CN1618278A - Technique for high-efficiency hereditary of wheat - Google Patents
Technique for high-efficiency hereditary of wheat Download PDFInfo
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- CN1618278A CN1618278A CNA2003101153453A CN200310115345A CN1618278A CN 1618278 A CN1618278 A CN 1618278A CN A2003101153453 A CNA2003101153453 A CN A2003101153453A CN 200310115345 A CN200310115345 A CN 200310115345A CN 1618278 A CN1618278 A CN 1618278A
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Abstract
An effiicent genetic transformation technique for wheat includes choosing the protelean ear at proper stage as explant, the induced culture of calli in the inducing culture medium, and the genetic transformation at the fast growth stage of said calli by use of gene gun. Its advantage is high transform efficiency up to 8.16%.
Description
Technical field:
The present invention relates to a kind of method that obtains the efficient genetic transformation of wheat.
Background technology:
In the research of transgenic wheat, particle bombardment is the main method that obtains the wheat transgenic plant at present.
Be immature embryo and derivative thereof by the particle gun transformation receptor of generally acknowledging and be widely adopted at present.But the genetic transformation efficiency of using this conversion system acquisition is on the low side, only can reach 0.1%-2.5%.And its transformation efficiency also will be subjected to genotypic influence, and especially, nearly 20% genotype is difficult to obtain regeneration plant.Therefore, the kind of some comprehensive agronomy shapeliness is because the reviviscence difference can't obtain transfer-gen plant, and the strong kind of minority reviviscence is not that production practices are needed or the application in production practices is very limited.
Summary of the invention:
The objective of the invention is to overcome the low shortcoming of existing conversion system transformation efficiency, set up and a kind ofly can obtain more transfer-gen plant, obtain the method for transformation of higher genetic transformation frequency.
The solution of the present invention is by selecting proper explant, adjust medium composition and supporting corrective measure, the external source genes of interest being imported wheat, to obtain higher genetic transformation frequency.
The present invention has selected for use the vegetable material of 3 kinds to study.Studied reaction and the wheat different explants particle gun transformation efficiency of wheat different explants to cultured in vitro.Discovery with the young fringe of suitable time (sterile lemma idiophase to female stamen original hase idiophase) as explant, carrying out callus induction on inducing culture cultivates, carry out particle gun at young fringe callus growth animated period and transform, can obtain higher genetic transformation frequency.
This method increases inducing and the elongation stage of a bud seedling in differentiation screening back, can reach purpose of the present invention better.
The concrete grammar step is as follows:
1. young fringe is inoculated on the inducing culture as explant;
2. the callus growth animated period bombards young fringe callus with particle gun;
3. above-mentioned callus is gone to and carry out the Basta resistance screening on the screening culture medium;
4. will go on the bud seedling elongation medium through the callus of screening;
5. transplanting seedling grows to 15cm and screens once more with Basta when high.
6. the transformant blade carries out the PCR detection.
The present invention has carried out stripped contrast culture to three genotypic in good time young fringes and corresponding immature embryo.The result shows: be higher than its corresponding immature embryo with in good time young fringe as the green seedling differentiation rate that explant carries out cultured in vitro.Show that carrying out genetic transformation with young fringe as explant can obtain higher green seedling differentiation rate.
Find that in the research of carrying out wheat different explants particle gun transformation efficiency during as the particle gun transformation receptor, genetic transformation efficiency is higher, can reach 2.53%～8.16% with young fringe callus; And with among the contrast experiment of immature embryo callus as the particle gun transformation receptor, capacitation does not get positive plant, or transformation efficiency has only 0.91%.Show the present invention with young fringe as explant inoculation and then carry out particle gun and transform and can obtain higher genetic transformation frequency.
The present invention research overcome the low shortcoming of transformation efficiency of conversion system in the past as the conversion system of transformation receptor with young fringe, can obtain more transfer-gen plant, obtain higher genetic transformation frequency, transformation efficiency can reach 8.16%, is a kind of transformation system of wheat cdna efficiently.
Embodiment:
Embodiment
One. materials and methods
1. vegetable material is prepared
Selection comprehensive agronomy proterties Gaocheng 8901, H6756 and H311 preferably is a test material, gets the young fringe that field sterile lemma to female stamen original hase forms the phase, uses 0.1%HgCl
2Sterilization, aseptic water washing.
2. medium
Induce and the subculture medium of callus are MS+2mg/l 2.4-D
It is MS+0.2mol/L sorbierite+0.2mol/L mannitol that height oozes medium
Induce screening culture medium MS+0.2mg/L 2,4-D+5mg/L Basta
Differentiation screening culture medium MS+5mg/L Basta
Bud seedling elongation medium MS+0.2mg/1NAA+1.5mg/1BA
Root media 1/2MS+0.2mg/L IAA
3. condition of culture
Callus induction is 25 ℃ of dark cultivations, and the callus differentiation culture is 26 ℃ of 14h/10h illumination cultivation.
4. the preparation of particulate bullet
Take by weighing the 60mg bronze in the 1.5ml centrifuge tube, add 1ml70% ethanol, mix with vortex mixer, the centrifugal supernatant that goes repeats to wash 3 times with sterile water, adds the aseptic glycerine of 1ml 50%, and uses the vortex mixing.Get in the aseptic centrifuge tube of bronze to of the above-mentioned preparation of 50 μ l, order adds 5 μ l plasmid DNA, 50 μ l2.5MCaCl
2With 50 μ l0.1M spermidines, with the mixture vortex, centrifugal, go supernatant.Respectively precipitate 1 time with 70% ethanol and 100% ethanol, at last with 48 μ l100% ethanol suspend again, discrete particles.
5.PCR testing conditions
Reaction system is 10 μ L, and reaction condition is 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 50s, 41 ℃ of renaturation 40s, 72 ℃ are extended 1min, circulate 35 times; 72 ℃ are extended 5min, 4 ℃ of preservations.
Two. test procedure
1. get the young fringe that Gaocheng 8901, H6756 and three kind sterile lemmas of H311 to female stamen original hase forms the phase, as explant; Use 0.1%HgCl before the inoculation
2Sterilized 8-10 minute, aseptic water washing 2-3 time separates young fringe and is inoculated on the inducing culture in super-clean bench.
2. inoculate about a week and young fringe callus is put the culture dish central authorities of oozing medium in height, the PDS-1000/He type particle gun bombardment of producing with Bio-Rad company after 6 hours at the callus growth animated period.Bombardment pressure is 1100psi, and vacuum is 27-28pa, and the distance between converting material and the little bullet is 7cm.
3. bombard back 18h, callus is gone to recover on the inducing culture to cultivate 10 days.
4. the callus after recovering to cultivate goes to induce and carries out the Basta resistance screening on the screening culture medium, goes to after 20 days on the differentiation screening culture medium and screens.
5. go on the bud seedling elongation medium after the differentiation screening, this medium can effectively impel a large amount of small and weak bud seedling growths, thereby significantly improves green seedling differentiation rate.
6. the green seedling of regenerating goes to (1/2MS+0.2mg/L IAA) strong sprout on the root media.The green seedling that will contain healthy and strong root system at last is through 0～4 ℃ of vernalization, acclimatization and transplants.
7. transplant seedling and grow to 15cm when high, the Basta solution that picks 100mg/L with cotton swab is smeared the blade of transformant, observes the downright bad situation of blade after 10 days.
8. clip has the blade of the transformant of Herbicid resistant, adopts the CTAB method to extract total DNA, carries out PCR and detects.
Three. experimental result
1. the wheat different explants is to the reaction of cultured in vitro
This test has been carried out cultured in vitro to three genotypic in good time young fringes and corresponding immature embryo, the results are shown in Table 1.
Table 1. different cultivars and explant cultured in vitro result
Variety name explant inoculation number goes out more, and number healing rate (%) changes the green seedling differentiation rate of the differentiation green seedling number of callus
(％)
The in good time young fringe 95 95 100 58 32 55.17 of H6756
Immature embryo 100 100 100 63 7 11.11
The in good time young fringe 80 80 100 70 32 45.71 of H311
Immature embryo 100 98 98 60 5 8.33
Gaocheng 8901 in good time young fringes 100 99 99 80 60 75
Immature embryo 100 100 100 80 55 68.75
Above result can find out, H6756 and H311 are respectively 55.17% and 45.71% with young fringe as the green seedling differentiation rate of explant cultured in vitro, and the green seedling differentiation rate of its corresponding immature embryo has only 11.11% and 8.33%; The Gaocheng green seedling differentiation rate of 8901 young fringes is 75%, is higher than the green seedling differentiation rate 68.75% of immature embryo.
Conclusion: in good time young fringe is higher than its corresponding immature embryo as the green seedling differentiation rate that explant carries out cultured in vitro, carries out genetic transformation with young fringe as explant and can obtain higher green seedling differentiation rate.
2. wheat different explants particle gun transformation efficiency
Bombard the embryo callus that embryo callus that H6756, H311 and the young fringe of 8,901 three kinds in Gaocheng (strain) induce and immature embryo are induced respectively with particle gun.
Among three groups of contrast experiments, genetic transformation efficiency is respectively 5.36% and 0,2.53% and 0,8.16% and 0.91%.Concrete outcome sees Table 2.
Table 2 different explants genetic transformation result
The positive strain rate of PCR is counted in the positive strain of variety name explant bombardment outer planting Basta resistance Basta resistant strain PCR
Body sum strain digit rate (%) (%)
The in good time young fringe 932 205 22.00 50 5.36 of H6756
Immature embryo 431 0000
The in good time young fringe 475 70 14.74 12 2.53 of H311
Immature embryo 199 0000
Gaocheng 8901 in good time young fringes 49 13 26.53 4 8.16
Immature embryo 881 440 49.94 8 0.91
As can be seen from the above results, with young fringe callus during as the particle gun transformation receptor, genetic transformation efficiency is higher, can reach 8.16%, minimum is 2.53%, and with the immature embryo callus during as the particle gun transformation receptor, transformation efficiency is the highest to have only 0.91%, and particularly H6756 and H311 all do not obtain positive plant.
Conclusion: with young fringe as explant inoculation and then carry out particle gun and transform and can obtain higher genetic transformation frequency.
Claims (3)
1. a grow wheat high-efficiency genetic transforming method, it is characterized in that the sterile lemma idiophase is arrived the wheat children tassel of female stamen original hase idiophase as explant, on inducing culture, carry out callus induction and cultivate, carry out particle gun at young fringe callus growth animated period and transform.
2. the described method of claim 1 increases inducing and the elongation stage of a bud seedling in young fringe callus differentiation screening back.
3. the described method of claim 2, the concrete grammar step is:
1) young fringe is inoculated on the inducing culture as explant;
2) callus growth animated period bombards young fringe callus with particle gun;
3) above-mentioned callus is gone to carry out the Basta resistance screening on the screening culture medium;
4) will go on the bud seedling elongation medium through the callus of screening;
5) transplanting seedling grows to 15cm and screens once more with Basta when high.
6) the transformant blade carries out the PCR detection.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2003101153453A CN1618278A (en) | 2003-11-20 | 2003-11-20 | Technique for high-efficiency hereditary of wheat |
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| CNA2003101153453A CN1618278A (en) | 2003-11-20 | 2003-11-20 | Technique for high-efficiency hereditary of wheat |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101748152B (en) * | 2010-01-22 | 2011-05-25 | 西北农林科技大学 | Wheat anther callus gene gun genetic transformation method |
| CN102952823A (en) * | 2012-11-30 | 2013-03-06 | 中国科学院植物研究所 | Wheat genetic transformation method |
| CN104429957A (en) * | 2014-11-26 | 2015-03-25 | 西北农林科技大学 | Culture method and application of genetic transformation receptors of young wheat ears |
-
2003
- 2003-11-20 CN CNA2003101153453A patent/CN1618278A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101748152B (en) * | 2010-01-22 | 2011-05-25 | 西北农林科技大学 | Wheat anther callus gene gun genetic transformation method |
| CN102952823A (en) * | 2012-11-30 | 2013-03-06 | 中国科学院植物研究所 | Wheat genetic transformation method |
| CN104429957A (en) * | 2014-11-26 | 2015-03-25 | 西北农林科技大学 | Culture method and application of genetic transformation receptors of young wheat ears |
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