CN104429967A - 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 and obtain regeneration plant field with tissue culture technique, carry out embryoid induction cultivation in particular to a kind of flower pesticide by the lily using A type, LA type as explant, then embryoid is carried out young shoot cultivation, method that monoploid regenerated plant culture obtains monoploid regeneration plant.
Background technology
Monoploid refers to the individuality or tissue with gametic chromosome number, and the individuality only having a chromosome set to form is called monoploid, and monoploid can obtain double haploid after nature or artificial doubling.Monoploid, double haploid material all have great importance in plant breeding and hereditary basis theoretical research, and can be used for Marker-assisted selection and genetic map drafting, is the receptor population of transgenosis the best.In the practices of breeding, utilize Doubled haploid breeding technology not only can obtain pure lines fast, shorten breeding cycle, and can obtain manyly making a variation widely, particularly recessive character is more easily showed, thus enriches the type of breeding basic material, accelerates breeding process.
The haploid a kind of Main Means of current acquisition is that in vitro anther culture obtains (two) monoploid regeneration plant.Generally obtain regeneration plant by anther culture and have two kinds of occurring modes, i.e. embryoid development pathway (direct development approach) and healing tissue development's approach (indrect development approach).Embryoid development pathway refers to that microspore direct development becomes the embryoid with radicle plumular axis plumule structure, and embryoid can grow into normal plant on common MS or 1/2MS medium.Healing tissue development's approach needs explant to be first placed in evoked callus on dedifferentiation medium, and then the callus derived is placed in the differentiation of differential medium being induced leaf bud, finally transfer to breaking up the indefinite bud obtained on seedling medium.Therefore, we are called for short embryoid development pathway for " one-step method ", and healing tissue development's approach is " two step method ".
The report being trained merit about lily flower pesticide published both at home and abroad has only 3 sections of (Han DongSheng 1997; Chu Yunxia 2001; Xiu-Li Han 2010), these 3 sections of documents are all the regeneration plants obtained by healing tissue development's approach, also do not see the report successfully obtaining lily (two) haplobiont via embryoid development pathway at present.Two kinds of methods are compared, and change the medium of newtype, not only reduce materials cost because " one-step method " does not need in the Induction Process of regeneration plant, and save about 1/3rd artificial.Under the background that qualified manpower resource worsening shortages and human cost constantly increase, " one-step method " has " two step method " unrivaled advantage in the extension process of industrialization.
Although (two) haploid breeding technology (that is: DH breeding technique) is as far back as being just born decades ago, up to now, the minute quantity plants such as corn, Chinese cabbage, rape are only had to have ripe DH breeding technique.Trace it to its cause, the difficult point of DH breeding technique is, plant is different, and abductive approach is different; In same plant, genotype is different, different to the induction mode reaction of the same terms.Existing similitude between plant, variant again.This just determines some successful experience can bring reference, and some successful experience can allow people go astray on the contrary.On the other hand, except genotype, successful DH breeding technique also will be forced the permutation and combination of concentration between processing mode and time, basal medium type, hormone kind and concentration, multiple hormone, carbon source kind and concentration, the impact of training method etc. series of factors, these factors of influence are relatively independent each other, therefore be only all in OK range at all factors of influence, just likely succeed.
Lily (Lilium spp.) is the perennial flowering bulb plant of Liliaceae lilium, is one of world-renowned commercial floriculture, occupies very consequence at Market of Fresh Cut Flower.In recent years, A type, LA type lily become lily type comparatively popular on domestic flowers market gradually.The new varieties of current lily get more and more, to the research of the excellent kind of economic characters with utilize the emphasis becoming Lilies breeding work gradually.Mainly there is two problems in hybrid lily breeding, one is that breeding cycle is long, and will experience the 2-3 year from planting seed to blooming, growth cycle is long; Two is interspecific cross difficulties.Lily gene element is A, L, O, T Four Great families, the problem of hybrid lily breeding ubiquity incompatibility, difficulty of being not only fertilized, and after fertilization is difficult to obtain seed, and therefore DH breeding technique can become the another kind of Main Means of Lilies breeding in theory.So the DH breeding technique of lily has great importance in cultivation independent intellectual property right kind.
Chinese patent " induction OT type lily flower pesticide obtains method and the medium thereof of (two) haplobiont " (patent No.: ZL201210479417.1, the applying date: on November 22nd, 2012, Shen Qing Publication day: on February 27th, 2013, authorized announcement date: on January 29th, 2014) disclose a kind of comprise low temperature, dark culturing, Heat thermostability induction OT type lily flower pesticide obtain the method for (two) haplobiont, and the inducing culture containing 2,4-D, 6-BA.But the method is only applicable to the lily of OT type, and be not suitable for the lily of A type, LA type.OT type lily heat-shock temperature in this patent is 35-37 (DEG C), 7-9 days, but the ability of A type and LA type lily flower pesticide tolerance low temperature is more weak, under this hot shock condition, microspore is all dead, cannot regeneration plant be obtained, the DH breeding method of applicable OT type is described and is not suitable for A type and LA type.Find A type through many experiments, LA type lily can tolerate the high temperature of 33 DEG C, but OT type lily is insensitive to the high temperature of 33 DEG C, so same can not be adopted to coerce mode.
Therefore, all need in scientific research and practice a kind of A of being applicable to type, LA type lily, improve selection and breeding efficiency, simultaneously also based on research provide desirable acceptor material, one-step method induction lily flower pesticide obtains the cultural method of DH plant.
Summary of the invention
The invention provides a kind of by the flower pesticide of the lily of A type, LA type is carried out embryoid induction cultivation as explant on inducing culture, then embryoid is transferred to the method for seedling medium being cultivated and obtaining regeneration plant.The present invention can induce the microspore development of flower pesticide inside to be complete monoploid or double haploid, for lily DH breeding provides the multiple breeding material isozygotied, create more cenospecies matter, improve selection and breeding efficiency, simultaneously also based on research provide desirable acceptor material.
The object of the invention is to be achieved through the following technical solutions:
One-step method induction lily flower pesticide obtains the cultural method of DH plant, comprises the following steps:
Anther pretreatment step: after lily bud is carried out chilling treatment, lift-off processing, disinfecting, 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 young shoot incubation step: the flower pesticide after described Stress treatment is placed in inducing culture, cultivate under 25 DEG C of dark conditions, obtains flower pesticide embryoid; Cultivate 1 ~ 2 week under described flower pesticide embryoid being transferred to illumination condition, obtain young shoot;
Regenerated plant culture step: described young shoot length being greater than 0.5cm is transferred on seedling medium, cultivates under 25 DEG C of illumination conditions, obtains regeneration plant in 6 ~ 8 weeks.
Further, described lily flower pesticide is from A type, LA type lily.
Further, described chilling treatment is, when described lily flower pesticide is from A type lily, is placed 1 day by lily bud in 4 ~ 6 DEG C; When described lily flower pesticide is from LA type lily, lily bud is placed 3 days in 4 ~ 6 DEG C.
Further, described Stress treatment is, when described lily flower pesticide is from A type lily, is cultivated 9 days by described pretreated flower pesticide under 33 DEG C of dark conditions; When described lily flower pesticide is from LA type lily, described pretreated flower pesticide is cultivated 3 days under 33 DEG C of dark conditions.
Further, in described flower pesticide embryoid and young shoot incubation step, under 25 DEG C of dark conditions, cultivated at least 2 weeks.
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 the solid culture medium that the edible soft white sugar of interpolation and agar obtain in 1/10MS minimal medium: wherein the final concentration of edible soft 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 running water.
Further, the implication of described 1/10MS minimal medium is, the content of macroelement, trace element, molysite and vitamin is 1/10 of standard MS minimal medium content.
Compared with the prior art the present invention has following beneficial effect:
1, the present invention adopts the flower pesticide of the lily of A type, LA type to carry out embryoid induction cultivation as explant, can obtain regeneration plant by young shoot.
2, the present invention adopts suitable inducing culture, and carries out Stress treatment before flower pesticide embryoid and young shoot are cultivated, and improves the induced efficiency of young shoot.
3, 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 lily is open, anther length is generally between 2-4 centimetre, and pollen amount is huge; The pigment contained due to lilium pollen is non-water-soluble, easily pollutes petal after loose powder, so had a strong impact on the sight of flower, is also difficult to clean when pollen rubs on clothing by people because of carelessness.And the time and efforts of manual removal's flower pesticide meeting at substantial.The kind of cultivating without pollen is one of direction of Lilies breeding, about the research of this respect is also little at present.Monoploid lily can not produce normal pollen, avoids the generation of problems, thus improves sight and the commodity of lily.
Accompanying drawing explanation
Fig. 1 is the photo of the flower pesticide embryoid that flower pesticide embryoid of the present invention and young shoot incubation step obtain.
Fig. 2 is the flow cytomery result figure of the regeneration plant of " Republican Party " lily obtained in embodiment 2; Wherein left figure represents monoploid, and right figure represents dliploid.
Fig. 3 is the flow cytomery result figure of the regeneration plant of " red core " lily obtained in embodiment 3; Wherein left figure represents monoploid, and right figure represents dliploid.
Embodiment
One-step method induction lily flower pesticide obtains the cultural method of DH plant, embryoid induction cultivation is carried out as explant by the flower pesticide of the lily using A type, LA type, again by the method that the embryoid of acquisition carries out young shoot cultivation, regenerated plant culture obtains regeneration plant, comprise the following steps:
Step one, to draw materials:
Gather each 20 of the bud being in mid-late uninucleate stage, the A type of dicaryotic phase, LA type lily; The bud morphological feature being in above-mentioned period is: bud is in green, and parcel is tight, flower pesticide dark green; The visible most microspore of microscopy is in mid-late uninucleate stage, and small part is in dicaryotic phase; Because genotype is different, bud length is also different, is within the scope of 2.5-3.5cm.
Wherein, A type lily cultivar comprises: lanzhou lily, the Republican Party (Gironde), shady deal (Blackout); LA type lily cultivar comprises: Baptiste sieve (Batistero), Da Nuo (Ceb Dazzle), red core (Fangio).
Step 2, anther pretreatment:
Bud is placed in closed container after carrying out chilling treatment, lift-off processing, disinfecting, obtains pretreated flower pesticide.
Chilling treatment is: when lily flower pesticide is A type, is placed 1 day by lily bud in 4-6 DEG C; When lily flower pesticide is LA type, lily bud is placed 3 days in 4-6 DEG C;
Lift-off processing is: before disinfecting, remove calyx and petal, after disinfecting, remove filigree;
Disinfect for: in superclean bench, with the bud 45 ~ 60 seconds of 75% alcohol-pickled removal calyx and petal, to vibrate sterilization 12 ~ 15 minutes with the liquor natrii hypochloritis of 2.5% afterwards, then use aseptic water washing 3 times, each 1 ~ 2 minute.
Step 3, flower pesticide Stress treatment:
After pretreated flower pesticide is removed filigree, the outside of belly lies in downwards on inducing culture, carries out Stress treatment, obtains the flower pesticide after Stress treatment.
Stress treatment is: when lily flower pesticide is from A type lily, cultivated 9 days by pretreated flower pesticide under 33 DEG C of dark conditions; When lily flower pesticide is from LA type lily, pretreated flower pesticide is cultivated 3 days under 33 DEG C of dark conditions.
Step 4, flower pesticide embryoid and young shoot are cultivated:
Flower pesticide after Stress treatment is placed in inducing culture, at least cultivate under 25 DEG C of dark conditions 2 weeks (such as in 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks etc. arbitrarily or arbitrarily between the two, be preferably 2 ~ 4 weeks) obtain white meat worm shape, length is the flower pesticide embryoid (as shown in Figure 1) of the maturation of more than 5mm; Again this flower pesticide embryoid to be transferred under illumination condition 1 ~ 2 week, obtain young shoot: after 1 week, color turns green, obviously can observe the formation of young shoot after 2 weeks.
The pulp furnish of inducing culture is: based on B5 minimal medium, add 6-benzyladenine (6-BA) 1.0 ~ 2.0mg/L (such as at 1.0mg/L, 1.2mg/L, 1.4mg/L, 1.5mg/L, 1.6mg/L, 1.8mg/L, in 2.0mg/L etc. arbitrarily or arbitrarily between the two), 2, 4-dichlorphenoxyacetic acid (2, 4-D) 0.5 ~ 2.0mg/L is (such as at 0.5mg/L, 1.0mg/L, 1.2mg/L, 1.4mg/L, 1.5mg/L, 1.6mg/L, 1.8mg/L, in 2.0mg/L etc. arbitrarily or arbitrarily between the two), the final concentration of sucrose is that 90 ~ 120g/L is (such as at 90g/L, 95g/L, 100g/L, 105g/L, 110g/L, 115g/L, in 120g/L etc. arbitrarily or arbitrarily between the two), the final concentration of agar is that 12 ~ 15g/L is (such as at 12g/L, 13g/L, 14g/L, in 15g/L etc. arbitrarily or arbitrarily between the two), pH5.8,
The composition of above-mentioned B5 minimal medium (not containing agar and carbohydrate) is:
Macroelement, comprising:
Potassium nitrate (KNO
3): 2500mg/L, magnesium sulfate (MgSO
47H
2o): 250mg/L, calcium chloride (CaCl
22H
2o): 750mg/L, ammonium sulfate ((NH
4)
2sO
4): 134mg/L, sodium dihydrogen phosphate (NaH
2pO
4h
2o): 150mg/L;
Trace element, comprising:
Potassium iodide (KI): 0.75mg/L, boric acid (H
3bO
3): 3mg/L, manganese sulphate (MnSO
4h
2o): 5mg/L, zinc sulphate (ZnSO
47H
2o): 2mg/L, sodium molybdate (Na
2moO
42H
2o): 0.25mg/L, cobalt chloride (CoCl
26H
2o): 0.025mg/L, copper sulphate (CuSO
45H
2o): 0.025mg/L;
Molysite, comprising:
Two ethylenediamine hydrate tetraacethyl disodium (Na
2-EDTA): 37.3mg/L, ferrous sulfate (FeSO
44H
2o): 27.8mg/L;
Organic, comprising:
Inositol: 100mg/L, nicotinic acid: 1mg/L, puridoxine hydrochloride: 1mg/L, thiamine: 10mg/L.
Step 5, regenerated plant culture:
Young shoot length being greater than 0.5cm is transferred on seedling (taking root) medium, cultivates under 25 DEG C of illumination conditions, and 3 ~ 4 weeks young shoots are grown up seedling, within 6 ~ 8 weeks, obtain regeneration plant.
Root media, in 1/10MS minimal medium, add the solid culture medium that edible soft white sugar and agar obtains: wherein the final concentration of edible soft white sugar be 15-30g/L (such as in 15g/L, 18g/L, 20g/L, 25g/L, 28g/L, 29g/L, 30g/L etc. arbitrarily or arbitrarily between the two, be preferably 25-30g/L), the final concentration of agar is 5-10g/L (such as in 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L etc. arbitrarily or arbitrarily between the two, be preferably 6-7g/L); PH5.8; The solvent of root media is running water; The implication of 1/10MS minimal medium is, the content of macroelement, trace element, molysite and vitamin is 1/10 of standard MS minimal medium content;
The composition of above-mentioned MS minimal medium (not containing agar and carbohydrate) is:
Macroelement, comprising:
Potassium nitrate (KNO
3): 1900mg/L, ammonium nitrate (NH
4nO
3): 1650mg/L, magnesium sulfate (MgSO
47H
2o): 370mg/L, potassium dihydrogen phosphate (KH
2pO
4): 170mg/L, calcium chloride (CaCl
22H
2o): 440mg/L;
Trace element, comprising:
Manganese sulphate (MnSO
4h
2o): 16.9mg/L, zinc sulphate (ZnSO
47H
2o): 8.6mg/L, boric acid (H
3bO
3): 6.2mg/L, potassium iodide (KI): 0.83mg/L, sodium molybdate (Na
2moO
42H
2o): 0.25mg/L, copper sulphate (CuSO
45H
2o): 0.025mg/L, cobalt chloride (CoCl
26H
2o): 0.025mg/L;
Molysite, comprising:
Two ethylenediamine hydrate tetraacethyl disodium (Na
2-EDTA): 37.3mg/L, ferrous sulfate (FeSO
44H
2o): 27.8mg/L;
Organic, comprising:
Glycine: 2.0mg/L, puridoxine hydrochloride: 0.5mg/L, thiamine: 0.1mg/L, nicotinic acid: 0.5mg/L, creatine: 100mg/L.
Obtained the cultural method of DH plant by one-step method induction lily flower pesticide, A type lily inductivity is 160-200%, and planting percent is 30-40%; LA type lily 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: the seedling number that after all embryoids (being greater than the embryoid of 0.5cm) are transferred to seedling medium, the first run obtains is divided by all embryoids number (comprising the embryoid being less than 0.5cm).
The flower pesticide number that it is noted that in indivedual bud in actual mechanical process is not 6 of standard, has plenty of 5 or 7; Or in same bud, some anther developments are normal, and some anther developments are abnormal; Or human factor causes flower pesticide number to change in operating process.
Step 6, detecting step: to the Ploidy detection of the regeneration plant obtained;
The regeneration plant obtained in step 5 is carried out Ploidy Identification, the FACSCalibur flow cytometer (Flow cytometric) of U.S. company BD is adopted to carry out Ploidy detection, and obtain data with CellQuest (BD) software, ModFit software analysis result.
Assay method operation is as follows:
Get parental plant in contrast, described regeneration plant is plant to be measured; Each 0.2 gram of the fresh blade getting adjoining tree and plant plantlet in vitro to be measured is respectively placed in the culture dish that diameter is 6cm, add 2ml lysate respectively, with the chopping of sharp blade, 100 order cell screen filtrations, collect filtrate, through 800 revs/min after centrifugal 6 minutes, discard supernatant, add 200 μ lPI (Propidiumiodide more respectively, propidium iodide, 50 μ g/ml) dye liquor carries out fluorescence labeling to nucleus DNA, be placed in dark condition after lower 20 minutes, carry out plant Ploidy Identification with flow cytometer.
In said method, the growth conditions of donor plant also has important decisive action to the success or failure of DH breeding, and the region that therefore the present invention is applicable to is the region similar to Beijing weather conditions; South gas epidemic disaster, all apparently higher than Beijing, may need to do suitable adjustment to cultural method; In addition, lily cultivar ploidy common on market is more various, and dliploid, triploid, tetraploid kind have.Whether microspore development is successful most important to cultivation for period, and may be subject to the impact of operator's subjectivity to the differentiation of developmental stage, and this also can cause inductivity to there will be larger change; Incubator variations in temperature, in 1-2 degree Celsius, does not have fatal impact to one-tenth embryo; Various places tap water quality is different, and edible sugar is difference slightly, but all can not cause too large impact to seedling; Monoploid seedling is slow, weak, suitably can improve the nutrient component in medium.
Said method adopts the flower pesticide of the lily of A type, LA type to carry out embryoid induction cultivation as explant, can obtain regeneration plant by young shoot;
Said method adopts suitable inducing culture, and carries out Stress treatment before flower pesticide embryoid and young shoot are cultivated, and improves the induced efficiency of young shoot;
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 lily is open, anther length is generally between 2-4 centimetre, and pollen amount is huge; The pigment contained due to lilium pollen is non-water-soluble, easily pollutes petal after loose powder, so had a strong impact on the sight of flower, is also difficult to clean when pollen rubs on clothing by people because of carelessness.And the time and efforts of manual removal's flower pesticide meeting at substantial.The kind of cultivating without pollen is one of direction of Lilies breeding, about the research of this respect is also little at present.Monoploid lily can not produce normal pollen, avoids the generation of problems, thus improves sight and the commodity of lily.
Further describe the present invention below by way of several specific embodiment, but the invention is not restricted to the concrete definition of these embodiments.
Embodiment 1:
The present embodiment is the cultural method of the one-step method induction lily flower pesticide acquisition DH plant of A type lily " Republican Party ", comprises the following steps:
(1), draw materials: gather and be in mid-late uninucleate stage, dicaryotic phase, length is each 20 of the bud of 2.5-3.5cm.
(2), anther pretreatment: bud is placed in closed container and places 1 day in 4-6 DEG C, remove calyx and petal; Again in superclean bench, with 75% alcohol-pickled 45 ~ 60 seconds, to vibrate sterilization 12 ~ 15 minutes with 2.5% liquor natrii hypochloritis afterwards, finally use aseptic water washing 3 times, each 1 ~ 2 minute, obtain pretreated flower pesticide.
(3), flower pesticide Stress treatment: after pretreated flower pesticide is removed filigree, the outside of belly lies in downwards on inducing culture, cultivates 9 days, obtain the flower pesticide after Stress treatment under 33 DEG C of dark conditions.
(4), flower pesticide embryoid and young shoot are cultivated: the flower pesticide after Stress treatment is placed in inducing culture, cultivates 4-6 week under 25 DEG C of dark conditions, obtain the flower pesticide embryoid of white meat worm shape; To be moved on under illumination condition 1 ~ 2 week again, obtained young shoot.
The pulp furnish of inducing culture is: based on B5 minimal medium, and add 6-BA 1.0mg/L, 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: young shoot length being greater than 0.5cm is transferred on seedling (taking root) medium, cultivates under 25 DEG C of illumination conditions, and young shoot is grown up seedling after 3 ~ 4 weeks, obtains regeneration plant in 6 ~ 8 weeks;
Root media is the solid culture medium that the edible soft white sugar of interpolation and agar obtain in 1/10MS minimal medium: wherein the final concentration of edible soft white sugar is 25-30g/L, and the final concentration of agar is 6-7g/L; PH5.8; The solvent of root media is running water.
In the present embodiment, A type lily " Republican Party " inductivity is 169.6%, and planting percent is 36.8%, and in regeneration plant, monoploid quantity is 7 strains, amphiploid quantity is 28 strains.
(6), detect: the regeneration plant of acquisition is carried out Ploidy Identification, the FACSCalibur flow cytometer (Flow cytometric) of U.S. company BD is adopted to carry out Ploidy detection, and obtain data with CellQuest (BD) software, ModFit software analysis result.
Embodiment 2:
The present embodiment is the cultural method of the one-step method induction lily flower pesticide acquisition DH plant of A type lily " Republican Party ", comprises the following steps:
(1), draw materials: gather and be in mid-late uninucleate stage, dicaryotic phase, length is each 20 of the bud of 2.5-3.5cm.
(2), anther pretreatment: bud is placed in closed container and places 1 day in 4-6 DEG C, remove calyx and petal; Again in superclean bench, with 75% alcohol-pickled 45 ~ 60 seconds, to vibrate sterilization 12 ~ 15 minutes with 2.5% liquor natrii hypochloritis afterwards, finally use aseptic water washing 3 times, each 1 ~ 2 minute, obtain pretreated flower pesticide.
(3), flower pesticide Stress treatment: after pretreated flower pesticide is removed filigree, the outside of belly lies in downwards on inducing culture, cultivates 9 days, obtain the flower pesticide after Stress treatment under 33 DEG C of dark conditions.
(4), flower pesticide embryoid and young shoot are cultivated: the flower pesticide after Stress treatment is placed in inducing culture, cultivates the flower pesticide embryoid that 4-6 week obtains white meat worm shape under 25 DEG C of dark conditions; To be moved on under illumination condition 1 ~ 2 week again, obtained young shoot.
The pulp furnish of inducing culture is: based on B5 minimal medium, and add 6-BA 2.0mg/L, 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: young shoot length being greater than 0.5cm is transferred on seedling (taking root) medium, cultivates under 25 DEG C of illumination conditions, and young shoot is grown up seedling after 3 ~ 4 weeks, obtains regeneration plant in 6 ~ 8 weeks.
Root media is the solid culture medium that the edible soft white sugar of interpolation and agar obtain in 1/10MS minimal medium: wherein the final concentration of edible soft white sugar is 25-30g/L, and the final concentration of agar is 6-7g/L; PH5.8; The solvent of root media is running water;
In the present embodiment, A type lily " Republican Party " inductivity is 176.8%, and planting percent is 37.4%, and in regeneration plant, monoploid quantity is 4 strains, amphiploid quantity is 33 strains.
(6), detect: the regeneration plant of acquisition is carried out Ploidy Identification, the FACSCalibur flow cytometer (Flow cytometric) of U.S. company BD is adopted to carry out Ploidy detection, and obtain data with CellQuest (BD) software, ModFit software analysis result.Fig. 2 is the flow cytomery result figure of the regeneration plant of " Republican Party " lily that the present embodiment obtains; Wherein left figure represents monoploid, and right figure represents dliploid.
Embodiment 3:
The present embodiment is the cultural method of the one-step method induction lily flower pesticide acquisition DH plant of LA type lily " red core ", comprises the following steps:
(1), draw materials: gather and be in mid-late uninucleate stage, dicaryotic phase, length is each 20 of the bud of 2.5-3.5cm.
(2), anther pretreatment: bud is placed in closed container and places 3 days in 4-6 DEG C, remove calyx and petal; Again in superclean bench, with 75% alcohol-pickled 45 ~ 60 seconds, to vibrate sterilization 12 ~ 15 minutes with 2.5% liquor natrii hypochloritis afterwards, finally use aseptic water washing 3 times, each 1 ~ 2 minute, obtain pretreated flower pesticide.
(3), flower pesticide Stress treatment: after pretreated flower pesticide is removed filigree, the outside of belly lies in downwards on inducing culture, cultivates 3 days, obtain the flower pesticide after Stress treatment under 33 DEG C of dark conditions.
(4), flower pesticide embryoid and young shoot are cultivated: the flower pesticide after Stress treatment is placed in inducing culture, cultivates the flower pesticide embryoid that 4-6 week obtains white meat worm shape under 25 DEG C of dark conditions; To be moved on under illumination condition 1 ~ 2 week again, obtained young shoot.
The pulp furnish of inducing culture is: based on B5 minimal medium, and add 6-BA 1.0mg/L, 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: young shoot length being greater than 0.5cm is transferred on seedling (taking root) medium, cultivates under 25 DEG C of illumination conditions, and young shoot is grown up seedling after 3 ~ 4 weeks, obtains regeneration plant in 6 ~ 8 weeks.
Root media is the solid culture medium that the edible soft white sugar of interpolation and agar obtain in 1/10MS minimal medium: wherein the final concentration of edible soft white sugar is 25-30g/L, and the final concentration of agar is 6-7g/L; PH5.8; The solvent of root media is running water;
In the present embodiment, LA type lily " red core " inductivity is 3.4%, and planting percent is 100%, and in regeneration plant, monoploid quantity is 1 strain, amphiploid quantity is 1 strain.
(6), detect: the regeneration plant of acquisition is carried out Ploidy Identification, the FACSCalibur flow cytometer (Flow cytometric) of U.S. company BD is adopted to carry out Ploidy detection, and obtain data with CellQuest (BD) software, ModFit software analysis result.
Fig. 3 is the flow cytomery result figure of the regeneration plant of " red core " lily that the present embodiment obtains; Wherein left figure represents monoploid, and right figure represents dliploid.
Embodiment 4:
The present embodiment is the cultural method of the one-step method induction lily flower pesticide acquisition DH plant of LA type lily " red core ", comprises the following steps:
(1), draw materials: gather and be in mid-late uninucleate stage, dicaryotic phase, length is each 20 of the bud of 2.5-3.5cm.
(2), anther pretreatment: bud is placed in closed container and places 3 days in 4-6 DEG C, remove calyx and petal; Again in superclean bench, with 75% alcohol-pickled 45 ~ 60 seconds, to vibrate sterilization 12 ~ 15 minutes with 2.5% liquor natrii hypochloritis afterwards, finally use aseptic water washing 3 times, each 1 ~ 2 minute, obtain pretreated flower pesticide.
(3), flower pesticide Stress treatment: after pretreated flower pesticide is removed filigree, the outside of belly lies in downwards on inducing culture, cultivates 3 days, obtain the flower pesticide after Stress treatment under 33 DEG C of dark conditions.
(4), flower pesticide embryoid and young shoot are cultivated: the flower pesticide after Stress treatment is placed in inducing culture, cultivates the flower pesticide embryoid that 4-6 week obtains white meat worm shape under 25 DEG C of dark conditions; To be moved on under illumination condition 1 ~ 2 week again, obtained young shoot.
The pulp furnish of inducing culture is: based on B5 minimal medium, and add 6-BA 2.0mg/L, 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: young shoot length being greater than 0.5cm is transferred on seedling (taking root) medium, cultivates under 25 DEG C of illumination conditions, and young shoot is grown up seedling after 3 ~ 4 weeks, obtains regeneration plant in 6 ~ 8 weeks.
Root media is the solid culture medium that the edible soft white sugar of interpolation and agar obtain in 1/10MS minimal medium: wherein the final concentration of edible soft white sugar is 25-30g/L, and the final concentration of agar is 6-7g/L; PH5.8; The solvent of root media is running water;
In the present embodiment, LA type lily " red core " inductivity is 15.5%, and planting percent is 66.7%, and in regeneration plant, monoploid quantity is 1 strain, amphiploid quantity is 5 strains.
(6), detect: the regeneration plant of acquisition is carried out Ploidy Identification, the FACSCalibur flow cytometer (Flow cytometric) of U.S. company BD is adopted to carry out Ploidy detection, and obtain data with CellQuest (BD) software, ModFit software analysis result.
Embodiment 5:
The present embodiment is the cultural method of the one-step method induction lily flower pesticide acquisition DH plant of LA type lily " red core ", comprises the following steps:
(1), draw materials: gather and be in mid-late uninucleate stage, dicaryotic phase, length is each 20 of the bud of 2.5-3.5cm.
(2), anther pretreatment: bud is placed in closed container and places 3 days in 4-6 DEG C, remove calyx and petal; Again in superclean bench, with 75% alcohol-pickled 45 ~ 60 seconds, to vibrate sterilization 12 ~ 15 minutes with 2.5% liquor natrii hypochloritis afterwards, finally use aseptic water washing 3 times, each 1 ~ 2 minute, obtain pretreated flower pesticide;
(3), flower pesticide Stress treatment: after pretreated flower pesticide is removed filigree, the outside of belly lies in downwards on inducing culture, cultivates 3 days, obtain the flower pesticide after Stress treatment under 33 DEG C of dark conditions.
(4), flower pesticide embryoid and young shoot are cultivated: the flower pesticide after Stress treatment is placed in inducing culture, cultivates the flower pesticide embryoid that 4-6 week obtains white meat worm shape under 25 DEG C of dark conditions; To be moved on under illumination condition 1 ~ 2 week again, obtained young shoot.
The pulp furnish of inducing culture is: based on B5 minimal medium, and add 6-BA 2.0mg/L, 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: young shoot length being greater than 0.5cm is transferred on seedling (taking root) medium, cultivates under 25 DEG C of illumination conditions, and young shoot is grown up seedling after 3 ~ 4 weeks, obtains regeneration plant in 6 ~ 8 weeks.
Root media is the solid culture medium that the edible soft white sugar of interpolation and agar obtain in 1/10MS minimal medium: wherein the final concentration of edible soft white sugar is 25-30g/L, and the final concentration of agar is 6-7g/L; PH5.8; The solvent of root media is running water;
In the present embodiment, LA type lily " red core " inductivity is 150%, and planting percent is 42.5%, and in regeneration plant, monoploid quantity is 3 strains, amphiploid quantity is 34 strains.
(6), detect: the regeneration plant of acquisition is carried out Ploidy Identification, the FACSCalibur flow cytometer (Flow cytometric) of U.S. company BD is adopted to carry out Ploidy detection, and obtain data with CellQuest (BD) software, ModFit software analysis result.
Claims (8)
1. one-step method induction lily flower pesticide obtains the cultural method of DH plant, it is characterized in that: comprise the following steps:
Anther pretreatment step:
After lily bud is carried out chilling treatment, lift-off processing, disinfecting, 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 young shoot 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; Cultivate 1 ~ 2 week under described flower pesticide embryoid being transferred to illumination condition, obtain young shoot;
Regenerated plant culture step:
Described young shoot length being greater than 0.5cm is transferred on seedling medium, cultivates and obtain regeneration plant under 25 DEG C of illumination conditions.
2. one-step method induction lily flower pesticide obtains the cultural method of DH plant according to claim 1, it is characterized in that: described lily flower pesticide is from A type, LA type lily.
3. according to claim 1 or 2, one-step method induction lily flower pesticide obtains the cultural method of DH plant, it is characterized in that: described chilling treatment is, when described lily flower pesticide is from A type lily, is placed 1 day by lily bud in 4 ~ 6 DEG C; When described lily flower pesticide is from LA type lily, lily bud is placed 3 days in 4 ~ 6 DEG C.
4. according to claim 1 or 2, one-step method induction lily flower pesticide obtains the cultural method of DH plant, it is characterized in that: described Stress treatment is, when described lily flower pesticide is from A type lily, described pretreated flower pesticide is cultivated 9 days under 33 DEG C of dark conditions; When described lily flower pesticide is from LA type lily, described pretreated flower pesticide is cultivated 3 days under 33 DEG C of dark conditions.
5. according to claim 1 or 2, one-step method induction lily flower pesticide obtains the cultural method of DH plant, it is characterized in that: in described flower pesticide embryoid and young shoot incubation step, cultivated at least 2 weeks under 25 DEG C of dark conditions.
6. according to claim 1 or 2, one-step method induction lily flower pesticide obtains the cultural method of DH plant, it is characterized in that:
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.
7. according to claim 1 or 2, one-step method induction lily flower pesticide obtains the cultural method of DH plant, it is characterized in that: described root media, the solid culture medium that the edible soft white sugar of interpolation and agar obtain in 1/10MS minimal medium: wherein the final concentration of edible soft 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 running water.
8. one-step method induction lily flower pesticide obtains the cultural method of DH plant according to claim 7, it is characterized in that: the implication of described 1/10MS minimal medium is, the content of macroelement, trace element, molysite and vitamin is 1/10 of standard MS minimal medium content.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105794653A (en) * | 2016-05-26 | 2016-07-27 | 江苏强农农业技术服务有限公司 | Anther culture detoxification method of edible lily |
| CN116806694A (en) * | 2023-08-16 | 2023-09-29 | 金陵科技学院 | Eggplant doubled haploid anther culture method |
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| CN101116424A (en) * | 2007-09-04 | 2008-02-06 | 云南省农业科学院花卉研究所 | Highly effective lily bulblet inducement culture method |
| JP4169213B2 (en) * | 2005-12-27 | 2008-10-22 | 国立大学法人山口大学 | Heterogeneous chromosome-added plant with the ability to transmit next-generation single chromosomes from different species |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP4169213B2 (en) * | 2005-12-27 | 2008-10-22 | 国立大学法人山口大学 | Heterogeneous chromosome-added plant with the ability to transmit next-generation single chromosomes from different species |
| CN101116424A (en) * | 2007-09-04 | 2008-02-06 | 云南省农业科学院花卉研究所 | Highly effective lily bulblet inducement culture method |
| 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 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105794653A (en) * | 2016-05-26 | 2016-07-27 | 江苏强农农业技术服务有限公司 | Anther culture detoxification method of edible lily |
| CN116806694A (en) * | 2023-08-16 | 2023-09-29 | 金陵科技学院 | Eggplant doubled haploid anther culture method |
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