CN107190019A - A kind of agriculture bacillus mediated sinocalamus latiflorus method for transformation - Google Patents
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- CN107190019A CN107190019A CN201710513446.8A CN201710513446A CN107190019A CN 107190019 A CN107190019 A CN 107190019A CN 201710513446 A CN201710513446 A CN 201710513446A CN 107190019 A CN107190019 A CN 107190019A
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Abstract
The invention discloses a kind of agriculture bacillus mediated sinocalamus latiflorus method for transformation, including the preparation of explant, Agrobacterium infect the preparation of bacterium solution, infect, screen, the induction of adventitious bud and root induction step.Go to infect sinocalamus latiflorus stem end explant using Agrobacterium, co-cultured in the NB culture mediums containing 22,4 D and 200 μM of acetosyringones of mg/L, by there are the steps such as screening finally to obtain transgenosis sinocalamus latiflorus.The present invention uses agriculture bacillus mediated Efficient Conversion system, the transformation system using sinocalamus latiflorus nutrition organs as the sinocalamus latiflorus of starting is established first, the system can break through the obstacle for overcoming traditional breeding method can not be applied in bamboo class, to provide premise by the molecular breeding of transgenic technology progress sinocalamus latiflorus in the future；With it, providing technical support in basic research field for research bamboo genoid function.
Description
Technical field
The invention belongs to Forest Tree Genetic Breeding field, and in particular to a kind of agriculture bacillus mediated sinocalamus latiflorus method for transformation.
Background technology
Sinocalamus latiflorus is grass family Bambusoideae Dendrocalamus sinocalamus latiflorus subgenus, is divided naturally in portions such as Fujian-Taiwan Guangdong, Guangxi Guizhou Yunnan
Cloth, is one of large-scale sympodial bamboo kind of the important double purposes of In South China and the southeastern coastal areas, with high economic valency
Value and social value.
With the development of the social economy, yield and quality requirement more and more higher of the people to bamboo wood, but corresponding therewith it is
Bamboo class breeding it is much delayed.Traditional genetic and breeding method can not be carried out in bamboo, main reasons is that Studies on Bamboo Flowering has
There are chronicity and uncertainty, it is impossible to carry out the hybridization between bamboo kind.Transgenic breeding is the important means in crop breeding, is led to
Cross transgenosis and carry out accurately gene regulation, the yield and quality of bamboo wood is greatly improved.
Research in terms of current sinocalamus latiflorus transgenosis is fewer.2014, Qiao et al. was built using anther tissue for explant
Regenerating system is erected, and on this basis by bacteriumcodAChannel genes are to sinocalamus latiflorus genome.This is that sinocalamus latiflorus transgenosis is ground
The unique report studied carefully.But by sinocalamus latiflorus flower pesticide originate transgenic technology exist there may be chimera, monoploid or
The problems such as being polyploid and relatively low conversion ratio.Therefore, by agriculture bacillus mediated method conversion by being cured that nutritive issue is produced
Injured tissue, the conversion for carrying out functional gene is to be improved the problem of must solving to sinocalamus latiflorus resource.
The content of the invention
It is an object of the invention in view of the shortcomings of the prior art, providing a kind of agriculture bacillus mediated sinocalamus latiflorus method for transformation.This
Invention inventor set up using sinocalamus latiflorus stem end as explant regenerating system on the basis of, utilize agriculture bacillus mediated method to carry out
The transgenic research of sinocalamus latiflorus.The present invention using this solves sinocalamus latiflorus transgenosis for a long time it is difficult the problem of, can be extensive
Functional gene identification and molecular breeding applied to sinocalamus latiflorus.
To achieve the above object, the present invention is adopted the following technical scheme that：
A kind of agriculture bacillus mediated sinocalamus latiflorus method for transformation, specifically includes following steps：
（1）The preparation of explant：The young tender stem end of the sinocalamus latiflorus being grown in greenhouse is taken, after washing, sterilizing, the small of 0.5-1cm is cut into
Section, the wherein position of tubercle are located at end wound；Stem end after cut-out is positioned on calli induction media, secretly trained
Support, cultivation temperature is 26 DEG C；After 1.5 months, after callus is induced, it is moved into callus proliferated culture medium, and at this
Cultivated in culture medium 7-8 months, produce flaxen embryo callus, and object is infected as Agrobacterium；
（2）Agrobacterium infects the preparation of bacterium solution：Agrobacterium containing target gene is rule and cultivated, is subsequently transferred to containing 50
In the YEB culture mediums of mg/L kanamycins, and stayed overnight in 28 DEG C of shaken cultivations, then according to 1:100 volume ratio will be cultivated
Base dilutes, and is added in the YEB culture mediums of the kanamycins containing 50 mg/L and 200 μM of acetosyringone, after
Continuous cultivate to OD is 0.6-0.8；Supernatant is removed after centrifugation, gained bacterium solution is suspended in containing 200 μM of acetosyringone
In AMM fluid nutrient mediums, it is 0.8 to be diluted to OD, obtains Agrobacterium and infects bacterium solution；
（3）Infect：By step（1）It is middle to breed the explant immersion step for embryo callus（2）Obtained Agrobacterium is infected
In bacterium solution, room temperature is placed 15 minutes after application of vacuum 15 minutes；The explant infected is taken out, is placed in sterile, with sterilizing
Filter paper blot surface bacterium solution, after be transferred to containing 2 mg/L 2, in the NB culture mediums of 4-D and 200 μM of acetosyringone, 25
DEG C, co-culture 4 days under dark condition；
（4）Screening：By step（3）The callus finished is co-cultured to clean 3-5 times in 400 mg/L carbenicillin, after
Screening and culturing is carried out in kanamycin-resistant callus tissue amplification culture medium, embryo callus is gradually formed, the induction for adventitious bud；It is described
The condition of screening and culturing is：Cultivated 4 months under 26 DEG C of dark condition；
（5）The induction of adventitious bud：By step（4）The callus that screening and culturing is obtained is placed into resistant budses inducing culture
Row adventitious bud induction culture, condition of culture is：Cultivation temperature is 26 DEG C, 70 μm of ol m-2 s-1, 16h is cultivated under illumination condition,
The lower culture of dark 8 hours；After adventitious bud induction culture, Elongation of adventitious bud culture, condition of culture are carried out in bud proliferated culture medium
For：16 h, 70 μm of ol m of intensity of illumination are cultivated under illumination condition-2 s-1；The formula of the bud proliferated culture medium is induced with bud and trained
The culture for supporting base is identical；
（6）Root induction：When the bud of regeneration grows into 3-4 centimeter lengths, the root induction culture for not containing antibiotic is transferred to
Cultivated in base, condition of culture is：Cultivation temperature is 26 DEG C, 70 μm of ol m-2 s-1, 16h is cultivated under illumination condition, it is dark
Lower culture 8 hours；Complete sinocalamus latiflorus transfer-gen plant is become after root induction, rear transplant is buried.
Step（1）Described in the formula of calli induction media be：MS+2,4-D 8 mg/L + IBA 0.5 mg/L+
The g/L sucrose of 250 mg/L PVP+3g/L sorbierites+30.
Step（1）Described in the formula of callus proliferated culture medium be：The g/L sucrose+250 of 3/4MS+3g/L sorbierites+30
mg/L PVP+2mg/L 2,4-D。
Step（2）Described in Agrobacterium strain be EHA105.
Step（4）Described in the formula of kanamycin-resistant callus tissue amplification culture medium be：The g/L sugarcanes of 3/4MS+3g/L sorbierites+30
The sugared mg/L carbenicillins of+250 mg/L PVP+2mg/L 2,4-D+35 mg/L hygromycin+400.
Step（5）Described in the formula of resistant budses inducing culture be：The mg/L carboxylics benzyl of 35 mg/L hygromycin+200 is blue or green
Mycin+MS culture mediums+the g/L of 0.5 mg/L+ agar powders of sucrose 30 g/L+ TDZ, 0.1 mg/L+NAA 8.
Step（6）Described in the formula of root induction culture medium be：The mg/L+30 g/L sucrose+35 of MS+IAA 1
Mg/L hygromycin.
The beneficial effects of the present invention are：
1）The present invention uses agriculture bacillus mediated Efficient Conversion system, and the transformation system of sinocalamus latiflorus is established first.The system energy
Enough break through overcomes the obstacle that traditional breeding method can not be applied in bamboo class；Sinocalamus latiflorus is carried out by transgenic technology for future
Molecular breeding provides premise；With it, providing technical support in basic research field for research bamboo genoid function；
2）The present invention is using the stem end of sinocalamus latiflorus as starting explant, to build transgenic line；Selecting the strain for infecting Agrobacterium is
EHA105 is to increase transformation efficiency；
3）Agrobacterium is infected in the preparation of bacterium solution, and the concentration for controlling Agrobacterium is OD 0.8, and use contains 200 μM of acetyl
The AMM fluid nutrient mediums of syringone, the Agrobacterium of the medium culture can effectively infect callus；
4）The culture medium of co-cultivation used is NB, and need to add 2,4-D 2mg/L and 200 μM of acetosyringones to be conducive to
While improving Agrobacterium infectivity, and excessive injury is not caused to plant tissue.
Brief description of the drawings
Fig. 1 is converted and qualification figure for agriculture bacillus mediated sinocalamus latiflorus, in figure：（a）Agrobacterium containing target gene, includes tide
Mycin resistant geneHPTIIAnd GUS；（b）The whole flow process of conversion：I infect before callus state；Ii kanamycin-resistant callus tissues are from browning
Parent in grow；Iii kanamycin-resistant callus tissue squamous subcultures；Iv kanamycin-resistant callus tissues progressively produce embryo callus subculture；V kanamycin-resistant callus tissues start differentiation
Produce bud point；Vi kanamycin-resistant callus tissue seedling differentiations；Vii regenerates seedling rooting；Viii regrowths bury；（c）PCR identifications produce 1044
Positive band；（d）Callus and regeneration stem after conversion are presented positive.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described, but the present invention is not limited only to these embodiments.
Embodiment
A kind of agriculture bacillus mediated sinocalamus latiflorus method for transformation, specifically includes following steps：
（1）The preparation of explant：The young tender stem end of the sinocalamus latiflorus being grown in greenhouse is taken, is positioned over after cut-out in detergent and soaks 60
Minute, after under running water rinse 2 hours.Rinsed with sterile water 5 is used after aseptically being soaked 1 minute in 75% ethanol
It is secondary, after sterilized 8 minutes with 0.1% life mercury again, then with aseptic water washing 6 times.1cm segment is cut at stem end after sterilizing, its
The position of middle tubercle is located at end wound；Stem end after cut-out is positioned on calli induction media, light culture, training is carried out
It is 26 DEG C to support temperature；After 1.5 months, after callus is induced, it is moved into callus proliferated culture medium, and in the culture medium
Middle culture 7 months, produces flaxen embryo callus（In Fig. 1（b）-i）, and infect object as Agrobacterium；
（2）Agrobacterium infects the preparation of bacterium solution：By the Agrobacterium EHA105 containing target gene Pcambia1301-GUS（In Fig. 1
's（a））Line culture, is subsequently transferred in the YEB culture mediums containing 50 mg/L kanamycins, and in 28 DEG C of shaken cultivation mistakes
Night, then according to 1:100 volume ratio dilutes culture medium, and is added to the kanamycins containing 50 mg/L and 200
μM acetosyringone YEB culture mediums in, continue cultivate to OD be 0.6；Supernatant is removed after centrifugation, gained bacterium solution is suspended
In the AMM fluid nutrient mediums containing 200 μM of acetosyringone, it is 0.8 to be diluted to OD, obtains Agrobacterium and infects bacterium solution；
（3）Infect：By step（1）It is middle to breed the explant immersion step for embryo callus（2）Obtained Agrobacterium is infected
In bacterium solution, application of vacuum（-0.8bar）Room temperature is placed 15 minutes after 15 minutes；The explant infected is taken out, sterile is placed in
In, blot surface bacterium solution with the filter paper of sterilizing, after be transferred to containing 2 mg/L 2, the NB cultures of 4-D and 200 μM of acetosyringone
In base, co-cultured 4 days under 25 DEG C, dark condition；
（4）Screening：By step（3）The callus that finishes is co-cultured to clean 4 times in 400 mg/L carbenicillin, after
Screening and culturing is carried out in kanamycin-resistant callus tissue amplification culture medium, embryo callus is gradually formed, the induction for adventitious bud；The sieve
Choosing culture condition be：Cultivated 4 months under 26 DEG C of dark condition；New resistant calli is had within 2 months or so from parent
In separate（In Fig. 1（b）-ii）, the time of 4 months or so gradually forms embryo callus subculture (in Fig. 1（b）-iii, -
iv)；
（5）The induction of adventitious bud：By step（4）The callus that screening and culturing is obtained is placed into resistant budses inducing culture
Row adventitious bud induction culture, condition of culture is：Cultivation temperature is 26 DEG C, 70 μm of ol m-2 s-1, 16h is cultivated under illumination condition,
The lower culture of dark 8 hours；After adventitious bud induction culture, Elongation of adventitious bud culture is carried out in bud proliferated culture medium（In Fig. 1
（b）-v, -vi）, condition of culture is：16 h, 70 μm of ol m of intensity of illumination are cultivated under illumination condition-2 s-1；
（6）Root induction：When the bud of regeneration grows into 3-4 centimeter lengths, the root induction culture for not containing antibiotic is transferred to
Cultivated in base, condition of culture is：Cultivation temperature is 26 DEG C, 70 μm of ol m-2 s-1, 16h is cultivated under illumination condition, it is dark
Lower culture 8 hours；Complete sinocalamus latiflorus transfer-gen plant is become after root induction（In Fig. 1（b）-vii）, transplant bury afterwards
（In Fig. 1（b）-viii）.
（7）Finally obtain 17 plants of positive seedlings, wherein 12 plants verified through PCR after be positive（In Fig. 1（c））, it is used
Primer is the specific primers of HPTII and 35S, and its sequence is：
(Forward: 5’-TGCCATCATTGCGATAAAGGAAAG-3’) and HPTII gene (Reverse: 5’-
CTGC TGCTCCATACAAGCCAACC-3’).The as shown by data of GUS dyeing, the callus after conversion（In Fig. 1（d）-
ii）With stem end（In Fig. 1（d）-iv）The positive signal of blueness is presented, and in control group（In Fig. 1（d）In-i and Fig. 1
（d）-iii）Positive signal is not occurred.
Step（1）Described in the formula of calli induction media be：MS+2,4-D 8 mg/L + IBA 0.5 mg/L+
The g/L sucrose of 250 mg/L PVP+3g/L sorbierites+30.
Step（1）Described in the formula of callus proliferated culture medium be：The g/L sucrose+250 of 3/4MS+3g/L sorbierites+30
mg/L PVP+2mg/L 2,4-D。
Step（4）Described in the formula of kanamycin-resistant callus tissue amplification culture medium be：The g/L sugarcanes of 3/4MS+3g/L sorbierites+30
The sugared mg/L carbenicillins of+250 mg/L PVP+2mg/L 2,4-D+35 mg/L hygromycin+400.
Step（5）Described in the formula of resistant budses inducing culture be：The mg/L carboxylics benzyl of 35 mg/L hygromycin+200 is blue or green
Mycin+MS culture mediums+the g/L of 0.5 mg/L+ agar powders of sucrose 30 g/L+ TDZ, 0.1 mg/L+NAA 8.
Step（6）Described in the formula of root induction culture medium be：The mg/L+30 g/L sucrose+35 of MS+IAA 1
Mg/L hygromycin.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, should all belong to the covering scope of the present invention.
Claims (7)
1. a kind of agriculture bacillus mediated sinocalamus latiflorus method for transformation, it is characterised in that：Specifically include following steps：
（1）The preparation of explant：The young tender stem end of the sinocalamus latiflorus being grown in greenhouse is taken, after washing, sterilizing, the small of 0.5-1cm is cut into
Section, the wherein position of tubercle are located at end wound；Stem end after cut-out is positioned on calli induction media, secretly trained
Support, cultivation temperature is 26 DEG C；After 1.5 months, after callus is induced, it is moved into callus proliferated culture medium, and at this
Cultivated in culture medium 7-8 months, produce flaxen embryo callus, and object is infected as Agrobacterium；
（2）Agrobacterium infects the preparation of bacterium solution：Agrobacterium containing target gene is rule and cultivated, is subsequently transferred to containing 50
In the YEB culture mediums of mg/L kanamycins, and stayed overnight in 28 DEG C of shaken cultivations, then according to 1:100 volume ratio will be cultivated
Base dilutes, and is added in the YEB culture mediums of the kanamycins containing 50 mg/L and 200 μM of acetosyringone, after
Continuous cultivate to OD is 0.6-0.8；Supernatant is removed after centrifugation, gained bacterium solution is suspended in containing 200 μM of acetosyringone
In AMM fluid nutrient mediums, it is 0.8 to be diluted to OD, obtains Agrobacterium and infects bacterium solution；
（3）Infect：By step（1）It is middle to breed the explant immersion step for embryo callus（2）Obtained Agrobacterium is infected
In bacterium solution, room temperature is placed 15 minutes after application of vacuum 15 minutes；The explant infected is taken out, is placed in sterile, with sterilizing
Filter paper blot surface bacterium solution, after be transferred to containing 2 mg/L 2, in the NB culture mediums of 4-D and 200 μM of acetosyringone, 25
DEG C, co-culture 4 days under dark condition；
（4）Screening：By step（3）The callus finished is co-cultured to clean 3-5 times in 400 mg/L carbenicillin, after
Screening and culturing is carried out in kanamycin-resistant callus tissue amplification culture medium, embryo callus is gradually formed, the induction for adventitious bud；It is described
The condition of screening and culturing is：Cultivated 4 months under 26 DEG C of dark condition；
（5）The induction of adventitious bud：By step（4）The callus that screening and culturing is obtained is placed into resistant budses inducing culture
Row adventitious bud induction culture, condition of culture is：Cultivation temperature is 26 DEG C, 70 μm of ol m-2 s-1, 16h is cultivated under illumination condition,
The lower culture of dark 8 hours；After adventitious bud induction culture, Elongation of adventitious bud culture, condition of culture are carried out in bud proliferated culture medium
For：16 h, 70 μm of ol m of intensity of illumination are cultivated under illumination condition-2 s-1；The formula of the bud proliferated culture medium is induced with bud and trained
The culture for supporting base is identical；
（6）Root induction：When the bud of regeneration grows into 3-4 centimeter lengths, the root induction culture for not containing antibiotic is transferred to
Cultivated in base, condition of culture is：Cultivation temperature is 26 DEG C, 70 μm of ol m-2 s-1, 16h is cultivated under illumination condition, it is dark
Lower culture 8 hours；Complete sinocalamus latiflorus transfer-gen plant is become after root induction, rear transplant is buried.
2. a kind of agriculture bacillus mediated sinocalamus latiflorus method for transformation according to claim 1, it is characterised in that：Step（1）Middle institute
The formula for the calli induction media stated is：MS+2,4-D 8 mg/L + IBA 0.5 mg/L+250 mg/L PVP+3g/L
The g/L sucrose of sorbierite+30.
3. a kind of agriculture bacillus mediated sinocalamus latiflorus method for transformation according to claim 1, it is characterised in that：Step（1）Middle institute
The formula for stating callus proliferated culture medium is：The mg/L PVP+2mg/L 2 of+30 g/L sucrose of 3/4MS+3g/L sorbierites+250,
4-D。
4. a kind of agriculture bacillus mediated sinocalamus latiflorus method for transformation according to claim 1, it is characterised in that：Step（2）Middle institute
The Agrobacterium strain stated is EHA105.
5. a kind of agriculture bacillus mediated sinocalamus latiflorus method for transformation according to claim 1, it is characterised in that：Step（4）Middle institute
The formula for the kanamycin-resistant callus tissue amplification culture medium stated is：The mg/L PVP+ of+30 g/L sucrose of 3/4MS+3g/L sorbierites+250
The mg/L carbenicillins of 2mg/L 2,4-D+35 mg/L hygromycin+400.
6. a kind of agriculture bacillus mediated sinocalamus latiflorus method for transformation according to claim 1, it is characterised in that：Step（5）Middle institute
The formula for the resistant budses inducing culture stated is：Mg/L carbenicillins+MS the culture mediums of 35 mg/L hygromycin+200+sucrose
The g/L of 30 g/L+ TDZ 0.1mg/L+ NAA, 0.5 mg/L+250 mg/L PVP+ agar powders 8.
7. a kind of agriculture bacillus mediated sinocalamus latiflorus method for transformation according to claim 1, it is characterised in that：Step（6）Middle institute
The formula for the root induction culture medium stated is：The mg/L hygromycin of 1 mg/L+30 g/L sucrose of MS+IAA+35.
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| CN110724689A (en) * | 2019-11-19 | 2020-01-24 | 福建农林大学 | Cas 9-mediated dendrocalamus latiflorus gene editing vector and application |
| CN113692969A (en) * | 2021-03-11 | 2021-11-26 | 吉林农业大学 | Tissue culture method of dendrocalamus latiflorus |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107630034A (en) * | 2017-11-16 | 2018-01-26 | 中国农业科学院麻类研究所 | A kind of agriculture bacillus mediated hemp transgenic method |
| CN110724689A (en) * | 2019-11-19 | 2020-01-24 | 福建农林大学 | Cas 9-mediated dendrocalamus latiflorus gene editing vector and application |
| CN110724689B (en) * | 2019-11-19 | 2021-03-26 | 福建农林大学 | Cas 9-mediated dendrocalamus latiflorus gene editing vector and application |
| CN113692969A (en) * | 2021-03-11 | 2021-11-26 | 吉林农业大学 | Tissue culture method of dendrocalamus latiflorus |
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