Lily open tissue-free genetic transformation method
By optimizing Agrobacterium-mediated lily transformation through an open tissue culture-free method and using scales for transformation in an open environment, the complex and time-consuming problems of lily breeding were solved, and efficient and simple breeding and gene function research were achieved.
Patent Information
- Application Number
- CN202510886744.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-30
- Publication Date
- 2025-09-12
AI Technical Summary
Existing technologies make it difficult to carry out genetic transformation of lilies efficiently and simply. The traditional breeding process is complex and time-consuming, and cannot meet the needs of scientific research and breeding.
An open tissue culture-free method is adopted. By optimizing the Agrobacterium-mediated transformation conditions, lily scales are used for transformation in an open bacterial environment. The recombinant vector is constructed by combining molecular cloning technology. After Agrobacterium infection and scale incision treatment, it is directly cultured in moist soil to simplify the operation process.
It improves the efficiency of lily breeding, shortens the breeding cycle, reduces costs, simplifies the operation process, and is suitable for gene function research and variety improvement of lily.
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Figure CN120624533A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of plant genetic transformation, and in particular to an open tissue culture-free genetic transformation method for lily. Background Art
[0002] lily( Lilium brownii var. viridulum Baker Lily is a perennial bulbous herb. Its bulbs are rich in nutrients such as protein, starch, vitamins, amino acids, and trace elements. It also contains a variety of medicinal ingredients, including lily saponins, colchicine, and polysaccharides. Therefore, lilies have both high edible and medicinal value. Lilies are also highly ornamental, and their aromatic oils are used as fragrances. Therefore, lilies have always been a key subject of scientific research and industrial development.
[0003] Research on gene function in lilies requires the use of transformation technologies for gene delivery, modification, or editing. More importantly, traditional lily breeding involves developing new varieties with superior traits through artificial hybridization and selection. However, this traditional lily breeding process is complex and time-consuming, potentially requiring 10-20 years to develop a single new lily variety. However, using transformation technologies to deliver, modify, or edit genes in lilies allows for precise control of specific traits and accelerates the breeding cycle.
[0004] Currently, lily transformation requires the use of lily tissue culture, performed under sterile conditions through methods such as Agrobacterium-mediated transformation and particle bombardment. The growth and differentiation of the tissue culture are then controlled using various nutrients and plant hormones. This process requires complex and tedious sterile manipulation and tissue culture procedures, consuming considerable time and labor.
[0005] In summary, the above methods can no longer meet the actual needs of scientific research and breeding for lily transformation technology, and it is urgent to develop an efficient, simple and suitable transformation method for lily. Summary of the Invention
[0006] In view of the shortcomings of the existing technology, the present invention provides an open tissue culture-free transformation method for lily.
[0007] The purpose of the present invention is to provide an open tissue culture-free transformation method for lily, the specific steps of the method are as follows: (1) Vector construction: molecular cloning technology is used to connect the target gene and the vector to obtain a recombinant vector containing the target gene; (2) Transformation and screening of Agrobacterium: The recombinant vector obtained in step (1) was transformed into Agrobacterium competent cells. The transformed Agrobacterium was added to TY liquid medium without antibiotics and cultured at 28°C for 2 h. The bacterial liquid was then spread on TY solid medium containing antibiotics corresponding to the vector resistance gene and cultured at 28°C in an inverted manner until a positive Agrobacterium single colony was grown. The formula of TY liquid medium was: 5 g / L tryptone, 3 g / L yeast extract, 10 mM calcium chloride; (3) Preparation of Agrobacterium for infection: Inoculate a single positive Agrobacterium colony into TY liquid culture medium containing antibiotics and culture at 28°C and 220 rpm for 16-40 h. Collect the Agrobacterium after centrifugation and resuspend it in an infection suspension. The infection suspension formula is as follows: add 10 mM MgCl2, 10 mM 2-(N-morpholino)ethanesulfonic acid, and 100 μM acetosyringone to sterile water, pH 6.0; inoculate the selected positive Agrobacterium colony into TY liquid culture medium and culture at 28°C and 220 rpm for 16-40 h. Then, aspirate the bacterial liquid and evenly spread it on TY solid culture medium containing the antibiotic corresponding to the vector resistance gene. Culture it upside down at 28°C until colonies grow for subsequent smearing of scale incisions. (4) Transformation operation: Select healthy, pest-free lily bulbs, cut them open at the base of the bulb, and cut them into single scales. Immerse the base of the cut lily scales in the prepared Agrobacterium infection suspension and leave them for 10-60 minutes. After the infection is completed, remove the scales, absorb the excess bacterial solution with sterile filter paper, and smear the cut at the base of the scales with the bacterial layer on the TY solid culture medium. (5) Embed or propagate the transformed lily scales in moist soil and water to keep the soil moist; (6) After 10-30 days of culture, bulges or small bulbs can be observed at the base of the scales. Phenotypes, reporter genes or PCR can be used for observation and screening to identify successfully transformed small bulbs, and then positive transformed plants can be formed.
[0008] Preferably, the vector construction in step (1) can be replaced by molecular cloning techniques such as enzyme ligation and Gibson assembly.
[0009] Preferably, in step (3), the Agrobacterium is resuspended in the infection suspension to adjust the bacterial concentration to OD 600 It is 0.5-0.6.
[0010] Preferably, the infection method in step (4) is vacuum infection in a vacuum pump at 0.03-0.09 MPa for 10 -60 s.
[0011] The present invention improves the transformation speed by optimizing the transformation conditions mediated by Agrobacterium and combining the regeneration characteristics of lily scales. The transformation is carried out directly using scales as materials in an open and bacterial environment, avoiding a complicated tissue culture process. The operation is simple and the cost is low. The lily breeding cycle is shortened, the breeding efficiency is improved, and it is easy to promote and apply. The present invention provides a new method for the study of gene function and variety improvement of lilies. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 This is an operational flow chart for genetic transformation of Lilium lanzhouense scales in Example 1.
[0013] Figure 2 This is a photo of the lily scale processing in step (5) of Example 1.
[0014] Figure 3 This is a photo of lily scales infected with Agrobacterium in step (6) of Example 1.
[0015] Figure 4 This is a photo of the vacuum infection of lily scales in step (6) of Example 2.
[0016] Figure 5 This is a photo of lily scale culture in step (7) of Example 1.
[0017] Figure 6 This is a photo of the positive plants screened after 10 days of cultivation in step (8) of Example 1, wherein the framed portion is the regenerated bulblet of the lily.
[0018] Figure 7 This is a photo of leaves extracted from the bulblets grown after about two months of cultivation in Example 1, wherein the framed portion is the regenerated bulblet of the lily, and 1 is the extracted leaf.
[0019] Figure 8 These are photos of the later stage of lily scale growth and formation. Under a fluorescence microscope, it was observed that the reporter gene was expressed in the growing bulblets and the emerging leaves. Among them, a and b are regenerated bulblets, and c is a leaf emerging from the regenerated bulblet. DETAILED DESCRIPTION
[0020] The present invention will be described in detail below with reference to specific embodiments. The following embodiments are intended to help those skilled in the art further understand the present invention, but are not intended to limit the present invention in any form.
[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0022] Unless otherwise specified, the reagents and equipment used in the following examples can be purchased through commercial channels. Example 1
[0023] (1) Vector construction: The GFP green fluorescent protein encoding gene is used as the reporter gene; the expression vector is cut with enzymes to make it linear, and the GFP reporter gene and the expression vector are connected using a ligase so that they are located downstream of the promoter.
[0024] The GFP gene transformation vector is designed as follows: Basic vector: binary vector; components: ① GFP gene + promoter, ② selection marker (such as an antibiotic resistance gene); GFP gene knock-in vector design (using CRISPR / Cas9 gene editing as an example): Basic vector: binary vector; components: ① Cas9 gene + promoter, ② sgRNA expression cassette (targeting insertion site), ③ homologous recombination donor DNA (containing GFP sequence + homology arms), ④ selection marker (such as an antibiotic resistance gene). The two technologies described above differ only in the components of the vector. For subsequent gene delivery and gene editing technologies, genetic transformation or gene editing can be achieved by simply replacing the vector components specific to the target gene (paying attention to issues such as promoter compatibility when replacing components).
[0025] (2) Transformation and screening of Agrobacterium: The recombinant vector was transformed into Agrobacterium K599 competent cells. The transformed Agrobacterium was added to TY liquid medium without antibiotics and shaken at 28°C for 2 h. The bacterial liquid was then spread onto TY solid medium containing 50 mg / L kanamycin (Kan) and incubated at 28°C in an inverted manner until a single positive Agrobacterium colony was grown.
[0026] (3) Preparation of Agrobacterium for infection: Pick a single colony with good morphology and inoculate it into TY liquid medium containing Kan (50 mg / L). Cultivate it at 28°C and 220 rpm for 16-40 hours. Centrifuge the cultured Agrobacterium at 25°C and 5000 rpm for 10 minutes to collect the cells. Resuspend the cells in infection buffer and adjust the concentration of the culture to OD 600 At 0.5-0.6, prepare an Agrobacterium infection suspension. Simultaneously, inoculate a single positive Agrobacterium colony screened into TY liquid medium and culture at 28°C, 220 rpm, with shaking for 16-24 hours. Then, pipette the bacterial suspension and spread an appropriate amount onto TY solid medium. Cultivate for 24 hours to form a uniform bacterial layer for subsequent smearing of the scale incision.
[0027] (4) Explant selection: Select lily bulbs that are free of pests and diseases, have tightly clasped scales, and have a neat and shiny appearance as propagation materials.
[0028] (5) Explant treatment: Use a sharp blade to cut the lily bulb at the base and cut it into single scales, trying not to damage the base, and remove the scales with disease and insect spots.
[0029] (6) Infection of lily scales: Immerse the base of the cut lily scales in the prepared Agrobacterium infection suspension and leave it for 10-60 minutes. After infection, remove the scales, absorb the excess bacterial solution with sterile filter paper, and then smear the cut at the base of the scales with the bacterial layer on the TY solid culture medium.
[0030] (7) Lily scale culture: Place the infected scales with the cut side facing downwards in a culture tray filled with moist soil. The scale planting depth should be approximately 1 / 3 of the scale length. The culture conditions are a temperature of 26°C, a light intensity of 1500-2000 lx, a light period of 16 hours per day, and a dark period of 8 hours per day.
[0031] (8) Screening of positive plants: After about 10 days of culture, bulges or small bulbs can be observed at the base of the scales. With the help of the GFP reporter gene carried by the vector, the small bulbs that have grown are regularly observed and screened using a fluorescence microscope.
[0032] (9) Positive plant culture: The positive plants screened out are cultured until positive transformed plants are formed.
[0033] The lily scales were transformed through the above operations, and the transformed scales were continuously cultured and observed under a stereo fluorescence microscope. Figure 8 As shown: a shows that the small bulbs (boxed area) regenerated from the genetically transformed mother scales (indicated by arrows) show GFP fluorescence signals under specific excitation light (the brighter area in the box of the fluorescence field image), indicating that the target gene is successfully expressed in the small bulbs regenerated after transformation.
[0034] b shows a comparison of GFP fluorescence in regenerated bulblets from untransformed (left) and transformed (right). Fluorescence microscopy revealed that the transformed group displayed GFP fluorescence signals (brighter areas within the frame) under specific excitation light, indicating successful expression of the target gene; no fluorescence signal was detected in the untransformed group.
[0035] c shows a comparison of GFP fluorescence in leaves extracted from regenerated bulblets from untransformed (left) and transformed (right). Green fluorescence signals were detected in the leaves of the transformed group (bright spots indicated by arrows in the figure), indicating successful expression of the target gene; no fluorescence signals were detected in the untransformed group. Example 2
[0036] This example is similar to Example 1, except that step (6) of lily scale infection is performed by immersing the base of the cut lily scale into the prepared Agrobacterium suspension. The infection is then performed in a vacuum pump at 0.03-0.09 MPa for 10-60 seconds. After infection, the scale is removed, and the excess bacterial solution is removed with sterile filter paper. The bacterial layer from TY solid culture medium is then applied to the cut base of the scale. Example 3
[0037] This example is similar to Example 1, except that the elements of the GFP gene conversion vector in step (1) are replaced with elements of the GFP gene knock-in vector (taking CRISPR / Cas9 gene editing as an example).
[0038] (1) Target site and sgRNA design: Select the knock-in site based on the target gene or genomic region. Retrieve the genomic sequence of the region and ensure that the nearby PAM (NGG) is rich and there are no important functional domains. Use online tools to predict multiple candidate sgRNAs, and prioritize 20 nt sequences with low off-target rates and good on-target efficiency.
[0039] (2) Construction of Cas9 expression cassette: plant promoter, Cas9 gene coding sequence and terminator.
[0040] (3) Construction of sgRNA expression cassette: U6 promoter, sgRNA (target sequence + tracrRNA scaffold) and terminator (poly T sequence).
[0041] (4) Construction of homology-directed repair (HDR) template: in order: left homology arm, 35S promoter, GFP gene, terminator, selection marker (such as Bar gene) and right homology arm.
[0042] (5) Construction of the Agrobacterium vector backbone: in order: replication origin, bacterial selection marker (such as Kan resistance gene) and other auxiliary elements (such as Vir gene-dependent auxiliary plasmid).
[0043] In this example, the GFP gene knock-in vector carried by Agrobacterium tumefaciens includes, in order: a Cas9 expression cassette, an sgRNA expression cassette, an HDR template, and an Agrobacterium vector backbone. The plant promoter is preferably the CaMV 35S promoter, but can also be the Ubiquitin promoter. The Cas9 can be derived from sources such as SpCas9 and SaCas9. Example 4
[0044] This example is similar to Examples 1 and 3, except that the constructed vector does not contain an HDR template. Instead, it relies on the non-homologous end joining (NHEJ) repair mechanism mediated by the CRISPR / Cas9 system to cause loss of target gene function by introducing base insertions or deletions.
[0045] The gene knockout vector carried by Agrobacterium tumefaciens consists, in order: a Cas9 expression cassette, a sgRNA expression cassette, and an Agrobacterium vector backbone. To improve knockout efficiency, a dual-sgRNA system can be constructed, targeting two different exon regions of the target gene, creating large deletions and enhancing the gene knockout effect. The plant promoter is preferably the CaMV 35S promoter, but can also be the Ubiquitin promoter. The Cas9 can be derived from sources such as SpCas9 and SaCas9. Example 5
[0046] This example is similar to Example 1, except for the vector construction in step (1): the target gene vector is constructed using Gateway recombination technology.
[0047] (1) Design primers and add attB1 / attB2: Design and add attB1 and attB2 recombination sites at both ends of the target sequence to be constructed (such as Cas protein coding region, sgRNA expression cassette, GFP reporter gene or screening marker gene), and the primers amplify the target fragment containing the attB region.
[0048] (2) BP recombination to generate Entry intermediate vector: The PCR product containing attB is recombined with the donor vector containing attP site under the action of BP cloning enzyme to replace the toxic gene on the donor vector and generate the Entry intermediate vector containing attL1 / attL2.
[0049] (3) LR recombination to generate expression vector: The above-mentioned Entry intermediate vector and the target sequence binary vector containing attR1 / attR2 (which already contains plant promoter and selection marker) are recombined under the action of LR cloning enzyme to finally obtain the expression vector for plant transformation. Example 6
[0050] This example is similar to Example 1, except for the vector construction in step (1): GoldenGate technology is used to construct the target gene vector.
[0051] Design functional modules such as Cas9, sgRNA, GFP reporter or selection marker, and pre-add BsaI / IIS restriction sites and specific sticky ends at both ends; Each modular DNA fragment, BsaI restriction endonuclease and DNA ligase are added to the reaction system at the same time. Through cyclic enzyme digestion-ligation reaction, the target fragment is precisely spliced into the binary vector skeleton, and finally a recombinant expression vector for plant transformation is obtained. Example 7
[0052] This example is similar to Example 1, except for the vector construction in step (1): the target gene vector is constructed using Gibson Assembly technology.
[0053] PCR amplify Cas9, sgRNA, HDR donor (e.g., GFP and screening cassette), and backbone modules, designing 20-40 bp homology overlap regions at both ends of each pair of fragments; All PCR fragments were mixed with Gibson enzyme mix (containing 5' exonuclease, DNA polymerase, and ligase) and incubated at 50°C for 15-60 minutes for a one-step reaction. Finally, multiple fragments were seamlessly spliced to obtain a recombinant expression vector for plant transformation.
[0054] Lily scales treated with the method provided by the present invention ultimately achieved positive transformation after culture. Observation using a stereofluorescence microscope demonstrated that the method provided by the present invention can successfully transform lilies. This present invention provides a complete, feasible, and simple method for lily transformation that requires no special equipment and can be performed under open, tissue culture-free conditions. The method provides an in-depth explanation of the technical principles, operational steps, and beneficial effects, providing strong technical support for scientific research and genetic modification of lilies.
[0055] The above is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the present technical field can make equivalent substitutions or changes based on the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, such as the gene delivery and transformation technology implemented in lily by the present method, gene editing technology based on CRISPR / Cas system (including gene knockout, gene knockin, gene expression increase or inhibition) and other operating means, as well as new lily germplasm or variety breeding plans derived from the above technologies and homologous technology improvements and expanded applications achieved based on the core principles of the present invention, all of which are deemed to fall within the scope of protection of the claims of the present invention.
Claims
1. A method for genetic transformation of lily without tissue culture, characterized in that The method comprises the following steps: (1) Vector construction: molecular cloning technology is used to connect the target gene and the vector to obtain a recombinant vector containing the target gene; (2) Transformation and screening of Agrobacterium: The recombinant vector obtained in step (1) was transformed into Agrobacterium competent cells. The transformed Agrobacterium was added to TY liquid medium without antibiotics and cultured at 28°C for 2 h. The bacterial liquid was then spread on TY solid medium containing antibiotics corresponding to the vector resistance gene and cultured at 28°C in an inverted manner until a positive Agrobacterium single colony was grown. The formula of TY liquid medium was: 5 g / L tryptone, 3 g / L yeast extract, 10 mM calcium chloride; (3) Preparation of infective Agrobacterium: Inoculate a single colony of positive Agrobacterium into TY liquid culture medium containing antibiotics and culture at 28°C and 220 rpm for 16-40 h. Collect Agrobacterium after centrifugation and resuspend it in an infection suspension. The infection suspension formula is: sterile water, 10 mM MgCl2, 10 mM 2-(N-morpholino)ethanesulfonic acid, 100 μM acetosyringone, pH 6.0; inoculate the selected positive Agrobacterium single colony into TY liquid culture medium and culture at 28°C and 220 rpm for 16-40 h. Then, aspirate the bacterial liquid and spread it on TY solid culture medium containing the antibiotic corresponding to the vector resistance gene. Culture it upside down at 28°C until colonies grow for subsequent smearing of scale incisions. (4) Transformation operation: Select healthy, pest-free lily bulbs, cut them open at the base of the bulb, and cut them into single scales. Immerse the base of the cut lily scales in the prepared Agrobacterium infection suspension and leave it for 10-60 minutes. Remove the scales, absorb the excess bacterial solution with sterile filter paper, and smear the cut at the base of the scales with the bacterial layer on the TY solid culture medium. (5) Embed or propagate the transformed lily scales in moist soil and water to keep the soil moist; (6) After 10-30 days of culture, bulges or small bulbs can be observed at the base of the scales. Phenotypes, reporter genes or PCR can be used for observation and screening to identify successfully transformed small bulbs, and then positive transformed plants can be formed.
2. The method for genetic transformation of lily without tissue culture according to claim 1, characterized in that The molecular cloning technology described in step (1) is an enzyme digestion technology: the vector is digested to linearize the vector, and the target gene and the vector are connected using a ligase or homologous recombination, thereby constructing and obtaining a recombinant vector containing the target gene.
3. The method for genetic transformation of lily without tissue culture according to claim 1, characterized in that The target gene described in step (1) includes a GFP reporter gene, a resistance gene, a CRISPR / Cas system component, and other functional expression or editing systems.
4. The method for genetic transformation of lily without tissue culture according to claim 1, characterized in that The molecular cloning technology described in step (1) is Gibson assembly, gene editing technology based on CRISPR / Cas system, GoldenGate\Gateway, etc.
5. The open tissue culture-free genetic transformation method for lily according to claim 1, characterized in that The infection method in step (4) is to perform vacuum infection in a vacuum pump at 0.03-0.09 MPa for 10 -60 s.
6. The open tissue culture-free genetic transformation method for lily according to claim 1, characterized in that Step (5) is to inoculate the infected scales with the cut facing downwards into a culture tray filled with moist soil. The scale planting depth is about 1 / 3 of the scale length. The culture conditions are temperature 26°C, light intensity 1500-2000lx, light time 16 h / d, and dark time 8 h / d.