CN105154383A - Separation method of microspores developed and enlarged by broccoli - Google Patents
Separation method of microspores developed and enlarged by broccoli Download PDFInfo
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- CN105154383A CN105154383A CN201510560195.XA CN201510560195A CN105154383A CN 105154383 A CN105154383 A CN 105154383A CN 201510560195 A CN201510560195 A CN 201510560195A CN 105154383 A CN105154383 A CN 105154383A
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Abstract
The invention discloses a separation method of microspores developed and enlarged by broccoli. The method comprises the following steps: taking broccoli buds, adding NLN-13 liquid induction medium after sterilization is performed under sterile environment, performing filtration after the mixture is smashed into suspension liquid, centrifuging filter liquor and adding the NLN-13 liquid induction medium into centrifuged sediment, and obtaining microspore suspension liquid; respectively loading the microspore suspension liquid into sterile petri dishes, performing heat shock in darkness, continuously placing in darkness for culturing at 23 to 27 DEG C, and then observing the developmental state of the microspores by a microscope, performing gradient filter by using a microstrainer, and obtaining the developed and enlarged microspores; eluting the developed and enlarged microspores into a centrifuge tube for centrifugation, poring out supernate, and placing the microspores and the centrifuge tube into liquid nitrogen for preservation and standby use. Filter screens with different bore diameters are adopted for filtering, thus obtaining fully developed microspores, and providing powerful guarantee to molecular biology and proteomics research in the development of microspore embryos.
Description
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to a kind of broccoli grow expand sporule be separated method.
Background technology
Broccoli (
brassicaoleraceal.var.
italica) having another name called Caulis et Folium Brassicae capitatae, broccoli, stem cabbage etc., the green bouquet formed with stem and side shoot top is for product, and nutritious, color tool is good, is world market special vegetables of a kind of name in very great demand.Current China broccoli main breed is mostly from abroad, and the kind with independent intellectual property right is deficient, main because breeding resources is few, is difficult to the kind prepared.Traditionally, inbreeding of more generation is separated generally needs the time of 5 ~ 6 years to obtain the stable parent material that isozygotys.Require great effort consuming time.And microspore-isolated culture method can obtain double haploid pure lines fast in 1 ~ 2 year, shorten the time of 3 ~ 4 years than traditional method.Therefore Elite inbred initiative and improve in breeding of new variety efficiency etc. there is vital role.Meanwhile, recessive character is also easy to express, and has enriched breeding resources.
Since nineteen eighty-two Lichter reported first obtains Microspore of Brassica napus regeneration plant, no matter cress, the especially microspores culture of rape, be at germ extraction rate, go out embryo amount, or embryo culture and regeneration plant aspect are all successful.Broccoli microspore-isolated culture, foreign study sees (1991) reports such as Takahata the earliest, domestic, is that Zhang Deshuan equals to succeed in the research of the kinds such as the green broccoli of bar for 1997.Subsequently, (2009), the Zhang Zhens superfine (2013,2014) such as (2005), Yuan Suxia such as Zhang Yanguo etc. (2005), Fang Shugui have also been made large quantity research, and culture efficiency obtains and improves to a certain extent.
About the study mechanism such as the molecular biology in sporule embryo development procedure, proteomics are less, trace it to its cause is that sporule volume is small, the test materials obtaining sufficient amount needs to do microspores culture test that is a large amount of, that repeat, and separates have difficulties growing the sporule of expanding and rudimentary sporule.The method adopting the micro-strainer of different pore size to filter according to sporule diameter can be simple and effective separate growing the sporule of expanding, thus provide enough test materialss for broccoli sporule fetal development study mechanism, there is important practice significance.
Summary of the invention
For the deficiencies in the prior art, the object of the present invention is to provide a kind of broccoli grow expand sporule be separated method, according to growth expand sporule with do not grow sporule diameter difference, the method adopting different pore size micro-strainer to filter can be simple and effective separate growing the sporule of expanding, thus provide analysis of experiments material for sporule fetal development study mechanism.
Broccoli grow expand sporule be separated a method, comprise the following steps:
Step 1, gets broccoli bud sterilising treatment in an aseptic environment, obtains aseptic bud;
Step 2, adds NLN-13 liquid inducing culture, filters after grinding to form suspension in aseptic bud, and filtrate is centrifugal, and adds NLN-13 liquid inducing culture in the precipitation after centrifugal, obtains microspore suspension;
Step 3, is dispensed into microspore suspension in sterile petri dish, and at being placed in dark 32 DEG C ~ 33 DEG C, Heat thermostability is after 1 ~ 3 day, for subsequent use;
Step 4, continues to be placed in dark by the sterile petri dish after Heat thermostability, cultivates 1 ~ 10 day, then uses microscopic examination microspore development situation, and utilize micro-strainer to carry out gradient filtration, must grow the sporule of expanding for 23 ~ 27 DEG C;
Step 5, be eluted in centrifuge tube centrifugal by growing the sporule distilled water expanded, incline supernatant liquor, is placed in liquid nitrogen saves backup together with centrifuge tube.
As preferably, it is early stage to double-core for late period that the bud of broccoli described in step 1 is in monokaryon, and petal and anther length ratio are 0.85 ~ 1.15.
As preferably, sterilising treatment described in step 1 is that green vegetables flower bud is first used sterilized solution sterilizing on aseptic operating platform, again with sterilized water concussion cleaning 3 ~ 5 times, described sterilized solution sterilizing is mass percentage concentration is the formulated sterilizing of 5.6% clorox 18 ~ 20 minutes, or be that 0.1% mercury chloride is formulated by mass percentage concentration, sterilizing 10 ~ 15 minutes.
As preferably, the inducing culture of NLN-13 described in step 2 is made up of NLN liquid nutrient medium 1L and sucrose 130g, pH6.0 ~ 6.2;
Described NLN liquid nutrient medium, in 1L, consists of: KNO
3125mg, Ca (NO
3)
24H
2o500mg, MgSO
47H
2o125mg, KH
2pO
4125mg, H
3bO
36.2mg, MnSO
4h
2o18.95mg, ZnSO
47H
2o8.6mg, Na
2moO
42H
2o0.25mg, CuSO
45H
2o0.025mg, CoCl
26H
2o0.025mg, vitaminB10 .5mg, vitamin B6 0.5mg, vitamin H 0.05mg, folic acid 0.5mg, Na
2eDTA37.3mg, FeSO
47H
2o27.8mg, inositol 100mg, glycine 2mg, nicotinic acid 5mg, L-glutaminate 800mg, gsh 30mg, vitamin B5 5mg, Serine 100mg, surplus is sterilized water.
As preferably, gradient filtration described in step 4 is by 23 ~ 27 DEG C of cultivations microspore suspension of 1 ~ 10 day in dark, first filter with 300 ~ 350 mesh filter screens, to remove the larger material of volume, filter with 450 ~ 600 mesh filter screens after filtrate suspends with NLN-13 liquid inducing culture, repeat must grow the sporule of expanding 2 ~ 3 times.
As preferably, grow the sporule of expanding described in step 4 and to refer to after Heat thermostability random observation 10 visuals field under 20 power microscopes, grow and expand sporule quantity and account for the ratio of whole sporule more than 5%.
As preferably, in step 4, gradient filtration is vacuum filtration, and vacuum tightness used is 20-50kpa.
beneficial effect
1, the present invention is directed to broccoli and grow the problem of sporule separation difficulty of expanding, provide a kind of according to growing and not growing sporule diameter difference, adopt the method for different pore size micro-strainer filtering separation, growth expand sporule with do not grow sporule and separate, thus obtain the test materials of sufficient amount.In the sporule test materials obtained by present method, grow the sporule quantity accounting of expanding and bring up to more than 80%, and do not contrasted only up to 15% by different pore size filter membrane gradient filtration.
2, by repeating microspores culture operation, micro-strainer filtering separation in a large number, the test materials of sufficient amount is obtained, for molecular biology, proteomics research provide guarantee.
Embodiment
embodiment 1
Step 1, get broccoli healthy growth, without the inflorescence of disease and pest as the donor plant of microspores culture, get petal and anther length on inflorescence than be 0.85, monokaryon late period is to the early stage bud of double-core, Bechtop puts into sterile petri dish broccoli bud, add sterilized solution, be put into shake on shaking table and carry out surface sterilization 20 minutes, after washing 3 times with aseptic pond again, for subsequent use, wherein, sterilized solution is mixed with by aqueous sodium hypochlorite solution 56ml/L, dehydrated alcohol 100ml/L, liquid detergent 10/L, sterilized water;
Step 2,12 aseptic buds are placed in sterile beaker by Bechtop, add 10mlNLN-13 liquid inducing culture, with tack glass stick squeeze broken, grind to form suspension, this suspension with the aseptic strainer filtering of 300 order in 50ml centrifuge tube, cover tightly, the centrifugal 3min of 900rpm/min, outwells supernatant liquor after centrifugal, in precipitation, add 10mlNLN-13 liquid inducing culture, again centrifugal 2 times as stated above, abandon supernatant liquor; Add NLN-13 liquid inducing culture 40ml, obtain microspore suspension, wherein, NLN-13 liquid inducing culture: NLN liquid nutrient medium 1L+ sucrose 130g/L, pH6.0, filtration sterilization;
It consists of: KNO
3125mg, Ca (NO
3)
24H
2o500mg, MgSO
47H
2o125mg, KH
2pO
4125mg, H
3bO
36.2mg, MnSO
4h
2o18.95mg, ZnSO
47H
2o8.6mg, Na
2moO
42H
2o0.25mg, CuSO
45H
2o0.025mg, CoCl
26H
2o0.025mg, vitaminB10 .5mg, vitamin B6 0.5mg, vitamin H 0.05mg, folic acid 0.5mg, Na
2eDTA37.3mg, FeSO
47H
2o27.8mg, inositol 100mg, glycine 2mg, nicotinic acid 5mg, L-glutaminate 800mg, gsh 30mg, vitamin B5 5mg, Serine 100mg and surplus sterilized water;
Step 3, microspore suspension be dispensed in 60mm sterile petri dish, every culture dish adds 4ml microspore suspension, adds a cover the sealing of rear parafilm film, to be placed in 32.5 DEG C of constant temperature biochemical cultivation cases, under dark condition Heat thermostability 1 day;
Step 4, cultivate 3 days at sterile petri dish after Heat thermostability being continued be placed in dark 25 DEG C, when microscopic examination finds sporule embryo development rate more than 5%, microspore suspension is first filtered with 300 mesh filter screens, to remove the larger material of volume, filter after filtrate suspends with NLN-13 liquid inducing culture with 450 mesh filter screens, the precipitation of acquisition is filtered with after the suspension of NLN-13 liquid inducing culture, repeat 2 times, the final precipitation obtained is the sporule of growth;
Step 5, be eluted in centrifuge tube centrifugal by growing the sporule distilled water expanded, incline supernatant liquor, centrifuge tube is placed in liquid nitrogen and saves backup.
result:in the sporule test materials obtained by present method, grow the sporule quantity accounting of expanding and bring up to 85%, and be not only 12% by the contrast of different pore size filter membrane gradient filtration.
embodiment 2
Except get petal and anther length on inflorescence in step 1 than be 0.95, monokaryon late period is to the early stage bud of double-core, sterilized solution is formulated by aqueous sodium hypochlorite solution 56ml/L+ dehydrated alcohol 100ml/L+ liquid detergent 10/L+ sterilized water, and sterilizing 18 minutes, then wash 4 times with aseptic pond;
On Bechtop, 15 aseptic buds are placed in sterile beaker in step 2, add 10mlNLN-13 liquid inducing culture, with tack glass stick squeeze broken, grind to form suspension; This suspension is with the aseptic strainer filtering of 300 order in 50ml centrifuge tube, and cover tightly, the centrifugal 3min of 900rpm/min, outwells supernatant liquor after centrifugal, in precipitation, add 10mlNLN-13 liquid inducing culture, more centrifugal 2 times as stated above, abandons supernatant liquor; Add NLN-13 liquid inducing culture 40ml, obtain microspore suspension, wherein, NLN-13 liquid inducing culture: NLN liquid nutrient medium 1L+ sucrose 130g/L, pH6.1, filtration sterilization;
Step 3, is dispensed into this microspore suspension in 60mm sterile petri dish, and every culture dish adds 4ml sporule mixing suspension, adds a cover the sealing of rear parafilm film, to be placed in 33 DEG C of constant temperature biochemical cultivation cases, under dark condition Heat thermostability 1 day; Rear taking-up continues to cultivate under being placed in 25 DEG C of constant incubators, dark condition;
Step 4, cultivate 6 days at sterile petri dish after Heat thermostability being continued be placed in dark 25 DEG C, when microscopic examination finds sporule embryo development rate more than 6%, microspore suspension is first filtered with 350 mesh filter screens, to remove the larger material of volume, filter after filtrate suspends with NLN-13 liquid inducing culture with 500 mesh filter screens, the precipitation of acquisition is filtered with after the suspension of NLN-13 liquid inducing culture, repeat 2 times, the final precipitation obtained is the sporule growing and expand.
Step 5, be eluted in centrifuge tube centrifugal by growing the sporule distilled water expanded, incline supernatant liquor, centrifuge tube is placed in liquid nitrogen and saves backup.
All the other operations embodiment 1 simultaneously.
Result: in the sporule test materials obtained by present method, is grown the sporule quantity accounting of expanding and brings up to 87%, and be not only 15% by the contrast of different pore size filter membrane gradient filtration.
embodiment 3
Except petal in step 1 and anther length than be 1.15, monokaryon late period, sterilized solution was made up of mercury chloride 10ml/L, liquid detergent 10,1000ml sterilized water to double-core early stage bud, and sterilizing 12 minutes, then washed 5 times with aseptic pond;
On Bechtop, 20 aseptic buds are placed in sterile beaker in step 2, add 10mlNLN-13 liquid inducing culture, with tack glass stick squeeze broken, grind to form suspension; This suspension is with the aseptic strainer filtering of 300 order in 50ml centrifuge tube, and cover tightly, the centrifugal 3min of 900rpm/min, outwells supernatant liquor after centrifugal, in precipitation, add 10mlNLN-13 liquid inducing culture, more centrifugal 2 times as stated above, abandons supernatant liquor; Add NLN-13 liquid inducing culture 40ml, obtain microspore suspension; Wherein, NLN-13 liquid inducing culture: NLN liquid nutrient medium 1L+ sucrose 130g/L, pH6.2, filtration sterilization;
Step 3, is dispensed into this microspore suspension in 90mm sterile petri dish, and every culture dish adds 10ml sporule mixing suspension, adds a cover the sealing of rear parafilm film, to be placed in 32.5 DEG C of constant temperature biochemical cultivation cases, under dark condition Heat thermostability 1 day;
Step 4, cultivate 8 days at sterile petri dish after Heat thermostability being continued be placed in dark 25 DEG C, when microscopic examination finds sporule embryo development rate more than 6%, microspore suspension is first filtered with 350 mesh filter screens, to remove the larger material of volume, filter after filtrate suspends with NLN-13 liquid inducing culture with 600 mesh filter screens, the precipitation of acquisition is filtered with after the suspension of NLN-13 liquid inducing culture, repeat 2 times, the final precipitation obtained is the sporule growing and expand.
Step 5, be eluted in centrifuge tube centrifugal by growing the sporule distilled water expanded, incline supernatant liquor, centrifuge tube is placed in liquid nitrogen and saves backup.
All the other operations embodiment 1 simultaneously.
result:in the sporule test materials obtained by present method, grow the sporule quantity accounting of expanding and bring up to 96%, and be not only 9% by the contrast of different pore size filter membrane gradient filtration.
As can be seen from the result of embodiment 1-3, the inventive method acquisition is grown the sporule quantity accounting of expanding and is brought up to more than 80%, and is not contrasted only up to 15% by different pore size filter membrane gradient filtration.By repeating microspores culture operation, micro-strainer filtering separation in a large number, obtain the test materials of sufficient amount, for molecular biology, proteomics research provide guarantee.
Claims (7)
1. broccoli grow expand sporule be separated a method, it is characterized in that, comprise the following steps:
Step 1, gets broccoli bud sterilising treatment in an aseptic environment, obtains aseptic bud;
Step 2, adds NLN-13 liquid inducing culture, filters after grinding to form suspension in aseptic bud, and filtrate is centrifugal, and adds NLN-13 liquid inducing culture in the precipitation after centrifugal, obtains microspore suspension;
Step 3, is dispensed into microspore suspension in sterile petri dish, and at being placed in dark 32 DEG C ~ 33 DEG C, Heat thermostability is after 1 ~ 3 day, for subsequent use;
Step 4, continues to be placed in dark by the sterile petri dish after Heat thermostability, cultivates 1 ~ 10 day, then uses microscopic examination microspore development situation, and utilize micro-strainer to carry out gradient filtration, must grow the sporule of expanding for 23 ~ 27 DEG C;
Step 5, be eluted in centrifuge tube centrifugal by growing the sporule distilled water expanded, incline supernatant liquor, is placed in liquid nitrogen saves backup together with centrifuge tube.
2. a kind of broccoli according to claim 1 is grown and is expanded the method that sporule is separated, and it is characterized in that: it is early stage to double-core for late period that the bud of broccoli described in step 1 is in monokaryon, and petal and anther length ratio are 0.85 ~ 1.15.
3. a kind of broccoli according to claim 1 is grown and is expanded the method that sporule is separated, it is characterized in that: sterilising treatment described in step 1 is that green vegetables flower bud is first used sterilized solution sterilizing on aseptic operating platform, again with sterilized water concussion cleaning 3 ~ 5 times, described sterilized solution sterilizing is mass percentage concentration is the formulated sterilizing of 5.6% clorox 18 ~ 20 minutes, or be that 0.1% mercury chloride is formulated by mass percentage concentration, sterilizing 10 ~ 15 minutes.
4. a kind of broccoli according to claim 1 is grown and is expanded the method that sporule is separated, and it is characterized in that: the inducing culture of NLN-13 described in step 2 is made up of NLN liquid nutrient medium 1L and sucrose 130g, pH6.0 ~ 6.2;
Described NLN liquid nutrient medium, in 1L, consists of: KNO
3125mg, Ca (NO
3)
24H
2o500mg, MgSO
47H
2o125mg, KH
2pO
4125mg, H
3bO
36.2mg, MnSO
4h
2o18.95mg, ZnSO
47H
2o8.6mg, Na
2moO
42H
2o0.25mg, CuSO
45H
2o0.025mg, CoCl
26H
2o0.025mg, vitaminB10 .5mg, vitamin B6 0.5mg, vitamin H 0.05mg, folic acid 0.5mg, Na
2eDTA37.3mg, FeSO
47H
2o27.8mg, inositol 100mg, glycine 2mg, nicotinic acid 5mg, L-glutaminate 800mg, gsh 30mg, vitamin B5 5mg, Serine 100mg, surplus is sterilized water.
5. a kind of broccoli according to claim 1 is grown and is expanded the method that sporule is separated, it is characterized in that: the gradient filtration described in step 4 is by 23 ~ 27 DEG C of cultivations microspore suspension of 1 ~ 10 day in dark, first filter with 300 ~ 350 mesh filter screens, to remove the larger material of volume, filter with 450 ~ 600 mesh filter screens after filtrate suspends with NLN-13 liquid inducing culture, repeat must grow the sporule of expanding 2 ~ 3 times.
6. a kind of broccoli according to claim 1 is grown and is expanded the method that sporule is separated, it is characterized in that: grow the sporule of expanding described in step 4 and to refer to after Heat thermostability random observation 10 visuals field under 20 power microscopes, grow and expand sporule quantity and account for the ratio of whole sporule more than 5%.
7. a kind of broccoli according to claim 1 is grown and is expanded the method that sporule is separated, and it is characterized in that: in step 4, gradient filtration is vacuum filtration, vacuum tightness used is 20-50kpa.
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Cited By (2)
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Application publication date: 20151216 |