CN107190019B - A kind of sinocalamus latiflorus method for transformation of mediated by agriculture bacillus - Google Patents
A kind of sinocalamus latiflorus method for transformation of mediated by agriculture bacillus Download PDFInfo
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- CN107190019B CN107190019B CN201710513446.8A CN201710513446A CN107190019B CN 107190019 B CN107190019 B CN 107190019B CN 201710513446 A CN201710513446 A CN 201710513446A CN 107190019 B CN107190019 B CN 107190019B
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Abstract
The invention discloses a kind of sinocalamus latiflorus method for transformation of mediated by agriculture bacillus, and preparation, Agrobacterium including explant 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, containing 2 mg/L 2, co-culturing in the NB culture medium of 4-D and 200 μM of acetosyringone, through having screening and etc. final obtain transgenosis sinocalamus latiflorus.The present invention uses the Efficient Conversion system of mediated by agriculture bacillus, it is established for the first time with the transformation system for the sinocalamus latiflorus that sinocalamus latiflorus nutrition organs is starting, the system can break through the obstacle for overcoming traditional breeding method that can not apply in bamboo class, to provide premise by the molecular breeding that transgenic technology carries out sinocalamus latiflorus in the future；In this way, providing technical support in basic research field for research bamboo genoid function.
Description
Technical field
The invention belongs to Forest Tree Genetic Breeding fields, and in particular to a kind of sinocalamus latiflorus method for transformation of mediated by agriculture bacillus.
Background technique
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 important one of the large-scale sympodial bamboo kind of double purposes of In South China and the southeastern coastal areas, has high economic valence
Value and social value.
With the development of the social economy, people are higher and higher to the yield and quality requirement of bamboo wood, but corresponding therewith it is
Much lag of bamboo class breeding.Traditional genetic and breeding method can not carry out in bamboo, main reasons is that Studies on Bamboo Flowering has
There are chronicity and uncertainty, the hybridization between bamboo kind can not be carried out.Transgenic breeding is the important means in crop breeding, is led to
It crosses transgenosis and carries out accurately gene regulation, the yield and quality of bamboo wood is greatly improved.
Research at present in terms of sinocalamus latiflorus transgenosis is fewer.2014, Qiao et al. was built using anther tissue for explant
Regenerating system is erected, and on this basis will be in bacteriumcodAChannel genes are to sinocalamus latiflorus genome.This is that sinocalamus latiflorus transgenosis is ground
The unique an example report studied carefully.But by the anther of sinocalamus latiflorus starting transgenic technology exist there may be chimera, monoploid or
The problems such as being polyploid and lower conversion ratio.Therefore, it is cured by the method conversion of mediated by agriculture bacillus by what nutritive issue generated
Injured tissue, the conversion for carrying out functional gene are to be improved to solve the problems, such as to sinocalamus latiflorus resource.
Summary of the invention
It is an object of the invention in view of the shortcomings of the prior art, providing a kind of sinocalamus latiflorus method for transformation of mediated by agriculture bacillus.This
Invention inventor establish using sinocalamus latiflorus stem end as explant regenerating system on the basis of, utilize mediated by agriculture bacillus method carry out
The transgenic research of sinocalamus latiflorus.The present invention solves the problems, such as that sinocalamus latiflorus transgenosis is difficult for a long time with this method, can be extensive
Functional gene identification and molecular breeding applied to sinocalamus latiflorus.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of sinocalamus latiflorus method for transformation of mediated by agriculture bacillus, specifically includes the following steps:
(1) preparation of explant: the sinocalamus latiflorus children's tender stem end being grown in greenhouse is taken to be cut into 0.5-1cm after washing, sterilizing
Segment, wherein the position of tubercle is located at end wound；Stem end after cutting is placed on calli induction media, is carried out
Dark culture, cultivation temperature are 26 DEG C；After 1.5 months, after inducing callus, it is moved into callus proliferated culture medium, and
It is cultivated in the culture medium 7-8 months, generates flaxen embryo callus, and infect object as Agrobacterium；
(2) Agrobacterium infects the preparation of bacterium solution: by containing target gene Agrobacterium cross culture, be subsequently transferred to containing
It in the YEB culture medium of 50 mg/L kanamycins, and stays overnight in 28 DEG C of shaken cultivations, then will be trained according to the volume ratio of 1:100
Base dilution is supported, and is added in the YEB culture medium of the kanamycins containing 50 mg/L and 200 μM of acetosyringone,
Continuing to cultivate to OD is 0.6-0.8；Supernatant is removed after centrifugation, and gained bacterium solution is suspended in the acetosyringone containing 200 μM
AMM fluid nutrient medium in, be diluted to OD be 0.8, obtain Agrobacterium and infect bacterium solution；
(3) it infects: the Agrobacterium that the explant that embryo callus is proliferated out in step (1) immersion step (2) is obtained
It infects in bacterium solution, is placed at room temperature for 15 minutes after being vacuum-treated 15 minutes；The explant infected is taken out, is placed in sterile, is used
The filter paper of sterilizing blots surface bacterium solution, after be transferred to containing 2 mg/L 2, in the NB culture medium of 4-D and 200 μM of acetosyringone,
It is co-cultured 4 days under 25 DEG C, dark condition；
(4) it screens: step (3) being co-cultured into the callus finished and clean 3-5 in the carbenicillin of 400 mg/L
Time, after screening and culturing is carried out in kanamycin-resistant callus tissue amplification culture medium, embryo callus is gradually formed, for luring for adventitious bud
It leads；The condition of the screening and culturing are as follows: cultivated 4 months under 26 DEG C of dark condition；
(5) callus that step (4) screening and culturing obtains the induction of adventitious bud: is placed into resistant buds induced medium
Middle carry out adventitious bud induction culture, condition of culture are as follows: cultivation temperature is 26 DEG C, 70 μm of ol m-2 s-1, cultivate under illumination condition
16h, dark lower culture 8 hours；After adventitious bud induction culture, Elongation of adventitious bud culture, culture are carried out in bud proliferated culture medium
Condition are as follows: 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 lured with bud
The culture for leading culture medium is identical；
(6) root induction: when regenerated bud grows into 3-4 centimeter length, it is transferred to the root induction without containing antibiotic
It is cultivated in culture medium, condition of culture are as follows: cultivation temperature is 26 DEG C, 70 μm of ol m-2 s-1, 16h is cultivated under illumination condition,
Dark lower culture 8 hours；Complete sinocalamus latiflorus transgenic plant is become after root induction, rear transplanting is buried.
The formula of calli induction media as described in step (1) are as follows: 8 mg/L+IBA of MS+2,4-D, 0.5 mg/L+
+ 30 g/L sucrose of 250 mg/L PVP+3g/L sorbierite.
The formula of callus proliferated culture medium described in step (1) are as follows:+30 g/L sucrose+250 of 3/4MS+3g/L sorbierite
mg/L PVP+2mg/L 2,4-D。
Agrobacterium strain described in step (2) is EHA105.
The formula of kanamycin-resistant callus tissue amplification culture medium described in step (4) are as follows:+30 g/L sugarcane of 3/4MS+3g/L sorbierite
+ 400 mg/L carbenicillin of sugared+250 mg/L PVP+2mg/L 2,4-D+35 mg/L hygromycin.
The formula of resistant buds induced medium described in step (5) are as follows:+200 mg/L carboxylic benzyl of 35 mg/L hygromycin is green
Mycin+MS culture medium+0.5 mg/L+ agar powder of sucrose 30 g/L+ TDZ, 0.1 mg/L+NAA, 8 g/L.
The formula of root induction culture medium described in step (6) are as follows: 1 mg/L+30 g/L sucrose+35 of MS+IAA
Mg/L hygromycin.
The beneficial effects of the present invention are:
1) present invention uses the Efficient Conversion system of mediated by agriculture bacillus, establishes the transformation system of sinocalamus latiflorus for the first time.The body
System can break through the obstacle for overcoming traditional breeding method that can not apply in bamboo class；To carry out fiber crops by transgenic technology in the future
The molecular breeding of bamboo provides premise；In this way, providing technology branch in basic research field for research bamboo genoid function
Support；
2) present invention constructs transgenic line to be starting explant with the stem end of sinocalamus latiflorus；The bacterium of Agrobacterium is infected in selection
Kind is EHA105 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 uses and contain 200 μM
The Agrobacterium of the AMM fluid nutrient medium of acetosyringone, the culture medium culture can effectively infect callus；
4) culture medium of the co-cultivation used in is NB, and need to add 2,4-D 2mg/L and 200 μM of acetosyringones to have
While conducive to Agrobacterium infectivity is improved, and excessive injury is not caused to plant tissue.
Detailed description of the invention
Fig. 1 converts for the sinocalamus latiflorus of mediated by agriculture bacillus and qualification figure, in figure :(a) the Agrobacterium containing target gene includes tide
Mycin resistant geneHPTIIAnd GUS；(b) convert whole flow process: i infect before callus state；Ii kanamycin-resistant callus tissue is from browning
Parent in grow；Iii kanamycin-resistant callus tissue squamous subculture；Iv kanamycin-resistant callus tissue gradually generates embryo callus subculture；V kanamycin-resistant callus tissue starts to break up
Generate bud point；Vi kanamycin-resistant callus tissue seedling differentiation；Vii regenerates seedling rooting；Viii regrowth buries；(c) PCR identification generates 1044
Positive band；(d) callus and regeneration stem after converting present positive.
Specific 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 sinocalamus latiflorus method for transformation of mediated by agriculture bacillus, specifically includes the following steps:
(1) preparation of explant: the sinocalamus latiflorus children's tender stem end being grown in greenhouse is taken, is placed in detergent and impregnates after cutting
60 minutes, after under tap water rinse 2 hours.It is floated after aseptically being impregnated 1 minute in 75% ethyl alcohol with sterile water
Wash 5 times, after sterilized 8 minutes with 0.1% life mercury again, then with aseptic water washing 6 times.The segment of 1cm is cut at stem end after sterilizing,
Wherein the position of tubercle is located at end wound；Stem end after cutting is placed on calli induction media, dark culture is carried out,
Cultivation temperature is 26 DEG C；After 1.5 months, after inducing callus, it is moved into callus proliferated culture medium, and in the culture
It cultivates 7 months, generates flaxen embryo callus ((b)-i in Fig. 1) in base, and infected pair as Agrobacterium
As；
(2) Agrobacterium infects the preparation of bacterium solution: by the Agrobacterium EHA105 containing target gene Pcambia1301-GUS
((a) in Fig. 1) scribing line culture, is subsequently transferred in the YEB culture medium containing 50 mg/L kanamycins, and vibrate in 28 DEG C
Then overnight incubation dilutes culture medium according to the volume ratio of 1:100, and be added to the card containing 50 mg/L that is mould
In the YEB culture medium of element and 200 μM of acetosyringone, continuing to cultivate to OD is 0.6；Supernatant is removed after centrifugation, by gained
Bacterium solution is suspended in the AMM fluid nutrient medium containing 200 μM of acetosyringone, and being diluted to OD is 0.8, is obtained Agrobacterium and is invaded
Microbiological contamination liquid；
(3) it infects: the Agrobacterium that the explant that embryo callus is proliferated out in step (1) immersion step (2) is obtained
It infects in bacterium solution, is vacuum-treated (- 0.8bar) and is placed at room temperature for 15 minutes after 15 minutes；The explant infected is taken out, nothing is placed in
In bacterium platform, blot surface bacterium solution with the filter paper of sterilizing, after be transferred to containing 2 mg/L 2, the NB of 4-D and 200 μM of acetosyringone
In culture medium, co-cultured 4 days under 25 DEG C, dark condition；
(4) it screens: step (3) being co-cultured into the callus finished and are cleaned 4 times in the carbenicillin of 400 mg/L,
Screening and culturing is carried out in kanamycin-resistant callus tissue amplification culture medium afterwards, gradually forms embryo callus, the induction for adventitious bud；Institute
State the condition of screening and culturing are as follows: cultivate 4 months under 26 DEG C of dark condition；Have within 2 months or so new resistant calli from
Separated in parent ((b)-in Fig. 1 ii), 4 months or so time gradually form embryo callus subculture ((b)-iii in Fig. 1,
-iv)；
(5) callus that step (4) screening and culturing obtains the induction of adventitious bud: is placed into resistant buds induced medium
Middle carry out adventitious bud induction culture, condition of culture are as follows: cultivation temperature is 26 DEG C, 70 μm of ol m-2 s-1, cultivate under illumination condition
16h, dark lower culture 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 are as follows: 16 h, 70 μm of ol m of intensity of illumination are cultivated under illumination condition-2 s-1；
(6) root induction: when regenerated bud grows into 3-4 centimeter length, it is transferred to the root induction without containing antibiotic
It is cultivated in culture medium, condition of culture are as follows: cultivation temperature is 26 DEG C, 70 μm of ol m-2 s-1, 16h is cultivated under illumination condition,
Dark lower culture 8 hours；Complete sinocalamus latiflorus transgenic plant is become after root induction (to be transplanted into after (b)-in Fig. 1 vii),
((b)-in Fig. 1 is viii) for soil.
(7) 17 plants of positive seedlings are finally obtained, wherein 12 plants are positive ((c) in Fig. 1) after PCR is verified, it is used
Primer is the primer of HPTII and 35S specificity, sequence are as follows:
(Forward: 5’-TGCCATCATTGCGATAAAGGAAAG-3’) and HPTII gene (Reverse:
5'-CTGC TGCTCCATACAAGCCAACC-3').GUS dyeing statistics indicate that, the callus after conversion is (in Fig. 1
(d)-ii) (positive signal of blue is iv) presented in (d)-in Fig. 1, and in control group (in (d)-i and Fig. 1 in Fig. 1 with stem end
(d)-iii) do not occur positive signal.
The formula of calli induction media as described in step (1) are as follows: 8 mg/L+IBA of MS+2,4-D, 0.5 mg/L+
+ 30 g/L sucrose of 250 mg/L PVP+3g/L sorbierite.
The formula of callus proliferated culture medium described in step (1) are as follows:+30 g/L sucrose+250 of 3/4MS+3g/L sorbierite
mg/L PVP+2mg/L 2,4-D。
The formula of kanamycin-resistant callus tissue amplification culture medium described in step (4) are as follows:+30 g/L sugarcane of 3/4MS+3g/L sorbierite
+ 400 mg/L carbenicillin of sugared+250 mg/L PVP+2mg/L 2,4-D+35 mg/L hygromycin.
The formula of resistant buds induced medium described in step (5) are as follows:+200 mg/L carboxylic benzyl of 35 mg/L hygromycin is green
Mycin+MS culture medium+0.5 mg/L+ agar powder of sucrose 30 g/L+ TDZ, 0.1 mg/L+NAA, 8 g/L.
The formula of root induction culture medium described in step (6) are as follows: 1 mg/L+30 g/L sucrose+35 of MS+IAA
Mg/L hygromycin.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (2)
1. a kind of sinocalamus latiflorus method for transformation of mediated by agriculture bacillus, it is characterised in that: specifically includes the following steps:
(1) preparation of explant: the sinocalamus latiflorus children's tender stem end being grown in greenhouse is taken to be cut into the small of 0.5-1cm after washing, sterilizing
Section, wherein the position of tubercle is located at end wound；Stem end after cutting is placed on calli induction media, is secretly trained
It supports, cultivation temperature is 26 DEG C；After 1.5 months, after inducing callus, it is moved into callus proliferated culture medium, and at this
It is cultivated in culture medium 7-8 months, generates flaxen embryo callus, and infect object as Agrobacterium；
(2) Agrobacterium infects the preparation of bacterium solution: the Agrobacterium containing target gene being crossed and is cultivated, is subsequently transferred to containing 50
It in the YEB culture medium of mg/L kanamycins, and stays overnight in 28 DEG C of shaken cultivations, then will be cultivated according to the volume ratio of 1:100
Base dilution, and be added in the YEB culture medium 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, and gained bacterium solution is suspended in the acetosyringone containing 200 μM
In AMM fluid nutrient medium, being diluted to OD is 0.8, obtains Agrobacterium and infects bacterium solution；
(3) it infects: the Agrobacterium that the explant for being proliferated out embryo callus in step (1) immersion step (2) obtains is infected
In bacterium solution, 15 minutes are placed at room temperature for after being vacuum-treated 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 medium of 4-D and 200 μM of acetosyringone, 25
DEG C, co-culture 4 days under dark condition；
(4) it screens: step (3) being co-cultured into the callus finished and are cleaned 3-5 times in the carbenicillin of 400 mg/L, after
Screening and culturing is carried out in kanamycin-resistant callus tissue amplification culture medium, gradually forms embryo callus, the induction for adventitious bud；It is described
The condition of screening and culturing are as follows: cultivated 4 months under 26 DEG C of dark condition；
(5) induction of adventitious bud: by the callus that step (4) screening and culturing obtains be placed into resistant buds induced medium into
Row adventitious bud induction culture, condition of culture are as follows: cultivation temperature is 26 DEG C, 70 μm of ol m-2 s-1, 16h is cultivated under illumination condition,
Dark lower culture 8 hours；After adventitious bud induction culture, Elongation of adventitious bud culture, condition of culture are carried out in bud proliferated culture medium
Are as follows: 16 h, 70 μm of ol m of intensity of illumination are cultivated under illumination condition-2 s-1；Formula and the bud induction of the bud proliferated culture medium are trained
The culture for supporting base is identical；
(6) root induction: when regenerated bud grows into 3-4 centimeter length, it is transferred to the root induction culture without containing antibiotic
It is cultivated in base, condition of culture are as follows: 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 transgenic plant is become after root induction, rear transplanting is buried；
The formula of calli induction media as described in step (1) are as follows: 8 mg/L+IBA of MS+2,4-D, 0.5 mg/L+250
+ 30 g/L sucrose of mg/L PVP+3g/L sorbierite；
The formula of callus proliferated culture medium described in step (1) are as follows:+250 mg/ of+30 g/L sucrose of 3/4MS+3g/L sorbierite
L PVP+2mg/L 2,4-D；
The formula of kanamycin-resistant callus tissue amplification culture medium described in step (4) are as follows:+30 g/L sucrose of 3/4MS+3g/L sorbierite+
+ 400 mg/L carbenicillin of 250 mg/L PVP+2mg/L 2,4-D+35 mg/L hygromycin；
The formula of resistant buds induced medium described in step (5) are as follows:+200 mg/L carbenicillin of 35 mg/L hygromycin+
30 g/L+ TDZ 0.1mg/L+ NAA of MS culture medium+sucrose, 0.5 mg/L+250 mg/L PVP+ agar powder, 8 g/L；
The formula of root induction culture medium described in step (6) are as follows: 1 mg/L+30 g/L sucrose of MS+IAA+35 mg/L tide
Mycin.
2. a kind of sinocalamus latiflorus method for transformation of mediated by agriculture bacillus according to claim 1, it is characterised in that: institute in step (2)
The Agrobacterium strain stated is EHA105.
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| CN110724689B (en) * | 2019-11-19 | 2021-03-26 | 福建农林大学 | Cas 9-mediated dendrocalamus latiflorus gene editing vector and application |
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| CN106854659A (en) * | 2015-12-08 | 2017-06-16 | 中国林业科学研究院亚热带林业研究所 | A kind of method of sinocalamus latiflorus genetic transformation and plant regeneration |
Non-Patent Citations (2)
| Title |
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| Enhanced cold stress tolerance of transgenic Dendrocalamus latiflorus Munro (Ma bamboo) plants expressing a bacterial CodA gene;Qiao, GR等;《IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT》;20140308;第50卷(第4期);第385-391页 |
| 利用农杆菌介导法获得转codA基因麻竹再生植株的研究;张玲等;《竹子研究汇刊》;20120215;第31卷(第1期);第1-6页 |
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