CN113293176A - Preparation method of pineapple agrobacterium transformation receptor and application of pineapple agrobacterium transformation receptor in pineapple transformation - Google Patents
Preparation method of pineapple agrobacterium transformation receptor and application of pineapple agrobacterium transformation receptor in pineapple transformation Download PDFInfo
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Abstract
The invention discloses a preparation method of a pineapple agrobacterium transformation receptor and application thereof in pineapple transformation, wherein the preparation method comprises the following steps: taking a pineapple plant as an explant, disinfecting the pineapple plant, inducing the generation of adventitious buds through an adventitious bud induction culture medium, so that in the process of generating the adventitious buds, tissue blocks are generated at the base part of the pineapple plant, then taking the tissue blocks, cutting the tissue blocks into small blocks, and placing the small blocks in a pineapple callus induction culture medium to prepare pineapple calluses, namely a pineapple agrobacterium transformation receptor; compared with the traditional pineapple callus induction and transformation method, the method obtains the sterile plant by optimizing the explant disinfection method, induces the pineapple callus on the basis of the tissue blocks, shortens the pineapple callus induction time, ensures that the induced pineapple callus has a good state, is beneficial to transformation and improves the transformation efficiency of the pineapple. The method of the invention has the advantages of simple required equipment, easy mastering of operation technology and wide development and application prospect.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering and plant cultivation, in particular to a preparation method of an agrobacterium pineapple transformation receptor and application thereof in pineapple transformation.
Background
Pineapple (Ananas comosus) is also called pineapple, pineapple peel, pineapple grass, pineapple and so on. The native tropical region of America, commonly known as pineapple, is one of the famous tropical fruits. The pineapple, the banana and the papaya are called as three tropical herbal fruit trees, and are tropical fruits which are popular among people and are used as fruits, vegetables and medicines, and the pineapple can and pineapple juice can be processed besides fresh food; in addition, pineapple is processed into byproducts, such as sugar, alcohol, monosodium glutamate, citric acid, etc. The pineapple is rich in nutrition, especially has the highest content of vitamin C, is sweet and slightly sour in taste and slightly cold in nature, has the effects of clearing away summer-heat, promoting the production of body fluid to quench thirst and promoting urination, and can be used for treating diseases such as sunstroke, fever, polydipsia, abdominal fullness and stuffiness, dyspepsia, dysuresia, dizziness and dim eyesight. In addition, the fruit juice also contains an enzyme similar to gastric juice, which can decompose protein and help digestion.
Pineapple is a plant with high economic and medicinal values. However, the problem of pineapple cold injury, namely that pineapples like warm and are contraindicated for cold, is actually urgently required to be solved in pineapple production at present. The pineapple yield in the next year is easy to obtain high and stable yield; on the contrary, the yield is obviously reduced in the next year due to cold winter, and the trend of low and unstable yield is shown. The overwintering temperature condition has great influence on the production of the pineapples and is a main meteorological factor for developing the production of the pineapples. Therefore, the cultivation of new varieties with cold resistance by transgenic technology is an urgent need for the development of pineapple production and is a fundamental way for improving pineapple yield.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for preparing an agrobacterium pineapple transformation receptor which is reliable in implementation, convenient in preparation, simple in required equipment and beneficial to transformation, and an application of the agrobacterium pineapple transformation receptor in pineapple transformation.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
a preparation method of an Agrobacterium pineapple transformation receptor comprises the following steps: taking a pineapple plant as an explant, sterilizing the pineapple plant, inducing the generation of adventitious buds through an adventitious bud induction culture medium, so that in the process of generating the adventitious buds, tissue blocks are generated at the base part of the pineapple plant, then taking the tissue blocks, cutting the tissue blocks into small blocks, and placing the small blocks in a pineapple callus induction culture medium to prepare pineapple calluses, namely the pineapple agrobacterium transformation receptors.
As a possible implementation manner, further, the pineapple plant is a young pineapple plant, which is washed clean by running water and then transferred to an aseptic environment for sterilization treatment, and the aseptic environment is maintained during the sterilization treatment.
As a possible embodiment, further, the sterilization treatment of the pineapple plant comprises: sterilizing with 75% ethanol for 30 s, cleaning with sterile water, and adding 0.1% HgCl2And (4) disinfecting for 6-8min, and then cleaning for at least 5 times by using sterile water, wherein the cleaning time is more than 1min each time.
As a possible implementation mode, the disinfected pineapple plant is further dried by absorbing excess water through sterilized sterile filter paper, then the explant is cut into the size of about 0.4-0.8cm by using a pair of tweezers and a knife which are subjected to sterile treatment, the explant is inoculated on a pineapple adventitious bud induction culture medium 3B0.2N, then the pineapple plant is placed under the condition of illumination for induction and differentiation to generate adventitious buds, and the adventitious buds are obtained after 20-30 days of treatment.
As a possible embodiment, further, when the adventitious bud grows into a plant, the base of the pineapple plant produces a tissue mass for inducing pineapple callus;
after the pineapple plants grow out of the adventitious buds, removing the browned parts of leaves and tissue blocks, taking the tissue blocks with the basal state meeting the preset requirement of the pineapple plants, cutting the tissue blocks into small blocks of 0.5cm, carrying out dark culture in a pineapple callus induction culture medium 2B0.2N5D, carrying out once propagation transfer in one month, removing the brown and white adventitious bud tissues in the process, and obtaining the pineapple callus meeting the preset state after propagation for 4-5 months, wherein the pineapple callus is used as an agrobacterium pineapple transformation receptor.
As a possible embodiment, further, the pineapple adventitious bud induction medium 3B0.2N comprises the following components in mass concentration:
MS 4.43g/L, sucrose 30g/L, plant gel 3g/L, 6-BA 3mg/L, NAA 0.2 mg/L;
the pH value is 5.6-5.8;
one of the component mixing schemes of the pineapple adventitious bud induction culture medium 3B0.2N is as follows: the mixture containing MS 4.43g/L, sucrose 30g/L, plant gel (phytagel) 3g/L and pH 5.6-5.8 is autoclaved and then added with 6-BA 3mg/L (mother liquor concentration 1mg/mL) and NAA 0.2mg/L (mother liquor concentration 1 mg/mL).
As a possible embodiment, further, the pineapple callus induction medium 2B0.2N5D comprises the following components in mass concentration:
MS 4.43g/L, sucrose 30g/L, plant gel 3g/L, 6-BA 2mg/L, NAA 0.2mg/L and 2, 4-D5 mg/L;
the pH value is 5.6-5.8;
one of the component mixing schemes of the pineapple callus induction culture medium 2B0.2N5D is as follows: the mixture containing MS 4.43g/L, sucrose 30g/L, plant gel (phytagel) 3g/L and pH 5.6-5.8 is autoclaved and then added with 6-BA 2mg/L (mother liquor concentration 1mg/mL), NAA 0.2mg/L (mother liquor concentration 1mg/mL) and 2, 4-D5 mg/L (mother liquor concentration 5 mg/mL).
As an implementation integration, the preparation method of the pineapple agrobacterium transformation receptor is realized by the following steps:
(1) obtaining of pineapple sterile plant and establishment of adventitious bud induction system
Firstly, selecting relatively young pineapple plants as explants, washing the pineapple plants clean by running water, then washing the pineapple plants clean by sterile water in a super-clean workbench, and then performing disinfection treatment;
placing the explant into a tissue culture bottle subjected to high-pressure sterilization, adding 75% alcohol (ensuring that the explant is submerged), sterilizing for 30 seconds, pouring out the alcohol, and cleaning for 3-5 times by using sterile water;
③ use 0.1% of HgCl2Sterilizing for 6-8min, cleaning with sterile water for at least 1min for 5 times to reduce HgCl2Poisoning of explants;
fourthly, absorbing excessive water by using filter paper sterilized by high pressure, cutting the explant into the size of about 0.4 to 0.8cm by using sterile tweezers and a knife, inoculating the explant on an adventitious bud induction culture medium 3B0.2N of the pineapple, placing the explant on a light irradiation culture medium for induction and differentiation, and obtaining the adventitious bud after 20 to 30 days.
(2) Establishment of pineapple callus induction system
And (2) after a large number of pineapple plants grow out from the adventitious buds obtained in the step (1), removing partial leaves and browned parts, cutting tissue blocks at the base parts of the pineapple plants, cutting the tissue blocks into small blocks of about 0.5cm, placing the small blocks in a callus induction culture medium 2B0.2N5D for dark culture, performing propagation transfer once a month, removing tissues and the like of the white adventitious buds which are browned and possibly appear in the process, and performing propagation for 4-5 months to obtain the pineapple callus with a better state.
The agrobacterium-mediated pineapple transformation method of the pineapple agrobacterium transformation receptor prepared based on the preparation method comprises the following steps:
(a) pre-culturing: removing brown stain around the callus of the pineapple and the white adventitious bud tissue, cutting into small pieces, and placing on a pineapple callus induction culture medium 2B0.2N5D for pre-culture for 24 h;
(b) infection: taking 1/2MS liquid culture medium, suspending agrobacterium containing marker gene in the liquid culture medium, taking the liquid culture medium as a staining solution, then placing pre-cultured pineapple callus into the staining solution for infection, firstly sucking the infected pineapple callus with sterile filter paper, and then drying in a super clean bench;
(c) co-culturing: placing the pineapple callus infected in the step (b) on a CM culture medium for 3 days;
(d) and (3) growth and culture: placing the pineapple callus processed in the step (c) on an RM culture medium for illumination culture, inducing and differentiating adventitious buds, and screening after 30 days to obtain adventitious buds;
(e) screening: transferring the adventitious bud cultured in the step (d) to an SM culture medium, screening for 20-40 days under illumination, and obtaining a survival green bud after screening;
(f) regeneration: transferring the green buds survived in the step (e) into an MS culture medium as plants, replacing the culture medium according to the growth condition of the plants according to preset conditions, transferring the plants into a tissue culture bottle for culture when the plants grow to a preset state, wherein roots of the plants grow out when the plants are cultured in the MS culture medium, and performing PCR verification when the plants grow to 3-5 cm;
(g) screening pineapple resistant plants through a dip-dyeing marker of agrobacterium tumefaciens, and then carrying out PCR identification verification on the pineapple resistant plants to obtain positive plants;
(h) hardening seedlings: putting the positive plants obtained in the step (g) into culture soil, and culturing in an incubator by illumination; and transferring the plant to a greenhouse after the plant grows to a preset state.
As a possible embodiment, further, in the step (a), the medium 2B0.2N5D in the preculture includes the following components in mass concentration:
MS 4.43g/L, sucrose 30g/L, plant gel 3g/L, 6-BA 2mg/L, NAA 0.2mg/L, 2, 4-D5 mg/L;
the pH value is 5.6-5.8;
the OD value of the staining solution in the step (b) is 0.6-0.8;
the infection method comprises the following steps: processing the pineapple callus for 5min under the vacuum-pumping pressure of 73 KPa; and then soaking the pineapple callus in the infection solution for 30-60min, pouring out the bacterial solution, sucking the bacterial solution on the surface of the pineapple callus by using sterile filter paper, and then blowing the pineapple callus in an ultra-clean bench for 20-30 min.
As a possible embodiment, further, the CM medium of step (c) comprises the following components in mass concentration:
MS 4.43g/L, sucrose 30g/L, plant gel 3g/L, AS 200 MuM, and glucose 4 g/L;
the pH was 5.8;
one of the component mixing schemes for CM media is as follows: the mixture containing MS 4.43g/L, sucrose 30g/L, plant gel (phytagel) 3g/L and pH 5.8 is autoclaved and then AS 200. mu.M and glucose 4g/L are added.
The RM medium in the step (d) comprises the following components in mass concentration:
MS 4.43g/L, sucrose 30g/L, plant gel 3g/L, 6-BA 1mg/L, NAA 0.1.1 mg/L;
the pH value is 5.6-5.8;
one of the component mixing schemes for RM media is as follows: the mixture containing MS 4.43g/L, sucrose 30g/L, plant gel (phytagel) 3g/L and pH 5.6-5.8 is autoclaved and added with 6-BA 1mg/L (mother liquor concentration 1mg/mL) and NAA 0.1mg/L (mother liquor concentration 1 mg/mL).
The SM culture medium in the step (e) comprises the following components in mass concentration:
MS 4.43g/L, sucrose 30g/L, plant gel 3g/L, hygromycin 15-20 mg/L;
the pH value is 5.6-5.8;
one of the component mixing schemes for SM medium is as follows: sterilizing the mixture containing MS 4.43g/L, sucrose 30g/L, plant gel (phybagel) 3g/L and pH 5.6-5.8 under high pressure, and adding hygromycin 15-20 mg/L.
By adopting the technical scheme, compared with the prior art, the invention has the beneficial effects that: the preparation method of the pineapple agrobacterium transformation receptor and the application of the pineapple agrobacterium transformation receptor in pineapple transformation provided by the scheme have the advantages of simple and easy operation, simple and efficient culture medium formula required by callus induction and high transformation efficiency of the marker gene, and can provide a method for genetic engineering research of pineapples.
The method has wide application value in the fields related to pineapple biotechnology, including pineapple genetic engineering, cell engineering, metabolic engineering, molecular breeding of pineapples and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a diagram of a sliced tissue mass placed on pineapple induced callus culture medium 2B0.2N5D in an example of the present invention.
FIG. 2 is a diagram of a tissue mass one month after placing pineapple induced callus culture medium 2B0.2N5D in the example of the present invention.
FIG. 3 is a graph of pineapple callus prepared for infection four months after being placed on 2B0.2N5D medium in an example of the present invention.
FIG. 4 is a graph of callus placed on RM medium after infection with Agrobacterium in an example of the invention.
FIG. 5 is a graph showing adventitious buds of callus differentiated on RM medium in the example of the present invention.
FIG. 6 is a graph showing adventitious buds differentiating callus on a selection medium in the example of the present invention.
FIG. 7 shows the adventitious bud selected in the example of the present invention growing into a plant.
FIG. 8 shows the expression vector pCAMBIA1305.2-XY13(MYB24) constructed in the present example.
FIG. 9 is a diagram showing the process of amplifying a marker gene fragment and constructing an overexpression recombinant vector in the example of the present invention.
FIG. 10 is a diagram showing the results of PCR identification of pineapple transgenic resistant plants in the examples of the present invention, in which 1-23 are pineapple plant samples and No. 24 is a positive control (primer HYG amplification).
FIG. 11 is a diagram showing the results of PCR identification of pineapple transgenic resistant plants in the example of the present invention, wherein 1-23 are pineapple plant samples, the first row is the amplification of XY13-VF1+ XY13-VR1 primer, and the second row is the amplification of XY13-VF2+ XY13-VR2 primer.
FIG. 12 is a GUS staining verification result diagram of pineapple transgenic positive plants in the embodiment of the invention.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. It is to be noted that the following examples are only illustrative of the present invention, and do not limit the scope of the present invention. Similarly, the following examples are only some but not all examples of the present invention, and all other examples obtained by those skilled in the art without any inventive work are within the scope of the present invention.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The following takes XS pineapple as an example to further illustrate the preparation of the pineapple agrobacterium transformation receptor and the pineapple agrobacterium transformation method provided by the invention.
A preparation method of a pineapple agrobacterium transformation receptor comprises the following steps:
(1) obtaining of pineapple sterile plant and establishment of adventitious bud induction system
Firstly, selecting relatively young pineapple plants as explants, washing the pineapple plants clean by running water, then washing the pineapple plants clean by sterile water in a super-clean workbench, and then performing disinfection treatment;
placing the explant into a tissue culture bottle subjected to high-pressure sterilization, adding 75% alcohol (ensuring that the explant is submerged), sterilizing for 30 seconds, pouring out the alcohol, and cleaning for 3-5 times by using sterile water;
③ use 0.1% of HgCl2Sterilizing for 6-8min, cleaning with sterile water for at least 1min for 5 times to reduce HgCl2Poisoning of explants;
fourthly, absorbing excessive water by using filter paper sterilized by high pressure, cutting the explant into the size of about 0.4 to 0.8cm by using sterile tweezers and a knife, inoculating the explant on an adventitious bud induction culture medium 3B0.2N of the pineapple, placing the explant on a light irradiation culture medium for induction and differentiation, and obtaining the adventitious bud after 20 to 30 days.
(2) Establishment of pineapple callus induction system
And (2) after a large number of pineapple plants grow out from the adventitious buds obtained in the step (1), removing partial leaves and browned parts, cutting tissue blocks at the base parts of the pineapple plants, cutting the tissue blocks into small blocks of about 0.5cm, placing the small blocks in a callus induction culture medium 2B0.2N5D for dark culture, performing propagation transfer once a month, removing tissues and the like of white adventitious buds which are browned and possibly appear in the process, performing propagation for 4-5 months to obtain pineapple calluses with better states, wherein the culture change process can be shown in one of figures 1 to 3.
In the scheme, varieties of the pineapples comprise MD2 and XS. In the scheme, the pineapple is preferably of XS variety.
The pineapple adventitious bud induction medium 3B0.2N in the step (1) comprises the following components: MS 4.43g/L, sucrose 30g/L, 6-BA 3mg/L (mother liquor concentration 1mg/mL), NAA 0.2mg/L (mother liquor concentration 1mg/mL), plant gel (phytagel) 3g/L, and pH is 5.6-5.8.
The pineapple callus induction culture medium 2B0.2N5D in the step (2) comprises the following components: MS 4.43g/L, sucrose 30g/L, plant gel (phytagel) 3g/L, pH 5.6-5.8, after autoclaving, add 6-BA 2mg/L (mother liquor concentration 1mg/mL), NAA 0.2mg/L (mother liquor concentration 1mg/mL), 2, 4-D5 mg/L (mother liquor concentration 5 mg/mL).
Based on the preparation method of the agrobacterium pineapple transformation receptor, the embodiment further provides a construction method of a marker gene vector on the basis of the preparation method.
The marker gene XY13(MYB24) to be transformed is subjected to vector construction, and the map of the marker gene and the vector construction is shown in FIG. 8.
The plant expression vector is named as a vector pCAMBIA1305.2, the vector is provided with a resistance gene HYG, a reporter gene on the vector is GUS, a promoter used in the vector pCAMBIA1305.2 is an XY13(MYB24) gene self promoter, the size of the promoter is 3416bp, the whole genome size of the marker gene is 5254bp, the marker gene can be subjected to fusion expression with the GUS gene, the function of the promoter is to start a transferred XY13(MYB24) gene sequence and further start the expression of the GUS gene, the expression condition of the marker gene in pineapple transformed plants can be observed conveniently, and the feasibility and the effectiveness of an agrobacterium-mediated transformation method in pineapple are further determined.
Primers were designed and PCR amplified to a fragment comprising the MYB24 promoter and the full-length gene, 8.7kb in size, with the following primer names and sequences:
PCR amplification yielded an about 8kb band of interest, consistent with the genomic size of XY13(MYB24) (FIG. 9A). After the desired band was recovered and purified, the expression vector pCambia1305.2 and the desired band were digested with BamH I and Sal I, respectively (see FIG. 9B). The linearized vector and the marker gene after double digestion are ligated by T4 ligase. The ligation product was transformed into E.coli, and the colony PCR verified that the result was correct (see FIG. 9C). Meanwhile, the PCR positive colonies are sent to a company for sequencing, and the comparison and sequencing result shows that the recombinant vector pCambia1305.2-1XY13(MYB24) is successfully constructed.
And (3) transforming the successfully constructed recombinant vector into agrobacterium, screening positive clones for shake bacteria, and storing bacterial liquid for transformation. The transformed Agrobacterium was shaken with LB liquid medium with a final concentration of 50mg/L kanamycin and 50mg/L rifampicin. Centrifuging the bacterial liquid in a 50mL centrifuge tube at 18 ℃ and 3500rpm for 10min, adding 1/2MS resuspension (liquid MS culture medium, formula: 1/2MS (2.215 g/L), sucrose (30 g/L), pH (about 5.8), autoclaving, and then placing to normal temperature) to ensure that the final OD600 value is about 0.5-0.8, and then infecting. The agrobacterium used in the embodiment of the invention has three strains of GV3101, LBA4404 and EHA 105.
Based on the pineapple agrobacterium transformation receptor prepared by the preparation method and the agrobacterium infection scheme, this embodiment further provides an agrobacterium-mediated pineapple transformation method based on the pineapple agrobacterium transformation receptor of the scheme, which is shown in one of fig. 4 to fig. 7 and includes the following steps:
1. pre-culturing: removing brown stain and white adventitious bud tissue around the callus of pineapple, cutting into small pieces, placing on pineapple callus induction culture medium 2B0.2N5D, and pre-culturing for 24 hr.
2. Infection: 1/2MS liquid culture medium suspending Agrobacterium containing marker gene, adjusting OD value to 0.6-0.8 with infection solution, vacuumizing the pineapple callus obtained by the preparation method of claim 1 with infection solution for 5min, and soaking for 30-60 min. The bacterial solution was decanted, and the infected calli were blotted dry with sterile filter paper and blown in a clean bench for about 20-30 min.
3. Co-culturing: and (3) placing the pineapple callus infected in the step (2) on a CM culture medium for culturing for 3 days.
4. And (3) growth and culture: and (3) placing the pineapple callus co-cultured in the step (3) on an RM culture medium for illumination culture, inducing and differentiating adventitious buds, and screening after about 30 days.
5. Screening: transferring the adventitious bud cultured in the step 4 to SM medium (Hygr15-20mg/L)) and screening for 20-40 days under illumination.
6. Regeneration: and (3) transferring the green buds screened and survived in the step (5) to an MS culture medium, replacing the culture medium according to the growth condition of the plants, transferring the larger plants into a tissue culture bottle for culture, growing roots of the plants in the MS culture medium, and carrying out PCR verification when the plants grow to 3-5 cm.
7. And carrying out PCR verification on the resistant pineapple plants.
(1) Primer design
The designed primer sequences are as follows:
(2) PCR verification of transgenic pineapple resistant plants
3 pairs of primers are used for verifying the transgenic pineapple positive plants obtained in the embodiment of the invention. Firstly, using hygromycin gene verification (the verification result is shown in figure 10) to obtain a plant with the hygromycin gene; the marker gene was then amplified using two additional pairs of primers for the marker gene (validation shown in FIG. 11). The No. 21 and No. 22 plants are verified to be transgenic positive plants by PCR.
(3) GUS (glucuronidase) staining verification of transgenic pineapple positive plant
GUS staining is carried out through reporter genes on the vectors, and the results show that the leaves of No. 21 and No. 22 positive plants can be stained (as shown in figure 12)
8. Hardening seedlings: putting the obtained positive plants into culture soil, and culturing in an incubator by illumination. And transferring to a greenhouse when the plants grow healthily.
Wherein the pre-culture medium 2B0.2N5D in the step 1 comprises the following components: the mixture of MS 4.43g/L, sucrose 30g/L, plant gel (phytagel) 3g/L and pH 5.6-5.8 is autoclaved and then added with 6-BA 2mg/L (mother liquor concentration 1mg/mL), NAA 0.2mg/L (mother liquor concentration 1mg/mL) and 2, 4-D5 mg/L (mother liquor concentration 5 mg/mL).
The CM medium in the step 3 comprises the following components: the mixture of MS 4.43g/L, sucrose 30g/L, plant gel (phybagel) 3g/L and pH 5.8 is autoclaved and then AS 200uM and glucose 4g/L are added.
The RM medium in the step 4 comprises the following components: the mixture of MS 4.43g/L, sucrose 30g/L, plant gel (phytagel) 3g/L and pH 5.6-5.8 is autoclaved and then added with 6-BA 1mg/L (mother liquor concentration 1mg/mL) and NAA 0.1mg/L (mother liquor concentration 1 mg/mL).
The SM culture medium in the step 5 comprises the following components: the mixture of MS 4.43g/L, sucrose 30g/L, plant gel (phybagel) 3g/L and pH 5.6-5.8 is autoclaved and added with hygromycin 15-20 mg/L.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (10)
1. A preparation method of a pineapple agrobacterium transformation receptor is characterized by comprising the following steps: taking a pineapple plant as an explant, sterilizing the pineapple plant, inducing the generation of adventitious buds through an adventitious bud induction culture medium, so that in the process of generating the adventitious buds, tissue blocks are generated at the base part of the pineapple plant, then taking the tissue blocks, cutting the tissue blocks into small blocks, and placing the small blocks in a pineapple callus induction culture medium to prepare pineapple calluses, namely the pineapple agrobacterium transformation receptors.
2. The method for preparing the agrobacterium pineapple transformation receptor according to claim 1, wherein the pineapple plant is a young pineapple plant, and the young pineapple plant is washed clean by running water and then is sterilized in a sterile environment, and the sterile environment is maintained in the sterilization process.
3. The method for preparing an agrobacterium pineapple transformation acceptor according to claim 1, wherein the disinfection treatment of the pineapple plant comprises: sterilizing with 75% ethanol for 30 s, cleaning with sterile water, and adding 0.1% HgCl2And (4) disinfecting for 6-8min, and then cleaning for at least 5 times by using sterile water, wherein the cleaning time is more than 1min each time.
4. The method for preparing the pineapple agrobacterium transformation acceptor according to claim 1, wherein the disinfected pineapple plant is further dried by sucking excess water through sterilized sterile filter paper, then the explant is cut into about 0.4-0.8cm by using a pair of sterile tweezers and a sterile knife, the explant is inoculated on a pineapple adventitious bud induction culture medium 3B0.2N, then the pineapple adventitious bud is induced and differentiated under the illumination condition to generate an adventitious bud, and the adventitious bud is obtained after 20-30 days of treatment.
5. The method for preparing an Agrobacterium Ananadis transformation receptor according to claim 1, wherein, when adventitious buds are generated and/or grown into a plant, the base of the pineapple plant generates a tissue mass for inducing callus of pineapple;
after the pineapple plants grow out of the adventitious buds, removing the browned parts of leaves and tissue blocks, taking the tissue blocks with the basal state meeting the preset requirement of the pineapple plants, cutting the tissue blocks into small blocks of 0.5cm, carrying out dark culture in a pineapple callus induction culture medium 2B0.2N5D, carrying out once propagation transfer in one month, removing the brown and white adventitious bud tissues in the process, and obtaining the pineapple callus meeting the preset state after propagation for 4-5 months, wherein the pineapple callus is used as an agrobacterium pineapple transformation receptor.
6. The method for preparing the agrobacterium pineapple transformation acceptor according to claim 4, wherein the pineapple adventitious bud induction medium 3B0.2N comprises the following components in mass concentration:
MS 4.43g/L, sucrose 30g/L, plant gel 3g/L, 6-BA 3mg/L, NAA 0.2 mg/L;
the pH value is 5.6-5.8.
7. The method for preparing the agrobacterium pineapple transformation receptor as set forth in claim 5, wherein the pineapple callus induction medium 2B0.2N5D comprises the following components in mass concentration:
MS 4.43g/L, sucrose 30g/L, plant gel 3g/L, 6-BA 2mg/L, NAA 0.2mg/L and 2, 4-D5 mg/L;
the pH value is 5.6-5.8.
8. Agrobacterium-mediated pineapple transformation method of the Agrobacterium pineapple Agrobacterium transformed acceptor obtained on the basis of the preparation process according to one of claims 1 to 7, characterized in that it comprises the following steps:
(a) pre-culturing: removing brown stain around the callus of the pineapple and the white adventitious bud tissue, cutting into small pieces, and placing on a pineapple callus induction culture medium 2B0.2N5D for pre-culture for 24 h;
(b) infection: taking 1/2MS liquid culture medium, suspending agrobacterium containing marker gene in the liquid culture medium, taking the liquid culture medium as a staining solution, then placing pre-cultured pineapple callus into the staining solution for infection, firstly sucking the infected pineapple callus with sterile filter paper, and then drying in a super clean bench;
(c) co-culturing: placing the pineapple callus infected in the step (b) on a CM culture medium for 3 days;
(d) and (3) growth and culture: placing the pineapple callus processed in the step (c) on an RM culture medium for illumination culture, inducing and differentiating adventitious buds, and screening after 30 days to obtain adventitious buds;
(e) screening: transferring the adventitious bud cultured in the step (d) to an SM culture medium, screening for 20-40 days under illumination, and obtaining a survival green bud after screening;
(f) regeneration: transferring the green buds survived in the step (e) into an MS culture medium as plants, replacing the culture medium according to the growth condition of the plants according to preset conditions, transferring the plants into a tissue culture bottle for culture when the plants grow to a preset state, wherein roots of the plants grow out when the plants are cultured in the MS culture medium, and performing PCR verification when the plants grow to 3-5 cm;
(g) screening pineapple resistant plants through a dip-dyeing marker of agrobacterium tumefaciens, and then carrying out PCR identification verification on the pineapple resistant plants to obtain positive plants;
(h) hardening seedlings: putting the positive plants obtained in the step (g) into culture soil, and culturing in an incubator by illumination; and transferring the plant to a greenhouse after the plant grows to a preset state.
9. The agrobacterium-mediated pineapple transformation method of an agrobacterium pineapple transformation receptor according to claim 8, wherein in step (a), the medium 2B0.2N5D in the preculture comprises the following components in mass concentration:
MS 4.43g/L, sucrose 30g/L, plant gel 3g/L, 6-BA 2mg/L, NAA 0.2mg/L, 2, 4-D5 mg/L;
the pH value is 5.6-5.8;
the OD value of the staining solution in the step (b) is 0.6-0.8;
the infection method comprises the following steps: processing the pineapple callus for 5min under the vacuum-pumping pressure of 73 KPa; and then soaking the pineapple callus in the infection solution for 30-60min, pouring out the bacterial solution, sucking the bacterial solution on the surface of the pineapple callus by using sterile filter paper, and then blowing the pineapple callus in an ultra-clean bench for 20-30 min.
10. The agrobacterium-mediated pineapple transformation method of an agrobacterium pineapple transformation receptor according to claim 8, wherein the CM medium of step (c) comprises the following components in mass concentration:
MS 4.43g/L, sucrose 30g/L, plant gel 3g/L, AS 200 MuM, and glucose 4 g/L;
the pH was 5.8;
the RM medium in the step (d) comprises the following components in mass concentration:
MS 4.43g/L, sucrose 30g/L, plant gel 3g/L, 6-BA 1mg/L, NAA 0.1.1 mg/L; the pH value is 5.6-5.8;
the SM culture medium in the step (e) comprises the following components in mass concentration:
MS 4.43g/L, sucrose 30g/L, plant gel 3g/L, hygromycin 15-20 mg/L;
the pH value is 5.6-5.8.
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