CN109182375B

CN109182375B - A kind of genetic transformation method of German iris

Info

Publication number: CN109182375B
Application number: CN201811199826.XA
Authority: CN
Inventors: 刘清泉; 张永侠; 原海燕; 黄苏珍; 唐君; 王玮琳; 杨永恒; 徐晓洋
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2018-09-26
Filing date: 2018-09-26
Publication date: 2022-02-15
Anticipated expiration: 2038-09-26
Also published as: CN109182375A

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

Genetic transformation method of German iris

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

1. a genetic transformation method of German iris, is characterized in that, step is as follows:

(1) get the shoot tip of German iris and inoculate it into callus induction medium, induce callus, and carry out subculture;

(2) get the German iris callus that step (1) obtains, place after dark cultivation on the proliferation medium, for subsequent use;

(3) the German iris callus obtained in step (2) is infected in the Agrobacterium infection solution containing the plasmid, and then placed on the co-cultivation medium for co-cultivation; the Agrobacterium strain used for infection It is EHA105, the Agrobacterium strain contains the plant expression vector PBI121, the Agrobacterium infection solution is added with 200mg/L acetosyringone, and the OD ₆₀₀ is 0.3-0.5; the co-cultivation medium formula is: MS+1.5mg/L2, 4-D+0.2mg/L6-BA+30g/L sucrose+6.0g/L agar powder, pH adjusted to 5.8;

The preparation method of the Agrobacterium infection solution is as follows: streak the Agrobacterium containing the plasmid on the YEB solid medium supplemented with 50 mg/L rifampicin, pick a single colony after culturing it in the dark at 28 °C for 3 days, and insert it into the medium containing 50 mg/L rifampicin. The YEB liquid medium of rifampicin was shaken at 28 ± 2 °C and 200-220 rpm for 16 hours under dark conditions; the cultured bacterial liquid was placed in a sterilized centrifuge tube, centrifuged at 6000 rpm for 5 min to collect the bacterial cells, and the bacteria were collected with 1/2 MS The solution was resuspended the bacterial cells, adjusted to an OD ₆₀₀ value of 0.3-0.5, and 200 mg/L acetosyringone was added to obtain an Agrobacterium infection solution;

(4) after washing the co-cultivated iris germanica callus in step (3), transfer it to an antibiotic-containing callus proliferation medium to screen the resistant callus; The medium contains 100mg/L of antibiotic G418 and 150mg/L of Timentin;

(5) Transfer the newly proliferated resistant callus in step (4) to the adventitious bud induction medium to induce adventitious buds; the adventitious bud induction medium formula is: MS+1.0mg/L6-BA+ 0.2mg/LNAA+150mg/LG418+150mg/L Timentin+30g/L sucrose+6g/L agar powder, pH 5.8;

(6) Cut off the adventitious buds of German iris and transfer them to a rooting medium to induce rooting; the rooting medium formula is: MS+0.2mg/LNAA+150mg/LG418+150mg/L Timentin+30g/ L sucrose+6g/L agar powder, pH 5.8;

(7) Take out the well-grown resistant seedlings from the medium, move them into nutrient soil, cut out the DNA of the new leaves of the resistant seedlings, carry out PCR amplification with carrier-specific primers, and screen the resistant seedlings with positive PCR amplification.

2. the genetic transformation method of German iris according to claim 1, is characterized in that, in step (1), described callus induction medium is: MS+1.5mg/L 2,4-D+0.2mg /L 6-BA+30g/L sucrose+6g/L agar powder, pH 5.8.

3. the genetic transformation method of German iris according to claim 1, is characterized in that, in step (2), described proliferation medium formula is: MS+1.0mg/L 2,4-D+0.1mg/L -BA+30g/L sucrose+6g/L agar powder, pH 5.8.

4. the genetic transformation method of German iris according to claim 1, is characterized in that in step (2), the callus of sub-generation 2-3 times is cut into small pieces of 2mm size and is placed on the proliferation medium.

5. the genetic transformation method of German iris according to claim 1, is characterized in that, in step (7), cut the new leaf that German iris resistance callus induces, extract DNA, utilize specific primer on PBI121 carrier to carry out PCR detection, screening positive transgenic seedlings.