CN109207514B - High-efficiency genetic transformation method of alpine rhododendron agrobacterium-mediated whole strain infection method - Google Patents
High-efficiency genetic transformation method of alpine rhododendron agrobacterium-mediated whole strain infection method Download PDFInfo
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Abstract
The invention discloses a high-efficiency genetic transformation method of a rhododendron lapponicum agrobacterium-mediated whole strain infection method. The method mainly comprises the following steps: the method comprises the steps of seed pre-culture, ultrasonic treatment, preparation of agrobacterium liquid, agrobacterium infection and vacuum filtration, co-culture, bacteria removal and cleaning, germination culture and GUS histochemical staining. The method has the advantages of simple operation, low experimental cost, short transformation period and high genetic transformation efficiency up to 61 percent, and can be applied to the genetic transformation of other rhododendron plants.
Description
Technical Field
The invention relates to the technical field of plant life science, in particular to a high-efficiency genetic transformation system which is invented by taking Rhododendron delavayi (Rhododendron delavayi), a Rhododendron delavayi, as an experimental material.
Background
Rhododendron (Rhododendron L.) is a famous garden ornamental plant in the world, and is one of the ten traditional flowers in China and the eight famous flowers in Yunnan province in China. Alpine rhododendron is a general name of original species of evergreen broad-leaved rhododendron and filial generations thereof, and gradually becomes a new appearance of Chinese rice glue-leaved azalea flowers and high-grade landscaping due to the fact that the alpine rhododendron is elegant in plant type, large in flower color and evergreen in four seasons. China has more than 320 rhododendron plants, accounts for 1/3 of the rhododendron plants in the world, and southwest and adjacent east Himalayan areas are the distribution center and the origin of evergreen rhododendron, but the breeding research on rhododendron starts late and is far behind the western countries, and the rhododendron varieties sold in the market of China and the total amount thereof are still in the embarrassing situation depending on foreign import to a great extent. Under the situation, researchers need to accelerate the process of genetic improvement and breeding research of rhododendron plants in addition to the conventional cross breeding research of rhododendron plants, but the mature and efficient genetic transformation technology of rhododendron needs to be overcome further. At present, related researches on rhododendron genetic transformation systems all depend on tissue culture means, research objects are limited to Azalea type rhododendron plants, and the bottleneck problems of system instability and low transformation efficiency are difficult to solve. The genetic transformation system of Rhododendron alpine has not been reported successfully so far. Therefore, a set of stable and efficient plant genetic transformation system is needed to be developed for rhododendron alpinum, and a basic technical support is provided for molecular genetic improvement and breeding research of rhododendron plants.
Disclosure of Invention
In order to solve the technical problems, the invention provides a high-efficiency genetic transformation method of a rhododendron lapponicum agrobacterium-mediated whole strain infection method.
The rhododendron plant has large seed quantity and small seed volume, and the seed harvesting is relatively convenient. The invention combines the seed characteristic of rhododendron plants, takes seeds as explants, adopts a method of ultrasonic treatment and vacuum filtration to establish a high-efficiency genetic transformation method of a rhododendron alpinum agrobacterium-mediated whole strain infection method in a non-tissue culture way, and comprises the following steps:
(1) seed pre-culture
Soaking sterilized Rhododendron lapponicum seed in GA solution with pH of 5.2 and containing 0.03g/L31/2MS liquid medium on a shaker at 25 deg.CCulturing at 120rpm with slow shaking for 24 hr, transferring to a dish containing 1/2MS liquid culture medium soaked filter paper, and pre-culturing at 25 + -2 deg.C in dark for 3 days;
(2) ultrasonic treatment
Transferring the pre-cultured seeds into a triangular flask filled with 1/2MS liquid culture medium, and treating for 15min under ultrasonic waves with working frequency of 35kHz and power of 100W;
(3) preparation of Agrobacterium infection liquid
Selecting single colony of Agrobacterium for activation, inoculating the activated bacterial liquid into YEB culture medium containing 100mg/L kanamycin and 50mg/L rifampicin, and performing amplification culture to OD600And (3) centrifuging at 4 ℃ and 400rpm at low speed to collect bacterial liquid 0.6-0.8, and suspending the collected bacterial liquid to OD by using 1/2MS liquid culture medium containing 5g/L of sucrose and 100uM of acetosyringone600Standing for 50-60 min, and then adding Silwett L-77 to obtain an agrobacterium infection solution, wherein the final concentration of the Silwett L-77 is 0.2% v/v;
(4) agrobacterium infection and vacuum filtration
Transferring the seeds treated by the ultrasonic waves in the step (2) to a triangular flask filled with the agrobacterium tumefaciens staining solution prepared in the step (3), covering a membrane cover, carrying out low-speed shaking culture on a shaking table at 28 ℃ and 120rpm for 30min, and then putting the triangular flask with the membrane cover removed into a vacuum pump for vacuum filtration;
(5) co-cultivation
After vacuum filtration, transferring the seeds to sterile filter paper for draining, and then transferring the seeds to 1/2MS solid culture medium containing 100uM acetosyringone for 3 days at 25 +/-2 ℃ in the dark;
(6) bacteria-removing cleaning
First use ddH2Cleaning the co-cultured seeds for 2-3 times by using O, and then using ddH containing 200mg/L of cefuroxime sodium2O, cleaning the seeds for 2-3 times, and then draining on sterile filter paper;
(7) germination culture
Sowing the seeds subjected to drying control in the step (6) on 1/2MS solid culture medium containing 200mg/L of cefuroxime sodium for germination culture at 25 +/-2 DEG CThe illumination intensity is 700 mu mol/m2S, 14 hours light, 10 hours dark daily;
(8) GUS histochemical staining method for detecting transgenic plant
And (4) after 5-6 leaves grow out from the germchit subjected to germination culture in the step (7), carrying out GUS histochemical staining on the complete plant with the root, detecting the rhododendron lapponicum transgenic plant, and simultaneously using the rhododendron lapponicum plant which is not infected by agrobacterium as a negative control material.
Further, the sterilization in step (1) is to apply ddH to the seeds2Cleaning for 1-2 times with O, soaking the seeds in 75% alcohol for 45 seconds, and then soaking in ddH2Cleaning for 1-2 times by using O; soaking the seeds in 2% v/v sodium hypochlorite solution containing Tween-20 for 15min, shaking up at intervals, and adding ddH2O, cleaning the seeds for 3-4 times; the 2% v/v sodium hypochlorite solution added with the Tween-20 is 2-3 drops of Tween-20 added into each 10 ml of 2% v/v sodium hypochlorite solution.
Further, the 1/2MS solid culture medium in the steps (5) and (7) is prepared by adding 7g agar powder into 1/2MS liquid culture medium per liter.
Further, the agrobacterium in the step (3) contains a plant expression vector, and the agrobacterium containing the plant expression vector is obtained by transfecting the plant expression vector into the agrobacterium through a liquid nitrogen repeated freeze-thaw method.
Further, the plant expression vector includes, but is not limited to, pCAMBIA 3301.
Further, the vacuum filtration in the step (4) is a filtration under 508mm Hg for 9 min.
Further, the alpine rhododendron is a Rhododendron delavayi Franch.
Further, said agrobacterium includes, but is not limited to, agrobacterium EHA 105.
Compared with the reported rhododendron plant genetic transformation system, the method has the following innovation points and beneficial effects:
1. fills the technical blank of the alpine rhododendron genetic transformation system.
2. The agrobacterium-mediated non-tissue culture approach genetic transformation method can be applied to genetic transformation of other rhododendron plants and is the first creation in rhododendron plants.
3. The seeds are used as explants, the characteristics of large seed quantity and small seeds of rhododendron lapponicum are fully utilized, and the convenience and reliability in material taking are higher compared with those of other tissue parts.
4. Compared with the genetic transformation method of a tissue culture approach, the method has the advantages of greatly shortened transformation period, simplicity and easiness in operation, low experiment cost and high transformation efficiency. The method of the invention can obtain positive transformant in 50-60 days of transformation period, and the genetic transformation efficiency is up to 61%.
5. For perennial woody plants, the whole plant infection of the seedling is directly carried out to generate a genetic transformation plant, which can greatly shorten the breeding process.
Drawings
FIG. 1: the Rhododendron delavayi plant, which normally germinated without genetic transformation, was a negative control.
FIG. 2: GUS staining result of Rhododendron delavayi genetic transformation positive strain 1.
FIG. 3: GUS staining result of Rhododendron delavayi genetic transformation positive strain 2.
FIG. 4: GUS staining result of Rhododendron delavayi genetic transformation positive line 3.
Detailed Description
The present invention is further illustrated by the following examples, each of which is a conventional method without specific description. Materials and reagents used in the following examples are commercially available.
The special equipment used by the invention is as follows:
an ultrasonic cleaner: shanghai Kegan ultrasonic instruments Inc. (model specification: SK2200LHC, 220V, 50/60 Hz).
A vacuum filtration device: a Millipore vacuum/pressure dual-purpose pump (model: WP6122050, 230V, 50Hz, 1.7A) is assembled with a Nalgene polycarbonate vacuum drying pot for use.
Experimental materials: rhododendron delavayi (Rhododendron delavayi) seed of Rhododendron delavayi.
Example 1
In this embodiment, taking Rhododendron delavayi (Rhododendron delavayi) as an example, the efficient genetic transformation method of the agrobacterium-mediated whole strain infection method includes the following steps:
(1) seed Sterilization and seed Pre-culture
① sterilizing seeds, and mixing the seeds with ddH2O Wash 2 times, then soak seeds in 75% v/v alcohol for 45 seconds, then soak with ddH2O cleaning for 2 times; soaking rhododendron lapponicum seeds in 2% v/v sodium hypochlorite solution added with Tween-20 for 15min, shaking up for 2-3 times, and finally, adding ddH2O, cleaning the seeds for 2-3 times; the 2% v/v sodium hypochlorite solution added with the Tween-20 is 2-3 drops of Tween-20 added into each 10 ml of 2% v/v sodium hypochlorite solution.
② seed pre-culture
Soaking sterilized Rhododendron delavayi Franch seed in GA with pH of 5.2 and containing 0.03g/L3The cultured cells were cultured in 1/2MS liquid medium on a shaker at 25 ℃ and 120rpm for 24 hours with slow shaking, and then transferred to a plate containing 1/2MS liquid medium-soaked filter paper, and pre-cultured in the dark at 25 ℃. + -. 2 ℃ for 3 days.
(2) Ultrasonic treatment
Transferring the pre-cultured Rhododendron delavayi Franch seeds into a triangular flask filled with 1/2MS liquid culture medium, and treating for 15min under ultrasonic waves with working frequency of 35kHz and power of 100W.
(3) Preparation of Agrobacterium infection liquid
Selecting single colony of Agrobacterium containing plasmid pCAMBIA3301, inoculating on YEB culture medium, activating and culturing at 28 deg.C and 220rpm under shaking for 24 hr, inoculating the activated bacterial liquid on YEB culture medium containing 100mg/L kanamycin and 50mg/L rifampicin, and performing amplification culture to OD600And (3) centrifuging at 4 ℃ and 4000rpm at 0.6-0.8, collecting bacterial liquid, and suspending the collected bacterial liquid to OD by using 1/2MS liquid culture medium containing 5g/L sucrose and 100uM acetosyringone600Standing for 50-60 min, and then adding Silwett L-77 to make the final concentration of the Silwett L-77 be 0.2% v/v to obtain an agrobacterium infection solution; the Agrobacterium used in this experiment containing plasmid pCAMBIA3301 was purchased containing CaMV 35The pCAMBIA3301 plant expression vector of GUS reporter gene driven by S promoter is obtained by introducing Agrobacterium EHA105 by liquid nitrogen repeated freeze thawing method. The bacterial resistance of the plant expression vector pCAMBIA3301 is kanamycin, and the plant resistance is glufosinate.
(4) Agrobacterium infection and vacuum filtration
And (3) transferring the seeds of the Rhododendron delavayi Franch treated by the ultrasonic wave in the step (2) into a triangular flask filled with the agrobacterium-infected liquid prepared in the step (3), covering a membrane cover, performing low-speed shaking culture on a shaking table at 28 ℃ and 120rpm for 30min, and then putting the triangular flask with the membrane cover removed into a vacuum pump to perform vacuum filtration for 9min under 508mm Hg.
(5) Co-cultivation
After vacuum filtration, transferring the Rhododendron delavayi Franch seeds to sterile filter paper for draining, and then transferring the Rhododendron delavayi Franch seeds to 1/2MS solid culture medium containing 100uM acetosyringone for dark culture at 25 +/-2 ℃ for 3 days; the 1/2MS solid culture medium is prepared by adding 7g agar powder into 1/2MS liquid culture medium per liter.
(6) Bacteria-removing cleaning
First use ddH2O washing the co-cultured Rhododendron delavayi seeds for 2 times, and adding ddH containing 200mg/L cefuromycin sodium2O washing the Rhododendron delavayi seeds for 3 times, and draining on sterile filter paper.
(7) Germination culture
Sowing the rhododendron delavayi seeds drained on the sterile filter paper in the step (6) on 1/2MS solid culture medium containing 200mg/L of cefamycin sodium, and germinating and culturing under the conditions that the temperature is 25 +/-2 ℃, the illumination intensity is 700 mu mol/m2 s, the illumination is 14 hours per day and the illumination is 10 hours in darkness; the 1/2MS solid culture medium is prepared by adding 7g agar powder into 1/2MS liquid culture medium per liter.
(8) GUS histochemical staining method for detecting transgenic plant
After 5-6 leaves grow out from the germchit of the Rhododendron delavayi cultivated in the step (7) (the seedling age is more than 50 days), a complete plant with roots is taken and subjected to GUS histochemical staining, meanwhile, a negative control experiment is carried out (the negative control material is the Rhododendron delavayi plant which is not infected by agrobacterium), and a transgenic plant of the Rhododendron delavayi is detected.
Calculation formula of genetic transformation efficiency:
genetic transformation efficiency (%) (number of positive transformants)/number of germinated seeds after infection
In this embodiment: 289 grains of Rhododendron delavayi Franch, 216 strains of complete plants with roots obtained by germination culture, and 131 strains of positive transformants which show blue color after GUS histochemical staining, and stable genetic transformants of Rhododendron delavayi Franch (figure 1-figure 4) are obtained, and the genetic transformation efficiency is as high as 61%.
Claims (8)
1. A high-efficiency genetic transformation method of an Agrobacterium alpine agrobacterium mediated whole strain infection method is characterized by comprising the following steps:
(1) seed pre-culture
Soaking sterilized Rhododendron lapponicum seed in GA 0.03g/L with pH =5.23In the 1/2MS liquid culture medium,
culturing on a shaking table at 25 deg.C and 120rpm for 24h with slow shaking, transferring to a dish containing 1/2MS liquid culture medium soaked filter paper, and pre-culturing in dark at 25 deg.C + -2 deg.C for 3 days;
(2) ultrasonic treatment
Transferring the pre-cultured seeds into a triangular flask filled with 1/2MS liquid culture medium, and treating for 15min under ultrasonic waves with working frequency of 35kHz and power of 100W;
(3) preparation of Agrobacterium infection liquid
Selecting single colony of Agrobacterium for activation, inoculating the activated bacterial liquid into YEB culture medium containing 100mg/L kanamycin and 50mg/L rifampicin, and performing amplification culture to OD600= 0.6-0.8, then 400rp at 4 ℃Collecting bacterial liquid by low speed centrifugation, and resuspending the collected bacterial liquid to OD with 1/2MS liquid culture medium containing 5g/L sucrose and 100 μ M acetosyringone600= 0.8-1.0, standing for 50-60 min, and adding Silwett L-77 to obtain an agrobacterium infection solution, wherein the final concentration of the Silwett L-77 is 0.2% v/v;
(4) agrobacterium infection and vacuum filtration
Transferring the seeds treated by the ultrasonic waves in the step (2) to a triangular flask filled with the agrobacterium tumefaciens staining solution prepared in the step (3), covering a membrane cover, carrying out low-speed shaking culture on a shaking table at 28 ℃ and 120rpm for 30min, and then putting the triangular flask with the membrane cover removed into a vacuum pump for vacuum filtration;
(5) co-cultivation
After the vacuum filtration is finished, transferring the seeds onto sterile filter paper for draining, and then transferring the seeds into 1/2MS solid culture medium containing 100 mu M acetosyringone to carry out dark culture at 25 +/-2 ℃ for 3 days;
(6) bacteria-removing cleaning
First use ddH2Cleaning the co-cultured seeds for 2-3 times by using O, and then using ddH containing 200mg/L of cefuroxime sodium2O, cleaning the seeds for 2-3 times, and then draining on sterile filter paper;
(7) germination culture
Sowing the seeds after the drying control in the step (6) on 1/2MS solid culture medium containing 200mg/L of cefuroxime sodium for germination culture at the temperature of 25 +/-2 ℃ and the illumination intensity of 700 mu mol/m2S, 14 hours light, 10 hours dark per day;
(8) GUS histochemical staining method for detecting transgenic plant
After 5-6 leaves grow out from the seedlings subjected to germination culture in the step (7), taking the complete plant with roots for GUS organization
And (4) carrying out chemical dyeing, detecting the rhododendron alpinum transgenic plant, and simultaneously using the rhododendron alpinum plant which is not infected by agrobacterium as a negative control material.
2. The method for efficient genetic transformation of Rhododendron lapponicum by Agrobacterium-mediated whole plant infection according to claim 1, wherein the Agrobacterium-mediated whole plant infection is a whole plant infectionThe method comprises the following steps: the sterilization in the step (1) is to use ddH to treat the seeds2Cleaning for 1-2 times with O, soaking the seeds in 75% alcohol for 45 seconds, and then soaking in ddH2Cleaning for 1-2 times by using O; soaking the seeds in 2% v/v sodium hypochlorite solution containing Tween-20 for 15min, shaking up at intervals, and adding ddH2O, cleaning the seeds for 3-4 times; the 2% v/v sodium hypochlorite solution added with the Tween-20 is 2-3 drops of Tween-20 added into each 10 ml of 2% v/v sodium hypochlorite solution.
3. The method for efficient genetic transformation of rhododendron lapponicum through agrobacterium-mediated whole strain infection according to claim 1, wherein the method comprises the following steps: the 1/2MS solid culture medium in the steps (5) and (7) is prepared by adding 7g agar powder into 1/2MS liquid culture medium per liter.
4. The method for efficient genetic transformation of rhododendron lapponicum through agrobacterium-mediated whole strain infection according to claim 1, wherein the method comprises the following steps: the agrobacterium in the step (3) contains a plant expression vector, and is obtained by transfecting the plant expression vector into agrobacterium through a freeze-thaw method.
5. The method for efficient genetic transformation of Rhododendron lapponicum by Agrobacterium-mediated whole strain infection according to claim 4, wherein the genetic transformation comprises: the plant expression vector includes, but is not limited to pCAMBIA 3301.
6. The method for high-efficiency genetic transformation of the Rhododendron lapponicum Agrobacterium-mediated whole-plant infection method according to claim 1,
the method is characterized in that: the vacuum filtration in the step (4) is a filtration under 508mm Hg for 9 min.
7. The method for high-efficiency genetic transformation of the Rhododendron lapponicum Agrobacterium-mediated whole-plant infection method according to claim 1,
the method is characterized in that: the alpine rhododendron is a Rhododendron delavayi Franch.
8. The method for efficient genetic transformation of Agrobacterium alpina mediated whole plant infection according to any one of claims 1 to 7, wherein the method comprises the steps of: such Agrobacterium include, but are not limited to Agrobacterium EHA 105.
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| CN111235176B (en) * | 2020-01-17 | 2020-12-01 | 北京农学院 | a genetic transformation system |
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| CN116369311A (en) * | 2023-04-04 | 2023-07-04 | 中国电建集团贵阳勘测设计研究院有限公司 | Preservation, germination and seedling raising method for improving germination rate of rhododendron alpine seeds |
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