CN102120980B - Method for culturing homozygous diploid carrot - Google Patents
Method for culturing homozygous diploid carrot Download PDFInfo
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- CN102120980B CN102120980B CN 201010548362 CN201010548362A CN102120980B CN 102120980 B CN102120980 B CN 102120980B CN 201010548362 CN201010548362 CN 201010548362 CN 201010548362 A CN201010548362 A CN 201010548362A CN 102120980 B CN102120980 B CN 102120980B
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Abstract
The invention discloses a method for culturing a homozygous diploid carrot, comprising the following steps: (1) carrying out heat shock treatment on a carrot microspore; (2) culturing the carrot microspore subjected to the heat shock treatment to obtain an embryoid; and (3) carrying out tissue culture on the embryoid to obtain a regeneration plant of the carrot. Experiments prove that the method for culturing the homozygous diploid carrot, which is provided by the invention, has the advantages of higher embryoid forming rate and higher survival rate. The method is suitable for culturing carrots of various gene types; and by utilizing method, the stable homozygous diploid of the carrot can be quickly obtained, and the carrot breeding age limit is greatly shortened to improve the carrot breeding efficiency and facilitate prompting the carrot breeding process. The method disclosed by the invention has a wide application prospect in the field of carrot breeding.
Description
Technical field
The present invention relates to a kind of method of cultivating the zygoid Radix Dauci Sativae.
Background technology
Radix Dauci Sativae is a kind of genetic storehouse narrow raise crop, particularly orange type, and germ plasm resource is more limited.389 parts of local variety resources that China collects have only 30.9% to be the orange type, and majority is yellow, red type.Along with domestic orange Radix Dauci Sativae cultivated area increases year by year, because the unicity of kind, continuous cropping for many years causes the existing large-area disease and pest in domestic some areas to take place, and therefore carries out the seed selection beginning of excellent resources initiative and good breeding material and must go.
The method of initiative new resources is many at present, like means such as distant hybirdization, tissue culture, radiation breeding, transgenics.Numerous results of study show that these methods have lot of advantages; Can cross over kind, belong in addition section between hybridization, initiative geneticist and the required special resource of breeder, but these resources be actual breeding use need be through for a long time; Many reach (Cai Xu more than 20 years; Plant genetics and breeding is learned, Beijing: Science Press, 1988.427-458; Liu Dajun, cytogenetics, Beijing: Chinese agriculture press, 1999).It is modern development cell engineering fast that Isolated microspore is cultivated; Can on unicellular level, obtain zygoid; Can in 2-3, obtain stable self-mating system, this has set up ripe culture technique system in crops such as barley, wheat, paddy rice, corn and Btassica.
NLN liquid nutrient medium, B5 medium and MS substratum are substratum commonly used in the tissue culture.
Summary of the invention
An object of the present invention is to provide a kind of method of cultivating the Radix Dauci Sativae embryoid.
The method of cultivation Radix Dauci Sativae embryoid provided by the present invention comprises the steps:
(1) heat shock of Radix Dauci Sativae sporule is handled;
(2) the Radix Dauci Sativae sporule after the heat shock processing is cultivated, obtained embryoid.
In the said process, the method that said heat shock is handled comprises the steps: said Radix Dauci Sativae sporule at 33 ℃ of held 2d.
In the said process, in the method that said heat shock is handled, said before 33 ℃ of held 2d; Comprise the steps: earlier said Radix Dauci Sativae sporule to be suspended in the NLN liquid nutrient medium; To wherein adding gac, obtain mixture again, with said mixture at 33 ℃ of held 2d.
In the said process, in the method that said heat shock is handled, in the said mixture, the proportioning of said Radix Dauci Sativae sporule, gac and NLN liquid nutrient medium is: (3-5) * 10
5Individual sporule: 1mg gac: (3ml-5ml) NLN liquid nutrient medium is specially 3 * 10
5Individual sporule: 1mg gac: 5ml NLN liquid nutrient medium, 4 * 10
5Individual sporule: 1mg gac: 4ml NLN liquid nutrient medium, 5 * 10
5Individual sporule: 1mg gac: 3ml NLN liquid nutrient medium or 3 * 10
5Individual sporule: 1mg activated carbon; 3ml NLN liquid nutrient medium.
In the said process, in the said step (2), the Radix Dauci Sativae sporule after the heat shock processing is cultivated, the method that obtains embryoid is shown in following I or the II:
I, the Radix Dauci Sativae microspores culture after comprising the steps: directly heat shock to be handled are to obtaining embryoid;
II, the Radix Dauci Sativae microspores culture after comprising the steps: earlier heat shock to be handled obtain callus, and cultured calli obtains embryoid again;
Among the said method I, the substratum that uses in the said cultivation is the NLN liquid nutrient medium; The temperature of said cultivation is 24 ℃-26 ℃ or 24 ℃ or 25 ℃ or 26 ℃, and said cultivation is dark the cultivation;
Among the said method II, the Radix Dauci Sativae microspores culture after said elder generation handles heat shock obtains in the step of callus, and the substratum that said cultivation is used is the NLN liquid nutrient medium; The temperature of said cultivation is 24 ℃-26 ℃ or 24 ℃ or 25 ℃ or 26 ℃, and said cultivation is dark the cultivation;
Among the said method II, said cultured calli again obtains in the step of embryoid, the substratum that uses in the said cultivation consist of MS+0.1mg/L2,4-D; The temperature of said cultivation is 24 ℃-26 ℃ or 24 ℃ or 25 ℃ or 26 ℃, illumination every day 16h in the said cultivation.
In the said process; In the said method; Said Radix Dauci Sativae sporule obtains according to the method that comprises the steps: the keep to the side Radix Dauci Sativae inflorescence of phase of monokaryon is cleaned, with 75% ethanol aqueous solution immersion 30S, with the aqueous solution soaking 15min of 10% Youxiaolin; Aseptic water washing 3 times, the inflorescence after being sterilized; Inflorescence after the sterilization is put into B5 medium, and the extruding bud discharges sporule, gets supernatant, and filter in 300 order apertures, collects filtrating, will filtrate with the centrifugal 4min of 1200rpm rotating speed, abandons supernatant, and collecting precipitation promptly obtains said Radix Dauci Sativae sporule.
In the said process, any in the sporule that said Radix Dauci Sativae sporule is following Radix Dauci Sativae:
By the dark reddish purple F1 that obtains with Radix Dauci Sativae F8-4-4 hybridization in Radix Dauci Sativae Shanxi for Radix Dauci Sativae,
Radix Dauci Sativae HCM,
The F1 that is obtained by Radix Dauci Sativae HCM and Radix Dauci Sativae BETA III hybridization is for Radix Dauci Sativae,
The F1 that is obtained by 2327 hybridization of Radix Dauci Sativae HCM and Radix Dauci Sativae is for Radix Dauci Sativae,
Five cun-1 self-mating systems in the black field of Radix Dauci Sativae,
Five cun-2 self-mating systems in the black field of Radix Dauci Sativae,
Radix Dauci Sativae 6366B self-mating system,
Radix Dauci Sativae 2327 self-mating systems,
Radix Dauci Sativae 3640B self-mating system,
Radix Dauci Sativae FN2-9 self-mating system,
Radix Dauci Sativae Amsterdam-1 self-mating system,
Radix Dauci Sativae Amsterdam-2 self-mating system,
Radix Dauci Sativae Amsterdam-3 self-mating system,
The F1 generation of Radix Dauci Sativae red hat selfing in early spring,
Radix Dauci Sativae 2006-295 self-mating system,
Radix Dauci Sativae Hn1117 self-mating system,
The five cun self-mating systems in the black field of Radix Dauci Sativae improvement,
Radix Dauci Sativae Korea S black field self-mating system,
Radix Dauci Sativae Amsterdam-3 self-mating system,
By five cun in the Radix Dauci Sativae super black field F4 that hybridization obtains with Radix Dauci Sativae HCM for Radix Dauci Sativae,
The F1 that is obtained by the dark reddish purple hybridization of Radix Dauci Sativae Little and Radix Dauci Sativae Shanxi is for Radix Dauci Sativae,
The F1 that is obtained by Radix Dauci Sativae PI421301 and Radix Dauci Sativae Amsterdam hybridization is for Radix Dauci Sativae,
The F2 that is obtained by two neat hybridization of Radix Dauci Sativae red hat in early spring and Radix Dauci Sativae is for Radix Dauci Sativae,
By the F2 of Radix Dauci Sativae HCM * obtain with the five cun hybridization in Radix Dauci Sativae super black field for Radix Dauci Sativae,
Radix Dauci Sativae White self-mating system,
Radix Dauci Sativae Nantes CA self-mating system,
Radix Dauci Sativae Chanteny self-mating system,
Radix Dauci Sativae strike Radix Dauci Sativae self-mating system,
Radix Dauci Sativae good luck self-mating system,
Radix Dauci Sativae 7262B self-mating system.
Another object of the present invention provides a kind of method of cultivating the Radix Dauci Sativae regeneration plant.
The method of cultivation Radix Dauci Sativae regeneration plant provided by the present invention comprises the steps: to obtain the Radix Dauci Sativae embryoid according to above-mentioned arbitrary said method for preparing the Radix Dauci Sativae embryoid, and the said embryoid of tissue culture obtains the Radix Dauci Sativae regrowth again.
In the above-mentioned cultivation Radix Dauci Sativae regeneration plant method, the temperature of said tissue culture is 24 ℃-26 ℃ or 24 ℃ or 25 ℃ or 26 ℃, and light application time every day of said tissue culture is 16 hours; The substratum that said tissue culture is used is the MS substratum.
In the above-mentioned cultivation Radix Dauci Sativae regeneration plant method, said Radix Dauci Sativae regrowth is triploid, tetraploid or zygoid.
Experiment showed, the method for cultivation Radix Dauci Sativae zygoid provided by the invention, become the embryo rate higher.This method is fit to the cultivation of several genes type Radix Dauci Sativae, especially can obtain stable Radix Dauci Sativae zygoid fast, greatly reduces the Radix Dauci Sativae breeding time limit, to improve the Radix Dauci Sativae breeding efficiency, helps to promote the Radix Dauci Sativae breeding process.The inventive method will have broad application prospects in the breeding field of Radix Dauci Sativae.
Description of drawings
Fig. 1 is the process of Isolated microspore to regeneration plant, and A is the embryoid that Isolated microspore forms, and B is the callus that Isolated microspore forms, and C is the regeneration plant that Isolated microspore forms.
Fig. 2 is cells were tested by flow cytometry ploidy result.A is monoploid peak figure, and B is diploid peak figure, and C is triploid peak figure.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
Embodiment 1, cultivation Radix Dauci Sativae regeneration plant
Material described in the table 1 document " Ou Chenggang, village scud, Zhao Zhiwei etc. Radix Dauci Sativae root thick heredity and the Heterosis Analysis thereof long with root. gardening journal; 2009,36 (1): 115-120. " and document " village scud, Pei Hongxia; Ou Chenggang etc. inducing of Radix Dauci Sativae sporule embryoid and callus. gardening journal; 2010,37 (10): 1613-1620. " in disclosed, the public can be from vegetable or flower institute of the Chinese Academy of Agricultural Sciences each material of acquisition.
In March, 2009, plant the kind root of experiment material shown in the table 1 in March, 2010 respectively, all according to document (chief editor of Vegetable & Flower Inst., Chinese Academy of Agriculture Science. the China's Vegetable cultivation. Beijing: agriculture press, 1987) described in method manage.
But prepare described in the concrete reference literature of NLN liquid nutrient medium " Lichter; of haploid plants fromisolated pollen of Brassica napus.Z.Pflanzen Physiol R.1982.Induction, 105:427-434. ".But the concrete reference literature of B5 medium and MS substratum " the bright compiling of Li Jun .2002. plant tissue culture study course. Beijing: China Agricultyre University Press " described in prepare.
One, the sterilization of Radix Dauci Sativae sporule
When the inflorescence of Radix Dauci Sativae material launches, choose the keep to the side little inflorescence of Radix Dauci Sativae of phase of monokaryon, its tea of packing into is leaked clean.On Bechtop, soak 30S, with the aqueous solution sterilization 15min of 10% (volumn concentration) Youxiaolin, aseptic water washing 3 times with 75% ethanol aqueous solution.
Inflorescence is put into mortar, add B5 medium, to discharge sporule, draw supernatant, be filtered to the 10ml centrifuge tube through 300 mesh filter screens with pestle extruding bud.1200rpm, centrifugal 4min abandons supernatant, and collecting precipitation promptly obtains sporule.
Two, heat shock is handled
The sporule deposition that step 1 is obtained suspends with the NLN liquid nutrient medium, and adjustment sporule density obtains microspore suspension (1 * 10
5Individual sporule/ml), every petridish adds the 3ml spore suspension, and to add 100 μ l concentration be 1% gac mother liquor, seals up petridish with sealing film.Place 33 ℃ to handle 2d in whole petridish.
In the mixture in the petridish, the proportioning of Radix Dauci Sativae sporule, gac and NLN liquid nutrient medium is: 3 * 10
5Individual sporule: 1mg gac: 3ml NLN liquid nutrient medium.
Change its proportioning into " 3 * 10
5Individual sporule: 1mg gac: 5ml NLN liquid nutrient medium, 4 * 10
5Individual sporule: 1mg gac: 4ml NLN liquid nutrient medium or 5 * 10
5Individual sporule: 1mg gac: 3ml NLN liquid nutrient medium ", obtain the result and " 3 * 10 of embryoid or callus
5Individual sporule: 1mg gac: 3mlNLN liquid nutrient medium " time do not have a significant difference.
Three, obtain embryoid
Method I (note is made approach E): the petridish after the heat shock processing is placed 25 ℃ of (actually operatings; Temperature can fluctuate 1 ℃; Promptly 25 ℃ ± 1 ℃), under the condition of dark, cultivate, begin to form embryoid (different gene material formation time is different) after 1-2 month;
Method II (note is made approach C): the petridish after heat shock is handled places 25 ℃ (actually operating, temperature can fluctuate 1 ℃, promptly 25 ℃ ± 1 ℃), after cultivating 1 month under the condition of dark, begins to form the callus of white; The callus of white is transferred to MS+0.1mg/L 2, on the 4-D substratum, 25 ℃ of temperature (in the actually operating; Temperature can fluctuate 1 ℃, promptly 25 ℃ ± 1 ℃), cultivate under the illumination every day 16h condition; Cultivated 1-2 month, and made callus be differentiated to form embryoid.
Count embryo or callus rate.Calculation formula is: germ extraction rate=the go out petridish number/petridish number of embryo.Callus rate=the obtain petridish number/petridish number of callus.
The result of regenerating and culturing is shown in Fig. 1 (A and B) and table 1.Can find out in 9 parts of materials that supplied to try in 2009, have 4 parts to produce sporule callus or embryoid from table 1, be respectively 70Q78,80E43, and 80Q52 and 80Q54, wherein 80Q54 generation ratio is the highest, reaches 20%.In supplying 40 parts of materials of examination in 2010, there are 25 parts to produce sporule callus or embryoid, 900C2 wherein, 90W78 reaches 53.8%, 60.0% and 88.9% respectively with the ratio of 900Q1 generation.
Four, obtain regrowth
The embryoid that step 3 is obtained is inoculated in the MS substratum, 25 ℃ of temperature (in the actually operating, temperature can fluctuate 1 ℃, promptly 25 ℃ ± 1 ℃), cultivates under the illumination every day 16h condition, obtains regrowth (Fig. 1 C).
Five, domestication is transplanted
Place natural lighting and room temperature (25 ℃) to take exercise 3-4 days down regeneration plant earlier, then tissue cultured seedling is transplanted to the Nursery that vermiculite is housed, (28 ℃ of day temperatures, 15 ℃ of evenings) cultivation obtains the Radix Dauci Sativae regeneration plant in heliogreenhouse.7,33 and 97 of 70Q78,80Q54 and 80E43 regeneration plants have been obtained.
Six, detect the ploidy of the regeneration plant that obtains
The 137 strain regeneration plants that adopt flow cytometer that 70Q78,80Q54 and 80E43 are formed carry out ploidy and measure., observe peak and confirm the plant ploidy as reference with common diploid HCM.
Result such as table 3 are with shown in Figure 2.It is respectively 42.9%, 63.6% and 17.5% that 70Q78,80Q54 and 80E43 produce the zygoid ratio, explains that the inventive method not only is fit to the filial generation material but also is fit to the self-mating system material, also shows still to have significant difference between the different genes type material." type " in the table refers to filial generation or self-mating system.
The table 1. different genotype Radix Dauci Sativae material of participating in the experiment
Table 3 regeneration plant ploidy qualification result
Embodiment 2, to the low temperature pre-treatment of Radix Dauci Sativae sporule
Prepare disinfectant Radix Dauci Sativae sporule according to method described in the embodiment 1.
The Isolated microspore of selecting 4 ℃ of low temperature that Radix Dauci Sativae is extracted carries out pre-treatment.The result is as shown in table 2; Not through in 7 parts of materials of cold pretreatment 6 parts of material production embryoids or callus being arranged; The all material of handling 3 days does not all produce embryoid or callus, and the part material of handling 1-2 days that has only forms embryoid or callus.Show that the low temperature pre-treatment is unfavorable for the generation of Radix Dauci Sativae embryoid and callus.
4 ℃ of low temperature pre-treatment of table 2 produce the influence (% of unit) of embryoid or callus to the Radix Dauci Sativae Isolated microspore
| Material number | 0 day | 1 day | 2 days | 3 days |
| 90220 | 15.2 | 16.7 | 0 | 0 |
| 90225 | 21.4 | 0 | 0 | 0 |
| 90251 | 15.4 | 0 | 0 | 0 |
| 900E4 | 6.7 | 0 | 0 | 0 |
| 900C2 | 26.7 | 13.3 | 21.4 | 0 |
| 900Q1 | 35.3 | 0 | 20.0 | 0 |
| 70Q50 | 0 | 0 | 0 | 0 |
Claims (8)
1. a method of cultivating the Radix Dauci Sativae embryoid comprises the steps:
(1) heat shock of Radix Dauci Sativae sporule is handled;
(2) the Radix Dauci Sativae sporule after the heat shock processing is cultivated, obtained embryoid;
Said heat shock is handled and is comprised the steps: earlier said Radix Dauci Sativae sporule to be suspended in the NLN liquid nutrient medium, to wherein adding gac, obtains mixture again, with said mixture 33 ℃ of held 2 days;
During said heat shock was handled, the proportioning of Radix Dauci Sativae sporule, gac and NLN liquid nutrient medium was: (3-5) * 10
5Individual sporule: 1mg gac: (3ml-5ml) NLN liquid nutrient medium;
In the said step (2), the Radix Dauci Sativae sporule after the heat shock processing is cultivated, the method that obtains embryoid is shown in following I or the II:
I, the Radix Dauci Sativae microspores culture after comprising the steps: directly heat shock to be handled are to obtaining embryoid;
II, the Radix Dauci Sativae microspores culture after comprising the steps: earlier heat shock to be handled obtain callus, and cultured calli obtains embryoid again;
Among the said method I, the substratum that uses in the said cultivation is the NLN liquid nutrient medium; The temperature of said cultivation is 24 ℃-26 ℃, and said cultivation is dark the cultivation;
Among the said method II, the Radix Dauci Sativae microspores culture after said elder generation handles heat shock obtains in the step of callus, and the substratum that said cultivation is used is the NLN liquid nutrient medium; The temperature of said cultivation is 24 ℃-26 ℃, and said cultivation is dark the cultivation;
Among the said method II, said cultured calli again obtains in the step of embryoid, the substratum that uses in the said cultivation consist of MS+0.1mg/L2,4-D; The temperature of said cultivation is 24 ℃-26 ℃, and illumination every day is 16 hours in the said cultivation;
In the sporule that said Radix Dauci Sativae sporule is following Radix Dauci Sativae any:
By the dark reddish purple F1 that obtains with Radix Dauci Sativae F8-4-4 hybridization in Radix Dauci Sativae Shanxi for Radix Dauci Sativae,
Radix Dauci Sativae HCM self-mating system,
The F1 that is obtained by Radix Dauci Sativae HCM and Radix Dauci Sativae BETA III hybridization is for Radix Dauci Sativae,
The F1 that is obtained by 2327 hybridization of Radix Dauci Sativae HCM and Radix Dauci Sativae is for Radix Dauci Sativae,
Five cun-1 self-mating systems in the black field of Radix Dauci Sativae,
Five cun-2 self-mating systems in the black field of Radix Dauci Sativae,
Radix Dauci Sativae 6366B self-mating system,
Radix Dauci Sativae 2327 self-mating systems,
Radix Dauci Sativae 3640B self-mating system,
Radix Dauci Sativae FN2-9 self-mating system,
Radix Dauci Sativae Amsterdam-1 self-mating system,
Radix Dauci Sativae Amsterdam-2 self-mating system,
Radix Dauci Sativae Amsterdam-3 self-mating system,
The F1 generation of Radix Dauci Sativae red hat selfing in early spring,
Radix Dauci Sativae 2006-295 self-mating system,
Radix Dauci Sativae Hn1117 self-mating system,
The five cun self-mating systems in the black field of Radix Dauci Sativae improvement,
Radix Dauci Sativae Korea S black field self-mating system,
By five cun in the Radix Dauci Sativae super black field F4 that hybridization obtains with Radix Dauci Sativae HCM for Radix Dauci Sativae,
The F1 that is obtained by the dark reddish purple hybridization of Radix Dauci Sativae Little and Radix Dauci Sativae Shanxi is for Radix Dauci Sativae,
The F1 that is obtained by Radix Dauci Sativae PI421301 and Radix Dauci Sativae Amsterdam hybridization is for Radix Dauci Sativae,
The F2 that is obtained by two neat hybridization of Radix Dauci Sativae red hat in early spring and Radix Dauci Sativae is for Radix Dauci Sativae,
By the F2 of Radix Dauci Sativae HCM * obtain with the five cun hybridization in Radix Dauci Sativae super black field for Radix Dauci Sativae,
Radix Dauci Sativae White self-mating system,
Radix Dauci Sativae Nantes CA self-mating system,
Radix Dauci Sativae Chanteny self-mating system,
Radix Dauci Sativae strike Radix Dauci Sativae self-mating system,
Radix Dauci Sativae good luck self-mating system,
Radix Dauci Sativae 7262B self-mating system.
2. method according to claim 1 is characterized in that: in the method that said heat shock is handled, in the said mixture, the proportioning of said Radix Dauci Sativae sporule, gac and NLN liquid nutrient medium is: 3 * 10
5Individual sporule: 1mg gac: 5ml NLN liquid nutrient medium, 4 * 10
5Individual sporule: 1mg gac: 4ml NLN liquid nutrient medium, 5 * 10
5Individual sporule: 1mg gac: 3ml NLN liquid nutrient medium or 3 * 10
5Individual sporule: 1mg gac: 3ml NLN liquid nutrient medium.
3. method according to claim 1 is characterized in that: among the said method I, the temperature of said cultivation is 24 ℃ or 25 ℃ or 26 ℃;
Among the said method II, the Radix Dauci Sativae microspores culture after said elder generation handles heat shock obtains in the step of callus, and the temperature of said cultivation is 24 ℃ or 25 ℃ or 26 ℃;
Among the said method II, said cultured calli again obtains in the step of embryoid, and the temperature of said cultivation is 24 ℃ or 25 ℃ or 26 ℃.
4. according to arbitrary described method among the claim 1-3; It is characterized in that: in the said method; Said Radix Dauci Sativae sporule obtains according to the method that comprises the steps: the keep to the side Radix Dauci Sativae inflorescence of phase of monokaryon is cleaned, with 75% ethanol aqueous solution immersion 30S, with the aqueous solution soaking 15min of 10% Youxiaolin; Aseptic water washing 3 times, the inflorescence after being sterilized; Inflorescence after the sterilization is put into B5 medium, and the extruding bud discharges sporule, gets supernatant, and filter in 300 order apertures, collects filtrating, will filtrate with the centrifugal 4min of 1200rpm rotating speed, abandons supernatant, and collecting precipitation promptly obtains said Radix Dauci Sativae sporule.
5. a method of cultivating the Radix Dauci Sativae regeneration plant comprises the steps: to obtain the Radix Dauci Sativae embryoid according to arbitrary said method among the claim 1-4, and the said embryoid of tissue culture obtains the Radix Dauci Sativae regrowth again.
6. method according to claim 5 is characterized in that: the temperature of said tissue culture is 24 ℃-26 ℃, and light application time every day of said tissue culture is 16 hours; The substratum that said tissue culture is used is the MS substratum.
7. method according to claim 6 is characterized in that: the temperature of said tissue culture is 24 ℃ or 25 ℃ or 26 ℃.
8. according to arbitrary described method among the claim 5-7, it is characterized in that: said Radix Dauci Sativae regrowth is triploid, tetraploid or zygoid.
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| 尹立荣等.胡萝卜游离小孢子培养技术研究.《园艺学报》.2009,第36卷1950. * |
| 管长志等.胡萝卜游离小孢子培养研究初报.《天津农业科学》.2006,第12卷(第2期),11-12. * |
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