CN115232832B - Molecular breeding method for obtaining new variety of transgenic Baimaigen from cutting seedlings - Google Patents
Molecular breeding method for obtaining new variety of transgenic Baimaigen from cutting seedlings Download PDFInfo
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- C12N15/8205—Agrobacterium mediated transformation
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- C12N15/821—Non-antibiotic resistance markers, e.g. morphogenetic, metabolic markers
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Abstract
The invention discloses a molecular breeding method for obtaining a new variety of transgenic Baimaigen from cutting seedlings. The method constructs a plant expression vector of a target gene according to the breeding purpose, takes regenerated bud leaves induced after detoxification of cutting seedlings as genetic transformation receptors, introduces the target gene into a hundred vein root genome by an agrobacterium tumefaciens mediated method, screens transformants by hygromycin during transformation callus induction and regeneration of transformed buds, and detects through PCR, southern hybridization, GUS staining and other modes to prove that the obtained resistant strains are transgenic plants and are successfully transplanted into soil. The method is not limited by the quantity and fertility of genetic transformation starting materials, and a large number of new species of the lotus seed roots of the transgenes can be obtained within 5 months, so that precious genetic materials are provided for the innovation of the germplasm resources of the lotus seed roots.
Description
Technical Field
The invention belongs to the technical field of plant molecular breeding, and particularly relates to a molecular breeding method for obtaining a new variety of transgenic Baimaigen from cutting seedlings.
Background
The plant of the genus Baimai (lotus root l.) of the genus Baimai of the family leguminosae is a perennial high quality grass, native to the warm region of two continents of europe, and is now widely planted as a high quality grass in countries developed in animal husbandry such as the united states, canada, new zealand, etc., and is also distributed in areas such as inner mongolia, northwest, southwest, south china, etc. It has the advantages of high yield, good quality, barren resistance, long green period, etc. And the microbial nitrogen-fixing agent has the characteristic of symbiotic nitrogen fixation with rhizobia, and has wide application prospect in the aspects of soil improvement, ecological environment restoration and the like. Therefore, the excellent grazing cultivation varieties are cultivated, so that the grazing cultivation varieties are popularized and planted as excellent pastures, and the pasture cultivation varieties in China can be effectively expanded; the cultivation of the Baimaigen variety with high nitrogen fixation efficiency in extreme environment has important significance for improving ecological environment, improving the utilization rate of domestic soil and sustainable agriculture.
Transgenic biotechnology is one of the most rapid important technologies applied to modern biotechnology, and gene editing technology with transgenic technology as a core will greatly enhance the wonderful aspects of cultivating good varieties of plants in the future.
Disclosure of Invention
In order to overcome the defect that seeds or anthers are required to be used as starting materials in the prior art for genetic transformation and regeneration of the Baimai roots, and the nutritional propagation materials cannot be utilized for genetic transformation and regeneration, the invention aims to provide a molecular breeding method for obtaining a new variety of transgenic Baimai roots from cutting seedlings.
The invention discloses a method for preparing transgenic plant, which comprises the steps of transforming agrobacterium with an expression vector carrying a target gene, co-culturing agrobacterium with a plant tissue culture technique-cultured Baimaigen explant, inserting the target gene into the genome of the explant, screening resistance elements, detecting resistance calli or resistance buds by a molecular detection method, obtaining a transformant strain which contains the target gene, obtaining a large number of transformants by a bud multiplication way, and obtaining a new transgenic Baimaigen variety after the transformants are cultivated, thereby realizing the purpose of the invention.
The molecular breeding method for obtaining the new variety of the transgenic Baimaigen from the cutting seedlings comprises the following steps:
a. culture of explants: cutting off the branch of the vein, cutting off leaves, carrying out detoxification, cutting the branch into small sections, wherein each small section at least comprises one node, cutting the small section branch into a regeneration bud induction culture medium for culture, and carrying out regeneration bud induction; taking small leaf blades on the regenerated buds after the regenerated buds grow to 3-5cm long, and cutting the small leaf blades into small blocks with the size of 0.5 multiplied by 0.5cm as explants;
b. target gene transformation explant and regeneration: soaking the explant in agrobacterium tumefaciens bacterial solution containing an expression vector carrying a target gene for 20min, and taking out the explant to remove redundant bacterial solution; then the explant is moved to a co-culture medium and cultured in darkness for 5 days; after washing, the explant is transferred to a resistant callus screening culture medium for culture, and resistant callus is induced; transferring the resistant callus to a resistant bud induction culture medium for culture, and updating the culture medium every 2 weeks to obtain regenerated resistant buds; then cutting resistant buds, inoculating the resistant buds into a root induction culture medium, and culturing and inducing rooting to obtain resistant plants;
c. and (3) molecular detection: carrying out PCR molecular detection, southern hybridization detection or RT-PCR detection by using the sequences of the screening marker gene, the reporter gene or the target gene on the expression vector, and detecting whether the resistant callus, the resistant bud or the resistant plant is transgenic material;
d. transplanting transgenic plants: and taking out the detected resistant plants containing the target genes from the culture medium, and transplanting the resistant plants to a culture medium to obtain the transgenic hundred vein roots.
Preferably, the detoxification of step a comprises the following steps: soaking the Baimai root branches in 75% ethanol water solution for 30s, washing the branches with sterile water, transferring the branches to a sterile water solution containing tween 20 with the final concentration of 0.1% and sodium hypochlorite with the final concentration of 2% by mass, soaking the branches for 15min, and washing the branches with sterile water for 5-6 times. The sterilization method can sterilize the hundred-vein root branches without damaging the hundred-vein root branches, and ensures the regeneration of the hundred-vein root cutting branches under the pollution-free condition.
Preferably, the step of immersing the explant in an agrobacterium tumefaciens bacterial solution containing an expression vector carrying a target gene for 20min comprises the following steps: agrobacterium tumefaciens containing an expression vector carrying a gene of interest is first suspended in 1/2B5 culture at pH5.2 containing acetosyringone at a final concentration of 2mg/LCulturing in medium, conventional culture to bacterial liquid OD 600 0.4 to 0.5, and then soaking the explant in the bacterial liquid for 20 minutes.
Preferably, the washing in the step b is to wash the co-cultured explant 5-6 times with a sterile aqueous solution containing Tween 20 with a final concentration of 0.1% by volume, and then placing the explant on sterile paper to suck the water.
Preferably, the regeneration bud induction medium is: 1/2B5 Medium was supplemented with 8g/L agar, 0.2mg/L6-BA, pH 5.5.
Preferably, the co-culture medium is: 1/10B5 medium was supplemented with 8g/L agar, 0.5 mg/L6-BA, 0.05mg/L NAA, 5mM MES buffer and 2mg/L acetosyringone, pH 5.5.
Preferably, the resistant callus screening medium is: 1/2B5 Medium 8g/L agar, 1 mg/L6-BA, 0.1mg NAA, 20mg hygromycin and 500mg cephalosporin were added at pH 5.5.
Preferably, the resistant bud induction medium is: 1/2B5 Medium was supplemented with 8g/L agar, 0.2mg/L6-BA, 10mM (NH) 4 ) 2 SO 4 10mM MES, 20mg/L hygromycin and 300mg/L cephalosporin, pH 5.5.
Preferably, the root induction medium is: 1/2B5 Medium 8g/L agar, 10g/L sucrose and 0.5mg/L NAA, pH 5.5 were added.
Preferably, the culture temperature in the steps a and b is 24+/-1 ℃, and the illumination culture conditions are 35 mu mol.m of illumination intensity in other tissue culture stages except co-culture -2 ·s -1 16h light/8 h dark.
Preferably, the agrobacterium tumefaciens containing the expression vector carrying the target gene is constructed by the following method: after inserting the target gene into the expression vector promoter, the constructed expression vector carrying the target gene is guided into agrobacterium tumefaciens by a freeze thawing method, and the agrobacterium tumefaciens containing the expression vector carrying the target gene is obtained through resistance screening and molecular detection verification.
Preferably, the expression vector is a pCAMBIA1301 vector, which contains a hygromycin phosphotransferase gene hpt as a hygromycin resistance screening gene; the agrobacterium tumefaciens is agrobacterium tumefaciens AGL1.
Preferably, the expression vector promoter is a ubiquitin promoter of Baimaigen or a CaMV35S promoter of tobacco cauliflower mosaic virus. Both promoters can well start the target gene to express in the Baimaigen. The Ubiquitin promoter belongs to a known promoter, and is a promoter with strong expression in various tissue types of the Baimaigen, and is derived from Baimaigen polyubiquitin gene (polyubiquitin gene). The CaMV35S promoter is also a known promoter, which is the constitutive promoter most commonly used in dicotyledonous plants, derived from the tobacco cauliflower mosaic virus.
The hygromycin content in the resistant callus screening culture medium and the resistant bud induction culture medium is preferably 20mg hygromycin per liter of culture medium, and under the screening pressure of the concentration, the resistant callus, the resistant bud and the resistant plant obtained by screening are all transgenic materials. Therefore, when the Baimaigen transgenic plant is performed, the screening pressure of 20mg/L hygromycin can ensure that the obtained resistant plant is the transgenic plant, and the workload of later detection and verification of whether the obtained resistant plant is the transgenic plant is reduced.
The invention has the following beneficial effects:
the molecular breeding method for rapidly obtaining a large number of new varieties of transgenic Baimai roots, which is established by the invention, can purposefully introduce exogenous genes to improve the characters of Baimai roots, thereby obtaining new varieties of Baimai roots with excellent characters.
Stable genetic transformation of the Baimaigen generally depends on the hypocotyl of the seedling. The invention uses the small leaf blade cut pieces of the regenerated buds induced by the cuttage branches as transformation receptors, the regenerated buds obtained by the tissue culture technology can be preserved aseptically for a long time under laboratory conditions, and can be proliferated by the tissue culture technology when the regenerated buds are needed, so that the source of the plant is not limited, the transgenic operation can be performed at any time, the step of needing a large amount of seeds for the transgenic operation is omitted, and an available method is provided for genetic transformation of sterile or seed yield-deficient hundred-pulse root mutant materials and the like. In the prior art, the tissue culture and gene transformation method of the Baimai root needs to take seeds to sprout seedlings or needs to take anthers as starting materials, and the inventor discovers a new Baimai root sterile mutant in the research process, but the tissue culture and gene transformation regeneration work of sterile mutant materials cannot be carried out at all by using the prior method. The inventor solves the problems of asexual propagation of the Baimai root sterile line and new variety cultivation, develops the molecular breeding method for obtaining the transgenic Baimai root new variety from the cutting seedling, and can be used for mass propagation and gene transformation of Baimai root materials which are free of flowers, sterile or can not be propagated by seeds due to quantity and other reasons; and meanwhile, transformation is not started until seeds come out, so long as seedlings can be used, and the propagation process is accelerated.
Repeated tests prove that the transformation frequency of the molecular breeding method can reach 15% -18%, and the required transgenic strain can be obtained by one transformation test operation because the explants used for transformation are easy to obtain in large quantity.
In the molecular breeding method, each transformed explant can generate a regeneration bud, the regeneration buds can be multiplied by transfer culture and subculture, and most of the regeneration buds can root and develop into normal plants, so that a large number of transgenic plants can be obtained within 5 months by applying the molecular breeding method, and most of the transgenic plants can be successfully transplanted into soil, thereby ensuring the success of the variety cultivation cultivated by the molecular breeding method and realizing the cultivation of new variety of the Baimaigen.
The molecular breeding method of the transgenic Baimai root overcomes the obstacle of the traditional breeding and cultivation method applied to Baimai root breeding, meets the urgent need of the current academic research on cultivation, screening, symbiotic nitrogen fixation and the like of Baimai root pasture with excellent quality and high nitrogen fixation efficiency, and provides reliable genetic materials for the innovation, research and sustainable development of Baimai root germplasm resources; has remarkable ecological, economic and social benefits in the field of forage grass variety cultivation and ecological environment improvement.
Drawings
FIG. 1 is a flow chart from cutting shoots to obtaining transgenic lines.
FIG. 2 is a process from the cutting of the Baimaigen shoots to the obtaining of transgenic lines; wherein, the graph A is the branch of cuttage; FIG. B shows regenerated shoots grown at stem nodes after three weeks of cultivation of cutting stems in regenerated shoot induction medium; panel C shows embryogenic callus induced from leaf explants; panel D shows shoots formed after 6 weeks of culture on resistant shoot induction medium; panel E shows the roots formed after 3 weeks of culture in root induction medium; panel F is a T0 transgenic line grown in vermiculite for 3 weeks.
FIG. 3 is an electrophoretogram of PCR-detected resistant calli, wherein WT is an untransformed strain, 1, 2, 3, 4 are transgenic lines obtained by transforming pCAMBIA1301 plasmid, and primers for the hpt gene are used for the detection.
FIG. 4 is an electrophoretogram of PCR detection of resistant shoots, wherein WT is an untransformed strain, 1, 2, 3, 4 are transgenic lines obtained by transforming pCAMBIA1301 plasmid, and primers of hpt gene are used for the detection.
FIG. 5 is an electrophoretogram of T0 transgenic plants after PCR detection of the resulting transgenic lines obtained by transforming pCAMBIA1301 plasmid with the primers of the hpt gene, wherein WT is untransformed, and 1, 2, 3, 4.
FIG. 6 is an electrophoretogram of a transgenic line detected by Southern blot, wherein WT is an untransformed strain, 1, 2, 3, 4 are the 4 transgenic lines T0 generation obtained by transforming the pCAMBIA1301 plasmid, and genomic DNA is digested with HindIII using the GUS gene as a probe.
FIG. 7 is a transgenic line expressing pLjTHIC in ljTHIC lethal mutant and its phenotypic verification using the method of example 1.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
The individual components of the media referred to in the examples below, such as Gamborg's B-5 medium salts (Sigma-Aldrich, cat# G5768) and vitamins (Sigma-Aldrich, cat# G5768), agar (Sigma-Aldrich, cat# A1296, 0.8% added to the solid medium), benzyladenine (BA), a-Naphthalene Acetic Acid (NAA), MES buffer, hygromycin, cephalosporin and acetosyringone, are commercially available. 1/10, 1/2B5 medium was prepared by reducing all components of B5 medium to 1/10, 1/2 content according to the instructions of Gamborg's B-5 medium salt.
Example 1
(1) Construction of the expression vector of the Gene plant of interest
Plasmid pCAMBIA1301 (commercially available from biological reagent company) containing the CaMV35S promoter of tobacco cauliflower mosaic virus followed by the beta-glucuronidase gene (gus reporter gene) and hygromycin phosphotransferase selection gene hpt was selected as an expression vector. When the transformation of the target gene is required, the target gene is inserted into the CaMV35S promoter of the pCAMBIA1301 vector, and then the subsequent operation is performed. In this example, a pCAMBIA1301 empty vector containing no target gene will be described as an example.
(2) Preparation of engineering strain containing target gene plant expression vector
The pCAMBIA1301 plasmid was introduced into Agrobacterium tumefaciens AGL1 by freeze thawing. The specific method comprises the following steps: 1. Mu.g of pCAMBIA1301 plasmid is taken and added into 200. Mu.L of competent cells dissolved on ice, mixed lightly, ice-bathed for 30min, quick frozen for 5min in liquid nitrogen, water-bathed for 5min at 37 ℃ and then rapidly ice-bathed for 2min, 800. Mu.l of YEP liquid medium is added, and the mixture is gently shaken for 4 to 6h at 28 ℃. And (3) coating 50-200 mu L of bacterial liquid on a selective medium YEP containing 50mg/L kanamycin and 50mg/L rifampicin, culturing for 2d at 28 ℃ in an inverted mode, and selecting single colony for detection. Then inoculating the transformed agrobacterium tumefaciens EHA105 into a YEP liquid culture medium containing kanamycin with the final concentration of 50mg/L and rifampicin with the final concentration of 50mg/L, culturing for 24 hours at the temperature of 28 ℃, centrifuging to remove bacterial liquid, suspending bacterial cells in a 1/2B5 culture medium (pH 5.2) containing acetosyringone with the final concentration of 2mg/L, and conventionally culturing until the bacterial liquid OD600 = 0.4-0.5 for later use.
(3) Culture of explants for transformation
Collecting the vigorous growth of Baimai root, cutting off leaves, washing with tap water, and adding 75% of the total volumeSoaking the branches in an ethanol aqueous solution for 30s, then quickly washing the branches with sterile water, transferring the branches into a sterile aqueous solution containing tween 20 with the final concentration of 0.1 percent and sodium hypochlorite with the final concentration of 2 percent for 15min, washing the branches with sterile water for 5 to 6 times, and cutting the branches into small sections with sterile blades, wherein each small section at least comprises one node; cutting the small branch into regenerated bud induction culture medium, and placing at 24+ -1deg.C with illumination intensity of 35 μmol.m -2 ·s -1 Culturing under 16h illumination/8 h darkness, inducing regeneration bud, and growing regeneration bud after 2-4 weeks; the regeneration bud induction culture medium is as follows: 1/2B5 medium added with 8g/L agar, 0.2mg/L6-BA (6-Benzylaminoprone) (the above added component concentrations were all the final concentrations after addition to the medium, and the other mediums described below were all expressed in the same manner), pH was 5.5. The induction rate of the explant induced regeneration bud reaches more than 90%. And (3) taking small leaf blades on the regenerated buds after the regenerated buds grow to 3-5cm long, cutting the small leaf blades on the regenerated buds into small blocks with the size of 0.5X0.5 cm, and using the small leaf blades as genetically transformed explants for agrobacteria dip-dyeing.
(4) Obtaining hygromycin resistant plants
Soaking the explant in the standby agrobacterium tumefaciens bacteria solution containing pCAMBIA1301 plasmid for 20min, taking out the explant, placing the explant on sterile paper, and sucking the redundant bacteria solution; then the explant is moved into a co-culture medium, placed at 24+/-1 ℃ and cultured for 5 days in the dark; the co-culture medium is as follows: 1/10B5 medium was supplemented with 8g/L agar, 0.5 mg/L6-BA, 0.05mg/LNAA (a-Naphthalene acetic acid), 5mM MES buffer and 2mg/L acetosyringone, pH 5.5. Then washing the co-cultured explant with sterile water solution containing Tween 20 with final concentration of 0.1% by volume for 5-6 times, placing the explant on sterile paper, drying the water, transferring the explant to a resistant callus screening culture medium, and placing the explant at 24+ -1deg.C under light intensity of 35 μmol.m -2 ·s -1 Culturing for 3-6 weeks under 16h illumination/8 h darkness to induce resistant callus; the resistant callus screening culture medium is as follows: 1/2B5 Medium 8g/L agar, 1 mg/L6-BA, 0.1mg NAA, 20mg hygromycin and 500mg cephalosporin were added at pH 5.5. The callus induction rate of the culture medium is 604%. Transferring the resistant callus to a resistant bud induction culture medium, and placing at 24+ -1deg.C under light intensity of 35μmol.m -2 ·s -1 Culturing under 16h illumination/8 h darkness, and renewing the culture medium every 2 weeks, wherein regeneration of resistant buds can be observed after 4-7 weeks; the resistant bud induction culture medium is as follows: 1/2B5 Medium was supplemented with 8g/L agar, 0.2mg/L6-BA, 10mM (NH) 4 ) 2 SO 4 10mM MES, 20mg/L hygromycin and 300mg/L cephalosporin, pH 5.5. With this medium, the induction rate from resistant calli to shoots was about 13%. Then cutting resistant buds and inoculating the resistant buds into root induction culture medium, wherein the temperature is 24+/-1 ℃ and the illumination intensity is 35 mu mol.m -2 ·s -1 Culturing under 16h illumination/8 h darkness, inducing rooting for 1-3 weeks, and then transplanting; the root induction culture medium is as follows: 1/2B5 Medium 8g/L agar, 10g/L sucrose and 0.5mg/L NAA, pH 5.5 were added. With this medium, the root induction rate exceeded 90%. The flow of genetic transformation is shown in FIG. 1. The tissue culture process is shown in FIG. 2.
(5) Molecular detection of transgenic plants
Since pCAMBIA1301 contains the GUS reporter gene, the expression of the GUS gene can be detected by staining according to the GUS staining method of Jefferson et al (1987). The resistant callus, resistant shoots and untransformed strain (which is not transformed into pCAMBIA1301 plasmid) are immersed in X-Gluc staining solution, incubated at 37 ℃ for 3-5 hours, decolorized with 70% ethanol, and blue staining is observed. The detection shows that the resistant callus of the pCAMBIA1301 plasmid transferred from the regenerated shoot leaf, regenerated shoot rhizome combination and regenerated shoot leaf stalk is blue, the resistant bud of the pCAMBIA1301 plasmid transferred is blue, and the untransformed strain without the pCAMBIA1301 plasmid transferred is not blue, which indicates that the gus gene has been successfully introduced into the hundred vein root and stably expressed in the transformed strain by the above method.
By a PCR method, a specific primer for screening the gene hpt is adopted:
HFw(5’-CGA TCT TAG CCA GAC GAG CGG GTT C-3’)，
HRe(5’-GCT GGG GCG TCG GTT TCC ACT ATC GG-3’)；
the amplification condition is 5min at 94 ℃; 30 cycles of 30 total cycles of 30s at 94 ℃, 30s at 60 ℃ and 1min at 72 ℃; finally, the mixture was extended at 72℃for 10min.
As shown in FIG. 3, the target fragment of 629bp was amplified from the resistant callus by the above PCR method, but not from the negative control callus (WT) without the pCAMBIA1301 plasmid. As shown in FIG. 4, the target fragment of 629bp was amplified from the resistant shoots by the PCR method described above, but not from the regenerated shoots without transformation with pCAMBIA 1301. As shown in FIG. 5, the target fragment of 629bp was amplified from the T0 generation transgenic line by the PCR method described above, but not amplified by the Wild Type (WT) without the pCAMBIA1301 plasmid.
Further, by the Southern hybridization method, as shown in FIG. 6, a clear hybridization band was observed in the T0 generation transgenic line with GUS as a probe, whereas no signal was observed in the untransformed line WT (the hundred roots which were not transformed with pCAMBIA1301 plasmid). The Southern hybridization results further demonstrate that the GUS gene was successfully transferred into the Baimai genome and that all T-DNA insertions in the transgenic lines were single copy insertions.
The experiment shows that all the resistant plants obtained in the example are transgenic materials which have been transferred into pCAMBIA1301 plasmid, which shows that the resistant plants obtained by 20mg/L hygromycin selection are transgenic materials. Therefore, when the Baimaigen transgenic plant is performed, the screening pressure of 20mg/L hygromycin can ensure that the obtained resistant plant is the transgenic plant, and the workload of detecting the transgenic plant in the later period is reduced.
(6) Transplanting of transgenic plants
The transformed plants which are screened by hygromycin and rooted are taken out of the culture medium, the culture medium is washed out, the transformed plants are planted in sterilized vermiculite, the sterilized vermiculite is covered by a preservative film, small holes are formed in the preservative film, the plants grow in a dark plant growth room at 22+/-1 ℃ for 16h illumination/8 h darkness, and 100% of the plants can survive. By the above method, a large number of transgenic plants can be obtained within 5 months.
Transgenic plants are obtained according to the method, and the transformation frequency can reach 15% -18%, and in the embodiment, the transformation frequency is 16%.
Example 2: application of the method of example 1 to ljthic lethal mutant
THIC (Phosphomethylpyrimidine synthase, encoding methyl pyrimidine phosphate synthase) is a key enzyme in the plant thiamine synthesis pathway, which after mutation, plants cannot grow normally, but are seedling-stage lethal (FIG. 7A), but mutants can grow normally when exogenous thiamine is sprayed. To verify the biological function of the Baimaigen THIC gene, we obtained a transgenic line expressing pLjTHIC in the ljTHIC mutant by the method of example 1 after spraying 0.1mM thiamine during the planting of the ljTHIC mutant, and so on, to obtain a certain amount of cuttable shoots, the expression of LjTHIC in the mutant can completely restore the phenotype of ljTHIC mutant seedling death, and the mutant not transformed into LjTHIC is still lethal (FIG. 7B).
Comparative example 1
This comparative example is essentially the same as example 1, except that the explant material is different.
Example 1 resistant callus induction was 60.4% using leaflet leaf cut pieces (0.5x0.5 cm size) of regeneration buds on detoxified cutting shoots as explants.
In this comparative example, stem sections (length of 0.5 cm) of regenerated shoots on detoxified cuttings were used as explants, and the other treatment methods, culture media at each stage, culture conditions, and the like were the same as in example 1, and the rate of induction of resistant calli at this time was only 33.33%.
Comparative example 2
The ability of the transgenic explants to regenerate is an important factor in assessing the transformation efficiency of the transgenic system.
As in example 1, the callus induction rates of regenerated buds were observed under different hormone concentration conditions using leaflet leaf cut pieces (0.5X0.5 cm size) as starting materials for transgenes. Callus induction was performed using three different hormone concentrations, namely, medium 1 was 1/2B5 medium supplemented with 8g/L agar, 1 mg/L6-BA and 0.1mg/L NAA (minimal medium and hormone concentrations of the resistant callus screening medium of example 1); medium 2 was 1/2B5 medium supplemented with 8g/L agar, 0.5 mg/L6-BA and 0.05mg/L NAA; medium 3 was 1/2B5 medium supplemented with 8g/L agar, 0.2mg/L6-BA and 0.05mg/L NAA. The callus induction efficiency was calculated with regenerated bud leaflets as explants. The results showed that the highest callus induction rate (60.4%) was obtained when 1 mg/L6-BA and 0.1mg/L NAA were added to the callus induction medium (i.e., medium 1), whereas the callus induction rate of medium 2 was only 22.9%, and the callus induction rate of medium 3 was only 20.8%.
Claims (2)
1. A molecular breeding method for obtaining a new variety of transgenic Baimaigen from cutting seedlings, which is characterized by comprising the following steps:
a. culture of explants: cutting off the branch of the vein, cutting off leaves, carrying out detoxification, cutting the branch into small sections, wherein each small section at least comprises one node, cutting the small section branch into a regeneration bud induction culture medium for culture, and carrying out regeneration bud induction; taking small leaf blades on the regenerated buds after the regenerated buds grow to 3-5cm length, and cutting the small leaf blades into small blocks with the size of 0.5 multiplied by 0.5cm to be used as explants; the regeneration bud induction culture medium is as follows: 1/2B5 medium was supplemented with 8g/L agar, 0.2mg/L6-BA, pH 5.5; the detoxification comprises the following steps: soaking the branch of the Baimai with 75% ethanol water solution for 30s, then washing the branch with sterile water, transferring the branch into sterile water solution containing Tween 20 with the final concentration of 0.1% and sodium hypochlorite with the final concentration of 2% for 15min, and then washing the branch with sterile water for 5-6 times;
b. target gene transformation explant and regeneration: agrobacterium tumefaciens containing an expression vector carrying a gene of interest was suspended in 1/2B5 medium containing acetosyringone at a final concentration of 2mg/L and pH5.2 and routinely cultured to a bacterial liquid OD 600 Soaking the explant in the bacterial liquid for 20min to obtain the residual bacterial liquid for the explant; the explants were then transferred to co-culture medium and cultured in darkness for 5d; flushing the co-cultivated explant for 5-6 times by using a sterile water solution containing Tween 20 with the final concentration of 0.1% by volume, then placing the explant on sterile paper to suck water, and transferring the explant to a resistant callus screening culture medium for culture to induce resistant callus; transferring the resistant callus to resistant bud induction medium for culturing, and renewing the medium every 2 weeks to obtainObtaining regenerated resistant buds; then cutting resistant buds, inoculating the resistant buds into a root induction culture medium, and culturing and inducing rooting to obtain resistant plants; the agrobacterium tumefaciens containing the expression vector carrying the target gene is constructed by the following method: after inserting a target gene into an expression vector promoter, introducing the constructed expression vector carrying the target gene into agrobacterium tumefaciens by a freeze thawing method, and obtaining the agrobacterium tumefaciens containing the expression vector carrying the target gene through resistance screening and molecular detection verification; the expression vector is pCAMBIA1301 vector, which contains hygromycin phosphotransferase genehptAs hygromycin resistance screening genes; the agrobacterium tumefaciens is agrobacterium tumefaciens AGL1; the co-culture medium is as follows: 1/10B5 medium was supplemented with 8g/L agar, 0.5 mg/L6-BA, 0.05mg/L NAA, 5mM MES buffer and 2mg/L acetosyringone, pH 5.5; the resistant callus screening culture medium is as follows: 1/2B5 medium was supplemented with 8g/L agar, 1 mg/L6-BA, 0.1mg/L NAA, 20mg/L hygromycin and 500mg/L cephalosporin, pH 5.5; the resistant bud induction culture medium is as follows: 1/2B5 Medium was supplemented with 8g/L agar, 0.2mg/L6-BA, 10mM (NH) 4 ) 2 SO 4 10mM MES, 20mg/L hygromycin and 300mg/L cephalosporin, pH 5.5; the root induction culture medium is as follows: 1/2B5 Medium 8g/L agar, 10g/L sucrose and 0.5mg/L NAA, pH 5.5;
c. and (3) molecular detection: carrying out PCR molecular detection, southern hybridization detection or RT-PCR detection by using the sequences of the screening marker gene, the reporter gene or the target gene on the expression vector, and detecting whether the resistant callus, the resistant bud or the resistant plant is transgenic material;
d. transplanting transgenic plants: taking out the detected resistant plants containing the target genes from the culture medium, and transplanting the resistant plants to a culture medium to obtain transgenic vein roots;
the culture temperature in the steps a and b is 24+/-1 o C, at other tissue culture stages except co-culture, the illumination culture condition is that the illumination intensity is 35 mu mol.m -2 ·s -1 16h light/8 h darkness.
2. The method of claim 1, wherein the expression vector promoter is the ubiquitin promoter of centella asiatica or the CaMV35S promoter of tobacco cauliflower mosaic virus.
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