CN101703004B - Method for wheat tissue culture - Google Patents
Method for wheat tissue culture Download PDFInfo
- Publication number
- CN101703004B CN101703004B CN2009102373621A CN200910237362A CN101703004B CN 101703004 B CN101703004 B CN 101703004B CN 2009102373621 A CN2009102373621 A CN 2009102373621A CN 200910237362 A CN200910237362 A CN 200910237362A CN 101703004 B CN101703004 B CN 101703004B
- Authority
- CN
- China
- Prior art keywords
- final concentration
- wheat
- medium
- growth hormone
- inducing culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for wheat tissue culture, which comprises the following steps: firstly, performing induction culture on a wheat germ to obtain an embryogenic callus; and secondly, performing differentiation culture on the embryogenic callus to obtain a wheat regenerated plantlet, wherein the method of the induction culture comprises the steps of successively performing induction cultures on the wheat germ with a culture medium containing auxin, a culture medium without the auxin, and the culture medium containing the auxin again to obtain the embryogenic callus. The method for the wheat tissue culture further improves the regeneration frequency of immature embryos and mature embryos of wheat, weakens the barriers caused by the conditions such as genotypes, physiological states and the like to the regeneration, provides a technical support for the cell engineering breeding, the genetic engineering breeding and the functional genomics research of the wheat, and has great significance for the biotechnology breeding and the functional genomics research of the wheat.
Description
Technical field
The present invention relates to the method for a grow wheat tissue culture.
Background technology
The tissue culture high-frequency regeneration is the basis of cell engineering breeding, genetic engineering breeding and functional genomics research; The regeneration of monocotyledons such as dicotyledon such as tobacco, arabidopsis and paddy rice, slender false brome grass is very easy to, thereby becomes the model plant of gene engineering research.By contrast, the wheat tissue culture is also unstable, receives the restriction of factors such as explant type, physiological status, genotype and medium to a great extent, has influenced the process of genetic engineering breeding and functional genomics research.So far, the explant that the wheat tissue culture is successful comprises rataria, young fringe, flower pesticide, mature embryo etc., and it is the easiest that rataria is cultivated, and anther culture and young fringe are cultivated and taken second place, and it is the most difficult that mature embryo is cultivated.Therefore, rataria is used as the ideal material of wheat tissue culture and transgenic research always, and callus induction rate and shoot regeneration frequency are higher relatively.The wheat mature embryo cultivation has obtained certain progress, and successfully is used for Agrobacterium-mediated Transformation and particle gun conversion.No matter be the tissue culture of which kind of explant of wheat; Conventional method is explant to be seeded in to contain 2.0mg/l 2 earlier; Dark condition is cultivated 4 all left and right sides evoked callus down on the medium of growth hormone such as 4-D or Dicamba; Transfer to then and remove 2.0mg/l 2, on the medium of growth hormone such as 4-D or Dicamba, illumination condition is the differentiation plant down.Utilize such technical system, different wheat varieties under the same conditions with same wheat genotypes in different physiological statuss, the culture effect significant difference, a lot of genotype can not successfully regenerate or regeneration rate very low, can not satisfy the needs of gene engineering research.
Summary of the invention
An object of the present invention is to provide the method for tissue culture of a grow wheat.
Wheat method for tissue culture provided by the present invention comprises the steps: earlier the wheat inducing culture to be become embryo callus; Again said embryo callus differentiation culture is become the wheat regeneration plant; The method of said inducing culture comprises the steps: said wheat is carried out inducing culture I, carries out inducing culture II, carries out inducing culture III with the medium that contains growth hormone again with the medium that does not contain growth hormone with the medium that contains growth hormone successively, obtains embryo callus.
In the said process, said wheat can be wheat immature embryo.
When said wheat was wheat immature embryo, said concrete the composition as follows of medium of containing growth hormone: by final concentration was the NH of 1650mg/L
4NO
3, final concentration is the KNO of 1900mg/L
3, final concentration is the CaCl of 440mg/L
22H
2O, final concentration are the MgSO of 370mg/L
47H
2O, final concentration are the KH of 1700mg/L
2PO
4, final concentration is that KI, the final concentration of 0.83mg/L is the H of 6.2mg/L
3BO
3, final concentration is the MnSO of 22.3mg/L
44H
2O, final concentration are the ZnSO of 8.6mg/L
47H
2O, final concentration are the Na of 0.25mg/L
2MoO
42H
2O, final concentration are the CuSO of 0.025mg/L
45H
2O, final concentration are the CoCl of 0.025mg/L
26H
2O, final concentration are the FeSO of 27.8mg/L
47H
2O, final concentration are the Na of 37.3mg/L
2-EDTA2H
2O, final concentration are that the sucrose of 30g/L, the vitamin B1 that final concentration is 10mg/L, glutamine, the final concentration that final concentration is 150mg/L are 2 of 2.0mg/L, and 4-D, final concentration are that the Phytagel (plant gel) of 2.4g/L forms; Said final concentration is the concentration of each material in the said medium that contains growth hormone.This pH value that contains the medium of growth hormone specifically can be 6.0.
When said wheat was wheat immature embryo, the consisting of of the said medium that does not contain growth hormone: except not containing 2, outside the 4-D, all the other formed identical with the composition of the above-mentioned medium that contains growth hormone.
When said wheat was wheat immature embryo, the composition of the medium of using in the said differentiation culture was specific as follows: be the NH of 1650mg/L by final concentration
4NO
3, final concentration is the KNO of 1900mg/L
3, final concentration is the CaCl of 440mg/L
22H
2O, final concentration are the MgSO of 370mg/L
47H
2O, final concentration are the KH of 1700mg/L
2PO
4, final concentration is that KI, the final concentration of 0.83mg/L is the H of 6.2mg/L
3BO
3, final concentration is the MnSO of 22.3mg/L
44H
2O, final concentration are the ZnSO of 8.6mg/L
47H
2O, final concentration are the Na of 0.25mg/L
2MoO
42H
2O, final concentration are the CuSO of 0.025mg/L
45H
2O, final concentration are the CoCl of 0.025mg/L
26H
2O, final concentration are the FeSO of 27.8mg/L
47H
2O, final concentration are the Na of 37.3mg/L
2-EDTA2H
2O, final concentration are the V of 1mg/L
B1, final concentration is the V of 0.5mg/L
B6, final concentration is that the nicotinic acid of 0.5mg/L, glycine that final concentration is 2mg/L, the inositol that final concentration is 100mg/L, the sucrose that final concentration is 20g/L, the Phytagel that final concentration is 2.4g/L are formed; Concentration in the medium that said final concentration is used in said differentiation culture for each material; The pH value of the medium of using in this differentiation culture is 6.0.
In the said process, said wheat immature embryo can be heart-shaped phase wheat immature embryo.
In the said process, when said wheat was wheat immature embryo, said wheat specifically can be in the wheat line 8601, wheat line HW642 or wheat breed Jimai 22.
In the said process, said wheat also can be wheat mature embryo.
In the said process, when said wheat was wheat mature embryo, the composition of the said medium that contains growth hormone is specific as follows: by final concentration was the NH of 1650mg/L
4NO
3, final concentration is the KNO of 1900mg/L
3, final concentration is the CaCl of 440mg/L
22H
2O, final concentration are the MgSO of 370mg/L
47H
2O, final concentration are the KH of 1700mg/L
2PO
4, final concentration is that KI, the final concentration of 0.83mg/L is the H of 6.2mg/L
3BO
3, final concentration is the MnSO of 22.3mg/L
44H
2O, final concentration are the ZnSO of 8.6mg/L
47H
2O, final concentration are the Na of 0.25mg/L
2MoO
42H
2O, final concentration are the CuSO of 0.025mg/L
45H
2O, final concentration are the CoCl of 0.025mg/L
26H
2O, final concentration are the FeSO of 27.8mg/L
47H
2O, final concentration are the Na of 37.3mg/L
2-EDTA2H
2O, final concentration are the MgCl of 0.75g/L
2Final concentration is the mannitol of 15g/L; Final concentration is the sorbierite of 15g/L; Final concentration is the plant gel of 2.4g/L; Final concentration is the glutaminase of 5mg/L; Final concentration is the caseinhydrolysate of 500mg/L; Final concentration is the glucose of 12g/L; Final concentration is the vitamin B1 of 10mg/L; Final concentration is the vitamin B6 of 1mg/L; Final concentration is the nicotinic acid of 1mg/L; Final concentration is the glycine of 2mg/L; Final concentration is the acetosyringone of 39mg/L; Final concentration is the Dicamba of 2mg/L; Final concentration is the AgNO of 4mg/L
3, final concentration is that the cysteine of 40mg/L, Vc that final concentration is 100mg/L form; Said final concentration is the concentration of each material in the said medium that contains growth hormone; This pH value that contains the medium of growth hormone specifically can be 6.0.
In the said process, when said wheat was wheat mature embryo, the consisting of of the said medium that does not contain growth hormone: except not containing the Dicamba, all the other formed identical with the composition of the said medium that contains growth hormone.
In the said process, when said wheat was wheat mature embryo, the composition of the medium of using in the said differentiation culture specifically can be following: be the NH of 1650mg/L by final concentration
4NO
3, final concentration is the KNO of 1900mg/L
3, final concentration is the CaCl of 440mg/L
22H
2O, final concentration are the MgSO of 370mg/L
47H
2O, final concentration are the KH of 1700mg/L
2PO
4, final concentration is that KI, the final concentration of 0.83mg/L is the H of 6.2mg/L
3BO
3, final concentration is the MnSO of 22.3mg/L
44H
2O, final concentration are the ZnSO of 8.6mg/L
47H
2O, final concentration are the Na of 0.25mg/L
2MoO
42H
2O, final concentration are the CuSO of 0.025mg/L
45H
2O, final concentration are the CoCl of 0.025mg/L
26H
2O, final concentration are the FeSO of 27.8mg/L
47H
2O, final concentration are the Na of 37.3mg/L
2-EDTA2H
2O, final concentration are that sucrose, the final concentration of 30g/L is the V of 10mg/L
B1, final concentration is the V of 1mg/L
B6, final concentration is that the nicotinic acid of 1mg/L, glycine that final concentration is 2mg/L, the glutamine that final concentration is 5mg/L, the IAA that final concentration is 0.2mg/L, the Phytagel that final concentration is 2.4g/L are formed; The pH value of the medium of using in this differentiation culture is 6.0.
In the said process, when said wheat was wheat mature embryo, said wheat specifically can be wheat line CB037.
In the said process, the time that the medium that said usefulness contains growth hormone carries out inducing culture I can be 7-9 days, is preferably 8 days; The time that the medium that said usefulness contains growth hormone carries out inducing culture III can be 7-9 days, is preferably 8 days; The time that the medium that said usefulness does not contain growth hormone carries out inducing culture II can be 6-14 days, further can be 6-12 days, specifically can be 10-14 days, is preferably 12 days.
In the said process; The medium that said usefulness contains growth hormone carries out medium that inducing culture I, said usefulness do not contain growth hormone and carries out the medium that inducing culture II and said usefulness contains growth hormone and carry out among the inducing culture III, and culture condition is 24 ℃-26 ℃ for dark, temperature.
In the general wheat group training; Inducing the formation callus stage all is to continue to handle with growth hormone; Wheat method for tissue culture of the present invention inducing the formation callus stage to adopt " growth hormone-interruption of growth element-growth hormone " training mode, improves the pick-up rate of the green seedling of wheat greatly.Experiment showed, in the wheat that it is 208.2% that 8601 ratarias are cultivated green seedling pick-up rate, conventional cultural method does not obtain regeneration plant (Fig. 1, Fig. 3, table 1); It is 25.3% that wheat Jimai 22 ratarias are cultivated green seedling pick-up rate, and conventional cultural method regeneration rate is merely 0.9% (Fig. 4); It is 211.3% that wheat HW642 rataria is cultivated green seedling pick-up rate, and conventional cultural method regeneration rate is merely 26.2% (Fig. 5); It is 67.1% that wheat CB037 mature embryo is cultivated green seedling pick-up rate, and conventional cultural method regeneration rate is 32.4% (Fig. 6); The inventive method makes green seedling pick-up rate improve 10-20 doubly.In 0-12 days scope of growth hormone Interrupt Process; Green seedling pick-up rate significantly increases (Fig. 1 with the prolongation of growth hormone break period; Table 1); Regeneration associated genes expressions such as catalase obviously strengthen with the prolongation of growth hormone break period, and 9 days expressions of growth hormone interruption are the highest, show that catalase gene and regeneration proterties have positive correlation (Fig. 2).The result shows that wheat immature embryo, mature embryo are handled through growth hormone was enough to the dedifferentiation of inducing wheat cell in 7-9 days; On the contrary, long period growth hormone processing has suppressed the expression of wheat regeneration associated genes and cell breaks up again, is unfavorable for that embryo callus forms, and has reduced green seedling pick-up rate.
The inventive method is through sport technique segments such as improvement cultivation programs; Wheat immature embryo and mature embryo regeneration frequency have further been improved; Overcome the obstacle of conditions such as genotype, physiological status to regeneration; Weaken the strong dependence of wheat tissue culture to factors such as genotype, physiological statuss; For the breeding of wheat cell engineering, genetic engineering breeding and functional genomics research provide technical support, improved operating efficiency greatly, significant for wheat biotechnology breeding and functional genomics research.
Description of drawings
Fig. 1. growth hormone batch process different time is to the influence of wheat immature embryo regeneration effect
Fig. 2. regeneration associated genes expression (hydrogen peroxide enzyme coding gene) in the growth hormone batch process different time wheat immature embryo callus
Fig. 3. utilize the present invention's technology and common technology to cultivate 8601 rataria regeneration effects comparison in the wheat
Fig. 4. utilize the present invention's technology and common technology to cultivate wheat Jimai 22 rataria regeneration effects relatively
Fig. 5. utilize the present invention's technology to cultivate wheat HW642 rataria regeneration effect relatively
Fig. 6. utilize the present invention's technology and common technology to cultivate wheat CB037 mature embryo regeneration effect relatively
Embodiment
Employed experimental technique is conventional 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.
The definition of wheat immature embryo: the wheat that strips on the tender seed of wheat ovary pollination back growth 13-14 days wheat children; The definition of wheat mature embryo: the wheat that the maturing stage strips from 2-3 month wheat seed of wheat plant results back storage.
" kind " is the genetic stocks of country or the authorization of the variety certification committee of provinces and cities, can be at suitable regional popularizing planting; " strain " for the height with good economical character of breeding units seed selection for genetic stocks, through becoming kind or carry out breeding and research and utilization after the authorization as variety source; In most cases also strain writing kind.
Wheat breed Jimai 22 (world agriculture, 2009,9:78) (Institute of Crop Science, Chinese Academy of Agricultural Science);
8601 (Xu Huijun etc., Acta Genetica Sinica, 1996,5:377-381) (Institute of Crop Science, Chinese Academy of Agricultural Science) in the wheat line;
Wheat line CB037 (Tao Lili, Chinese Academy of Agricultural Sciences's master thesis, in June, 2009) (Institute of Crop Science, Chinese Academy of Agricultural Science);
Wheat line HW642 (Zhang Zengyan etc., Acta Agronomica Sinica, 2002,28 (4): 486-491) (Institute of Crop Science, Chinese Academy of Agricultural Science);
Genetic resources title: wheat breed Jimai 22.The acquiring way of genetic resources: I, genetic resources are taken from plant, II, obtain manner: give.Direct sources: acquisition time in August, 2007; Non-acquisition mode, supplier's Name or Designation Crop Inst. of shandong Prov. Agriculture science Academy, China Shandong Province, supplier country of living in, supplier's contact method (No. 28 Crop Inst. of shandong Prov. Agriculture science Academy in mulberry field, Jinan City, Shandong Province road).Primary source is unclear.
Genetic resources title: in the wheat line 8601.The acquiring way of genetic resources: I, genetic resources are taken from: plant, II, obtain manner: national germplasm storehouse.Direct sources: acquisition time in March, 2006; Non-acquisition mode; Supplier's Name or Designation Institute of Crop Science, Chinese Academy of Agricultural Science crop germplasm resource is preserved the center; BeiJing, China city, supplier country of living in, No. 12 Institute of Crop Science, Chinese Academy of Agricultural Science in supplier's contact method Zhongguancun South Street, Beijing City.Primary source is unclear.
Genetic resources title: wheat line CB037.The acquiring way of genetic resources: I, genetic resources are taken from: plant, II, obtain manner: other (seed selection of inventor seminar is also preserved).Direct sources: 2006; Non-acquisition mode, supplier's Name or Designation Institute of Crop Science, Chinese Academy of Agricultural Science, supplier country of living in: BeiJing, China city, No. 12 Institute of Crop Science, Chinese Academy of Agricultural Science in supplier's contact method Zhongguancun South Street, Beijing City.Primary source is unclear.
Genetic resources title: wheat line HW642.The acquiring way of genetic resources: I, genetic resources are taken from: plant, II, obtain manner: other (seed selection of inventor seminar is also preserved).Direct sources: acquisition time 2005; Non-acquisition mode, supplier's Name or Designation Institute of Crop Science, Chinese Academy of Agricultural Science, supplier country of living in: BeiJing, China city, No. 12 Institute of Crop Science, Chinese Academy of Agricultural Science in supplier's contact method Zhongguancun South Street, Beijing City; Primary source is unclear.
Embodiment 1, carry out tissue culture with wheat immature embryo
In the present embodiment, be experimental subjects with following wheat breed respectively: in the wheat 8601, wheat HW642 and wheat Jimai 22.The tissue culture method step of each wheat lines is following:
One, the acquisition of wheat immature embryo
With in the wheat 8601, wheat HW642 and wheat Jimai 22 be planted in Xia Fan base, Guyuan, Hebei (during the 4-7 month wheat growth temperature 10-25 ℃); Bloom and pollinate back 13-14 days; Collect in the wheat 8601, the rataria (heart-shaped phase, size is 1.0-1.2mm) of wheat HW642 and wheat Jimai 22.
Two, tissue culture
1, inducing culture obtains embryo callus
Above-mentioned wheat immature embryo is seeded in 8 days (dark, 25 ℃) of cultivation on the following SD2 medium, obtains callus I; Callus is transferred to following SD0 medium culture 3,6,9 or 12 days (dark, 25 ℃) (in 8601 done the experiment of cultivating 3,6,9 or 12 days, 22 of HW642 and Jimais were cultivated 12 days), obtain callus II; Again callus II is transferred to following SD2 medium culture 8 days (dark, 25 ℃), obtain embryo callus III.
2, differentiation culture obtains the wheat regeneration plant
Embryo callus III is transferred to 20-25 days (intensity of illumination is 3500Lx, and temperature is 25 ℃) of cultivation on the following FHCK medium, and differentiation obtaining wheat seedling.
Record inoculation rataria number, wheat seedling number, frequency of embryonic callus induction, callus differentiation rate, green seedling pick-up rate that the embryo callus number that statistics obtains, callus differentiation number, differentiation obtain.
Frequency of embryonic callus induction (%)=(embryo callus is counted ÷ inoculation rataria number) * 100
Callus differentiation rate (%)=(the differentiation callus is counted ÷ inoculation rataria number) * 100
Green seedling pick-up rate (%)=(break up green seedling and count ÷ inoculation rataria number) * 100
Each medium all adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes.
The SD2 medium is formed: by final concentration is the NH of 1650mg/L
4NO
3, final concentration is the KNO of 1900mg/L
3, final concentration is the CaCl of 440mg/L
22H
2O, final concentration are the MgSO of 370mg/L
47H
2O, final concentration are the KH of 1700mg/L
2PO
4, final concentration is that KI, the final concentration of 0.83mg/L is the H of 6.2mg/L
3BO
3, final concentration is the MnSO of 22.3mg/L
44H
2O, final concentration are the ZnSO of 8.6mg/L
47H
2O, final concentration are the Na of 0.25mg/L
2MoO
42H
2O, final concentration are the CuSO of 0.025mg/L
45H
2O, final concentration are the CoCl of 0.025mg/L
26H
2O, final concentration are the FeSO of 27.8mg/L
47H
2O, final concentration are the Na of 37.3mg/L
2-EDTA2H
2O, final concentration are that the sucrose of 30g/L, the vitamin B1 that final concentration is 10mg/L, glutamine, the final concentration that final concentration is 150mg/L are 2 of 2.0mg/L, and 4-D, final concentration are that the Phytagel (plant gel) of 2.4g/L forms; Said final concentration is the concentration of each material in said SD2 medium; The pH value of SD2 medium is 6.0.Above composition adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes.
The SD0 medium: except not containing 2, outside the 4-D, other composition and consumption and SD2 medium are identical.
The FHCK medium is formed: by final concentration is the NH of 1650mg/L
4NO
3, final concentration is the KNO of 1900mg/L
3, final concentration is the CaCl of 440mg/L
22H
2O, final concentration are the MgSO of 370mg/L
47H
2O, final concentration are the KH of 1700mg/L
2PO
4, final concentration is that KI, the final concentration of 0.83mg/L is the H of 6.2mg/L
3BO
3, final concentration is the MnSO of 22.3mg/L
44H
2O, final concentration are the ZnSO of 8.6mg/L
47H
2O, final concentration are the Na of 0.25mg/L
2MoO
42H
2O, final concentration are the CuSO of 0.025mg/L
45H
2O, final concentration are the CoCl of 0.025mg/L
26H
2O, final concentration are the FeSO of 27.8mg/L
47H
2O, final concentration are the Na of 37.3mg/L
2-EDTA2H
2O, final concentration are the V of 1mg/L
B1, final concentration is the V of 0.5mg/L
B6, final concentration is that the nicotinic acid of 0.5mg/L, glycine that final concentration is 2mg/L, the inositol that final concentration is 100mg/L, the sucrose that final concentration is 20g/L, the Phytagel that final concentration is 2.4g/L are formed; Said final concentration is the concentration of each material in said FHCK medium; The pH value of FHCK medium is 6.0.
Above composition adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes.
Three, control group: 2,4-D continues to handle contrast
Method: identical wheat immature embryo is seeded on the SD2 medium dark condition cultivates about 4 weeks down; Induce and obtain callus; Transfer to then on the FHCK medium, (intensity of illumination is 3500Lx, 25 ℃ of temperature) differentiation obtains the wheat regeneration plant under the illumination condition.
3 repetitions are established in experiment.The result takes the mean.In the tissue culture situation of 8601 wheat immature embryos as shown in table 1.
8601 green seedling pick-up rates are 208.2% (Fig. 1, Fig. 3, table 1) in the wheat, and control group does not obtain regeneration plant; Wheat Jimai 22 green seedling pick-up rates are 25.3%, and the green seedling pick-up rate of control group is merely 0.9% (Fig. 4); The green seedling pick-up rate of wheat HW642 is 211.3%, and the green seedling pick-up rate of control group is merely 26.2% (Fig. 5).
Among Fig. 1, A: contrast (growth hormone continues to handle); B: growth hormone intermittently 3 days; C: growth hormone intermittently 6 days; D: growth hormone intermittently 9 days; E: growth hormone intermittently 12 days.
Among Fig. 3, among Fig. 4, among Fig. 5, A is a control group, and B is the inventive method group.
The regeneration situation of different intermittent times of 8601 wheat immature embryo growth hormone in the table 1.
| The growth hormone batch process time (my god) | Inoculation rataria number | The embryo callus number | Differentiation callus number | Break up green seedling number | Frequency of embryonic callus induction (%) | Callus differentiation rate (%) | Green seedling pick-up rate (%) |
| 0 | 182 | 0 | 0 | 0 | 0 | 0 | 0 |
| 3 | 204 | 16 | 0 | 0 | 7.8 | 0 | 0 |
| 6 | 232 | 135 | 109 | 177 | 58.2 | 47.0 | 76.3 |
| 9 | 146 | 90 | 88 | 181 | 61.6 | 60.3 | 124.0 |
| 12 | 232 | 196 | 186 | 483 | 84.5 | 80.2 | 208.2 |
Four, catalase gene expression analysis in 8601 callus in
For 8601 (they being plain 3,6,9,12 days processing of interruption of growth) in the different growth hormone batch process, on differential medium, get callus, place liquid nitrogen flash freezer rapidly, and preserve the usefulness that supplies the RNA extraction for-70 ℃.
(Tiangen Beijing) carries out the total RNA of material and extracts the explanation of method reference reagent box to adopt the TRIzol kit.Reverse transcription is according to PrimeScript
TM1
StStrand cDNA Synthesis (TaKaRa, Dalian) kit carries out.
As interior mark, adopt semi-quantitative RT-PCR to carry out the template homogenization with wheat Tubulin, the template that homogeneous is good is carried out the expression analysis of catalase gene immediately.
The pcr amplification condition of template homogeneous: 94 ℃ of sex change 4min, 28 circulations comprise 94 ℃ of sex change 30s, 61 ℃ of annealing 30s, 72 ℃ are extended 20s, and last 72 ℃ are extended 10min;
Catalase gene RT-PCR amplification condition: 94 ℃ of sex change 4min, 25 circulations comprise 94 ℃ of sex change 30s, 55-62 ℃ of annealing 30s, 72 ℃ are extended 20s, and last 72 ℃ are extended 10min; 1% agarose gel electrophoresis separates the purpose fragment, and (BioRad USA) carries out quantitative analysis to adopt Quantity-One discovery series.
Tubulin interior label primer: 5 '-TCCTCCTATGCCCCAGTGATC-3 ',
5’-ACTCATCGCCCTCATCACCG-3’。
Catalase gene primer: 5 '-CAAGGGCTTCTTCGAGGTCAC-3 ',
5’-TGTAGAAGGTCCACTCCGGGTAG-3’。
The result is as shown in Figure 2, and (0d: expression growth hormone continues to handle (contrast); 3d, 6d, 9d, 12d: represented growth hormone respectively intermittently 3 days, 6 days, 9 days and 12 days).Show that catalase regeneration associated genes expression obviously strengthens with the prolongation of growth hormone break period, it is the highest that growth hormone interrupts 9 days expressions, shows that catalase regeneration associated genes and regeneration proterties have positive correlation.
Embodiment 2, carry out tissue culture with wheat mature embryo
In the present embodiment, be experimental subjects with kind CB037 respectively.The tissue culture method step is following:
One, tissue culture
1, wheat seed disinfects
The ripe kind successively with after the 70% alcohol sterilization 10 minutes, 25% clorox sterilization 25 minutes, aseptic water washing 5 times of wheat CB037 soaked 15-18 hour inferior daily 15% clorox sterilize 15 minutes, aseptic water washing 5 times in sterile water.
2, mature embryo is induced into embryo callus
On the seed of disinfecting, mature embryo is scraped broken with vaccinating lancet; Be seeded in and cultivate 8 days (dark, 25 ℃), evoked callus on the following A di medium; Then callus is transferred to and cultivated 12 days (dark on the following A d0 medium that removes Dicamba; 25 ℃), callus is transferred on the Adi medium that contains Dicamba cultivated 8 days again, obtain embryo callus.
3, differentiation culture obtains the wheat regeneration plant
Embryo callus is transferred to 20-25 days (intensity of illumination is 3500Lx, and temperature is 25 ℃) of cultivation on the following XCFH medium, and differentiation obtaining the wheat regeneration plant.
Record inoculation mature embryo number, wheat regrowth number and shoot regeneration frequency that the statistics differentiation obtains.
The Adi medium is formed: by final concentration is the NH of 1650mg/L
4NO
3, final concentration is the KNO of 1900mg/L
3, final concentration is the CaCl of 440mg/L
22H
2O, final concentration are the MgSO of 370mg/L
47H
2O, final concentration are the KH of 1700mg/L
2PO
4, final concentration is that KI, the final concentration of 0.83mg/L is the H of 6.2mg/L
3BO
3, final concentration is the MnSO of 22.3mg/L
44H
2O, final concentration are the ZnSO of 8.6mg/L
47H
2O, final concentration are the Na of 0.25mg/L
2MoO
42H
2O, final concentration are the CuSO of 0.025mg/L
45H
2O, final concentration are the CoCl of 0.025mg/L
26H
2O, final concentration are the FeSO of 27.8mg/L
47H
2O, final concentration are the Na of 37.3mg/L
2-EDTA2H
2O, final concentration are the MgCl of 0.75g/L
2Final concentration is the mannitol of 15g/L; Final concentration is the sorbierite of 15g/L; Final concentration is the Phytagel (plant gel) of 2.4g/L; Final concentration is the glutaminase of 5mg/L; Final concentration is the caseinhydrolysate of 500mg/L; Final concentration is the glucose of 12g/L; Final concentration is the vitamin B1 of 10mg/L; Final concentration is the vitamin B6 of 1mg/L; Final concentration is the nicotinic acid of 1mg/L; Final concentration is the glycine of 2mg/L; Final concentration is the acetosyringone of 39mg/L; Final concentration is the Dicamba of 2mg/L; Final concentration is the AgNO of 4mg/L
3, final concentration is that the cysteine of 40mg/L, Vc that final concentration is 100mg/L form; Said final concentration is the concentration of each material in said Adi medium; The pH value of Adi medium is 6.0.Above composition adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes.
The Ad0 medium: except not containing the Dicamba, other composition and manner of formulation and sterilization method and Adi medium are identical.
The XCFH medium is formed: by final concentration is the NH of 1650mg/L
4NO
3, final concentration is the KNO of 1900mg/L
3, final concentration is the CaCl of 440mg/L
22H
2O, final concentration are the MgSO of 370mg/L
47H
2O, final concentration are the KH of 1700mg/L
2PO
4, final concentration is that KI, the final concentration of 0.83mg/L is the H of 6.2mg/L
3BO
3, final concentration is the MnSO of 22.3mg/L
44H
2O, final concentration are the ZnSO of 8.6mg/L
47H
2O, final concentration are the Na of 0.25mg/L
2MoO
42H
2O, final concentration are the CuSO of 0.025mg/L
45H
2O, final concentration are the CoCl of 0.025mg/L
26H
2O, final concentration are the FeSO of 27.8mg/L
47H
2O, final concentration are the Na of 37.3mg/L
2-EDTA2H
2O, final concentration are that sucrose, the final concentration of 30g/L is the V of 10mg/L
B1, final concentration is the V of 1mg/L
B6, final concentration is that the nicotinic acid of 1mg/L, glycine that final concentration is 2mg/L, the glutamine that final concentration is 5mg/L, the IAA that final concentration is 0.2mg/L, the Phytagel that final concentration is 2.4g/L are formed; The pH value of XCFH medium is 6.0.Above composition adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes.
Two, control group: Dicamba continues to handle contrast
The broken tissue of mature embryo is seeded on the Adi medium dark condition to be cultivated about 4 weeks down; Induce and obtain callus; Transfer to then on the XCFH medium that removes Dicamba growth hormone, (intensity of illumination is 3500Lx, and temperature is 25 ℃) differentiation obtains the wheat regeneration plant under the illumination condition.
3 repetitions are established in experiment.The result takes the mean.
In the inventive method group, the green seedling pick-up rate of wheat CB037 is 67.1% (Fig. 6-B); In the control group, the green seedling pick-up rate of wheat CB037 is 32.4% (Fig. 6-A).
Claims (4)
1. the method for tissue culture of a grow wheat comprises the steps: earlier the wheat inducing culture to be become embryo callus; Again said embryo callus differentiation culture is become the wheat regeneration plant; The method of said inducing culture comprises the steps:
Said wheat is carried out inducing culture I with the medium that contains growth hormone successively; Incubation time is 7 days-9 days; Medium with not containing growth hormone carries out inducing culture II, and incubation time is 6 days-14 days, carries out inducing culture III with the medium that contains growth hormone again; Incubation time is 7 days-9 days, obtains embryo callus;
Said wheat is wheat immature embryo or wheat mature embryo;
Said concrete the composition as follows of medium of containing growth hormone of the said wheat immature embryo of inducing culture: by final concentration is the NH of 1650mg/L
4NO
3, final concentration is the KNO of 1900mg/L
3, final concentration is the CaCl of 440mg/L
22H
2O, final concentration are the MgSO of 370mg/L
47H
2O, final concentration are the KH of 1700mg/L
2PO
4, final concentration is that KI, the final concentration of 0.83mg/L is the H of 6.2mg/L
3BO
3, final concentration is the MnSO of 22.3mg/L
44H
2O, final concentration are the ZnSO of 8.6mg/L
47H
2O, final concentration are the Na of 0.25mg/L
2MoO
42H
2O, final concentration are the CuSO of 0.025mg/L
45H
2O, final concentration are the CoCl of 0.025mg/L
26H
2O, final concentration are the FeSO of 27.8mg/L
47H
2O, final concentration are the Na of 37.3mg/L
2-EDTA2H
2O, final concentration are that the sucrose of 30g/L, the vitamin B1 that final concentration is 10mg/L, glutamine, the final concentration that final concentration is 150mg/L are 2 of 2.0mg/L, and 4-D, final concentration are that the plant gel of 2.4g/L is formed; Said final concentration contains the concentration in the medium of growth hormone for each material the said wheat immature embryo of inducing culture said; The said wheat immature embryo of inducing culture said do not contain the consisting of of medium of growth hormone: except not containing 2, outside the 4-D, all the other are formed, and to contain the composition of medium of growth hormone identical with the said wheat immature embryo of inducing culture said;
The wheat that adopts during the said wheat immature embryo of inducing culture is in the wheat line 8601, wheat line HW642 or wheat breed Jimai 22;
Said concrete the composition as follows of medium of containing growth hormone of the said wheat mature embryo of inducing culture: by final concentration is the NH of 1650mg/L
4NO
3, final concentration is the KNO of 1900mg/L
3, final concentration is the CaCl of 440mg/L
22H
2O, final concentration are the MgSO of 370mg/L
47H
2O, final concentration are the KH of 1700mg/L
2PO
4, final concentration is that KI, the final concentration of 0.83mg/L is the H of 6.2mg/L
3BO
3, final concentration is the MnSO of 22.3mg/L
44H
2O, final concentration are the ZnSO of 8.6mg/L
47H
2O, final concentration are the Na of 0.25mg/L
2MoO
42H
2O, final concentration are the CuSO of 0.025mg/L
45H
2O, final concentration are the CoCl of 0.025mg/L
26H
2O, final concentration are the FeSO of 27.8mg/L
47H
2O, final concentration are the Na of 37.3mg/L
2-EDTA2H
2O, final concentration are the MgCl of 0.75g/L
2Final concentration is the mannitol of 15g/L; Final concentration is the sorbierite of 15g/L; Final concentration is the Phytagel plant gel of 2.4g/L; Final concentration is the glutaminase of 5mg/L; Final concentration is the caseinhydrolysate of 500mg/L; Final concentration is the glucose of 12g/L; Final concentration is the vitamin B1 of 10mg/L; Final concentration is the vitamin B6 of 1mg/L; Final concentration is the nicotinic acid of 1mg/L; Final concentration is the glycine of 2mg/L; Final concentration is the acetosyringone of 39mg/L; Final concentration is the Dicamba of 2mg/L; Final concentration is the AgNO of 4mg/L
3, final concentration is that the cysteine of 40mg/L, Vc that final concentration is 100mg/L form; Said final concentration contains the concentration in the medium of growth hormone for each material the said wheat mature embryo of inducing culture said; The said wheat mature embryo of inducing culture said do not contain the consisting of of medium of growth hormone: except not containing the Dicamba, all the other are formed, and to contain the composition of medium of growth hormone identical with the said wheat mature embryo of inducing culture said;
The wheat that adopts during the said wheat mature embryo of inducing culture is wheat line CB037.
2. method according to claim 1 is characterized in that: said wheat immature embryo is heart-shaped phase wheat immature embryo.
3. method according to claim 1 is characterized in that: the time that the medium that said use contains growth hormone carries out inducing culture I is 8 days; The time that the medium that said use contains growth hormone carries out inducing culture III is 8 days; The time that the medium that said use does not contain growth hormone carries out inducing culture II is 12 days.
4. method according to claim 1; It is characterized in that: the medium that said usefulness contains growth hormone carries out medium that inducing culture I, said usefulness do not contain growth hormone and carries out the medium that inducing culture II and said usefulness contains growth hormone and carry out among the inducing culture III, and culture condition is 24 ℃-26 ℃ for dark, temperature.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102373621A CN101703004B (en) | 2009-11-16 | 2009-11-16 | Method for wheat tissue culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102373621A CN101703004B (en) | 2009-11-16 | 2009-11-16 | Method for wheat tissue culture |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101703004A CN101703004A (en) | 2010-05-12 |
| CN101703004B true CN101703004B (en) | 2012-06-20 |
Family
ID=42373321
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009102373621A Expired - Fee Related CN101703004B (en) | 2009-11-16 | 2009-11-16 | Method for wheat tissue culture |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101703004B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103210843A (en) * | 2013-04-09 | 2013-07-24 | 南京农业大学 | High-frequency roegneria kamoji immature embryo callus induction and regeneration cultivation method |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103131729A (en) * | 2011-11-24 | 2013-06-05 | 中国农业科学院作物科学研究所 | Production method of no-false-positive transgenic wheat |
| CN102599059B (en) * | 2012-03-28 | 2013-09-04 | 中国农业科学院作物科学研究所 | Method for improving tissue culture regeneration rate of wheat genotype immature embryo with low regeneration capacity |
| CN102668982B (en) * | 2012-05-04 | 2013-06-26 | 四川农业大学 | Tissue culture method of mature wheat embryo |
| CN102668983B (en) * | 2012-05-23 | 2013-08-28 | 四川农业大学 | Tissue culture method of mature embryo of aegilops tauschii |
| CN103444532B (en) * | 2013-09-04 | 2015-06-03 | 中国农业科学院作物科学研究所 | Tissue culture method for increasing regeneration rate of wheat immature embryos |
| CN103444533B (en) * | 2013-09-04 | 2015-06-03 | 中国农业科学院作物科学研究所 | Tissue culture method for increasing plant regeneration rate of wheat immature embryos |
| CN111657149A (en) * | 2020-07-09 | 2020-09-15 | 山东农业大学 | Tissue culture method for improving regeneration rate of mature wheat embryos |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101265481A (en) * | 2008-05-09 | 2008-09-17 | 中国农业科学院作物科学研究所 | Culture medium for increasing wheat mature embryo regeneration ratio and conversion method for agrobacterium tumefaciens thereof |
| CN101265480A (en) * | 2008-05-09 | 2008-09-17 | 中国农业科学院作物科学研究所 | Conversion method for overcoming agrobacterium tumefaciens mediated wheat immature embryo browning and special culture medium for the same |
-
2009
- 2009-11-16 CN CN2009102373621A patent/CN101703004B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101265481A (en) * | 2008-05-09 | 2008-09-17 | 中国农业科学院作物科学研究所 | Culture medium for increasing wheat mature embryo regeneration ratio and conversion method for agrobacterium tumefaciens thereof |
| CN101265480A (en) * | 2008-05-09 | 2008-09-17 | 中国农业科学院作物科学研究所 | Conversion method for overcoming agrobacterium tumefaciens mediated wheat immature embryo browning and special culture medium for the same |
Non-Patent Citations (3)
| Title |
|---|
| 周朴华，等.荻幼穗愈伤组织诱导及无性系的建立.《湖南农学院学报》.1992,第18卷(第3期),第569-573页. * |
| 朱至清,等.大麦和普通小麦杂种再生植株的形态和染色体变化.《遗传学报》.1985,第12卷(第6期),第430-433页. * |
| 高俊山，等.不同组织培养途径对小麦再生能力的研究.《激光生物学报》.2003,第12卷(第6期),第406-411页. * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103210843A (en) * | 2013-04-09 | 2013-07-24 | 南京农业大学 | High-frequency roegneria kamoji immature embryo callus induction and regeneration cultivation method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101703004A (en) | 2010-05-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101703004B (en) | Method for wheat tissue culture | |
| Gomes et al. | Regeneration of somatic embryos of oil palm (Elaeis guineensis) using temporary immersion bioreactors | |
| Shen et al. | Micropropagation of herbaceous peony (Paeonia lactiflora Pall.) | |
| CN101548647B (en) | Tree peony callus induction method | |
| Premvaranon et al. | In vitro studies to produce double haploid in Indica hybrid rice | |
| CN110301357A (en) | A kind of the regeneration seedling establishment method and special culture media of sponge gourd Unfertilized Ovaries embryoid | |
| CN105494108B (en) | A kind of tissue culture and rapid propagation method of fast-growing American elm | |
| CN103155862B (en) | Sinocalamus latiflorus flower pesticide inductor embryo the method obtaining regeneration plant | |
| CN103385173B (en) | A kind of method of efficient acquisition sealwort spirit lily zygoid seedling | |
| CN1311739C (en) | Cotton tissue culturing method using cotyledon petiole as explant | |
| CN102172220B (en) | Method for inducing calluses of stems and leaves of tree peony | |
| CN101011028A (en) | Breeding method of chrysanthemum haploid | |
| CN108522270B (en) | Method for improving detoxification rate and hardening-seedling survival rate based on cucumber anther culture | |
| CN104322370A (en) | Bitter gourd in vitro rapid propagation method | |
| Li et al. | Highly effective organogenesis and somatic embryogenesis of Clivia | |
| CN106106178A (en) | A kind of method for tissue culture of confection Rhizoma Iridis Tectori | |
| CN109735538A (en) | A kind of carrier and its preparation method and application improving forest Strawberry Leaves regeneration efficiency | |
| CN106811471A (en) | Application of the paddy rice SPL7 genes in plant type is regulated and controled | |
| CN1799332A (en) | Method for preparing plant haploid embryo and haploid plant | |
| CN107950394A (en) | A kind of method for tissue culture of tree peony rataria | |
| CN105638468B (en) | Efficient inducing soybean cotyledonary node explant produces the method and culture medium of Multiple Buds | |
| Herrera et al. | Colchicine-induced microspore embryogenesis in coffee | |
| CN102144563B (en) | Method for inducing calluses of peony anther | |
| CN104620988B (en) | Method for obtaining cucumber regenerated plantlet by inducing gynogenesis by virtue of culture of unfertilized ovules | |
| CN101015281A (en) | Regeneration method of Japanese lawngrass |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120620 Termination date: 20171116 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |