CN114921491B - Method for transient expression of agrobacterium-mediated paeonia lactiflora - Google Patents

Method for transient expression of agrobacterium-mediated paeonia lactiflora Download PDF

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CN114921491B
CN114921491B CN202210684960.9A CN202210684960A CN114921491B CN 114921491 B CN114921491 B CN 114921491B CN 202210684960 A CN202210684960 A CN 202210684960A CN 114921491 B CN114921491 B CN 114921491B
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孙晓梅
葛佳媛
裴新辉
费日雯
赵晴雯
霍丽茹
康雪宁
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Shenyang Agricultural University
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Abstract

The application provides a method for transient expression of agrobacterium-mediated paeonia lactiflora, which comprises the following steps: completely immersing the target paeonia lactiflora seedlings in a preset conversion solution, and carrying out treatment with the negative pressure intensity of 10 to obtain target paeonia lactiflora tissue culture seedlings and seedlings; infecting the target paeonia lactiflora tissue culture Miao Zhendang for 24 hours in the dark at 28 ℃, and carrying out oscillation infection on the paeonia lactiflora seedlings for 12 hours to obtain target paeonia lactiflora tissue culture seedlings after infection and paeonia lactiflora seedlings after infection; respectively inoculating the infected target peony tissue culture seedling and the infected peony seedling into a solid co-culture medium and fine sand, and culturing for 3 days under the dark condition at 25 ℃ to obtain the cultured target peony tissue culture seedling and the cultured seedling; GUS staining is carried out on the cultured target paeonia lactiflora tissue culture seedlings and the seedlings, so that the transient over-expression of the target genes is completed. The method does not depend on expensive experimental equipment, and has low cost; the conversion process is simple and quick, and the whole process only needs 4-5 days; the conversion rate can reach 93.3%, and powerful technical support is provided for analysis of peony gene functions.

Description

Method for transient expression of agrobacterium-mediated paeonia lactiflora
Technical Field
The application relates to the technical field of plant genetic transformation, in particular to a method for transient expression of agrobacterium-mediated paeonia lactiflora.
Background
Paeonia lactiflora (Paeonia lactiflora pall.) is a perennial root herb flower of Paeonia genus of Paeoniaceae family, is a traditional flower of China, is widely introduced as it has medicinal value and is an economic plant providing excellent ornamental value, is an important garden application flower, and is deeply favored by people.
With the increasing growing area, the environmental challenges that paeonia will face are more complex, so that research on the functions of the genes related to the resistance of paeonia has important significance. At present, the common gene function research is to carry out stable inheritance on a target gene in the plant to obtain a transgenic plant. In the aspect of paeonia, although research on tissue culture of paeonia has been greatly advanced at home and abroad at present, the problems that explants are easy to pollute, brown and vitrify, callus is difficult to redifferentiate, robust cluster seedlings are difficult to induce, and a mature genetic transformation system is difficult to root are not established yet exist, so that gene function multi-utilization mode plants of paeonia, such as tobacco, arabidopsis and the like, are researched at home and abroad at present.
The transient expression of the plant can realize transient high-level expression of the target gene in a short time, and compared with stable transformation, the transient expression of the target gene does not depend on chromosome integration of heterologous DNA, and the transient expression of the plant is simpler and faster to operate and has low cost, so that the technology becomes a powerful tool for researching the plant gene function without a regeneration system. Common methods of transient transformation are particle bombardment transformation, PEG-mediated protoplast transformation, plant viral vector-mediated transformation and agrobacterium-mediated transformation, which offer high transformation efficiency compared to other transformation methods, because the intercellular space is one third of the plant tissue volume, agrobacterium is actively transported to the intercellular space by osmosis, which provides an efficient way for agrobacterium to enter most plant cells, allowing efficient transfer of T-DNA, and which does not require instrumentation, while allowing handling of large numbers of plants, and multiple transient expression analyses with several transgene constructs on one leaf. Currently, this technology has been successfully applied to many plants such as arabidopsis thaliana, birch, willow, poplar, upland cotton, china rose, cotton, and the like. However, research on establishing a transient expression system in paeonia has not been reported yet.
Disclosure of Invention
In view of the above, the present application aims to provide a method for transient expression of paeonia lactiflora mediated by agrobacterium, which solves the problems of paeonia lactiflora in gene function research to a certain extent, and provides a method for fast and efficiently realizing transient expression of target genes in paeonia lactiflora seedlings, which is used for functional verification research of genes related to resistance to paeonia lactiflora.
The embodiment of the application provides a method for transient expression of agrobacterium-mediated paeonia lactiflora, which comprises the following steps:
immersing target paeonia lactiflora seedlings in a preset transformation solution completely, and carrying out treatment with negative pressure intensity of 10 to obtain target paeonia lactiflora tissue culture seedlings and seedlings, wherein the target paeonia lactiflora seedlings are paeonia lactiflora tissue culture seedlings obtained by germinating embryo of hybrid seeds of paeonia lactiflora and paeonia lactiflora in a starting culture medium for 30 days and seedlings growing 2-3cm buds through sand storage, and the preset transformation solution is obtained by carrying out secondary activation OD (optical density) on the preset transformation solution 600 The agrobacterium solution with the value of 1.2 is centrifuged and resuspended in the transformation solution containing AS and surfactant;
infecting the target paeonia lactiflora tissue culture Miao Zhendang for 24 hours in the dark at 28 ℃, and carrying out oscillation infection on the paeonia lactiflora seedlings for 12 hours to obtain target paeonia lactiflora tissue culture seedlings after infection and paeonia lactiflora seedlings after infection;
respectively inoculating the infected target peony tissue culture seedling and the infected peony seedling into a solid co-culture medium and fine sand, and culturing for 3 days under the dark condition at 25 ℃ to obtain the cultured target peony tissue culture seedling and the cultured seedling;
GUS staining is carried out on the cultured target paeonia lactiflora tissue culture seedlings and the seedlings, so that the transient over-expression of the target genes is completed.
Optionally, the OD 600 The agrobacterium solution with the value of 1.2 is obtained by performing secondary activation in 100mL of LB liquid medium containing 50mg/L Kan and 50mg/L Rif, wherein the LB liquid medium consists of yeast extract powder, tryptone and NaCl, 5g of yeast extract powder, 10g of tryptone and 10g of NaCl are added into each liter, and the pH value is 7.0; the plant expression vector is pCAMBIA1301 containing GUS reporter gene; the agrobacterium strain is EHA105; culturing the bacterial liquid at 28 ℃ at 200rpm in a dark way; bacterial liquid OD 600 The values were measured using a spectrophotometer; the conversion liquid is MS, MES, mgCl 2 、AS、Tween-20 and sucrose, wherein MS 0.214g, MES 1mM, mgCl are added per 100mL 2 2mM, AS 200. Mu. M, tween-20.01% and sucrose 3g, pH 5.8.
Alternatively, the solid co-culture medium is composed of MS, GA 3 6-BA, AS, sucrose and agar, wherein MS 2.14g, sucrose 30g, AS 200 mu M, GA are added per liter 3 0.5mg, 6-BA1.0mg and agar 6.5g, pH 5.8.
Optionally, the step of performing GUS staining on the cultured target paeonia lactiflora tissue culture seedlings and the seedlings to complete transient over-expression of the target genes comprises the following steps:
immersing the cultured target paeonia lactiflora tissue culture seedlings and the seedlings in GUS staining solution, and performing GUS histochemical staining to complete transient over-expression of the target genes.
In order to make the above objects, features and advantages of the present application more comprehensible, preferred embodiments accompanied with figures are described in detail below.
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In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered limiting the scope, and that other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a GUS staining photograph of a peony tissue culture seedling;
FIG. 2 is a GUS staining photograph of a peony seedling;
FIG. 3 is the effect of germination time on transformation efficiency of Paeonia lactiflora tissue culture Miao Shunshi;
FIG. 4 is the effect of bacterial fluid concentration on transformation efficiency of peony tissue culture Miao Shunshi;
FIG. 5 is the effect of infection time on transformation efficiency of Paeonia lactiflora tissue culture Miao Shunshi;
FIG. 6 is the effect of AS concentration on transformation efficiency of paeonia lactiflora tissue culture Miao Shunshi;
FIG. 7 is the effect of co-culture time on transformation efficiency of paeonia lactiflora tissue culture Miao Shunshi;
FIG. 8 is the effect of negative pressure intensity on transformation efficiency of paeonia lactiflora tissue culture Miao Shunshi;
FIG. 9 is the effect of Tween-20 concentration on transformation efficiency of paeonia lactiflora tissue culture Miao Shunshi;
FIG. 10 is the effect of infection time on transient transformation efficiency of peony seedlings;
FIG. 11 is the expression level of stress-resistant gene in transient over-expressed Paeonia lactiflora seedlings under abiotic stress.
Detailed Description
For the purposes of making the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is apparent that the described embodiments are only some embodiments of the present application, but not all embodiments. The components of the embodiments of the present application, which are generally described and illustrated in the figures herein, may be arranged and designed in a wide variety of different configurations. Thus, the following detailed description of the embodiments of the present application, as provided in the accompanying drawings, is not intended to limit the scope of the application, as claimed, but is merely representative of selected embodiments of the application. Based on the embodiments of the present application, every other embodiment that a person skilled in the art would obtain without making any inventive effort is within the scope of protection of the present application.
First, application scenarios applicable to the present application will be described. The application can be applied to the functional verification research of the genes related to the peony stress resistance.
A method for agrobacterium-mediated transient expression of paeonia lactiflora, comprising:
immersing target paeonia lactiflora seedlings in a preset transformation solution completely, and carrying out treatment with negative pressure intensity of 10 to obtain target paeonia lactiflora tissue culture seedlings and seedlings, wherein the target paeonia lactiflora seedlings are paeonia lactiflora tissue culture seedlings obtained by germinating embryo of hybrid seeds of paeonia lactiflora and paeonia lactiflora in a starting culture medium for 30 days and seedlings growing 2-3cm buds through sand storage, and the preset transformation solution is obtained by carrying out secondary activation OD (optical density) on the preset transformation solution 600 Agrobacterium solution with a value of 1.2 was centrifuged and resuspended in AS-and surfactant-containingA conversion solution;
infecting the target paeonia lactiflora tissue culture Miao Zhendang for 24 hours in the dark at 28 ℃, and carrying out oscillation infection on the paeonia lactiflora seedlings for 12 hours to obtain target paeonia lactiflora tissue culture seedlings after infection and paeonia lactiflora seedlings after infection;
respectively inoculating the infected target peony tissue culture seedling and the infected peony seedling into a solid co-culture medium and fine sand, and culturing for 3 days under the dark condition at 25 ℃ to obtain the cultured target peony tissue culture seedling and the cultured seedling;
GUS staining is carried out on the cultured target paeonia lactiflora tissue culture seedlings and the seedlings, and transient over-expression of the target genes is completed as shown in figures 1-2.
Optionally, the OD 600 The agrobacterium solution with the value of 1.2 is obtained by performing secondary activation in 100mL of LB liquid medium containing 50mg/L Kan and 50mg/L Rif, wherein the LB liquid medium consists of yeast extract powder, tryptone and NaCl, 5g of yeast extract powder, 10g of tryptone and 10g of NaCl are added into each liter, and the pH value is 7.0; the plant expression vector is pCAMBIA1301 containing GUS reporter gene; the agrobacterium strain is EHA105; culturing the bacterial liquid at 28 ℃ at 200rpm in a dark way; bacterial liquid OD 600 The values were measured using a spectrophotometer; the conversion liquid is MS, MES, mgCl 2 AS, tween-20 and sucrose, wherein MS 0.214g, MES 1mM, mgCl are added per 100mL 2 2mM, AS 200. Mu. M, tween-20.01% and sucrose 3g, pH 5.8.
Alternatively, the solid co-culture medium is composed of MS, GA 3 6-BA, AS, sucrose and agar, wherein MS 2.14g, sucrose 30g, AS 200 mu M, GA are added per liter 3 0.5mg, 6-BA1.0mg and agar 6.5g, pH 5.8.
Optionally, the step of performing GUS staining on the cultured target paeonia lactiflora tissue culture seedlings and the seedlings to complete transient over-expression of the target genes comprises the following steps:
immersing the cultured target paeonia lactiflora tissue culture seedlings and the seedlings in GUS staining solution, and performing GUS histochemical staining to complete transient over-expression of the target genes.
A method for transient expression of Paeonia lactiflora mediated by agrobacterium comprises the steps of taking Paeonia lactiflora tissue culture seedlings and seedlings AS explants, screening optimal explant germination time, bacterial liquid concentration, infection time, AS concentration, co-culture time, negative pressure intensity and Tween-20 concentration of transient transformation of Paeonia lactiflora by a GUS histochemical staining method, so that the transient expression rate of Paeonia lactiflora tissue culture seedlings reaches 76.6%, and the transient expression rate of Paeonia lactiflora seedlings reaches 93.3%.
The method comprises the following steps:
selecting peony seedlings suitable for germination time:
obtaining peony tissue culture seedlings: soaking hybrid seeds of radix Paeoniae powder herba Artemisiae Anomalae and radix Curcumae powder herba Polygoni Avicularis in warm water for 48 hr, removing seed coat, sterilizing with 75% alcohol in ultra clean bench for 30s, washing with sterile water for 2 times, and adding 0.1% HgCl 2 Sterilizing the solution for 5-6min, washing with sterile water for 3-4 times each for 1min, sucking water with sterile filter paper, taking out embryo, inoculating to start-up culture medium (MS+0.5mg/L GA) 3 +1.0mg/L6-BA+30g/L sucrose+6.5 g/L agar, pH=5.8), and growing for 30 days under the conditions of 16h/d illumination time, 2000-3000lx illumination intensity and 24-26 ℃;
obtaining peony seedlings: washing hybrid seeds of Paeonia lactiflora and Polygonum aviculare with washing powder for 20min, repeatedly washing with clear water until no foam is produced, soaking the seeds in water for 3 hr, retaining the submerged plump seeds, and adding 0.5% KMnO 4 Sterilizing for 40min, washing with clear water to remove all residues, soaking seeds in 45deg.C warm water for 24 hr, changing new 45deg.C warm water to continue soaking for 24 hr, cleaning seeds, and discarding floating seeds, wherein the rest seeds are used for sand storage. Filling a gauze at the bottom of the flowerpot, paving a layer of broken stone with the size of soybean and sand with the thickness of 1cm, uniformly mixing seeds and sand according to the volume ratio of 1:3, putting into the pot, watering a proper amount, covering the pot mouth with plastic cloth, tying with rubber band, culturing in an environment of 15-20 ℃, observing once every 5 days, putting into a refrigerator of 4 ℃ for germination acceleration when the root length of the seeds is 3-4cm, taking out after two months, putting into the environment of 15-20 ℃, and taking out seedlings with buds of 2-3cm for test after 3-7 days;
in a shaking box at 28 ℃, the agrobacterium liquid (containing plant expressionCarrier) was subjected to secondary activation in 100mL of LB liquid medium containing 50mg/LKan and 50mg/L Rif at 200rpm, and cultured until the bacterial liquid OD was obtained 600 The pellet was collected by centrifugation at 5000rpm for 10min at 1.2 and resuspended in conversion solution (MS+1 mM MES+2mM MgCl) 2 +200 μM as+30g/L sucrose+0.01% tween-20, ph=5.8);
immersing the obtained Paeonia lactiflora tissue culture seedling and seedling completely in a 20mL syringe filled with a conversion liquid, sealing an injection port, drawing for 10 times, carrying out negative pressure treatment 3-5cm each time, and pouring the obtained Paeonia lactiflora tissue culture seedling and seedling back into a wide-mouth conical flask filled with the conversion liquid;
infecting the treated paeonia lactiflora tissue culture Miao Zhendang for 24 hours in the dark at 28 ℃ and infecting the paeonia lactiflora seedlings for 12 hours;
inoculating the infected radix Paeoniae tissue culture seedling into solid co-culture medium (MS+0.5mg/L GA) 3 +1.0 mg/L6-BA+200. Mu.M AS+30g/L sucrose+6.5 g/L agar, pH=5.8), seedlings were planted in fine sand and co-cultured for 3 days in dark conditions at 25 ℃;
preparing GUS staining solution by using a GUS staining kit of Zhongkeruitai, immersing the obtained Paeonia lactiflora seedlings in the GUS staining solution, and then placing the Paeonia lactiflora seedlings in a constant temperature incubator at 37 ℃ for 24 hours in a dark place. The seedlings were decolorized with 75% alcohol and then transferred to 95% alcohol, during which 2-3 times of alcohol were changed until chlorophyll was completely removed, and the blue-stained seedlings were considered positive transient transformed seedlings.
The invention takes the peony tissue culture seedling and the seedling as explants, and the tissue culture seedling and the seedling are derived from seeds, so that the test material is convenient to obtain, is not limited by seasons, can be researched all year round, is easy to disinfect, is simple to operate and has lower pollution rate, wherein the seeds for culturing the seedling can be stored with a large amount of sand in the same time, so that the sufficiency of the transformation material is ensured, and all the test materials for later gene function research can be prepared at the same time, thereby greatly shortening the test period. Negative pressure treatment is introduced, so that the target gene is efficiently and transiently overexpressed in the paeonia lactiflora seedlings, and the transient conversion rate of the paeonia lactiflora is improved by 72%. AS is added into the transformation solution, and Tween-20 accounting for 0.01 percent is also added, so that the permeability of peony seedling cells is improved, and the agrobacteria is helped to infiltrate and plantThe object cell gap, and further the transient transformation efficiency is improved. Compared with other surfactants, the Tween-20 has low price and high cost performance, and can save test cost. GA with concentration of 0.5mg/L is added into the solid co-culture medium 3 And 1.0mg/L of 6-BA, can induce the growth of tissue culture seedlings, is beneficial to the infiltration of agrobacterium and improves the instant transformation efficiency. The method does not depend on expensive experimental equipment, and has low cost; the conversion process is simple and quick, and the whole process only needs 4-5 days; the conversion rate can reach 93.3%, and powerful technical support is provided for analysis of peony gene functions.
Example 1:
(1) Selecting 30 days, 45 days and 60 days of peony tissue culture seedlings as explants, and taking 30 strains of tissue culture seedlings at each germination time for infection;
(2) In a shaking box at 28 ℃, the agrobacterium tumefaciens bacterial liquid (containing plant expression vector) is subjected to secondary activation in 100mL of LB liquid culture medium containing 50mg/L Kan and 50mg/L Rif at 200rpm, and is cultured until bacterial liquid OD 600 The pellet was collected by centrifugation at 5000rpm for 10min at 1.2 and resuspended in conversion solution (MS+1 mM MES+2mM MgCl) 2 +200 μΜ as+30g/L sucrose, ph=5.8);
(3) Immersing the paeonia lactiflora tissue culture seedlings in the transformation liquid completely, and carrying out oscillation infection for 12 hours under the dark condition at 28 ℃;
(4) Inoculating the infected radix Paeoniae tissue culture seedling into solid co-culture medium (MS+0.5mg/L GA) 3 +1.0 mg/L6-BA+200. Mu.M AS+30g/L sucrose+6.5 g/L agar, pH=5.8), co-culturing for 3 days at 25℃under dark conditions;
(5) Preparing GUS staining solution by using a GUS staining kit of Zhongkeruitai, placing the co-cultured Paeonia lactiflora tissue culture Miao Jinmei in the GUS staining solution, and then placing the Paeonia lactiflora tissue culture Miao Jinmei in a constant temperature incubator at 37 ℃ for 24 hours in a dark place. Decolorizing the seedlings with 75% alcohol, transferring into 95% alcohol, and replacing 2-3 times alcohol until chlorophyll is completely removed, wherein blue-stained seedlings are regarded as positive instant transformed seedlings according to the formula: GUS transient conversion (%) =number of GUS staining positive explants/total number of agrobacterium infection explants x 100%, statistical transient conversion, the above experiment was repeated three times.
(6) As can be seen from FIG. 3, the transformation rate of 30-day-germinated paeonia lactiflora tissue culture Miao Shunshi is highest, and therefore 30-day-germinated paeonia lactiflora tissue culture seedlings are selected as explants in the later experiments.
Example 2:
(1) Selecting a peony tissue culture seedling sprouting for 30 days as an explant for infection;
(2) In a shaking box at 28 ℃, the agrobacterium tumefaciens bacterial liquid (containing plant expression vector) is subjected to secondary activation in 100mL LB liquid culture medium containing 50mg/L Kan and 50mg/L Rif at 200rpm, and bacterial liquid OD 600 Culturing to 0.8, 1.0, 1.2, 1.4, centrifuging at 5000rpm for 10min to collect bacterial blocks, and re-suspending in a transformation solution (MS+1 mM MES+2mM MgCl) 2 +200 μΜ as+30g/L sucrose, ph=5.8);
(3) Immersing the paeonia lactiflora tissue culture seedlings in the transformation liquid completely, and carrying out oscillation infection for 12 hours under the dark condition at 28 ℃;
(4) Inoculating the infected radix Paeoniae tissue culture seedling into solid co-culture medium (MS+0.5mg/L GA) 3 +1.0 mg/L6-BA+200. Mu.M AS+30g/L sucrose+6.5 g/L agar, pH=5.8), co-culturing for 3 days at 25℃under dark conditions;
(5) Preparing GUS staining solution by using a GUS staining kit of Zhongkeruitai, placing the co-cultured Paeonia lactiflora tissue culture Miao Jinmei in the GUS staining solution, and then placing the Paeonia lactiflora tissue culture Miao Jinmei in a constant temperature incubator at 37 ℃ for 24 hours in a dark place. Decolorizing the seedlings with 75% alcohol, transferring into 95% alcohol, and replacing 2-3 times alcohol until chlorophyll is completely removed, wherein blue-stained seedlings are regarded as positive instant transformed seedlings according to the formula: GUS transient conversion (%) =number of GUS staining positive explants/total number of agrobacterium infection explants x 100%, statistical transient conversion, the above experiment was repeated three times.
(6) As can be seen from FIG. 4, when OD 600 When the value is 1.2, the transient transformation efficiency is highest, so that the optimal bacterial liquid concentration of the peony transient expression system is 1.2.
Example 3:
(1) Selecting a peony tissue culture seedling sprouting for 30 days as an explant for infection;
(2) In a shaking box at 28 ℃, the agrobacterium tumefaciens bacteria liquid (containing plant expression vector) is added into 100mL containing 50mg/L Kan and 50mPerforming secondary activation in LB liquid medium with g/L Rif at 200rpm, and culturing until bacterial liquid OD 600 The pellet was collected by centrifugation at 5000rpm for 10min at 1.2 and resuspended in conversion solution (MS+1 mM MES+2mM MgCl) 2 +200 μΜ as+30g/L sucrose, ph=5.8);
(3) Immersing the paeonia lactiflora tissue culture seedlings in the transformation liquid completely, and carrying out oscillation infection for 8 hours, 12 hours, 24 hours and 48 hours under the dark condition at 28 ℃;
(4) Inoculating the infected radix Paeoniae tissue culture seedling into solid co-culture medium (MS+0.5mg/L GA) 3 +1.0 mg/L6-BA+200. Mu.M AS+30g/L sucrose+6.5 g/L agar, pH=5.8), co-culturing for 3 days at 25℃under dark conditions;
(5) Preparing GUS staining solution by using a GUS staining kit of Zhongkeruitai, placing the co-cultured Paeonia lactiflora tissue culture Miao Jinmei in the GUS staining solution, and then placing the Paeonia lactiflora tissue culture Miao Jinmei in a constant temperature incubator at 37 ℃ for 24 hours in a dark place. Decolorizing the seedlings with 75% alcohol, transferring into 95% alcohol, and replacing 2-3 times alcohol until chlorophyll is completely removed, wherein blue-stained seedlings are regarded as positive instant transformed seedlings according to the formula: GUS transient conversion (%) =number of GUS staining positive explants/total number of agrobacterium infection explants x 100%, statistical transient conversion, the above experiment was repeated three times.
(6) As can be seen from FIG. 5, the transient transformation efficiency is highest when the tissue culture seedling is infected for 24 hours, and thus, the optimal infection time of the peony transient expression system is 24 hours.
Example 4:
(1) Selecting a peony tissue culture seedling sprouting for 30 days as an explant for infection;
(2) In a shaking box at 28 ℃, the agrobacterium tumefaciens bacterial liquid (containing plant expression vector) is subjected to secondary activation in 100mL of LB liquid culture medium containing 50mg/L Kan and 50mg/L Rif at 200rpm, and is cultured until bacterial liquid OD 600 The pellet was collected by centrifugation at 5000rpm for 10min at 1.2 and resuspended in conversion solution (MS+1 mM MES+2mM MgCl) 2 +100, 150, 200, 250 μΜ as+30g/L sucrose, ph=5.8);
(3) Immersing the peony tissue culture seedling in the transformation liquid completely, and oscillating and infecting for 24 hours under the dark condition at 28 ℃;
(4) After infection ofThe peony tissue culture seedling is inoculated into a solid co-culture medium (MS+0.5mg/L GA) 3 +1.0 mg/L6-BA+200. Mu.M AS+30g/L sucrose+6.5 g/L agar, pH=5.8), co-culturing for 3 days at 25℃under dark conditions;
(5) Preparing GUS staining solution by using a GUS staining kit of Zhongkeruitai, placing the co-cultured Paeonia lactiflora tissue culture Miao Jinmei in the GUS staining solution, and then placing the Paeonia lactiflora tissue culture Miao Jinmei in a constant temperature incubator at 37 ℃ for 24 hours in a dark place. Decolorizing the seedlings with 75% alcohol, transferring into 95% alcohol, and replacing 2-3 times alcohol until chlorophyll is completely removed, wherein blue-stained seedlings are regarded as positive instant transformed seedlings according to the formula: GUS transient conversion (%) =number of GUS staining positive explants/total number of agrobacterium infection explants x 100%, statistical transient conversion, the above experiment was repeated three times.
(6) As can be seen from FIG. 6, 200. Mu.M AS was added to the transformation solution, and the number of successfully blue-stained peony tissue culture seedlings was maximized, so that the optimal AS concentration of the peony transient expression system was 200. Mu.M.
Example 5:
(1) Selecting a peony tissue culture seedling sprouting for 30 days as an explant for infection;
(2) In a shaking box at 28 ℃, the agrobacterium tumefaciens bacterial liquid (containing plant expression vector) is subjected to secondary activation in 100mL of LB liquid culture medium containing 50mg/L Kan and 50mg/L Rif at 200rpm, and is cultured until bacterial liquid OD 600 The pellet was collected by centrifugation at 5000rpm for 10min at 1.2 and resuspended in conversion solution (MS+1 mM MES+2mM MgCl) 2 +200 μΜ as+30g/L sucrose, ph=5.8);
(3) Immersing the peony tissue culture seedling in the transformation liquid completely, and oscillating and infecting for 24 hours under the dark condition at 28 ℃;
(4) Inoculating the infected radix Paeoniae tissue culture seedling into solid co-culture medium (MS+0.5mg/L GA) 3 +1.0 mg/L6-BA+200. Mu.M AS+30g/L sucrose+6.5 g/L agar, pH=5.8), co-cultivation for 2 days, 3 days, 4 days, 5 days under dark conditions at 25 ℃;
(5) Preparing GUS staining solution by using a GUS staining kit of Zhongkeruitai, placing the co-cultured Paeonia lactiflora tissue culture Miao Jinmei in the GUS staining solution, and then placing the Paeonia lactiflora tissue culture Miao Jinmei in a constant temperature incubator at 37 ℃ for 24 hours in a dark place. Decolorizing the seedlings with 75% alcohol, transferring into 95% alcohol, and replacing 2-3 times alcohol until chlorophyll is completely removed, wherein blue-stained seedlings are regarded as positive instant transformed seedlings according to the formula: GUS transient conversion (%) =number of GUS staining positive explants/total number of agrobacterium infection explants x 100%, statistical transient conversion, the above experiment was repeated three times.
(6) As can be seen from FIG. 7, transient transformation efficiency is highest when co-culturing for 3 days, and thus, optimal co-culturing time of the peony transient expression system is 3 days.
Example 6:
(1) Selecting a peony tissue culture seedling sprouting for 30 days as an explant for infection;
(2) In a shaking box at 28 ℃, the agrobacterium tumefaciens bacterial liquid (containing plant expression vector) is subjected to secondary activation in 100mL of LB liquid culture medium containing 50mg/L Kan and 50mg/L Rif at 200rpm, and is cultured until bacterial liquid OD 600 The pellet was collected by centrifugation at 5000rpm for 10min at 1.2 and resuspended in conversion solution (MS+1 mM MES+2mM MgCl) 2 +200 μΜ as+30g/L sucrose, ph=5.8);
(3) Immersing radix Paeoniae tissue culture seedling in 20mL syringe filled with conversion solution, sealing injection port, drawing for 0 times, 5 times, 10 times, and 15 times, each time drawing for 3-5cm, performing negative pressure treatment, and pouring into wide-mouth conical flask filled with conversion solution;
(4) Infecting the paeonia lactiflora tissue culture Miao Zhendang subjected to negative pressure treatment for 24 hours under the dark condition at 28 ℃;
(5) Inoculating the infected radix Paeoniae tissue culture seedling into solid co-culture medium (MS+0.5mg/L GA) 3 +1.0 mg/L6-BA+200. Mu.M AS+30g/L sucrose+6.5 g/L agar, pH=5.8), co-culturing for 3 days at 25℃under dark conditions;
(6) Preparing GUS staining solution by using a GUS staining kit of Zhongkeruitai, placing the co-cultured Paeonia lactiflora tissue culture Miao Jinmei in the GUS staining solution, and then placing the Paeonia lactiflora tissue culture Miao Jinmei in a constant temperature incubator at 37 ℃ for 24 hours in a dark place. Decolorizing the seedlings with 75% alcohol, transferring into 95% alcohol, and replacing 2-3 times alcohol until chlorophyll is completely removed, wherein blue-stained seedlings are regarded as positive instant transformed seedlings according to the formula: GUS transient conversion (%) =number of GUS staining positive explants/total number of agrobacterium infection explants x 100%, statistical transient conversion, the above experiment was repeated three times.
(7) As can be seen from FIG. 8, when the negative pressure intensity is 10, the transient transformation efficiency of paeonia lactiflora is highest and can reach 63.3%, which is 1.72 times that of the paeonia lactiflora which is not subjected to negative pressure treatment, so that the optimal negative pressure intensity of the paeonia lactiflora transient expression system is 10.
Example 7:
(1) Selecting a peony tissue culture seedling sprouting for 30 days as an explant for infection;
(2) In a shaking box at 28 ℃, the agrobacterium tumefaciens bacterial liquid (containing plant expression vector) is subjected to secondary activation in 100mL of LB liquid culture medium containing 50mg/L Kan and 50mg/L Rif at 200rpm, and is cultured until bacterial liquid OD 600 The pellet was collected by centrifugation at 5000rpm for 10min at 1.2 and resuspended in conversion solution (MS+1 mM MES+2mM MgCl) 2 +200 μM as+0, 0.005%, 0.01%, 0.015%, 0.02% tween-20+30g/L sucrose, ph=5.8);
(3) Immersing radix Paeoniae tissue culture seedling completely in 20mL syringe filled with conversion liquid, sealing injection port, drawing for 10 times, carrying out negative pressure treatment 3-5cm each time, and pouring into wide-mouth conical flask filled with conversion liquid;
(4) Infecting the paeonia lactiflora tissue culture Miao Zhendang subjected to negative pressure treatment for 24 hours under the dark condition at 28 ℃;
(5) Inoculating the infected radix Paeoniae tissue culture seedling into solid co-culture medium (MS+0.5mg/L GA) 3 +1.0 mg/L6-BA+200. Mu.M AS+30g/L sucrose+6.5 g/L agar, pH=5.8), co-culturing for 3 days at 25℃under dark conditions;
(6) Preparing GUS staining solution by using a GUS staining kit of Zhongkeruitai, placing the co-cultured Paeonia lactiflora tissue culture Miao Jinmei in the GUS staining solution, and then placing the Paeonia lactiflora tissue culture Miao Jinmei in a constant temperature incubator at 37 ℃ for 24 hours in a dark place. Decolorizing the seedlings with 75% alcohol, transferring into 95% alcohol, and replacing 2-3 times alcohol until chlorophyll is completely removed, wherein blue-stained seedlings are regarded as positive instant transformed seedlings according to the formula: GUS transient conversion (%) =number of GUS staining positive explants/total number of agrobacterium infection explants x 100%, statistical transient conversion, the above experiment was repeated three times.
(7) As can be seen from FIG. 9, when the concentration of Tween-20 was 0.01%, the transient transformation efficiency was significantly improved, and it was able to reach 73.3%, which is 1.08 times that of the group without Tween-20, so that the optimal concentration of Tween-20 in the transient expression system of Paeonia lactiflora was 0.01%.
Example 8:
(1) Selecting seedlings which grow 2-3cm buds through sand storage as explants for infection;
(2) In a shaking box at 28 ℃, the agrobacterium tumefaciens bacterial liquid (containing plant expression vector) is subjected to secondary activation in 100mL of LB liquid culture medium containing 50mg/L Kan and 50mg/L Rif at 200rpm, and is cultured until bacterial liquid OD 600 The pellet was collected by centrifugation at 5000rpm for 10min at 1.2 and resuspended in conversion solution (MS+1 mM MES+2mM MgCl) 2 +200 μM as+0.01% tween-20+30g/L sucrose, ph=5.8);
(3) Immersing radix Paeoniae seedling completely in 20mL syringe filled with conversion solution, sealing injection port, drawing for 10 times, carrying out negative pressure treatment 3-5cm each time, and pouring into wide-mouth conical flask filled with conversion solution;
(4) Oscillating and infecting the peony seedlings subjected to negative pressure treatment for 8 hours, 12 hours and 24 hours in the dark at the temperature of 28 ℃;
(5) Planting the infected paeonia lactiflora seedlings into fine sand, and co-culturing for 3 days under the dark condition at 25 ℃;
(6) Preparing GUS staining solution by using a GUS staining kit of Zhongkeruitai, immersing the co-cultured Paeonia lactiflora seedlings in the GUS staining solution, and then placing the Paeonia lactiflora seedlings in a constant temperature incubator at 37 ℃ for 24 hours in a dark place. Decolorizing the seedlings with 75% alcohol, transferring into 95% alcohol, and replacing 2-3 times alcohol until chlorophyll is completely removed, wherein blue-stained seedlings are regarded as positive instant transformed seedlings according to the formula: GUS transient conversion (%) =number of GUS staining positive explants/total number of agrobacterium infection explants x 100%, statistical transient conversion, the above experiment was repeated three times.
(7) As can be seen from FIG. 10, when the peony seedlings are used as explants, the optimal infection time is 12 hours, and the instantaneous conversion rate can reach 93.3%.
Example 9:
(1) Selecting a paeonia lactiflora tissue culture seedling sprouting for 30 days and a seedling sprouting for 2-3cm from a sand reservoir as explants for infection;
(2) The agrobacterium containing recombinant expression vectors of empty load and peony stress-resistant genes are subjected to secondary activation in 100mL LB liquid medium containing 50mg/L Kan and 50mg/L Rif, and OD is cultivated at 28 ℃ at 200rpm 600 The pellet was collected by centrifugation at 5000rpm for 10min at a value of 1.2 and resuspended in conversion solution (MS+1 mM MES+2mM MgCl) 2 +200 μM as+30g/L sucrose+0.01% tween-20, ph=5.8);
(3) Immersing Paeonia lactiflora seedling completely in 20mL syringe filled with conversion solution, sealing injection port, drawing for 10 times, carrying out negative pressure treatment 3-5cm each time, and pouring into wide-mouth conical flask filled with conversion solution;
(4) Infecting the paeonia lactiflora tissue culture Miao Zhendang subjected to negative pressure treatment for 24 hours in the dark at 28 ℃ and infecting the paeonia lactiflora seedlings for 12 hours;
(5) Inoculating the infected radix Paeoniae tissue culture seedling into solid co-culture medium (MS+0.5mg/L GA) 3 +1.0 mg/L6-BA+200. Mu.M AS+30g/L sucrose+6.5 g/L agar, pH=5.8), seedlings were planted in fine sand and co-cultured for 3 days in dark conditions at 25 ℃;
(6) And (3) carrying out abiotic stress treatment on the transient transgenic seedlings, sampling at stress for 0h and 2h in each abiotic stress treatment, quick-freezing by liquid nitrogen, and preserving at-80 ℃ for later use. Using qRT-PCR technique and 2 -ΔΔCt The expression level of the gene was analyzed by the method of (C). The above test was repeated three times.
(7) As can be seen from fig. 11, in the normal growth state, there is no significant difference between the expression level of the stress-resistance gene in the control seedling (transient transformed empty paeonia lactiflora seedling) and the transient transgenic seedling, while the expression level of the stress-resistance gene in the control seedling and the transient transgenic seedling is significantly improved after the abiotic stress treatment, and the expression level of the stress-resistance gene in the transient transgenic seedling is significantly higher than that of the control seedling. The method shows that the peony stress-resistant genes PlGPAT, plDHN2 and PlHD-Zip are successfully and transiently overexpressed in the peony seedlings, and the established agrobacterium-mediated peony transient expression system can be applied to the functional research of the peony stress-resistant genes.
It will be clear to those skilled in the art that, for convenience and brevity of description, specific working procedures of the above-described systems, apparatuses and units may refer to corresponding procedures in the foregoing method embodiments, and are not repeated herein.
In the several embodiments provided in this application, it should be understood that the disclosed systems, devices, and methods may be implemented in other manners. The above-described apparatus embodiments are merely illustrative, for example, the division of the units is merely a logical function division, and there may be other manners of division in actual implementation, and for example, multiple units or components may be combined or integrated into another system, or some features may be omitted, or not performed. Alternatively, the coupling or direct coupling or communication connection shown or discussed with each other may be through some communication interface, device or unit indirect coupling or communication connection, which may be in electrical, mechanical or other form.
The units described as separate units may or may not be physically separate, and units shown as units may or may not be physical units, may be located in one place, or may be distributed on a plurality of network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.
In addition, each functional unit in each embodiment of the present application may be integrated in one processing unit, or each unit may exist alone physically, or two or more units may be integrated in one unit.
The functions, if implemented in the form of software functional units and sold or used as a stand-alone product, may be stored in a non-volatile computer readable storage medium executable by a processor. Based on such understanding, the technical solution of the present application may be embodied essentially or in a part contributing to the prior art or in a part of the technical solution, in the form of a software product stored in a storage medium, including several instructions for causing a computer device (which may be a personal computer, a server, or a network device, etc.) to perform all or part of the steps of the methods described in the embodiments of the present application. And the aforementioned storage medium includes: a U-disk, a removable hard disk, a Read-Only Memory (ROM), a random access Memory (Random Access Memory, RAM), a magnetic disk, or an optical disk, or other various media capable of storing program codes.
Finally, it should be noted that: the foregoing examples are merely specific embodiments of the present application, and are not intended to limit the scope of the present application, but the present application is not limited thereto, and those skilled in the art will appreciate that while the foregoing examples are described in detail, the present application is not limited thereto. Any person skilled in the art may modify or easily conceive of the technical solution described in the foregoing embodiments, or make equivalent substitutions for some of the technical features within the technical scope of the disclosure of the present application; such modifications, changes or substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present application, and are intended to be included in the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims (3)

1. A method for transient agrobacterium-mediated peony expression, comprising:
immersing the target paeonia lactiflora seedlings in a preset transformation solution completely, and carrying out treatment with the negative pressure intensity of 10 to obtain target paeonia lactiflora tissue culture seedlings and seedlings, wherein the target paeonia lactiflora seedlings are paeonia lactiflora tissue culture seedlings obtained by germinating embryo of hybrid seeds of paeonia lactiflora and paeonia lactiflora in a starting culture medium for 30 days and seedlings growing 2-3cm buds through sand storage, and the preset transformation solution is obtained by carrying out secondary activation OD (optical density) on the preset transformation solution 600 The agrobacterium solution with the value of 1.2 is centrifuged and resuspended in the transformation solution containing acetosyringone and surfactant;
infecting the target paeonia lactiflora tissue culture Miao Zhendang for 24 hours in the dark at 28 ℃, and carrying out oscillation infection on the paeonia lactiflora seedlings for 12 hours to obtain target paeonia lactiflora tissue culture seedlings after infection and paeonia lactiflora seedlings after infection;
respectively inoculating the infected target peony tissue culture seedling and the infected peony seedling into a solid co-culture medium and fine sand, and culturing for 3 days under the dark condition at 25 ℃ to obtain the cultured target peony tissue culture seedling and the cultured seedling;
GUS staining is carried out on the cultured target paeonia lactiflora tissue culture seedlings and the seedlings, so that transient over-expression of the target genes is completed;
the OD is 600 The agrobacterium solution with the value of 1.2 is obtained by performing secondary activation in 100mL of LB liquid medium containing 50mg/LKan and 50mg/LRif, wherein the LB liquid medium consists of yeast extract, tryptone and NaCl, 5g of yeast extract, 10g of tryptone and 10g of NaCl are added into each liter, and the pH value is 7.0; the plant expression vector containsGUSpCAMBIA1301 of the reporter; the agrobacterium strain is EHA105; culturing the bacterial liquid at 28 ℃ at 200rpm in a dark way; bacterial liquid OD 600 The values were measured using a spectrophotometer; the conversion liquid is MS, MES, mgCl 2 AS, tween-20 and sucrose, wherein MS 0.214g, MES 1mM, mgCl are added per 100mL 2 2mM, AS 200. Mu. M, tween-20.01% and sucrose 3g, pH 5.8.
2. The method of agrobacterium-mediated transient peony expression of claim 1, wherein the solid co-culture medium is composed of MS, GA 3 6-BA, AS, sucrose and agar, wherein MS 2.14g, sucrose 30g, AS 200 mu M, GA are added per liter 3 0.5mg, 6-BA1.0mg and agar 6.5g, pH 5.8.
3. The method for transient expression of agrobacterium-mediated peony of claim 1, wherein the step of performing GUS staining on the cultured target peony tissue culture seedling and the seedlings to complete transient overexpression of the target gene comprises:
immersing the cultured target paeonia lactiflora tissue culture seedlings and the seedlings in GUS staining solution, and performing GUS histochemical staining to complete transient over-expression of the target genes.
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