CN109182375B - Genetic transformation method of German iris - Google Patents
Genetic transformation method of German iris Download PDFInfo
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- CN109182375B CN109182375B CN201811199826.XA CN201811199826A CN109182375B CN 109182375 B CN109182375 B CN 109182375B CN 201811199826 A CN201811199826 A CN 201811199826A CN 109182375 B CN109182375 B CN 109182375B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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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- 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
Abstract
The invention discloses an agrobacterium tumefaciens-mediated genetic transformation method of iris germanica, and belongs to the technical field of plant genetic transformation. The method mainly takes stem tip callus of iris germanica as a transformation receptor, and obtains a transgenic strain through the processes of agrobacterium infection transformation, resistant callus screening, redifferentiation, PCR detection of a resistant plant and the like. The method provided by the invention has the advantages of simple operation and high genetic transformation rate, and has great theoretical significance and application and popularization value for genetic transformation, gene function research and new germplasm creation of German iris and other iris plants.
Description
Technical Field
The invention belongs to the technical field of construction of plant genetic transformation systems, and particularly relates to a genetic transformation method of German iris.
Background
The German Iris (Iris germanica L.) is perennial herb of Iridaceae, is a famous ornamental flower, has the characteristics of large flower, peculiar flower type, bright color, evergreen four seasons and the like besides the beard and hair with changeable color on the ribs in petals, and has huge development space and prospect in landscaping, pot culture and ground cover application. Many researches on hybridization breeding, tissue culture and rapid propagation, cut flower preservation and the like of the German iris are carried out at home and abroad, and the current research on genetic improvement of the German iris by utilizing the genetic engineering technology is only reported. Deeply researching the related characters and stress resistance molecular mechanisms of iris plants such as iris germanica and the like, excavating related genes and further cultivating new iris germanica varieties with excellent characters (flower development, stress resistance and the like) have important significance. The establishment of genetic transformation system of iris germanica is a key means for revealing the molecular mechanism of development and stress resistance of iris germanica, and can greatly promote the innovative cultivation process of new germplasm of iris plants such as iris germanica and the like.
Disclosure of Invention
Aiming at the defects of the traditional breeding technology, the invention aims to provide a genetic transformation method of the German iris which can be used in the molecular breeding process of the German iris.
In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:
a method for obtaining a transgenic plant by transforming German iris callus comprises the steps of induction and enrichment culture of German iris callus, pre-culture of callus, preparation of agrobacterium strain containing a plant expression vector, activation of agrobacterium liquid and preparation of agrobacterium infection liquid, agrobacterium infection and co-culture, agrobacterium bacteria removal and induction of adventitious bud regeneration, culture and rooting of adventitious buds, screening of antibiotic positive seedlings, transplantation of antibiotic positive seedlings, PCR identification and the like; the method comprises the following steps:
1. induction and proliferation of iris germanica callus: taking the stem tip part of the German iris, cleaning the stem tip part with a cleaning solution, and transferring the stem tip part to an ultra-clean workbench for disinfection. Then, the stem tip was cut into small pieces and placed on a callus induction medium, and the callus was induced under light irradiation and subcultured. The callus induction culture medium comprises: MS +1.5mg/L2, 4-D +0.2mg/L6-BA +30g/L sucrose +6g/L agar powder, and the pH value is 5.8.
2. Pre-culturing callus tissues: cutting the callus of 2-3 subcultures into small pieces of 2mm, placing on proliferation culture medium, dark culturing for 5-7 days; the formula of the proliferation culture medium is as follows: MS +1.0mg/L2, 4-D +0.1mg/L6-BA +30g/L sucrose +6g/L agar powder, and the pH value is 5.8.
3. Bacterial liquid activity of agrobacteriumPreparing a chemical and agrobacterium infection solution: streaking plasmid-containing Agrobacterium on YEB solid culture medium containing rifampicin, dark culturing at 28 deg.C for 3d, selecting single colony, inoculating into YEB liquid culture medium containing rifampicin, shake culturing at 28 + -2 deg.C in dark for 16h, centrifuging, collecting thallus, resuspending thallus with 1/2MS solution, adjusting to OD600Adding acetosyringone (200mg/L) with a value of 0.3-0.5 to obtain an agrobacterium infection solution; the agrobacterium strain used for infection is EHA105, and the agrobacterium strain contains a plant expression vector PBI 121;
4. agrobacterium infection callus: infecting the precultured iris germanica callus in an agrobacterium infection solution containing plasmids for 3min, then airing, then placing on a co-culture medium, and co-culturing under a dark condition; 5. removing bacteria of agrobacterium and screening resistant callus: cleaning the callus of the iris germanica co-cultured in the step 4 with sterile water, transferring the callus to a callus proliferation culture medium containing G418(100mg/L) and Tim (150mg/L), culturing for 3 weeks at the temperature of 25 +/-2 ℃ and the light cycle of 14/10h, and screening resistant callus;
6. the regeneration of adventitious buds induced by the resistant callus: transferring the newly proliferated resistant callus in the step 5 to an adventitious bud induction culture medium containing G418(150mg/L) and Tim (150mg/L) to induce the generation of adventitious buds, wherein the culture conditions are the same as those of the step 5, and differentiating the adventitious buds after culturing for 4 weeks; the adventitious bud induction culture medium comprises the following components in parts by weight: MS +1.0mg/L of 6-BA +0.2mg/LNAA +150mg/L G418+150mg/L of timentin +30g/L of cane sugar +6g/L of agar powder, and the pH value is 5.8.
7. And (3) rooting of resistant buds: when the adventitious bud of the German iris is grown to 3-4cm, the bud is cut and transferred to a rooting culture medium containing G418(150mg/L) and Tim (150mg/L), the culture conditions are the same as the step 6, and the bud is rooted after 3 weeks of culture; the rooting medium comprises the following components in parts by weight: MS +0.2mg/L NAA +150mg/L G418+150mg/L timentin +30g/L sucrose +6g/L agar powder, and the pH value is 5.8.
8. Transplanting of resistant seedlings and transgenic PCR identification: and taking out the well-grown resistant seedlings from the culture medium, transferring the well-grown resistant seedlings into nutrient soil, and culturing the well-grown resistant seedlings in a greenhouse. Shearing new leaves induced by the resistant callus of the iris germanica, extracting DNA, carrying out PCR detection by using a specific primer on a PBI121 carrier, and screening positive transgenic seedlings.
Compared with the prior art, the application has the following beneficial effects:
the invention provides a construction method of a genetic transformation system of an ornamental flower Iris germanica. The method takes iris germanica stem tip callus as a transformation receptor and carries out genetic transformation through agrobacterium mediation. The stem tip callus selected by the invention has the inductivity of 82%, strong differentiation and regeneration capacity, good infection effect of agrobacterium tumefaciens and 50% genetic transformation efficiency. The genetic transformation system of the iris germanica established by the invention can be used for genetic improvement of the iris germanica and innovative cultivation of new germplasm, and also provides theoretical and technical reference basis for genetic transformation, molecular improvement and the like of other iris plants.
According to the invention, two antibiotics (G418 and Tim) are adopted for screening and genome PCR identification, so that a positive transgenic plant is successfully obtained, and the positive rate is 50%.
Drawings
FIG. 1: iris germanica shoot tip callus;
FIG. 2: screening the German iris resistance callus;
FIG. 3: differentiation of german iris resistant calli;
FIG. 4: rooting the resistant plants;
FIG. 5: and (3) PCR identification of the transgenic plant.
M in fig. 5: DNA Marker, 1-4: resistance strains; 5: a plasmid positive control; 6 wild type strain negative control.
Detailed Description
The invention will now be further illustrated by reference to the following examples, which are included to facilitate a better understanding of the invention but are not intended to limit the invention thereto.
Example 1
The invention relates to a method for constructing a genetic transformation system of German iris, which comprises the following materials and reagents: iris germanica stem tip, agrobacterium tumefaciens (EHA105), plasmid (PBI 121); anhydrous ethanol, mercuric chloride, MS culture medium, 2, 4-dichlorophenoxyacetic acid (2, 4-D), alpha-naphthylacetic acid (NAA), 6-benzylaminopurine (6-BA), rifampicin (Rif), Acetosyringone (AS), geneticin (G418), timentin (Tim), sucrose and agar powder. The main equipment is as follows: illumination incubator, superclean bench, centrifuge, PCR amplification appearance.
The method comprises the following steps:
1. induction and proliferation of iris germanica callus: digging 2-3 years old German iris plant from nursery garden and bringing it back to laboratory, cutting off leaves and rootstocks, keeping distance about 1cm from top to bottom of stem tip, soaking in 10% detergent for 10min, then washing with running water for 1h, moving to super clean bench, disinfecting with 75% ethanol for 45s, washing with sterile water for 2 times, then disinfecting with 0.1% mercuric chloride for 10min, and washing with sterile water for 5 times. Subsequently, explants with shoot tips cut to 2X 2mm size were placed on callus induction medium after water uptake with sterile filter paper and cultured with light at 25. + -. 2 ℃. Wherein the callus induction culture medium comprises the following components in percentage by weight: MS +2, 4-D (1.5mg/L) +6-BA (0.2mg/L) + sucrose (30g/L) + agar powder (6g/L), pH 5.8; after 30 days of induction, the cells were transferred to a growth medium and cultured for growth at a subculture cycle of 30 days (FIG. 1).
2. Pre-culturing callus tissues: cutting the callus of 2-3 subcultures into small pieces of 2mm, placing on proliferation culture medium, and culturing at 25 + -2 deg.C in dark for 5-7 d; wherein the callus proliferation culture medium comprises the following components in percentage by weight: MS +2, 4-D (1.0mg/L) +6-BA (0.1mg/L) + sucrose (30g/L) + agar powder (6.0g/L), and adjusting pH to 5.8;
3. activating agrobacterium liquid and preparing agrobacterium infection liquid: marking out agrobacterium containing plasmid on YEB solid culture medium added with rifampicin (50mg/L), culturing in dark at 28 ℃ for 3d, picking out single colony, inoculating to YEB liquid culture medium containing rifampicin (50mg/L), and performing shake culture at 28 +/-2 ℃ and 200-; placing the cultured bacteria liquid in sterilized centrifuge tube, centrifuging at 6000rpm for 5min to collect thallus, resuspending thallus with 1/2MS solution, and adjusting to OD600Adding acetosyringone (200mg/L) with a value of 0.3-0.5 to obtain an agrobacterium infection solution; the agrobacterium strain used for infection was EHA105 (containing plant expression vector PBI 121).
4. Agrobacterium infection callus: selecting a yellow-white relatively dry German iris callus with a compact structure, infecting the callus in an agrobacterium infection solution for 3min, sucking the bacterium solution by using sterile filter paper, placing the bacterium solution on a co-culture medium, and culturing the bacterium solution for 3d at 25 ℃ under a dark condition; wherein, the formula of the co-culture medium is as follows: MS +2, 4-D (1.5mg/L) +6-BA (0.2mg/L) + sucrose (30g/L) + agar powder (6.0g/L), and adjusting pH to 5.8;
5. removing bacteria of agrobacterium and screening resistant callus: washing the callus of the iris germanica co-cultured in the step 4 with sterile water for 5 times, then sucking the callus with sterile filter paper, transferring the callus to a callus proliferation culture medium, and culturing for 3 weeks at the temperature of 25 +/-2 ℃ and the photoperiod of 14/10 h; the callus proliferation culture medium comprises the following components in percentage by weight: MS +2, 4-D (1.0mg/L) +6-BA (0.1mg/L) + sucrose (30G/L) + agar powder (6.0G/L), wherein antibiotic G418(100mg/L) and timentin Tim (150mg/L) are added, and the pH is adjusted to 5.8; as shown in FIG. 2, the resistant callus grows on the resistance selection medium for a certain period of time and new callus is grown from the resistant callus, while the non-resistant callus gradually browns and dies.
6. The regeneration of adventitious buds induced by the resistant callus: transferring the newly proliferated callus (white) in the step 5 to an adventitious bud induction culture medium under the same culture conditions, and after culturing for 4 weeks, differentiating adventitious buds (figure 3); the adventitious bud induction culture medium comprises the following components in parts by weight: MS + NAA (0.2mg/L) +6-BA (1.0mg/L) + sucrose (30G/L) + agar powder (6.0G/L), wherein antibiotics G418(150mg/L) and Tim (150mg/L) are added, and the pH is adjusted to 5.8;
7. and (3) rooting of resistant buds: when the adventitious bud of the German iris is grown to 3-4cm, the bud is cut and transferred to a rooting culture medium under the same culture conditions, and the bud is cultured for 3 weeks and then rooted (figure 4); the rooting medium comprises the following components in parts by weight: MS + NAA (0.2mg/L) + sucrose (30G/L) + agar powder (6.0G/L), wherein antibiotics G418(150mg/L) and Tim (150mg/L) are added, and the pH is adjusted to 5.8;
8. transplanting of resistant seedlings and transgenic PCR identification: after the resistant seedling root system grows well, taking out the resistant seedling root system without damaging the root, washing the residual culture medium and transferring the residual culture medium into soil. Randomly taking 4 transgenic lines, cutting new leaves after 1-2 new leaves grow out from seedlings, extracting DNA by a CTAB method, carrying out PCR amplification by using PBI121 vector specific primers (GUS-F: 5 ' -GCGGTAACAAGAAAGGGATC-3 and NOS-R: 5'-AATCATCGCAAGACCGGC-3'), and screening resistant seedlings with positive PCR amplification. As shown in FIG. 5, in 4 randomly selected resistant strains, 2 seedlings detected PCR amplification products with the size of about 250bp, which was consistent with the expected size, thereby confirming that the foreign gene is integrated into the genome of the German iris and establishing the genetic transformation system of the German iris.
The agrobacterium-mediated stem tip callus transformation method established by the invention has the characteristics of simple and convenient operation, rapidness, high efficiency and the like. According to the invention, two antibiotics (G418 and Tim) are adopted for screening and genome PCR identification, so that a positive transgenic plant is successfully obtained, and the positive rate is 50%. In conclusion, the invention establishes the genetic transformation method of the German iris plant with simple operation, high efficiency and reliability by taking the stem tip of the German iris as the explant, and the plasmid containing the foreign gene can be efficiently transferred into the German iris by the method to obtain the positive transgenic German iris plant.
Example 2
Well-grown stem tips, tender ovaries, tender buds and root tips are taken as explants respectively, the rest of processing steps are the same as those in example 1, the contamination rate, callus induction rate, callus state and color of the explants are counted at 45d respectively, and the results are shown in Table 1.
TABLE 1 Effect of different explants on callus induction of Iris germanica
The data are analyzed, and the iris germanica stem tip is taken as the explant, so that the callus induction rate is up to 82%, the embryogenesis is good, the materials are easy to obtain, and the like.
Example 3
The treatment method of example 1 was used, except that acetosyringone and OD of the Agrobacterium infection solution600The results of the statistical positive rate are shown in Table 2.
TABLE 2 different acetosyringone concentrations and OD600Influence of value of (d) on conversion effect
Analysis of the data shows that acetosyringone in the agrobacterium infection liquid of the invention is 200mg/L and OD600The highest positive rate was obtained when the value of (2) was 0.4.
Finally, the above-described embodiments are intended only to illustrate the technical solution of the present invention and not to limit the scope of the invention, and although the present invention has been described in detail by way of the above-described examples, it will be understood by those skilled in the art that various changes and modifications in form and detail may be made thereto without departing from the scope of the invention defined by the appended claims. The protection scope of the present invention should be subject to the appended claims.
Claims (5)
1. A genetic transformation method of iris germanica is characterized by comprising the following steps:
(1) inoculating the stem tip of the German iris into a callus induction culture medium, inducing the callus, and carrying out subculture;
(2) placing the callus of the iris germanica obtained in the step (1) on a proliferation culture medium for dark culture for later use;
(3) infecting the callus of the iris germanica obtained in the step (2) in an agrobacterium infection solution containing plasmids, and then placing the infected tissue on a co-culture medium for co-culture; the agrobacterium strain used for infection is EHA105, the agrobacterium strain contains a plant expression vector PBI121, 200mg/L acetosyringone is added into an agrobacterium infection solution, and OD is6000.3-0.5; the formula of the co-culture medium is as follows: MS +1.5mg/L2, 4-D +0.2mg/L6-BA +30g/L sucrose +6.0g/L agar powder, and the pH is adjusted to 5.8;
the preparation method of the agrobacterium infection solution comprises the following steps: streaking Agrobacterium containing plasmid on YEB solid culture medium containing 50mg/L rifampicin, culturing at 28 deg.C in dark for 3d, picking out single colony, inoculating to the medium containing rifampicin50mg/L rifampicin YEB liquid culture medium, under the dark condition 28 + -2 ℃, 200-; placing the cultured bacteria liquid in sterilized centrifuge tube, centrifuging at 6000rpm for 5min to collect thallus, resuspending thallus with 1/2MS solution, and adjusting to OD600Adding 200mg/L acetosyringone to obtain agrobacterium infection solution with the value of 0.3-0.5;
(4) cleaning the callus of the iris germanica co-cultured in the step (3), transferring the callus to a callus proliferation culture medium containing antibiotics, and screening resistant callus; the culture medium for screening the German iris resistance callus contains 100mg/L of antibiotic G418 and 150mg/L of timentin;
(5) transferring the newly proliferated resistant callus in the step (4) to an adventitious bud induction culture medium to induce the generation of adventitious buds; the adventitious bud induction culture medium comprises the following components in parts by weight: MS +1.0mg/L6-BA +0.2mg/L LNAA +150mg/LG418+150mg/L timentin +30g/L sucrose +6g/L agar powder, and the pH value is 5.8;
(6) cutting and transferring adventitious bud buds of the German iris to a rooting culture medium, and inducing to root; the rooting medium comprises the following components in parts by weight: MS +0.2mg/LNAA +150mg/LG418+150mg/L timentin +30g/L sucrose +6g/L agar powder, and the pH value is 5.8;
(7) taking out the well-grown resistant seedlings from the culture medium, transferring the well-grown resistant seedlings into nutrient soil, shearing new leaf DNA of the resistant seedlings, performing PCR amplification by using a carrier specific primer, and screening the resistant seedlings with positive PCR amplification.
2. The method for genetic transformation of iris germanica according to claim 1, wherein in step (1), said callus induction medium is: MS +1.5mg/L2, 4-D +0.2mg/L6-BA +30g/L sucrose +6g/L agar powder, and the pH value is 5.8.
3. The method for genetic transformation of iris germanica according to claim 1, wherein in step (2), said propagation medium is formulated as: MS +1.0mg/L2, 4-D +0.1mg/L6-BA +30g/L sucrose +6g/L agar powder, and the pH value is 5.8.
4. The method of genetic transformation of iris germanica according to claim 1, wherein in the step (2), the callus of the subculture is cut into 2 mm-sized pieces and placed on the propagation medium for 2 to 3 times.
5. The genetic transformation method of iris germanica according to claim 1, wherein in step (7), new leaves induced from resistant callus of iris germanica are cut, DNA is extracted, PCR detection is performed using specific primers on PBI121 vector, and positive transgenic seedlings are selected.
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| Genetic Transformation of Iris germanica Mediated by Agrobacterium tumefaciens;Zoran Jeknic等;《J. AMER. SOC. HORT. SCI.》;19991231;第124卷(第(6)期);摘要、方法部分 * |
| 德国鸢尾的组织培养;黄苏珍等;《江苏林业科技》;20000930;第27 卷;方法，讨论部分 * |
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