CN112175991A - Genetic transformation method of creeping glumes - Google Patents
Genetic transformation method of creeping glumes Download PDFInfo
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- CN112175991A CN112175991A CN202011163653.3A CN202011163653A CN112175991A CN 112175991 A CN112175991 A CN 112175991A CN 202011163653 A CN202011163653 A CN 202011163653A CN 112175991 A CN112175991 A CN 112175991A
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Abstract
The invention relates to a genetic transformation method of creeping glume, and relates to the field of plant culture by using genetic engineering. The method successfully transforms the glufosinate-ammonium resistance gene into bentgrass plants by taking glufosinate-ammonium as a screening agent and taking a target plasmid with the glufosinate-ammonium resistance gene. The invention realizes the regeneration and genetic transformation of creeping bentgrass, has simple and easy operation, successfully transforms the glufosinate resistance gene to the bentgrass plant through an optimized regeneration system, obtains a positive strain resisting glufosinate, promotes the molecular breeding and genetic improvement of creeping bentgrass, and cultivates a good variety with market value.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of plant genetic engineering, in particular to a genetic transformation method of creeping glume-cutting.
[ background of the invention ]
Creeping bentgrass (agrostis. stolonifera L.) belongs to the genus bentgrass, and is a perennial herbaceous plant. The fertilizer is distributed in the northeast, northwest and west of China, as well as the west and Zhejiang. The lawn has strong cold resistance, drought resistance, barren resistance, shade resistance and pruning resistance, fine and compact leaves and strong transverse spreading capability, and is widely used for sports field lawns and ornamental lawns. Nonetheless, creeping bentgrass is often threatened by a variety of weeds, some of which are competitive and in severe cases result in a substantial reduction in yield to the point of losing economic benefit.
Glufosinate ammonium (phosphinothricin) is a broad-spectrum contact-type biocidal herbicide developed successfully by egeft, formerly germany (post-homed bayer corporation) in the 80 s of the 20 th century. Glufosinate belongs to phosphonic acid herbicides and can inhibit glutamine synthetase in plant nitrogen metabolic pathways, so that metabolism of plants is interfered, and the plants die.
The transgenic technology is one of the current breeding means, and the technology is used for carrying out variety improvement on turfgrass, so that the accurate improvement of target characters can be realized. The establishment of a high-frequency regeneration system and a high-efficiency genetic transformation technical system can greatly promote the molecular breeding and genetic improvement of the species and cultivate excellent varieties with higher market value.
However, there is no successful case in the prior art for genetic transformation of creeping bentgrass by combining glufosinate-resistant screening with transgenic technology, so that a novel genetic transformation method needs to be researched to promote molecular breeding and genetic improvement of creeping bentgrass and culture a good variety with higher market value.
[ summary of the invention ]
Aiming at the defects of the prior art, the invention provides a genetic transformation method of creeping bentgrass, which successfully transforms glufosinate resistance genes into bentgrass plants and promotes molecular breeding and genetic improvement of the species.
In view of the above, the present invention aims to provide a genetic transformation method of creeping glume: glufosinate ammonium is used as a screening agent to carry out genetic transformation on creeping glume-cut.
According to one embodiment of the invention, said genetic transformation comprises in particular the following steps: sterilizing creeping bentgrass seeds, inducing callus, selecting embryonic callus and pre-culturing, infecting and co-culturing by agrobacterium containing target plasmids, and then obtaining glufosinate-resistant bentgrass plants through screening culture, callus differentiation, rooting, seedling hardening and transplanting; the target plasmid carries a glufosinate-ammonium resistance gene.
According to one embodiment of the present invention, the target plasmid is pCAMBIA 3301.
According to one embodiment of the invention, the step of sterilizing the seeds comprises: the unshelled creeping bentgrass seeds are soaked in 75% alcohol for 1 minute, and then 50% 84 solution is added for disinfection and soaking for 10 minutes.
According to one embodiment of the invention, the callus induction step uses a medium comprising 6.6mg/L of 2,4-D and 0.5mg/L of 6-BA.
According to one embodiment of the invention, the agrobacterium infection time is 30 min.
According to one embodiment of the invention, the selection culture comprises a first selection culture and a second selection culture, and the concentration of glufosinate in the second selection culture is 8 mg/L.
According to one embodiment of the invention, the callus differentiation step uses a medium comprising 6-BA at a concentration of 1.0 mg/L.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention realizes the regeneration and genetic transformation of creeping bentgrass, has simple and easy operation, successfully transforms the glufosinate resistance gene to the bentgrass plant through an optimized regeneration system, obtains a positive strain resisting glufosinate, promotes the molecular breeding and genetic improvement of creeping bentgrass, and cultivates a good variety with market value.
2. The genetic transformation method provided by the invention has high callus induction rate which reaches 92.3%, the formation proportion of the resistance callus after optimization reaches 54.8%, and the optimal proportion of callus differentiation into seedlings reaches 81.2%.
3. The genetic transformation method provided by the invention has high transformation positive rate which reaches 80.9%.
4. The genetic transformation method provided by the invention has a short period, and a transgenic regeneration plant can be obtained in 4-5 months.
[ description of the drawings ]
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a map of a target plasmid pCAMBIA3301 of the present invention.
FIG. 2 is a schematic representation of the stages of genetic transformation of the creeping, shear-strand glumes of the present invention.
[ detailed description ] embodiments
The following examples are intended to illustrate the invention without limiting its scope. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.
The invention provides a genetic transformation method of creeping glume, which specifically comprises the steps of successfully transforming glufosinate-resistance genes into bentgrass plants by taking glufosinate as a screening agent and target plasmids with glufosinate-resistance genes, so as to obtain glufosinate-resistance positive strains, and particularly, through optimizing a regeneration system, the callus induction rate is high and reaches 92.3%, the formation proportion of the optimized resistant callus reaches 54.8%, the callus differentiation proportion reaches 81.2%, the transformation period is shortened to 5 months, the average positive rate of the genetic transformation method is 80.9%, and the period is only 4-5 months. Has better prospect for promoting the molecular breeding and genetic improvement of creeping bentgrass and cultivating good varieties with more market value.
According to the specific embodiment of the present invention, pCAMBIA3301 is selected as the target plasmid with glufosinate-ammonium resistance gene, and other genes may be added as vectors to pCAMBIA3301 plasmid according to the common knowledge in the art.
According to a particular embodiment of the invention, the method for the genetic transformation of stoloniferous glumes involves the following culture media:
inducing a callus culture medium: 4.43g/L MS, 30g/L sucrose, 500mg/L casein hydrosate, 0-8.8 mg/L2, 4-D, 0-1 mg/L6-BA, 2g/L phytagel, and the PH is 5.7; preferably, the concentration of 2,4-D is 6.6mg/L and the concentration of 6-BA is 0.5 mg/L.
Embryogenic callus selection medium: 4.43g/L MS, 30g/L sucrose, 500mg/L casein hydrosate, 0-8.8 mg/L2, 4-D, 0-1 mg/L6-BA, 2g/L phytagel, and the PH is 5.7.
And (3) carrying out dip dyeing and co-culture on agrobacterium:
a) pre-culture medium: 4.43g/L MS, 30g/L sucrose, 500mg/L casein hydrosate, 0-8.8 mg/L2, 4-D, 0-1 mg/L6-BA, 100 mu mol/L AS and 2g/L phytagel, and the PH is 5.7.
b) First round screening medium: 4.43g/L MS, 30g/L sucrose, 500mg/L casein hydrosate, 0-8.8 mg/L2, 4-D, 0-1 mg/L6-BA, 125mg/L cefotaxime, 250mg/L carbenicillin and 2g/L phytagel, and the PH is 5.7.
c) Second round screening medium: 4.43g/L MS, 30g/L sucrose, 500mg/L casein hydrosate, 0-8.8 mg/L2, 4-D, 0-1 mg/L6-BA, 250mg/L cefotaxime, 2-10 mg/L phosphinothricin and 2g/L phytagel, wherein the PH is 5.7.
Callus differentiation medium: 4.43g/L MS, 30g/L sucrose, 100mg/L myo-inositol, 0.3-1.2 mg/L6-BA, 250mg/L cefotaxime, 8mg/L phosphinothricin and 2g/L Phytagel. Preferably, the concentration of 6-BA is 1.0 mg/L.
Rooting culture medium: 2.2g/L MS, 20g/L sucrose, 125mg/L cefotaxime, 4mg/L phosphinothricin, 3g/L phytagel.
Example 1 Sterilization treatment of seeds
The seed of creeping bentgrass is very little, and outer quilt fine hair need adopt abrasive paper to rub gently and remove fine hair, plays the purpose of peel simultaneously, the subsequent disinfection of being convenient for is handled. And (3) disinfecting seeds on a superclean workbench: soaking in 75% (v/v) alcohol for 1 minute, pouring out the alcohol, adding 50% (v/v)84 solution, soaking for 6, 10 and 14 minutes respectively, cleaning for 5 times by using sterile water, transferring the seeds to sterilized 0.1% agarose solution, uniformly sucking, uniformly spreading on an MS culture medium by using a pipette, sealing by using a sealing film, placing in a culture chamber, culturing in the dark for 2 weeks, and then counting the germination rate. The result shows that the 84 solution can effectively control the pollution after being sterilized for 10 minutes, the germination rate reaches 79.3 percent, and the germination rate of the seeds is influenced and gradually reduced along with the prolonging of the treatment time. Accordingly, sterilization of the 84 solution for 10 minutes is considered to be the optimal treatment time.
TABLE 1 seed Sterilization treatment
Example 2 Induction of callus
After the seeds were subjected to the above-described optimal sterilization treatment, the seeds were transferred to a sterilized 0.1% agarose solution, pipetted uniformly, and laid flat on the MS medium. The culture medium comprises the following components: 4.43g/L MS, 30g/L sucrose, 500mg/L casein hydrosate, 0-8.8 mg/L2, 4-D, 0-1 mg/L6-BA, 2g/L phytagel, wherein the pH is 5.7, the mixture is placed in a culture room after being sealed by a sealing film, and is cultured for 6 weeks in the dark at the temperature of 25 ℃. Wherein the concentration of 2,4-D is set to be 2.2mg/L, 4.4mg/L, 6.6mg/L and 8.8mg/L4 types, and the concentration of 6-BA is set to be 0.5mg/L and 1.0mg/L2 types. After 4 weeks, the callus induction condition is counted, and the result shows that when the concentration of 2,4-D is 6.6mg/L and the concentration of 6-BA is 0.5mg/L, the callus induction rate reaches 92.3 percent, and meanwhile, the callus is compact in structure and bright yellow in color and is in a better growth state, so that the concentration combination is selected as the optimal formula for callus induction.
TABLE 2 Effect of different hormone formulations on callus induction and growth
Example 3 determination of concentration of resistant callus selection
Selecting calli with good growth state and consistent size, and transferring the calli to a subculture medium (the culture medium comprises 4.43g/L MS, 30g/L sucrose, 500mg/L casein hydrosate, 6.6 mg/L2, 4-D, 0.5 mg/L6-BA, 2-10 mg/L phosphinothricin, 2g/L phytagel, and the pH is 5.7). Wherein the concentration of the glufosinate-ammonium is 2mg/L, 4mg/L, 6mg/L, 8mg/L and 10mg/L respectively. And (3) observing the growth state of the callus after dark culture for two weeks at the temperature of 25-28 ℃, and finding that when the concentration of the glufosinate-ammonium reaches 8mg/L, the callus turns brown and grows stagnant, so that the callus is a relatively proper screening concentration.
Example 4 callus preculture, Agrobacterium Dip and Co-culture, selection of resistant callus
And (3) performing experiments on seed disinfection and callus induction according to the optimal conditions, and performing agrobacterium infection after the callus growth reaches about 0.5 mm. One day before the infection, the embryogenic callus was divided into 1-2 mm pieces and pre-cultured on an induction medium containing AS (100. mu. mol/L) (medium composition: 4.43g/L MS, 30g/L sucrose, 500mg/L casein hydrosate, 6.6 mg/L2, 4-D, 0.5 mg/L6-BA, 100. mu. mol/L AS, 2g/L phytagel, pH 5.7). One day before the infection, the agrobacterium strain of pCAMBIA3301 plasmid (plasmid map is shown in figure 1) is streak-cultured on LB culture medium (kanamycin is added to the final concentration of 50mg/L), the agrobacterium is scraped into suspension culture solution by the next day, and the concentration is made to be 0.4(660nm) after the agrobacterium is evenly sucked and beaten. Transferring the pre-cultured callus to a triangular flask, pouring the prepared agrobacterium liquid, and soaking for 15, 30, 45 and 60 minutes respectively, wherein the period can be properly shaken. And (4) pouring off the bacterial liquid, sucking off the redundant bacterial liquid on the surface, airing on an ultra-clean workbench for 2 hours, transferring to a co-culture medium (the composition of the culture medium is the same as that of the pre-culture medium), and culturing in the dark for 2-3 days at the temperature of 25-28 ℃.
The calli were harvested from the co-culture medium, washed 3 times with sterile water, blotted to remove surface water, transferred to Medium for selection 1 (medium composition: 4.43g/L MS, 30g/L sucrose, 500mg/L casein hydrosate, 6.6 mg/L2, 4-D, 0.5 mg/L6-BA, 125mg/L cefotaxime, 250mg/L carbenicillin, 2g/L phytagel, pH 5.7) and cultured in the dark for 2 weeks.
Transferring the callus onto a screening 2 culture medium (the culture medium comprises 4.43g/L MS, 30g/L sucrose, 500mg/L casein hydrosate, 6.6 mg/L2, 4-D, 0.5 mg/L6-BA, 250mg/L cefotaxime, 8mg/L phosphinothricin, 2g/L phytagel and pH 5.7), carrying out dark culture at the temperature of 25-28 ℃, and replacing the screening culture medium every three weeks until resistant callus grows out.
Finally, the number of the resistance calluses growing out after different dip dyeing time is counted, and the proportion of the resistance calluses in the dip dyed calluses is calculated. The results show (table 3) that the resistant callus percentage was high up to 54.8% after 30 minutes of agrobacterium-disseminated while the growth of agrobacterium was easily controlled, which is the optimal treatment time.
TABLE 3 Effect of different Dip-dyeing times on resistant callus formation
Example 5 callus differentiation
And transferring the resistant callus to a differentiation medium ((4.43g/L MS, 30g/L sucrose, 100mg/L myo-inositol, 0.3-1.2 mg/L6-BA, 250mg/L cefotaxime, 8mg/L phosphinothricin and 2g/L Phytagel) for plant regeneration, and counting the regeneration frequency according to the budding situation after three weeks, wherein the 6-BA concentration is respectively set to be 0.3, 0.5, 0.8, 1.0 and 1.2mg/L, so as to screen out the optimal hormone concentration, and the result shows that the callus differentiation rate is the highest (figure 2.C) and the callus differentiation rate is 81.2% when the 6-BA concentration is 1.0 mg/L.
TABLE 4 Effect of different concentrations of 6-BA bentgrass seed callus differentiation
Transferring the regenerated buds with the length of 1-3 cm to a rooting medium (the medium comprises 2.2g/L MS, 20g/L sucrose, 125mg/L cefotaxime, 4mg/L phosphinothricin and 3g/L phytagel) for rooting culture. Exercising the rooted regenerated plant after four weeks, cleaning the culture medium at the root, transplanting the plant into the matrix soil, and appropriately shading to survive. FIG. 2 shows that 39 positive transformants were obtained after molecular testing during the regeneration process from seed disinfection (FIG. 2.A), callus induction (FIG. 2.B), differentiation (FIG. 2C) to rooting (FIG. 2D).
Example 6 Positive conversion
Three experiments are carried out according to the genetic transformation method of the creeping glume, and 350 grains of creeping glume seeds are repeatedly processed in each experiment. The result shows that the average positive rate of the transformed plants is 80.9%.
TABLE 5 conversion Positive Rate statistics
Example 7 genetic transformation cycle
Experiments are carried out according to a general genetic transformation method of creeping glume-cutting, transgenic positive plants need 6-7 months, the time of the second stage of callus induction and screening is shortened by optimizing a regeneration system, and the whole transformation period is shortened to 4-5 months.
The invention is not limited solely to that described in the specification and embodiments, and additional advantages and modifications will readily occur to those skilled in the art, so that the invention is not limited to the specific details, representative embodiments, and illustrative examples shown and described herein, without departing from the spirit and scope of the general concept as defined by the appended claims and their equivalents.
Claims (8)
1. A genetic transformation method of creeping glume is characterized in that glufosinate is used as a screening agent to carry out genetic transformation on the creeping glume.
2. The transformation method of claim 1, comprising the steps of: sterilizing creeping bentgrass seeds, inducing callus, selecting embryonic callus, infecting and co-culturing by agrobacterium containing target plasmids, screening and culturing, callus differentiating, rooting, hardening seedlings and transplanting to obtain glufosinate-resistant bentgrass plants;
wherein the target plasmid carries a glufosinate-ammonium resistance gene.
3. The transformation method according to claim 2, wherein the target plasmid is pCAMBIA 3301.
4. The transformation method according to claim 2, wherein the step of sterilizing the seeds comprises: the husked creeping bentgrass seeds are soaked in 75% (v/v) alcohol for 1 minute, and then 50% (v/v)84 solution is added for disinfection and soaking for 10 minutes.
5. The transformation method according to claim 2, wherein the callus induction step uses a medium comprising 6.6 mg/L2, 4-D and 0.5 mg/L6-BA.
6. The transformation method according to claim 2, wherein the agrobacterium infection time is 30 min.
7. The transformation method according to claim 2, wherein the selection culture comprises a first selection culture and a second selection culture, and the concentration of glufosinate in the second selection culture is 8 mg/L.
8. The transformation method according to claim 2, wherein the callus differentiation step uses a medium comprising 6-BA at a concentration of 1.0 mg/L.
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| CN113755521B (en) * | 2021-07-29 | 2024-02-06 | 上海市农业科学院 | Construction method of agrobacterium-mediated strawberry 'sweet Charles' genetic transformation system |
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| CN113755521B (en) * | 2021-07-29 | 2024-02-06 | 上海市农业科学院 | Construction method of agrobacterium-mediated strawberry 'sweet Charles' genetic transformation system |
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