CN114410678B - Method for establishing agrobacterium-mediated Chinese narcissus efficient genetic transformation system - Google Patents
Method for establishing agrobacterium-mediated Chinese narcissus efficient genetic transformation system Download PDFInfo
- Publication number
- CN114410678B CN114410678B CN202210162166.8A CN202210162166A CN114410678B CN 114410678 B CN114410678 B CN 114410678B CN 202210162166 A CN202210162166 A CN 202210162166A CN 114410678 B CN114410678 B CN 114410678B
- Authority
- CN
- China
- Prior art keywords
- callus
- medium
- agrobacterium
- culture
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
- C12N15/821—Non-antibiotic resistance markers, e.g. morphogenetic, metabolic markers
- C12N15/8212—Colour markers, e.g. beta-glucoronidase [GUS], green fluorescent protein [GFP], carotenoid
-
- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Cell Biology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for establishing an agrobacterium-mediated Chinese narcissus efficient genetic transformation system, which comprises the following steps: plant material pretreatment, explant sterilization, callus induction, infection liquid preparation, agrobacterium infection, co-culture, screening culture and GUS staining detection. The invention uses the Chinese narcissus ovary as a transformation material, establishes a high-efficiency genetic transformation system of the Chinese narcissus through the optimization of the agrobacterium type, the callus pre-culture time and the kanamycin concentration, has high transformation efficiency, and lays a foundation for the creation of Chinese narcissus planting resources and the improvement of varieties.
Description
Technical Field
The invention relates to a Chinese narcissus genetic transformation method in the field of biotechnology, in particular to a method for establishing an agrobacterium-mediated Chinese narcissus efficient genetic transformation system by taking callus generated by an induction ovary as a receptor.
Background
Chinese narcissus (Narcissus tazetta var. Chinensis) is a perennial bulb plant of Amaryllidaceae, and the main cultivation area is Fujian Zhangzhou, shanghai Chongming, zhejiang Zhoushan, which is one of the ten flowers of Chinese traditional. The flower has ornamental value due to the characteristics of beautiful flower shape, elegant flower fragrance, strong adaptability, easy carving and the like; but the flower color is single, the flowering period is short, and the development of the narcissus industry is limited. Improving the flower color and prolonging the flowering period of the colchicine are one of the important research targets of the vast gardening breeders. At present, the study on the Chinese narcissus-induced callus system at home and abroad is deeper, but most of the existing genetic transformation systems are limited by genotypes and can only be expressed instantaneously, and the positive rate of stable genetic transformation is extremely low, so that experiments on the basis of the former are needed to search for a proper genetic transformation system.
The agrobacterium Ti plasmid can carry exogenous gene to be inserted into plant genome through homologous recombination, and has the advantages of fixed copy number, stable offspring, simple inheritance and the like. Therefore, in plant transgenesis, agrobacterium-mediated methods are often employed. The Chinese narcissus has the advantages of few varieties, lack of wild resources and difficult sexual hybridization breeding of homologous triploid, so that the breeding means for improving the characters of flowering phase, flower color, flower fragrance, flower shape and the like through genetic engineering has strong feasibility and high application value. At present, the research on establishing a genetic transformation system of high-efficiency Chinese narcissus generally has the problems of low transformation efficiency, incapability of stable inheritance, poor regeneration capability and the like. The invention aims to solve the problems and lays a foundation for further cultivating new narcissus species with long flowering period, rich fragrance and rich color.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for establishing an agrobacterium-mediated Chinese narcissus efficient genetic transformation system.
In order to achieve the above object, the present invention adopts the following technical solutions:
a method of establishing an agrobacterium-mediated high efficiency genetic transformation system for chinese narcissus, the method comprising the steps of:
(1) Ball seed pretreatment: placing the three-year-old Chinese narcissus seed balls at 4 ℃ and refrigerating for 4 weeks in a dark place;
(2) Explant sterilization: taking out the complete ovary after the complete scape is disinfected;
(3) Callus induction: cutting ovary into small pieces, placing on Z1 preculture medium, and at 25+ -2deg.C, photoperiod 16h/d and illumination intensity 50 [ mu ] mol m -2 s -1 Is cultured under the condition of (1) for 60d;
(4) Preparation of the dip dyeing liquid: take 2. Mu.L OD 600 Monoclonal agrobacterium solution=0.8 was transferred to 2mL YEP liquid medium, and after shaking culture at 28 ℃ and 200rpm for 36-48h, 20 μl of the solution was transferred to 20mL YEP liquid medium, and shaking culture at 28 ℃ and 200rpm for overnight until OD was reached 600 Up to 0.8, the cells were collected by centrifugation and rinsed with 1/2MS medium to remove residual YEP liquid medium, and resuspended with 100. Mu.M acetosyringone-containing 1/2MS medium to give a bacterial suspension OD 600 Up to 0.4-0.5, oscillating the bacterial suspension at 140rpm and 28 ℃ for 2-4h to obtain dyeing liquor;
(5) Infection: immersing the callus with good growth condition obtained by induction into an aggressive dyeing liquid, and oscillating for 20min at room temperature and 20 rpm;
(6) Co-cultivation: after infection is finished, taking out the callus, spreading the callus on sterile filter paper, airing, and then clamping the callus on filter paper infiltrated by a Z1 liquid culture medium, and performing dark co-culture at 28 ℃ for 3d;
(7) End of co-cultivation and screening: after the end of the co-culture with 3d, ddH was used 2 Washing callus 5 times by O, washing once by using a Z1 liquid culture medium, spreading on sterile filter paper, airing, and transferring to R1 regeneration culture, wherein the temperature is 25+/-2 ℃, the photoperiod is 16h/d, and the illumination intensity is 50 mu mol m -2 s -1 30d, during which a resistant plant is obtained once every 20 days;
(8) The obtained resistant callus is detected by GUS staining, and the resistant callus with blue color reaction is identified as positive callus, which indicates successful genetic transformation of Chinese narcissus.
Further, the disinfection method comprises the following steps: soaking the whole flower with 1g/L carbendazim solution for 15min, soaking with 75% ethanol solution for 1min, soaking with 1% NaClO and 0.1% Tween-20 mixed solution for 8min, and washing with sterile water for 5 times for 30s each time.
Further, the above-mentioned Z1 preculture medium was based on MS medium, and 9. 9mg/L of 6-benzylaminopurine, 3. 3mg/L of 1-naphthylacetic acid, 0.16. 0.16g/L of adenine sulfate and 30g/L of sucrose were added.
Further, the agrobacterium is EHA105 agrobacterium, which contains a plant expression vector pGWB344 and carries an kanamycin resistance gene and a reporter gene GUS.
Further, the Z1 liquid culture medium takes MS culture medium as basic culture medium, and 400mg/L of cephalosporin and 50mg/L of kanamycin are added.
Further, the ddH described above 2 O contained 400mg/L of cephalosporin and 50mg/L of kanamycin.
Furthermore, the YEP liquid culture medium contains 50mg/L of spectinomycin and 100mg/L of rifampicin.
Further, the R1 regeneration medium was based on MS medium, and 3mg/L of 6-benzylaminopurine, 9mg/L of 1-naphthylacetic acid, 0.16. 0.16g/L of adenine sulfate, 30g/L of sucrose and 50mg/L of kanamycin were added thereto.
Furthermore, the method for establishing the agrobacterium-mediated Chinese narcissus efficient genetic transformation system can be applied to narcissus breeding.
The invention has the remarkable advantages that:
(1) The invention adopts agrobacterium-mediated method to treatGUSThe gene is transferred into the Chinese narcissus, and by optimizing the infection conditions such as the callus induction time of the sub-room, the culture medium, the concentration of the strain, the bacterial liquid, the infection time, the co-culture time, the concentration of cefmetacin and the like, an agrobacterium-mediated high-efficiency stable genetic transformation system of the Chinese narcissus is established, and a foundation is laid for the establishment of planting resources and the improvement of varieties of the Chinese narcissus.
(2) The invention selects the ovary-induced callus as a transformation material, and has the advantages of convenient material acquisition of the ovary-induced callus, simple operation, less symbiotic bacteria, difficult pollution, strong activity of the callus, capability of inducing to directly differentiate adventitious buds to form regenerated plants, capability of avoiding variation or chimerism generation in the regeneration process, high regeneration rate, low false positive rate, and capability of obtaining complete plants in about 3 months after shortening the period, thereby greatly improving the genetic transformation efficiency.
Detailed Description
In order to facilitate the understanding of the present invention, the construction of the Agrobacterium-mediated genetic transformation system of Narcissus chinensis Jin Zhanyin' is further described by the following examples, but the present invention is not limited thereto.
The Agrobacterium tumefaciens strain used in the examples was EHA105, contained a plant expression vector pGWB344, carrying a reporter geneGUS。
The formulations of the MS medium, 1/2MS medium and YEP liquid medium used in the examples are well known to those skilled in the art.
Example 1 determination of the invasive Strain (GV 3101 was used as a control strain)
(1) Pretreatment of plant materials: three-year-old commercial seed balls (the production place is Fujian Zhangzhou) which are not subjected to water culture are placed in the Chinese narcissus Jin Zhanyin and refrigerated for 4 weeks at the temperature of 4 ℃ in a dark place.
(2) Explant sterilization: taking out the complete flower bud after the scales of the refrigerated seed balls are separated layer by layer but not broken bud, soaking the flower calyx in 1g/L carbendazim solution for 15min, then soaking the flower calyx in 75% ethanol solution for 1min, and then soaking the flower calyx in a mixed solution of 1% NaClO and 0.1% Tween-20 for 8min; finally, the mixture is washed with sterile water for 5 times each for 30 seconds. The sterilized flower bud is transversely cut at the basal part of the bud, the bud is longitudinally cut off but can damage florets, the florets are obtained, the auxiliary crowns of the perianth above each floret ovary are cut off, and the tissues of the ovary and below are reserved.
(3) Callus induction: cutting ovary into small pieces with length and width of 2-3mm, placing on Z1 preculture medium, and controlling temperature to 25+ -2deg.C, photoperiod 16h, and illumination intensity to 50 μmol/m 2 /s 2 Is cultured under the condition of (1) for 60d; wherein, the Z1 preculture medium is based on MS medium, and is added with 9 mg/L6-benzylaminopurine, 3 mg/L1-naphthylacetic acid, 0.16g/L adenine sulfate and 30g/L sucrose.
(4) Preparation of the dip dyeing liquid: bacterial solutions (OD) of two positive strains verified for EHA105 and GV3101 600 =0.8) transferring 2 μl into 2mL of YEP liquid medium containing 50mg/L spectinomycin and 100mg/L rifampicin, and shake culturing at 28deg.C and 200rpm for 36-48 hr; then transferring 20. Mu.L of bacterial liquid into 20mL of YEP liquid culture medium containing 50mg/L of spectinomycin and 100mg/L of rifampicin, and culturing at 28 ℃ under shaking at 200rpm overnight until reaching OD 600 The cells were collected by centrifugation at 5000rpm for 20min to 0.8 and washed with 1/2MS medium to remove residual YEP medium, and resuspended in 1/2MS medium containing 100. Mu.M acetosyringone to give a bacterial suspension OD 600 Reaching 0.4-0.5, oscillating the two bacterial suspensions at 140rpm and 28 ℃ for 2-4h to obtain infectionAnd (3) liquid.
(5) Infection: immersing the callus obtained by the preculture into an invasion solution, oscillating for 20min at room temperature and 200rpm, then co-culturing for 3 days at 25 ℃ in dark places, flushing with sterile water for 5 times each for 30s, and ending the invasion.
(6) Co-cultivation: after infection is finished, taking out the callus, spreading the callus on sterile filter paper, airing, and then clamping the callus on the filter paper infiltrated by the Z1 liquid culture medium, and performing dark co-culture for 3d at 28 ℃; wherein, the Z1 liquid culture medium takes MS culture medium as basic culture medium, and 400mg/L of cephalosporin and 50mg/L of kanamycin are added.
(7) End of co-cultivation and screening: after the end of the 3d co-culture, the culture was incubated with ddH containing 400mg/L of cephalosporin and 50mg/L of kanamycin 2 Washing callus 5 times by O, washing once by using a Z1 liquid culture medium, spreading on sterile filter paper, airing, and carrying out regeneration culture by using R1, wherein the temperature is 25+/-2 ℃, the photoperiod is 16h, and the illumination intensity is 50 mu mol m -2 s -1 For 30 days, and the survival is the resistant callus once every 20 days; wherein, the Z1 liquid culture medium takes MS culture medium as basic culture medium, and 400mg/L of cephalosporin and 50mg/L of kanamycin are added; wherein, the R1 regeneration culture medium takes MS culture medium as basic culture medium, and is added with 3 mg/L6-benzylaminopurine, 9 mg/L1-naphthylacetic acid, 0.16g/L adenine sulfate and 30g/L sucrose.
(8) GUS histochemical staining analysis: GUS staining is carried out on the resistant callus according to a conventional method, the resistant callus with blue color reaction is identified as positive callus, and the GUS transient expression rate is counted.
The GUS transient expression rate is shown in Table 1. As can be seen from Table 1, under the same infection conditions, the infection efficiency of the EHA105 strain is far higher than that of GV3101, and it is worth mentioning that about 8.8% of tissue-extracted genomic DNA in the EHA 105-infected callus can be amplified by PCR to obtain GUS gene fragment. Although the fraction of fragment calli amplified was less than 10%, 8.8% of the currently reported Narcissus transformation system was the highest.
TABLE 1 Effect of different strains on the callus transformation efficiency of the sub-room-induced Chinese narcissus Jin Zhanyin plants
EXAMPLE 2 determination of Pre-culture time of ovary-induced callus
(1) Pretreatment of plant materials: three-year-old commercial seed balls (the production place is Fujian Zhangzhou) which are not subjected to water culture are placed in the Chinese narcissus Jin Zhanyin and refrigerated for 4 weeks at the temperature of 4 ℃ in a dark place.
(2) Explant sterilization: taking out the complete flower bud after the scales of the refrigerated seed balls are separated layer by layer but not broken bud, soaking the flower calyx in 1g/L carbendazim solution for 15min, then soaking the flower calyx in 75% ethanol solution for 1min, and then soaking the flower calyx in a mixed solution of 1% NaClO and 0.1% Tween-20 for 8min; finally, washing 3 times with sterile water for 30s each time; the sterilized flower bud is transversely cut at the basal part of the bud, the bud is longitudinally cut but can damage florets, the florets are obtained, the auxiliary crowns of the perianth above each floret ovary are cut off, and the tissues of the ovary and below are reserved.
(3) Callus induction: cutting ovary into small pieces of 2-3mm, placing on Z1 preculture medium, respectively at 25+ -2deg.C, photoperiod 16h, and illumination intensity 50 μmol m -2 s -1 Culturing for 0d, 20d, 60d; wherein, the Z1 preculture medium is based on MS medium, and is added with 9 mg/L6-benzylaminopurine, 3 mg/L1-naphthylacetic acid, 0.16g/L adenine sulfate and 30g/L sucrose.
(4) Preparing an aggressive dyeing liquid: mu.L of EHA105 Agrobacterium solution (OD) 600 =0.8) was transferred to 2mL of YEP liquid medium containing 50mg/L of spectinomycin and 100mg/L of rifampicin, and shake-cultured at 28 ℃ at 200rpm for 36-48h; then transferring 20. Mu.L of bacterial liquid into 20mL of YEP liquid culture medium containing 50mg/L of spectinomycin and 100mg/L of rifampicin, and culturing at 28 ℃ under shaking at 200rpm overnight until reaching OD 600 The cells were collected by centrifugation at 5000rpm for 20min to 0.8 and used1/2MS Medium the cells were rinsed to remove residual YEP Medium and resuspended in 1/2MS Medium containing 100. Mu.M acetosyringone to give a bacterial suspension OD 600 And (3) reaching 0.4-0.5, and oscillating and re-suspending the bacterial suspension for 2-4 hours at the temperature of 28 ℃ at 140rpm to obtain the dyeing liquid.
(5) Infection: the callus tissues with different preculture time periods are respectively immersed into the invasion solution, and are oscillated for 20min at room temperature and 200rpm, then co-cultured for 3 days at the temperature of 25 ℃ in the dark, washed for 5 times with sterile water for 30s each time, and the invasion is ended.
(6) Co-cultivation: after the infection is finished, the callus is taken out and laid on sterile filter paper, and after the callus is dried in the air, the callus is clamped on the filter paper infiltrated by the liquid Z1 culture medium, and the callus is co-cultured in the dark at 28 ℃ for 3d.
(7) End of co-cultivation and screening: after the end of the 3d co-culture, the culture was incubated with ddH containing 400mg/L of cephalosporin and 50mg/L of kanamycin 2 Washing callus for 5 times by O, washing with Z1 liquid culture medium, spreading on filter paper, air drying, and regenerating with R1 at 25+ -2deg.C, photoperiod 16h, and illumination intensity 50 [ mu ] mol m -2 s -1 For 30 days, and the survival is the resistant callus once every 20 days; wherein, the Z1 liquid culture medium takes MS culture medium as basic culture medium, and 400mg/L of cephalosporin and 50mg/L of kanamycin are added; wherein, the R1 regeneration culture medium takes MS culture medium as basic culture medium, and is added with 3 mg/L6-benzylaminopurine, 9 mg/L1-naphthylacetic acid, 0.16g/L adenine sulfate and 30g/L sucrose.
(8) GUS histochemical staining analysis: GUS staining is carried out on the resistant callus according to a conventional method, the resistant callus with blue color reaction is identified as positive callus, and the GUS transient expression rate is counted.
The GUS transient expression rate is shown in Table 2. As can be seen from Table 2, the GUS transient expression rate was substantially increased with the extension of the preculture time, the GUS transient expression rate was the highest in 60d preculture and was 66.67%, the preculture was 20d, and the conversion rate was 46.67%, so 60d was the optimal ovary-induced callus culture time.
TABLE 2 influence of ovary-induced callus Pre-culture time on the conversion efficiency of Chinese Narcissus Jin Zhanyin station' callus
EXAMPLE 3 determination of the minimum concentration of Kana Screen transgenic callus
(1) Pretreatment of plant materials: three years of Chinese narcissus Jin Zhanyin seed balls (the production place is Fujian Zhangzhou) which are not subjected to water culture are placed at 4 ℃ and refrigerated for 4 weeks in a dark place.
(2) Explant sterilization: taking out the whole scape from the scales of the refrigerated seed balls which are separated layer by layer but cannot be broken, soaking the scape in 1g/L carbendazim solution for 15min, then soaking the scape in 75% ethanol solution for 1min, and then soaking the scape in a mixed solution of 1% NaClO and 0.1% Tween-20 for 8min; finally, washing with sterile water for 5 times for 30s each time; the sterilized flower bud is transversely cut at the basal part of the bud, the bud is longitudinally cut but can damage florets, the florets are obtained, the auxiliary crowns of the perianth above each floret ovary are cut off, and the tissues of the ovary and below are reserved.
(3) Callus induction: cutting ovary into small pieces of 2-3mm, placing on Z1 preculture medium, and controlling temperature at 25+ -2deg.C, photoperiod 16h, and illumination intensity at 50 μmol m -2 s -1 Is cultured under the condition of (1) for 60d; wherein, the Z1 preculture medium is based on MS medium, and is added with 9 mg/L6-benzylaminopurine, 3 mg/L1-naphthylacetic acid, 0.16g/L adenine sulfate and 30g/L sucrose.
(4) Preparation of the dip dyeing liquid: bacterial solutions (OD) of two positive strains verified for EHA105 and GV3101 600 =0.8) transferring 2 μl into 2mL of YEP liquid medium containing 50mg/L spectinomycin and 100mg/L rifampicin, and shake culturing at 28deg.C and 200rpm for 36-48 hr; then transferring 20. Mu.L of bacterial liquid into 20mL of YEP liquid culture medium containing 50mg/L of spectinomycin and 100mg/L of rifampicin, and culturing at 28 ℃ under shaking at 200rpm overnight until reaching OD 600 The cells were collected by centrifugation at 5000rpm for 20min to 0.8 and washed with 1/2MS medium to remove residual YEP medium, and resuspended in 1/2MS medium containing 100. Mu.M acetosyringone to give a bacterial suspension OD 600 Reaching 0.4-0.5, and oscillating the two bacterial suspensions at 140rpm and 28 ℃ for 2-4 hours to obtain the dyeing liquor.
(5) Infection: immersing the callus obtained by the preculture into an invasion solution, oscillating for 20min at room temperature and 200rpm, then co-culturing for 3 days at 25 ℃ in dark places, flushing with sterile water for 5 times each for 30s, and ending the invasion.
(6) Co-cultivation: after infection is finished, taking out the callus, spreading the callus on sterile filter paper, airing, and then clamping the callus on the filter paper infiltrated by the Z1 liquid culture medium, and performing dark co-culture for 3d at 28 ℃; wherein, the Z1 liquid culture medium takes MS culture medium as basic culture medium, and 400mg/L of cephalosporin and 50mg/L of kanamycin are added.
(7) End of co-cultivation and screening: after the end of the 3d co-culture, the culture was incubated with ddH containing 400mg/L of cephalosporin and 50mg/L of kanamycin 2 Washing callus 5 times with O, washing with Z1 liquid culture medium, spreading on sterile filter paper, air drying, and regenerating R1 culture with kanamycin (0 mg/L, 20 mg/L, 50mg/L, 70mg/L, 90 mg/L, 100 mg/L) at 25+ -2deg.C, photoperiod 16h, and illumination intensity 50 μmol m -2 s -1 The culture is carried out for 30 days, every 20 days, the surviving callus is the resistant callus, and the callus regeneration rate is counted; wherein, the Z1 liquid culture medium takes MS culture medium as basic culture medium, and 400mg/L of cephalosporin and 50mg/L of kanamycin are added; wherein, the R1 regeneration culture medium takes MS culture medium as basic culture medium, and is added with 3 mg/L6-benzylaminopurine, 9 mg/L1-naphthylacetic acid, 0.16g/L adenine sulfate and 30g/L sucrose.
(8) GUS histochemical staining analysis: GUS staining was performed on resistant calli according to conventional methods, and resistant calli with blue color reaction were identified as positive calli.
The callus regeneration rate is shown in Table 3. As can be seen from table 3, with increasing kanamycin concentration, the callus regeneration rate significantly decreased, and at a time of a=0.05, it was examined that 50mg/L was not significantly different from 70mg/L, 100mg/L results, so 50mg/L was the lowest concentration for screening. At the same time, too high a concentration of kanamycin also has an inhibitory effect on resistant calli, which makes them slow to grow.
TABLE 3 Effect of Kan at different concentrations on the callus regeneration of the sub-chamber-induced Chinese narcissus Jin Zhanyin
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (2)
1. A method for establishing an agrobacterium-mediated Chinese narcissus efficient genetic transformation system is characterized by comprising the following steps: the method comprises the following steps:
(1) Ball seed pretreatment: placing the three-year-old Chinese narcissus seed balls at 4 ℃ and refrigerating for 4 weeks in a dark place;
(2) Explant sterilization: taking out the complete ovary after the complete scape is disinfected;
(3) Callus induction: cutting ovary into small pieces, placing on Z1 preculture medium, and culturing at 25+ -2deg.C for 16 hr/d under light cycle with illumination intensity of 50μmol.m -2 ·s -1 Is cultured under the condition of (1) for 60d;
(4) Preparation of the dip dyeing liquid: take 2. Mu.L OD 600 Monoclonal agrobacterium solution=0.8 was transferred to 2mL YEP liquid medium, and after shaking culture at 28 ℃ and 200rpm for 36-48h, 20 μl of the solution was transferred to 20mL YEP liquid medium, and shaking culture was carried out at 28 ℃ and 200rpm overnight until OD was reached 600 Up to 0.8, the cells were collected by centrifugation and rinsed with 1/2MS medium to remove residual YEP liquid medium, and resuspended with 100. Mu.M acetosyringone-containing 1/2MS medium to give a bacterial suspension OD 600 Up to 0.4-0.5, bacterial suspensionOscillating for 2-4 hours at 140rpm and 28 ℃ to obtain dyeing liquor;
(5) Infection: immersing the callus with good growth condition obtained by induction into an aggressive dyeing liquid, and oscillating for 20min at room temperature and 200 rpm;
(6) Co-cultivation: after infection is finished, taking out the callus, spreading the callus on sterile filter paper, airing, and then clamping the callus on the filter paper infiltrated by the Z1 liquid culture medium, and performing dark co-culture for 3d at the temperature of 28 ℃;
(7) End of co-cultivation and screening: after 3d co-cultivation is completed, ddH is used 2 Washing callus 5 times with O, washing with Z1 liquid culture medium, spreading on sterile filter paper, air drying, and transferring to R1 regeneration culture at 25+ -2deg.C with photoperiod 16h/d and illumination intensity of 50mu mol.m -2 ·s -1 For 30d, and subculturing every 20 days to obtain resistant callus;
(8) The obtained resistant callus is detected by GUS staining, and the resistant callus with blue color reaction is identified as positive callus, which indicates successful genetic transformation of the Chinese narcissus;
the disinfection method comprises the following steps: soaking the complete scape in 1g/L carbendazim solution for 15min, soaking in 75% ethanol solution for 1min, soaking in 1% NaClO/0.1% Tween-20 mixed solution for 8min, and washing with sterile water for 5 times for 30s each time;
the Z1 preculture medium is based on an MS medium, and 9mg/L of 6-benzylaminopurine, 3mg/L of 1-naphthylacetic acid, 0.16g/L of adenine sulfate and 30g/L of sucrose are added;
the agrobacterium is EHA105 agrobacterium which contains a plant expression vector pGWB344 and carries an kanamycin resistance gene and a reporter gene GUS;
the Z1 liquid culture medium takes an MS culture medium as a basic culture medium, and 400mg/L of cephalosporin and 50mg/L of kanamycin are added;
the ddH 2 O contains 400mg/L of cephalosporin and 50mg/L of kanamycin;
the YEP liquid culture medium contains 50mg/L of spectinomycin and 100mg/L of rifampicin;
the R1 regeneration medium is based on an MS medium, and is added with 3mg/L of 6-benzylaminopurine, 9mg/L of 1-naphthylacetic acid, 0.16g/L of adenine sulfate, 30g/L of sucrose and 50mg/L of kanamycin.
2. The use of a method for establishing an agrobacterium-mediated high-efficiency genetic transformation system of colchicine in colchicine breeding according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210162166.8A CN114410678B (en) | 2022-02-22 | 2022-02-22 | Method for establishing agrobacterium-mediated Chinese narcissus efficient genetic transformation system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210162166.8A CN114410678B (en) | 2022-02-22 | 2022-02-22 | Method for establishing agrobacterium-mediated Chinese narcissus efficient genetic transformation system |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114410678A CN114410678A (en) | 2022-04-29 |
| CN114410678B true CN114410678B (en) | 2023-08-18 |
Family
ID=81262272
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210162166.8A Active CN114410678B (en) | 2022-02-22 | 2022-02-22 | Method for establishing agrobacterium-mediated Chinese narcissus efficient genetic transformation system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114410678B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011139431A1 (en) * | 2010-05-06 | 2011-11-10 | Pioneer Hi-Bred International, Inc. | Maize acc synthase 3 gene and protein and uses thereof |
| CN105950652A (en) * | 2016-06-06 | 2016-09-21 | 福建农林大学 | Bi-induction homologous recombination mark-free vector plasmid and application thereof |
-
2022
- 2022-02-22 CN CN202210162166.8A patent/CN114410678B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011139431A1 (en) * | 2010-05-06 | 2011-11-10 | Pioneer Hi-Bred International, Inc. | Maize acc synthase 3 gene and protein and uses thereof |
| CN105950652A (en) * | 2016-06-06 | 2016-09-21 | 福建农林大学 | Bi-induction homologous recombination mark-free vector plasmid and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 水仙SWEET糖转运蛋白的cDNA克隆及其功能的初步研究;魏培宁;《中国优秀硕士学位论文全文数据库 农业科技辑》;D048-326 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114410678A (en) | 2022-04-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Damm et al. | Regeneration of fertile plants from protoplasts of different Arabidopsis thaliana genotypes | |
| CN108456690B (en) | Efficient genetic transformation and rapid identification method for brassica napus | |
| US5792927A (en) | Genetically transformed rose plants and methods for their production | |
| WO2013097343A1 (en) | Method for breeding poplar haploid | |
| CN104770294B (en) | A kind of protocorm based on the sprouting of iris seed is the breeding method of acceptor | |
| AU2009209502B2 (en) | Regeneration and mass propagation of Jatropha curcas through somatic embryogenesis | |
| EP0536327A4 (en) | ||
| CN114058640A (en) | Efficient agrobacterium-mediated sugarcane genetic transformation method | |
| EP0582603A4 (en) | Carnation plants and methods for their transformation and propagation | |
| CN114410678B (en) | Method for establishing agrobacterium-mediated Chinese narcissus efficient genetic transformation system | |
| CN110305894B (en) | Rapid and efficient catalpa bungei genetic transformation method | |
| CN108901844B (en) | Method for constructing lycoris genus genetic transformation system | |
| CN113755521B (en) | Construction method of agrobacterium-mediated strawberry 'sweet Charles' genetic transformation system | |
| Subroto et al. | Tissue culture media optimization and genetic transformation of Jatropha curcas genotype Jatromas cotyledon explants | |
| KR20100053456A (en) | A method for producing transformed rice plant removed selection marker | |
| JP2008259497A (en) | Method for creating transformant of domestic variety of soybean through agrobacterium and method for acquiring seed of current generation and progeny of transformant in short period of time | |
| Kim et al. | Improvement of ornamental characteristics in Rehmannia elata through Agrobacterium rhizogenes-mediated transformation | |
| CN104017823B (en) | Screening method for fast and effectively reducing false positive rate of peanut genetic transformation plant | |
| CN103146748A (en) | Agrobacterium mediated rose bud transgenosis infecting method | |
| CN106047921A (en) | Growth media for genetic modification of diploid strawberries and genetic modification method adopting growth media | |
| Lombari et al. | Agrobacterium-mediated in vitro transformation | |
| Terakawa et al. | Improvement of regeneration and transformation systems for Cyclamen persicum using somatic embryo culture | |
| CN113875589B (en) | Preparation method and application of cold-resistant cauliflower germplasm | |
| CN115786232B (en) | Quick suspension culture and genetic transformation method for saussurea involucrata cells | |
| CN116590333B (en) | Method for establishing papaya genetic transformation system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |