阅读说明：本技术 农杆菌介导的甜椒遗传转化方法 (The pimento genetic transforming method of mediated by agriculture bacillus ) 是由 冀芦沙 苏振华 王华森 于 2018-10-24 设计创作，主要内容包括：本发明提供一种根癌农杆菌介导的甜椒遗传转化方法,所述方法至少包括以下步骤：提供甜椒外植体；提供含有目的基因的根癌农杆菌,侵染甜椒外植体；选择培养；生芽培养：生根培养。采用本方法或的甜椒遗传转化率高。(The present invention provides a kind of Agrobacterium tumefaciens mediated pimento genetic transforming method, and the method at least includes the following steps: providing pimento explant；Agrobacterium tumefaciems containing target gene is provided, pimento explant is infected；Selection culture；It sprouts culture: culture of rootage.Using this method or pimento genetic transformation rate it is high.)

1. a kind of Agrobacterium tumefaciens mediated pimento genetic transforming method, which is characterized in that the method includes at least following step It is rapid:

(1) pimento explant is provided；

(2) Agrobacterium tumefaciems containing target gene is provided, pimento 3~7min of explant is infected, co-cultures 36~72h, trains altogether Dark culture is used when supporting, 6-benzyl aminopurine concentration is 3~5mgL-1 in culture medium, the concentration of α-naphthylacetic acid is 0.15~ 0.25mg·L^-1, the concentration of acetosyringone is less than 200mmol/L；

(3) selection culture；6-benzyl aminopurine concentration is 3~5mgL in culture medium^-1, the concentration of α-naphthylacetic acid is 0.15~ 0.25mg·L^-1, kanamycins concentration is 45~75mgL^-1, the concentration of cephalosporin is 300~500mgL^-1；

(4) sprout culture: kanamycins concentration is 45~75mgL in culture medium^-1, the concentration of 6-benzyl aminopurine is 3~ 5mg·L^-1, the concentration of α-naphthylacetic acid is 0.15~0.25mgL^-1, concentration is 300~500mgL^-1Cephalosporin or Concentration is 100~300mgL^-1Ticarcillin/Clavulanate Acid；

(5) culture of rootage: the concentration of α-naphthylacetic acid is 3~5mgL in culture medium^-1, the concentration of 6-benzyl aminopurine is 0.15~ 0.25mg·L^-1, concentration is 300~500mgL^-1Cephalosporin or concentration be 100~300mgL^-1Ticarcillin/Clavulanate Acid.

2. Agrobacterium tumefaciens mediated pimento genetic transforming method according to claim 1, it is characterised in that: the method Used in whole culture medium pH be 5.6~6.0.

3. Agrobacterium tumefaciens mediated pimento genetic transforming method according to claim 1, it is characterised in that: the method Used in culture medium be all MS culture medium.

4. Agrobacterium tumefaciens mediated pimento genetic transforming method according to claim 1, it is characterised in that: the step (2) 23~26 DEG C of temperature in, dark culture, 36~72h of incubation time.

5. Agrobacterium tumefaciens mediated pimento genetic transforming method according to claim 1, it is characterised in that: the step (3) 23~26 DEG C of temperature in, illumination condition 1500~2200lux, 14-18h/d, 10~17d of incubation time.

6. Agrobacterium tumefaciens mediated pimento genetic transforming method according to claim 1, it is characterised in that: the step (4) 23~26 DEG C of temperature in, intensity of illumination 1500~2200lux, 14-18h/d, 10~17d of incubation time.

7. Agrobacterium tumefaciens mediated pimento genetic transforming method according to claim 1, it is characterised in that: the step (5) 23~26 DEG C of temperature in, intensity of illumination 1500~2200lux, 14-18h/d, 10~17d of incubation time.

8. Agrobacterium tumefaciens mediated pimento genetic transforming method according to claim 1, it is characterised in that: the cephalo Any one or a few in Cephalothin Sodium, ampicillin, carbenicillin, erythromycin, Ceftriaxone Sodium of rhzomorph.

9. Agrobacterium tumefaciens mediated pimento genetic transforming method according to claim 1, it is characterised in that: the step It (1) further include to pimento explant by preculture.

10. Agrobacterium tumefaciens mediated pimento genetic transforming method according to claim 1, it is characterised in that: the step Suddenly in (1) pimento explant use cotyledonary node top separate living tissue.

Technical field

The present invention relates to a kind of pimento genetic transforming methods of mediated by agriculture bacillus.

Background technique

With flourishing for life science, molecular biology and genetic engineering also achieve significant progress.Crown gall agriculture Bacillus (Agrobacterium tumefaciens) mediate exogenous gene transforming method, due to its can import large fragment DNA and Quiding gene copy number is low, high conversion and expression effect is good, arouses great concern, simultaneously because its to instrument and Operating technology requirement is lower, is widely used in plant genetic engineering, and for studying unknown gene function, various gene regulations are former The mechanism etc. of part.At present it is known that 80% genetically modified plants be to be completed by mediated by agriculture bacillus.

Pimento (scientific name: Capsicum annuum var.grossum) is commonly called as bell pepper, green pepper, pimento, TaiWan, China The also referred to as big cylinder of words is young, is a mutation for Solanaceae Capsicum capsicum, is distributed in the north and south various regions of China's Mainland, belongs to " inhuman Work introducing and planting " type plant.

Using the pimento transgenosis of mediated by agriculture bacillus, there has been no document reports.

Summary of the invention

In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide a kind of pimento of mediated by agriculture bacillus something lost Method for transformation is passed, for filling up blank in the prior art.

In order to achieve the above objects and other related objects, the present invention provides a kind of Agrobacterium tumefaciens mediated pimento heredity turn Change method, the method at least include the following steps:

(1) pimento explant is provided；

(2) Agrobacterium tumefaciems containing target gene is provided, pimento 3~7min of explant is infected, co-cultures 36~72h, Dark culture is used when co-cultivation, 6-benzyl aminopurine concentration is 3~5mgL in culture medium^-1, the concentration of α-naphthylacetic acid is 0.15 ~0.25mgL^-1, the concentration of acetosyringone is less than 200mmol/L；

(3) selection culture；6-benzyl aminopurine concentration is 3~5mgL in culture medium^-1, the concentration of α-naphthylacetic acid is 0.15 ~0.25mgL^-1, kanamycins (Kanamycin Kan) concentration is 45~75mgL^-1, the concentration of cephalosporin is 300~ 500mg·L^-1；

(4) sprout culture: kanamycins (Kanamycin Kan) concentration is 45~75mgL in culture medium^-1, 6- benzyl ammonia The concentration of base purine is 3~5mgL^-1, the concentration of α-naphthylacetic acid is 0.15~0.25mgL^-1, concentration is 300~500mg L^-1Cephalosporin or concentration be 100~300mgL^-1Ticarcillin/Clavulanate Acid；

(5) culture of rootage: the concentration of α-naphthylacetic acid is 3~5mgL in culture medium^-1, the concentration of 6-benzyl aminopurine is 0.15~0.25mgL^-1, concentration is 300~500mgL^-1Cephalosporin or concentration be 100~300mgL^-1Spy Mei Ting.

Further, the pimento explant by prior art culture or can pass through commercially available acquisition.

Utilize prior art culture pimento explant operating method are as follows: by pimento seed soaking, flushing, disinfection, cultivating Inoculated and cultured on base.Condition of culture can be 23~26 DEG C, 16h/d illumination cultivation, is not grown cotyledon after 6~12d, had The aseptic seedling of cotyledonary node.Cut explant of the cotyledonary node as genetic transformation.

Agrobacterium tumefaciems Agrobacterium tumefaciens

Ticarcillin/Clavulanate Acid is compound preparation, component are as follows: ticarcillin sodium and potassium clavulanate.

Further, in the step (1) pimento explant use cotyledonary node top separate living tissue.

Further, the pH of whole culture medium used in the method is 5.6~6.0.More preferably 5.8.

Further, culture medium used in the method is all MS culture medium.MS culture medium can be commercially available, such as Shanghai Yi Kaer Bioisystech Co., Ltd EK180112 (article No.).

Further, the step (1) further includes to pimento explant by preculture.

Further, the culture medium that the preculture uses is MS culture medium, dark culture, incubation time 36-72h, temperature 23-26 DEG C of degree.

Further, 23~26 DEG C of temperature in the step (2), dark culture, 36~72h of time, optimum condition is temperature 25 DEG C, incubation time 48h.

Further, 23~26 DEG C of temperature in the step (3), illumination condition 1500~2200lux, 14-18h/d, when Between 10~17d, optimum condition be 25 DEG C of temperature, illumination condition 2000lux, 16h/d, incubation time 15d.

Further, 23~26 DEG C of temperature in the step (4), intensity of illumination 1500~2200lux, 14-18h/d, when Between 10~17d, optimum condition be 25 DEG C of temperature, illumination condition 2000lux, 16h/d, incubation time 15d.

Further, 23~26 DEG C of temperature in the step (5), intensity of illumination 1500~2200lux, 14-18h/d, when Between 10~17d, optimum condition be 25 DEG C of temperature, illumination condition 2000lux, 16h/d, incubation time 15d.

Further, the cephalosporin is selected from Cephalothin Sodium (Cef), ampicillin (Amp), carbenicillin (Carb), erythromycin (E), any one or a few in Ceftriaxone Sodium (Ceftriaxone sodium).More preferably head Spore thiophene sodium.

As described above, Agrobacterium tumefaciens mediated pimento genetic transforming method of the invention, has the advantages that

Genetic transformation is carried out to pimento using this method, it is high-efficient.

Detailed description of the invention

Fig. 1 is shown as photo when embodiment 1 co-cultures.

Fig. 2 is shown as the photo of generation callus after the selection culture of embodiment 1.

Fig. 3 is shown as embodiment 1 and sprouts the photo that callus when cultivating germinates.

Fig. 4 is shown as photo when 1 culture of rootage of embodiment.

Fig. 5 is shown as the photo after embodiment 1 is transplanted.

Fig. 6 is shown as the photo of 1 leaf expression green fluorescence of embodiment.

Fig. 7 is shown as an other photo for 1 leaf expression green fluorescence of embodiment.

Specific embodiment

Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from Various modifications or alterations are carried out under spirit of the invention.

1 material source of table

Callus induction rate (%)=(the explant number/total explant number for generating callus) × 100%；

Average each callus resistant buds number=resistant buds number sum/callus sum.

After target gene in plasmid (1301GFP) imports pimento cell by Agrobacterium, pimento expression green can be made Fluorescin.

The pH of culture medium is 5.8 in following embodiment.