CN116515900A - Acer truncatum genetic transformation method and application - Google Patents
Acer truncatum genetic transformation method and application Download PDFInfo
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- CN116515900A CN116515900A CN202310520286.5A CN202310520286A CN116515900A CN 116515900 A CN116515900 A CN 116515900A CN 202310520286 A CN202310520286 A CN 202310520286A CN 116515900 A CN116515900 A CN 116515900A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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Abstract
Acer truncatum Bunge is taken as a woody plant with extremely high ornamental value, and the establishment of a genetic transformation system is important to the research of the gene function of the woody plant and the genetic engineering breeding. According to the invention, the 'Lihong' acer truncatum bunge stem is used as a test material, the acer truncatum embryo callus is induced to generate, and the acer truncatum embryo callus is transformed through agrobacterium mediation, so that an acer truncatum genetic transformation system is established, and the optimal antibacterial culture medium of the acer truncatum embryo callus is provided: WPM+30g/L sucrose+8 g/L agar+0.5 g/L acid hydrolyzed casein+0.2 mg/L KT+1.0mg/L TDZ+0.5mg/L IBA+0.1mg/L GA 3 +200mg/L Carbenicillin +200mg/L Cephalosporium, the result obtained by using the bacteriostatic medium is: the browning rate of embryogenic callus is obviously lowerThe antibacterial rate and the survival rate are reduced and increased; after screening by the screening culture medium, the obtained callus has the lowest browning rate, pollution rate and death rate and keeps vigorous growth.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a genetic transformation method of Acer truncatum and application thereof.
Background
Acer truncatum (ACer truncatum Bunge), which belongs to Aceraceae, is a very common afforestation tree species in China, japan and Korea, has application in Europe and North America, is an important autumn leaf tree species, and has very high ecological and ornamental value (Ma Qiayuue et al 2020). In the breeding process of the variety over 20 years, a new variety of Acer truncatum (Acer truncatum 'Lihong') is obtained, and the variety can be uniformly reddened in Beijing low-altitude urban areas in autumn. Every autumn 'Lihong' leaves turn from green to yellow and then quickly turn into uniform haematochrome, and the process is continued from 10 late to 11 late, so that the ornamental character defect of Acer truncatum applied to urban areas is overcome. The 'Lihong' acer truncatum has good economic and social benefits after being popularized by urban greening.
However, the red leaf ornamental period of 'Lihong' Acer truncatum in autumn is shorter, and the economic and social benefits cannot be fully reflected. In order to provide theoretical support for developing molecular improvement breeding of the Acer truncatum leaf color character in the later period, the 'Lihong' Acer truncatum with longer ornamental period is expected to be cultivated. So far, a good genetic transformation system is established for a plurality of plants, and a foundation is laid for subsequent gene function research and improved breeding.
The success of plant gene transformation is mainly dependent on a perfect genetic transformation system, so that exogenous genes can be effectively introduced into recipient cells and expressed. Zhu Yongxing et al established a poplar polygenic genetic transformation system using the 'Ning Yang No. 1' as the acceptor material, and co-transformed SOS1, SOS2, SOS3 to obtain 14 PCR positive lines (Zhu Yongxing et al 2015). Researchers succeed in establishing a good corn genetic transformation system by taking the stem node and bud tip growing points of the corn young embryo and the aseptic seedling as explants respectively, and a foundation is provided for improved breeding of corn (Xing Xiaolong and the like 2016; li Jingong and the like 2018; zhang Man and the like 2016). Zhang Xiying et al used the leaves and stems of the aseptic seedlings of potato as explants, transferred the RNA interference type genes of the anti-potato X virus and Y virus into the potato by the agrobacterium mediation method, and established a high-efficiency potato genetic transformation system by researching the influence of different transformation conditions and influencing factors such as the pre-culture of the explants, the concentration of bacterial liquid, the infection time, the co-culture and the like on the genetic transformation of the potato (Zhang Xiying and 2019). The millet mature embryo is used AS a receptor material, a millet regeneration and transformation system is optimized in terms of plant hormone concentration, anti-browning agent, agrobacterium tumefaciens bacteria liquid concentration, infection time, acetosyringone (AS) concentration and the like, and a set of millet genetic transformation system (Li Yanfang and 2019) is initially established by Li Yanfang. At present, two stable genetic transformation systems are established for raspberries, wherein the system 1 is used for directly differentiating cluster buds of leaves, the system 2 is used for dedifferentiating the leaves to form callus tissues and then differentiating the callus tissues into buds, and compared with the system 1, the system 1 has higher transformation efficiency, and has important significance for researching biological exploration and improved breeding research in aspects of gene functions, character formation mechanisms and the like (Liu Huan et al 2018). Cui Yangong and other researchers take wild jujube leaves as receptors, an agrobacterium-mediated method is adopted to introduce SPDS genes into wild jujube genome, and an agrobacterium tumefaciens-mediated wild jujube leaf efficient genetic transformation system is successfully established through optimization, so that a foundation is provided for jujube genetic engineering breeding and germplasm innovation (Cui Yangong and other 2018).
Because of the particularity of woody plants, the genetic transformation system of woody plants, especially arbor, is very difficult to study, agrobacterium-mediated transformation is adopted at present according to the plant type and the growth characteristics of the woody plants, a plurality of influencing factors and problems exist in the transformation process, and although many researchers have conducted researches on the genetic transformation system of acer truncatum, a truly effective genetic transformation system of acer truncatum is not established, such as the past research on transforming acer truncatum somatic cells into embryogenic callus, and the result is obtained: the browning/death rate of the callus is higher, the induction survival rate is lower, so far, no perfect genetic transformation system is established for the acer truncatum, which greatly limits the development of gene function research related to the excellent properties of the acer truncatum and also prevents the progress of molecular improvement breeding of the acer truncatum. Based on the problems, the invention provides a genetic transformation method and application of Acer truncatum.
Furthermore, there are differences in one aspect due to understanding to those skilled in the art; on the other hand, since the applicant has studied a lot of documents and patents while making the present invention, the text is not limited to details and contents of all but it is by no means the present invention does not have these prior art features, but the present invention has all the prior art features, and the applicant remains in the background art to which the right of the related prior art is added.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a bacteriostatic culture medium for genetic transformation of Acer truncatum, which comprises the following components: WPM, sucrose, agar, acid hydrolyzed casein, and hormone composition, wherein,
each liter of medium contains the hormone composition:
0.2-0.5 mg kinetin, 1.0mg thidiazuron, 0.5mg indolebutyric acid and 0.1mg gibberellin.
Preferably, the bacteriostatic medium further comprises carbenicillin and cephalosporin.
Preferably, the bacteriostatic medium comprises 200mg of carbenicillin per liter.
Preferably, the bacteriostatic medium comprises 200mg of cephalosporin per liter.
The invention provides a genetic transformation method of Acer truncatum, which comprises the following steps:
sterilizing Acer truncatum explant, and culturing to obtain embryogenic callus;
carrying out co-culture, antibacterial culture and resistance screening on the embryogenic callus after infection by using recombinant agrobacterium;
the bacteriostasis culture medium for bacteriostasis culture comprises: WPM, sucrose, agar, acid hydrolyzed casein, and hormone composition, wherein,
each liter of medium contains the hormone composition:
0.2-0.5 mg kinetin, 1.0mg thidiazuron, 0.5mg indolebutyric acid and 0.1mg gibberellin.
Preferably, the bacteriostatic medium further comprises carbenicillin and cephalosporin.
Preferably, the bacteriostatic medium comprises 200mg of carbenicillin per liter.
Preferably, the bacteriostatic medium comprises 200mg of cephalosporin per liter.
Preferably, the screening medium of the resistance screening comprises: WPM basal medium composed of WPM, sucrose, agar and acid hydrolyzed casein, wherein,
the screening medium also contained per liter:
0.2mg of kinetin, 1.0mg of thidiazuron, 0.5mg of indolebutyric acid, 0.1mg of gibberellin, 300mg of carbenicillin, 200mg of cephalosporin and 10-25 mg of hygromycin.
The invention also provides a genetic transformation method of Acer truncatum, which comprises the following steps:
obtaining stem segments of acer truncatum aseptic seedlings, inoculating the stem segments into a callus induction culture medium for culture to obtain primary callus;
selecting healthy primary callus, inoculating the primary callus to an embryogenic callus culture medium for culture to obtain embryogenic callus;
selecting the embryogenic callus with vigorous growth activity, inoculating the embryogenic callus into a proliferation culture medium, and inducing proliferation of the embryogenic callus;
carrying out co-culture and antibacterial culture on the healthy embryogenic callus after infection by using recombinant agrobacterium;
inoculating the embryogenic callus subjected to bacteriostasis culture into a screening culture medium for culture to obtain a resistant callus;
the culture medium for bacteriostasis culture comprises: WPM, sucrose, agar, acid hydrolyzed casein, and hormone composition, wherein,
the hormone composition comprises:
kinetin, thidiazuron, indolebutyric acid, gibberellin.
Preferably, the hormone composition consists of the following components in mass concentration:
0.2-0.5 mg/L kinetin, 1.0mg/L thidiazuron, 0.5mg/L indolebutyric acid, 0.1mg/L Gibberellin (GA) 3 )。
Preferably, the culture medium for bacteriostatic culture also comprises carbenicillin and cephalosporin.
According to a preferred embodiment, the bacteriostatic medium is: WPM+30g/L sucrose+8 g/L agar+0.5 g/L acid hydrolyzed casein+0.2 mg/L Kinetin (KT) +1.0mg/L Thidiazuron (TDZ) +0.5mg/L indolebutyric acid (IBA) +0.1mg/L Gibberellin (GA) 3 ) +200mg/L carbenicillin+200 mg/L cephalosporin.
Preferably, the method of infestation is as follows:
and (3) placing the embryogenic callus into a bacterial liquid containing the recombinant agrobacterium for infection.
Preferably, the recombinant agrobacterium is obtained by introducing agrobacterium from a recombinant expression vector comprising a gene of interest.
Preferably, the recombinant expression vector is competent to transform agrobacterium GV3101 by freeze-thawing.
Preferably, the embryogenic callus inoculated into the propagation medium is green granular.
Preferably, the embryogenic callus inoculated in the proliferation medium is embryogenic callus that is induced for 2 weeks.
The invention also provides a screening culture medium for screening the resistant callus of Acer truncatum, which is characterized by comprising the following components in percentage by weight: WPM, sucrose, agar, acid hydrolyzed casein, and hormone composition, wherein,
the hormone composition comprises the following components in mass concentration:
0.2mg/L kinetin, 1.0mg/L thidiazuron, 0.5mg/L indolebutyric acid, 0.1mg/L gibberellin, 300mg/L carbenicillin, 200mg/L cephalosporin, 10-25 mg/L hygromycin.
According to a preferred embodiment, the screening medium is WPM+30g/L sugarcaneSugar+8 g/L agar+0.5 g/L acid hydrolyzed casein+0.2 mg/L KT+1.0mg/L TDZ+0.5mg/L IBA+0.1mg/L GA 3 +300mg/L carbenicillin+200 mg/L cephalosporin+25 mg/L hygromycin.
The invention provides an antibacterial culture medium for genetic transformation of acer truncatum, an acer truncatum genetic transformation method or application of screening culture for screening of resistant callus of acer truncatum based on woody plant gene function research and genetic engineering breeding.
Drawings
FIG. 1 shows the culture of the acer truncatum aseptic seedling provided by the invention;
FIG. 2 is a process of a first infection test of Agrobacterium tumefaciens to Acer truncatum stem infection test provided by the present invention;
FIG. 3 shows the growth state of the agrobacterium tumefaciens provided by the invention after the antibacterial medium is replaced in a first infection test of the stem section infection test of Acer truncatum;
FIG. 4 is a second infection test procedure of the infection test of Acer truncatum on the stem segment by the Agrobacterium tumefaciens provided by the present invention;
FIG. 5 is a process of a first infection test of the infection test of Acer truncatum callus by the Agrobacterium tumefaciens provided by the invention;
FIG. 6 is a second infection test procedure of the infection test of Acer truncatum callus by Agrobacterium tumefaciens provided by the present invention;
FIG. 7 is a process of preculture of embryogenic callus of a third infection test of Agrobacterium tumefaciens to Acer truncatum callus provided by the present invention;
FIG. 8 is a process of co-culturing embryogenic callus of a third batch of infection test of Acer truncatum callus infection test by Agrobacterium tumefaciens provided by the invention;
FIG. 9 is a result of antibacterial culture of embryogenic callus of a third batch of infection test of Acer truncatum callus infection test by Agrobacterium tumefaciens provided by the invention;
FIG. 10 shows the result of screening culture of embryogenic callus resistance in the third infection test of Agrobacterium tumefaciens to Acer truncatum callus infection test provided by the present invention;
FIG. 11 is a graph showing statistics of browning rate, bacteriostasis rate and survival rate after bacteriostasis culture of three infection tests provided by the invention;
FIG. 12 is a screen for working concentration of hygromycin as provided by the invention;
FIG. 13 is a graph showing the statistical result of the survival rate of calli at different hygromycin concentrations provided by the invention;
FIG. 14 is an RNA electrophoretogram of uninfected and transgenic calli provided by the invention;
FIG. 15 is an Actin internal standard electrophoretogram of uninfected and transgenic calli provided by the invention;
FIG. 16 is a diagram of RT-PCR electrophoresis of uninfected calli and transgenic calli provided by the present invention.
Detailed Description
The following detailed description refers to the accompanying drawings.
According to the invention, the 'Lihong' Acer Truncatum Bunge is taken as a research object, the culture medium and the culture condition are systematically optimized and the Agrobacterium tumefaciens infection receptor material is adjusted to establish an Acer Truncatum Bunge genetic transformation system from the start of the induction of the explant, and a carrier is provided for the functional verification of leaf color associated candidate genes, so that a material foundation is laid for the research of the leaf color molecular regulation and control mechanism of the Acer Truncatum Bunge, and the preparation is also made for the improvement breeding of the later-stage Acer Truncatum Bunge molecules, so that the research has important significance.
Examples
1 materials and methods
1.1 materials
1.1.1 preparation of Acer Truncatum Bunge aseptic seedlings
Selecting full Acer truncatum seeds, removing fruit wings, and air drying under natural light. The seeds are put into a beaker, two drops of the detergent are dripped into the beaker to be washed for more than 3 times, and then the seeds are repeatedly washed and soaked for 4 hours by tap water, and water is changed for a plurality of times at random. Pouring the cleaned seeds into a sterile culture bottle, and adding sterile water for cleaning for 3min; sterilizing with 75% disinfectant for 1min, and cleaning with sterile water for 2 times and 1 time for 3min; finally, adding 20% sodium hypochlorite solution for rinsing for 10min, and cleaning with sterile water for 3 times and 1 time for 3min, and continuously shaking the culture flask to make shoe-shaped gold ingotThe maple seeds are fully sterilized; repeating the sterilization process of the sodium hypochlorite solution once; finally, the seeds are soaked in sterile water for 10min. The treated seeds were blotted with sterile filter paper to remove epidermal moisture and inoculated on MS minimal medium (1/2MS+3% sucrose+0.5 g/L hydrolyzed casein+7 g/L agar+0.1 mg/L GA) 3 Ph 5.8). Dark culturing at 15 deg.c for 1 week, and transferring the germinated seed to culture room for culturing. The seeds generally germinate for about 1 week, and 3-5 weeks after germination form seedlings with the length of 5-7 cm, as shown in figure 1.
1.1.2 basal Medium, hormone and culture conditions
The basal medium used in this experiment was 1/2MS (Table 1) and WPM (Table 2). After preheating an electronic balance, MS (or WPM) powder, sucrose and acid hydrolyzed casein are sequentially weighed into a clean beaker and ddH is added 2 0, stirring the mixed solution by using a magnetic stirrer, and adjusting the pH value of the solution to 5.8 by using 1mol/L NaOH after the medicine is completely dissolved. Finally, adding the agar powder, and then placing the mixture into an autoclave to be autoclaved for 20min at the high temperature of 121 ℃. Cooling to room temperature, adding corresponding plant hormone, shaking carefully, and pouring into square culture dish or sterile culture bottle.
Table 1 1/2MS basal Medium configuration Table
TABLE 2 WPM basal Medium configuration Table
The auxin used in this experiment was: IBA, NAA and 2,4-D; cytokinin: 6-BA, TDZ and KT; at the same time use GA 3 . And dissolving each hormone according to the dissolving characteristics, filtering, sterilizing, and finally sub-packaging and freezing for later use. The temperature of the culture room is (24+/-2) DEG C, the light/dark period is 16/8h, and the illumination intensity is about 5000 Lux.
1.1.3 Strain and plasmid
Strains: coli FastT1 competence was purchased from Norwegian corporation; agrobacterium tumefaciens GV3101 is competent available from the Alternaria company. Plasmid: TOPO cloning plasmid vector pCE2-TA/Blunt-Zero was purchased from Novain; the plant overexpression vector pCAMBIA1305.1 was stored in the laboratory.
1.1.4 Agrobacterium tumefaciens preparation and transformation
The competent Agrobacterium tumefaciens GV3101 and plasmid pCAMBIA1305-GST894-GFP were taken and placed on ice to complete the plasmid transformation procedure according to the instructions. And (3) taking single bacterial colonies which grow well on an LB solid medium containing antibiotics, carrying out colony PCR verification, picking 2-3 bacterial plaques after verification is correct, placing the bacterial plaques in 20ml of LB liquid medium containing antibiotics, shaking the bacterial plaques at 28 ℃ for 48 hours, and using the shaking bacterial liquid for transforming Acer truncatum stems or embryogenic calli. Preferably, the antibiotics are kanamycin and rifampicin.
1.2 method
1.2.1 Induction and proliferation of Acer Truncatum Bunge embryogenic callus
The stem segment of the aseptic seedling of Acer truncatum Bunge is inoculated on a 1/2MS basic culture medium containing 2.0 mg/L2, 4-D, 0.3 mg/L6-BA and 0.5mg/L NAA to induce callus. And inoculating the healthy callus on the embryogenic callus induction medium: 1/2MS+3.0 mg/L6-BA+2.0 mg/L TDZ+0.5mg/L IBA+0.1mg/L GA 3 Embryogenic callus was induced.
Selecting green granular embryogenic callus with vigorous activity induced for 2 weeks on WPM base culture medium, cutting into 0.Scm 3 The left and right small blocks are inoculated on a proliferation culture medium, and are cultivated in a culture room under illumination to induce proliferation of embryogenic callus. The formula of the culture medium is as follows: WPM+0.5g/L acid hydrolyzed casein+1.0 mg/L TDZ+0.5mg/L IBA+0.1mg/L GA 3 +3mg/L6-BA+1.0mg/L KT++30g/L sucrose+8 g/L agar. And (3) through repeated multiplication culture, selecting embryogenic callus with healthy growth and good state as a receptor material for an agrobacterium tumefaciens infection test.
1.2.2DNA extraction, clone primer design and PCR reaction system
Total RNA of Acer truncatum leaves was extracted using a kit (Tiangen Biochemical technology Co., ltd.). And taking total RNA with good integrity, and removing the contained Acer truncatum DNA by RNase-Free DNase treatment. cDNA was synthesized using a kit (Novain Co.), and the whole procedure was strictly followed according to the product instructions.
Based on the known genomic data of 'Lihong' Acer Truncatum Bunge, the Primer premier 6.0 software was used to design the upstream and downstream primers for amplifying the Open Reading Frame (ORF) of the cDNA fragment of interest, GST894-F:5'-GGATCCATGGCAGGCATCAAAAT-3', GST894-R:5'-CTCGAGCTTCTTGCTTTGCAAAG-3' and amplifying the gene sequence by using the obtained Acer Truncatum Bunge leaf cDNA as template and using Norpraise Hi-Fi enzyme. RT-PCR amplification System: high fidelity enzyme 2X Phanta Max Master Mix (Dye Plus) 25. Mu.L, 2. Mu.L each of the upstream and downstream primers, 1ng of cDNA template, ddH 2 0 was added to the total system at 50. Mu.L. RT-PCR reaction procedure: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing for 30s, extension at 72 ℃ for 1min, 30 cycles altogether, and final extension at 72 ℃ for 5min; preserving at-20 ℃. The PCR amplification product was separated by agarose gel electrophoresis, and then a target band was selected, recovered using a kit (Tiangen Biochemical technology Co., ltd.) and the ligation vector pCE2-TA/Blunt-Zero (Noruzan Co.), was transformed into competent E.coli Fast-T1 (Noruzan Co.). And (3) performing PCR identification by using bacterial liquid to obtain positive clones, selecting positive clones which are correctly connected through PCR verification, and sending the positive clones to Beijing Rui Biotechnology limited company for sequencing. After the sequencing is correct, the plasmid is extracted by shaking, and the pCE2-TA/Blunt-Zero and pCAMBIA1305.1 vector sequences are respectively digested by double enzymes according to the pre-designed digestion sites. And separating the enzyme fragments, recovering the target gene and the vector, and connecting the target gene with the pCAMBIA1305.1 vector by using T4 DNA ligase to successfully construct the pCAMBIA1305.1-GST894 expression vector.
Adding T4 DNA ligase into the recovered target fragment and linearization vector, placing in a PCR instrument, setting the temperature of the PCR instrument to 25 ℃, connecting for 0.5h, transforming competent cells of the escherichia coli, carrying out positive verification, transforming the recombinant plasmid without error into competent agrobacterium GV3101 by a freeze thawing method, and preserving bacterial liquid at the temperature of-80 ℃ for later use.
1.2.3 infection of Acer truncatum Bunge by Agrobacterium tumefaciens
(1) Pre-culturing: placing young stem segments (about 1cm cut) of aseptic Acer Truncatum Bunge seedlings in the preparation methodOn the medium, the culture was pre-incubated for 1 day under dark conditions. The specific culture medium is as follows: (1) WPM+30g/L sucrose+8 g/L agar+0.5 g/L acid hydrolyzed casein+0.2 mg/L KT+1.0mg/L TDZ+0.5mg/L IBA+0.1mg/L GA 3 The method comprises the steps of carrying out a first treatment on the surface of the (2) 1/2MS+30g/L sucrose+8 g/L agar+0.5 g/L acid hydrolyzed casein+2 mg/L2, 4-D+0.3 mg/L6-BA+0.5 mg/L NAA.
(2) Shaking: a small piece of agrobacterium plaque of the transformed target plasmid is picked up and put into 20ml of liquid LB containing antibiotics, the bacteria are shaken at a temperature of 28 ℃ and at a speed of 200rpm, and the bacteria liquid is taken out when the bacteria liquid is shaken until the OD600 = 0.6-0.8.
(3) And (3) bacterial collection: the bacteria were collected at 4000rpm for 10min in a centrifuge tube, and the supernatant was discarded.
(4) And (3) bacterial washing: first batch: WPM liquid medium; second batch: the cells were resuspended in 1/2MS liquid medium, centrifuged at 4000rpm for 10min, the supernatant discarded and repeated 3 times.
(5) And (5) resuspension: after re-suspending the cells with ((1) WPM; (2)1/2 MS) liquid medium to OD600 = 0.6-0.8, 100. Mu. Mol/L Acetosyringone (AS) and 0.025% Silwet-77 were added, shaken well and allowed to stand.
(6) Infection: putting Acer truncatum stem segments and the re-suspension bacteria liquid prepared in the previous step into a sterile wide-mouth bottle, and shaking the bottle for 10min at the temperature of 28 ℃ and at the speed of 100 rpm.
(7) Co-cultivation: the residual bacterial liquid on the stem segment is sucked by the sterile filter paper and then placed in embryogenic callus induction medium added with 100 mu mol/L AS, and the culture is performed in dark at 25 ℃ for 2 days. The specific culture medium is as follows: (1) WPM+30g/L sucrose+8 g/L agar+0.2 mg/L KT+1.0mg/L TDZ+0.5mg/L IBA+0.1mg/L GA 3 +100. Mu. Mol/L AS; (2) 1/2MS+30g/L sucrose+8 g/L agar+2 mg/L2, 4-D+0.3 mg/L6-BA+0.5 mg/L NAA+100. Mu. Mol/L AS.
(8) Stem segment cleaning: washing with sterile water for 2min, washing with ((1) WPM; (2)1/2 MS) liquid medium +200mg/L carbenicillin +400mg/L cephalosporin for 3 times, and washing with sterile water.
(9) And (3) bacteriostasis culture: the stem segments are sucked by the sterile filter paper and placed in a bacteriostatic culture medium added with 200mg/L of carbenicillin and 200mg/L of cephalosporin for illumination culture, and the culture days depend on the growth condition of the stem segments. The specific culture medium formula is as follows: (1) WPM+30g/L sucrose+8 g/L agar+0.5g/L acid hydrolyzed casein+0.2 mg/L KT+1.0mg/L TDZ+0.5mg/L IBA+0.1mg/L GA 3 +200mg/L carbenicillin+200 mg/L cephalosporin; (2) 1/2MS+30g/L sucrose+8 g/L agar+0.5 g/L acid hydrolyzed casein+2 mg/L2, 4-D+0.3 mg/L6-BA+0.5 mg/L NAA+200mg/L carbenicillin+200 mg/L Cephalosporium.
(10) Resistance screening: culture medium (WPM+30 g/L sucrose+8 g/L agar+0.5 g/L acid hydrolyzed casein+3 mg/L6-BA+1.0mg/L TDZ+0.5mg/L IBA+0.1mg/L GA) 3 ) 300mg/L of carbenicillin, 200mg/L of cephalosporin and hygromycin are added to screen positive stem segments, wherein the hygromycin is subjected to experiments by setting four concentration gradients, and the survival rate is counted after 7 days of culture.
( In this section, (1) represents a first batch of infestation tests; (2) represent a second batch of infestation tests )
1.2.4 infection of Agrobacterium tumefaciens with Acer Truncatum Bunge embryogenic callus
(1) Pre-culturing: cutting the embryogenic callus with vigorous growth ((1) 0.5 cm) 3 ；②0.5cm 3 ；③1cm 3 ) Is placed on a preculture medium with or without hormone added and precultured in the dark ((1)2 days); (2) for 2 days; (3) 1 day). The formula of the culture medium comprises: (1) WPM+0.1mol/L mannitol+8 g/L agar+30 g/L sucrose; (2) WPM+30g/L sucrose+8 g/L agar+0.5 g/L acid hydrolyzed casein+0.5 mg/L KT+2 mg/L6-BA+1.0mg/L TDZ+0.5mg/L IBA+0.1mg/L GA 3 The method comprises the steps of carrying out a first treatment on the surface of the (3) WPM+30g/L sucrose+8 g/L agar+0.5 g/L acid hydrolyzed casein+0.2 mg/L KT+1.0mg/L TDZ+0.5mg/L IBA+0.1mg/L GA 3 。
(2) Shaking: a small piece of agrobacterium plaque of the transformed target plasmid is picked up and put into 20ml of liquid LB containing antibiotics, the bacteria are shaken at a temperature of 28 ℃ and at a speed of 200rpm, and the bacteria liquid is taken out when the bacteria liquid is shaken until the OD600 = 0.6-0.8.
(3) And (3) bacterial collection: the bacteria were collected at 4000rpm for 10min in a centrifuge tube, and the supernatant was discarded.
(4) And (3) bacterial washing: bacteria were resuspended in ((1)1/2 MS), (2) WPM), (3) WPM liquid medium, centrifuged at 4000rpm for 10min, the supernatant discarded and repeated 3 times.
(5) And (5) resuspension: after the cells were resuspended in liquid medium ((1)1/2 MS), (2) WPM), (3) WPM) to OD600 = 0.6-0.8, 100. Mu. Mol/L Acetosyringone (AS) and 0.025% Silwet-77 were added, shaken well and allowed to stand.
(6) Infection: placing Acer truncatum embryo callus and the bacterial solution prepared in the last step into a sterile wide-mouth bottle, and shaking the bottle for 10min at 28 ℃ and 100 rpm.
(7) Co-cultivation: the residual bacterial liquid on the embryogenic callus was blotted by sterile filter paper and placed in embryogenic callus induction medium supplemented with 100. Mu. Mol/L AS, and dark cultured at 25℃for ((1) 60h; 2) 48h (3) 48 h). The specific culture medium formula is as follows: (1) WPM+30g/L sucrose+8 g/L agar+3 mg/L6-BA+1.0mg/L TDZ+0.5mg/L IBA+0.1mg/L GA 3 +100. Mu. Mol/L AS; (2) WPM+30g/L sucrose+8 g/L agar+0.5 mg/L KT+2 mg/L6-BA+1.0 mg/LTDZ+0.5mg/L IBA+0.1mg/L GA 3 +100. Mu. Mol/L AS; (3) WPM+30g/L sucrose+8 g/L agar+0.2 mg/L KT+1.0mg/L TDZ+0.5mg/L IBA+0.1mg/LGA 3 +100μmol/L AS。
(8) Embryogenic callus washing: washing with sterile water for 2min, washing with ((1)1/2 MS), (2) WPM, (3) WPM) liquid medium +200mg/L carbenicillin +400mg/L cephalosporin for 3 times, and finally washing with sterile water.
(9) And (3) bacteriostasis culture: the embryogenic callus was blotted dry with sterile filter paper and placed in embryogenic callus induction medium supplemented with 200mg/L carbenicillin and 200mg/L cephalosporin for light culture, the number of days of culture depending on the proliferation rate and browning rate of embryogenic callus. The specific culture medium formula is as follows: (1) WPM+30g/L sucrose+8 g/L agar+0.5 g/L acid hydrolyzed casein+3 mg/L6-BA+1.0 mg/LTDZ+0.5mg/L IBA+0.1mg/L GA 3 +200mg/L carbenicillin+200 mg/L cephalosporin; (2) WPM+30g/L sucrose+8 g/L agar+0.5 g/L acid hydrolyzed casein+0.5 mg/L KT+2 mg/L6-BA+1.0mg/L TDZ+0.5mg/L IBA+0.1mg/L GA 3 +200mg/L carbenicillin+200 mg/L cephalosporin; (3) WPM+30g/L sucrose+8 g/L agar+0.5 g/L acid hydrolyzed casein+0.2 mg/L KT+1.0mg/L TDZ+0.5mg/L IBA+0.1mg/L GA 3 +200mg/L carbenicillin+200 ma/L cephalosporin.
(10) Resistance screening: culture medium (WPM+30 g/L sucrose+8 g/L agar+0.5 g/L acid hydrolyzed casein+3 mg/L6-BA+1.0mg/L TDZ+0.5mg/L IBA+0.1mg/L GA) 3 ) Adding 300mg/L carbenicillin, 200mg/L cephalosporin and hygromycin to screen positive callus, wherein hygromycin is arrangedFour concentration gradients were tested and survival was counted after 7 days of incubation.
( In this section, (1) represents a first batch of infestation tests; (2) representing a second set of infestation tests; (3) represent a third batch of infestation tests )
1.2.4 identification of transgenic Positive calli
Selecting embryogenic callus which is not browned and has good growth condition after resistance screening, placing the embryogenic callus on an ultra-clean bench, cutting half of the embryogenic callus by forceps and a surgical knife, placing the embryogenic callus in a tinfoil bag, quickly placing the embryogenic callus in liquid nitrogen for quick freezing, and then placing the embryogenic callus in a refrigerator at the temperature of minus 80 ℃ for storage so as to facilitate the subsequent RNA extraction and use. The remaining half of embryogenic callus was still placed on secondary medium for continued culture while uninfected embryogenic callus was cut as a control, and three samples were taken for each of the infested and uninfected samples. Firstly, sterilizing the needed tools such as a mortar, a grinding rod and the like at high temperature, airing, adding the materials into liquid nitrogen, taking the materials out of a refrigerator at the temperature of-80 ℃ and rapidly putting the materials into the liquid nitrogen. Grinding the embryogenic callus into powder in a mortar by using a grinding rod, and rapidly filling into a centrifuge tube for standby. Total RNA extraction was performed simultaneously on infected and uninfected embryogenic calli with reference to the day root total RNA extraction kit instructions. The procedure was performed with reference to the Northey cDNA reverse transcription kit. The Acinetobacter Truncatum Actin gene is used as an internal reference, an upstream primer and a downstream primer are designed, and the addition amount of cDNA is adjusted according to the brightness of an action band on a gel chart until the brightness of amplified actions is consistent. To further identify whether embryogenic callus is transferred into target gene, specific GFP tag genes in target gene GST894 and over-expression vector pCAMBIA1305 are selected, and upstream and downstream primers (see Table 3) are designed according to the gene sequence, and gene sequence amplification is carried out by using infected and uninfected embryogenic callus cDNA as a template and using fast Taq enzyme.
TABLE 3 RT-primer Table for PCR
2 results and analysis
2.1 infection of Acetobacter tumefaciens with Acetobacter acer
2.1.1 infection of Acer truncatum Bunge by Agrobacterium tumefaciens
The gene over-expression vector pCAMBIA1305-GST894-GFP is transferred into agrobacterium GV3101 to directly infect the stem segment of Acer truncatum, and the growth state needs to be observed at any time when the plant is in the antibacterial culture stage.
In the first test result, the stem segments are cultivated in a bacteriostatic culture medium after being infected, and bacteriostatic cultivation is selected in a callus proliferation culture medium added with 200mg/L of carbenicillin and 200mg/L of cephalosporin, as shown in fig. 2, wherein fig. 2a is a stem segment of Acer truncatum that is pre-cultivated for 0 day, and fig. 2b is a stem segment of Acer truncatum that is pre-cultivated for 1 day; FIG. 2c is a Acer truncatum stem segment co-cultivated for 0 day, and FIG. 2d is a Acer truncatum stem segment co-cultivated for 2 days; FIG. 2e shows a Acer truncatum stem section cultivated for 0 day with bacteriostasis, FIG. 2f shows a Acer truncatum stem section cultivated for 7 days with bacteriostasis, FIG. 2g shows a Acer truncatum stem section cultivated for 14 days with bacteriostasis, and FIG. 2h shows a Acer truncatum stem section cultivated for 21 days with bacteriostasis. The antibacterial effect is still good, no pollution is caused, but the stem section does not induce callus, and the callus is gradually browned.
In the later stage, the stem segments in the bacteriostasis stage are transferred into a callus induction culture medium, and the state is improved, as shown in fig. 3, fig. 3a is the stem segments of the acer truncatum after the culture medium is replaced in the first infection experiment, fig. 3b is the stem segments of the acer truncatum after the culture medium is replaced in the first infection experiment and cultured for 2 days, the stem segments expand after 2 days, callus is generated at the cut parts at the two ends of each stem segment, large-area browning does not continuously occur, and the whole growth condition is good.
In the second test result, the stem segments are selected to be directly placed on a callus induction medium in the bacteriostasis stage, as shown in fig. 4, wherein fig. 4a is a stem segment of Acer truncatum which is pre-cultured for 0 day, and fig. 4b is a stem segment of Acer truncatum which is pre-cultured for 1 day; FIG. 4c shows the Acer truncatum stem segment co-cultivated for 0 day, and FIG. 4d shows the Acer truncatum stem segment co-cultivated for 2 days; fig. 4e shows the acer truncatum stem section after 0 day of antibacterial culture, fig. 4f shows the acer truncatum stem section after 3 days of antibacterial culture, fig. 4g shows the acer truncatum stem section after 6 days of antibacterial culture, fig. 4h shows the acer truncatum stem section after 9 days of antibacterial culture, and the acer truncatum stem section has obvious expansion, light green color and good overall growth. Callus appears at the incision, and the browning phenomenon at the current stage is less, and the subsequent continuous observation is needed.
2.1.2 infection of Acer truncatum callus by Agrobacterium tumefaciens
The gene overexpression vector pCAMBIA1305-GST894-GFP is transferred into agrobacterium GV3101 to directly infect the embryogenic callus of Acer truncatum, the growth condition of the embryogenic callus needs to be observed every day during bacteriostasis culture, and the embryogenic callus with serious pollution needs to be transferred in time and the bacteriostasis efficiency is counted. The transformed embryogenic calli were screened for resistance after bacteriostasis culture and the optimum screening concentration was determined by testing hygromycin concentration gradients.
In the first test results, as shown in FIG. 5, FIG. 5a is embryogenic callus inoculated by preculture, FIG. 5b is embryogenic callus by preculture for 2 days, FIG. 5c is embryogenic callus by co-culture, FIG. 5d is embryogenic callus by co-culture for 60 hours, FIG. 5e is embryogenic callus by antibacterial culture, FIG. 5f is embryogenic callus by antibacterial culture for 1 day, FIG. 5g is embryogenic callus by antibacterial culture for 3 days, and FIG. 5h is embryogenic callus by antibacterial culture for 5 days. After dark culture treatment for 2 days, the embryogenic callus has slight browning on the surface, but the embryogenic callus has good overall state and keeps bright green or yellowish green color. After being infected by agrobacterium, the embryogenic callus has no obvious change in the process of inoculation, and after 60h of co-culture, the surface has obvious browning phenomenon, the activity is obviously reduced, which indicates that the growth of the embryogenic callus is greatly affected by the agrobacterium infection. After the embryogenic callus is degerming and then subjected to bacteriostasis culture, the agrobacterium gradually wraps the embryogenic callus from day 3 to day 5, and the embryogenic callus is completely browned and dead.
In the second test results, as shown in FIG. 6, FIG. 6a is embryogenic callus inoculated by preculture, FIG. 6b is embryogenic callus by preculture for 2 days, FIG. 6c is embryogenic callus by co-culture, FIG. 6d is embryogenic callus by co-culture for 2 days, FIG. 6e is embryogenic callus by antibacterial culture, FIG. 6f is embryogenic callus by antibacterial culture for 1 day, FIG. 6g is embryogenic callus by antibacterial culture for 3 days, and FIG. 6h is embryogenic callus by antibacterial culture for 5 days. After the second batch of embryogenic callus is infected by agrobacterium, the result is similar to the first batch, and after 2 days of dark culture treatment, the embryogenic callus has slight browning phenomenon on the surface, but the overall state is good, and the color still keeps bright green or yellowish green. After being infected by agrobacterium, the bacteria have no obvious change in the process of inoculation, and after 2 days of co-culture, the surface has obvious browning phenomenon and the activity is obviously reduced. After the embryogenic callus is degerming, bacteriostasis culture is carried out, and the embryogenic callus is gradually wrapped by agrobacterium from day 3 to day 5, so that the embryogenic callus is completely browned and dead.
In the third experimental result, as shown in FIG. 7, FIGS. 7a to c show embryogenic calli inoculated by preculture, and FIGS. 7e to g show embryogenic calli precultured for 1 day. After the embryogenic callus is subjected to the pre-culture treatment for 1 day, the surface of the embryogenic callus has slight browning phenomenon, but the embryogenic callus is in a good overall state, and the embryogenic callus still keeps bright green in color.
FIG. 8a shows embryogenic callus inoculated by co-culture, and FIG. 8b shows embryogenic callus co-cultured for 2 days. The embryogenic callus has good state before co-culture after infection, and after 2 days of co-culture treatment, the comparison of fig. 8a and 8b can show that the embryogenic callus has obvious browning phenomenon and still keeps activity.
As shown in FIG. 9, FIG. 9a shows embryogenic callus inoculated in bacteriostatic culture, FIG. 9b shows embryogenic callus cultured in bacteriostatic culture for 7 days, and FIG. 9c shows embryogenic callus cultured in bacteriostatic culture for 14 days. The embryogenic callus and agrobacterium are co-cultured and then subjected to degerming treatment, and inoculated on a bacteriostatic culture medium after degerming, and after 14 days of bacteriostatic culture, the embryogenic callus has a large area of browning phenomenon, but the surface of the embryogenic callus has fine white granular callus, and the embryogenic callus can still keep activity.
As shown in FIG. 10, FIG. 10a shows embryogenic callus inoculated by screening culture, the concentration of hygromycin in the screening medium is 20mg/L, FIG. 10b shows embryogenic callus by screening culture for 7 days, the concentration of hygromycin in the screening medium is 20mg/L, FIG. 10c shows embryogenic callus inoculated by screening culture, the concentration of hygromycin in the screening medium is 25mg/L, FIG. 10d shows embryogenic callus by screening culture for 7 days, and the concentration of hygromycin in the screening medium is 25mg/L. After the embryogenic callus is subjected to bacteriostasis culture and is cultured on a hygromycin resistance screening medium containing 20mg/L for 7 days, a small part of embryogenic callus is completely browned and dead, and the rest part of embryogenic callus is browned and still keeps activity, so that the embryogenic callus can be used for subsequent PCR identification. The remaining embryogenic calli remained viable after transfer to screening medium containing 25mg/L hygromycin for 7 days.
Statistical analysis is performed on the bacteriostasis condition after embryogenic callus infection, as shown in fig. 11, the left side of fig. 11 is the statistical result of the browning rate of the three infection tests, and the abscissas 1, 2 and 3 respectively represent the first infection test, the second infection test and the third infection test, and the ordinates represent the browning rate of the three infection tests. From the statistics, it is possible to: the browning rate of the first infection test is close to 70%, the browning rate of the second infection test is close to 60%, and the browning rate of the third infection test is close to 15% due to the change of the culture medium; the browning rates of the first infection test, the second infection test and the third infection test show a gradually decreasing trend, wherein the browning rate of the third infection test is obviously lower than that of the first infection test and the second infection test (P < 0.05).
In fig. 11, the statistical results of the bacteriostasis rates of the three infection tests are shown in the horizontal coordinates 1, 2 and 3, which respectively show the bacteriostasis rates of the first infection test, the second infection test and the third infection test, and the vertical coordinates show the bacteriostasis rates of the three infection tests. From the statistics, it is possible to: the bacteriostasis rate of the first infection test is close to 70%, the bacteriostasis rate of the second infection test is about 85%, and the bacteriostasis rate of the third infection test is about 90%; the bacteriostasis rate of the first infection test, the second infection test and the third infection test shows a gradually rising trend.
Fig. 11 shows the statistical results of the survival rate of the three infection tests, wherein the abscissa of 1, 2 and 3 represents the survival rate of the first infection test, the second infection test and the third infection test, respectively, and the ordinate represents the survival rate of the three infection tests. From the statistics, it is possible to: the survival rate of the first infection test and the second infection test is 0, and the survival rate of the third infection test reaches 90% or more than 90%. In the three-time infection test, the browning rate is gradually reduced due to the change of the culture medium, the browning rate in the third infection is obviously lower than that in the previous two tests (P is less than 0.05), the bacteriostasis rate is gradually increased, and the embryogenic callus differentiation culture medium is more beneficial to the growth of callus, so that the further infection of agrobacterium is resisted, and the bacteriostasis rate and the survival rate are increased.
Screening for hygromycin working concentrations as shown in FIG. 12, FIG. 12a shows embryogenic callus inoculated at 10mg/L hygromycin (Hyg), FIG. 12b shows embryogenic callus cultured at 10mg/L hygromycin, FIG. 12c shows embryogenic callus inoculated at 15mg/L hygromycin, FIG. 12d shows embryogenic callus cultured at 15mg/L hygromycin for 7 days, FIG. 12e shows embryogenic callus inoculated at 20mg/L hygromycin, FIG. 12f shows embryogenic callus cultured at 20mg/L hygromycin for 7 days, FIG. 12g shows embryogenic callus inoculated at 25mg/L hygromycin, and FIG. 12h shows embryogenic callus cultured at 25mg/L hygromycin for 7 days. The embryogenic callus has no obvious change after 7 days of growth on a culture medium containing 10mg/L hygromycin, and the embryogenic callus starts to brown and die along with the increase of the hygromycin concentration, which indicates that part of embryogenic callus blocks are not infected successfully and do not have the capacity of resisting hygromycin pressure. FIG. 13 is a graph showing the statistics of the survival rate of embryogenic calli at various hygromycin concentrations; the abscissa represents the hygromycin concentration, and the ordinate represents the survival rate of embryogenic callus at different hygromycin concentrations, wherein the hygromycin concentration is 10mg/L, 15mg/L, 20mg/L and 25mg/L. As can be seen from FIG. 13, one-way analysis of variance shows that the survival rate of calli on the medium containing 20mg/L and 25mg/L hygromycin is significantly lower than that of other treatments (P < 0.05) and still maintained above 60%, indicating that 25mg/L hygromycin is more suitable for screening transgenic embryogenic calli of Acer truncatum.
2.2 identification of transgenic Positive calli
Six calli which are not brown and well grown after transgene screening are selected as experimental materials, RNA is extracted by taking uninfected calli as a control, and the RNA extraction quality is detected by agarose gel electrophoresis, as shown in figure 14, 8 lanes are shared from left to right, the 1 st lane and the 2 nd lane are RNA of uninfected embryogenic calli CK 1 and CK2, and the 3 rd lane to the 8 th lane are RNA of 6 embryogenic calli after transgene screening, and according to the result, the method can be used for obtaining: RNA strips of the callus after transgene screening are bright and clear, and no diffusion phenomenon exists. And detecting the purity and concentration of the RNA by using an ultra-micro spectrophotometer, and keeping the RNA for later use after meeting the requirements.
And (3) taking cDNA obtained by reverse transcription of the extracted RNA as a template, carrying out gene cloning by using fast Taq enzyme according to a designed primer of Acinetobacter Actin gene, and regulating the template amplification usage amount for a plurality of times after the gel electrophoresis result shows that the length meets the expectations until the target bands of amplification of all samples are basically consistent, as shown in figure 15.
The same template amount is amplified by using an action, gene cloning is carried out by using fast Taq enzyme according to the designed GFP primer and GST894 primer, gel electrophoresis shows that 6 calli screened by hygromycin after transgenosis all contain GFP labels, and the transgenic GST894 is over-expressed, which indicates that the 6 calli are positive calli, as shown in figure 16.
Conclusion 3
Although the antibacterial state can be achieved after the stem section of Acer truncatum Bunge is infected by the agrobacterium tumefaciens, the stem section does not continuously induce callus or regenerate adventitious buds, so that the stem section is not suitable for direct agrobacterium infection.
The key point of infection of agrobacterium tumefaciens to embryogenic callus of Acer truncatum Bunge is bacteriostasis culture, and three experiments prove that the optimal bacteriostasis culture medium is WPM+30g/L sucrose+8g/L agar+0.5 g/L acid hydrolyzed casein+0.2 mg/L KT+1.0mg/L TDZ+0.5mg/L IBA+0.1mg/L GA 3 +200mg/L Carboxylic penicillin+200 mg/L Cephalosporium, followed by hydrolysis of casein+0.2 mg/L KT+1.0mg/L TDZ+0.5mg/L IBA+0.1mg/L GA in WPM+30g/L sucrose+8 g/L agar+0.5 g/L acid 3 Screening with a screening culture medium of +300mg/L carbenicillin +200mg/L cephalosporin +25mg/L hygromycin, wherein the obtained embryogenic callus has the lowest browning rate, pollution rate and death rate and keeps vigorous growth.
6 embryogenic calli which grow vigorously after hygromycin resistance screening are selected, RNA is extracted together with uninfected embryogenic calli, cDNA obtained through reverse transcription is used as a template, the target genes GFP and CST894 amplified by the corresponding primers are expressed through gel electrophoresis, and GST894 endogenous genes are expressed excessively, so that the 6 calli are positive calli.
Discussion 4
According to the invention, the stem segments and embryogenic callus of Acer truncatum are respectively infected by agrobacterium tumefaciens, wherein the two infected stem segments are in a bacteriostasis stage, the comparison shows that the stem segments are directly placed in a primary callus induction culture medium of Acer truncatum, the bacteriostasis effect is good, meanwhile, embryogenic callus continuously grows, the browning condition of embryogenic callus is continuously concerned, and the subsequent transgenic embryogenic callus screening is carried out when the embryogenic callus which is not browned and has good growth condition is grown. The embryogenic callus is used as an infection material, and the verification shows that the target gene is successfully transferred into the embryogenic callus, which means that the genetic transformation system of the embryogenic callus of Acer truncatum is partially completed, effective bacteriostasis can be realized in the infection process, and the proliferation can be continued although the embryogenic callus is partially browned. Therefore, in the future experiments, the key point is still to continuously verify and optimize the optimal condition for genetic transformation of the embryogenic callus of Acer truncatum, so that the problems of difficult germination of the embryogenic callus and the like are solved.
It should be noted that the above-described embodiments are exemplary, and that a person skilled in the art, in light of the present disclosure, may devise various solutions that fall within the scope of the present disclosure and fall within the scope of the present disclosure. It should be understood by those skilled in the art that the present description and drawings are illustrative and not limiting to the claims. The scope of the invention is defined by the claims and their equivalents. The description of the invention encompasses multiple inventive concepts, such as "preferably," "according to a preferred embodiment," or "optionally," all means that the corresponding paragraph discloses a separate concept, and that the applicant reserves the right to filed a divisional application according to each inventive concept. Throughout this document, the word "preferably" is used in a generic sense to mean only one alternative, and not to be construed as necessarily required, so that the applicant reserves the right to forego or delete the relevant preferred feature at any time.
Claims (10)
1. A bacteriostatic medium for genetic transformation of acer truncatum, said bacteriostatic medium comprising: WPM, sucrose, agar, acid hydrolyzed casein, and hormone composition, wherein,
each liter of medium contains the hormone composition:
0.2-0.5 mg kinetin, 1.0mg thidiazuron, 0.5mg indolebutyric acid and 0.1mg gibberellin.
2. The bacteriostatic medium according to claim 1, wherein said bacteriostatic medium further comprises carbenicillin and cephalosporin.
3. Bacteriostatic medium according to claim 1 or 2, characterized in that it comprises 200mg of carbenicillin per liter of bacteriostatic medium.
4. A bacteriostatic medium according to any one of claims 1 to 3, characterized in that it comprises 200mg of cephalosporin per liter of bacteriostatic medium.
5. A genetic transformation method of acer truncatum, comprising the following steps:
sterilizing Acer truncatum explant, and culturing to obtain embryogenic callus;
carrying out co-culture, antibacterial culture and resistance screening on the embryogenic callus after infection by using recombinant agrobacterium;
it is characterized in that the method comprises the steps of,
the bacteriostasis culture medium for bacteriostasis culture comprises: WPM, sucrose, agar, acid hydrolyzed casein, and hormone composition, wherein,
each liter of medium contains the hormone composition:
0.2-0.5 mg kinetin, 1.0mg thidiazuron, 0.5mg indolebutyric acid and 0.1mg gibberellin.
6. The method of claim 5, wherein the bacteriostatic medium further comprises carbenicillin and cephalosporin.
7. The method according to claim 5 or 6, characterized in that the bacteriostatic medium comprises 200mg of carbenicillin per liter.
8. The method according to any one of claims 5 to 7, wherein 200mg of cephalosporin per liter of bacteriostatic medium is contained.
9. The method according to any one of claims 5 to 8, wherein the screening medium of the resistance screening comprises: WPM basal medium composed of WPM, sucrose, agar and acid hydrolyzed casein, wherein,
the screening medium also contained per liter:
0.2mg of kinetin, 1.0mg of thidiazuron, 0.5mg of indolebutyric acid, 0.1mg of gibberellin, 300mg of carbenicillin, 200mg of cephalosporin and 10-25 mg of hygromycin.
10. The bacteriostatic culture medium according to claims 1-4, the genetic transformation method of acer truncatum according to claims 5-9 for the study of the gene function of woody plants and the application of genetic engineering breeding.
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