CN103320377A

CN103320377A - Culture medium for cultivating transgenic plant

Info

Publication number: CN103320377A
Application number: CN2012100783816A
Authority: CN
Inventors: 漆小泉; 赵素珍
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2012-03-22
Filing date: 2012-03-22
Publication date: 2013-09-25
Anticipated expiration: 2032-03-22
Also published as: CN103320377B

Abstract

The invention discloses a culture medium for cultivating a transgenic plant. The culture medium group comprises a callus induction culture medium, a subculture culture medium, a co-culture culture medium, a screening culture medium and a regeneration culture medium. Experiment results show that: with a transformation system and a transformation method of the present invention, average every 100 brachypodium distachyon immature embryos can produce about 103 positive transformation seedlings, wherein a positive seedling transformation rate achieves 103.4%, and is far higher than positive seedling transformation rates of the traditional and grain model plant rice, and the highest rice positive seedling transformation rate is 47.6% (Chinese Bulletin of Botany, 2008, 25(3),322-331). With the efficient agrobacterium transformation system of the brachypodium distachyon, the important technology means is provided for temperate zone and grain plant gene function researches.

Description

A kind of substratum for cultivating transgenic plant

Technical field

The present invention relates to biological technical field, relate in particular to a kind of substratum for cultivating transgenic plant.

Background technology

False bromegrass formal name used at school two fringe false bromegrasses (Brachypodium distachyon (L.) P.Beauv), have another name called Purple False Brome, originate from Mediterranean Sea and Middle East, belong to Agrostidoideae (Pooideae) false bromegrass and belong to (Brachypodieae), in phyletic evolution, be in same evolution branch with temperate zone and cereal crop (Temperate cereals), grass kind plant (Meadow and Forage grasses).Because false bromegrass plant short and small (15-20cm), genome only has 272Mb, and self-pollination, breeding time short (8-12 week), easily cultivation has developed the model plant that becomes a kind of new temperate zone and cereal, grass kind plant in recent years rapidly.In February, 2010, international false bromegrass started the genome sequencing analytical work (http://www.brachypodium.org) that the council (The International Brachypodium Initiative) has finished two fringe false bromegrass Bd21.As the model plant of studying temperate zone and cereal, grass kind plant gene function, efficiently Agrobacterium transformation system is most important fast.

The Agrobacterium-mediated Transformation of false bromegrass is studied with regard to beginning as far back as calendar year 2001 in the world, but used genotype is different, and average conversion efficiency very low (Draper, J etc., 2001).The John Vogel of U.S. USDA Western Regional Research Center in 2008, with false bromegrass strain Bd21-3 as acceptor, make average conversion efficiency reach 37%, used callus induction substratum is: LS salt, 3% sucrose, 2,4-D 11.25 μ M, 0.2% plant gel, however Bd21-3 has very big-difference (Vogel with Bd21 on genetic background, J etc., 2008).The Philippe Vain of Britain John Innes Centre has set up efficient transformation system with order-checking strain Bd21, and this system is take the MS substratum as minimum medium, and Totomycin is the foliage filter screening mark, complex operation, and be not suitable for domestic application.

Summary of the invention

An object of the present invention is to provide a kind of substratum group for cultivating the transgenosis false bromegrass.

Substratum group provided by the invention is comprised of calli induction media, subculture medium, common culture medium, screening culture medium and regeneration culture medium;

Described calli induction media is to add 2,4-D, maltose, altheine, inositol, caseinhydrolysate, proline(Pro) and vitamin in the MS minimum medium, obtains calli induction media; Wherein, described 2, the final concentration of 4-D in described calli induction media is 2.5mg/L, the final concentration of described maltose in described calli induction media is 30mg/L, the final concentration of described altheine in described calli induction media is 150mg/L, the final concentration of described inositol in described calli induction media is 0.2g/L, the final concentration of described caseinhydrolysate in described calli induction media is 0.5g/L, the final concentration of described proline(Pro) in described calli induction media is 700mg/L, and the final concentration of described vitamin in described calli induction media is 1.5mg/L;

Described subculture medium is to add 2,4-D and 6-BA in the MS minimum medium, obtains subculture medium; Wherein, described 2, the final concentration of 4-D in described subculture medium is 2.5mg/L, and the final concentration of described 6-BA in described subculture medium is 0.01mg/L,

Described altogether culture medium is to add glucose and Syringylethanone in the MS minimum medium, obtains common culture medium; Wherein, the final concentration of described glucose in described altogether culture medium is 10g/L, and the final concentration of described Syringylethanone in described altogether culture medium is 200 μ M,

Described screening culture medium is to add paromycin and Ticarcillin/Clavulanate Acid in the MS minimum medium, obtains screening culture medium; Wherein, the final concentration of described paromycin in described screening culture medium is 25mg/L-50mg/L, and the final concentration of described Ticarcillin/Clavulanate Acid in described screening culture medium is 125mg/L-225mg/L;

Described regeneration culture medium is to add cytokinin in the MS minimum medium, obtains regeneration culture medium; Wherein, the final concentration of described cytokinin in described regeneration culture medium is 0.2mg/L.

In the above-mentioned substratum group, described screening culture medium is following screening culture medium I or screening culture medium II:

Among the described screening culture medium I, the final concentration of described paromycin in described screening culture medium I is 25mg/L, and the final concentration of described Ticarcillin/Clavulanate Acid in described screening culture medium I is 225mg/L;

Among the described screening culture medium II, the final concentration of described paromycin in described screening culture medium II is 50mg/L, and the final concentration of described Ticarcillin/Clavulanate Acid in described screening culture medium II is 125mg/L.

In the above-mentioned substratum group, all also comprise peptizer in the various described substratum, described peptizer is specially agar, and the concentration of described agar in described various substratum is 7g/L.

Another object of the present invention provides a kind of method for preparing above-mentioned substratum group.

Method provided by the invention comprises the steps:

1) the described calli induction media in the above-mentioned substratum group of preparation, described subculture medium, described altogether culture medium, described screening culture medium and described regeneration culture medium;

2) with step 1) the described calli induction media that obtains, described subculture medium, described altogether culture medium, described screening culture medium and described regeneration culture medium independent packaging, obtain the substratum group.

In the aforesaid method, described calli induction media is according to the preparation of the method that comprises the steps: with described 2,4-D, described maltose, described altheine, described inositol, described caseinhydrolysate, described proline(Pro) and described vitamin are added in the MS minimum medium according to described separately final concentration and obtain;

Described subculture medium is according to the preparation of the method that comprises the steps: with described 2,4-D and described 6-BA are added in the MS minimum medium according to described separately final concentration and obtain;

Described altogether culture medium is according to the method preparation that comprises the steps: described glucose and described Syringylethanone are added in the MS minimum medium according to described separately final concentration obtain;

Described screening culture medium is according to the preparation of the method that comprises the steps: described paromycin and described Ticarcillin/Clavulanate Acid are added in the MS minimum medium according to described separately final concentration obtain;

Described regeneration culture medium is according to the preparation of the method that comprises the steps: described cytokinin is added in the MS minimum medium according to described final concentration obtains.

In the aforesaid method, all also add peptizer in various described substratum, described peptizer is specially agar, and the concentration of described agar in described various substratum is 7g/L.

The application of above-mentioned substratum group in cultivating transgenic plant also is the scope of protection of the invention; In the above-mentioned application, described plant is specially two fringe false bromegrasses.

The 3rd purpose of the present invention provides a kind of method of cultivating transgenic plant.

Method provided by the invention comprises the steps:

1) with cultivating in the described calli induction media of plant explants in above-mentioned substratum group, obtains embryo callus;

2) with step 1) cultivate in the described subculture medium of embryo callus in above-mentioned substratum group that obtain, obtain subculture embryo callus piece; 3) goal gene is imported step 2) behind the subculture embryo callus piece that obtains, in common culture medium, cultivate, obtain common cultured calli piece;

4) with step 3) the common cultured calli piece that obtains cultivates in described screening culture medium I and described screening culture medium II successively, obtains resistant calli;

5) with step 4) resistant calli that obtains cultivates in regeneration culture medium, namely obtains transgenic plant.

In the aforesaid method, step 1) in, described cultivation is 25 ℃ of dark cultivations for 3 week-4 weeks; Described cultivation is specially 25 ℃ of dark cultivations for 4 weeks;

Step 2) in, described cultivation is succeeding transfer culture 2-3 time; Described each succeeding transfer culture is the 25 ℃ of dark 2-3 of cultivation weeks; Described cultivation is specially succeeding transfer culture 3 times; Described each succeeding transfer culture is 25 ℃ of dark cultivations for 3 weeks;

Step 3) in, described goal gene imports step 2) the subculture embryo callus piece that obtains is for to be immersed in described subculture embryo callus piece in the restructuring Agrobacterium bacterium liquid that contains goal gene, and the temperature of described immersion is 23 ℃-28 ℃; The time of described immersion is 8-10min, and the temperature of described immersion is specially 25 ℃, and the time of described immersion is specially 10min;

Described in common culture medium, the cultivation as secretly cultivating 3 days at 25 ℃;

Step 4) in, the described cultivation at described screening culture medium I is to cultivate the dark 2-3 of cultivation week at 25 ℃, and described being specially at 25 ℃ in described screening culture medium I cultivation cultivated dark the cultivation for 2 weeks;

The described cultivation as cultivating secretly at 25 ℃ at described screening culture medium II cultivated for 4 weeks;

Step 5) in, the described cultivation in regeneration culture medium is 3 week-5 weeks of 25 ℃ of illumination cultivation, and the described time of cultivating in regeneration culture medium was specially for 3 weeks, and described intensity of illumination is 6000Lux, and described periodicity of illumination is illumination in 16 hours, 8 hours dark.

In the aforesaid method, step 1) in, described explant is rataria, described plant is two fringe false bromegrasses;

In described step 2) and step 3) between also comprise described subculture embryo callus piece pre-incubated step in described subculture medium, described preculture is 25 ℃ of dark cultivations 3-4 days, described preculture is specially 25 ℃ of dark cultivations 4 days;

Step 3) in, the bacterium liquid that the described restructuring Agrobacterium bacterium liquid that contains goal gene contains described goal gene for suspending restructuring Agrobacterium obtains, the described restructuring Agrobacterium that contains described goal gene is specially pK7G2D is imported the recombinant bacterium that obtains in the agrobacterium tumefaciens.

Of the present invention experimental results show that, the substratum that utilization of the present invention provides and cultural method carry out goal gene and transform two fringe false bromegrasses, detect average per 100 two fringe false bromegrass ratarias and just can produce approximately 103 positive transformation seedlings, positive seedling transformation efficiency reaches 103.4%, is higher than traditional and cereal model plant paddy rice far away.The peak rate of conversion of the positive seedling of paddy rice be 47.6% (BULLETIN OF BOTANY Vol., 2008,25 (3), 322-331).The efficient Agrobacterium-mediated Transformation system of false bromegrass provides important technique means for temperate zone and cereal grass gene functional research.

Description of drawings

Fig. 1 is the schema that transgenosis two fringe false bromegrasses obtain

Embodiment

Employed experimental technique is ordinary method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

MS minimum medium used among the following embodiment is comprised of solvent and solute, and solvent is water, and solute is specifically filled a prescription as follows:

The solute of table 1.MS minimum medium

Macroelement	Concentration (gL in the substratum ^-1)
		NH ₄NO ₃	1.65
KNO ₃	1.9
		KH ₂PO ₄	0.17
MgSO ₄.7H ₂O	0.37
		CaCl ₂	0.44
Trace element	Concentration (mgL in the substratum ^-1)
		FeSO ₄.7H ₂O	27.8
Na ₂EDTA	37.3
		MnSO ₄.4H ₂O	22.3
ZnSO ₄.4H ₂O	8.6
		H ₃BO ₃	6.2
KI	0.83
		Na ₂MoO ₄.2H ₂O	0.25
CuSO ₄.5H ₂O	0.025
		CoCl ₂.6H ₂O	0.025
Organic composition	Concentration (mgL in the substratum ^-1)

Glycine	2.0
		Vitamin	0.4
Pyridoxine hydrochloride	0.5
		Nicotinic acid	0.5
Inositol	100

The preparation of embodiment 1, substratum

1, calli induction media

With 2,4-D (2, the 4-dichlorphenoxyacetic acid), maltose, altheine, inositol, caseinhydrolysate, proline(Pro), vitamin, agar adds in the MS minimum medium, obtain calli induction media, wherein, 2, the final concentration of 4-D in calli induction media is 2.5mg/L, the final concentration of maltose in calli induction media is 30mg/L, the final concentration of altheine in calli induction media is 150mg/L, the final concentration of inositol in calli induction media is 0.2g/L, the final concentration of caseinhydrolysate in calli induction media is 0.5g/L, the final concentration of proline(Pro) in calli induction media is 700mg/L, the final concentration of vitamin in calli induction media is 1.5mg/L, the final concentration of agar in calli induction media is 7g/L, and transferring pH with the sodium hydroxide of 1M concentration or hydrochloric acid is 5.8.

2, subculture medium

With 2,4-D, 6-BA (6-benzyl aminopurine), agar add in the MS minimum medium, obtain subculture medium, wherein, the final concentration of 2,4-D in subculture medium is 2.5mg/L, and the final concentration of 6-BA in subculture medium is 0.01mg/L, the final concentration of agar in calli induction media is 7g/L, and transferring pH with the sodium hydroxide of 1M concentration or hydrochloric acid is 5.8.

3, be total to culture medium

Glucose, Syringylethanone, agar are added in the MS minimum medium, obtain common culture medium, wherein, the final concentration of glucose in common culture medium is 10g/L, the final concentration of Syringylethanone in common culture medium is 200 μ M, the final concentration of agar in calli induction media is 7g/L, and transferring pH with the sodium hydroxide of 1M concentration or hydrochloric acid is 5.2.

4, screening culture medium I

Screening culture medium I: paromycin, Timetin (Ticarcillin/Clavulanate Acid), agar are added in the MS minimum medium, obtain screening culture medium I, the final concentration of paromycin in screening culture medium I is 25mg/L, the final concentration of Timetin in screening culture medium I is 225mg/L, the final concentration of agar in calli induction media is 7g/L, is 5.8 with sodium hydroxide or the hydrochloric acid adjust pH of 1M concentration.

Screening culture medium II: paromycin, Timetin (Ticarcillin/Clavulanate Acid), agar are added in the MS minimum medium, obtain screening culture medium II, the final concentration of paromycin in screening culture medium II is 50mg/L, the final concentration of Timetin in screening culture medium II is 125mg/L, the final concentration of agar in calli induction media is 7g/L, is 5.8 with sodium hydroxide or the hydrochloric acid adjust pH of 1M concentration.

5, regeneration culture medium

With KT (cytokinin, kinetin, chemical name KT (or n6-furfuryladenine).), agar adds in the MS minimum medium, obtains regeneration culture medium, wherein, the final concentration of KT in regeneration culture medium is 0.2mg/L, the final concentration of agar in calli induction media is 7g/L, transferring pH with the sodium hydroxide of 1M concentration or hydrochloric acid is 5.8.

The acquisition of embodiment 2, transgenosis two fringe false bromegrasses

The schema that transgenosis two fringe false bromegrasses obtain as shown in Figure 1, wherein, A is that two fringe false bromegrass Bd21, B are that two fringe false bromegrass Bd21 ratarias, C are that callus of induce training, D are that succeeding transfer culture, E are that foreign DNA infects callus lines, F is that fluorescigenic callus lines, G are callus regeneration seedling, the positive regrowth of H.

One, explant obtains

Two fringe false bromegrasses (Brachypodium distachyon (L.) P.Beauv) Bd21 (Wang Honggui, Wang Baoli, Lin Chentao, Fu Yongfu, the morphological observation of two fringe false bromegrass Bd21, northwest agricultural journal, 6 phases in 2007, the morphological observation of two fringe false bromegrass Bd21, the public can obtain from Institute of Botany, Chinese Academy of Sciences).

The plantation of Bd21: vernalization: false bromegrass seed room temperature was placed down in thieving paper vernalization 3-5 days (apparent temperature and decide), bud is long to be transplanted to flowerpot during to 1-2cm, if want to improve percentage of germination, will plant skin and remove.Do not bury soil during transplanting excessively dark, water after the cultivation.Culture condition: 22 degree, illumination in 16 hours, intensity of illumination reaches 6000Lux, 8 hours dark.

Take away the Bd21 children fringe of flower about three weeks of pollination in 70% alcohol disinfecting 7min, aseptic water washing for several times; Push clever shell aside with hand (band sterile gloves), the silk noil tweezer is chosen the Bd21 rataria.

Two, the preparation of EHA105/pK7G2D bacteria suspension

1) pK7G2D carrier

With kalamycin resistance, band green fluorescence labelled protein GFP, its construction process is as follows:

The carrier is carrier that uses has pK7WG2D.1, pK7GWIWG2D (II) .0 and pENTR4 (to be the Gateway Destination vector network address of Invitrogen company: http://gateway.psb.ugent.be/index).

The concrete construction process of pK7G2D carrier:

The first step: with HindIII segment (the p35S promotor among pK7GWIWG2D (II) .0,700bp) downcut, be connected to the pK7WG2D.1 carrier that removes the HindIII segment, obtain the toxalbumin with ccd, promotor changes the pK7WG2D.1 carrier of p35S, called after pK7.1 into;

Second step: EcoRI single endonuclease digestion pENTR4 carrier, remove toxalbumin, called after pENTR4-ccd;

The 3rd step: permutoid reaction occurs in pK7.1 and pENTR4-ccd under LR recombinase (Invitrogen, Cat.No.11791-100) effect, remove the ccd toxalbumin, becomes for the simple insertion vector pK7G2D that transforms false bromegrass.

2) bacterial strain agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105

Change carrier pK7G2D over to agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 (Hooykaas PJ, Schilperoort RA.Agrobacterium and plant genetic engineering.Plant Mol Biol.1992May; 19 (1): 15-38, the public can obtain from Institute of Botany, Chinese Academy of Sciences.) in, wherein carry the EHA105 called after EHA105/pK7G2D of carrier pK7G2D, add Rifampin (50mg/L), spectinomycin (100mg/L) screening in solid medium MGL cultivation, obtaining transformant is positive recombinant bacterium, called after EHA105/pK7G2D.

Cultivate the solid medium MGL prescription of Agrobacterium EHA105/pK7G2D:

Treat that substratum is cooled to add 200 μ M AS (Syringylethanone)+75mg/L spectinomycins, the 50mg/L Rifampin before being down flat plate about 60 degree.

3) preparation of EHA105/pK7G2D bacteria suspension

Outstanding bacterium liquid AAM-CAS is prepared as follows:

With a large amount of (CaCl of AAM ₂.2H ₂O 1.5g/L, KH ₂PO ₄1.2g/L, MgSO ₄.7HO ₂2.5g/L, KCl 29.5g/L)), organic (glycine 7.5mg/L, vitamin 0.1mg/L, pyridoxine hydrochloride 0.5mg/L of AAM, nicotinic acid 0.5mg/L, glutamine 876mg/L, aspartic acid 266mg/L, arginine 174mg/L), B5 trace (specifically seeing the following form 2), caseinhydrolysate 0.5g/L, sucrose 10g/L, N.F,USP MANNITOL 10g/L, transferring pH with the sodium hydroxide of 1M concentration or hydrochloric acid is 5.2, adds Syringylethanone 200 μ M before the outstanding bacterium.

Table 2 is B5 trace ingredients

The single bacterium colony of picking EHA105/pK7G2D is in 10ml MGL substratum (kantlex 100mg/L, Rifampin 50mg/L), 28 ℃ of shaking culture are to logarithmic phase, be transferred to 28 ℃ of shaking culture of same fresh culture with 1: 100 ratio again, make about OD600=0.5 the Agrobacterium that obtains activating; The centrifugal 5min of Agrobacterium 4000g that activation is good outwells substratum, with the resuspended thalline of isopyknic outstanding bacterium liquid AAM-CAS, obtains the EHA105/pK7G2D bacteria suspension.

Three, transform

1, callus of induce is cultivated

Above-mentioned one rataria that obtains (explant) is placed the calli induction media that is obtained by embodiment 1,25 ℃ of dark cultivations for 4 weeks obtain embryo callus.

2, succeeding transfer culture

Selecting dry and comfortable embryo callus piece (diameter 2-3 millimeter) from the above-mentioned embryo callus that newly induces transfers 25 ℃ of 3 weeks of dark cultivation in the fresh subculture medium that is obtained by embodiment 1, subculture 3 times obtains subculture embryo callus piece (diameter 2-3 millimeter).

3, preculture

Before conversion, again subculture embryo callus piece is transferred to 25 ℃ of dark cultivations 4 days of fresh subculture medium, obtains the preculture callus lines.

4, cultivate altogether

4 days preculture callus lines of preculture (is infected callus, it is 3 preculture callus lines that an embryo callus can be grown at least) immerse by the two EHA105/pK7G2D bacteria suspensions that obtain and contaminate (immersion) (room temperature, 25 ℃) 10min, transfer to common culture medium after blotting bacteria suspension with sterilized water again, cultivated altogether 3 days under 25 ℃ of dark; Obtain common cultured calli piece.

5, screening and culturing

Directly forward the common cultured calli piece of above-mentioned acquisition to by a screening culture medium I who obtains 25 ℃ of dark cultivations for 2 weeks; Transfer to 25 ℃ of dark cultivations for 2 weeks by a screening culture medium II who obtains, fluorescence is dissected the callus lines (green point) that Microscopic observation is chosen green-emitting fluorescence.Each green point is continued dark the cultivation for 2 weeks 25 ℃ of screening culture medium II again, and the callus lines of statistics survival is resistant calli.

If the kanamycin-resistant callus tissue growth is slower, can be in a fresh screening culture medium II screening and culturing generation.

6, regeneration is cultivated

Above-mentioned resistant calli is transferred to regeneration culture medium, in 25 ℃ of illumination differentiation culture (intensity of illumination 6000Lux, periodicity of illumination illumination in 16 hours, 8 hours dark), cultivates and 3 weeks differentiated seedling, the quantity of the seedling that statistics obtains.

Again the seedling that obtains is screened under the fluorescence anatomical lens, the positive regrowth of green-emitting fluorescence is transgenosis two fringe false bromegrass seedlings, above-mentioned positive regrowth is transplanted to the illumination cultivation chamber cultivates (22 ℃, illumination in 18 hours), obtain transgenosis two fringe false bromegrass plant.

Four, checking transformation efficiency

1, callus of induce rate

The embryo callus result that the statistics callus of induce obtains such as following table 1, calculates inductivity (embryo callus/explant) and reaches 75%.

Table 1 false bromegrass rataria embryo callus subculture inductivity

2, callus instantaneous conversion efficient

In the step of above-mentioned screening and culturing, fluorescence is dissected the callus lines (green point) of Microscopic observation green-emitting fluorescence and the per-cent of preculture callus lines (be also referred to as and infect callus) is callus instantaneous conversion efficient.

The result is as shown in table 2, and callus instantaneous conversion efficient reaches 176.9% (greater than 100% being because one is infected on the callus, growing several green points).

Table 2 false bromegrass callus piece instantaneous conversion efficient

The culture dish numbering	Infect callus	Green point	The mean fluorecence piece
				1	32	54
2	28	48
				3	25	31
4	24	86
				5	28	58
6	25	45
				7	26	65

8	30	38
				9	26	29
10	25	33
				11	24	38
12	25	49
				13	24	31
Amount to	342	605	605/342＝1.77

3, the fluorescence callus lines is stable into motility rate

Each Agrobacterium of screening after twice is infected the fluorescence tissue block that produces on the callus piece, and after these fluorescence tissue block continued to cultivate, unstable the losing that has only had stable integration could become positive callus.

Statistics is in the step that above-mentioned screening culture medium II continues to cultivate, and the per-cent of the callus lines of the callus lines of survival and green-emitting fluorescence is that the fluorescence callus lines is stable into motility rate, and the result is as shown in table 3:

Table 3 fluorescence callus piece is stable into motility rate

4, regeneration rate

All seedling quantity that callus lines (the being resistant calli) differentiation culture of statistics survival produces, again the positive regrowth of green-emitting fluorescence in the above-mentioned seedling is counted, calculate regeneration rate, regeneration rate is the callus lines of all seedling quantity/survivals;

The result is as shown in table 4:

Table 4 regeneration rate

5, calculate transformation efficiency

According to above-mentioned experiment statistics result, begin to calculate transformation efficiency from sugarcane explants through callus induction, per 100 ratarias can induce 75 embryo callus subcultures, a callus is through 3 succeeding transfer culture, can breed into 3 (by minimum calculating) and be used for the preculture callus lines (infecting callus) that Agrobacterium is infected, the callus lines of 1.77 green-emitting fluorescence of every callus average generation, the callus lines stable growth rate of green-emitting fluorescence is 45.8%, regeneration rate is 66.7%, in regrowth, remove 15% without the fluorescence seedling, therefore the transformation efficiency of the positive seedling of fluorescence is calculated as:

75％x 3x 176.9％x 45.8％x 66.7％x(100％-15％)＝103.4％

Per 100 ratarias are described, through callus of induce, conversion, finally can access 122 transgenic seedlings, substratum of the present invention and method transformation efficiency are high.

247 explants of the present invention finally can obtain 255 transgenosiss, two fringe false bromegrass plant (the actual count result conforms to calculation result).

If calculate transformation efficiency to infect the callus piece, then:

Transformation efficiency=176.9%x 45.8%x 66.7%x (100%-15%)=45.9%.

Claims

1. a substratum group that is used for cultivating the transgenosis false bromegrass is comprised of calli induction media, subculture medium, common culture medium, screening culture medium and regeneration culture medium;

2. substratum group according to claim 1 is characterized in that:

Described screening culture medium is following screening culture medium I or screening culture medium II:

3. substratum group according to claim 1 and 2 is characterized in that:

All also comprise peptizer in the various described substratum, described peptizer is specially agar, and the concentration of described agar in described various substratum is 7g/L.

4. a method for preparing arbitrary described substratum group among the claim 1-3 comprises the steps:

1) the described calli induction media in arbitrary described substratum group, described subculture medium, described altogether culture medium, described screening culture medium and described regeneration culture medium among the preparation claim 1-3;

5. method according to claim 4 is characterized in that:

Described calli induction media is according to the preparation of the method that comprises the steps: with described 2,4-D, described maltose, described altheine, described inositol, described caseinhydrolysate, described proline(Pro) and described vitamin are added in the MS minimum medium according to described separately final concentration and obtain;

Described subculture medium is according to the preparation of the method that comprises the steps: with described 2,4-D and described 6-BA are added in the MS minimum medium according to described final concentration separately and obtain;

Described altogether culture medium is according to the method preparation that comprises the steps: described glucose and described Syringylethanone are added in the MS minimum medium according to described final concentration separately obtain;

Described screening culture medium is according to the preparation of the method that comprises the steps: described paromycin and described Ticarcillin/Clavulanate Acid are added in the MS minimum medium according to described final concentration separately obtain;

6. it is characterized in that according to claim 4 or 5 described methods:

All also add peptizer in various described substratum, described peptizer is specially agar, and the concentration of described agar in described various substratum is 7g/L.

7. the application of arbitrary described substratum group in cultivating transgenic plant among the claim 1-3; Described plant is specially two fringe false bromegrasses.

8. a method of cultivating transgenic plant comprises the steps:

1) plant explants is cultivated in the described calli induction media in arbitrary described substratum group in claim 1-3, obtain embryo callus;

2) with step 1) embryo callus that obtains cultivates in the described subculture medium in arbitrary described substratum group in claim 1-3, obtains subculture embryo callus piece;

3) goal gene is imported step 2) behind the subculture embryo callus piece that obtains, in common culture medium, cultivate, obtain common cultured calli piece;

9. method according to claim 8 is characterized in that:

Step 1) in, described cultivation is 25 ℃ of dark cultivations for 3 week-4 weeks;

Step 3) in, described goal gene imports step 2) the subculture embryo callus piece that obtains is for to be immersed in described subculture embryo callus piece in the restructuring Agrobacterium bacterium liquid that contains described goal gene, and the temperature of described immersion is 23 ℃-28 ℃; The time of described immersion is 8-10min, and the temperature of described immersion is specially 25 ℃, and the time of described immersion is specially 10min;

10. it is characterized in that according to claim 8 or 9 described methods:

Step 1) in, described explant is rataria, and described plant is two fringe false bromegrasses;