subculture in root media, it is long to transplanting to Turnover Box strong sprout, 15~20 days after 8cm after bloom control
After be transplanted into crop field.
Embodiment one
The sucrose solution concentration employed in efficient Wheat Anther Culture Callus method to illustrate the invention is to be adapted to maintain flower pesticide life
Power, we have been configured different sucrose solution concentration to observe the plasmolysis phenomenon of pollen cell.
To make separatory sucrose solution concentration close to the osmotic pressure of wheat anther, prevent flower pesticide water swelling or dehydration from withering
Here, pollen vitality in flower pesticide is maintained, we test the sucrose solution concentration isotonic to pollen cell, using various concentrations
The pollen that sucrose solution immersion is separate, soak time is 4 hours, observes the plasmolysis phenomenon of pollen cell, as a result as follows
Table one.
According to the experimental result of table one, there is cell membrane because Wheat Pollen is extracellular, it is impossible to it was observed that water suction phenomenon, but
The sucrose solution of 18% concentration is the Cmax of Wheat Pollen cell not dehydration, therefore it is concluded that 18% sucrose solution most connects
The osmotic pressure of nearly Wheat Pollen cell, and because sucrose also needs to be inoculated into the wheat anther after flower pesticide not damaged, and separation
Fiber differentiation on the SM of high concentration, thus 18% concentration of selection the medium solution that is centrifuged as flower pesticide of sucrose solution,
Pollen cell dehydration or water suction are not resulted in, the vitality of pollen cell can be effectively maintained.
Embodiment two
The most suitable centrifugal force employed in efficient Wheat Anther Culture Callus method to illustrate the invention is to be adapted to flower pesticide to separate demand,
We are provided with different centrifugal rotational speeds to observe centrifugal effect.
Due to different Wheat Tissue organs because density is of different sizes, with different sedimentation coefficients；To search out energy
Most suitable centrifugal force needed for efficiently separating wheat anther, the broken wheat head after 4.0-4.4mm/ sections of mechanical dissection is added 18% by us
300-400rpm concussions 8-10min on shaking table is placed in after sucrose solution, centrifugation point is then carried out using the centrifugal force of different rotating speeds
From（20 DEG C, 8min）, observe centrifuge results, such as table two.
By table two it can be found that the grain husk in cracked wheat fringe can be efficiently separated out using the centrifugation of 12000-14000rpm rotating speeds
Shell and branch stalk impurity, centrifugation goes out pure wheat anther from wheatear fragment, to the wheatear fragment after mechanical dissection
After 14000rpm 8min centrifugations, can clearly find that wheat anther is separated.
Embodiment three
The creativeness of the efficient Wheat Anther Culture Callus method for providing to illustrate the invention, now by the wheat anther of present invention acquisition and often
The wheat anther that rule tremble the separation of medicine method carries out same Fiber differentiation, compares the two inductivity difference, as a result such as table three.
By table three it can be found that the present invention provide technology separate wheat anther Callus induction rate with tremble what medicine method was separate
Simultaneously there was no significant difference for wheat anther Callus induction rate, and therefore, it is possible to illustrate, separating flower pesticide using this technology method can be preferable
Maintenance separate the vitality of flower pesticide, it is adaptable to further inoculated and cultured is used.The wheat that will be isolated using this technology route
Flower pesticide carries out Fiber differentiation and goes out excellent anther callus, further differentiation culture, can differentiate eugonic flower training
Seedling.
The present invention is crushed by way of creatively taking machine cuts to wheat anther, then according to specific need
Fragment is separated using different size of centrifugal force, successfully extract pure wheat anther, and by optimize from
Heart medium and sterile working is used, successfully got the wheat anther that flushes and further flower is trained successfully, obtained strong
Strong bloom control；Flower training operating efficiency is greatly improved while improving operational ton.
Above example is only explanation technological thought of the invention, it is impossible to limit protection scope of the present invention with this, every
According to technological thought proposed by the present invention, any change done on the basis of technical scheme each falls within the scope of the present invention
Within；The technology that the present invention is not directed to can be realized by prior art.
Claims (9)
1. a kind of efficient Wheat Anther Culture Callus method, it is characterised in that：The step of colored culture method, is as follows：
（1）Take fringe and pretreatment；
（2）The pretreated wheat head is sterilized；
（3）The wheat head is aseptically carried out into mechanical dissection, the wheat head of chopping is poured into centrifuge tube and shaken after sealing；
（4）Centrifuge tube after preliminary concussion is put into high speed freezing centrifuge, centrifugation obtains in vitro flower pesticide；
（5）In vitro flower pesticide is inoculated with and Fiber differentiation is gone out anther callus；
（6）Differentiation, subculture and transplanting, complete the flower training process.
2. efficient Wheat Anther Culture Callus method according to claim 1, it is characterised in that：The detailed step of the colored culture method
It is as follows：
（1）Take fringe and pretreatment：
Taking the fringe time is typically chosen the fine day morning 9:00-11:00, after materials, with 75% ethanol surface sterilization, the yarn for then moistening
Cloth wraps up the whole wheat head, keeps wheat head moistening, is put into refrigerator, Cold pretreatment 2-5 days at 3-5 DEG C；
（2）The pretreated wheat head is sterilized：
After pretreatment, the wheat head is cut, the wheat head is soaked in 2~3min for the treatment of in 75% alcohol, then shift in super the wheat head
In net workbench, with after the gauze wrapped wheat head sterilized with the mercuric chloride that concentration is 0.1% sterilize and between or shake, sterilized water floats
Wash 3~4 times；
（3）The wheat head is aseptically carried out into mechanical dissection, the wheat head of chopping is poured into centrifuge tube and shaken after sealing：
The wheat head is aseptically carried out into mechanical dissection, the wheat head that 20-30g is shredded is poured into and is contained 60-80ml sucrose solutions
100 milliliters of centrifuge tubes in, cover centrifugation and lid and tightly closed centrifuge tube headkerchief position with preservative film, from superclean bench
Middle taking-up centrifuge tube, is placed in 300-400rpm concussions 8-10min on shaking table；
（4）Centrifuge tube after preliminary concussion is put into high speed freezing centrifuge, centrifugation obtains in vitro flower pesticide：
Centrifuge tube is put into high speed freezing centrifuge and is centrifuged, after the completion of then gently take out centrifuge tube, it is possible to find broken grain husk
Shell and branch stalk are deposited to centrifugation bottom of the tube, and flower pesticide swims in liquid level, and centrifuge tube is gently put back in superclean bench, aseptic behaviour
Make, gently tear rupture disk, open centrifugation lid, upper strata flower pesticide is poured into and fills sterilized sucrose solution container；
（5）In vitro flower pesticide is inoculated with and Fiber differentiation is gone out anther callus：
With the mesh screen spoon agitation sucrose solution in the suitable aperture for sterilizing, make flower pesticide dispersed, then scoop out so that flower pesticide is equal
It is even to be distributed on mesh screen spoon, drop is controlled out, flower pesticide is knocked in the blake bottle equipped with inducing culture, 27-29 DEG C of light culture,
Evoked callus；
（6）Differentiation, subculture and transplanting, complete the flower training process：
After after anther callus diameter 1.0-1.5cm, transfer in optical culture on differential medium, differentiate flower training seedling, treat
Bloom control height of seedling to reach transfers after 3cm carry out squamous subculture in root media, long to transplanting to Turnover Box after 8cm after bloom control
Strong sprout, crop field is transplanted into after 15~20 days.
3. efficient Wheat Anther Culture Callus method according to claim 1 and 2, it is characterised in that：The step（1）In take fringe
The boot stage wheat children tassel of mid-late uninucleate stage is in as standard takes fringe with microspore development.
4. efficient Wheat Anther Culture Callus method according to claim 1 and 2, it is characterised in that：The step（2）In 0.1%
Mercuric chloride sterilization time be 10-12min.
5. efficient Wheat Anther Culture Callus method according to claim 1 and 2, it is characterised in that：The step（3）In the wheat head
The distance for carrying out mechanical dissection is 4.0-4.4mm/ sections.
6. efficient Wheat Anther Culture Callus method according to claim 1 and 2, it is characterised in that：The step（4）In centrifugation
Machine parameter is set to：Centrifugal rotational speed 12000-14000rpm, centrifugation time 8-10 minutes, 10-20 DEG C of centrifuging temperature.
7. efficient Wheat Anther Culture Callus method according to claim 1 and 2, it is characterised in that：The step（3）With（4）In
Sucrose solution mass fraction be 18%.
8. efficient Wheat Anther Culture Callus method according to claim 1 and 2, it is characterised in that：The step（5）In it is suitable
The aperture of the mesh screen spoon in aperture is 36-42 mesh.
9. efficient Wheat Anther Culture Callus method according to claim 1 and 2, it is characterised in that：The step（6）In light training
Foster condition is：27~29 DEG C of temperature, intensity of illumination 1500-2000lx, the dark 12h of photoperiod 12h/.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107711504A (en) * | 2017-11-09 | 2018-02-23 | 上海市农业科学院 | A kind of method that haynaldia villosa homozygosis regeneration plant is obtained by Anther Culture |
| CN114711108A (en) * | 2022-04-01 | 2022-07-08 | 上海市农业科学院 | Method for rapidly determining development period of rice microspore |
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| CN101554136A (en) * | 2009-05-11 | 2009-10-14 | 西北农林科技大学 | Wheat anther one-step seedling culture method |
| CN104705191A (en) * | 2015-04-04 | 2015-06-17 | 陈丁龙 | Wheat anther differentiation culture medium formula |
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| CN101554136A (en) * | 2009-05-11 | 2009-10-14 | 西北农林科技大学 | Wheat anther one-step seedling culture method |
| CN104705191A (en) * | 2015-04-04 | 2015-06-17 | 陈丁龙 | Wheat anther differentiation culture medium formula |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107711504A (en) * | 2017-11-09 | 2018-02-23 | 上海市农业科学院 | A kind of method that haynaldia villosa homozygosis regeneration plant is obtained by Anther Culture |
| CN114711108A (en) * | 2022-04-01 | 2022-07-08 | 上海市农业科学院 | Method for rapidly determining development period of rice microspore |
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