CN106884020A - A kind of birch transgenic method rapidly and efficiently - Google Patents
A kind of birch transgenic method rapidly and efficiently Download PDFInfo
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- CN106884020A CN106884020A CN201710283893.9A CN201710283893A CN106884020A CN 106884020 A CN106884020 A CN 106884020A CN 201710283893 A CN201710283893 A CN 201710283893A CN 106884020 A CN106884020 A CN 106884020A
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Abstract
A kind of birch transgenic method rapidly and efficiently, it is related to a kind of method of plant transgene.The invention aims to solve, existing white birch stable conversion efficiency is low and transformation period too long of problem.The solution of the present invention is：Choose the white birch tissue-cultured seedling that height of seedling is 5~6cm, Fiber differentiation callus；It is transferred to during the Agrobacterium tumefaciems containing objective gene sequence infects liquid, after vacuumize process, light culture；Resistance seedling screening is carried out, the resistance seedling of screening is transferred to culture in the root media for adding selective agent and bacteriostatic agent, after Molecular Detection is proved to be successful, that is, completed.Compared with prior art, average conversion improves 2.71 times.And it is the explant transformation period to shorten more than 1/3 to infect transformation period ratio blade and stem with callus.Present invention application transgenic field.
Description
Technical field
The present invention relates to a kind of method of plant transgene.
Background technology
White birch (Betula platyphylla) is a kind of deciduous tree of Betula (Betula Linn).In Ya Zhoudong
Portion blazons distribution.Light, cold-resistant, vitality is extremely strong, is to recover vegetation, the excellent afforestation tree of conserve forests moisture and soil
Kind, while being again an important excellent economic tree of medicinal and fiber.But the white birch traditional breeding method cycle is long, and genetic improvement enters
Journey is slow, it is impossible to transform proterties according to breeding objective to meet growing society need.And in genetic engineering breeding technology,
Existing birch transgenic is mainly explant is directly converted, because white birch regenerates and conversion explant tool tissue used
Specificity, transformation period be very long etc., and to often result in its transformation efficiency relatively low.Additionally, these transformation technologies are usually because the length of Agrobacterium
Phase participates in causing acceptor material yellow serious, and regeneration is difficult, and these turn into where " bottleneck " of restriction white birch molecular breeding process.
So it is most important to white birch gene functional research and genetic improvement to find a kind of quick, stabilization, efficient genetic transforming method.
The innovation one of this experiment is that the Agrobacterium carried out on the basis of white birch callus infects conversion, this avoid
The transformation time for directly being brought using explant is long, the low puzzlement of transformation efficiency.White birch explant is directly carried out in path for transformation
2-3 months is needed from the explant for infecting to resistant budses are obtained, is converted from explant to obtaining resistance using white birch callus
Bud needs 1 month or so, substantially reduces transformation time.Two be the increase in negative pressure (vacuumizing) process the step of, Negative pressure
Can improve white birch stable conversion efficiency.The use white birch callus of the invention with first in white birch use vacuum extraction
Technology is combined the efficiency that white birch stable conversion is greatly facilitated.Therefore the white birch stable conversion system after optimization can be largely
Transgene efficiency is improved, shortens the transgenosis time.
The content of the invention
It is that quick research is white the present invention is to solve existing white birch stable conversion efficiency is low and transformation period too long of problem
Birch gene function, the transfer-gen plant offer platform for obtaining objective trait improvement.White birch genetic transformation is that original white birch is turned
Change system it is perfect.
A kind of birch transgenic method rapidly and efficiently of the invention, it is followed the steps below：
First, the white birch tissue-cultured seedling that height of seedling is 5~6cm is chosen, explant blade and stem section is placed in superclean bench
In calli induction media, Fiber differentiation 7~10 days；
2nd, the callus for step one being grown in calli induction media takes out, and is transferred to containing objective gene sequence
Agrobacterium tumefaciems infect liquid, after 5~10min is vacuumized in vavuum pump, be placed on the culture medium of co-cultivation, dark processing
48h；Wherein, liquid OD is infected600It is 0.6~0.8,
3rd, the callus after step 2 dark processing is placed on the transgenic resistance screening and culturing medium for adding bacteriostatic agent
In screening and culturing is carried out under 25 DEG C of illumination conditions 40~50 days, white birch resistance seedling is obtained；
4th, the white birch resistance seedling of step 3 is transferred in the root media for adding selective agent and bacteriostatic agent and cultivates 37-42
After it, Molecular Detection checking is carried out, after detection checking is correct, that is, show to obtain transgenosis white birch seedling.
Beneficial effects of the present invention：
The method of the present invention has carried out callus induction treatment by selecting the white birch tissue-cultured seedling of approximate physiological state.This place
The problems such as reason mode avoids the white birch explant direct cycle for converting long and white birch explant selection difficulty.(note：Selection
White birch blade excessively it is young it is tender easily infected by Agrobacterium, it is necessary to blade carried out daily or every other day it is degerming, it is this excessively young tender
Blade is easy to be wrapped up in and dead, the resistance seedling after being difficult to be converted by bacterium bag.Though the white birch blade excessively aging of selection be difficult by
Agrobacterium is infected, but blade differentiation callus ability, it is not easy to obtain the successful resistance seedling of conversion.The stem section of white birch
There is a problem of similar blade, but the topmost problem of stem section is that stem section carries bud point, and substantial amounts of false positive seedling can be caused to produce,
Transformation efficiency is low.Application vavuum pump vacuumizes the treatment of 5-10min on the basis of white birch callus.The treatment can make containing mesh
Gene infect liquid and the callus of acceptor white birch is fully contacted, transformation efficiency can be greatly improved.In being total to for factors above
White birch stable conversion efficiency can be improved under same-action.Present invention transferase 45 in screening and degerming culture medium, obtains 19 altogether
Resistant budses, conversion ratio is 9.5%, and compared with white birch blade is infected, average conversion improves 4.75 times；It is white with directly infecting
Birch explant-stem section is compared, and average conversion improves 3.8 times, and compared with white birch callus is infected, average conversion improves
2.71 times.And it is the explant transformation period to shorten more than 1/3 to infect transformation period ratio blade and stem with callus.
Brief description of the drawings
Fig. 1 is the white birch tissue-cultured seedling photo of squamous subculture in embodiment one；
Fig. 2 is the squamous subculture white birch tissue-cultured seedling photo of 40 days in embodiment one；
Fig. 3 is the callus photo after calli induction media induction in embodiment one；
Fig. 4 is the callus photo after calli induction media induction in embodiment one；
Fig. 5 be in embodiment one callus that the white birch early stage resistance grown on rear screening and culturing medium is infected by genes of interest is indefinite
Bud photo；
Fig. 6 is that the white birch resistance that is grown on screening and culturing medium after being infected by genes of interest of callus is not in embodiment one
Normal bud tufted seedling photo；
Fig. 7 is the white birch resistance seedling photo on the root media of the added with antibiotic in embodiment one.
Specific embodiment
Specific embodiment one：A kind of birch transgenic method rapidly and efficiently of present embodiment, it is according to following
What step was carried out：
First, the white birch tissue-cultured seedling that height of seedling is 5~6cm is chosen, explant blade and stem section is placed in superclean bench
In calli induction media, Fiber differentiation 7~10 days；
2nd, the callus for step one being grown in calli induction media takes out, and is transferred to containing objective gene sequence
Agrobacterium tumefaciems infect liquid, after 5~10min is vacuumized in vavuum pump, be placed on the culture medium of co-cultivation, dark processing
48h；Wherein, liquid OD is infected600It is 0.6~0.8,
3rd, the callus after step 2 dark processing is placed on the transgenic resistance screening and culturing medium for adding bacteriostatic agent
In screening and culturing is carried out under 25 DEG C of illumination conditions 40~50 days, white birch resistance seedling is obtained；
4th, the white birch resistance seedling of step 3 is transferred in the root media for adding selective agent and bacteriostatic agent and cultivates 37-42
After it, Molecular Detection checking is carried out, after detection checking is correct, that is, show to obtain transgenosis white birch seedling.
Specific embodiment two：Present embodiment from unlike specific embodiment one：Taking root described in step one
Culture medium contains WPM (Woody Plant medium), the NAA (1-naphthylacetic acid) of 0.2mg/L, the sugarcane of 30g/L
Sugar and 3.0mg/L Phytagel (gellan gum), pH=5.8.Other are identical with specific embodiment one.
Specific embodiment three：Present embodiment from unlike specific embodiment one：Callus described in step one
Inducing culture is containing WPM (Woody Plant medium), the 6-BA (6-benzyladenine) of 0.8mg/L, 0.5mg/L
GA3The NAA (1-naphthylacetic acid) of (Gibberellic acid A3), 0.02mg/L, the sucrose of 30g/L and
The Phytagel (gellan gum) of 3.0mg/L, pH=5.8.Other are identical with specific embodiment one.
Specific embodiment four：Present embodiment from unlike specific embodiment one：Common training described in step 2
Foster culture medium contains WPM (Woody Plant medium), the 6-BA (6-benzyladenine) of 1.0mg/L, the sugarcane of 30g/L
The Phytagel (gellan gum), pH=5.8 of sugar and 3.0mg/L.Other are identical with specific embodiment one.
Specific embodiment five：Present embodiment from unlike specific embodiment one：Infecting described in step 2
Liquid contains WPM (Woody Plant medium), 150 μM of As (acetosyringone, Acetosyringone), the sucrose of 30g/L,
PH=5.8.Other are identical with specific embodiment one.
Specific embodiment six：Present embodiment from unlike specific embodiment one：Screening described in step 3
Culture medium containing WPM (Woody Plant medium), the 6-BA (6-benzyladenine) of 1.0mg/L, the sucrose of 30g/L,
The Phytagel (gellan gum) of 3.0mg/L, selective agent and inhibitor, pH=5.8.Other are identical with specific embodiment one.
Specific embodiment seven：Present embodiment from unlike specific embodiment six：Selective agent is 30~50mg/L
Kanamycins, inhibitor is the ammonia benzyl mycin of 300~500mg/L.Other are identical with specific embodiment six.
Specific embodiment eight：Present embodiment from unlike specific embodiment one：Taking root described in step 4
Culture medium contains WPM (Woody Plant medium), the NAA (1-naphthylacetic acid) of 0.2mg/L, the sugarcane of 30g/L
Sugar, the Phytagel of 3.0mg/L, selective agent and inhibitor, pH=5.8.Other are identical with specific embodiment one.
Specific embodiment nine：Present embodiment from unlike specific embodiment eight：Selective agent is 30~50mg/L
Kanamycins, inhibitor is the ammonia benzyl mycin of 300~500mg/L.Other are identical with specific embodiment eight.
Specific embodiment ten：Present embodiment from unlike specific embodiment one：The resistance seedling of step 4 selection
Plant height is 1.5~2.0cm.Other are identical with specific embodiment one.
Specific embodiment 11：Present embodiment from unlike specific embodiment one：Step 4 carries out molecule inspection
The seedling strain plant height for testting card is 5~6cm.Other are identical with specific embodiment one.
Present invention is not limited only to the content of the respective embodiments described above, the group of one of them or several specific embodiments
Contract sample can also realize the purpose of invention.
Beneficial effects of the present invention are verified by following examples：
Embodiment 1
The white birch stable conversion method of the present embodiment, is carried out according to the following steps：
First, it is the white birch tissue-cultured seedling of 5-6cm to choose height of seedling, is placed in more explant blade and stem section in superclean bench
In hindering inducing culture, Fiber differentiation 7-10 days.(note：When selecting external body-leaf, stem, should select tender, the plump blade of children and
Sturdy stem section.)
2nd, the callus grown in calli induction media in step one is taken out and is transferred quickly to added with containing BpGRP
The Agrobacterium tumefaciems of gene overexpression carrier pROKII-BpGRP1 (infects the OD of liquid Agrobacterium in infecting liquid600It is 0.6-0.8).
It is placed in negative pressure -0.1mpa in vavuum pump and vacuumizes 5-10min, is subsequently placed on the culture medium of co-cultivation, 28 DEG C, dark processing
48h.(note：Callus after infecting does not need aseptic water washing, passes directly to co-culture on culture medium and cultivates.) wherein infect
The preparation method of liquid is to take Agrobacterium tumefaciems of the 50uL containing pROKII-BpGRP1 to be added in 10mL LB fluid nutrient mediums, 28 DEG C
200rpm overnight incubations, to OD600 about 0.6~0.8,100uL bacterium solutions are then taken from 10mL bacterium solutions and are rejoined to 50mL LB
In fluid nutrient medium, 28 DEG C of 200rpm are cultivated to OD600After 0.6~0.8, normal temperature centrifuge 3000G centrifugations are placed in, remove supernatant,
Add isometric re-suspension liquid (note：Re-suspension liquid contains WPM, 30g/L sucrose) resuspended thalline.
3rd, the callus after dark processing in step 2 is moved to and is put on the screening and culturing medium containing selective agent and bacteriostatic agent
In 28 DEG C/25 DEG C (day/night), 14h- illumination/10h- is dark, and light intensity is close to 500mmol m-2s-1Greenhouse in sieved
(callus after dark processing grows, it is necessary to using bacteriostatic agent after aseptic water washing 2-3 times if any a large amount of Agrobacteriums for choosing culture
Such as concentration be 500mg/L Carbenicillin or concentration for 500mg/L Timentin water is washed one time again, with nothing
Bacterium filter paper suck dry moisture goes to screening and culturing in screening and culturing medium.Directly it is transferred to containing selective agent if being grown without a large amount of Agrobacteriums
Screening and culturing in the screening and culturing medium of (30mg/L Kanamycin) and bacteriostatic agent (500mg/L Carbenicillin).), 20
It or so grows resistance adventitious bud, and resistance adventitious bud grows to plant height 1.5-2.0cm resistance seedlings after 20-30 days.
4th, the plant height 1.5~2.0cm white birch resistance seedlings in step 3 are transferred to plus selective agent (50mg/L
Kanamycin) and in the root media of bacteriostatic agent (300mg/L Carbenicillin), produced not after about one week
Determine root, plant height is up to carrying out Molecular Detection checking after 30~35 days during 5~6cm.Show to obtain transgenosis after detection checking is correct
White birch seedling.
The white birch tissue-cultured seedling of squamous subculture is as illustrated in fig. 1 and 2 in the present embodiment.Fig. 1 is that the white birch tissue culture of just subculture is small
Seedling, white birch culturing young plants now can't be used for the stable conversion of white birch；Fig. 2 is white birch tissue-cultured seedling of the subculture after 40-45 days,
White birch tissue-cultured seedling now can be used for the genetic transformation of white birch；Fig. 3 and Fig. 4 is the explant-stem section and blade of white birch in callus
State after being cultivated 10 days on inducing culture；Fig. 5 is white birch callus by the over-express vector containing BpGRP genes
PROKII-BpGRP1 bacillus is infected after liquid infects, and the callus state of 15 days is grown on screening and culturing medium, now begins with early stage
Resistance adventitious bud is produced；Fig. 6 is white birch callus in invading by the over-express vector pROKII-BpGRP1 containing BpGRP genes
After dye liquor infects, on screening and culturing medium grow 20-30 days after state, now resistance adventitious bud grown to 1-1.5cm；Fig. 7 is
Resistance tufted seedling is transplanted into the root media containing selective agent and bacteriostatic agent to grow up seedling.
Embodiment 2
This embodiment scheme is used chooses height for the white birch tissue-cultured seedling of 5-6cm takes the 2nd or the 3rd healthy and strong blade use
In infecting for Agrobacterium, then it is placed in screening and culturing in screening and culturing medium.Other are same as Example 1.
The present embodiment is reference examples.
Embodiment 3
This embodiment scheme use selection growing way it is good, highly for the white birch tissue-cultured seedling of 5-6cm is cut into 1-1.5cm long shoots
Section infects for Agrobacterium, then is placed in screening and culturing in screening and culturing medium.Other are same as Example 1.
The present embodiment is reference examples.
Embodiment 4
The callus that it is good that this embodiment scheme uses selection state is transferred quickly to cross table added with gene containing BpGRP
Up to carrier pROKII-BpGRP1 Agrobacterium tumefaciems infect liquid in (infect liquid OD600It is 0.6-0.8) infected.Infect and finish
It is placed on the culture medium of co-cultivation afterwards, dark processing 36h.Other are same as Example 1.
The present embodiment is reference examples.
By embodiment 1 compared with the reference examples of embodiment 2,3 and 4, embodiment 1 is in screening and degerming culture medium
Upper transferase 45, obtains 19 resistant budses altogether, and conversion ratio is 9.5%, and compared with Example 2, average conversion improves 4.75
Times；Compared with Example 3, average conversion improves 3.8 times；Compared with Example 4, average conversion improves 2.71 times.
And it is the explant transformation period to shorten more than 1/3 to infect transformation period ratio blade and stem with callus.
Claims (10)
1. a kind of birch transgenic method rapidly and efficiently, it is characterised in that it is followed the steps below：
First, the white birch tissue-cultured seedling that height of seedling is 5~6cm is chosen, explant blade and stem section is placed in callus in superclean bench
In inducing culture, Fiber differentiation 7~10 days；
2nd, the callus for step one being grown in calli induction media takes out, and is transferred to the root containing objective gene sequence
Cancer Agrobacterium is infected in liquid, after 5~10min is vacuumized in vavuum pump, is placed on the culture medium of co-cultivation, dark processing 48h；
Wherein, liquid OD is infected600It is 0.6~0.8,
3rd, the callus after step 2 dark processing is placed on the transgenic resistance screening and culturing medium for adding bacteriostatic agent in 25
Screening and culturing is carried out under DEG C illumination condition 40~50 days, obtain white birch resistance seedling；
4th, the white birch resistance seedling of step 3 is transferred in the root media for adding selective agent and bacteriostatic agent and is cultivated 37~42 days
Afterwards, Molecular Detection checking is carried out, after detection checking is correct, that is, shows to obtain transgenosis white birch seedling.
2. a kind of birch transgenic method rapidly and efficiently according to claim 1, it is characterised in that described in step one
Root media containing WPM, 0.2mg/L NAA, 30g/L sucrose and 3.0mg/L Phytagel, pH=5.8.
3. a kind of birch transgenic method rapidly and efficiently according to claim 1, it is characterised in that described in step one
Calli induction media containing WPM, 0.8mg/L 6-BA, 0.5mg/L GA3, 0.02mg/L NAA, 30g/L sucrose and
The Phytagel of 3.0mg/L, pH=5.8.
4. a kind of birch transgenic method rapidly and efficiently according to claim 1, it is characterised in that described in step 2
Co-cultivation culture medium containing WPM, 1.0mg/L 6-BA, 30g/L sucrose and the Phytagel of 3.0mg/L, pH=5.8.
5. a kind of birch transgenic method rapidly and efficiently according to claim 1, it is characterised in that described in step 2
Infect liquid containing WPM, the sucrose of 150 μM As, 30g/L, pH=5.8.
6. a kind of birch transgenic method rapidly and efficiently according to claim 1, it is characterised in that described in step 3
The sucrose of 6-BA, 30g/L of the screening and culturing medium containing WPM, 1.0mg/L, the Phytagel of 3.0mg/L, selective agent and inhibitor,
PH=5.8.
7. a kind of birch transgenic method rapidly and efficiently according to claim 1, it is characterised in that selective agent is 30~
The kanamycins of 50mg/L, inhibitor is the ammonia benzyl mycin of 300~500mg/L.
8. a kind of birch transgenic method rapidly and efficiently according to claim 1, it is characterised in that described in step 4
The sucrose of NAA, 30g/L of the root media containing WPM, 0.2mg/L, the Phytagel of 3.0mg/L, selective agent and inhibitor,
PH=5.8.
9. a kind of birch transgenic method rapidly and efficiently according to claim 1, it is characterised in that selective agent is 30~
The kanamycins of 50mg/L, inhibitor is the ammonia benzyl mycin of 300~500mg/L.
10. a kind of birch transgenic method rapidly and efficiently according to claim 1, it is characterised in that step 4 selection
Resistance seedling plant height is 1.5~2.0cm.
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