CN115386591B - Molecular breeding method of single herba Cichorii - Google Patents
Molecular breeding method of single herba Cichorii Download PDFInfo
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Abstract
The invention discloses a molecular breeding method of single herba Cichorii. The invention takes leaves, petioles and stems of aseptic seedlings as transformation receptors, and introduces a target gene into a single herba oroxyli genome by a method of mediating agrobacterium tumefaciens by using a constructed plant expression vector containing the target gene, and differentiates the target gene into regenerated plants by a bud organogenesis way; the transformants were screened by adding the corresponding, appropriate concentration of the selection marker gene during the induction of the transformed shoots, elongation of the transformed shoots and rooting, and the obtained resistant strains were confirmed to be transformants by Southern hybridization, northern hybridization and GUS staining. The whole operation process of the method is 4 months, 100 leaf explants are operated at a time, transgenic plant lines of about 11 plant lines can be obtained on average, more than 20 buds can be generated from each transgenic leaf through 6 weeks of culture, the rooting rate of regenerated plants is 100%, and the survival rate of the transgenic plants transplanted to soil is 100%.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a molecular breeding method for rapidly obtaining a large number of transgenic single-locus chicory novel germplasm.
Background
The single-seat chicory (Metabriggsia ovalifolia W.T.Wang) is a plant of the genus Sonchus of the family Sonchaceae, is a special plant distributed in areas under the Guangxi (napo, jing and Huanjiang three counties), guizhou (Libo) and Yunnan elevation 1100 meters limestone forest in China, has important scientific research values in the aspects of evolution of the plant system of the family Sonchaceae, the growth and development regulation mechanism of special habitat plants, the maintenance of ecological species diversity and the like, has extremely high economic development value in the aspect of the resource development and utilization of horticultural plants, but due to the requirement of special habitat, the plant is listed in China as I-grade protection plant and in red directory of the endangered species of the world natural protection alliance. The method has the advantage that the plant resources of the single herba Cichorii are saved.
Transgenic biotechnology is undoubtedly one of the most rapid important technologies applied to modern biotechnology, has unique advantages in the aspect of cultivating excellent varieties of plants, and particularly has limited application research of traditional technologies such as hybridization, introduction domestication, bud mutation, physicochemical mutagenesis and the like on plants which can complete life history under special habitats, so that the transgenic technology plays an extremely important role in molecular breeding of the plants.
At present, single seat chicory transgenic technology research papers and patent reports are not found. Therefore, the development of the molecular breeding technology of the single-seat chicory has important scientific research and production significance.
Disclosure of Invention
Aiming at the defect that the single-endive transgenic technology does not exist in the prior art, the invention provides a molecular breeding method for rapidly obtaining a large number of transgenic single-endive, a large number of novel single-endive germplasm can be obtained within 4 months, and the novel single-endive germplasm for early flowers is created by using the method, so that the blank of the genetic transformation breeding technology of the single-endive at home and abroad is filled, the urgent need of the research on endangered scientific mechanism and ornamental resource development on the genetic transformation technology of the single-endive is satisfied, and the method has remarkable scientific, ecological, economic and social benefit significance in the conservation of germplasm resources, the maintenance of species diversity and the sustainable development of the single-endive.
The invention relates to a molecular breeding method of single herba Cichorii, which comprises the following steps:
S1, culturing of explants for transformation: the explant is leaf, petiole or stem of a single plant regenerated from herba Sonchi Oleracei; the regenerated plants are prepared through the following steps: taking single-seat chicory young leaves, sterilizing, cutting into sections with the length of 0.5-0.8cm, inoculating to a bud induction culture medium, culturing to generate green callus, then developing into buds, cutting out buds when the buds reach 1cm, transferring to a rooting culture medium for culturing, and inducing rooting to obtain regenerated plants;
The bud induction culture medium is MS culture medium added with 1mg/L BA, 0.1mg/L NAA, 30g/L sucrose and 8g/L agar, and the pH is 5.8;
the rooting culture medium is a 1/2MS culture medium, 30g/L sucrose and 8g/L agar are added, and the pH value is 5.8;
S2, transforming to obtain a resistant plant: soaking the explant prepared in the step S1 in agrobacterium tumefaciens bacterial solution containing a plant expression vector carrying a target gene for 25-35 minutes, taking out the explant, sucking the excessive bacterial solution, transferring the explant into a co-culture medium, culturing in darkness for 3 days, taking out the co-cultured explant, rinsing, transferring the explant into a resistant bud induction screening medium I, culturing under light, transferring the resistant bud into a fresh resistant bud induction medium II after the resistant bud grows, transferring the resistant bud into a resistant bud rooting medium when the resistant bud reaches 1cm, culturing under light, and elongating the resistant bud and generating adventitious roots to obtain a resistant plant;
The co-culture medium is MS culture medium added with 1mg/L BA, 0.1mg/L NAA, 20mg/L acetosyringone, 30g/L sucrose and 8g/L agar, and the pH is 5.5;
the resistant bud induction screening culture medium I is MS culture medium added with 1mg/L BA, 0.1mg/L NAA, 15-30mg/L hygromycin, 500mg/L cephalosporin, 30g/L sucrose and 8g/L agar, and the pH is 5.8;
The resistant bud induction screening culture medium II is prepared by adding 1mg/L BA, 0.1mg/L NAA, 15-30mg/L hygromycin, 250mg/L cephalosporin, 30g/L sucrose and 8g/L agar into MS culture, and the pH value is 5.8;
The rooting culture medium of the resistant buds is 1/2MS culture medium, 25-30mg/L hygromycin, 250mg/L cephalosporin, 30g/L sucrose and 8g/L agar are added, and the pH is 5.8;
s3, molecular detection of resistant plants: detecting whether the gene is transferred into a single locus of the herba Sonchi arvensis genome by utilizing the sequence of a screening marker gene, a reporter gene or a target gene on a plant expression vector, detecting the expression level of the target gene by GUS staining or Northern hybridization, and detecting whether a resistant bud or a resistant plant is transgenic material;
S4, transplanting transgenic plants: and removing the detected resistant plants confirmed to contain the target genes from the culture flask, washing off the root culture medium, and planting the root culture medium in a pot filled with a culture medium to obtain transgenic plants successfully transplanted.
Preferably, the disinfection in the step S1 is that after the young leaves are washed clean by tap water, the young leaves are put into a carbendazim aqueous solution with the mass fraction of 0.1 percent for soaking for 5 to 10 minutes, and the young leaves are taken out and washed clean by tap water; soaking in 75% ethanol water solution for 30 seconds, washing with sterile water for 3 times, transferring young leaves into 0.1% mercuric chloride water solution containing 0.05% Tween 80 for 12 minutes, washing with sterile water for 6 times, and then placing the leaves on sterile filter paper to absorb water.
Preferably, the rinsing in the step S2 is to rinse the co-cultured explant with a Tween 80 sterile water solution with the mass fraction of 0.05% for 5-6 times, then rinse the explant with a cephalosporin sterile water solution with the mass fraction of 500mg/L for one time, and place the explant on sterile paper to absorb water.
Preferably, in the step S2, the explant is immersed in agrobacterium tumefaciens bacterial solution containing a plant expression vector carrying a target gene for 25 minutes.
Preferably, in the step S2, 1mg/L BA, 0.1mg/L NAA, 15mg/L hygromycin, 500mg/L cephalosporin, 30g/L sucrose and 8g/L agar are added to the MS culture medium, and the pH is 5.8; the resistant bud induction screening culture medium II is MS culture and added with 1mg/L BA, 0.1mg/L NAA, 15mg/L hygromycin and 250mg/L cephalosporin, 30g/L sucrose and 8g/L agar, and the pH is 5.8.
Preferably, in the step S2, 25mg/L hygromycin, 250mg/L cephalosporin, 30g/L sucrose and 8g/L agar are added to the 1/2MS culture medium, and the pH is 5.8.
Preferably, the culture temperature in the steps S1 and S2 is 23-26 ℃.
Preferably, the agrobacterium tumefaciens containing the plant expression vector carrying the target gene is constructed by the following method: after inserting the target gene into the expression vector promoter, introducing the constructed plant expression vector carrying the target gene into agrobacterium tumefaciens by a freeze thawing method, and carrying out resistance screening to obtain the agrobacterium tumefaciens containing the plant expression vector carrying the target gene.
Preferably, the agrobacterium tumefaciens bacteria solution containing the plant expression vector carrying the target gene is prepared by the following steps: agrobacterium tumefaciens containing a plant expression vector carrying a target gene is first suspended in an MS medium containing 20mg/L acetosyringone at a pH of 5.2 and cultured until od600=0.4-0.5.
Preferably, the plant expression vector is a pCAMBIA1301 vector (which contains a β -glucosidase reporter gene gus driven by a CaMV 35S promoter and a hygromycin phosphotransferase selection gene hpt as selection genes), or a p1390Ubi vector. The p1390Ubi vector was constructed by excision of the Ubiquitin promoter from plasmid pAHC27 using HindIII and BamHI and insertion of the promoter fragment at the HindIII and BamHI sites of plasmid pCAMBIA1390, which contains the hygromycin phosphotransferase selection gene hpt driven by the CaMV 35S promoter as the selection gene and the maize Ubiquitin promoter. The target gene may be inserted after the CaMV 35S promoter or the Ubiquitin promoter of the above vector according to the study requirement.
The MS culture medium is an internationally-used culture medium, and the components and the configuration method are shown in Toshio Murashige,Folke Skoog(1962)ARevised Medium For Rapid GrowthAnd BioAssays With Tobacco Tissue Cultures.Physiollogia Plantarum,15:473-497.1/2MS culture medium which is a culture medium for halving each element in the MS culture medium.
The invention adopts plant tissue culture technology to obtain a large number of single-seat sowthistle aseptic seedlings for gene transformation; according to the actual research or breeding target, constructing a plant expression vector containing a target gene driven by CaMV35S promoter of tobacco cauliflower mosaic virus or a Ubiquitin promoter of corn and a screening marker gene, introducing the expression vector into agrobacterium tumefaciens, co-culturing the agrobacterium tumefaciens with leaves, petioles and stem explants, screening obtained resistant buds and resistant plants by using antibiotics corresponding to the screening marker gene, and proving to be transgenic materials by GUS staining and Southern hybridization detection technology. And (3) cutting leaves, petioles and stem node explants from the aseptic seedlings, and carrying out agrobacterium tumefaciens infection until a transplantable transgenic plant is obtained, wherein the whole operation process is 4 months. 100 leaf explants are operated at a time, transgenic plant lines of about 11 plant lines can be obtained on average, more than 20 buds can be generated from each transgenic leaf after 6 weeks of culture, the rooting rate of regenerated plants is 100%, and the survival rate of the transgenic plants transplanted to soil is 100%.
The method can obtain a large number of transgenic single-seat chicory germplasm in 4 months, meets the urgent need of the current single-seat chicory biological research and cultivation technology, provides an effective research method and precious research materials for the research of the innovation, research and development of single-seat chicory germplasm resources, cultivation of excellent new varieties and the like, and has remarkable scientific, ecological, economic and social benefit significance.
The invention has the following advantages:
(1) Accelerated scientific research of Sonchus arvensis
The transgenic technology is an important means of plant scientific research, and can provide necessary research technology for the scientific research of gene functions, development, propagation, characteristics and the like of the single herba cichorii, and promote the scientific research of the endangered species of the single herba cichorii.
(2) New variety of genetically improved single-seat chicory is quickly created
The single herba cichorii has peculiar flower color, a large amount of novel germplasm of the single herba cichorii can be obtained within 4 months by using the method of the invention, a series of mutants can be created, the properties of the single herba cichorii can be purposefully improved according to breeding, landscape or ecological requirements, and the ornamental gardening value of the single herba cichorii can be developed.
(3) Efficient single-seat chicory tissue culture plant regeneration system
The bud induction of the single herba Sonchi Oleracei of the former people adopts TDZ, BA, NAA, IBA with high concentration, and the bud rooting adopts NAA and IBA simultaneously. The single-seat chicory tissue culture plant regeneration system established by the invention is very efficient: 100% of leaves, petioles and stem nodes cut from the aseptic seedlings cultured by the method can induce buds in the bud induction culture medium provided by the method, and the bud induction culture medium only adopts BA and NAA; the rooting rate of the regenerated buds in the rooting culture medium is 100%, and the rooting culture medium is free of hormone; 100% of regenerated plants can successfully transplant into soil for survival.
(4) Simple culture of explants for transformation
The method provided by the invention can be used for aseptically preserving aseptic seedlings for a long time under laboratory conditions, the source of explants is not limited, and transgenic operation can be performed at any time.
(5) Method for strictly and effectively screening transformed plants
The resistant plants obtained by 25mg/L hygromycin screening are transgenic materials proved by GUS staining, southern hybridization and Northern hybridization experiments. This ensures that the resistant plants obtained by this technical method are transgenic plants, reducing the effort of molecular detection at a later stage.
(6) High conversion efficiency and good repeatability
The technical method provided by the invention proves that the conversion rate is 11% -15.2% through repeated experiments (Table 4). Since the explants used for transformation are readily available in large numbers, a large number of desired transgenic lines can be obtained in one transformation experimental run.
(7) High breeding efficiency
Through the operation, each positive transformation explant can generate cluster buds, the cluster buds can be multiplied by successive generations, more than 20 buds can be formed by 1 leaf in each 1 successive multiplication period (21 days), and the cluster buds can root and develop into plants, so that a large number of transgenic plants can be obtained in 4 months by applying the method disclosed by the invention, 100% of the transgenic plants can be successfully transplanted into soil, and the success of the cultivation of varieties cultivated by a molecular breeding method is ensured.
Drawings
FIG. 1 is a schematic representation of the T-DNA region of a plant expression vector; wherein 35S pro is the promoter of CaMV35S of tobacco cauliflower mosaic virus; ubi is the Ubiquitin promoter of maize.
FIG. 2 shows the resistant plexus shoots transformed with pCAMBIA1301 when inoculated in the resistant shoot induction screening medium for 42 days.
FIG. 3 shows the results of transient expression detection of GUS gene from leaf explants of Cichorium intybus after 3 days of co-culture with Agrobacterium tumefaciens EHA105/pCAMBIA 1301.
FIG. 4 shows resistant cluster buds (42 days; panel A) and resistant buds (80 days; panel B) of pCAMBIA1301 with GUS staining showing blue color.
FIG. 5 shows untransformed regenerated plants without GUS staining (Panel A) and resistant regenerated plants with GUS staining transformed with pCAMBIA1301 (Panel B).
FIG. 6 shows the result of Southern hybridization to detect hpt in pCAMBIA1301 transformants; wherein WT is an untransformed strain, exhibiting no hybridization signal; t1, T2, T3, T4, T5 are pCAMBIA1301 transformants, all with a hybridization signal and all being different transformants.
FIG. 7 shows the phenotype of Hd3a plants at day 22 (panel A), day 40 (panel B), day 67 (panel C), day 90 (panel D) flowering in vermiculite matrix between transplanting growths (22.+ -.1 ℃), 1 month transplanting (panel E), wild WT regenerated plants with no flowering at 1 year, and plants transformed with pCAMBIA1301 (panel F).
FIG. 8 shows the expression of Hd3a detected by Southern hybridization (panel A) and Hd3a detected by Northern hybridization (panel B); wherein the WT is an untransformed strain showing no detection of a hybridization signal specific for Hd3a and no expression of Hd3 a; h1, H2, H3 and H4 are Hd3a transformed plants, and correspond to A, B, C, D plant samples in FIG. 7 respectively.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Example 1
The molecular breeding method of the single herba Cichorii comprises the following steps:
a. Culture of explants for transformation
Taking young leaves of the single-seat herba oroxyli with good growth, washing the young leaves with tap water, soaking the young leaves in 0.1 mass percent carbendazim aqueous solution for 5 to 10 minutes, taking the young leaves out, and washing the young leaves with tap water. Soaking in 75% ethanol water solution for 30 seconds, washing with sterile water for 3 times, transferring young leaves into 0.1% mercuric chloride water solution containing 0.05% Tween 80 for 12 minutes, washing with sterile water for 6 times, and then placing the leaves on sterile filter paper to absorb water. The leaves were cut into 0.6cm sections for transformation experiments or individually inoculated into shoot induction medium, which was MS medium supplemented with 1mg/L BA, 0.1mg/L NAA, 30g/L sucrose and 8g/L agar, pH5.8. The culture is carried out under the conditions of illumination for 12 h/day at the temperature of 24+/-1 ℃ and the illumination for 50 mu mol.m –2·s–1. Culturing for about 21 days, wherein green callus appears, the callus develops into buds, the buds are cut out and transferred into a rooting culture medium when the buds are about 1cm high, and 30g/L sucrose and 8g/L agar are added into the rooting culture medium which is 1/2MS culture medium, and the pH value is 5.8; the 1/2MS culture medium refers to a culture medium obtained by halving all elements in the MS culture medium. Rooting induction is carried out under the same condition. Then, 1 subculture is carried out for 21 days, and buds with the height of about 1cm are cut off and transferred to rooting culture medium for culture. Leaves were excised from a sterile seedling about 1.5cm high as explants for transformation.
B. preparation of engineering strain containing target gene plant expression vector
The pCAMBIA1301 vector (pCAMBIA 1301 vector contains beta-glucosidase reporter gene gus driven by CaMV 35S promoter and hygromycin phosphotransferase screening gene hpt as screening genes, the target genes can be placed behind CaMV 35S promoter according to the breeding target requirement, and FIG. 1, the beta-glucosidase reporter gene gus is used as experimental test gene) is introduced into Agrobacterium tumefaciens EHA105 in this example, then inoculated in YEP medium containing 50mg/L kanamycin and 50mg/L rifampicin, cultured for 24 hours at 28 ℃, bacterial cell suspension is collected by centrifugation, bacterial cell suspension is carried out in MS medium (pH5.2) containing 20mg/L acetosyringone, and thus Agrobacterium tumefaciens EHA105 bacterial liquid containing the pCAMBIA1301 vector is obtained and named Agrobacterium tumefaciens EHA105/pCAMBIA1301.
C. Obtaining regenerated plants containing hygromycin
The leaf explant is soaked in agrobacterium tumefaciens EHA105/pCAMBIA1301 bacterial solution for 25 minutes, then the explant is taken out, the residual bacterial solution is sucked and dried on sterile paper, and then the explant is transferred to a co-culture medium, wherein the co-culture medium is MS culture medium, 1mg/L BA, 0.1mg/L NAA, 20mg/L acetosyringone, 30g/L sucrose and 8g/L agar are added, the pH is 5.5, and the culture is carried out in dark for 3 days at 25+/-1 ℃. Taking out the co-cultured explant, flushing 5-6 times by using a Tween 80 sterile water solution with the mass fraction of 0.05%, flushing once by using a cephalosporin sterile water solution with the mass fraction of 500mg/L, placing the explant on sterile paper, sucking water, and transferring the explant to a resistant bud induction screening culture medium I, wherein the resistant bud induction screening culture medium I is MS culture medium, and 1mg/L BA, 0.1mg/L NAA, 15mg/L hygromycin, 500mg/L cephalosporin, 30g/L sucrose and 8g/L agar are added into the resistant bud induction screening culture medium I, and the pH is 5.8. Culturing at 24+ -1deg.C under light for 21 days.
The explants with resistant shoots were transferred to resistant shoot induction screening Medium II, which added 1mg/L BA, 0.1mg/L NAA, 15mg/L hygromycin and 250mg/L cephalosporin, 30g/L sucrose and 8g/L agar for MS culture, pH5.8. Culturing was carried out at 24.+ -. 1 ℃ for 21 days under light (FIG. 2). Every 21 days, fresh resistant bud induction screening culture medium II is replaced, when the height of the resistant buds is 1cm, the culture medium is transferred to a resistant bud rooting culture medium, 25mg/L hygromycin, 250mg/L cephalosporin, 30g/L sucrose and 8g/L agar are added to the resistant bud rooting culture medium which is 1/2MS culture medium, pH is 5.8, and the culture is carried out under light at 24+/-1 ℃, so that the elongation of the resistant buds and the generation of adventitious roots can be observed. Thus, a hygromycin resistant regenerated plant (resistant plant) transformed with pCAMBIA1301 was obtained.
D. 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). Leaf explants, resistant buds, untransformed regenerated plants and transformed resistant plants after co-culturing with agrobacterium tumefaciens EHA105/pCAMBIA1301 for 3 days are respectively immersed in X-Gluc staining solution, kept at 37 ℃ for 3-5 hours, decolorized with 70% ethanol, and blue staining is observed. It can be seen that there was a distinct blue spot on the surface of the explant (FIG. 3) after 3 days of co-cultivation with Agrobacterium tumefaciens EHA105/pCAMBIA1301, indicating that Agrobacterium tumefaciens EHA105/pCAMBI A1301 was able to adsorb and dip the single-seat chicory explant, both resistant shoots (FIG. 4) and resistant plants (FIG. 5) were blue, whereas untransformed regenerated plants (FIG. 5) were not blue, indicating that the gus gene had been successfully introduced into the single-seat chicory and stably expressed in the transformant by the above procedure. Specific primers for screening gene hpt are used: HFw (5'-CGATCTTAGCCAGA CGAGCGGGTTC-3') and HRe (5'-GCTGGGGCG TCGGTTTCCACTATCGG-3') were synthesized and hybridization signals were observed in resistant plants T1, T2, T3, T4, T5 by Southern hybridization, and the banding patterns were different (FIG. 6), indicating that T1, T2, T3, T4, T5 were all different transgenic lines, and that untransformed strain WT (wild type) had no signal, again indicating that by this method, the hpt gene had been successfully introduced into the genome of the single seat of Sonchus arvensis.
E. Transplanting of transgenic regenerated plants
The non-transgenic plant and the transgenic plant with the length higher than 2cm are removed from a culture flask, root culture medium is washed off, the plant is planted in a pot filled with vermiculite, the plant is covered by a preservative film, small holes are formed in the preservative film, and the plant is uncovered after 5 days, so that the survival rate reaches 100%. After 31 days of field planting, the growth conditions of the two are observed and compared, and as shown in fig. 7E and F, the growth conditions of the hygromycin resistant regenerated plant transformed with pCAMBIA1301 are basically the same as those of the regenerated plant of the untransformed plant, which indicates that the obtained pCAMBIA1301 does not obviously influence the growth and phenotype of the plant.
By using the method of the embodiment, leaf explants are cut from aseptic seedlings to be subjected to agrobacterium tumefaciens infection to obtain transgenic plants which can be transplanted, and the whole operation process is 4 months. 100 leaf explants are operated at a time, transgenic plant lines of about 11 plant lines can be obtained on average, more than 20 buds can be generated from each transgenic leaf after 6 weeks of culture, the rooting rate of regenerated plants is 100%, and the survival rate of the transgenic plants transplanted to soil is 100%.
Regarding the concentration of hygromycin screening agent in the resistant bud induction medium:
Example 2
The procedure of this example is identical to the rest of the procedure of example 1, except that: the concentration of hygromycin in the resistant bud induction screening medium I, II used in this example was 10mg/L, and the corresponding screening effect is shown in Table 1.
Example 3
The procedure of this example is identical to the rest of the procedure of example 1, except that: the hygromycin concentration in the resistant bud induction screening medium I, II used in this example was 20mg/L, and the corresponding screening effect is shown in Table 1.
Example 4
The procedure of this example is identical to the rest of the procedure of example 1, except that: the hygromycin concentration in the resistant bud induction screening medium I, II used in this example was 30mg/L, and the corresponding screening effect is shown in Table 1.
Comparative example 1
This comparative example is identical to the rest of the procedure of example 1, except that: the explants used in this comparative example were stem cut pieces (plants with a stem width of about 0.5cm were cut transversely and stem flakes of about 0.1cm were cut transversely as explants) and the corresponding screening effects are shown in Table 1.
Comparative example 2
This comparative example is identical to the rest of the procedure of example 1, except that: the explants used in this comparative example were petioles (petioles cut transversely to a length of about 0.5cm were explants) and the corresponding screening results are shown in Table 1.
Parallel cultures were performed according to the methods of example 1, example 2, example 3, example 4, comparative example 1 and comparative example 2, respectively, and the culture time was 42d, and the results were counted and shown in Table 1.
TABLE 1 effects of different hygromycin concentrations on leaf explant bud induction
| Grouping | Hygromycin concentration (mg/L) | Sample size (number of explants) | Bud growth after 42 days (%) |
| Example 1 | 15 | 75 | 0 |
| Example 2 | 10 | 75 | 5.3 |
| Example 3 | 20 | 75 | 0 |
| Example 4 | 30 | 75 | 0 |
| Comparative example 1 | 15 | 20 | 0 |
| Comparative example 2 | 15 | 20 | 0 |
As can be seen from Table 1, the hygromycin dose was 15-30mg/L and no shoots were produced after 42 days of culture, indicating that 15mg/L hygromycin could be used for transformation screening of leaf, stem and petiole explants in the resistant shoot induction screening medium I, II.
Regarding agrobacterium infection time:
Example 5
The procedure of this example is identical to the rest of the procedure of example 1, except that: in this example, the time for immersing the explant in the bacterial solution of Agrobacterium tumefaciens EHA105/pCAMBIA1301 in step c was 15 minutes, and the corresponding efficiency of obtaining resistant shoots is shown in Table 2.
Example 6
The procedure of this example is identical to the rest of the procedure of example 1, except that: the time for immersing the explant in the Agrobacterium tumefaciens EHA105/pCAMBIA1301 bacterial solution in step c was 35 minutes, and the corresponding efficiency of obtaining resistant shoots is shown in Table 2.
Comparative example 3
This comparative example is identical to the rest of the procedure of example 1, except that: the explants used in this comparative example were stem sections (plants with a stem width of about 0.5cm were cut and stem flakes of about 0.1cm were cut transversely as explants), and the corresponding efficiencies of obtaining resistant shoots are shown in Table 2.
Comparative example 4
This comparative example is identical to the rest of the procedure of example 1, except that: the explants used in this comparative example were petioles (petioles with a length of about 0.5cm cut transversely were explants) and the corresponding efficiency of obtaining resistant shoots is shown in Table 2.
Comparative example 5
This comparative example is identical to the rest of the procedure of example 1, except that: the explants used in this comparative example were obtained from the 3 rd leaves of plants grown in vermiculite at a height of 10cm from top to bottom, and the disinfection method was the same as that of the young leaves in step a of example 1, and the corresponding efficiency of obtaining resistant shoots is shown in Table 2.
Parallel cultures were performed according to the methods of example 1, example 5, example 6, comparative example 3, comparative example 4 and comparative example 5, respectively, and the results are shown in Table 2.
TABLE 2 Effect of different Agrobacterium infection times on the yield of resistant shoots
As can be seen from Table 2, the Agrobacterium infection time was 25-35min, the efficiency of transient expression of GUS in the explants was higher than 70%, the generation rate of resistant shoots was highest in example 1, example 6 and comparative example 3, and the positive rate of GUS in the resistant shoots was highest in example 1. Comparative example 5, probably because the explant is derived from the sterilized leaf, the explant is damaged, affecting the adsorption effect of agrobacterium. In summary, the leaves, stems and petioles of the tissue culture aseptic seedlings are used as the explants infected by agrobacterium, and transgenic buds can be obtained, wherein the transformation efficiency obtained by the leaves of the tissue culture seedlings is highest, because the stems and petioles of the single herba cichorii are extremely short, the explants obtained in the wild are sterilized to cause damage to the explants, and the opposite leaves of the tissue culture seedlings are large, so that the tissue culture seedling leaves are used as the transformation explants with the best effect.
Regarding the concentration of hygromycin screening agent in the rooting medium:
Example 7
The procedure of this example is identical to the rest of the procedure of example 1, except that: the hygromycin concentration in the rooting medium of the resistant shoots used in this example was 15mg/L, and the corresponding efficiency in obtaining rooting resistant plants is shown in Table 3.
Example 8
The procedure of this example is identical to the rest of the procedure of example 1, except that: the hygromycin concentration in the rooting medium of the resistant shoots used in this example was 30mg/L, and the corresponding efficiency in obtaining rooting resistant plants is shown in Table 3.
Parallel cultures were performed according to the methods of example 1, example 7 and example 8, respectively, and the results are shown in Table 3.
TABLE 3 Effect of different concentrations of hygromycin on rooting of resistant shoots
As can be seen from Table 3, the hygromycin concentration in the rooting medium is 25-30mg/L, and the rooting resistant plants can be grown, and the GUS positive rate is 100%, so that the selection dose of hygromycin in the rooting medium of the resistant buds is 25mg/L, the obtained resistant plants are positive, and the workload of molecular detection is reduced.
Example 9
The transgenic method of the single herba Cichorii established in example 1 is adopted to introduce a flowering gene Hd3a derived from rice into a genome of the single herba Cichorii, so that early flowering of the single herba Cichorii is promoted, and the method for molecular breeding of the single herba Cichorii is utilized to create novel early flowering germplasm of the single herba Cichorii.
The procedure of this example is substantially the same as that of example 1, except that:
a. Preparing engineering strain containing target gene plant expression vector: preparation of agrobacterium tumefaciens EHA105/p1390UbiHd a bacterial liquid
With the flowering gene Hd3a derived from rice as a target gene, we constructed a plant expression vector of Hd3a driven by the Ubiquitin promoter derived from maize by the following method. The Ubiquitin promoter was excised from plasmid pAHC27 using HindIII and BamHI, and the fragment was inserted into the HindIII and BamHI sites of plasmid pCAMBIA1390 to construct plasmid p1390Ubi. Extracting mRNA of rice leaves, reversely transcribing the mRNA into cDNA, and designing a primer Hd3aFw according to the sequence of a rice Hd3a gene (GeneBank accession number: os06g 0157700): 5' -AATGAATTC (EcoRI site) ATGGCCG GAAGTGGCAGGGAC-3 and Hd3aRe:5'-AATGAATTC (EcoRI site) CTAGGGGTAGACC CTCCTG-3', amplifying Hd3a fragment from cDNA, cloning the fragment into pGEM-T vector, inserting the fragment into EcoRI site of plasmid p1390Ubi after sequencing verification, and obtaining constructed plasmid p1390UbiHd a after enzyme digestion and sequencing verification, wherein the specific structure is shown in figure 1. The plasmid also contains a selectable marker gene hpt, which can be used to select transformed cells. P1390UbiHd a was introduced into Agrobacterium tumefaciens EHA105 by freeze thawing, then inoculated into YEP medium containing 50mg/L kanamycin and 50mg/L rifampicin, cultured at 28℃for 24 hours, bacterial cells were collected by centrifugation, and suspended in MS medium (pH 5.2) containing 20mg/L acetosyringone, OD600 = 0.4-0.5, thereby obtaining Agrobacterium tumefaciens EHA105 bacterial liquid containing p1390UbiHd a vector, designated Agrobacterium tumefaciens EHA105/p1390Ubi Hd3a.
B. phenotype of Hd3 a-transformed regenerated plants
Hd3a transgenic plants showed early flowering, and FIGS. 7A-D show the phenotype of the transgenic plants when they had been shifted to flowering at 22, 40, 67 and 90 days between growth, whereas WT untransformed and pCAMBIA1301 plants were not flowering during 1 year of cultivation between growth (FIGS. 7E-F).
C. molecular detection of Hd3 a-transformed plants
The specific primer Hd3aFw of Hd3a is adopted to carry out 5'-ATGGCCGGAAGTGGCAGGGAC-3'; hd3aRe:5'-CTAGGGGTAGACCCTCCTG-3' synthetic probe, hybridization signals were observed by Southern hybridization in the early flowering Hd3a transgenic plants A, B, C, D, and the banding patterns were different (FIG. 8A), indicating that A, B, C, D were different transgenic lines, and that the untransformed strain WT (wild type) had no signal, indicating that the Hd3a gene had been successfully introduced into the genome of the single seat of Sonchus arvensis by the above method. Northern hybridization (FIG. 8B) showed that Hd3a expression signal was detected in Hd3a transgenic plants A, B, C, D, whereas the untransformed strain (WT) had no Hd3a expression. It was demonstrated that expression of the Hd3a gene promotes early flowering in Hd3a transgenic single-seat chicory plants.
The parallel operation of 3 times of conversion was carried out according to the method of example 1 and example 9, and the conversion efficiency results are shown in Table 4.
TABLE 4 comparison of conversion efficiencies for different batches
| Grouping | Batch of | Sample size (leaf explant number) | PCR amplification of hpt Positive Rate (%) |
| Example 1 | 1 | 73 | 11 |
| Example 1 | 2 | 69 | 14.5 |
| Example 1 | 3 | 74 | 14.9 |
| Example 9 | 1 | 66 | 15.2 |
| Example 9 | 2 | 82 | 12.2 |
| Example 9 | 3 | 79 | 11.4 |
As can be seen from Table 4, the single-endive transgenic method established by the invention has good repeatability and the transformation frequency is more than 11%.
Claims (7)
1. A molecular breeding method of single herba oroxyli, which is characterized by comprising the following steps:
s1, culturing of explants for transformation: the explant is a leaf of a single plant regenerated from herba Sonchi Oleracei; the regenerated plants are prepared through the following steps: taking single-seat chicory young leaves, sterilizing, cutting into sections with the length of 0.5-0.8cm, inoculating to a bud induction culture medium, culturing to generate green callus, then developing into buds, cutting out buds when the buds reach 1cm, transferring to a rooting culture medium for culturing, and inducing rooting to obtain regenerated plants;
The bud induction culture medium is MS culture medium added with 1mg/L BA, 0.1mg/L NAA, 30g/L sucrose and 8g/L agar, and the pH is 5.8;
the rooting culture medium is a 1/2MS culture medium, 30g/L sucrose and 8g/L agar are added, and the pH value is 5.8;
The disinfection is that after the young leaves are washed clean by tap water, the young leaves are put into a carbendazim aqueous solution with the mass percent of 0.1 percent for soaking for 5 to 10 minutes, and the young leaves are taken out and washed clean by tap water; soaking for 30 seconds by using an ethanol water solution with the volume fraction of 75%, flushing for 3 times by using sterile water, transferring young leaves into an aqueous solution with the mass fraction of 0.1% of mercury and 0.05% of Tween 80, soaking for 12 minutes, flushing for 6 times by using the sterile water, and then placing the leaves on sterile filter paper to absorb water;
s2, transforming to obtain a resistant plant: soaking the explant prepared in the step S1 in agrobacterium tumefaciens bacterial solution containing a plant expression vector carrying a target gene for 25 minutes, taking out the explant, sucking the excessive bacterial solution, transferring the explant into a co-culture medium, culturing in darkness for 3 days, taking out the co-cultured explant, rinsing, transferring the explant into a resistant bud induction screening culture medium I, culturing under light, transferring the resistant bud into a fresh resistant bud induction culture medium II after the resistant bud grows, transferring the resistant bud into a resistant bud rooting culture medium when the resistant bud reaches 1cm, culturing under light, and elongating the resistant bud and generating adventitious roots to obtain a resistant plant;
The co-culture medium is MS culture medium added with 1mg/L BA, 0.1mg/L NAA, 20mg/L acetosyringone, 30g/L sucrose and 8g/L agar, and the pH is 5.5;
the resistant bud induction screening culture medium I is MS culture medium added with 1mg/L BA, 0.1mg/L NAA, 15mg/L hygromycin, 500mg/L cephalosporin, 30g/L sucrose and 8g/L agar, and the pH is 5.8;
the resistant bud induction screening culture medium II is MS culture medium added with 1mg/L BA, 0.1mg/L NAA, 15mg/L hygromycin and 250mg/L cephalosporin, 30g/L sucrose and 8g/L agar, and the pH is 5.8;
The rooting culture medium of the resistant buds is a 1/2MS culture medium, 25mg/L hygromycin, 250mg/L cephalosporin, 30g/L sucrose and 8g/L agar are added, and the pH is 5.8;
The rinsing is to rinse the co-cultured explant with a Tween 80 sterile water solution with the mass fraction of 0.05% for 5-6 times, then rinse the explant with a cephalosporin sterile water solution with the mass fraction of 500mg/L once, and place the explant on sterile paper to absorb water;
s3, molecular detection of resistant plants: detecting whether the gene is transferred into a single locus of the herba Sonchi arvensis genome by utilizing the sequence of a screening marker gene, a reporter gene or a target gene on a plant expression vector, detecting the expression level of the target gene by GUS staining or Northern hybridization, and detecting whether a resistant bud or a resistant plant is transgenic material;
S4, transplanting transgenic plants: and removing the detected resistant plants confirmed to contain the target genes from the culture flask, washing off the root culture medium, and planting the root culture medium in a pot filled with a culture medium to obtain transgenic plants successfully transplanted.
2. The method according to claim 1, wherein the culture temperature in steps S1 and S2 is 23 to 26 ℃.
3. The method of claim 1, wherein the agrobacterium tumefaciens containing the plant expression vector carrying the target gene is constructed by: after inserting the target gene into the expression vector promoter, introducing the constructed plant expression vector carrying the target gene into agrobacterium tumefaciens by a freeze thawing method, and carrying out resistance screening to obtain the agrobacterium tumefaciens containing the plant expression vector carrying the target gene.
4. The method according to claim 1, wherein the agrobacterium tumefaciens bacterial solution containing the plant expression vector carrying the target gene is prepared by: agrobacterium tumefaciens containing a plant expression vector carrying a target gene is first suspended in an MS medium containing 20mg/L acetosyringone at a pH of 5.2 and cultured until od600=0.4-0.5.
5. The method of claim 1, wherein the plant expression vector is pCAMBIA1301, pCAMBIA1390 or p1390Ubi vector.
6. Use of the molecular breeding method of mono-endive as claimed in any one of claims 1-5 for obtaining early flowering mono-endive new varieties by transgenesis.
7. The use according to claim 6, wherein the Hd3a gene of rice is transferred as a target gene into the genome of Sonchus arvensis.
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5349124A (en) * | 1988-04-25 | 1994-09-20 | Monsanto Company | Insect-resistant lettuce plants |
| CN1606623A (en) * | 2000-11-21 | 2005-04-13 | 南洋理工大学 | Production of stilbenes in transgenic plants and the method of producing thereof |
| CN101940159A (en) * | 2010-06-30 | 2011-01-12 | 中国科学院华南植物园 | Molecular breeding method for quickly obtaining large number of new transgenic Saussurea involucrata varieties |
| CN101965800A (en) * | 2010-11-19 | 2011-02-09 | 中国科学院华南植物园 | Tissue culture breeding method for metabriggsia ovalifolia |
| CN102771396A (en) * | 2012-08-14 | 2012-11-14 | 四川省草原科学研究院 | Method for propagating chicory transgenosis plant |
| CN102776233A (en) * | 2012-08-14 | 2012-11-14 | 四川省草原科学研究院 | Method for culturing witloof high-quality new species by using plant genetic engineering technology |
| CN103947524A (en) * | 2014-04-24 | 2014-07-30 | 中国科学院华南植物园 | Breeding method for inducing adventitious buds and roots by cutting petioles of metabriggsia ovalifolia |
| CN105063086A (en) * | 2015-08-21 | 2015-11-18 | 中国科学院华南植物园 | Molecular breeding method fast obtaining large number of transgenosis sedum lineare new species |
| CN106755083A (en) * | 2016-12-22 | 2017-05-31 | 中国科学院华南植物园 | A kind of pocket orchid Agrobacterium Ovary injection transgenic method |
| CN113396821A (en) * | 2021-06-15 | 2021-09-17 | 西北农林科技大学 | Tissue culture method for agrobacterium-mediated transformation of lettuce |
| CN116445535A (en) * | 2023-04-17 | 2023-07-18 | 四川省农业科学院园艺研究所 | Agrobacterium-mediated lettuce genetic transformation method and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004108903A2 (en) * | 2003-06-06 | 2004-12-16 | Arborgen, Llc | Plant transformation and selection |
| JP5343460B2 (en) * | 2008-09-03 | 2013-11-13 | 住友化学株式会社 | Plant flowering time control method |
| GB201003702D0 (en) * | 2010-03-05 | 2010-04-21 | Univ Warwick | Molecular engineering of a floral inducer for crop improvement |
-
2022
- 2022-09-05 CN CN202211079084.3A patent/CN115386591B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5349124A (en) * | 1988-04-25 | 1994-09-20 | Monsanto Company | Insect-resistant lettuce plants |
| CN1606623A (en) * | 2000-11-21 | 2005-04-13 | 南洋理工大学 | Production of stilbenes in transgenic plants and the method of producing thereof |
| CN101940159A (en) * | 2010-06-30 | 2011-01-12 | 中国科学院华南植物园 | Molecular breeding method for quickly obtaining large number of new transgenic Saussurea involucrata varieties |
| CN101965800A (en) * | 2010-11-19 | 2011-02-09 | 中国科学院华南植物园 | Tissue culture breeding method for metabriggsia ovalifolia |
| CN102771396A (en) * | 2012-08-14 | 2012-11-14 | 四川省草原科学研究院 | Method for propagating chicory transgenosis plant |
| CN102776233A (en) * | 2012-08-14 | 2012-11-14 | 四川省草原科学研究院 | Method for culturing witloof high-quality new species by using plant genetic engineering technology |
| CN103947524A (en) * | 2014-04-24 | 2014-07-30 | 中国科学院华南植物园 | Breeding method for inducing adventitious buds and roots by cutting petioles of metabriggsia ovalifolia |
| CN105063086A (en) * | 2015-08-21 | 2015-11-18 | 中国科学院华南植物园 | Molecular breeding method fast obtaining large number of transgenosis sedum lineare new species |
| CN106755083A (en) * | 2016-12-22 | 2017-05-31 | 中国科学院华南植物园 | A kind of pocket orchid Agrobacterium Ovary injection transgenic method |
| CN113396821A (en) * | 2021-06-15 | 2021-09-17 | 西北农林科技大学 | Tissue culture method for agrobacterium-mediated transformation of lettuce |
| CN116445535A (en) * | 2023-04-17 | 2023-07-18 | 四川省农业科学院园艺研究所 | Agrobacterium-mediated lettuce genetic transformation method and application thereof |
Non-Patent Citations (6)
| Title |
|---|
| Establishment of an efficient Agrobacterium tumefaciens-mediated leaf disc transformation of Thellungiella halophila;Hong-Qing Li等;Plant Cell Rep;第26卷(第10期);1785-1789 * |
| Use of Chenopodium murale L. transgenic hairy root in vitro culture system as a new tool for allelopathic assays;Nevena Mitic等;Journal of Plant Physiology;第169卷(第12期);1203-1211 * |
| 南昆山莪术的组培快繁技术研究;张施君等;北方园艺(第4期);161-163 * |
| 根癌农杆菌介导的叶用莴苣基因转化***的建立;罗淑萍, 张智俊, 秋田求;新疆农业大学学报(01);30-33 * |
| 根癌农杆菌介导的高赖氨酸蛋白基因转化叶用莴苣的研究;朱路英, 刘玲, 张振贤;应用与环境生物学报(01);40-43 * |
| 甜蛋白基因MBLII对莴苣的遗传转化;刘敬梅, 陈大明, 陈杭;园艺学报(03);246-250 * |
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