CN104429967B - Cultivation method for obtaining a DH plant by inducing greenish lily flower anther through one-step method - Google Patents
Cultivation method for obtaining a DH plant by inducing greenish lily flower anther through one-step method Download PDFInfo
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Abstract
The invention belongs to the field of obtaining a regenerating plant by using a tissue culturing technology, and particularly relates to a cultivation method for obtaining a DH plant by inducing greenish lily flower anther through a one-step method. According to the invention, greenish lily flower anther is subjected to the steps of pretreatment, stress treatment, anther embryoid and bud cultivation, regenerating plant cultivation and the like, thus obtaining the regenerating plant. According to the invention, micropores in the anther can be induced to be developed to be an integral haploid or double-haploid plant, so as to provide a variety of homozygous breeding materials for greenish lily flower DH breeding, more novel germplasms can be created, the selecting and breeding efficiency is improved, and an ideal acceptor material is provided for basic research. The cultivation method is simple and convenient to operate, the cultivation medium is low in component cost, and the application value is high.
Description
Technical field
The invention belongs to obtaining regeneration plant field with tissue culture technique, particularly to a kind of by by a type, la type
The flower pesticide of Bulbus Lilii carries out embryoid induction culture as explant, then embryoid is carried out plumelet culture, monoploid regeneration plant
Cultivate the method obtaining monoploid regeneration plant.
Background technology
Monoploid refers to individuality or the tissue with gametic number, the individual title that only one chromosome set is constituted
For monoploid, monoploid can get dihaploid after nature or artificial doubling.Monoploid, dihaploid material are in plant
All have great importance in breeding and hereditary basiies theoretical research, can be used for Marker-assisted selection and genetic map is drawn, be
The optimal receptor population of transgenic.In the practices of breeding, pure lines not only can quickly be obtained using Doubled haploid breeding technology, contracting
Short breeding cycle, and more wider variations can be obtained, particularly recessive character is easier to be showed, thus abundant
The type of breeding basic material, accelerates breeding process.
A kind of currently acquired haploid Main Means are that in vitro Anther Culture obtains (double) monoploid regeneration plant.Typically
Obtaining regeneration plant by Anther Culture has two kinds of occurring modes, i.e. embryoid development pathway (direct development approach) and wound healing group
Knit development pathway (indrect development approach).Embryoid development pathway refers to that sporidiole direct development becomes with radicle plumular axis plumule
The embryoid of structure, embryoid can grow into normal plant in common ms or 1/2ms culture medium.Healing tissue development's approach
Need for explant to be initially positioned at callus induction in dedifferentiation culture medium, then again the calluss deriving are placed in
The differentiation of leaf bud is induced on division culture medium, the adventitious bud finally obtaining differentiation is transferred in seedling culture medium.Therefore, we
Abbreviation embryoid development pathway is " one-step method ", and healing tissue development's approach is " two step method ".
The report being trained work(with regard to Bulbus Lilii flower pesticide published both at home and abroad has only 3 (han dong sheng
1997；Chu Yunxia 2001；Xiu-Li Han 2010), this 3 documents are all the regeneration plants being obtained by healing tissue development's approach,
There is presently no and see the report successfully obtaining Bulbus Lilii (double) haplobiont via embryoid development pathway.Two kinds of methods are compared
Relatively, change the culture medium of new type because " one-step method " does not need in the Induction Process of regeneration plant, not only reduce materials
Cost, and save about 1/3rd artificial.Constantly increase in qualified manpower resource worsening shortages and human cost
Under high background, " one-step method " suffers from " two step method " unrivaled advantage in the extension process of industrialization.
Although (double) haploid breeding technology (that is: dh breeding technique) has just been born early in decades ago, so far
Till, only the very small amount plant such as Semen Maydiss, Chinese cabbage, Brassica campestris L has the dh breeding technique of maturation.Trace it to its cause, dh breeding technique
Difficult point be, plant is different, abductive approach is different；In same plant, genotype is different, anti-to the induction mode of the same terms
Should difference.Existing similarity between plant, but variant.This just determines some successful experiences is to bring reference
, and some successful experiences can allow people go astray on the contrary.On the other hand, in addition to genotype, a successful dh breeding skill
The row of concentration between art processing mode also to be forced and time, basal medium type, hormone kind and concentration, multiple hormone
Row combination, carbon source kind and concentration, the impact of training method etc. series of factors, these factors of influence are relatively only each other
Vertical, therefore only in all factors of influence all in OK range, being possible to succeed.
Bulbus Lilii (lilium spp.) is Liliaceae lilium perennial flowering bulb plant, is world-renowned commodity flower
One of grass, occupies highly important status in Market of Fresh Cut Flower.In recent years, a type, la type Bulbus Lilii are increasingly becoming domestic flowers market
On more popular Bulbus Lilii type.At present the new varieties of Bulbus Lilii get more and more, the research of kind excellent to economic characters and profit
With being increasingly becoming the emphasis of Lilies breeding work.Hybrid lily breeding is primarily present two problems, and one is that breeding cycle is long, from kind
Son is seeded into bloom and will experience the 2-3 year, and growth cycle is long；Two is that intervarietal hybridization is difficult.Bulbus Lilii gene element is a, l, o, t
The Four Great families, the problem of hybrid lily breeding generally existing incompatibility, not only it is fertilized difficult, and after fertilization is difficult to obtain and plants
Son, therefore dh breeding technique can become another kind of Main Means of Lilies breeding in theory.So the dh breeding technique of Bulbus Lilii
Have great importance in terms of cultivating independent intellectual property right kind.
Chinese patent " induction ot type Bulbus Lilii flower pesticide obtains method and its culture medium of (double) haplobiont " (patent No.:
Zl201210479417.1, the applying date: on November 22nd, 2012, Shen Qing Publication day: on 2 27th, 2013, authorized announcement date:
On January 29th, 2014) disclose a kind of induction ot type Bulbus Lilii flower pesticide acquisition (double) including low temperature, dark culturing, Heat thermostability
The method of haplobiont, and its contain 2, the inducing culture of 4-d, 6-ba.But, the method is only applicable to the hundred of ot type
Close, be not particularly suited for a type, the Bulbus Lilii of la type.Ot type Bulbus Lilii heat-shock temperature in this patent is 35-37 (DEG C), and 7-9 days equal
Can, but the ability that a type and la type Bulbus Lilii flower pesticide tolerate low temperature is weaker, under this hot shock condition, sporidiole all dead it is impossible to
To regeneration plant, illustrate that the dh breeding method being suitable for ot type is not appropriate for a type and la type.Find a type, la through many experiments
Type Bulbus Lilii can tolerate 33 DEG C of high temperature, but ot type Bulbus Lilii is insensitive to 33 DEG C of high temperature, so the same side of body can not be adopted
Urgent mode.
Therefore, scientific research and practice in be required to a kind of be applied to a type, la type Bulbus Lilii, improve selection and breeding efficiency,
Simultaneously also based on research provide preferable acceptor material, one-step method induce Bulbus Lilii flower pesticide obtain dh plant cultural method.
Content of the invention
The present invention provide a kind of by the flower pesticide of a type, the Bulbus Lilii of la type is carried out on inducing culture as explant
Embryoid induction is cultivated, then embryoid is transferred to the method that in seedling culture medium, culture obtains regeneration plant.The present invention is permissible
Microspore development within induction flower pesticide is complete monoploid or double haploid, provides multiple pure for Bulbus Lilii dh breeding
The breeding material closing, creates more new germplasm, improves selection and breeding efficiency, simultaneously also based on study and provide
Preferably acceptor material.
The purpose of the present invention is achieved through the following technical solutions:
One-step method induces Bulbus Lilii flower pesticide to obtain the cultural method of dh plant, comprises the following steps:
Anther pretreatment step: after Bulbus Lilii alabastrum is carried out chilling treatment, lift-off processing, disinfects, obtain pretreatment
Flower pesticide afterwards；
Flower pesticide Stress treatment step: described pretreated flower pesticide is positioned over inducing culture, carries out Stress treatment, obtain
Flower pesticide to after Stress treatment；
Flower pesticide embryoid and plumelet incubation step: the flower pesticide after described Stress treatment is placed in inducing culture, in 25 DEG C
Cultivate under dark condition, obtain flower pesticide embryoid；Described flower pesticide embryoid is transferred under illumination condition and cultivates 1～2 week, obtain
Plumelet；
Regenerated plant culture step: the described plumelet that length is more than 0.5cm is transferred in seedling culture medium, in 25 DEG C of light
According under the conditions of cultivate, obtain regeneration plant within 6～8 weeks.
Further, described Bulbus Lilii flower pesticide is derived from a type, la type Bulbus Lilii.
Further, described chilling treatment is, when described Bulbus Lilii flower pesticide is derived from a type Bulbus Lilii, by Bulbus Lilii alabastrum in 4～6
DEG C place 1 day；When described Bulbus Lilii flower pesticide is derived from la type Bulbus Lilii, Bulbus Lilii alabastrum is placed 3 days in 4～6 DEG C.
Further, described Stress treatment is, when described Bulbus Lilii flower pesticide is derived from a type Bulbus Lilii, will be described pretreated
Flower pesticide is cultivated 9 days under 33 DEG C of dark conditions；When described Bulbus Lilii flower pesticide is derived from la type Bulbus Lilii, by described pretreated flower pesticide
Cultivate 3 days under 33 DEG C of dark conditions.
Further, in described flower pesticide embryoid and plumelet incubation step, at least 2 weeks are cultivated under 25 DEG C of dark conditions.
Further, described inducing culture is, based on b5 minimal medium, add 6-benzyladenine 1.0～
2.0mg/l, 2,4- dichlorphenoxyacetic acid 0.5～2.0mg/l, the final concentration of sucrose is 90～120g/l, and the final concentration of agar is
12～15g/l.
Further, described root media, is that the edible soft plantation white sugar of interpolation and agar obtain in 1/10ms minimal medium
To solid medium: the final concentration of wherein edible soft plantation white sugar is 25～30g/l, and the final concentration of agar is 6～7g/l；Ph value is
5.8；The solvent of described root media is tap water.
Further, described 1/10ms minimal medium is meant that, a great number of elements, trace element, iron salt and vitamin
Content be the 1/10 of standard ms minimal medium content.
Compared with the prior art the present invention has the advantages that
1st, the present invention carries out embryoid induction culture using the flower pesticide of a type, the Bulbus Lilii of la type as explant, can pass through
Plumelet obtains regeneration plant.
2nd, the present invention adopts suitable inducing culture, and carries out at stress before flower pesticide embryoid and plumelet culture
Reason, improves the induced efficiency of plumelet.
3rd, the present invention is easy and simple to handle, and medium component is cheap, and using value is high.
4., the present invention has very important significance in Lilies breeding field: when Flos Lilii viriduli is open, anther length typically exists
Between 2-4 centimetre, pollen amount is huge；Because the pigment that lilium pollen contains is non-water-soluble, after loose powder, easily pollute petal, institute
To have had a strong impact on the sight of flower, it also is difficult to clean when people's imprudence rubs pollen on medicated clothing.And manual removal
Flower pesticide can take a substantial amount of time and energy.The kind cultivating WUHUAFEN is one of direction of Lilies breeding, relevant this respect
Research is at present also seldom.Monoploid Bulbus Lilii can not produce normal pollen, it is to avoid the generation of problems, thus improve hundred
The sight closed and commodity.
Brief description
The photo of the flower pesticide embryoid that Fig. 1 is the flower pesticide embryoid of the present invention and plumelet incubation step obtains.
Fig. 2 is the flow cytomery result figure of the regeneration plant of " Republican Party " Bulbus Lilii obtaining in embodiment 2；Wherein
Left figure represents monoploid, and right figure represents diploid.
Fig. 3 is the flow cytomery result figure of the regeneration plant of " red core " Bulbus Lilii obtaining in embodiment 3；Wherein left
Figure represents monoploid, and right figure represents diploid.
Specific embodiment
One-step method induce Bulbus Lilii flower pesticide obtain dh plant cultural method, by using a type, the Bulbus Lilii of la type flower pesticide as
Explant carries out embryoid induction culture, then the embryoid of acquisition is carried out plumelet culture, regenerated plant culture obtain regeneration plant
The method of strain, comprises the following steps:
Step one, draw materials:
Collection is in each 20 of the alabastrum of mid-late uninucleate stage, a type of dicaryotic phase, la type Bulbus Lilii；It is in the flower in above-mentioned period
Flower bud morphological characteristic is: alabastrum is in green, and parcel is tight, flower pesticide dark green；Microscopy visible overwhelming majority sporidiole is in monokaryon and leans on
Bian Qi, small part is in dicaryotic phase；Because genotype is different, alabastrum length is also different, is in the range of 2.5-3.5cm.
Wherein, a type lily cultivar includes: lanzhou lily, the Republican Party (gironde), shady deal (black out)；La type hundred
Close kind to include: Baptiste sieve (batistero), Da Nuo (ceb dazzle), red core (fangio).
Step 2, anther pretreatment:
Alabastrum is placed in hermetic container after carrying out chilling treatment, lift-off processing, disinfecting, obtains pretreated flower
Medicine.
Chilling treatment is: when Bulbus Lilii flower pesticide is a type, Bulbus Lilii alabastrum is placed 1 day in 4-6 DEG C；When Bulbus Lilii flower pesticide is la
During type, Bulbus Lilii alabastrum is placed 3 days in 4-6 DEG C；
Lift-off processing is: removed calyx and petal before disinfecting, removes filigree after disinfecting；
Disinfect for: in superclean bench, with the 75% alcohol-pickled alabastrum removing calyx and petal 45～60 seconds,
Afterwards with 2.5% liquor natrii hypochloritises' vibration sterilization 12～15 minutes, then use aseptic water washing 3 times, 1～2 minute every time.
Step 3, flower pesticide Stress treatment:
Pretreated flower pesticide is removed the outside of belly after filigree lie in downwards on inducing culture, carry out Stress treatment, obtain
Flower pesticide to after Stress treatment.
Stress treatment is: when Bulbus Lilii flower pesticide is derived from a type Bulbus Lilii, pretreated flower pesticide is trained under 33 DEG C of dark conditions
Support 9 days；When Bulbus Lilii flower pesticide is derived from la type Bulbus Lilii, pretreated flower pesticide is cultivated 3 days under 33 DEG C of dark conditions.
Step 4, flower pesticide embryoid and plumelet culture:
Flower pesticide after Stress treatment is placed in inducing culture, at least cultivates 2 weeks under 25 DEG C of dark conditions (such as 2
In week, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks etc. any or any between the two, preferably 2～4 weeks) obtain white meat worm shape
, length be more than 5mm maturation flower pesticide embryoid (as shown in Figure 1)；Again this flower pesticide embryoid is transferred to illumination condition
Lower 1～2 week, obtain plumelet: after 1 week, color turns green, after 2 weeks, can substantially observe the formation of plumelet.
The proportioning raw materials of inducing culture are: based on b5 minimal medium, add 6-benzyladenine (6-ba) 1.0
～2.0mg/l (appoints such as in 1.0mg/l, 1.2mg/l, 1.4mg/l, 1.5mg/l, 1.6mg/l, 1.8mg/l, 2.0mg/l etc.
Meaning or arbitrarily between the two), 2,4- dichlorphenoxyacetic acids (2,4-d) 0.5～2.0mg/l (such as 0.5mg/l, 1.0mg/l,
In 1.2mg/l, 1.4mg/l, 1.5mg/l, 1.6mg/l, 1.8mg/l, 2.0mg/l etc. arbitrarily or arbitrarily between the two), sucrose
Final concentration is that 90～120g/l (appoints such as in 90g/l, 95g/l, 100g/l, 105g/l, 110g/l, 115g/l, 120g/l etc.
Meaning or arbitrarily between the two), the final concentration of agar is that 12～15g/l (appoints such as in 12g/l, 13g/l, 14g/l, 15g/l etc.
Meaning or arbitrarily between the two), ph5.8；
The composition of above-mentioned b5 minimal medium (without agar and saccharide) is:
A great number of elements, comprising:
Potassium nitrate (kno3): 2500mg/l, magnesium sulfate (mgso4·7h2O): 250mg/l, calcium chloride (cacl2·2h2O):
750mg/l, ammonium sulfate ((nh4)2so4): 134mg/l, sodium dihydrogen phosphate (nah2po4·h2O): 150mg/l；
Trace element, comprising:
Potassium iodide (ki): 0.75mg/l, boric acid (h3bo3): 3mg/l, manganese sulfate (mnso4·h2O): 5mg/l, zinc sulfate
(znso4·7h2O): 2mg/l, sodium molybdate (na2moo4·2h2O): 0.25mg/l, cobaltous chloride (cocl2·6h2O): 0.025mg/
L, copper sulfate (cuso4·5h2O): 0.025mg/l；
Iron salt, comprising:
Two ethylenediamine hydrate tetraacethyl disodium (na2- edta): 37.3mg/l, ferrous sulfate (feso4·4h2O):
27.8mg/l；
It is organic, comprising:
Inositol: 100mg/l, nicotinic acid: 1mg/l, pyridoxine hydrochloride: 1mg/l, thiamine: 10mg/l.
Step 5, regenerated plant culture:
The plumelet that length is more than 0.5cm is transferred in seedling (taking root) culture medium, cultivates, 3 under 25 DEG C of illumination conditions
～4 weeks plumelets are grown up seedling, obtain regeneration plant within 6～8 weeks.
Root media, is to add the solid culture that edible soft plantation white sugar and agar obtain in 1/10ms minimal medium
Base: the final concentration of wherein edible soft plantation white sugar is 15-30g/l (such as in 15g/l, 18g/l, 20g/l, 25g/l, 28g/l, 29g/
In l, 30g/l etc. arbitrarily or arbitrarily between the two, preferably 25-30g/l), the final concentration of agar is 5-10g/l (such as in 5g/
In l, 6g/l, 7g/l, 8g/l, 9g/l, 10g/l etc. arbitrarily or arbitrarily between the two, preferably 6-7g/l)；ph5.8；Take root training
The solvent of foster base is tap water；1/10ms minimal medium is meant that, a great number of elements, trace element, iron salt and vitamin
Content is the 1/10 of standard ms minimal medium content；
The composition of above-mentioned ms minimal medium (without agar and saccharide) is:
A great number of elements, comprising:
Potassium nitrate (kno3): 1900mg/l, ammonium nitrate (nh4no3): 1650mg/l, magnesium sulfate (mgso4·7h2O):
370mg/l, potassium dihydrogen phosphate (kh2po4): 170mg/l, calcium chloride (cacl2·2h2O): 440mg/l；
Trace element, comprising:
Manganese sulfate (mnso4·h2O): 16.9mg/l, zinc sulfate (znso4·7h2O): 8.6mg/l, boric acid (h3bo3):
6.2mg/l, potassium iodide (ki): 0.83mg/l, sodium molybdate (na2moo4·2h2O): 0.25mg/l, copper sulfate (cuso4·5h2O):
0.025mg/l, cobaltous chloride (cocl2·6h2O): 0.025mg/l；
Iron salt, comprising:
Two ethylenediamine hydrate tetraacethyl disodium (na2- edta): 37.3mg/l, ferrous sulfate (feso4·4h2O):
27.8mg/l；
It is organic, comprising:
Glycine: 2.0mg/l, pyridoxine hydrochloride: 0.5mg/l, thiamine: 0.1mg/l, nicotinic acid: 0.5mg/l, creatine:
100mg/l.
Bulbus Lilii flower pesticide is induced to obtain the cultural method of dh plant by one-step method, a type Bulbus Lilii inductivity is 160-200%,
Planting percent is 30-40%；La type Bulbus Lilii inductivity is 2-200%, and planting percent is 30-100%.
The computational methods of inductivity: embryoid number is divided by being multiplied by absolutely after flower pesticide number；
The computational methods of planting percent: all embryoids (more than the embryoid of 0.5cm) are transferred to first after seedling culture medium
Take turns the seedling number obtaining divided by all embryoids number (including the embryoid less than 0.5cm).
It is noted that the flower pesticide number in indivedual alabastrums is not 6 of standard in actual mechanical process, have plenty of 5
Or 7；Or in same alabastrum, some anther developments are normal, and some anther developments are abnormal；Or it is artificial in operating process
Factor causes flower pesticide number to change.
Step 6, detecting step: the Ploidy detection to the regeneration plant obtaining；
The regeneration plant obtaining in step 5 is carried out Ploidy Identification, using the facscalibur streaming of bd company of the U.S.
Cell instrument (flow cytometric) carries out Ploidy detection, and obtains data, modfit software with cellquest (bd) software
Analysis result.
Assay method operation is as follows:
Take parental plant as comparison, described regeneration plant is plant to be measured；Take adjoining tree and plant group to be measured respectively
Each 0.2 gram of the fresh blade of seedlings cultivating is placed in the culture dish of a diameter of 6cm, is separately added into 2ml lysate, is cut with sharp blade
Broken, 100 mesh cell screen clothes filter, and collect filtrate, after 800 revs/min of centrifugations 6 minutes, discard supernatant, then are separately added into
200 μ lpi (propidiumiodide, propidium iodide, 50 μ g/ml) dye liquor carries out fluorescent labeling to nucleus dna, is placed in dark
Under the conditions of after 20 minutes, carry out plants ploidy identification with flow cytometer.
In said method, the growth conditions of donor plant also have important decisive action to the success or failure of dh breeding, therefore originally
The region that invention is suitable for is the region similar to Beijing weather conditions；Southern gas epidemic disaster obviously higher than Beijing it may be necessary to
Cultural method is done with suitable adjustment；In addition, common lily cultivar ploidy on market is more various, diploid, triploid, four
Times body kind has.Microspore development period, whether success was most important to culture, and the differentiation to developmental stage may be subject to
To the subjective impact of operator, this will also result in inductivity and larger change occur；Incubator temperature change is at 1-2 degree Celsius
Interior, there is no fatal impact to becoming embryo；Various places tap water quality is different, edible sugar slightly difference, but all seedling will not be made
Cheng Tai great affects；Monoploid seedling is slow, weak, can properly increase the nutritional labeling in culture medium.
Said method carries out embryoid induction culture using the flower pesticide of a type, the Bulbus Lilii of la type as explant, can pass through
Plumelet obtains regeneration plant；
Said method adopts suitable inducing culture, and carries out at stress before flower pesticide embryoid and plumelet culture
Reason, improves the induced efficiency of plumelet；
Said method is easy and simple to handle, and medium component is cheap, and using value is high；
Said method has very important significance in Lilies breeding field: when Flos Lilii viriduli is open, anther length typically exists
Between 2-4 centimetre, pollen amount is huge；Because the pigment that lilium pollen contains is non-water-soluble, after loose powder, easily pollute petal, institute
To have had a strong impact on the sight of flower, it also is difficult to clean when people's imprudence rubs pollen on medicated clothing.And manual removal
Flower pesticide can take a substantial amount of time and energy.The kind cultivating WUHUAFEN is one of direction of Lilies breeding, relevant this respect
Research is at present also seldom.Monoploid Bulbus Lilii can not produce normal pollen, it is to avoid the generation of problems, thus improve hundred
The sight closed and commodity.
Further describe the present invention below by way of several specific embodiments, but the invention is not restricted to these embodiments
Be specifically defined.
Embodiment 1:
The present embodiment is that the one-step method of a type Bulbus Lilii " Republican Party " induces Bulbus Lilii flower pesticide to obtain the cultural method of dh plant, bag
Include following steps:
(1), draw materials: collection is in mid-late uninucleate stage, dicaryotic phase, length is each 20 of the alabastrum of 2.5-3.5cm.
(2), anther pretreatment: alabastrum is placed in hermetic container and places 1 day in 4-6 DEG C, remove calyx and petal；Exist again
In superclean bench, with 75% alcohol-pickled 45～60 seconds, vibrate 12～15 points of sterilization with 2.5% liquor natrii hypochloritises afterwards
Clock, finally uses aseptic water washing 3 times, 1～2 minute every time, obtains pretreated flower pesticide.
(3), flower pesticide Stress treatment: pretreated flower pesticide is removed the outside of belly after filigree and lies in downwards inducing culture
On, cultivate 9 days under 33 DEG C of dark conditions, obtain the flower pesticide after Stress treatment.
(4), flower pesticide embryoid and plumelet culture: the flower pesticide after Stress treatment is placed in inducing culture, in 25 DEG C of dark
Under the conditions of culture 4-6 week, obtain the flower pesticide embryoid of white meat worm shape；It is moved into, 1～2 week under illumination condition, obtaining children again
Bud.
The proportioning raw materials of inducing culture are: based on b5 minimal medium, interpolation 6-ba 1.0mg/l, and 2,4-d
1.0mg/l, the final concentration of sucrose is 120g/l, and the final concentration of agar is 14g/l, ph5.8.
(5), regenerated plant culture: the plumelet that length is more than 0.5cm is transferred in seedling (taking root) culture medium, in 25 DEG C
Cultivate under illumination condition, after 3～4 weeks, plumelet is grown up seedling, obtains regeneration plant within 6～8 weeks；
Root media, is to add the solid culture that edible soft plantation white sugar and agar obtain in 1/10ms minimal medium
Base: the final concentration of wherein edible soft plantation white sugar is 25-30g/l, and the final concentration of agar is 6-7g/l；ph5.8；Root media molten
Agent is tap water.
In the present embodiment, a type Bulbus Lilii " Republican Party " inductivity is 169.6%, and planting percent is 36.8%, single in regeneration plant
Times body quantity is 7 plants, amphiploid quantity is 28 plants.
(6), detect: the regeneration plant of acquisition is carried out Ploidy Identification, using the facscalibur streaming of bd company of the U.S.
Cell instrument (flow cytometric) carries out Ploidy detection, and obtains data, modfit software with cellquest (bd) software
Analysis result.
Embodiment 2:
The present embodiment is that the one-step method of a type Bulbus Lilii " Republican Party " induces Bulbus Lilii flower pesticide to obtain the cultural method of dh plant, bag
Include following steps:
(1), draw materials: collection is in mid-late uninucleate stage, dicaryotic phase, length is each 20 of the alabastrum of 2.5-3.5cm.
(2), anther pretreatment: alabastrum is placed in hermetic container and places 1 day in 4-6 DEG C, remove calyx and petal；Exist again
In superclean bench, with 75% alcohol-pickled 45～60 seconds, vibrate 12～15 points of sterilization with 2.5% liquor natrii hypochloritises afterwards
Clock, finally uses aseptic water washing 3 times, 1～2 minute every time, obtains pretreated flower pesticide.
(3), flower pesticide Stress treatment: pretreated flower pesticide is removed the outside of belly after filigree and lies in downwards inducing culture
On, cultivate 9 days under 33 DEG C of dark conditions, obtain the flower pesticide after Stress treatment.
(4), flower pesticide embryoid and plumelet culture: the flower pesticide after Stress treatment is placed in inducing culture, in 25 DEG C of dark
Under the conditions of culture obtain the flower pesticide embryoid of white meat worm shape 4-6 week；It is moved into, 1～2 week under illumination condition, obtaining children again
Bud.
The proportioning raw materials of inducing culture are: based on b5 minimal medium, interpolation 6-ba 2.0mg/l, and 2,4-d
2.0mg/l, the final concentration of sucrose is 120g/l, and the final concentration of agar is 14g/l, ph5.8.
(5), regenerated plant culture: the plumelet that length is more than 0.5cm is transferred in seedling (taking root) culture medium, in 25 DEG C
Cultivate under illumination condition, after 3～4 weeks, plumelet is grown up seedling, obtains regeneration plant within 6～8 weeks.
Root media, is to add the solid culture that edible soft plantation white sugar and agar obtain in 1/10ms minimal medium
Base: the final concentration of wherein edible soft plantation white sugar is 25-30g/l, and the final concentration of agar is 6-7g/l；ph5.8；Root media molten
Agent is tap water；
In the present embodiment, a type Bulbus Lilii " Republican Party " inductivity is 176.8%, and planting percent is 37.4%, single in regeneration plant
Times body quantity is 4 plants, amphiploid quantity is 33 plants.
(6), detect: the regeneration plant of acquisition is carried out Ploidy Identification, using the facscalibur streaming of bd company of the U.S.
Cell instrument (flow cytometric) carries out Ploidy detection, and obtains data, modfit software with cellquest (bd) software
Analysis result.The flow cytomery result figure of the regeneration plant of " Republican Party " Bulbus Lilii that Fig. 2 obtains for the present embodiment；Wherein
Left figure represents monoploid, and right figure represents diploid.
Embodiment 3:
The present embodiment is that the one-step method of la type Bulbus Lilii " red core " induces Bulbus Lilii flower pesticide to obtain the cultural method of dh plant, including
Following steps:
(1), draw materials: collection is in mid-late uninucleate stage, dicaryotic phase, length is each 20 of the alabastrum of 2.5-3.5cm.
(2), anther pretreatment: alabastrum is placed in hermetic container and places 3 days in 4-6 DEG C, remove calyx and petal；Exist again
In superclean bench, with 75% alcohol-pickled 45～60 seconds, vibrate 12～15 points of sterilization with 2.5% liquor natrii hypochloritises afterwards
Clock, finally uses aseptic water washing 3 times, 1～2 minute every time, obtains pretreated flower pesticide.
(3), flower pesticide Stress treatment: pretreated flower pesticide is removed the outside of belly after filigree and lies in downwards inducing culture
On, cultivate 3 days under 33 DEG C of dark conditions, obtain the flower pesticide after Stress treatment.
(4), flower pesticide embryoid and plumelet culture: the flower pesticide after Stress treatment is placed in inducing culture, in 25 DEG C of dark
Under the conditions of culture obtain the flower pesticide embryoid of white meat worm shape 4-6 week；It is moved into, 1～2 week under illumination condition, obtaining children again
Bud.
The proportioning raw materials of inducing culture are: based on b5 minimal medium, interpolation 6-ba 1.0mg/l, and 2,4-d
1.0mg/l, the final concentration of sucrose is 120g/l, and the final concentration of agar is 14g/l, ph5.8.
(5), regenerated plant culture: the plumelet that length is more than 0.5cm is transferred in seedling (taking root) culture medium, in 25 DEG C
Cultivate under illumination condition, after 3～4 weeks, plumelet is grown up seedling, obtains regeneration plant within 6～8 weeks.
Root media, is to add the solid culture that edible soft plantation white sugar and agar obtain in 1/10ms minimal medium
Base: the final concentration of wherein edible soft plantation white sugar is 25-30g/l, and the final concentration of agar is 6-7g/l；ph5.8；Root media molten
Agent is tap water；
In the present embodiment, la type Bulbus Lilii " red core " inductivity is 3.4%, and planting percent is 100%, monoploid in regeneration plant
Quantity is 1 plant, amphiploid quantity is 1 plant.
(6), detect: the regeneration plant of acquisition is carried out Ploidy Identification, using the facscalibur streaming of bd company of the U.S.
Cell instrument (flow cytometric) carries out Ploidy detection, and obtains data, modfit software with cellquest (bd) software
Analysis result.
The flow cytomery result figure of the regeneration plant of " red core " Bulbus Lilii that Fig. 3 obtains for the present embodiment；Wherein left
Figure represents monoploid, and right figure represents diploid.
Embodiment 4:
The present embodiment is that the one-step method of la type Bulbus Lilii " red core " induces Bulbus Lilii flower pesticide to obtain the cultural method of dh plant, including
Following steps:
(1), draw materials: collection is in mid-late uninucleate stage, dicaryotic phase, length is each 20 of the alabastrum of 2.5-3.5cm.
(2), anther pretreatment: alabastrum is placed in hermetic container and places 3 days in 4-6 DEG C, remove calyx and petal；Exist again
In superclean bench, with 75% alcohol-pickled 45～60 seconds, vibrate 12～15 points of sterilization with 2.5% liquor natrii hypochloritises afterwards
Clock, finally uses aseptic water washing 3 times, 1～2 minute every time, obtains pretreated flower pesticide.
(3), flower pesticide Stress treatment: pretreated flower pesticide is removed the outside of belly after filigree and lies in downwards inducing culture
On, cultivate 3 days under 33 DEG C of dark conditions, obtain the flower pesticide after Stress treatment.
(4), flower pesticide embryoid and plumelet culture: the flower pesticide after Stress treatment is placed in inducing culture, in 25 DEG C of dark
Under the conditions of culture obtain the flower pesticide embryoid of white meat worm shape 4-6 week；It is moved into, 1～2 week under illumination condition, obtaining children again
Bud.
The proportioning raw materials of inducing culture are: based on b5 minimal medium, interpolation 6-ba 2.0mg/l, and 2,4-d
2.0mg/l, the final concentration of sucrose is 120g/l, and the final concentration of agar is 14g/l, ph5.8.
(5), regenerated plant culture: the plumelet that length is more than 0.5cm is transferred in seedling (taking root) culture medium, in 25 DEG C
Cultivate under illumination condition, after 3～4 weeks, plumelet is grown up seedling, obtains regeneration plant within 6～8 weeks.
Root media, is to add the solid culture that edible soft plantation white sugar and agar obtain in 1/10ms minimal medium
Base: the final concentration of wherein edible soft plantation white sugar is 25-30g/l, and the final concentration of agar is 6-7g/l；ph5.8；Root media molten
Agent is tap water；
In the present embodiment, la type Bulbus Lilii " red core " inductivity is 15.5%, and planting percent is 66.7%, single times in regeneration plant
Body quantity is 1 plant, amphiploid quantity is 5 plants.
(6), detect: the regeneration plant of acquisition is carried out Ploidy Identification, using the facscalibur streaming of bd company of the U.S.
Cell instrument (flow cytometric) carries out Ploidy detection, and obtains data, modfit software with cellquest (bd) software
Analysis result.
Embodiment 5:
The present embodiment is that the one-step method of la type Bulbus Lilii " red core " induces Bulbus Lilii flower pesticide to obtain the cultural method of dh plant, including
Following steps:
(1), draw materials: collection is in mid-late uninucleate stage, dicaryotic phase, length is each 20 of the alabastrum of 2.5-3.5cm.
(2), anther pretreatment: alabastrum is placed in hermetic container and places 3 days in 4-6 DEG C, remove calyx and petal；Exist again
In superclean bench, with 75% alcohol-pickled 45～60 seconds, vibrate 12～15 points of sterilization with 2.5% liquor natrii hypochloritises afterwards
Clock, finally uses aseptic water washing 3 times, 1～2 minute every time, obtains pretreated flower pesticide；
(3), flower pesticide Stress treatment: pretreated flower pesticide is removed the outside of belly after filigree and lies in downwards inducing culture
On, cultivate 3 days under 33 DEG C of dark conditions, obtain the flower pesticide after Stress treatment.
(4), flower pesticide embryoid and plumelet culture: the flower pesticide after Stress treatment is placed in inducing culture, in 25 DEG C of dark
Under the conditions of culture obtain the flower pesticide embryoid of white meat worm shape 4-6 week；It is moved into, 1～2 week under illumination condition, obtaining children again
Bud.
The proportioning raw materials of inducing culture are: based on b5 minimal medium, interpolation 6-ba 2.0mg/l, and 2,4-d
0.5mg/l, the final concentration of sucrose is 120g/l, and the final concentration of agar is 14g/l, ph5.8.
(5), regenerated plant culture: the plumelet that length is more than 0.5cm is transferred in seedling (taking root) culture medium, in 25 DEG C
Cultivate under illumination condition, after 3～4 weeks, plumelet is grown up seedling, obtains regeneration plant within 6～8 weeks.
Root media, is to add the solid culture that edible soft plantation white sugar and agar obtain in 1/10ms minimal medium
Base: the final concentration of wherein edible soft plantation white sugar is 25-30g/l, and the final concentration of agar is 6-7g/l；ph5.8；Root media molten
Agent is tap water；
In the present embodiment, la type Bulbus Lilii " red core " inductivity is 150%, and planting percent is 42.5%, single times in regeneration plant
Body quantity is 3 plants, amphiploid quantity is 34 plants.
(6), detect: the regeneration plant of acquisition is carried out Ploidy Identification, using the facscalibur streaming of bd company of the U.S.
Cell instrument (flow cytometric) carries out Ploidy detection, and obtains data, modfit software with cellquest (bd) software
Analysis result.
Claims (5)
1. one-step method induce Bulbus Lilii flower pesticide obtain dh plant cultural method it is characterised in that: comprise the following steps:
Anther pretreatment step:
After Bulbus Lilii alabastrum is carried out chilling treatment, lift-off processing, disinfects, obtain pretreated flower pesticide；
Flower pesticide Stress treatment step:
Described pretreated flower pesticide is positioned over inducing culture, carries out Stress treatment, obtain the flower pesticide after Stress treatment；
Flower pesticide embryoid and plumelet incubation step:
Flower pesticide after described Stress treatment is placed in inducing culture, cultivates under 25 DEG C of dark conditions, obtain flower pesticide embryoid；
Described flower pesticide embryoid is transferred under illumination condition and cultivates 1～2 week, obtain plumelet；
Regenerated plant culture step:
The described plumelet that length is more than 0.5cm is transferred in seedling culture medium, cultivates and regenerated under 25 DEG C of illumination conditions
Plant；
Described Stress treatment is, when described Bulbus Lilii flower pesticide is derived from a type Bulbus Lilii, by described pretreated flower pesticide in 33 DEG C of dark
Under the conditions of cultivate 9 days；When described Bulbus Lilii flower pesticide is derived from la type Bulbus Lilii, by described pretreated flower pesticide in 33 DEG C of dark conditions
Lower culture 3 days.
2. according to claim 1 one-step method induce Bulbus Lilii flower pesticide obtain dh plant cultural method it is characterised in that: described
Chilling treatment is, when described Bulbus Lilii flower pesticide is derived from a type Bulbus Lilii, Bulbus Lilii alabastrum to be placed 1 day in 4～6 DEG C；When described Flos Lilii viriduli
When medicine is derived from la type Bulbus Lilii, Bulbus Lilii alabastrum is placed 3 days in 4～6 DEG C.
3. according to claim 1 one-step method induce Bulbus Lilii flower pesticide obtain dh plant cultural method it is characterised in that: described
In flower pesticide embryoid and plumelet incubation step, cultivate at least 2 weeks under 25 DEG C of dark conditions.
4. according to claim 1 one-step method induce Bulbus Lilii flower pesticide obtain dh plant cultural method it is characterised in that:
Described inducing culture is, based on b5 minimal medium, interpolation 6-benzyladenine 1.0～2.0mg/l, and 2,4- bis-
Chlorophenoxyacetic acid 0.5～2.0mg/l, the final concentration of sucrose is 90～120g/l, and the final concentration of agar is 12～15g/l.
5. according to claim 1 one-step method induce Bulbus Lilii flower pesticide obtain dh plant cultural method it is characterised in that: described
Seedling culture medium, is to add the solid medium that edible soft plantation white sugar and agar obtain in 1/10ms minimal medium；Wherein eat
It is 25～30g/l with the final concentration of soft plantation white sugar, the final concentration of agar is 6～7g/l, ph value is 5.8；Described seedling culture medium
Solvent is tap water；
Described 1/10ms minimal medium is meant that, the content of a great number of elements, trace element, iron salt and vitamin is standard
The 1/10 of ms minimal medium content.
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| CN102939900A (en) * | 2012-11-22 | 2013-02-27 | 北京市农林科学院 | Method for obtaining (diploid) haploid plant through induction of OT type lily anther and culture medium thereof |
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| CN102939900A (en) * | 2012-11-22 | 2013-02-27 | 北京市农林科学院 | Method for obtaining (diploid) haploid plant through induction of OT type lily anther and culture medium thereof |
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