Genetic transformation methods for Primulina species
By obliquely infecting and sterilizing the petioles of *Gentiana danxiaensis* with *Agrobacterium rhizogenes* K599, adventitious buds were directly obtained and transplanted for culture. This solved the problems of complex operation and low efficiency in the genetic transformation of *Gentiana danxiaensis*, and realized a simple and efficient genetic transformation method.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
- Filing Date
- 2026-04-21
- Publication Date
- 2026-06-30
AI Technical Summary
Traditional genetic transformation methods for *Geranium danxiaense* rely on tissue culture, which is complex, time-consuming, has low transformation efficiency, and is prone to decay.
Agrobacterium rhizogenes K599 was used to infect obliquely cut petioles of *Gentiana danxiaensis*. Adventitious buds were directly obtained by covering with moist substrate and sterilization treatment, and then transplanted into a mixed substrate for culture to achieve genetic transformation.
No tissue culture is required, the operation is simple, the conversion rate is high, and the cycle is short. Positive plants can be obtained in just 40 to 50 days, and the conversion efficiency is increased by more than 60%.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant genetic engineering technology, specifically to the field of plant genetic transformation technology, and more specifically, to a method for genetic transformation of Primulina plants without tissue culture. Background Technology
[0002] *Primulina danxiaensis* is an endemic species of the genus *Primulina* in the family Gesneriaceae, found only in Danxia landform areas. It possesses extremely high ornamental value and ecological conservation significance. *Primulina danxiaensis* grows to a height of 2-10 cm, blooms profusely from May to June, and produces pale yellow flowers. Due to its scarcity in the wild and low natural reproductive efficiency, improving its traits and expanding its propagation scale through genetic transformation technology is of great importance for species conservation and resource utilization.
[0003] Traditional genetic transformation methods for *Geranium danxiaense* typically rely on tissue culture techniques. While this method can achieve genetic transformation, the leaves of *Geranium danxiaense* are brittle and thin, and contain symbiotic endophytic bacteria, making the tissue culture process highly susceptible to decay or contamination, resulting in low transformation efficiency and poor quality of transformants. Furthermore, tissue culture techniques are complex to operate and have long cycles. Summary of the Invention
[0004] Based on this, the purpose of this invention is to provide a genetic transformation method for Primulina plants that does not require tissue culture, which has a high transformation rate, is simple to operate, and has a short cycle.
[0005] The specific technical solutions for achieving the above-mentioned objectives are as follows.
[0006] This invention provides a method for genetic transformation of Primula species, comprising the following steps:
[0007] (1) Slant cut the petiole of Primrose, soak the petiole with leaves in Agrobacterium rhizogenes K599 infection solution for infection, and then cover the cut of the petiole with moist planting material for culture; the Agrobacterium rhizogenes K599 is transformed with a recombinant vector carrying the target gene.
[0008] (2) After adventitious buds grow from the cut of the petiole, sterilize the petiole and the planting material;
[0009] (3) When the adventitious buds grow to a height of 2-3 cm, separate the seedlings from the petioles and transplant them into a mixed substrate for cultivation until a complete plant is obtained.
[0010] Based on years of research and exploration into the genetic transformation of *Primulina danxiaensis*, the inventors of this invention discovered a novel method for genetic transformation of *Primulina danxiaensis*. This method utilizes a specific concentration of *Agrobacterium rhizogenes* K599, which transforms recombinant vectors carrying the target gene, as an infection solution. The petioles of obliquely cut *Primulina danxiaensis* are directly infected before transplantation. This "petiole oblique cutting-infection-transplantation" model successfully established a genetic transformation method for *Primulina danxiaensis*. This method requires no tissue culture, is simple to operate, fills the gap in non-tissue culture transgenic methods for the *Primulina* genus, and has high transformation efficiency, allowing for direct screening of genetically transformed plants. Therefore, the method of this invention provides a simple and efficient approach for the study of the biological characteristics and genetic engineering breeding of this genus, possessing significant scientific research and application value.
[0011] The genetic transformation method of this invention has a short cycle, requiring only 40 to 50 days to obtain positive plants, which greatly shortens the time for obtaining transgenic plants (more than 60% shorter than conventional methods). Attached Figure Description
[0012] Figure 1 This is a map of the recombinant vector, where the blue box indicates the inserted target gene.
[0013] Figure 2 This is a schematic diagram of a petiole cut at an oblique angle.
[0014] Figure 3 Image showing the infection of petioles with K599 Agrobacterium tumefaciens solution.
[0015] Figure 4 Positive plants of *Agrobacterium danxiaense* obtained by infecting petioles with K599 Agrobacterium infection solution.
[0016] Figure 5 Electrophoresis diagram for PCR identification of positive *Danxia florida* plants.
[0017] Figure 6 This is a schematic diagram of a complete cut at the leaf base.
[0018] Figure 7 This image shows a leaf infected with K599 Agrobacterium infection solution.
[0019] Figure 8 Positive plants of Danxia small flower were obtained by infecting leaves with K599 Agrobacterium infection solution.
[0020] Figure 9 Image showing the infection of petioles with EHA105 Agrobacterium tumefaciens solution.
[0021] Figure 10 Positive plants of *Agrobacterium danxiaense* obtained by infecting petioles with EHA105 Agrobacterium tumefaciens solution. Detailed Implementation
[0022] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.
[0023] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.
[0024] Unless otherwise specified, all examples were conducted under standard experimental conditions or as recommended in the manufacturer's instructions. All raw materials used were commercially available and readily available.
[0025] In some embodiments of the present invention, a method for genetic transformation of Primula species is disclosed, comprising the following steps:
[0026] (1) Slant cut the petiole of Primrose, soak the petiole with leaves in Agrobacterium rhizogenes K599 infection solution for infection, and then cover the cut of the petiole with moist planting material for culture; the Agrobacterium rhizogenes K599 is transformed with a recombinant vector carrying the target gene.
[0027] (2) After adventitious buds grow from the cut of the petiole, sterilize the petiole and the planting material;
[0028] (3) When the adventitious buds grow to a height of 2-3 cm, separate the seedlings from the petioles and transplant them into a mixed substrate for cultivation until a complete plant is obtained.
[0029] The target gene described in this invention refers to a specific gene sequence selected for isolation, manipulation, transfer, or research in genetic transformation experiments. Theoretically, it can originate from any organism (plant, animal, microorganism), and can also be artificially synthesized or modified genes. In this invention, the target gene comes from *Primulina macrocarpa*. It typically encodes a protein with a specific function or a regulatory RNA. The ultimate goal of genetic transformation experiments is to induce the expression of this target gene in the recipient organism (producing its encoded protein or RNA), thereby conferring a new trait or function upon the recipient.
[0030] In one embodiment, the angle of the bevel cut in step (1) is 40°~50°, and the length of the bevel cut is about 0.3 cm~0.5 cm.
[0031] In one embodiment, the OD600 of the infiltration solution in step (1) is 0.8 to 1.2.
[0032] In one embodiment, the OD600 of the infiltration solution in step (1) is 0.9~1.1.
[0033] In one embodiment, the OD600 of the infiltration solution in step (1) is 0.95~1.05.
[0034] In one embodiment, the infection time in step (1) is 10 h to 12 h.
[0035] In one embodiment, the substrate described in steps (1) and (2) is perlite.
[0036] In one embodiment, the culture conditions in step (1) are: 24℃~26℃, 13 h~15 h / 9 h~11 h light / dark cycle.
[0037] In one embodiment, the plasmid used in the recombinant vector in step (1) includes 35S and a reporter gene. Plasmids that can transform plants can be used to construct recombinant vectors.
[0038] In one embodiment, the reporter gene is a Ruby gene, a GFP gene, or a GUS gene.
[0039] In one embodiment, the plasmid used for the recombinant vector is pHDE-35S:Ruby.
[0040] In one embodiment, step (2) involves sterilizing the petioles and substrate with sterile water containing 450 mg / L to 550 mg / L Cef.
[0041] In one embodiment, the mixed matrix in step (3) is peat, perlite and volcanic rock in a volume ratio of 0.8~1.2:0.8~1.2:0.8~1.2.
[0042] In one embodiment, the cultivation conditions in step (3) are: humidity 75%~85%, 24℃~26℃, light / dark cycle of 13 h~15 h / 9 h~11 h, and 28 days~32 days.
[0043] In one embodiment, the Primulina species is *Primulina danxiaensis*.
[0044] The plasmid vector pHDE-35S (containing the reporter gene RUBY, which enables transgenic plants to express betaine, thus causing them to exhibit a visible purple-red phenotype, used to observe and verify the effectiveness of genetic transformation) used in the following examples was purchased from Miaoling Company. Agrobacterium rhizogenes K599 competent cells and Agrobacterium tumefaciens EHA105 were purchased from Shanghai Weidi Biotechnology Co., Ltd.
[0045] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
[0046] Example 1: Construction and transformation of recombinant vectors carrying the target gene
[0047] This embodiment uses the target gene APETALA 2 as an example, which is precisely cloned into the pHDE-35S vector backbone (containing a Ruby reporter gene, purchased from Miaoling Company, as shown in the figure). Figure 1 The cloning sites downstream of the 35S promoter and upstream of the NOS terminator were used to obtain a recombinant vector carrying the target gene. The target gene APETALA 2 was constitutively expressed under the regulation of the 35S promoter.
[0048] Specifically, the following steps are included:
[0049] 1. Modification of pHDE-35S carrier
[0050] Using pHDE-35S DNA as a template, PCR amplification was performed using SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 as primers, respectively, to add promoter and terminator to the pHDE-35S vector.
[0051] The amplification system is shown in Table 1. The amplification program is as follows: 98℃, 3min pre-denaturation → 95℃, 30s (35 cycles) → 58℃, 30s annealing → 72℃, 5min extension → 4℃ storage.
[0052] SEQ ID NO: 1: 5'-actagatccccaaacaagcttCGCGCCAAGCTTTGAGAC-3'
[0053] SEQ ID NO:2: 5'-ccgcagcgatcgcatccatggTCTAGAAGCCGTCCCCGTG-3'
[0054] SEQ ID NO:3: 5'-actagatccccaaacaagcttGATCGTTCAAACATTTGGCAATAA-3'
[0055] SEQ ID NO:4: 5'-tgttgaaaagtctcaaagcttGATCTAGTAACATAGATGACACCGCG-3'
[0056] Table 1
[0057]
[0058] 2. Linearization treatment of the modified pHDE-35S carrier
[0059] The modified pHDE-35S vector was double-digested with SacII and XbaI enzymes to obtain the linearized pHDE-35S vector.
[0060] 3. Obtaining the target gene APETALA 2
[0061] RNA was extracted from Primula macrocarpa, and cDNA was obtained by reverse transcription. The cDNA library was then amplified using specific primers (SEQ ID NO:5 and SEQ ID NO:6) (the amplification system is shown in Table 1, and the amplification procedure is the same as in step 1). The product was recovered to obtain the target gene APETALA 2 fragment.
[0062] SEQ ID NO:5:5'-acacgggggacggcttctagaGCAGTTTTTTCCGGTGGATG-3'
[0063] SEQ ID NO:6: 5'-catatacgcccggagccgcggGTGAGTATATATATATATATATATTTTAAAAAATAAAGGG-3'
[0064] 4. The linearized pHDE-35S vector from step 2 was ligated with the target gene APETALA 2 fragment from step 3 at 16°C overnight. The ligation system is shown in Table 2.
[0065] Table 2
[0066]
[0067] 5. Transform the ligation product from step 4 into competent E. coli cells.
[0068] Take 5 μL of the ligation product from step 3, add 100 μL of DH5α competent cells, and incubate on ice for 30 min; heat shock at 42℃ for 90 s, then immediately incubate on ice for 2-3 min; add 900 μL of antibiotic-free LB liquid medium, and revive at 37℃ and 200 rpm for 1 h on a shaker; take 100-200 μL of the revived bacterial culture, spread it on LB solid plates containing 50 mg / mL spectinomycin (Spe); incubate upside down at 37℃ for 12-16 h, and screen for single colonies.
[0069] 5. Verification of positive clones
[0070] Single colonies were picked and amplified by PCR using APETALA 2 specific primers (SEQ ID NO:5 and SEQ ID NO:6). Electrophoresis was performed to detect the presence of the target size band. PCR-positive colonies were inoculated into 5 mL of LB broth containing 50 mg / mL spectinomycin (Spe) and incubated at 37°C for 12 h. Plasmids were extracted using a plasmid miniprep kit. The plasmids were sequenced, and the sequencing results were compared with the APETALA 2 gene reference sequence to confirm the absence of base mutations and the correct reading frame. The correctly sequenced positive clones (i.e., the recombinant vector carrying the target gene APETALA 2) were added to glycerol and stored at -80°C.
[0071] Example 2: Transformation of the recombinant vector into Agrobacterium strain
[0072] In this embodiment, the recombinant vector obtained in Example 1 is transformed into Agrobacterium rhizogenes K599, specifically including the following steps:
[0073] 1. Take the competent Agrobacterium rhizogenes K599 cells stored at -80℃ and place them at room temperature for 5 min. After they partially thaw, take 2 μL of the recombinant vector constructed in Example 1 and add it to 100 μL of the thawed Agrobacterium rhizogenes K599 competent cell culture. Gently aspirate or tap to mix thoroughly. Place on ice for 30 min, freeze with liquid nitrogen for 5 min, and then quickly transfer to a 37℃ water bath for 5 min. Finally, place in ice for 5 min.
[0074] 2. Add 700 μL of antibiotic-free YEP liquid culture medium and place it on a shaker at 28°C for 3 h at a speed of 180 rpm.
[0075] 3. After the culture is completed, centrifuge at 6000 rpm for 1 min, discard 700 μL of supernatant, leaving about 100 μL of supernatant. Gently aspirate the supernatant with a pipette tip to resuspend the bacterial cells, spread them on YEP plates containing the corresponding antibiotics (K599 strain has Kan resistance, and the recombinant vector has Spe resistance), and incubate upside down in a 28℃ incubator for 3 days until white, smooth Agrobacterium single colonies grow on the plates.
[0076] 4. Randomly pick single colonies with a pipette tip and place them in a centrifuge tube containing 1 mL of YEP liquid medium with kanamycin resistance. Mix well and incubate at 28°C and 180 rpm for 10 h until turbidity is observed. Then, use the bacterial culture as a template for PCR.
[0077] PCR reaction system: 2×Rapid Taq Master Mix 5.0 μL, Forward prime (SEQ ID NO:5) 0.4 μL, Reverse primer (SEQ ID NO:6) 0.4 μL, Bacteria solution 2.0 μL, ddH2O 2.2 μL.
[0078] The PCR reaction program was as follows: 95℃ for 3 min, 1 cycle; 95℃ for 10 s, 55℃ for 15 s, 72℃ for 1 min, 34 cycles; 72℃ for 3 min, 1 cycle. After agarose gel electrophoresis, strains with a positive 35S promoter sequence were identified as Agrobacterium rhizogenes K599 strains successfully transformed into the recombinant vector.
[0079] Example 3: Genetic transformation method of *Gesneria danxiaensis*
[0080] This embodiment obtained transgenic positive plants of *Gentiana danxiaensis* through genetic transformation, specifically including the following steps:
[0081] 1. Pick a single colony of *Agrobacterium rhizogenes* K599 transformed with the recombinant vector obtained in Example 2, inoculate it into 5 mL of liquid YEP medium, and culture it on a shaker at 28°C and 180 rpm until the logarithmic growth phase. Then, take 1 mL of the bacterial culture and add it to 50 mL of liquid YEP medium and continue culturing until the logarithmic growth phase. Centrifuge the bacterial culture at 5000 rpm for 3 min, discard the supernatant, and resuspend the bacterial cells in sterile water containing 100 μmol / L acetylsyleugenol. Adjust the OD of the resuspended culture. 600 The value is increased to 1.0 to obtain the Agrobacterium infection solution.
[0082] 2. Make oblique cuts on the petiole of fresh leaves of *Gynostemma pentaphyllum*. Figure 2 The oblique cut angle is 45°, and the length of the oblique cut surface is about 0.5 cm. The leaf is retained as a nutrient donor, and the petiole is immersed in 2 mL of Agrobacterium infection solution ( Figure 3 After being placed in the dark for 10 hours, the cut surface was covered with moist substrate (perlite), spread flat on a petri dish, and co-cultured at 25°C with a light / dark cycle of 14 / 10 hours.
[0083] 3. After adventitious buds grow from the petiole cut, spray the leaf segments and planting medium with sterile water containing 500 mg / L Cef (cephalosporin) for sterilization. When the adventitious buds grow to a height of 2-3 cm, separate the seedlings from the leaves with tweezers and transplant them into a mixed substrate of peat moss, perlite, and volcanic rock in a volume ratio of 1:1:1 to continue growth. Maintain an ambient humidity of 80% and a temperature of 25℃, and conduct a 14 / 10 hour light / dark cycle culture for 30 days.
[0084] 4. Phenotypic observation of seedlings reveals a red phenotype at the roots. Figure 4 Leaf tissue was taken from seedlings, and genomic DNA was extracted using the Plant Direct PCR Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.). PCR molecular detection was performed using APETALA 2 specific primers (SEQ ID NO:5 and SEQ ID NO:6). The results are as follows: Figure 5 As shown, the stripes on the seedlings are clear positive bands, indicating that transgenic plants of Danxia small flower have been obtained.
[0085] Example 4 Genetic transformation method of *Gesneria danxiaensis*
[0086] In this embodiment, transgenic positive plants of *Gentiana danxiaensis* were obtained through genetic transformation, except for the OD of the *Agrobacterium* infection solution in step 1. 600 The value was 0.8, and all other steps were the same as in Example 3. PCR molecular detection showed that the seedlings showed clear positive bands, indicating that transgenic Danxia small flower plants had been obtained.
[0087] Example 5 Genetic transformation method of *Gesneria danxiaensis*
[0088] In this embodiment, transgenic positive plants of *Gentiana danxiaensis* were obtained through genetic transformation, except for the OD of the *Agrobacterium* infection solution in step 1. 600 The value was 1.2, and all other steps were the same as in Example 3. PCR molecular detection showed that the seedlings showed clear positive bands, indicating that transgenic Danxia small flower plants had been obtained.
[0089] Comparative Example 1
[0090] This comparative example obtained transgenic positive plants of *Gentiana danxiaensis* through genetic transformation, except that in step 2, all fresh leaves of *Gentiana danxiaensis* were completely cut off at the leaf base. Figure 6 The leaves were soaked in Agrobacterium infection solution for infection. Figure 7 Except for the steps described in Example 3, all other steps are the same. The obtained positive Danxia small flower plants are as follows: Figure 8 As shown, from Figure 8 It can be seen that the obtained positive plants do not have the red root / bud phenotype.
[0091] Comparative Example 2
[0092] This comparative example obtained transgenic positive plants of *Gentiana danxiaensis* through genetic transformation, except for the OD of the *Agrobacterium* infection solution in step 1. 600 The value was 0.8, and all other steps were the same as in Comparative Example 1. The obtained positive plants did not have the red root / bud phenotype.
[0093] Comparative Example 3
[0094] This comparative example obtained transgenic positive plants of *Gentiana danxiaensis* through genetic transformation, except for the OD of the *Agrobacterium* infection solution in step 1. 600 The value was 1.2, and all other steps were the same as in Comparative Example 1. The obtained positive plants did not have the red root / bud phenotype.
[0095] Comparative Example 4
[0096] This comparative example provides a genetic transformation method for *Lysimachia danxiaensis*, which involves infecting *Agrobacterium tumefaciens* EHA105 with a recombinant vector (construction method as in Example 2) using the same method as in Example 3.
[0097] Phenotypic observation of seedlings was not very effective because the base of the stem of *Gentiana danxiaensis* is naturally red, making visual observation difficult. Figure 10 A portion of leaf tissue (3*3mm) from the seedlings was taken for PCR molecular detection (using a direct amplification PCR kit). The seedlings showed clear positive bands, indicating that transgenic plants of Danxia small flower had been obtained.
[0098] Comparative Example 5
[0099] This comparative example provides a genetic transformation method for *Geranium danxiaense*, using *Agrobacterium tumefaciens* EHA105 transformed with a recombinant vector for infection, in addition to the OD of the *Agrobacterium* infection solution. 600 The value was 0.8, and all other steps were the same as in Comparative Example 4. Visual inspection of the base revealed no red root / bud phenotype. PCR molecular detection showed clear positive bands in the seedlings, indicating the successful acquisition of transgenic Danxia florets.
[0100] Comparative Example 6
[0101] This comparative example provides a genetic transformation method for *Geranium danxiaense*, using *Agrobacterium tumefaciens* EHA105 transformed with a recombinant vector for infection, in addition to the OD of the *Agrobacterium* infection solution. 600 The value was 1.2, and all other steps were the same as in Comparative Example 4. Visual inspection of the base revealed no red root / bud phenotype. PCR molecular detection showed clear positive bands in the seedlings, indicating the successful acquisition of transgenic Danxia florets.
[0102] The infection effects of Examples 3-5 and Comparative Examples 1-6 were statistically analyzed, and the results are shown in Table 3.
[0103] Table 3
[0104]
[0105] As can be seen from the results in Table 3:
[0106] 1. When the infecting Agrobacterium is Agrobacterium rhizogenes K599 and the infection site is the petiole, the three ODs... 600 Positive seedlings were obtained under gradient conditions (Examples 3-5), with transformation rates ranging from 54% to 65%. All positive seedlings exhibited the characteristic phenotype of red roots, indicating successful expression of the Ruby reporter gene driven by the 35S promoter. The positive seedlings were genuine transformed seedlings, with no false positives. The transformation rate increased with OD... 600 It shows a trend of first rising and then falling: OD 600 The conversion rate is highest (65%) when OD = 1.0; 600 The conversion rate decreased slightly at concentrations of 0.8 and 1.2 (to 54% and 55%, respectively). This pattern aligns with the general characteristics of Agrobacterium infection: excessively low bacterial concentrations result in insufficient bacterial cells and low infection efficiency; excessively high concentrations lead to browning and death of plant cells, thus reducing conversion efficiency. Therefore, OD... 600 =1.0 is the optimal concentration for K599 to infect petioles.
[0107] 2. When the infecting Agrobacterium is Agrobacterium rhizogenes K599 and the infection site is the leaf base, the three OD values... 600 The conversion rate was 0% under the gradient (Comparative Examples 1-3), and no red root / bud phenotype was observed. This indicates that the leaf base is not a suitable infection recipient site for this plant material. We analyzed that this may be related to the high degree of cell differentiation in the leaf base tissue, making it difficult for Agrobacterium to attach and infect, or the strong intracellular defense response.
[0108] 3. When the infecting Agrobacterium is Agrobacterium tumefaciens EHA105 and the infection site is the petiole, the three ODs... 600 Positive seedlings were also obtained under gradient conditions (Comparative Examples 4-6), with conversion rates ranging from 51% to 60%, and at OD... 600 The transformation rate peaked at a concentration of 1.0 (60%). This concentration effect is consistent with that of K599, indicating that the two strains have similar infection concentration requirements. However, none of the positive seedlings showed a red root / bud phenotype, and no red phenotype of the Ruby reporter gene was observed. This phenomenon may be due to the plasmid only integrating the resistance selection marker gene and not carrying the complete Ruby reporter gene expression cassette, representing incomplete integration of the vector fragment; or the Ruby reporter gene, although integrated into the plant genome, was not effectively expressed due to the infection characteristics of the EHA105 strain or the plant's endogenous gene silencing mechanism. Therefore, although the EHA105 strain can produce resistant positive seedlings, the lack of the Ruby phenotype makes it unsuitable for transformation experiments in this system.
[0109] Based on the above results, for *Dendrocalamus danxiaensis*, strain K599-petiole infection-OD 600 =1.0 mode is the optimal combination for achieving efficient transformation of recombinant vectors, with high transformation efficiency and no false positives.
[0110] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0111] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.
Claims
1. A method for genetic transformation of Primula species, characterized in that, Includes the following steps: (1) Slant cut the petiole of Primrose, soak the petiole with leaves in Agrobacterium rhizogenes K599 infection solution for infection, and then cover the cut of the petiole with moist planting material for culture; the Agrobacterium rhizogenes K599 is transformed with a recombinant vector carrying the target gene. (2) After adventitious buds grow from the cut of the petiole, sterilize the petiole and the planting material; (3) When the adventitious buds grow to a height of 2-3 cm, separate the seedlings from the petioles and transplant them into a mixed substrate for cultivation until a complete plant is obtained.
2. The genetic transformation method for Primula species according to claim 1, characterized in that, The angle of the oblique cut in step (1) is 40°~50°.
3. The genetic transformation method for Primula plants according to claim 1, characterized in that, The OD600 of the infiltration solution in step (1) is 0.8~1.2, preferably 0.9~1.1, and more preferably 0.95~1.
05.
4. The genetic transformation method for Primula species according to claim 1, characterized in that, The plasmid used in the recombinant vector described in step (1) includes 35S and a reporter gene; Preferably, the reporter gene is a Ruby gene, a GFP gene, or a GUS gene; Preferably, the plasmid used in the recombinant vector is pHDE-35S:Ruby.
5. The genetic transformation method for Primula species according to claim 1, characterized in that, The infection time described in step (1) is 10 h to 12 h.
6. The genetic transformation method for Primula species according to claim 1, characterized in that, The substrate mentioned in steps (1) and (2) is perlite.
7. The genetic transformation method for Primula species according to claim 1, characterized in that, The culture conditions described in step (1) are: 24℃~26℃, 13 h~15 h / 9 h~11 h light / dark cycle; And / or, the cultivation conditions described in step (3) are: humidity 75%~85%, 24℃~26℃, light / dark cycle of 13 h~15 h / 9 h~11 h, and 28 days~32 days.
8. The genetic transformation method for Primula species according to claim 1, characterized in that, In step (2), the petioles and substrate are sterilized using sterile water containing 450 mg / L to 550 mg / L Cef.
9. The genetic transformation method for Primula plants according to claim 1, characterized in that, The mixed matrix mentioned in step (3) is peat, perlite and volcanic rock in a volume ratio of 0.8~1.2:0.8~1.2:0.8~1.
2.
10. The genetic transformation method for Primula species according to any one of claims 1 to 9, characterized in that, The Primulina species mentioned is *Primulina danxiaensis*.