CN112175991B - Genetic transformation method for creeping cut-off glume - Google Patents
Genetic transformation method for creeping cut-off glume Download PDFInfo
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- CN112175991B CN112175991B CN202011163653.3A CN202011163653A CN112175991B CN 112175991 B CN112175991 B CN 112175991B CN 202011163653 A CN202011163653 A CN 202011163653A CN 112175991 B CN112175991 B CN 112175991B
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
Abstract
The invention relates to a genetic transformation method of creeping cut-off glume, and relates to the field of plant culture by utilizing genetic engineering. The method uses glufosinate as a screening agent and uses a target plasmid with a glufosinate resistance gene to successfully transform the glufosinate resistance gene into bentgrass plants. The invention realizes the regeneration and genetic transformation of creeping bentgrass, has simple and easy operation, successfully transforms the glufosinate-ammonium resistance gene into bentgrass plants through an optimized regeneration system, obtains a positive line of glufosinate-ammonium resistance, promotes the molecular breeding and genetic improvement of creeping bentgrass, and cultures excellent varieties with more market value.
Description
[ field of technology ]
The invention relates to the technical field of plant genetic engineering, in particular to a genetic transformation method for creeping shear-strand glume.
[ background Art ]
Creeping bentgrass (agrotis stolonifera l.) belongs to the genus bentgrass of the family gramineae and is a perennial herb. Is distributed in northeast, north China, northwest, jiangxi, zhejiang and other areas of China. The plant has strong cold resistance, drought resistance, barren resistance, shadow resistance and pruning resistance, and the plant has fine and compact blades and strong transverse spreading capability, so that the plant is widely used for sports field lawns and ornamental lawns. However, creeping bentgrass is often threatened by a variety of weeds, some of which are highly competitive and severely affected, resulting in a significant yield loss, and hence a loss of economic efficiency.
Glufosinate (glufosinate ammonium, phosphinothricin) was a broad-spectrum contact-killing herbicide developed successfully in the 80 s of the 20 th century by the germany Ai Gefu company (postascribed bayer company). Glufosinate belongs to phosphonic herbicides and can inhibit glutamine synthetase in plant nitrogen metabolic pathways, thereby interfering with plant metabolism and causing plant death.
The transgenic technology is one of the current breeding means, and the technology is used for improving the variety of turf grass, so that the precise improvement of target characters can be realized. The establishment of a high-frequency regeneration system and a high-efficiency genetic transformation technology system greatly promotes the molecular breeding and genetic improvement of the species and cultivates good varieties with higher market value.
However, in the prior art, there is no successful case of genetic transformation of creeping bentgrass by combining glufosinate-resistant screening with transgenic technology, so that it is necessary to research a novel genetic transformation method to promote molecular breeding and genetic improvement of creeping bentgrass and cultivate a good variety with more market value.
[ invention ]
Aiming at the defects of the prior art, the invention provides a genetic transformation method of creeping agrostis, which successfully transforms a glufosinate-ammonium resistance gene into bentgrass plants and promotes molecular breeding and genetic improvement of the species.
In view of this, the object of the present invention is to propose a method for genetic transformation of creeping shear-strands: glufosinate is used as a screening agent to carry out genetic transformation on creeping shear-strand glume.
According to one embodiment of the invention, the genetic transformation specifically comprises the steps of: sterilizing creeping bentgrass seeds, inducing callus, selecting embryogenic callus, pre-culturing, infecting and co-culturing agrobacterium containing target plasmid, screening, culturing, callus differentiation, rooting, hardening off and transplanting to obtain glufosinate-resistant bentgrass plants; the target plasmid carries a glufosinate-ammonium resistance gene.
According to one embodiment of the invention, the plasmid of interest is pCAMBIA3301.
According to one embodiment of the invention, the seed disinfection treatment comprises the following steps: the creeping bentgrass seeds after shelling are soaked in 75% alcohol for 1 minute, and then are added with 50% 84 solution for disinfection and soaking for 10 minutes.
According to one embodiment of the invention, the medium used in the step of inducing callus comprises 6.6 mg/L2, 4-D and 0.5 mg/L6-BA.
According to one embodiment of the invention, the agrobacterium infection time is 30min.
According to one embodiment of the invention, the screening culture comprises a first round of screening culture and a second round of screening culture, wherein the concentration of glufosinate in the second round of screening culture is 8mg/L.
According to one embodiment of the invention, the callus differentiation step employs a medium comprising 6-BA at a concentration of 1.0mg/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-ammonium resistance gene into bentgrass plants through an optimized regeneration system, obtains a positive line of glufosinate-ammonium resistance, promotes the molecular breeding and genetic improvement of creeping bentgrass, and cultures excellent varieties with more market value.
2. The genetic transformation method provided by the invention has higher callus induction rate reaching 92.3%, the formation proportion of the resistance callus reaches 54.8% after optimization, and the optimal proportion of the callus differentiated 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 the transgenic regenerated plant can be obtained within 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 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 invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a map of the plasmid pCAMBIA3301 of the present invention.
FIG. 2 is a schematic representation of the stages of genetic transformation of creeping femtocells according to the invention.
[ detailed description ] of the invention
The following examples are illustrative of the invention but do not limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The invention provides a genetic transformation method of creeping glume, which uses glufosinate as a screening agent, uses a target plasmid with glufosinate resistance genes to successfully transform the glufosinate resistance genes into bentgrass plants, and obtains a positive strain resistant to glufosinate, particularly, by optimizing a regeneration system, the callus induction rate is higher and reaches 92.3%, the formation proportion of resistant callus after optimization reaches 54.8%, the callus differentiation proportion reaches 81.2%, the transformation period is shortened to 5 months, the average transformation positive rate of the genetic transformation method is 80.9%, and the period is only 4-5 months. Has better prospect for promoting molecular breeding and genetic improvement of creeping bentgrass and cultivating good varieties with higher market value.
According to a specific embodiment of the present invention, the target plasmid with the glufosinate resistance gene is selected from pCAMBIA3301, and other genes may be added to the pCAMBIA3301 plasmid as a vector according to common knowledge in the art.
According to a specific embodiment of the invention, the genetic transformation method of creeping shear-strands involves the following medium:
callus induction medium: 4.43g/L MS, 30g/L cross, 500mg/L casein hydrolysate, 0-8.8 mg/L2, 4-D, 0-1 mg/L6-BA, 2g/L phytagel, pH=5.7; preferably, the 2,4-D concentration is 6.6mg/L and the 6-BA concentration is 0.5mg/L.
Embryogenic callus selection medium: 4.43g/L MS, 30g/L cross, 500mg/L casein hydrolysate, 0-8.8 mg/L2, 4-D, 0-1 mg/L6-BA, 2g/L phytagel, pH=5.7.
Agrobacteria dip-dyeing and co-culturing:
a) Pre-culture medium: 4.43g/L MS, 30g/L cross, 500mg/L casein hydrolysate, 0-8.8 mg/L2, 4-D, 0-1 mg/L6-BA, 100. Mu. Mol/L AS, 2g/L phytagel, pH=5.7.
b) First round screening media: 4.43g/L MS, 30g/L cross, 500mg/L casein hydrolysate, 0-8.8 mg/L2, 4-D, 0-1 mg/L6-BA, 125mg/L cefotaxime, 250mg/L carbenicillin, 2g/L phytagel, pH=5.7.
c) Second round of screening media: 4.43g/L MS, 30g/L cross, 500mg/L casein hydrolysate, 0-8.8 mg/L2, 4-D, 0-1 mg/L6-BA, 250mg/L cefotaxime, 2-10 mg/L phosphinothricin, 2g/L phytagel, pH=5.7.
Callus differentiation medium: 4.43g/L MS, 30g/L sucrose, 100mg/L myo-inosol, 0.3-1.2 mg/L6-BA, 250mg/L cefotaxime, 8mg/L phosphinothricin, 2g/L Phytagel. Preferably, the 6-BA concentration is 1.0mg/L.
Rooting medium: 2.2g/L MS, 20g/L cross, 125mg/L cefotaxime, 4mg/L phosphinothricin, 3g/L phytagel.
EXAMPLE 1 seed Disinfection treatment
The creeping bentgrass seeds are very small, and the velvet is coated, so that the velvet is removed by slightly rubbing with sand paper, and the purposes of shelling and facilitating the subsequent disinfection treatment are achieved. Seed disinfection is carried out on an ultra-clean workbench: soaking in 75% (v/v) alcohol for 1 min, pouring out alcohol, adding 50% (v/v) 84 solution, soaking for 6, 10 and 14 min respectively, washing with sterile water for 5 times, transferring seeds to sterilized 0.1% agarose solution, sucking uniformly, spreading on MS culture medium uniformly with a pipettor, sealing with a sealing film, placing in a culture chamber, culturing in darkness at 25-28deg.C for 2 weeks, and counting germination rate. The result shows that the pollution can be effectively controlled by sterilizing the 84 solution for 10 minutes, the germination rate reaches 79.3 percent, and the germination rate of seeds is affected and gradually reduced along with the extension of the treatment time. Accordingly, a solution of 84 sterilized for 10 minutes is considered to be the optimal treatment time.
TABLE 1 seed disinfection treatment
EXAMPLE 2 callus induction
After the optimal disinfection treatment, the seeds are transferred to a sterilized 0.1% agarose solution, and evenly spread on an MS culture medium by a liquid transfer device after being sucked and beaten evenly. The culture medium comprises the following components: 4.43g/L MS, 30g/L cross, 500mg/L casein hydrolysate, 0-8.8 mg/L2, 4-D, 0-1 mg/L6-BA, 2g/L phytagel, PH=5.7, sealing the sealing film, placing in a culture room, culturing at 25 ℃ in dark for 6 weeks. Wherein the concentration of 2,4-D is set to 2.2mg/L, 4.4mg/L, 6.6mg/L and 8.8mg/L of 4 species, and the concentration of 6-BA is set to 0.5mg/L and 1.0mg/L of 2 species. The results of statistics of the callus induction conditions after 4 weeks show that the callus induction rate reaches 92.3% when the concentration of 2,4-D is 6.6mg/L and the concentration of 6-BA is 0.5mg/L, and the callus structure is compact and the callus is bright yellow in color and is a good growth state, so that the concentration combination is selected as the optimal formula of callus induction.
TABLE 2 influence of different hormone formulations on callus induction and growth
EXAMPLE 3 determination of the concentration of resistant callus screens
The calli with better growth state and consistent size are selected and transferred to a secondary culture medium (medium composition: 4.43g/L MS, 30g/L cross, 500mg/L casein hydrolysate, 6.6 mg/L2, 4-D, 0.5 mg/L6-BA, 2-10 mg/L phosphinothricin, 2g/L phytagel, PH=5.7). Wherein the concentration of the glufosinate is 2mg/L, 4mg/L, 6mg/L, 8mg/L and 10mg/L respectively. The growth state of the callus is observed after dark culture for two weeks at the temperature of 25-28 ℃, and when the concentration of glufosinate reaches 8mg/L, the callus turns brown and growth stagnates, so that the callus is a proper screening concentration.
EXAMPLE 4 Pre-culture of callus, agrobacterium infection and Co-culture, selection of resistant callus
Seed disinfection and callus induction are carried out according to the optimal conditions, and agrobacterium infection is carried out after the callus growth reaches about 0.5 mm. The day before the dip-plating, embryogenic callus was divided into 1-2 mm pieces and placed on induction medium containing AS (100. Mu. Mol/L) for preculture (medium composition: 4.43g/L MS, 30g/L sucrose, 500mg/L casein hydrolysate, 6.6 mg/L2, 4-D, 0.5 mg/L6-BA, 100. Mu. Mol/L AS, 2g/L phytagel, pH=5.7). The day before the dip-dyeing, the Agrobacterium strain of pCAMBIA3301 plasmid (plasmid map, see FIG. 1) was streaked on LB medium (kanamycin was added to a final concentration of 50 mg/L), and the Agrobacterium was scraped into the suspension medium the next day, and after pipetting and mixing, the concentration was brought to OD=0.4 (660 nm). The precultured calli were transferred to a triangular flask, poured into a prepared agrobacterium solution, and soaked for 15, 30, 45 and 60 minutes, respectively, during which time they were properly shaken. Pouring out the bacterial liquid, sucking the superfluous bacterial liquid on the surface, air-drying for 2 hours on an ultra-clean workbench, 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 25-28 ℃.
The calli were collected from the co-culture medium, washed 3 times with sterile water, surface moisture was blotted off, and transferred to a screening 1 medium (medium composition: 4.43g/L MS, 30g/L sucrose, 500mg/L casein hydrolysate, 6.6 mg/L2, 4-D, 0.5 mg/L6-BA, 125mg/L cefotaxime, 250mg/L carbicillin, 2g/L phytagel, pH=5.7) for 2 weeks in darkness.
The calli were transferred to Screen 2 medium (medium composition: 4.43g/L MS, 30g/L sucrose, 500mg/L casein hydrolysate, 6.6 mg/L2, 4-D, 0.5 mg/L6-BA, 250mg/L cefotaxime, 8mg/L phosphinothricin, 2g/L phytagel, pH=5.7) and dark cultured at 25-28℃with screen medium changed every three weeks until resistant calli developed.
Finally, counting the number of the resistant calli growing out after different dip-dyeing time, and calculating the proportion of the resistant calli to the dip-dyed calli. The results show (Table 3) that the resistant calli were higher, reaching 54.8%, after 30 minutes of Agrobacterium infection, while the Agrobacterium growth was easier to control, being the optimal treatment time.
TABLE 3 influence of different Time of exhaust on resistant callus formation
EXAMPLE 5 callus differentiation
The resistant calli were transferred to differentiation medium ((4.43 g/L MS, 30g/L sucrose, 100mg/L myo-inosol, 0.3-1.2 mg/L6-BA, 250mg/L cefotaxin, 8mg/L phosphinothricin, 2g/L Phytagel) for plant regeneration, and three weeks later, regeneration frequencies were counted according to the budding conditions, wherein the 6-BA concentration was set to 0.3, 0.5, 0.8, 1.0 and 1.2mg/L, respectively, to thereby select the optimal hormone concentration.
TABLE 4 influence of different concentrations of 6-BA bentgrass seed callus differentiation
The regenerated shoots growing to a height of 1-3 cm are transferred to rooting medium (medium composition: 2.2g/L MS, 20g/L cross, 125mg/L cefotaxime, 4mg/L phosphinothricin, 3g/L phytagel) for rooting culture. Hardening seedlings of the rooted regenerated plants after four weeks, cleaning a culture medium of the roots, transplanting the culture medium into matrix soil, and carrying out proper shading treatment to survive. FIG. 2 shows the regeneration process from seed disinfection (FIG. 2. A), callus induction (FIG. 2. B), differentiation (FIG. 2C) to rooting (FIG. 2D), after molecular detection, 39 positive transformants were obtained.
EXAMPLE 6 conversion Positive Rate
And carrying out three experiments according to the genetic transformation method of the creeping glumes, and repeatedly treating 350 creeping glumes seeds in each experiment. The results showed that the average positive rate of transformed plants was 80.9%.
TABLE 5 conversion Positive statistics
EXAMPLE 7 genetic transformation cycle
According to the genetic transformation method of general creeping glume, the experiment is carried out, and the transgenic positive plant is obtained in 6-7 months, and the invention shortens the time of the second stage of callus induction and screening by optimizing a regeneration system, and shortens the whole transformation period to 4-5 months.
The present invention is not limited to the details and embodiments described herein, and further advantages and modifications may readily be achieved by those skilled in the art, so that the present invention is not limited to the specific details, representative solutions and illustrative examples shown and described herein, without departing from the spirit and scope of the general concepts defined by the claims and the equivalents.
Claims (2)
1. A method for genetic transformation of creeping shear-strands, comprising the steps of: sterilizing creeping bentgrass seeds, inducing callus, selecting embryogenic callus, subculturing, pre-culturing, infecting and co-culturing agrobacterium containing target plasmid, screening, culturing, callus differentiation, rooting, hardening off and transplanting to obtain glufosinate-ammonium-resistant bentgrass plants;
wherein the target plasmid carries a glufosinate-ammonium resistance gene; the infection time of the agrobacterium is 30min;
the seed disinfection treatment steps are as follows: soaking the shelled creeping bentgrass seeds in 75% (v/v) alcohol for 1 min, and then adding 50% (v/v) 84 solution for disinfection and soaking for 10 min;
the culture medium adopted in the step of inducing the callus comprises the following components: 4.43g/L MS, 30g/L cross, 500mg/L casein hydrolysate, 6.6 mg/L2, 4-D, 0.5 mg/L6-BA, 2g/L phytagel, pH=5.7;
the culture medium adopted in the subculture step comprises the following components: 4.43g/L MS, 30g/L cross, 500mg/L casein hydrolysate, 6.6 mg/L2, 4-D, 0.5 mg/L6-BA, 8mg/L phosphinothricin, 2g/L phytagel, pH=5.7;
the culture medium adopted in the pre-culture step comprises the following components: 4.43g/L MS, 30g/L cross, 500mg/L casein hydrolysate, 6.6 mg/L2, 4-D, 0.5 mg/L6-BA, 100. Mu. Mol/L AS, 2g/L phytagel, pH=5.7;
the culture medium adopted in the co-culture step is the same as that adopted in the pre-culture step;
the screening culture comprises a first round of screening culture and a second round of screening culture, wherein the culture medium adopted by the first round of screening culture comprises the following components: 4.43g/L MS, 30g/L cross, 500mg/L casein hydrolysate, 6.6 mg/L2, 4-D, 0.5 mg/L6-BA, 125mg/L cefotaxime, 250mg/L carbicillin, 2g/L phytagel, pH=5.7;
the culture medium used for the second round of screening culture comprises the following components: 4.43g/L MS, 30g/L sucrose, 500mg/L casein hydrolysate, 6.6 mg/L2, 4-D, 0.5 mg/L6-BA, 250mg/L cefotaxime, 8mg/L phosphinothricin, 2g/L phytagel, pH=5.7;
the composition of the culture medium adopted in the callus differentiation step is as follows: 4.43g/L MS, 30g/L sucrose, 100mg/L myo-inosol, 1.0 mg/L6-BA, 250mg/L cefotaxime, 8mg/L phosphinothricin, 2g/L Phytagel;
the rooting step adopts a culture medium comprising the following components: 2.2g/L MS, 20g/L cross, 125mg/L cefotaxime, 4mg/L phosphinothricin, 3g/L phytagel.
2. The transformation method according to claim 1, wherein the target plasmid is pCAMBIA3301.
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