CN109997693B - Genetic transformation method of alfalfa - Google Patents
Genetic transformation method of alfalfa Download PDFInfo
- Publication number
- CN109997693B CN109997693B CN201910275841.6A CN201910275841A CN109997693B CN 109997693 B CN109997693 B CN 109997693B CN 201910275841 A CN201910275841 A CN 201910275841A CN 109997693 B CN109997693 B CN 109997693B
- Authority
- CN
- China
- Prior art keywords
- alfalfa
- culture
- explant
- culture medium
- bud
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- A01H4/001—Culture apparatus 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
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention discloses a genetic transformation method of alfalfa. The invention provides an alfalfa genetic transformation method, which is characterized in that the stem tip or tissue containing the stem tip of alfalfa is used as an explant to carry out genetic transformation. Compared with the prior art, the invention has the advantages that: the method has the advantages of short transformation period and high efficiency. The rapid and efficient induction system for the stem tip of the alfalfa established by the invention can greatly shorten the transformation period, can induce a large number of cluster buds, and provides an important technical platform and support for the research of an efficient propagation system and a genetic transformation system of the alfalfa.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture and genetic transformation, and particularly relates to a genetic transformation method of alfalfa.
Background
Alfalfa (Medicago sativa), also known as alfalfa, is a perennial legume grass. Because the forage grass is widely distributed in the continental Oya and various countries in the world, and meanwhile, the stem leaves are tender and delicious, are deeply favored by various livestock and have the reputation of 'the king of the forage grass'. The alfalfa produced by conventional breeding cannot meet the social requirements, and transgenic genetic breeding is needed to directionally improve the quality and the stress resistance of the alfalfa. However, the alfalfa genetic transformation technology has been limited by many factors because of the difficulties in the field of plant genetic engineering. The common alfalfa genetic transformation method is to obtain a transgenic line by inducing callus and an indirect organ differentiation way, but the method has the troubles of long consumption period, difficult callus differentiation, low regeneration frequency and the like. How to increase the inducing speed of the alfalfa multiple shoots and the positive conversion rate is a technical bottleneck. Therefore, a method for directly obtaining organ differentiation paths is needed to obtain a large amount of excellent alfalfa regeneration differentiation seedlings in a short period.
Disclosure of Invention
In order to solve the problems of long transformation period and low differentiation rate of the purple flower in the prior art and fill up the technical blank, the invention provides the following technical scheme:
the invention provides an alfalfa genetic transformation method, which is characterized in that the stem tip or tissue containing the stem tip of alfalfa is directly used as an explant (callus induction is not needed) to carry out genetic transformation.
In the above method, the method comprises the steps of:
1) transfecting exogenous DNA molecules to the stem tip or tissues containing the stem tip of the alfalfa, and culturing to obtain an infected explant;
specifically, the stem tip or the tissue containing the stem tip of the alfalfa is infected in agrobacterium containing exogenous DNA molecules for 1 h;
2) inducing and culturing the infected explant in a cluster bud induction culture medium containing a screening agent to obtain a cluster bud explant;
3) carrying out elongation culture on the explant with the cluster buds in a resistant bud elongation culture medium to obtain the explant with the resistant buds;
4) and (3) carrying out rooting culture on the bud explants with the resistance in a rooting culture medium to obtain regenerated alfalfa plants, thereby realizing alfalfa genetic transformation.
In the method, the cluster bud induction culture medium containing the screening agent comprises a basic culture medium for tissue culture, 20-30g/L of sucrose, 1.4-1.6mg/L of 6-benzylaminopurine, 0.35-0.45mg/L of the screening agent and 200-300mg/L of bacteriostatic agent.
In the method, the resistant bud elongation culture medium comprises a basic culture medium for tissue culture, 20-30g/L of sucrose, 0.05-0.15mg/L of indoleacetic acid, 0.4-0.6mg/L of gibberellin, 0.3-0.5mg/L of screening agent and 200-300mg/L of bacteriostatic agent;
or the rooting culture medium comprises a basic culture medium for tissue culture, 10-20g/L of sucrose, 0.4-0.6mg/L of indoleacetic acid, 0.3-0.5mg/L of screening agent and 100-200mg/L of bacteriostatic agent.
In the above method, each of the culture media further comprises a coagulant;
or the basic culture medium for tissue culture is a liquid basic culture medium for tissue culture;
or the cluster bud induction culture medium containing the screening agent consists of a basic culture medium for tissue culture, 20-30g/L of sucrose, 1.4-1.6mg/L of 6-benzylaminopurine, 0.35-0.45mg/L of the screening agent, 200-300mg/L of bacteriostatic agent and 6-7g/L of coagulant;
or the resistant bud elongation culture medium consists of a basic culture medium for tissue culture, 20-30g/L of sucrose, 0.05-0.15mg/L of indoleacetic acid, 0.4-0.6mg/L of gibberellin, 0.3-0.5mg/L of screening agent, 200-300mg/L of bacteriostatic agent and 6-7g/L of coagulant;
or the rooting culture medium consists of a basic culture medium for tissue culture, 10-20g/L of sucrose, 0.4-0.6mg/L of indoleacetic acid, 0.3-0.5mg/L of screening agent, 100-200mg/L of bacteriostatic agent and 6-7g/L of coagulant;
the cluster bud induction culture medium containing the screening agent consists of a basic culture medium for tissue culture, 25g/L of sucrose, 1.5mg/L of 6-benzylaminopurine, 0.4mg/L of the screening agent, 250mg/L of bacteriostatic agent and 6.5g/L of coagulant;
or the resistant bud elongation culture medium consists of a basic culture medium for tissue culture, 25g/L of cane sugar, 0.1mg/L of indoleacetic acid, 0.5mg/L of gibberellin, 0.4mg/L of screening agent, 250mg/L of bacteriostatic agent and 6.5g/L of coagulator; (ii) a
Or the rooting culture medium consists of a basic culture medium for tissue culture, 15g/L of cane sugar, 0.5mg/L of indoleacetic acid, 0.4mg/L of screening agent, 150mg/L of bacteriostatic agent and 6.5g/L of coagulant.
The minimal medium may be MS medium or MSB medium. In the examples of the present invention, MSB medium is used.
In the method, the coagulant is agarose;
or, the pH value of each culture medium is 5.7-5.9; in particular to 5.8;
or, the screening agent is glufosinate-n;
or the bacteriostatic agent is timentin. Under the screening condition and the bacteriostatic condition, the explant can effectively and rapidly grow.
In the method, in the step 1), the transfection time is 1h, the infection time is too short, and agrobacterium is not easy to integrate into an explant, so that the infection efficiency is low; the infection time is too long, and the explants are easy to return bacteria in the subsequent culture process, so that the explants die.
The culture is dark culture for 4 days, and the culture temperature is 24-26 ℃; the culture is carried out for 4 days, which is beneficial to effectively controlling the pollution of agrobacterium and the survival efficiency of explants and can ensure higher transformation efficiency.
In the step 2), the conditions of the induction culture are 24-26 ℃, the humidity is 50-70%, the illumination intensity is 1500-; under the culture condition, the fast growth of the explant is facilitated.
In the step 3), the conditions of the extension culture are 24-26 ℃, the humidity is 50-70%, the illumination intensity is 1500-;
in the step 4), the starting time of the rooting culture of the bud explants with the resistance in the rooting culture medium is when the buds of the bud explants with the resistance extend to 4-5 cm;
or, the rooting culture conditions are 24-26 ℃, the humidity is 50-70%, the illumination intensity is 1500-.
In the method, the tissue of the alfalfa containing the stem tip is an alfalfa explant with a apical meristem;
the alfalfa explant with the apical meristem consists of one cotyledon and alfalfa stem tip;
the alfalfa explants with apical meristem were prepared as follows: soaking the alfalfa seeds in water to obtain the imbibed alfalfa seeds; and then taking the alfalfa explants with apical meristematic tissues in the alfalfa seeds after imbibition.
In the method, the alfalfa variety is longdong alfalfa or jinhuang alfalfa. The invention also provides a kit for alfalfa genetic transformation.
The kit provided by the invention comprises a substance for obtaining the stem tip or tissue containing the stem tip of alfalfa, a clustered shoot induction culture medium containing a screening agent, a resistant shoot elongation culture medium and a rooting culture medium in the method;
or the application of the kit in alfalfa genetic transformation.
The invention selects and uses the direct organ differentiation way of alfalfa, successfully obtains the alfalfa seedlings successfully transformed within 2 months, and improves the genetic transformation efficiency to 15 percent. The method is applied to related genetic transformation research and production work, and provides an important technical platform and support for transgenic research.
Compared with the prior art, the invention has the advantages that: the method has the advantages of short transformation period and high efficiency. In addition, the sterilized alfalfa is placed in a greenhouse for imbibition culture, so that the germination culture time can be shortened, and the consumption of a germination culture medium can be reduced; the proportion of each component of the induction culture medium can provide excellent explants for genetic transformation, a large number of excellent and robust differentiated buds can be induced after induction, the obtaining time is 15-20 days, and the induction culture medium has the advantages of low pollution rate and good differentiated bud state. Compared with the callus induction way in the prior art, the method needs 30-50 days, the established rapid and efficient induction system for the stem tip of the alfalfa can greatly shorten the transformation period, can induce a large number of cluster buds, and provides an important technical platform and support for the research of an efficient propagation system and a genetic transformation system of the alfalfa.
Drawings
FIG. 1 shows the induction of clumpy shoots after 15 days on clumpy shoot induction medium (left) and the elongation of resistant shoots after 15 days on elongation medium (right) using the method of the present invention in alfalfa King.
FIG. 2 shows the induction of multiple shoots after 15 days on multiple shoot induction medium (left) and the elongation of resistant shoots after 15 days on elongation medium (right) by the method of the present invention.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 high-efficiency and quick genetic transformation method of alfalfa
1. Preparation of sterile explants
In an ultraclean workbench, 100 selected alfalfa seeds with golden queen are placed into a sterilized 50ml centrifuge tube, an appropriate amount of ethanol water solution with the volume fraction of 75% is added for sterilization for 3min, then the alfalfa seeds are sterilized by 15% hydrogen peroxide for 15min, then the alfalfa seeds are washed by sterilized water for 4 times, finally, an appropriate amount of sterilized water is added to immerse the seeds, and the seeds are placed in a greenhouse with the temperature of 25 ℃ for imbibition for 24 h.
Taking out the bloated golden queen seeds on an ultraclean workbench by using forceps, placing the bloated golden queen seeds in a sterilized culture dish, peeling off seed coats, cutting off a part of hypocotyls by using a scalpel, and longitudinally cutting along the midline of the hypocotyls to obtain alfalfa explants (tissues containing stem tips) with apical meristems.
The alfalfa explant with the apical meristem consists of one cotyledon and alfalfa stem tip; after transformation, the shoot tip portion was directly differentiated into a seedling through a direct organ differentiation pathway.
2. Transformation of
1) Preparation of agrobacterium and infection liquid
Taking required strain EHA105/pCAMBIA3301 from-80 deg.C refrigerator, streaking on YEP resistant plate, inverting, and culturing in dark at 28 deg.C for 2 days; inoculating 2 cultured single colonies into 2mL YEP liquid culture medium (added with proper antibiotics) and performing shaking culture at 28 ℃ and 250rpm for 16h overnight; taking the once activated bacterial liquid, mixing the once activated bacterial liquid with the ratio of 1: 1000, adding a fresh YEP liquid culture medium (containing antibiotics) and carrying out shaking culture at 28 ℃ and 250rpm for 16h until OD600 is equal to 0.714, centrifugally collecting bacterial liquid precipitate, and suspending the collected agrobacterium liquid to OD600 to 0.781 by using an MS liquid culture medium to obtain bacterial liquid suspension;
EHA105/pCAMBIA3301 is a recombinant strain obtained by transferring pCAMBIA3301 vector into EHA105, wherein pCAMBIA3301 vector is purchased from wuhan transduced biologies laboratories ltd, and contains bar gene, catalog No.: VT 4012; EHA105 was purchased from beijing, washington, catalog No.: GX 0133-100.
2) Genetic transformation of
1 cut was made by scalpel at the top of the explant (alfalfa cotyledons with apical meristem) obtained at 1; then placing the explant into the bacterial liquid suspension obtained in the step 1) for infection for 1h, transferring the explant into an MSB solid minimal medium (shown in table 1) for dark culture for 4d, wherein the culture temperature is 25 ℃, and obtaining the infected explant;
3) screening and rooting of resistant shoots
Transferring the infected explant in the step 2) into a cluster bud induction culture medium containing glufosinate, and placing the explant in a tissue culture room for induction culture at 25 ℃ (illumination intensity is 2000lux, humidity is 60%, illumination is 16 hours every day, and darkness is 8 hours) for 15d (as shown in the left side of a picture 1) to obtain the explant with cluster buds;
transferring the explant with the cluster buds into a culture medium for elongation culture of the resistant buds (25 ℃, induction culture, illumination intensity of 2000lux, humidity of 60%, 16 hours of illumination each day, 8 hours of darkness) for 25d (as shown in the right part of the figure 1) to obtain the explant with the resistant buds;
and when the resistant bud stem of the explant with the resistant bud extends to 4cm (namely the resistant bud stem is subjected to extension culture for about 25 d), transferring the explant into a rooting culture medium for induction culture at 25 ℃ for 20d (the illumination intensity is 2000lux, the humidity is 60%, the illumination is 16 hours every day, and the darkness is 8 hours), and obtaining the T0 generation alfalfa seedlings.
The above-mentioned cluster bud induction culture medium containing glufosinate-containing consists of MSB liquid minimal medium, 25g/L sucrose, 1.5 mg/L6-benzylaminopurine, 0.4mg/L glufosinate-containing, 250mg/L timentin (Shanghai Probiota, catalog number: A600950-0001) and 6.5g/L agar, pH value is 5.8;
each 1L of the glufosinate-containing cluster bud induction medium is prepared according to the following method: adding 25g/L of sucrose, 1.5mg/L of 6-benzylaminopurine, 0.4mg/L of glufosinate-ammonium, 250mg/L of timentin and 6.5g/L of agar into each component of the MSB liquid minimal medium shown in the table 1 according to the concentration, and fixing the volume to 1L by using water;
the culture medium for resistant bud elongation consists of MSB liquid minimal medium, 25g/L sucrose, 0.1mg/L indoleacetic acid, 0.5mg/L gibberellin, 0.4mg/L glufosinate, 250mg/L timentin (Shanghai bio-engineering, product catalog number: A600950-0001) and 6.5g/L agar, and the pH value is 5.8;
each 1L of the medium for elongation of resistant shoots was prepared as follows: adding 25g/L of sucrose, 0.1mg/L of indoleacetic acid, 0.5mg/L of gibberellin, 0.4mg/L of glufosinate-ammonium, 250mg/L of timentin and 6.5g/L of agar into each component of the MSB liquid minimal medium shown in the table 1 according to the concentration of the sucrose, the indole acetic acid, the gibberellin, the glufosinate-ammonium, the timentin and the agar, and fixing the volume to 1L by using water;
the components of the rooting culture medium consist of an MSB liquid minimal medium, 15g/L of cane sugar, 0.5mg/L of indoleacetic acid, 0.4mg/L of screening agent, 150mg/L of timentin and 6.5g/L of agar, and the pH value is 5.8.
Each 1L of rooting medium components were prepared as follows: the components of the MSB liquid minimal medium shown in the table 1 are added according to the concentration, 15g/L of sucrose, 0.5mg/L of indoleacetic acid, 0.4mg/L of screening agent, 150mg/L of timentin and 6.5g/L of agar, and the volume is fixed to 1L by water.
The MSB liquid minimal medium was obtained by dissolving solutes at respective concentrations shown in Table 1 in water at the concentrations shown in Table 1.
MSB solid minimal medium consisted of solute, water and agarose at each concentration shown in Table 1, the agarose concentration was 6.5 g/L.
Table 1 shows the solutes and the corresponding concentrations in the liquid MSB minimal medium
3. Detection of
Hardening the T0 generation alfalfa seedlings, opening the tissue culture box of the alfalfa seedlings, adding 10ml of distilled water, and culturing for 16 hours in a non-light 25 ℃ tissue culture room. Taking alfalfa leaves, extracting DNA of the leaves, and detecting a bar gene of a plant resistance vector by using a PCR method. The detection primer is
F1 (5'-TAACATGGTGGAGCACGACA-3') and R1 (5'-TTGACCGTGCTTGTCTCGAT-3') (primers synthesized by Shanghai Biotech). And calculating the conversion positive rate of the alfalfa according to the PCR result.
The alfalfa seedlings of the T0 generation which are positive are amplified to 500 bp.
In the example, 50 explants are selected in total, after genetic transformation, 42 explants have resistance buds extending and successfully rooting in the culture medium, 123T 0 alfalfa seedlings are obtained in total, and 2.9 resistance buds grow on each explant on average; for the obtained 123T 0 alfalfa seedlings, 19 PCR tests were positive, and the positive rate was 15.4% (19/123 × 100%).
The induction rate was 25 days with an average of 2.9 per explant.
Example 2 high-efficiency and quick genetic transformation method of alfalfa
1. Preparation of sterile explants
In an ultraclean workbench, 100 selected alfalfa seeds with golden queen are placed into a sterilized 50ml centrifuge tube, an appropriate amount of ethanol water solution with the volume fraction of 75% is added for sterilization for 3min, then the alfalfa seeds are sterilized by 15% hydrogen peroxide for 15min, then the alfalfa seeds are washed by sterilized water for 4 times, finally, an appropriate amount of sterilized water is added to immerse the seeds, and the seeds are placed in a greenhouse with the temperature of 25 ℃ for imbibition for 24 h.
Taking out the bloated golden queen seeds on an ultraclean workbench by using forceps, placing the bloated golden queen seeds in a sterilized culture dish, peeling off seed coats, cutting off a part of hypocotyls by using a scalpel, and longitudinally cutting along the midline of the hypocotyls to obtain alfalfa explants with apical meristems.
The alfalfa explant with the apical meristem consists of one cotyledon and alfalfa stem tip; after transformation, the shoot tip portion was directly differentiated into a seedling through a direct organ differentiation pathway. .
2. Transformation of
1) Preparation of agrobacterium and infection liquid
Taking the required strain EHA105/pCAMBIA3301 from-80 deg.C refrigerator, streaking on YEP resistant plate, inverting the plate, and culturing in dark at 28 deg.C for 2 days; inoculating 3 cultured single colonies into 2mL YEP liquid culture medium (added with proper antibiotics) and performing shaking culture at 250rpm for 16h at 28 ℃; taking the once activated bacterial liquid, mixing the once activated bacterial liquid with the ratio of 1: 1000 adding fresh YEP liquid culture medium (containing antibiotics) and carrying out shaking culture at 28 ℃ and 250rpm for 16h until OD600 is 0.698, centrifuging and collecting bacterial liquid precipitate, and suspending the collected agrobacterium liquid to OD 600-0.807 by using MS liquid culture medium;
2) genetic transformation
1 cut was made by scalpel at the top of the explant (alfalfa cotyledons with apical meristem) obtained at 1; then placing the explant into the bacterial liquid suspension obtained in the step 1) for infection for 1h, transferring the explant into an MSB solid minimal medium (shown in table 1) for dark culture for 4d, wherein the culture temperature is 25 ℃, and obtaining the infected explant;
3) screening and rooting of resistant shoots
Transferring the explant subjected to the dark culture in the step 2) into a cluster bud induction culture medium containing glufosinate, and placing the explant in a tissue culture room for induction culture at 25 ℃ (the illumination intensity is 2000lux, the humidity is 60%, the illumination is 16 hours every day, and the darkness is 8 hours) for 22d (as shown in the left side of the figure 1), so as to obtain the explant with cluster buds;
transferring the explant with the cluster buds into a culture medium for elongation culture of the resistant buds for 21d (25 ℃, induction culture, illumination intensity of 2000lux, humidity of 60%, 16 hours of illumination and 8 hours of darkness per day) (as shown in the right part of figure 1), and obtaining the explant with the resistant buds;
and when the resistant bud stem of the explant with the resistant bud extends to 4cm, transferring the explant with the resistant bud to a rooting culture medium for induction culture at 25 ℃ (the illumination intensity is 2000lux, the humidity is 60%, the illumination is 16 hours every day, and the darkness is 8 hours) for 20 days, and obtaining the T0 generation alfalfa seedlings.
The above-mentioned cluster bud induction culture medium containing glufosinate-containing consists of MSB liquid minimal medium, 25g/L sucrose, 1.5 mg/L6-benzylaminopurine, 0.4mg/L glufosinate-containing, 250mg/L timentin (Shanghai Probiota, catalog number: A600950-0001) and 6.5g/L agar, pH value is 5.8;
each 1L of the glufosinate-containing cluster bud induction medium is prepared according to the following method: adding 25g/L of sucrose, 1.5mg/L of 6-benzylaminopurine, 0.4mg/L of glufosinate-ammonium, 250mg/L of timentin and 6.5g/L of agar into each component of the MSB liquid minimal medium shown in the table 1 according to the concentration, and fixing the volume to 1L by using water;
the culture medium for resistant bud elongation consists of MSB liquid minimal medium, 25g/L sucrose, 0.1mg/L indoleacetic acid, 0.5mg/L gibberellin, 0.4mg/L glufosinate, 250mg/L timentin (Shanghai bio-engineering, product catalog number: A600950-0001) and 6.5g/L agar, and the pH value is 5.8;
each 1L of the medium for elongation of resistant shoots was prepared as follows: adding 25g/L of sucrose, 0.1mg/L of indoleacetic acid, 0.5mg/L of gibberellin, 0.4mg/L of glufosinate-ammonium, 250mg/L of timentin and 6.5g/L of agar into each component of the MSB liquid minimal medium shown in the table 1 according to the concentration of the sucrose, the indole acetic acid, the gibberellin, the glufosinate-ammonium, the timentin and the agar, and fixing the volume to 1L by using water;
the components of the rooting culture medium consist of an MSB liquid minimal medium, 15g/L of cane sugar, 0.5mg/L of indoleacetic acid, 0.4mg/L of screening agent, 150mg/L of timentin and 6.5g/L of agar, and the pH value is 5.8.
Each 1L of rooting medium components were prepared as follows: the components of the MSB liquid minimal medium shown in the table 1 are added according to the concentration, 15g/L of sucrose, 0.5mg/L of indoleacetic acid, 0.4mg/L of screening agent, 150mg/L of timentin and 6.5g/L of agar, and the volume is fixed to 1L by water.
3. Detection of
Hardening the T0 generation alfalfa seedlings, opening the tissue culture box of the alfalfa seedlings, adding 10ml of distilled water, and culturing for 16 hours in a non-light 25 ℃ tissue culture room. Taking alfalfa leaves, extracting DNA of the leaves, and detecting a bar gene of a plant resistance vector by using a PCR method. The detection primers used were F1 (5'-TAACATGGTGGAGCACGACA-3') and R1 (5'-TTGACCGTGCTTGTCTCGAT-3') (primers synthesized by Shanghai Biotech). And calculating the conversion positive rate of the alfalfa according to the PCR result.
The alfalfa seedlings of the T0 generation which are positive are amplified to 500 bp.
In this example, a total of 50 explants were selected, and after genetic transformation, 38 explants had resistant shoots that extended and successfully rooted in the medium, resulting in a total of 125T 0 alfalfa seedlings. On average, 3.2 resistant buds outgrow from each explant, and 19 PCR detections of 125T 0 alfalfa seedlings were performed, wherein the positive rate was 15.2% (19/125 × 100%).
Example 3 high-efficiency and quick genetic transformation method of alfalfa
1. Preparation of sterile explants
Placing 100 selected Longdong alfalfa seeds in a sterilized 50ml centrifuge tube in a superclean bench, adding a proper amount of 75% ethanol by volume, sterilizing for 4min, then sterilizing for 13min with 15% hydrogen peroxide, washing for 5 times with sterile water, finally adding a proper amount of sterile water to submerge the seeds, and placing the seeds in a greenhouse at 25 ℃ for imbibition for 24 h;
on a clean bench, the well-imbibed Longdong alfalfa seeds were placed in a sterile petri dish with forceps, the seed coat was peeled off, a portion of the hypocotyl was cut with a scalpel, and the cut was made longitudinally along the centerline of the hypocotyl to obtain alfalfa explants with apical meristems.
The alfalfa explant with the apical meristem consists of one cotyledon and alfalfa stem tip; after transformation, the shoot tip portion was directly differentiated into a seedling through a direct organ differentiation pathway. .
2. Transformation of
1) Preparation of agrobacterium and infection liquid
Taking required strain EHA105/pCAMBIA3301 from-80 deg.C refrigerator, streaking on YEP resistant plate, inverting, and culturing in dark at 28 deg.C for 2 days; inoculating 2 cultured single colonies into 2mL YEP liquid culture medium (added with proper antibiotics) and performing shaking culture at 28 ℃ and 250rpm for 16h overnight; taking the once activated bacterial liquid, mixing the once activated bacterial liquid with the ratio of 1: 1000 adding fresh YEP liquid culture medium (containing antibiotics) and carrying out shaking culture at 28 ℃ and 250rpm for 16h until OD600 is 0.801, centrifuging to collect bacterial liquid precipitate, and suspending the collected Agrobacterium liquid to OD 600-0.789 by using MS liquid culture medium;
2) genetic transformation of
The scalpel made 2 cuts at the top of the explant (alfalfa cotyledons with apical meristem) obtained at 1; then placing the explant into the bacterial liquid suspension obtained in the step 1) for infection for 1h, transferring the explant into an MSB culture medium for dark culture for 4d, wherein the culture temperature is 25 ℃, and obtaining the infected explant;
3) screening and rooting of resistant shoots
Transferring the infected explant in the step 2) into a cluster bud induction culture medium containing glufosinate, and placing the explant in a tissue culture room for induction culture at 25 ℃ (illumination intensity is 2000lux, humidity is 60%, illumination is 16 hours every day, and darkness is 8 hours) for 19d (as shown in the left side of figure 2) to obtain the explant with cluster buds;
transferring the explant with the cluster buds into a resistant bud elongation culture medium for elongation culture for 25d (25 ℃ induction culture, illumination intensity of 2000lux, humidity of 60%, illumination for 16 hours per day and darkness for 8 hours) (as shown on the right of figure 2) to obtain the explant with the resistant buds;
and when the resistant bud stem of the explant with the resistant bud extends to 5cm, transferring the explant with the resistant bud to a rooting culture medium for induction culture at 25 ℃ (the illumination intensity is 2000lux, the humidity is 60%, the illumination is 16 hours every day, and the darkness is 8 hours) for 20 days, and obtaining the T0 generation alfalfa seedlings.
The cluster bud induction culture medium containing glufosinate-containing consists of MSB liquid minimal medium, 25g/L sucrose, 1.5 mg/L6-benzylaminopurine, 0.4mg/L glufosinate-containing, 250mg/L timentin (Shanghai bio-engineering, catalog number: A600950-0001) and 6.5g/L agar, and the pH value is 5.8;
each 1L of the glufosinate-containing cluster bud induction medium is prepared according to the following method: adding 25g/L of sucrose, 1.5mg/L of 6-benzylaminopurine, 0.4mg/L of glufosinate-ammonium, 250mg/L of timentin and 6.5g/L of agar into each component of the MSB liquid minimal medium shown in the table 1 according to the concentration, and fixing the volume to 1L by using water;
the culture medium for resistant bud elongation consists of MSB liquid minimal medium, 25g/L sucrose, 0.1mg/L indoleacetic acid, 0.5mg/L gibberellin, 0.4mg/L glufosinate, 250mg/L timentin (Shanghai bio-engineering, product catalog number: A600950-0001) and 6.5g/L agar, and the pH value is 5.8;
each 1L of the medium for elongation of resistant shoots was prepared as follows: adding 25g/L of sucrose, 0.1mg/L of indoleacetic acid, 0.5mg/L of gibberellin, 0.4mg/L of glufosinate-ammonium, 250mg/L of timentin and 6.5g/L of agar into each component of the MSB liquid minimal medium shown in the table 1 according to the concentration of the sucrose, the indole acetic acid, the gibberellin, the glufosinate-ammonium, the timentin and the agar, and fixing the volume to 1L by using water;
the components of the rooting culture medium consist of an MSB liquid minimal medium, 15g/L of cane sugar, 0.5mg/L of indoleacetic acid, 0.4mg/L of screening agent, 150mg/L of timentin and 6.5g/L of agar, and the pH value is 5.8.
Each 1L of rooting medium components were prepared as follows: the components of the MSB liquid minimal medium shown in the table 1 are added according to the concentration, 15g/L of sucrose, 0.5mg/L of indoleacetic acid, 0.4mg/L of screening agent, 150mg/L of timentin and 6.5g/L of agar, and the volume is fixed to 1L by water.
3. Detection of
Hardening the T0 generation alfalfa seedlings, opening the tissue culture box of the alfalfa seedlings, adding 10ml of distilled water, and culturing for 16 hours in a non-light 25 ℃ tissue culture room. Taking alfalfa leaves, extracting DNA of the leaves, and detecting a bar gene of a plant resistance vector by using a PCR method. The detection primers used were F1 (5'-TAACATGGTGGAGCACGACA-3') and R1 (5'-TTGACCGTGCTTGTCTCGAT-3') (primers synthesized by Shanghai Biotech). And calculating the conversion positive rate of the alfalfa according to the PCR result.
The alfalfa seedlings of the T0 generation which are positive are amplified to 500 bp.
In the example, 50 explants are selected in total, after genetic transformation, resistant buds of 35 explants are elongated and successfully rooted in a culture medium, 110T 0 alfalfa seedlings are obtained in total, and 3.1 resistant buds grow on each explant on average; the obtained 110T 0 alfalfa seedlings were subjected to PCR detection, and 16 of the seedlings were positive, with a positive rate of 14.5% (16/110 × 100%).
Example 4 high-efficiency and fast genetic transformation method of alfalfa
1. Preparation of sterile explants
Placing 100 selected Longdong alfalfa seeds in a sterilized 50ml centrifuge tube in a superclean bench, adding a proper amount of 75% ethanol by volume, sterilizing for 3min, then sterilizing for 15min with 15% hydrogen peroxide, cleaning for 3 times with sterile water, finally adding a proper amount of sterile water to submerge the seeds, and placing the seeds in a greenhouse at 25 ℃ for imbibition for 24 h;
on a clean bench, the well-imbibed Longdong alfalfa seeds were placed in a sterile petri dish with forceps, the seed coat was peeled off, a portion of the hypocotyl was cut with a scalpel, and the cut was made longitudinally along the centerline of the hypocotyl to obtain alfalfa explants with apical meristems.
The alfalfa explant with the apical meristem consists of one cotyledon and alfalfa stem tip; after transformation, the shoot tip portion was directly differentiated into a seedling through a direct organ differentiation pathway. .
2. Transformation of
1) Preparation of agrobacterium and infection liquid
Taking the required strain EHA105/pCAMBIA3301 from-80 deg.C refrigerator, streaking on YEP resistant plate, inverting the plate, and culturing in dark at 28 deg.C for 2 days; inoculating 3 cultured single colonies into 2mL YEP liquid culture medium (added with proper antibiotics) and performing shaking culture at 250rpm for 16h at 28 ℃; taking the once activated bacterial liquid, mixing the once activated bacterial liquid with the ratio of 1: 1000 adding fresh YEP liquid culture medium (containing antibiotics) and culturing at 28 ℃ and 250rpm for 16h under shaking until OD600 is 0.751, centrifuging to collect bacterial liquid precipitate, and suspending the collected Agrobacterium liquid to OD 600-0.805 by using MS liquid culture medium;
2) genetic transformation of
The scalpel made 2 cuts at the top of the explant (alfalfa cotyledons with apical meristem) obtained at 1; then placing the explant into the bacterial liquid suspension obtained in the step 1) for infection for 1h, and transferring the explant into an MSB culture medium for dark culture for 4d (the culture temperature is 25 ℃), so as to obtain the infected explant;
3) screening and rooting of resistant shoots
Transferring the explant subjected to the dark culture in the step 2) into a cluster bud induction culture medium containing glufosinate, and placing the explant in a tissue culture room for induction culture at 25 ℃ (illumination intensity of 2000lux, humidity of 60%, illumination for 16 hours and darkness for 8 hours every day) for 20d (as shown in the left of figure 2) to obtain the explant with cluster buds;
transferring the explant with the cluster buds into a resistant bud elongation culture medium for elongation culture for 24d (25 ℃ induction culture, illumination intensity of 2000lux, humidity of 60%, 16 hours of illumination and 8 hours of darkness per day) (as shown on the right of figure 2), and obtaining the explant with the resistant buds;
and when the resistant bud stem of the explant with the resistant bud extends to 5cm, transferring the explant with the resistant bud to a rooting culture medium for induction culture at 25 ℃ (the illumination intensity is 2000lux, the humidity is 60%, the illumination is 16 hours every day, and the darkness is 8 hours) for 18d, and obtaining the T0 generation alfalfa seedlings.
The above-mentioned cluster bud induction culture medium containing glufosinate-containing consists of MSB liquid minimal medium, 25g/L sucrose, 1.5 mg/L6-benzylaminopurine, 0.4mg/L glufosinate-containing, 250mg/L timentin (Shanghai Probiota, catalog number: A600950-0001) and 6.5g/L agar, pH value is 5.8;
each 1L of the glufosinate-containing cluster bud induction medium is prepared according to the following method: adding 25g/L of sucrose, 1.5mg/L of 6-benzylaminopurine, 0.4mg/L of glufosinate-ammonium, 250mg/L of timentin and 6.5g/L of agar into each component of the MSB liquid minimal medium shown in the table 1 according to the concentration, and fixing the volume to 1L by using water;
the culture medium for resistant bud elongation consists of MSB liquid minimal medium, 25g/L sucrose, 0.1mg/L indoleacetic acid, 0.5mg/L gibberellin, 0.4mg/L glufosinate, 250mg/L timentin (Shanghai bio-engineering, product catalog number: A600950-0001) and 6.5g/L agar, and the pH value is 5.8;
each 1L of the medium for elongation of resistant shoots was prepared as follows: adding 25g/L of sucrose, 0.1mg/L of indoleacetic acid, 0.5mg/L of gibberellin, 0.4mg/L of glufosinate-ammonium, 250mg/L of timentin and 6.5g/L of agar into each component of the MSB liquid minimal medium shown in the table 1 according to the concentration of the sucrose, the indole acetic acid, the gibberellin, the glufosinate-ammonium, the timentin and the agar, and fixing the volume to 1L by using water;
the components of the rooting culture medium consist of an MSB liquid minimal medium, 15g/L of cane sugar, 0.5mg/L of indoleacetic acid, 0.4mg/L of screening agent, 150mg/L of timentin and 6.5g/L of agar, and the pH value is 5.8.
Each 1L of rooting medium components were prepared as follows: the components of the MSB liquid minimal medium shown in the table 1 are added according to the concentration, 15g/L of sucrose, 0.5mg/L of indoleacetic acid, 0.4mg/L of screening agent, 150mg/L of timentin and 6.5g/L of agar, and the volume is fixed to 1L by water.
3. Detection of
Hardening the T0 generation alfalfa seedlings, opening the tissue culture box of the alfalfa seedlings, adding 10ml of distilled water, and culturing for 16 hours in a non-light 25 ℃ tissue culture room. Taking alfalfa leaves, extracting DNA of the leaves, and detecting a bar gene of a plant resistance vector by using a PCR method. The detection primers used were F1 (5'-TAACATGGTGGAGCACGACA-3') and R1 (5'-TTGACCGTGCTTGTCTCGAT-3') (primers synthesized by Shanghai Biotech). And calculating the conversion positive rate of the alfalfa according to the PCR result.
The alfalfa seedlings of the T0 generation which are positive are amplified to 500 bp.
In this example, a total of 50 explants were selected, and after genetic transformation, 40 explants in the culture medium had resistant shoots that extended and successfully rooted, and a total of 113T 0 alfalfa seedlings were obtained. On average, 2.8 resistant buds outgrow from each explant, and 17 PCR detections of 113T 0 alfalfa seedlings were performed, wherein the positive rate was 15.0% (17/113 × 100%).
Claims (4)
1. A method for genetic transformation of alfalfa, the method comprising the steps of:
1) transfecting the tissue of alfalfa containing a stem tip with an exogenous DNA molecule, and culturing to obtain an infected explant;
2) inducing and culturing the infected explant in a cluster bud induction culture medium containing a screening agent to obtain a cluster bud explant;
3) carrying out elongation culture on the explant with the cluster buds in a resistant bud elongation culture medium to obtain the explant with the resistant buds;
4) rooting and culturing the bud explants with the resistance in a rooting culture medium to obtain regenerated alfalfa plants, and realizing alfalfa genetic transformation;
the cluster bud induction culture medium containing the screening agent consists of an MSB liquid minimal medium, 25g/L of sucrose, 1.5mg/L of 6-benzylaminopurine, 0.4mg/L of the screening agent, 250mg/L of bacteriostatic agent and 6.5g/L of coagulant;
the resistant bud elongation culture medium consists of an MSB liquid minimal medium, 25g/L of cane sugar, 0.1mg/L of indoleacetic acid, 0.5mg/L of gibberellin, 0.4mg/L of screening agent, 250mg/L of bacteriostatic agent and 6.5g/L of coagulant;
the rooting culture medium consists of an MSB liquid minimal medium, 15g/L of sucrose, 0.5mg/L of indoleacetic acid, 0.4mg/L of screening agent, 150mg/L of bacteriostatic agent and 6.5g/L of coagulant;
the tissue of the alfalfa containing the stem tip is an alfalfa explant with a apical meristem;
the alfalfa explant with the apical meristem consists of one cotyledon and alfalfa stem tip;
the screening agent is glufosinate-butyl;
the bacteriostatic agent is timentin.
2. The method of claim 1, wherein:
the coagulant is agarose;
the pH value of each culture medium is 5.7-5.9.
3. The method of claim 1, wherein:
in the step 1), the transfection time is 1 h; the culture is dark culture for 4 days, and the culture temperature is 24-26 ℃;
in the step 2), the conditions of the induction culture are 24-26 ℃, the humidity is 50-70%, the illumination intensity is 1500-;
in the step 3), the conditions of the extension culture are 24-26 ℃, the humidity is 50-70%, the illumination intensity is 1500-;
in the step 4), the initiation time of the rooting culture of the bud explants with the resistance in the rooting culture medium is when the buds of the bud explants with the resistance extend to 4-5 cm;
the rooting culture conditions are 24-26 ℃, the humidity is 50-70%, the illumination intensity is 1500-.
4. A method according to any one of claims 1-3, characterized in that: the alfalfa variety is Longdong alfalfa or Jinhuanghou alfalfa.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910275841.6A CN109997693B (en) | 2019-04-08 | 2019-04-08 | Genetic transformation method of alfalfa |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910275841.6A CN109997693B (en) | 2019-04-08 | 2019-04-08 | Genetic transformation method of alfalfa |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109997693A CN109997693A (en) | 2019-07-12 |
| CN109997693B true CN109997693B (en) | 2022-04-05 |
Family
ID=67170260
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910275841.6A Active CN109997693B (en) | 2019-04-08 | 2019-04-08 | Genetic transformation method of alfalfa |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109997693B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115735770B (en) * | 2022-11-29 | 2023-08-18 | 上饶师范学院 | Ultralow-temperature preservation method for broccoli by embedding vitrification method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106916847A (en) * | 2017-03-21 | 2017-07-04 | 中国农业科学院北京畜牧兽医研究所 | A kind of method for improving alfalfa genetic transformation efficiency |
| CN108531504A (en) * | 2018-04-04 | 2018-09-14 | 辽宁大学 | A method of efficiently formulating low content of lignin alfalfa using genetic engineering means |
-
2019
- 2019-04-08 CN CN201910275841.6A patent/CN109997693B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106916847A (en) * | 2017-03-21 | 2017-07-04 | 中国农业科学院北京畜牧兽医研究所 | A kind of method for improving alfalfa genetic transformation efficiency |
| CN108531504A (en) * | 2018-04-04 | 2018-09-14 | 辽宁大学 | A method of efficiently formulating low content of lignin alfalfa using genetic engineering means |
Non-Patent Citations (2)
| Title |
|---|
| 农杆菌介导的耐盐多基因转化紫花苜蓿及分子检测;王静等;《农业生物技术学报》;20170825;第25卷(第8期);摘要，第1257页左栏第1段、右栏第1.1.3节，第1258页第1.2.1节、第1.2.2节，第1259页右栏第2.1节，第1260页左栏第1段，第1258页左栏第5-6行 * |
| 新疆大叶紫花苜蓿两种再生体系的比较;施莉娟等;《东北农业大学学报》;20100425;第41卷(第4期);摘要，第97页第3节 讨论与结论，第95页第1.3.2.1节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109997693A (en) | 2019-07-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5272072A (en) | Method for preparing transformed plant | |
| AU721423B2 (en) | Process for propagation and/or selection of plant material | |
| CN108338073A (en) | A kind of method for tissue culture of capsicum | |
| CN111893133A (en) | Agrobacterium-mediated cabbage heart genetic transformation method | |
| CN108795971B (en) | Application of miR159 in changing plant root system morphology | |
| Moghaieb et al. | Plant regeneration from hypocotyl and cotyledon explant of tomato (Lycopersicon esculentum Mill.) | |
| Agarwal et al. | Comparison of genetic transformation in Morus alba L. via different regeneration systems | |
| CN107022561B (en) | Culture medium and culture method for cultivating transgenic corn | |
| CA2505499C (en) | Methods of plant regeneration and transformation | |
| CN110172476B (en) | Method for establishing genetic transformation and regeneration system of Rhododendron delavayi Franch | |
| CN109997693B (en) | Genetic transformation method of alfalfa | |
| EP0536327A4 (en) | ||
| WO2002067652A2 (en) | Novel mutilple shoot proliferation and regeneration system for plants | |
| CN107012162B (en) | Agrobacterium-mediated cotton embryo tip rapid transformation method | |
| KR20100053456A (en) | A method for producing transformed rice plant removed selection marker | |
| Kose et al. | Agrobacterium-Mediated) Transformation of Cucumber (Cucumis Sativus L.) and Plant Regeneration | |
| AU778695B2 (en) | Novel method for the generation and selection of transgenic linseed/flax plants | |
| CN113637701A (en) | Method for establishing wild rice genetic transformation system | |
| Suri et al. | High frequency regeneration and Agrobacterium tumefaciens-mediated transformation of broccoli (Brassica oleracea var. italica) | |
| CN114606258B (en) | Genetic transformation method of feeding bermuda grass | |
| CN103098714A (en) | Method for improving regeneration in vitro and conversion rate of cucumber cotyledonary node | |
| AU2005330877B2 (en) | Method of constructing transgenic ice plant | |
| CN115851736A (en) | Promoter specifically expressed in flower organ of brassica napus and application thereof | |
| CN111549164A (en) | Molecular marker of tomato environmental phosphorus deficiency related gene and application thereof | |
| Louzada et al. | Production of transgenic Valencia orange suspension cells to be used as donors for chromosome transfer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20220310 Address after: 102206 B401, 4th floor, block B, building 1, No. 27, shengshengyuan Road, Life Science Park, Changping District, Beijing Applicant after: Beijing qiheshengke Biotechnology Co.,Ltd. Address before: 301700 first floor on the east side of building C10, venture headquarters base on the north of Fuyuan Road, Wuqing Development Zone, Wuqing District, Tianjin Applicant before: TIANJIN GENOVO BIOTECHNOLOGY Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |