CN103190346A - Method for constructing corn reproduction system taking coleoptile section as explant - Google Patents
Method for constructing corn reproduction system taking coleoptile section as explant Download PDFInfo
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- CN103190346A CN103190346A CN2013101407472A CN201310140747A CN103190346A CN 103190346 A CN103190346 A CN 103190346A CN 2013101407472 A CN2013101407472 A CN 2013101407472A CN 201310140747 A CN201310140747 A CN 201310140747A CN 103190346 A CN103190346 A CN 103190346A
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Abstract
The invention relates to a method for constructing a corn reproduction system taking a coleoptile section as an explant, and belongs to the field of agricultural biotechnology and crop breeding. The method mainly comprises the following steps of: (1) placing sterilized corn seeds into a sprouting culture medium for sprouting, and obtaining seedlings; (2) cutting the coleoptile sections from the seedlings, and inducing callus tissues; (3) performing transgenerational propagation on the callus tissues; (4) performing embryoid inducing on the callus tissues; and (5) performing differentiate seedlings on the embryoid. According to the method, obtaining of the materials is not limited by the seasons, and the propagation efficiency is high; and the method can be applied to large-scale genetic transforming of corns and propagation of haploid materials.
Description
Technical field
Technical field under the present invention is agricultural biological technical field and crop breeding field.
Background technology
Corn is one of the world five big crops, all occupies critical role in China and even global grain-production.In improving corn per unit area yield all multifactor, the effect of breeding of new variety accounts for 40%.Tremendous development along with Protocols in Molecular Biology, beginnings such as transgenic technology, monoploid technology play an important role in the corn variety seed selection, they have many unique advantages that are different from traditional breeding technology, therefore have been subjected to domestic and international breeding work person's great attention.
Transgenic technology is according to breeding objective, utilize the modern biotechnology means from the donor biology, to separate genes of interest, import the acceptor crop through DNA reorganization and genetic transformation, through the transgenic line of screening acquisition stably express, select to breed transgenosis new varieties or new germ plasm in conjunction with field trial and land for growing field crops.The transgenic breeding biggest advantage is that specific aim is very strong, can import in the crop of needs improvement, to reach its intended purposes according to the suitable gene of the directed selection of objective trait.Application by transgenic technology, not only can promote the efficiency of research and development of traditional breeding method, and promoted the breeding research and development accuracy rate, reduce blindness and repetition and waste, enriched traditional breeding technique, make march toward new step of the industry development level of crop breeding.At present, transgenic breeding has obtained great success in crops such as soybean, corn, cotton and rape, and wherein, transgenic corns has become second largest genetically modified crops of global cultivated area that are only second to genetically engineered soybean.
When research and development genetically modified crops new varieties, one of most important sport technique segment is exactly the crop genetic transformation technology.The genetic transforming method that corn is commonly used mainly is particle bombardment and agrobacterium-mediated transformation, and these two kinds of method for transformation all need at first to set up the corn regenerating system.
For a long time, conventional technology such as pedigree method, backcross method, multiple cross and recurrent selection are the main means of improvement corn inbred line, cultivated many elite hybrids that can adapt to various ecotopes, but there are shortcomings such as breeding cycle length in conventional genetic improvement always.Chase in 1949 have proposed the method for haploid induction choosing system.Principle is to utilize the haplobiont of abiogenesis or artificial culture, the liploid plant through manually or naturally doubling to obtain to isozygoty, therefrom seed selection inbred line again.Utilize the haploid breeding technology only to need the DH system that 2 generations just can obtain isozygotying, greatly shortened breeding cycle, improved breeding efficiency.Over nearly 10 years, some corn seed companies of states such as U.S. Monsanto Company, German KWS company and Russia are all attempting utilizing monoploid to bring out and are carrying out inbred line breeding, and wherein the technology with German KWS company is ripe relatively.Approximately can produce 1500-2000 pure lines according to incompletely statistics every year.Yet corn monoploid doubles with the probability of artificial doubling all lower naturally, and the genotype that a part is good is lost owing to doubling to get nowhere.If corn monoploid technology and corn tissue's cultivation regeneration techniques are combined, obtain monoploid by the parthenogenesis approach, using-system is cultivated regeneration techniques and monoploid is expanded numerous, so just can guarantee that more monoploid is doubled, reduce as far as possible owing to add losing of the low breeding material that causes of multiplying power.
Can find out that from above statement in corn gene technology and the monoploid technology, the foundation that tissue is cultivated regenerating system all is one of its crucial technology.The used material of corn regenerating system can be divided into rataria and non-rataria type, wherein the former comprises that also the embryo callus of directly inducing with rataria or rataria carries out the foundation of regenerating system, and the latter comprises and directly regenerating as material with shoot apical meristem and the embryo callus that utilizes mature embryo, blade, stem apex etc. to induce etc.The embryo callus that rataria and rataria are induced is simple to operate, and the plant regeneration ratio is easier to, and is acceptor material the most frequently used in the corn gene.But the used acceptor material genotype of transgenosis limits seriously, the rataria material is subject to season and the greenhouse experiment restriction, can not transform by the continual high-throughout corn that carries out, and has significant limitation in commercial application.In the material of non-rataria type, though directly easy with shoot apical meristem conversion and regeneration, and not limited by genotype, conversion ratio is low, and the regeneration plant of acquisition is chimera mostly, and the offspring need do a large amount of screening operations.And adopt materials such as mature embryo, blade, stem apex difficulty induce embryo callus, regeneration is difficulty.The tissue that we set up as material based on maize bud scale joint is cultivated regenerating system relatively and the conversion of maize immature embryos, transformation efficiency is lower slightly, but because it is not subjected to the restriction of greenhouse experiment and the season of growth, easy and simple to handle, can realize high flux, continual genetic transformation, thereby in the commercialization transgenosis research and development of corn, has sizable application potential.In the monoploid application facet, will be behind the corn monoploid seed sprouting by coleoptile joint evoked callus, and then obtain regeneration plant, can effectively expand numerously to monoploid, double to provide wide variety of materials for haploid.Compare with regenerating systems such as mature embryo, blade, stem apexs, the efficient of maize bud scale joint induced embryonic callus is higher, therefore can obtain a large amount of monoploid in the corn haploid breeding rapidly, lays a solid foundation for accelerating breeding process.
Summary of the invention
The object of the present invention is to provide a kind of is the corn renovation process of explant with the coleoptile joint, may further comprise the steps:
1) corn seed with sterilization places the germination medium to cultivate, and sprouts to obtain seedling;
2) from above-mentioned steps 1) the seedling that obtains cut the coleoptile joint, be inoculated on the callus inducing medium evoked callus and subculture and cultivate;
3) with above-mentioned steps 2) in the callus that obtains place the embryoid induction medium, obtain the maize calli embryoid;
4) with above-mentioned steps 3) in the callus embryoid that obtains place regeneration culture medium, obtain regrowth;
5) with above-mentioned steps 4) in the regrowth that obtains place root media, obtain to have the corn regeneration plant of 3-4 bar root;
Above-mentioned germination medium is MS+ maltose 40g/L+ enzymatic hydrolysis casein 0.1g/L+ glutamine 0.5g/L+2-morpholino b acid 2g/L+MgCl
26H2O 1.6g/L+ ascorbic acid 0.1g/L+6-BA 3mg/L+ picloram 0.01g/L;
The callus of induce medium is MS+ sucrose 30g/L+ Cobastab
10.5mg/L+ enzymatic hydrolysis casein 0.5g/L+ proline 1.5g/L+ silver nitrate 5mg/L+2,4-D 1.5-5mg/L+ picloram 0-0.001mg/L;
The embryoid induction medium is MS+ sucrose 30g/L+ proline 0.7g/L+6-BA 2mg/L;
Regeneration culture medium is MS+ sucrose 20g/L+ inositol 0.1g/L.
Step 2) the coleoptile joint described in refers to the apical meristem of seedling projection.
Step 2) it is that the callus that will obtain is cultivated propagation, the callus that obtains breeding at callus inducing medium that the subculture described in is cultivated.
The regeneration plant that step 6) obtains need carry out being transplanted in the soil after the hardening again.
Major technique effect of the present invention is:
1) the coleoptile joint that adopts of the present invention can be germinateed by mature seed at any time and obtain, and is not subject to seasonal restrictions, and also need not in the winter time as rataria in the greenhouse or numerous the obtaining in south, therefore draws materials when setting up regenerating system conveniently, expense is cheap;
2) the coleoptile joint evoked callus efficient that adopts of invention is than outer planting heights such as mature embryo, blades, and single coleoptile joint can obtain callus 2-3 time, realizes easily that therefore large-scale plant regeneration, the numerous effect of expansion are obvious;
3) invention employing coleoptile saves the I type callus state of inducing and will change into II type callus easily significantly better than the callus that is obtained by mature embryo, blade, thus seedling differentiation, and regeneration efficiency is higher, has good application prospects.
4) seed gramineous has the special construction of coleoptile outside plumule, and its growth plays guide and protective effect to plumule rapidly during seed germination.Therefore, the present invention not only is applied to the regenerating system of corn, also can be applicable to have the regenerating system of the gramineous plants of coleoptile special construction, as wheat, paddy rice etc.
Description of drawings
Fig. 1 is the seed of corn rudiment.
Fig. 2 is the maize bud scale joint that cuts.
The callus that Fig. 3 induces for the coleoptile joint.
Fig. 4 is that the expansion of callus is numerous.
Fig. 5 is the embryoid of callus induction.
Fig. 6 is the regrowth that differentiates.
Fig. 7 is taken root for regeneration plant.
Fig. 8 is that the regeneration plant of taking root is transplanted in the nutritive cube.
Embodiment
The invention will be further described below in conjunction with concrete enforcement, but the present invention is not limited to following examples.
Among the following embodiment, if no special instructions, be conventional method.
Inducing and plant regeneration of embodiment one corn seed coleoptile joint callus
1. seed sterilization treatment
At first the foreign material in the corn seed are cleaned out, are chosen full complete seed, then it is put in the container with distilled water and cleans 3 times, during concuss with the residue of cleaning attaching surface.With 75% alcohol-pickled 3 minutes, the clorox with concentration 5.25% soaked 20 minutes then on super-clean bench, at last with aqua sterilisa flushing 5-6 time.
2. germinate and cultivate
Seed after the sterilization is placed the germination medium, attention will with the embryo of seed up level be inoculated in the germination medium.In culturing room (16h/d illumination, light intensity 80-100 μ E/m
2/ s, temperature 26-28 ℃) cultivated 7-10 days.
Above-mentioned germination medium is to add following material in the MS minimal medium: maltose 40g/L, enzymatic hydrolysis casein 0.1g/L, glutamine 0.5g/L, MES(2-morpholino b acid) 2g/L, MgCl
26H2O 1.6g/L, ascorbic acid 0.1g/L, 6-BA 3mg/L, picloram 0.01g/L.
3. the coleoptile joint cuts
The seedling that has grown is scaled off, put into the empty ware of a sterilization, downcut seedling coleoptile joint zone each 0.5cm up and down with scalpel.The stem section vertically is divided into two.
4. callus induces
Coleoptile is saved wound face contact medium, be put in the callus of induce medium, 10-16 material/ware cultivated (16h/d illumination, light intensity 80-100 μ E/m in the illumination cultivation chamber
2/ s, 28 ℃ of temperature).Irregularly carry out the excision that coleoptile saves big bud during the callus of induce, 2-4 went to embryo callus in the new callus of induce medium after week.28 ℃ of dark cultivations 2-4 week can induce callus.
Above-mentioned callus of induce medium is to add following material in the MS minimal medium: sucrose 30g/L, VB
1(Cobastab
1) 0.5mg/L, enzymatic hydrolysis casein 0.5g/L, proline 1.5g/L, silver nitrate 5mg/L, 2,4-D 1.5-5mg/L, picloram 0-0.001mg/L.
5. plant regeneration
The callus that induces is gone in the embryoid induction medium 28 ℃ of dark cultivations 7 days.To organize piece to go in the regeneration culture medium, 1-6 callus/ware, 16h/d illumination, light intensity 80-100 μ E/m
2/ s cultivates 4-6 week for 28 ℃.The seedling morsel of growing thickly that bears again is placed on and carries out the subculture cultivation in the culture dish (90*25mm).The seedling morsel of growing thickly that bears again is placed on and carries out culture of rootage in the root media, 16h/d illumination, light intensity 80-100 μ E/m
2/ s cultivated about 10 days, the seedling that grows 3-4 bar root is carried out hardening for 28 ℃.
Above-mentioned embryoid induction medium is to add following material in the MS minimal medium: sucrose 30g/L, proline 0.7g/L, 6-BA 2mg/L.
Above-mentioned regeneration culture medium is to add following material in the MS minimal medium: sucrose 20g/L, inositol 0.1g/L.
Root media is the conventional corn seedling medium of growing thickly.
6. data statistics
We have chosen 11 corn varieties (being) and have carried out the experiment (table 1) of coleoptile joint, and these 11 corn varieties (being) all can be purchased and obtain.Obtain 2310 coleoptiles joint, 268 nascent callus altogether, expanded numerous 14588 secondary callus that gone out.Wherein 4 kinds (being) such as DH382, PH2VK, PH6WC, Zheng 58 have formed 509 embryoids altogether, have finally obtained 840 regeneration plants.
The average inductivity of this 11 corn varieties (being) callus is 12%, and that the highest is Hi II A, and inductivity can reach 52%, but finally fails to obtain regeneration plant.The regeneration average efficiency that wherein can obtain 4 kinds (being) of regeneration plant is that 1.65(is that each embryoid can form 1.65 seedling).
The comparison of the different corn variety coleoptile joint of table 1 evoked callus
Embodiment two different kinds of culture medium are to the influence of maize bud scale joint callus induction rate
MS and N6 medium are two kinds of the most frequently used type of culture medium during corn tissue cultivates, and callus of induce rate and the regeneration efficiency difference of different cultivars in these two kinds of medium is bigger.We have chosen callus induction rate height or regeneration efficiency, and PH2VK, PHB1M, PH6WC carry out different kinds of culture medium to the experiment of coleoptile joint callus induction rate as experiment material preferably.
Experimental result sees Table 2, and the MS-genotype is that the used medium of this genotype is the MS medium in the table, and the N6-genotype is that the used medium of this genotype is the N6 medium.We have obtained 702 coleoptile joints altogether, have induced 164 callus, and average inductivity is 23.4%.In the MS medium, induce efficient to be higher than N6.
The different kinds of culture medium of table 2 are to the comparison of maize bud scale joint evoked callus
Claims (4)
1. one kind is the corn renovation process of explant with the coleoptile joint, may further comprise the steps:
1) corn seed with sterilization places the germination medium to cultivate, and sprouts to obtain seedling;
2) from above-mentioned steps 1) the seedling that obtains cut the coleoptile joint, be inoculated on the callus inducing medium evoked callus and subculture and cultivate;
3) with above-mentioned steps 2) in the callus that obtains place the embryoid induction medium, obtain the maize calli embryoid;
4) with above-mentioned steps 3) in the callus embryoid that obtains place regeneration culture medium, obtain regrowth;
5) with above-mentioned steps 4) in the regrowth that obtains place root media, obtain to have the corn regeneration plant of 3-4 bar root;
Above-mentioned germination medium is MS+ maltose 40g/L+ enzymatic hydrolysis casein 0.1g/L+ glutamine 0.5g/L+2-morpholino b acid 2g/L+MgCl
26H2O 1.6g/L+ ascorbic acid 0.1g/L+6-BA 3mg/L+ picloram 0.01g/L;
The callus of induce medium is MS+ sucrose 30g/L+ Cobastab
10.5mg/L+ enzymatic hydrolysis casein 0.5g/L+ proline 1.5g/L+ silver nitrate 5mg/L+2,4-D 1.5-5mg/L+ picloram 0-0.001mg/L;
The embryoid induction medium is MS+ sucrose 30g/L+ proline 0.7g/L+6-BA 2mg/L;
Regeneration culture medium is MS+ sucrose 20g/L+ inositol 0.1g/L.
2. the method for claim 1 is characterized in that step 2) described in coleoptile joint refer to the apical meristem of seedling projection.
3. method as claimed in claim 2 is characterized in that step 2) described in subculture to cultivate be that the callus that will obtain is cultivated propagation, the callus that obtains breeding at callus inducing medium.
4. method as claimed in claim 3 is characterized in that the regeneration plant that step 6) obtains need carry out being transplanted in the soil after the hardening again.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103444525A (en) * | 2013-08-15 | 2013-12-18 | 广东省农业科学院作物研究所 | Hormone-free corn callus regeneration method |
| CN104026017A (en) * | 2014-06-20 | 2014-09-10 | 四川农业大学 | Culture method of corn haplobiont |
| CN104130058A (en) * | 2014-07-24 | 2014-11-05 | 吉林省农业科学院 | High-efficiency inducing subculture medium for young corn embryos and preparation method thereof |
| CN106538390A (en) * | 2016-12-06 | 2017-03-29 | 百奥森（江苏）食品安全科技有限公司 | A kind of poison-removing method for grains such as Semen Maydiss, Semen Tritici aestivis |
| CN109156362A (en) * | 2018-10-30 | 2019-01-08 | 四川施邦威生物科技发展有限公司 | A kind of corn training seedling method for culturing seedlings |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103444525A (en) * | 2013-08-15 | 2013-12-18 | 广东省农业科学院作物研究所 | Hormone-free corn callus regeneration method |
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| CN104130058A (en) * | 2014-07-24 | 2014-11-05 | 吉林省农业科学院 | High-efficiency inducing subculture medium for young corn embryos and preparation method thereof |
| CN104130058B (en) * | 2014-07-24 | 2016-08-17 | 吉林省农业科学院 | A kind of maize immature embryos efficiently induces subculture medium and preparation method |
| CN106538390A (en) * | 2016-12-06 | 2017-03-29 | 百奥森（江苏）食品安全科技有限公司 | A kind of poison-removing method for grains such as Semen Maydiss, Semen Tritici aestivis |
| CN109156362A (en) * | 2018-10-30 | 2019-01-08 | 四川施邦威生物科技发展有限公司 | A kind of corn training seedling method for culturing seedlings |
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Application publication date: 20130710 |