CN102071215B - Method for transforming in-vitro soybean growing points with gene gun - Google Patents
Method for transforming in-vitro soybean growing points with gene gun Download PDFInfo
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- CN102071215B CN102071215B CN201010564705A CN201010564705A CN102071215B CN 102071215 B CN102071215 B CN 102071215B CN 201010564705 A CN201010564705 A CN 201010564705A CN 201010564705 A CN201010564705 A CN 201010564705A CN 102071215 B CN102071215 B CN 102071215B
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Abstract
The invention discloses a method for transforming in-vitro soybean growing points with a gene gun, belonging to the technical field of bioengineering. The method is characterized by making bullets with carriers containing target genes, loading the bullets into the gene gun, then bombarding aseptic seedlings and performing inoculation induction to obtain T0-generation plants; performing rooting induction and seedling hardening by opening a vessel lid on the T0-generation plants, and then inducing the plants to bloom and fruit to obtain T1-generation seeds; and finally transplanting the T1-generation seeds to a phytotron or large fields to obtain T2-generation plants and homozygous positive transformant thereof. The method has the following advantage: by utilizing the characteristic that the plants prepared from the in-vitro soybean growing points are relatively easy to regenerate, the transformation efficiency can be obviously improved.
Description
Technical field
What the present invention relates to is a kind of method of bioengineering field, specifically is a kind of soybean isolated growth point gene rifle method for transformation.
Background technology
Soybean is a fifth-largest crop in the world today, is only second to wheat, paddy rice, corn and barley.China is soybean big producing country, also is consumption big country, all wants a large amount of genetically engineered soybeans of import every year.Compared with developed countries, China's soybean scientific and technological level is backward relatively, and soybean quality lacks international competitiveness.Strengthening the autonomous research and the utilization of genetically engineered soybean, is the developing direction that China should adhere in the soybean industry from now on.The principal element that the China of restriction at present soybean transgene technology further develops is; Agriculture bacillus mediated soybean cotyledon node, hypocotyl; The imperfection of transgene receptor plant regeneration systems such as callus, most regeneration plant deformities can not be extended, taken root; Last dead, cause transgene efficiency low.
Retrieval through to the prior art document finds that Wang Ping etc. have delivered " particle bombardment imports the GmDREB gene research of soybean " on " soybean science ", obtained 4 positive transformants.The transgene receptor that uses in this article is somatic embryo.Somatic embryo inducement receives genotypic restriction, has only a few kind to induce successfully at present.The plant regeneration frequency of somatic embryo is also very low in addition, so transformation efficiency is very low.
Summary of the invention
The present invention is directed to the above-mentioned deficiency that prior art exists; A kind of soybean isolated growth point gene rifle method for transformation is provided; Abundant with quantity; The soybean isolated growth point that is exposed to the outside be an acceptor, through particle gun with goal gene importing wherein, in progeny seed, screens then and obtains the positive transformant that isozygotys.Because soybean isolated growth point plant regeneration ratio is easier to, and can significantly improve transformation efficiency.
The present invention realizes that through following technical scheme the present invention installs on the particle gun through processing bullet with comprising the goal gene carrier, bombards aseptic seedling then, induces through inoculation, gets T0 for plant; To refine seedling to T0 after induce it to blossom and bear fruit for plant through root induction and uncork then, obtain T1 for seed; At last T1 is arrived phytotron or big Tanaka for the seed transfer, obtain T2, specifically comprise the steps: for the plant and the positive transformant that isozygotys thereof
Step 1 carries out disinfection to the soybean seeds surface with chlorine, is inoculated into then in the seed germination substratum, obtains aseptic seedling;
Complete and the healthy no scab of the kind skin of described soybean seeds.
Step 2 is peeled off abundant vegetative point, is paved with in the glass culture dish;
Described vegetative point is meant chooses that to have 4-6 sheet leaf primordium and length be 0.4-0.6 centimetre aseptic seedling, pushs leaf primordium aside and makes vegetative point be exposed to the aseptic seedling outside.
Step 3 is processed bullet with comprising the goal gene carrier, installs on the particle gun, bombards central part 3-4 time of petridish then;
Described bullet is meant: the diameter of parcel target dna is the gold granules of 1.5 μ m, and 60 μ g bronzes encapsulate 1 μ g DNA.
Described particle gun adopts BD81-GJ-1000 type gas electron gun; The metal particle that this particle gun will encapsulate foreign DNA quickens; Make it pass cell walls and get in the cell; Exogenous DNA molecule is sent in nucleus or the organoid, be incorporated on karyomit(e) or the cellular genome, thus the transfer of realization foreign DNA.
The described goal gene carrier that comprises is plant expression vector PBI221;
Step 4 is inoculated into into the seedling substratum with the vegetative point after the particle gun bombardment, induces into seedling, obtains T0 for plant;
Described one-tenth seedling substratum is for adding the MS substratum of 0.5mg/L 6-benzyl aminopurine.
Described one-tenth seedling substratum is for adding the 1/2MS substratum of 1.0mg/L Plant hormones regulators,gibberellins.
Described MS substratum adopts Murashige and the disclosed substratum of Skoog (1962), and the MS substratum is existing
Field such as biology and agricultural have a wide range of applications.Concrete composition is following:
Described 1/2MS substratum is the MS substratum that macroelement reduces by half.
Step 5 changes T0 over to root induction in the culturing bottle of the usefulness of taking root for plant;
The described culturing bottle that uses of taking root does, 20 centimetres high, and the tubbiness bottle that diameter is 8 centimetres gathers interior lactide and processes with degradable, is filled with root media in this culturing bottle;
Described root media is for adding 0.3mg/L indolebutyric acid 1/2MS substratum;
Step 6, in T0 generation, taken root plant uncork refining seedling after 3-5 days, and transfer and induces it to blossom and bear fruit in phytotron, obtains T1 for seed;
Described refining seedling is meant: open culturing bottle and take exercise by seedling is middle in the controlled environment chamber, make it adapt to physical environment as early as possible.
Step 7, T1 to phytotron or big Tanaka, obtains T2 for plant for the seed transfer;
Described transfer is meant to phytotron: with T0 for plant together with the degradable culturing bottle, transfer is in the soil of phytotron together.
Step 8 is carried out Molecular Identification and transgenic functional verification to T2 for plant, thereby is obtained the positive transformant that isozygotys.
Compared with prior art, the advantage that the present invention has is: 1. vegetative point is little to the influence of acceptor material as transformation receptor, has very high plant regeneration frequency, can improve transformation efficiency; 2. saved the Agrobacterium immersion, cultivated altogether, steps such as degerming, easy and simple to handle, degree of controllability is high.
Embodiment
Elaborate in the face of embodiments of the invention down, present embodiment provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment being to implement under the prerequisite with technical scheme of the present invention.
The soybean seeds of present embodiment is from this laboratory.
Present embodiment comprises the steps:
Step 1, the chlorine that generates with the reaction of 75ml Youxiaolin and 25ml concentrated hydrochloric acid is inoculated in the seed germination substratum (being MS+0.5mg/L 6-BA) of the MS that adds the 0.5mg/L 6-benzyl aminopurine soybean seeds surface sterilization 1 hour then, obtains aseptic seedling;
Step 2 is peeled off abundant band 4-6 sheet leaf primordium, and length is 0.4-0.6 centimetre vegetative point, pushs leaf primordium aside and makes vegetative point be exposed to the outside, and then these vegetative point being put into a diameter is in 5-6 centimetre the glass culture dish, is paved with at the bottom of the whole ware.
Step 3 is wrapping on the bullet with the carrier that comprises goal gene, installs on the particle gun, bombards central part 3-4 time of petridish then;
Step 4, it (is 1/2MS+1.0mg/L GA that the vegetative point after the particle gun bombardment is inoculated into the 1/2MS one-tenth seedling substratum that adds 1.0mg/L Plant hormones regulators,gibberellins
3) in, induce into seedling, obtain T0 for plant;
Step 5 changes T0 in the root media (being 1/2MS+0.3mg/L IBA) that adds 0.3mg/L indolebutyric acid 1/2MS for plant, root induction, and lactide was processed in culturing bottle gathered with degradation material;
Step 6, in T0 generation, taken root plant uncork refining seedling after 3-5 days, in phytotron, and induces it to blossom and bear fruit together with the transfer of degradable culturing bottle, obtains T1 for seed;
Step 7, T1 for planting seed in the controlled environment chamber in, obtain T2 for plant;
Step 8 is carried out PCR and Northern detection and transgenic functional verification to T2 for plant, thereby is obtained the positive transformant that isozygotys.
Present embodiment is sterilized to 100 soybean seeds, obtains after the aseptic seedling microscopically and peels off and obtain being with 4 leaf primordium, and length is 0.4-0.6 centimetre vegetative point.Particle gun has obtained 10 positive transformants that isozygoty after transforming.
Claims (1)
1. a soybean isolated growth point gene rifle method for transformation is characterized in that, comprises the steps:
Step 1; The chlorine that generates with the reaction of 75ml Youxiaolin and 25ml concentrated hydrochloric acid was to soybean seeds surface sterilization 1 hour; Be inoculated into then in the seed germination substratum, obtain aseptic seedling, said seed germination substratum is for adding the MS substratum of 0.5mg/L 6-benzyl aminopurine;
Step 2 is peeled off abundant band 4-6 sheet leaf primordium, and length is 0.4-0.6 centimetre vegetative point, pushs leaf primordium aside and makes vegetative point be exposed to the outside, and then these vegetative point being put into a diameter is in 5-6 centimetre the glass culture dish, is paved with at the bottom of the whole ware;
Step 3 is wrapping on the bullet with the carrier that comprises goal gene, installs on the particle gun, bombards central part 3-4 time of petridish then;
Step 4 is inoculated into into the vegetative point after the particle gun bombardment in the seedling substratum, induces into seedling, obtains T0 for plant, and said one-tenth seedling substratum is for adding the 1/2MS substratum of 1.0mg/L Plant hormones regulators,gibberellins, and said 1/2MS substratum is the MS substratum that macroelement reduces by half;
Step 5; Change T0 in the root media over to for plant, root induction, lactide was processed in culturing bottle gathered with degradation material; Said root media is for adding the 1/2MS substratum of 0.3mg/L indolebutyric acid, and said 1/2MS substratum is the MS substratum that macroelement reduces by half;
Step 6, in T0 generation, taken root plant uncork refining seedling after 3-5 days, in phytotron, and induces it to blossom and bear fruit together with the transfer of degradable culturing bottle, obtains T1 for seed;
Step 7, T1 for planting seed in the controlled environment chamber in, obtain T2 for plant;
Step 8 is carried out PCR and Northern detection and transgenic functional verification to T2 for plant, thereby is obtained the positive transformant that isozygotys.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270353A (en) * | 2007-03-19 | 2008-09-24 | 福建农林大学 | Method for quickly acquiring transgenic sugarcane by using pmi gene |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101270353A (en) * | 2007-03-19 | 2008-09-24 | 福建农林大学 | Method for quickly acquiring transgenic sugarcane by using pmi gene |
Non-Patent Citations (4)
| Title |
|---|
| Dennis E.McCabe et al..STABLE TRANSFORMATION OF SOYBEAN(GLYCINE MAX) BY PARTICLE ACCELERATION.《BIOTECHNOLOGY》.1988,第6卷第923页右栏第18行-第924页第2行，第925页左栏倒数第23行-倒数第5行. * |
| 王萍等.基因枪法将GmDREB基因导入大豆的研究.《大豆科学》.2007,第26卷(第3期),第315-318页. * |
| 王萍等.大豆转基因体系的研究进展.《遗传》.2004,第26卷(第6期),第969-976页. * |
| 程林梅等.大豆生物工程研究进展.《大豆科学》.2001,第20卷(第1期),全文. * |
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