CN109984041B - Azalea transgenic method with leaves as explants - Google Patents
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- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 33
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- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 12
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- 238000012258 culturing Methods 0.000 claims description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 33
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- 241000894006 Bacteria Species 0.000 claims description 8
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- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 5
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- 238000006243 chemical reaction Methods 0.000 claims description 2
- 239000012883 rooting culture medium Substances 0.000 claims description 2
- 238000002054 transplantation Methods 0.000 claims 1
- 230000009466 transformation Effects 0.000 abstract description 17
- 230000015572 biosynthetic process Effects 0.000 abstract description 6
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 239000000463 material Substances 0.000 abstract description 3
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a rhododendron transgenic method taking leaves as explants, belonging to the technical field of molecular biology. The method uses the leaves as explants, and the explants are directly subjected to agrobacterium infection transformation after disinfection and culture, so that time is saved compared with the method that the callus is infected by agrobacterium after the callus is obtained. The callus induction culture medium is used in the processes of preculture, co-culture and selective culture after the disinfection of the explant, and is beneficial to the formation and redifferentiation of the callus, and the same culture medium is used from the formation of the callus and the differentiation of the cluster buds to the proliferation of the cluster buds, so that the complicated procedure of configuring different culture media in the tissue culture process is omitted, and the tissue culture time, the manpower and the material resources are saved.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a rhododendron transgenic method taking leaves as explants.
Background
China is the country with the most varieties of rhododendrons in the world, and the southwest region of China is the hometown of the world recognized rhododendrons. The rhododendron is commonly used as a landscape plant or a gardening potted plant, is used for greening and beautifying public garden environment and indoor and outdoor colors, and has extremely high ornamental value. At present, the domestic research on rhododendron is focused on the fields of resource investigation, morphological classification, sporopollen, ecology, distribution, phytochemistry and the like, and the research on the aspect of molecular biology is slow. The establishment of a genetic transformation system has great significance for promoting the development of rhododendron molecular biology. The reported genetic transformation of rhododendrons is mainly the infection of stem segments by agrobacterium-mediated genetic transformation and the transformation of leaves by gene gun method. The callus formation and plant regeneration efficiency is low when the stem section is used as the explant for genetic transformation, and the requirements on gene gun instrument equipment are high and the cost is high.
The invention provides a rhododendron transgenic method taking leaves as explants, which improves the transformation efficiency, reduces the transformation cost and provides convenience for molecular biology research of rhododendron.
Disclosure of Invention
The invention provides a rhododendron transgenic method taking leaves as explants, aiming at the problems of low genetic transformation efficiency and high cost of the existing rhododendron. The method uses the leaves as explants, and the explants are directly subjected to agrobacterium infection transformation after disinfection and culture, so that time is saved compared with the method that the callus is infected by agrobacterium after the callus is obtained. The callus induction culture medium is used in the processes of preculture, co-culture and selective culture after the disinfection of the explant, and is beneficial to the formation and redifferentiation of the callus, and the same culture medium is used from the formation of the callus and the differentiation of the cluster buds to the proliferation of the cluster buds, so that the complicated procedure of configuring different culture media in the tissue culture process is omitted, and the tissue culture time, the manpower and the material resources are saved.
In order to realize the purpose, the invention adopts the following technical scheme:
a rhododendron transgenic method using leaves as explant includes such steps as using the leaves of rhododendron as explant, cutting the disinfected leaves into small blocks, pre-culturing in callus inducing culture medium, taking out the leaves, infecting by Agrobacterium, co-culturing, selective culturing and secondary selective culturing, rooting culturing, transplanting and identifying transgenic plant.
A rhododendron transgenic method taking leaves as explants comprises the following steps: leaf collection and disinfection, pre-culture, agrobacterium infection, co-culture, selective culture, subculture selection culture, rooting culture and transplanting, and transgenic plant identification.
A rhododendron transgenic method taking leaves as explants comprises the following specific steps:
(1) Leaf collection and disinfection: taking young and tender branches with leaves of rhododendron, washing for 30-50 min by using tap water, sucking surface water drops to be dry by using filter paper, then putting the branches into a beaker, disinfecting for 45 s by using 75vol% alcohol in a super clean workbench, and cleaning twice by using sterile water; at 100 mL of 0.1wt% mercuric chloride (HgC 1) 2 ) Soaking for 6-8 min, washing with sterile water for 5-6 times, soaking in sterile water, cutting into 0.5 cm × 0.5 cm square, and making 2-3 cuts in the middle of the blade.
(2) Pre-culturing: inoculating the leaf surface cut in the step (1) on a callus induction culture medium, and culturing in the dark at 4 ℃ for 2-3 days.
(3) Infection of agrobacterium: adding the GV3101 Agrobacterium liquid carrying expression vector into liquid LB culture medium containing Kan of 100 ug/ml, rif of 50 ug/ml and Spec of 100 ug/ml in the ratio of 1-2 vol%, 28 deg.C, 180 rpmCultured to OD 600 Transferring the bacterial liquid into a 50 mL sterile centrifuge tube for 5 min at 4000 rpm at room temperature to remove supernatant, re-suspending the bacterial cells with BME liquid culture medium with pH of 5.6, and diluting to OD 600 And (3) 0.5 to 0.8, adding AS with the final concentration of 20 mg/L for conversion, taking out the leaf explant pre-cultured in the step (2) in a small sterile culture dish in an ultraclean workbench, pouring prepared re-suspension bacterial liquid, soaking for 20 min, and removing surface bacterial liquid by using sterile filter paper.
(4) Co-culturing: inoculating the infected leaves in the step (3) on a leaf callus induction culture medium padded with a layer of sterile filter paper, and culturing in the dark at 25 ℃ for 2-3 days.
(5) Selecting and culturing: and (5) washing the leaves co-cultured in the step (4) with sterile water containing Cef with the final concentration of 600 mg/L for 3 times, washing the leaves with sterile water without antibiotics for 5 min for two times, sucking dry water with filter paper, transferring the leaves to a callus induction culture medium containing Kan with the final concentration of 60 mg/L, culturing the leaves in dark for 15 days, culturing the leaves in light for 40 to 50 days, differentiating the explants to generate resistant adventitious buds, starting to unfold the leaves, and culturing the leaves at the temperature of 25 ℃.
(6) Subculture selection culture: transferring the resistant adventitious bud differentiated in the step (5) into a new selective culture medium containing Kan with the final concentration of 60 mg/L for subculture propagation at the culture temperature of 25 ℃, subculturing once every 30 days, and performing rooting culture after 2-3 subcultures.
(7) Rooting culture and transplanting: and (4) cutting off the adventitious bud after the adventitious bud cultured by the subculture selection in the step (6) grows to be more than 2 cm, and inserting the cut adventitious bud into a rooting culture medium containing Kan with the final concentration of 60 mg/L to perform rooting culture, wherein the culture temperature is 25 ℃, and adventitious roots grow out in 14 to 20 days.
(8) Identification of transgenic plants: and after rooting culture for 4-8 weeks, opening a culture bottle cap, hardening seedlings for 2 days, transplanting the tissue culture seedlings into a culture medium, and observing and detecting whether transgenosis succeeds or not through PCR, GUS dyeing and GFP.
In the above method, the callus induction medium is: BME +10 mg/L ZT +10 mg/L IAA, pH5.6.
In the method, the rooting medium is BME plus 1 mg/L IAA, and the pH value is 5.6.
Further, in the above method, the expression vector in the step (3) carriesGUSGenes andGFPgenes, and Kan resistance genes.
The invention has the beneficial effects that:
1. the method uses leaf as explant, and has wide source without tissue culture. The disinfection and the direct agrobacterium infection after the culture of the explant save more time compared with the infection of the callus by the agrobacterium after the callus is obtained.
2. The callus induction culture medium is used in the processes of pre-culture, co-culture and selective culture after explant disinfection, and is beneficial to callus formation and redifferentiation, and the same culture medium is used in the processes, so that the complex procedure of configuring different culture media in the tissue culture process is omitted, and the tissue culture time, manpower and material resources are saved.
Description of the drawings:
FIG. 1 rooting of resistant plants.
FIG. 2 shows the results of transgenic plant detection. Panel A shows GUS staining results, and the right most panel shows a non-transgenic control; the GFP observation result of the picture B, 1 is a transgenic plant, 2 is a non-transgenic contrast, and the arrow points to the edge of a non-transgenic leaf; the C picture is an electrophoresis detection result, M is DL2000 marker, and 1-10 are resistance plants to be detected; -is a negative control, + is a positive control, and H is a water control.
Detailed Description
The present invention will be further illustrated with reference to the following examples, but the present invention is not limited thereto.
Example 1
A rhododendron transgenic method taking leaves as explants comprises the following specific steps:
(1) Leaf collection and disinfection: taking young and tender branches with leaves of the 'Zhuangyuan red' rhododendron, and washing for 45 min by using tap water. Sucking surface water drops with filter paper, putting the filter paper into a beaker, disinfecting the beaker for 45 s with 75vol% alcohol in a clean bench, and cleaning the beaker with sterile water twice; at 100 mL of 0.1wt% mercuric chloride (HgC 1) 2 ) Soaking for 7 min, and cleaning with sterile waterAfter washing 5 times, the tube was immersed in sterile water. The leaves were cut into 0.5 cm × 0.5 cm squares in sterile water, after which 2 cuts were made in the middle of the leaves.
(2) Pre-culturing: inoculating the leaf surface cut in the step (1) on a callus induction culture medium (BME +10 mg/L ZT +10 mg/L IAA, pH5.6), and culturing in the dark at 4 ℃ for 2 days.
(3) Infection of agrobacterium: taking a vector carrying pK7FWG2-GUS expression vector (the vector carriesGUSGenes andGFPgenes, and Kan resistance genes) [ Karimi, M.; inze, D.; depicker, A. Gateway vectors for Agrobacterium-mediated Plant transformation. Trends Plant Sci. 2002, 7, 193-195.]The GV3101 Agrobacterium solution of (2) was added to a liquid LB medium (containing 100. Mu.g/ml Kan, 50. Mu.g/ml Rif and 100. Mu.g/ml Spec at a final concentration of 1.5 vol%) and cultured at 28 ℃ and 180 rpm until OD600 became 0.8. Transferring the bacterial liquid into a 50 mL sterile centrifuge tube, centrifuging at 4000 rpm for 5 min at room temperature, removing supernatant, re-suspending the thallus with BME liquid culture medium (pH 5.6), and diluting to OD 600 0.6, while AS was added at a final concentration of 20 mg/L for transformation. And (3) taking the explant pre-cultured in the step (2) out of a clean bench, putting the explant into a small sterile culture dish, pouring prepared heavy suspension bacteria liquid, soaking for 20 min, and absorbing surface bacteria liquid by using sterile filter paper.
(4) Co-culturing: the infected leaves in step (3) were inoculated on leaf callus induction medium (BME +10 mg/L ZT +10 mg/L IAA, pH 5.6) on which a layer of filter paper was laid, and cultured in the dark at 25 ℃ for 3 days.
(5) Selecting and culturing: the leaves after co-cultivation were washed 3 times with sterile water containing Cef at a final concentration of 600 mg/L for 5 min each time, washed 3 times with sterilized water without antibiotics, blotted dry with filter paper, and transferred to callus induction medium (BME +10 mg/L ZT +10 mg/L IAA, pH 5.6) with 60 mg/L Kan added. Culturing in dark for 15 days, and culturing at 25 deg.C under light. After 45 days the explants differentiated resistant adventitious buds and the leaves had begun to spread.
(6) Subculture selection culture: transferring the resistant adventitious bud differentiated in the step (5) into a new selection culture medium (BME +10 mg/L ZT +10 mg/L IAA, pH 5.6) containing 60 mg/L Kan at the final concentration for subculture propagation at the temperature of 25 ℃, subculturing once every 30 days, and carrying out rooting culture after 2 subcultures.
(7) Rooting culture and transplanting: when the adventitious bud cultured by the subculture selection in the step (6) grows to 2 cm, the cut adventitious bud is cut off and inserted into a rooting medium (BME +1 mg/L IAA, pH 5.6) containing Kan at a final concentration of 60 mg/L for rooting culture at a culture temperature of 25 ℃ for 18 days to grow adventitious roots (FIG. 1).
(8) Identification of transgenic plants: after rooting culture for 4 weeks, opening a culture bottle cap, hardening seedlings for 2 days, and then transplanting the tissue culture seedlings into a culture medium. The success of the transgene was examined by PCR, GUS staining and GFP observation. The PCR detection result shows that the transgenic plant can be obtained by the method, the GUS dyeing and GFP observation results also prove that the transgenic plant is positive, and the detection result is shown in figure 2.
Example 2
A rhododendron transgenic method taking leaves as explants comprises the following specific steps:
(1) Leaf collection and disinfection: taking young and tender branches with leaves of the 'Zhuangyuan red' rhododendron, and washing for 30 min by using tap water. Sucking surface water drops with filter paper, putting the surface water drops into a beaker, disinfecting the beaker with 75vol% alcohol for 45 s in a superclean bench, and cleaning the beaker with sterile water twice; at 100 mL of 0.1wt% mercuric chloride (HgC 1) 2 ) Soaking in sterile water for 6 min, washing with sterile water for 6 times, and soaking in sterile water. The leaves were cut into 0.5 cm × 0.5 cm squares in sterile water, after which 2 cuts were made in the middle of the leaves.
(2) Pre-culturing: inoculating the cut leaf surface in the step (1) on a callus induction culture medium (BME +10 mg/L ZT +10 mg/L IAA, pH5.6), and culturing in the dark at 4 ℃ for 2 days.
(3) Infection of agrobacterium: taking an expression vector carrying pK7FWG2-GUS (the vector carriesGUSGenes andGFPgenes, and Kan resistance genes) [ Karimi, M.; inze, D.; depicker, A. Gateway vectors for Agrobacterium-mediated Plant transformation. Trends Plant Sci. 2002, 7, 193-195.]The GV3101 Agrobacterium solution of1vol% was added to liquid LB medium (containing Kan at a final concentration of 100. Mu.g/ml, 50. Mu.g/ml Rif and 100. Mu.g/ml Spec) and cultured at 28 ℃ and 180 rpm until OD600 became 0.8. Transferring the bacterial liquid into a 50 mL sterile centrifuge tube, centrifuging at 4000 rpm for 5 min at room temperature, removing supernatant, re-suspending the thallus with BME liquid culture medium (pH 5.6), and diluting to OD 600 0.5, while AS was added to a final concentration of 20 mg/L for transformation. And (3) taking the explants pre-cultured in the step (2) out of a clean bench, putting the explants into a small sterile culture dish, pouring prepared heavy suspension bacteria liquid, soaking for 20 min, and absorbing surface bacteria liquid by using sterile filter paper.
(4) Co-culturing: inoculating the infected leaves in the step (3) on a leaf callus induction culture medium (BME +10 mg/L ZT +10 mg/L IAA, pH5.6) on which a layer of filter paper is laid, and carrying out dark culture at 25 ℃ for 3 days.
(5) Selecting and culturing: the co-cultured leaves were washed 3 times with sterile water containing Cef at a final concentration of 600 mg/L for 5 min each time, washed 3 times with sterilized water without antibiotics, and then dried by filter paper, and transferred to callus induction medium (BME +10 mg/L ZT +10 mg/L IAA, pH 5.6) to which Kan was added at a final concentration of 60 mg/L. Culturing in dark for 15 days, and culturing in light at 25 deg.C. After 40 days the explants differentiated resistant adventitious buds and the leaves had begun to spread.
(6) Subculture selection culture: transferring the resistant adventitious bud differentiated in the step (5) into a new selection culture medium (BME +10 mg/L ZT +10 mg/L IAA, pH 5.6) containing 60 mg/L Kan at the final concentration for subculture propagation at the temperature of 25 ℃, subculturing once every 30 days, and carrying out rooting culture after 2 subcultures.
(7) Rooting culture and transplanting: when the adventitious bud cultured by the subculture selection in the step (6) grows to 3 cm, cutting off and inserting the cut adventitious bud into a rooting medium (BME +1 mg/L IAA, pH 5.6) containing Kan with the final concentration of 60 mg/L to perform rooting culture, wherein the culture temperature is 25 ℃, and the adventitious root grows out in 15 days.
(8) Identification of transgenic plants: after rooting culture for 4 weeks, opening a culture bottle cap, hardening seedlings for 2 days, and then transplanting the tissue culture seedlings into a culture medium. The success of the transgene was examined by PCR, GUS staining and GFP observation. The PCR detection result shows that the transgenic plant can be obtained by the method, and the GUS dyeing and GFP observation results also prove that the transgenic plant is positive.
Example 3
A rhododendron transgenic method taking leaves as explants comprises the following specific steps:
(1) Leaf collection and disinfection: taking tender and leafy branches of the 'Zhuangyuan red' rhododendron, and washing for 50min by using tap water. Sucking surface water drops with filter paper, putting the filter paper into a beaker, disinfecting the beaker for 45 s with 75vol% alcohol in a clean bench, and cleaning the beaker with sterile water twice; at 100 mL of 0.1wt% mercuric chloride (HgC 1) 2 ) Soaking in sterile water for 8min, washing with sterile water for 5 times, and soaking in sterile water. The leaves were cut into 0.5 cm × 0.5 cm squares in sterile water, after which 3 cuts were made in the middle of the leaves.
(2) Pre-culturing: inoculating the leaf surface cut in the step (1) on a callus induction culture medium (BME +10 mg/L ZT +10 mg/L IAA, pH5.6), and culturing in the dark at 4 ℃ for 2 days.
(3) Infection of agrobacterium: taking a vector carrying pK7FWG2-GUS expression vector (the vector carriesGUSGenes andGFPgenes, and Kan resistance genes) [ Karimi, M.; inze, D.; depicker, A. Gateway vectors for Agrobacterium-mediated Plant transformation. Trends Plant Sci. 2002, 7, 193-195.]The GV3101 Agrobacterium solution of (2 vol%) was added to a liquid LB medium (containing 100. Mu.g/ml Kan, 50. Mu.g/ml Rif and 100. Mu.g/ml Spec) and cultured at 28 ℃ and 180 rpm until OD600 became 0.8. Transferring the bacterial liquid into a 50 mL sterile centrifuge tube, centrifuging at 4000 rpm for 5 min at room temperature, removing supernatant, re-suspending the thallus with BME liquid culture medium (pH 5.6), and diluting to OD 600 0.8, while adding AS at a final concentration of 20 mg/L for transformation. And (3) taking the explant pre-cultured in the step (2) out of a clean bench, putting the explant into a small sterile culture dish, pouring prepared heavy suspension bacteria liquid, soaking for 20 min, and absorbing surface bacteria liquid by using sterile filter paper.
(4) Co-culturing: inoculating the infected leaves in the step (3) on a leaf callus induction culture medium (BME +10 mg/L ZT +10 mg/L IAA, pH5.6) on which a layer of filter paper is laid, and carrying out dark culture at 25 ℃ for 3 days.
(5) Selecting and culturing: the leaves after co-cultivation were washed 3 times with sterile water containing Cef at a final concentration of 600 mg/L for 5 min each time, washed 3 times with sterilized water without antibiotics, blotted dry with filter paper, and transferred to callus induction medium (BME +10 mg/L ZT +10 mg/L IAA, pH 5.6) with 60 mg/L Kan added. Culturing in dark for 15 days, and culturing in light at 25 deg.C. After 50 days the explants differentiated resistant adventitious buds and the leaves had begun to spread.
(6) Subculture selection culture: transferring the resistant adventitious bud differentiated in the step (5) into a new selective culture medium (BME +10 mg/L ZT +10 mg/L IAA, pH 5.6) containing 60 mg/L Kan at the final concentration for subculture propagation at the temperature of 25 ℃, subculturing once every 30 days, and performing rooting culture after 3 subculturing.
(7) Rooting culture and transplanting: when the adventitious bud cultured by the subculture selection in the step (6) grows to 2.5 cm, cutting off and inserting the cut adventitious bud into a rooting medium (BME +1 mg/L IAA, pH 5.6) containing Kan with the final concentration of 60 mg/L to perform rooting culture, wherein the culture temperature is 25 ℃, and the adventitious root grows out after 20 days.
(8) Identification of transgenic plants: after rooting culture for 4 weeks, opening a culture bottle cap, hardening the seedlings for 2 days, and then transplanting the tissue culture seedlings into a culture medium. The success of the transgene was examined by PCR, GUS staining and GFP observation. The PCR detection result shows that the transgenic plant can be obtained by the method, and the GUS dyeing and GFP observation results also prove that the transgenic plant is positive.
As can be seen from the above, the rhododendron transgenic method using leaves as explants can obtain transgenic plants within 6 months, has high transformation efficiency, and is a relatively high-efficiency rhododendron transgenic method.
Claims (2)
1. A rhododendron transgenic method taking leaves as explants is characterized in that: the method takes rhododendron mosaic as an explant, cuts disinfected leaves into small blocks, puts the small blocks on a callus induction culture medium for pre-culture, takes out the leaves for agrobacterium infection, then carries out co-culture, selective culture and subculture selection culture, and carries out transplantation and identification of transgenic plants after rooting culture;
the method comprises the following specific steps:
(1) Leaf collection and disinfection: taking young and tender branches with leaves of rhododendron, washing for 30-50 min by using tap water, sucking surface water drops to be dry by using filter paper, then putting the branches into a beaker, disinfecting for 45 s by using 75vol% alcohol in a super clean workbench, and cleaning twice by using sterile water; soaking in 100 mL of 0.1wt% mercuric chloride for 6-8 min, washing with sterile water for 5-6 times, soaking in sterile water, cutting the leaves into 0.5 cm × 0.5 cm squares in sterile water, and making 2-3 cuts in the middle of the leaves;
(2) Pre-culturing: inoculating the cut leaves in the step (1) with leaf surfaces facing upwards to a callus induction culture medium BME +10 mg/L ZT +10 mg/L IAA at the pH value of 5.6, and carrying out dark culture at the temperature of 4 ℃ for 2-3 days;
(3) Infection of agrobacterium: adding the GV3101 Agrobacterium liquid carrying the expression vector into a liquid LB culture medium containing 100 ug/ml Kan, 50 ug/ml Rif and 100 ug/ml Spec in a ratio of 1-2 vol%, culturing at 28 deg.C and 180 rpm to OD 600 To 0.8, transferring the bacterial liquid into a 50 mL sterile centrifuge tube, centrifuging at 4000 rpm for 5 min at room temperature, removing supernatant, resuspending the thallus with BME liquid culture medium with pH5.6, and diluting to OD 600 0.5 to 0.8, adding AS with the final concentration of 20 mg/L for conversion, taking out the leaf explant pre-cultured in the step (2) in a small sterile culture dish in an ultraclean workbench, pouring prepared heavy suspension bacteria liquid, soaking for 20 min, and removing surface bacteria liquid by using sterile filter paper;
(4) Co-culturing: inoculating the infected leaves in the step (3) on a leaf callus induction culture medium BME +10 mg/L ZT +10 mg/L IAA padded with a layer of sterile filter paper, and culturing in the dark at 25 ℃ for 2-3 days at the pH of 5.6;
(5) Selecting and culturing: washing the leaves co-cultured in the step (4) with sterile water containing Cef with the final concentration of 600 mg/L for 3 times, 5 min each time, washing with antibiotic-free sterile water twice, sucking dry water with filter paper, transferring to a callus induction culture medium BME +10 mg/L ZT +10 mg/L IAA with the final concentration of 60 mg/L Kan, pH5.6, culturing in dark for 15 days, then culturing in light for 40-50 days, at the moment, differentiating resistant adventitious buds from the explant, and starting to develop the leaves, wherein the culture temperature is 25 ℃;
(6) Subculture selection culture: transferring the resistant adventitious bud differentiated in the step (5) into a new selection culture medium BME +10 mg/L ZT +10 mg/L IAA with a final concentration of 60 mg/L Kan, carrying out subculture propagation at the pH of 5.6, carrying out subculture once every 30 days at the culture temperature of 25 ℃, and carrying out rooting culture after 2-3 subcultures;
(7) Rooting culture and transplanting: cutting off and inserting a rooting culture medium BME +1 mg/L IAA containing Kan with the final concentration of 60 mg/L when the adventitious bud cultured by the subculture selection in the step (6) grows to be more than 2 cm, and carrying out rooting culture at the pH value of 5.6, wherein the culture temperature is 25 ℃, and adventitious roots grow out in 14-20 days;
(8) Identification of transgenic plants: and after rooting culture for 4-8 weeks, opening a culture bottle cap, hardening seedlings for 2 days, transplanting the tissue culture seedlings into a culture medium, and observing and detecting whether transgenosis succeeds or not through PCR, GUS dyeing and GFP.
2. The method for transgenic rhododendron with leaves as explants according to claim 1, wherein the expression vector in step (3) carriesGUSGenes andGFPgenes, and Kan resistance genes.
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