Non-tissue culture dependent transformation method for promoting effective transformation of potatoes by using growth factors

Abstract

The invention belongs to the technical field of agricultural biology, and discloses a non-tissue culture dependent transformation method for promoting effective transformation of potatoes by using growth factors. The invention discloses a reagent composition. The reagent composition comprises an infection solution and a bud inducing screening solution, wherein the infection liquid is prepared from acetosyringone, dithiothreitol and plant hormones; the bud inducing screening liquid comprises plant hormones, glufosinate-methyl and regeneration factors. The invention provides a reagent composition. The reagent composition can be used for non-tissue culture dependent genetic transformation of potatoes. By using the reagent composition, the non-tissue culture dependent genetic transformation step of the potatoes can be simplified, the conditions of chimera and false positive of transformants are reduced, and the transformation efficiency is remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a non-tissue culture-dependent transformation method that utilizes growth factors to promote the effective transformation of potatoes. Background Technology

[0002] Potatoes are an important global food, feed, and industrial raw material, making molecular biology research and variety improvement of them extremely valuable. Potatoes exhibit multiple ploid types: cultivated varieties are predominantly tetraploid, but diploid and triploid varieties also exist. The polyploid genetic characteristics and high heterozygosity present significant challenges to traditional breeding methods. Genetic transformation technology can efficiently introduce single or multiple exogenous genes into superior lines with minimal impact on the genetic background, thus greatly accelerating the variety improvement of these two crops.

[0003] Potatoes are generally transformed using traditional genetic transformation techniques: in a sterile environment, exogenous DNA is directly introduced into plant tissues using methods such as Agrobacterium-mediated transformation, electroporation, gene guns, or chemical substances. After tissue culture, regenerated transgenic seedlings are obtained. Agrobacterium-mediated transformation is the most widely used method because it is convenient and simple to use, and the introduced DNA fragments are complete with a low copy number. However, these traditional methods have significant drawbacks: first, transformation efficiency is greatly affected by genotype; second, deformed seedlings are prone to occur after tissue culture, and diploid regenerated plants often exhibit chromosome doubling; third, the use requires strict sterility control, and the operation is delicate and complex, resulting in high costs and difficulties in widespread adoption.

[0004] In recent years, researchers have developed highly efficient genetic transformation technologies that are not tissue culture-dependent. These technologies can overcome genotype and species barriers to achieve efficient transformation at low cost, greatly compensating for the shortcomings of traditional methods. For example, the RAPID (Regenerative Activity-dependent in Planta Injection Delivery) transformation system can be applied to potatoes: transgenic seedlings are obtained by injecting Agrobacterium tumefaciens near the bud point of dormant shoots in potato tubers. However, because the transgenic seedlings obtained through this method may contain chimeras, and whether the transgene can be propagated asexually is unknown, and because this Agrobacterium injection method is time-consuming, labor-intensive, and material-intensive, this technology has significant advantages, but further improvement and optimization are still needed.

[0005] Therefore, this application is hereby submitted. Summary of the Invention

[0006] The first aspect of the present invention is to provide a reagent composition.

[0007] The second aspect of the present invention aims to provide the use of the reagent composition of the first aspect of the present invention in plant genetic transformation or in the preparation of products for plant genetic transformation.

[0008] A third aspect of the present invention is to provide a product.

[0009] The fourth aspect of this invention is to provide a method for genetic transformation of potatoes.

[0010] The fifth aspect of this invention aims to provide the application of the potato genetic transformation method of the fourth aspect of this invention in potato breeding.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a reagent composition comprising an infection solution and a bud selection solution; wherein, The inoculation solution includes acetylsuccinone, dithiothreitol, and plant hormones; The bud selection solution includes plant hormones, glufosinate, and regeneration factors.

[0012] In some embodiments of the present invention, the final concentration of acetylsuccinone in the dyeing solution is 50-300 mM.

[0013] In some preferred embodiments of the present invention, the final concentration of acetylsylgenin in the infiltration solution is 100–200 µM, such as any value or a range formed by any combination of 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200 µM.

[0014] In some embodiments of the present invention, the final concentration of the dithiothreitol in the infection solution is 100-300 mg / L.

[0015] In some preferred embodiments of the present invention, the final concentration of the dithiothreitol in the infection solution is 100 to 200 mg / L, such as any value or a range formed by any two of 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200 mg / L.

[0016] Since potatoes need to be cut open during genetic transformation, and they are prone to browning and rotting after being cut open, this invention adds dithiothreitol to the infection solution, which can effectively resist browning.

[0017] In some embodiments of the present invention, the final concentration of the plant hormone in the infection solution is 1 to 8 mg / L.

[0018] In some preferred embodiments of the present invention, the final concentration of the plant hormone in the infection solution is 1 to 5 mg / L, such as any value of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mg / L or a range formed by any two of them.

[0019] In some embodiments of the present invention, the infection solution further includes a culture medium.

[0020] In some embodiments of the present invention, the culture medium includes MS culture medium.

[0021] In some embodiments of the present invention, the culture medium contains sucrose.

[0022] In some embodiments of the present invention, the final concentration of sucrose in the culture medium is 5% to 15%.

[0023] In some embodiments of the present invention, the final concentration of sucrose in the culture medium is 8% to 12%, such as any value of 8%, 9%, 10%, 11%, 12%, or a range formed by any two of them.

[0024] In some embodiments of the present invention, the pH value of the culture medium is 5.4 to 6.0.

[0025] In some embodiments of the present invention, the plant hormone includes at least one of zeatin, indole-3-acetic acid, gibberellin, and 6-benzylaminopurine.

[0026] In some embodiments of the present invention, the plant hormone in the infection solution is zeatin. Adding zeatin to the infection solution can effectively improve the cell differentiation and sprouting ability of potato tubers.

[0027] In some embodiments of the present invention, the plant hormones in the bud selection solution include zeatin, indole-3-acetic acid, gibberellin, and 6-benzylaminopurine. These plant hormones can effectively induce adventitious buds to form from tubers in the soil.

[0028] In some embodiments of the present invention, the final concentration of zeatin in the bud selection solution is 1 to 10 mg / L.

[0029] In some preferred embodiments of the present invention, the final concentration of zeatin in the bud selection solution is 1 to 6 mg / L, such as any value of 1, 2, 3, 4, 5, 6 mg / L or a range formed by any two of them.

[0030] In some embodiments of the present invention, the final concentration of indole-3-acetic acid in the bud selection solution is 0.5 to 5 mg / L.

[0031] In some preferred embodiments of the present invention, the final concentration of indole-3-acetic acid in the bud selection solution is 1 to 3 mg / L, such as any value or a range formed by any two of 1, 1.2, 1.5, 1.7, 1.9, 2.1, 2.3, 2.5, 2.7, 2.9, and 3 mg / L.

[0032] In some embodiments of the present invention, the final concentration of the gibberellin in the bud selection solution is 0.01 to 1 mg / L.

[0033] In some preferred embodiments of the present invention, the final concentration of the gibberellin in the bud selection solution is 0.05 to 0.3 mg / L, such as any value of 0.05, 0.1, 0.15, 0.2, 0.25, 0.3 mg / L or a range formed by any two of them.

[0034] In some embodiments of the present invention, the final concentration of 6-benzylaminopurine in the bud selection solution is 0.01–2 mg / L.

[0035] In some preferred embodiments of the present invention, the final concentration of 6-benzylaminopurine in the bud selection solution is 0.1 to 1 mg / L, such as any value or a range formed by any two of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1 mg / L.

[0036] In some embodiments of the present invention, the regeneration factor is StREF1, and the amino acid sequence of StREF1 is shown in SEQ ID NO:1.

[0037] In some embodiments of the present invention, the final concentration of StREF1 in the bud selection solution is 80-200 mM.

[0038] In some preferred embodiments of the present invention, the final concentration of StREF1 in the bud selection solution is 90 to 150 mM, such as any value or a range formed by any two of 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, and 150 mM.

[0039] StREF1 has the function of improving the regeneration ability and genetic transformation efficiency of potatoes.

[0040] In some embodiments of the present invention, the final concentration of glufosinate in the bud-inducing screening solution is 1–20 mg / L.

[0041] In some preferred embodiments of the present invention, the final concentration of glufosinate in the bud-inducing screening solution is 4 to 15 mg / L, such as any value or a range formed by any two of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 mg / L.

[0042] glufosinate is very important in the sprout selection solution. Although kanamycin, hygromycin and glufosinate can all be used for potato screening in tissue culture-dependent genetic transformation (cultured in a sterile environment), in the inventors' preliminary experiments, only glufosinate showed a significant screening effect under non-tissue culture-dependent conditions (general conditions of soil cultivation).

[0043] In some embodiments of the present invention, the bud selection solution further includes glutamine and asparagine. Glutamine and asparagine can inhibit the plant's defense response and increase the infection success rate of Agrobacterium.

[0044] In some embodiments of the present invention, the final concentration of glutamine in the bud-inducing screening solution is 10-150 mg / L.

[0045] In some embodiments of the present invention, the final concentration of glutamine in the bud-inducing screening solution is 20 to 70 mg / L, such as any value of 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 mg / L or a range formed by any two of them.

[0046] In some embodiments of the present invention, the final concentration of the asparagine in the bud selection solution is 10-150 mg / L.

[0047] In some embodiments of the present invention, the final concentration of the asparagine in the bud-inducing screening solution is 20 to 70 mg / L, such as any value or a range formed by any two of 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, and 70 mg / L.

[0048] A second aspect of the invention provides the use of the reagent composition of the first aspect of the invention in plant genetic transformation or in the preparation of products for plant genetic transformation.

[0049] In some embodiments of the present invention, the plant includes at least one of potato, sweet potato, taro, and yam; preferably potato.

[0050] In some embodiments of the present invention, the genetic transformation is a tissue culture-independent genetic transformation.

[0051] A third aspect of the present invention provides a product comprising the reagent composition of the first aspect of the present invention.

[0052] In some embodiments of the present invention, the product includes reagents or kits.

[0053] In some embodiments of the present invention, the product can be used for plant genetic transformation, such as potato breeding.

[0054] A fourth aspect of the present invention provides a method for genetic transformation of potatoes, comprising the step of treating potatoes with a reagent composition of the first aspect of the present invention or a product of the third aspect of the present invention.

[0055] In some embodiments of the present invention, the potato genetic transformation method includes the following steps: (1) Cut potatoes without dormant buds and with skin into pieces and punch holes to obtain explants; (2) The explant is placed in the staining solution of the first aspect of the present invention for vacuum permeation treatment, wherein the staining solution contains Agrobacterium that can express heterologous genes; (3) After the explants are taken out and dried, they are planted in the substrate for co-culture, and then transferred to alternating light / dark conditions for culture. Sprouting is induced by the bud-inducing screening solution in the first aspect of the present invention to obtain transgenic potatoes.

[0056] For explant preparation, simply wash fresh potatoes (no sterilization required), and cut them into chunks (e.g., 1-2 cm long and wide, 0.3-0.6 cm thick) from areas without dormant buds. The chunks must contain the skin. Preliminary testing revealed that only tubers containing the skin can be induced to sprout adventitious buds by the applied regeneration factor; tissues without the skin cannot be induced to sprout.

[0057] Multiple small holes are deeply inserted into each side of the potato tuber using a needle, allowing the infection solution to enter through these holes and fill the entire tuber tissue.

[0058] In some embodiments of the present invention, the vacuum permeation treatment is performed by drawing a vacuum and maintaining a pressure of 0.08 MPa to 0.1 MPa for 3 to 5 minutes, followed by slow release, and repeating the treatment 2 to 4 times.

[0059] In some embodiments of the present invention, the total time of the vacuum permeation treatment is 10 to 20 minutes, such as any value or a range formed by any two of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 minutes.

[0060] In some embodiments of the present invention, after the vacuum permeation treatment, the explants are further immersed in the immersion solution for a time of 5 to 30 minutes, such as any value or a range formed by any two of the following: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 minutes.

[0061] In some embodiments of the present invention, the co-culture conditions are: co-culture at 20–28°C for 3–6 days under dark conditions.

[0062] In some embodiments of the present invention, the photoperiod of the light / dark conditions is day:night = 16h:8h, with incubation at 20–28°C and 65%–60% humidity.

[0063] In some embodiments of the present invention, the OD value of Agrobacterium in the infiltration solution is 0.2 to 1, such as any value or a range formed by any two of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.

[0064] In some embodiments of the present invention, the substrate comprises soil.

[0065] In some embodiments of the present invention, the sprouting screening solution is applied to a substrate in which potatoes are planted.

[0066] Irrigation with the sprout selection solution can improve the sprouting rate of potatoes and effectively screen for false positives. Tests showed that potato seedlings identified as non-GMO by PCR all died after irrigation with the selection solution, reducing the workload of subsequent molecular screening to obtain GMO seedlings. Furthermore, without selective pressure, the plants become undetectable of transgenic fragments after 1-2 months, allowing the sprout selection solution to reduce the chimerism rate of GMO seedlings, thus ensuring that newly grown tissues still contain transgenic fragments.

[0067] A fifth aspect of the present invention provides the application of the potato genetic transformation method of the fourth aspect of the present invention in potato breeding.

[0068] The beneficial effects of this invention are: This invention provides a reagent composition for tissue culture-independent genetic transformation of potatoes. Using this reagent composition simplifies the tissue culture-independent genetic transformation process of potatoes, reduces the occurrence of chimeras and false positives in transformants, and significantly improves transformation efficiency.

[0069] This invention provides a tissue culture-independent genetic transformation method for potatoes, comprising removing dormant buds from tubers, inducing adventitious buds in Agrobacterium-infected cells within the tubers using plant hormones and growth factors such as regeneration factor peptides, and then adding a bud-inducing screening solution for selection. This reduces the occurrence of chimeras and false positives in transformants, thereby improving transformation efficiency. Tests have shown that transgenic materials transformed using this method do not exhibit deformed seedlings and can be propagated asexually through tubers. The method provided by this invention is simple, safe, and easy to master: no special treatment such as sterilization of explants (potato tubers) is required; infection is accomplished simply through vacuuming and soaking; and the bud induction and screening processes are achieved by irrigating with a bud-inducing screening solution containing growth factors. The entire transformation process does not require controlled environmental cleanliness and can be carried out under normal conditions. Therefore, using this technology for genetic transformation of potatoes not only has low technical barriers and operating costs but is also easy to promote and scale up, possessing broad application prospects.

[0070] Compared with the existing tissue culture-independent high-efficiency genetic transformation technology (RAPID), which requires the use of syringes for infection, RAPID has the following disadvantages: (1) If syringe injection is used, the bacterial solution needs to be completely injected under the skin of the potato tuber and filled, which requires professional skills. Moreover, multiple injections are required for each potato tuber, which means that it takes more than 30 seconds to inject each potato tuber. The operation is time-consuming and labor-intensive. In addition, if there is no protection during the operation, the solution is easy to splash into the eyes and cause damage to the operator's eyes. Compared with this technology, the present invention improves the injection method by using vacuum and soaking to replace it, making the whole process easier to operate, scalable, safe and time-saving. In addition, it also reduces the consumption of syringes and achieves the effect of environmental protection. (2) RAPID potatoes need to use dormant buds (eyes), while the present invention deliberately avoids the use of dormant buds (eyes) to avoid Agrobacterium infection of the already developed bud tissue with multiple cells, which would result in the seedlings growing from this bud tissue being transgenic chimeras, making it difficult for transgenics to be propagated. Then, this invention uses tuber tissue without dormant buds (eyes) as explants. After infection with Agrobacterium, one or more infected cells are induced to grow into seedlings using growth hormones, thereby reducing the probability of chimerism and making transgenes effectively heritable. (3) Compared with RAPID, this invention adds the application of screening agents to maintain a certain selection pressure throughout the transformation process, so as to reduce the chimerism rate of transgenic seedlings and improve the efficiency of obtaining transgenic seedlings. Attached Figure Description

[0071] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The comparison results of REF1 sequences in different Solanaceae species (tomato, potato, euryale, etc.).

[0072] Figure 2 This is a diagram of the construction of the transformation vector (pSY81 plasmid) of the present invention; wherein, (a) is a map of the pSY81 plasmid, and (b) is a schematic diagram of the construction of the pSY81 plasmid.

[0073] Figure 3 This invention provides a non-tissue culture-dependent genetic transformation method for promoting effective potato transformation using growth factors; wherein, (a) is a schematic diagram of the explant preparation and infection process, (b) is a schematic diagram of the tuber bud induction and screening treatment, and (c) is a cultivation diagram of the transgenic seedlings obtained in Example 5.

[0074] Figure 4 Resistance genes in potato seedling transformants and offspring of potato seedlings obtained through asexual reproduction bar With target gene dsRED The fragment detection results are shown in the figure. In the figure, a+b represents the resistance gene. bar c+d represents the target gene dsRED WT represents wild-type potato, St1 and St2 represent transgenic seedlings from Examples 3 and 5, respectively, and St1-1 and St2-1 represent asexual offspring that germinate after tuber formation from transgenic seedlings from Examples 3 and 5, respectively.

[0075] Figure 5 The expression of GFPuv and dsRED in the roots of transgenic potato plants is shown. The scale bar is 2 mm. Detailed Implementation

[0076] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0077] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0078] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0079] Example 1: Synthesis of StREF1, a regeneration factor for potatoes To improve the transformation efficiency of potatoes, this embodiment predicted and synthesized a potato-specific StREF1 for use in genetic transformation testing. The specific method is as follows: The REF1 peptide located at the C-terminus of tomato SlPep precursor PROPEP (SlPROPEP) was found to enhance the regeneration ability and genetic transformation efficiency of tomatoes under aseptic conditions in tissue culture (Yang W, Zhai H, Wu F, et al. (2024). Peptide REF1 is a local woundsignal promoting plant regeneration. Cell, 187, 30). By comparing SlPROPEP sequences with those in the ClusteredNR database using NCBI blastP, four sequences with the highest similarity to SlPROPEP were obtained. These sequences were derived from wild tomato species in the Solanaceae family (…). Solanum pennellii Cultivated potatoes ( Solanum tuberosum ), wild potato ( Solanum pinnatisectum ), European and American ( Solanum dulcamara These sequences were compared in DNAMAN, and the REF1 sequence (ATERRGRPPSRPKVGSGPPPQNN, SEQ ID NO:1) located at the C-terminus of the predicted potato StPROPEP sequence was identified. This sequence is named StREF1. StREF1 is conserved in cultivated and wild potatoes, but differs from the REF1 of tomatoes (including wild tomatoes) by one amino acid and from the REF1 of European potatoes by five amino acids. Figure 1 ).

[0080] Since the specific sequence of StREF1 has not been reported in previous studies, and REF1 is a small peptide that is easily degraded in normal environments, whether the StREF1 predicted in this example can function under non-sterile, non-tissue culture-dependent transformation conditions remains to be tested. The StREF1 peptide was synthesized by Shanghai Taopu Biotechnology Co., Ltd. and used for subsequent genetic transformation.

[0081] Example 2 A tissue-culture-dependent genetic transformation method for promoting efficient potato transformation using growth factors includes the following steps: (1) Construction of transformation vector In preliminary experiments, the effectiveness of kanamycin (Kan), hygromycin, and phosphinothricin (PPT) as screening agents for potato seedlings induced without tissue culture dependence was tested. Only PPT showed a screening effect; therefore, this invention selected bar (bialaphos resistance gene) as the resistance marker gene in the vector design. For ease of observation, this invention added a gene sequence encoding P2A-GFPuv after the bar gene. Figure 2 The target gene was selected as the reporter gene dsRED, which is controlled by the potato polyubiquitin promoter. Figure 2 ).

[0082] Specific methods for constructing the carrier: Using the plasmid ZP04-GFPuv available in our laboratory as a template, a 0.8 kb GFPuvP2A fragment was cloned. The existing pSY83 plasmid from our research center (whose backbone is pCambia1300, resistance gene is bar, and resistance gene control elements are a 35S promoter and a 35S terminator) was cut using Mlu1 and Acc651, and the GFPuvP2A fragment was ligated via in-fusion to obtain the plasmid pS83-GFPuvP2A. Using yellow-fleshed potato DNA as a template, a 1.2kb potato polyubiquitin promoter (PStubi) was cloned. Then, using the pJL210A plasmid from the research center as a template, a 0.7kb dsRed fragment was cloned. Through overlap PCR, a 1.9kb PStubi-dsRED fragment was amplified. This fragment was then infused into the vector backbone of pS83-GFPuvP2A, which had been cleaved with Bsu36I and Xba1, to obtain the pSY81 plasmid. Figure 2 ).

[0083] All PCR amplifications related to vector construction were performed using KOD FX high-fidelity enzyme (TOYOBO, Japan), while in-fusion ligation was performed using the In-Fusion® HD Cloning Kit (Clontech, USA).

[0084] The nucleotide sequence of the pSY81 plasmid is shown in SEQ ID NO:2.

[0085]

[0086] (2) Genetic transformation Plasmid transformation of Agrobacterium: Take 1 μL of pSY81 plasmid and mix it with 50 μL of LB4404 (not limited to LB4404) competent cells, then add it to an electrode cup and transform using Gene Pulser Xcell. TM Electroporation was performed using a Bio-Rad system (USA). The electroporated mixture was added to 200 μL of LB liquid medium and incubated at 28°C and 200 rpm for 2.5 h. 50 μL of the bacterial culture was then plated onto LB solid medium plates containing 50 mg / L Rifampicin and 50 mg / L Kan, and incubated upside down at 28°C for 3 days. Single colonies were picked and incubated overnight in 5 mL of LB liquid medium containing 50 mg / L Rifampicin and 50 mg / L Kan to obtain Agrobacterium containing the target plasmid. The bacterial culture was stored at -80°C for later use.

[0087] Potato transformation: Agrobacterium containing the target plasmid was streaked onto a plate. Two days later, single colonies were collected and spread onto a plate or shaken. On the second day, the Agrobacterium was collected and the cells were resuspended in the infection solution. The OD of the infection solution was then measured. 600 The pH was adjusted to around 0.5 for later use. In this invention, the composition of the inoculum was 1 / 4 MS (Murashige & Skoog Basal Medium with Vitamins, PhytoTechLabs, US, catalog number M519) (containing 10 g / L sucrose and pH 5.8), 100 µM–200 µM acetosyringone (AS), 150 mg / L 1,4-Dithiothreitol (DTT), and 2 mg / L zeatin.

[0088] For explant preparation, simply wash fresh potatoes (no sterilization required), select areas without dormant buds, and cut them into pieces 1-2 cm long and wide, and about 0.5 cm thick. Figure 3 (a)). The tubers used as explants must contain an epidermis (preliminary tests showed that only tubers containing an epidermis could be induced to sprout adventitious buds by the applied growth factor; tissues without an epidermis could not be induced to sprout). Subsequently, multiple small holes were deeply pierced on each side of the tuber using a needle to allow the infection solution to enter through these holes and fill the entire tuber tissue. Next, these tubers were placed in the infection solution and maintained at a pressure of 0.08 MPa–0.1 MPa for 3–5 minutes, then slowly released. This process was repeated three times, followed by soaking for 10 minutes. The tubers were then removed and the surface of the bacterial solution was dried.

[0089] Place the potato tubers skin-side up in the soil and cover them with a thin layer of soil. Then, incubate these tubers in the dark for 4 days at a temperature of 25℃ ± 2℃. After co-cultivation, the tubers were moved to a greenhouse at 25℃ ± 2℃ and humidity of about 60%, with a photoperiod of day:night = 16h:8h. Figure 3 (b) Each tuber was irrigated weekly with 2 mL of the sprout selection solution. The sprout selection solution contained the following: plant hormone H solution (containing 4 mg / L Zeatin and 1.5 mg / L indole-3-aceticacid (IAA), 0.2 mg / L Gibberellin A3 (GA3), and 0.5 mg / L 6-Benzylaminopurine (6-BA). Preliminary experiments showed that this solution was effective in inducing adventitious buds from tubers in the soil), 6 mg / L selection agent (PPT), 50 mg / L glutamine (Gln), 50 mg / L asparagine (Asn) (the addition of 50 mg / L Gln and Asn can inhibit the plant's defense response and increase the infection success rate of Agrobacterium) and 100 mM StREF1 (Example 1). Transgenic seedlings were obtained after two months of sprout selection.

[0090] Example 3 A non-tissue culture-dependent genetic transformation method for promoting effective potato transformation using growth factors is disclosed. Compared with Example 2, the only difference is the composition of the sprout selection solution during potato transformation. Specifically, the sprout selection solution used in this example consists of: plant hormone H solution (containing 4 mg / L Zeatin, 1.5 mg / L indole-3-acetic acid, 0.2 mg / L gibberellin and 0.5 mg / L 6-benzylaminopurine) and 6 mg / L PPT.

[0091] Example 4 A non-tissue culture-dependent transformation method for promoting effective potato transformation using growth factors is disclosed. Compared with Example 2, the only difference lies in the composition of the sprout induction screening solution during potato transformation. Specifically, the sprout induction screening solution used in this example consists of: plant hormone H solution (containing 4 mg / L Zeatin, 1.5 mg / L indole-3-acetic acid, 0.2 mg / L gibberellin and 0.5 mg / L 6-benzylaminopurine), 6 mg / L PPT and 100 mM StREF1.

[0092] Example 5 A non-tissue culture-dependent genetic transformation method for promoting effective potato transformation using growth factors is disclosed. Compared with Example 2, the only difference lies in the composition of the sprout selection solution during potato transformation. Specifically, the sprout selection solution used in this example consists of: plant hormone H solution (containing 4 mg / L Zeatin, 1.5 mg / L indole-3-acetic acid, 0.2 mg / L gibberellin and 0.5 mg / L 6-benzylaminopurine) and 6 mg / L PPT, 50 mg / L Gln and 50 mg / L Asn.

[0093] Example 6 A non-tissue culture-dependent genetic transformation method for promoting efficient potato transformation using growth factors is disclosed. Compared with Example 2, the only difference is the composition of the infection solution during potato transformation. Specifically, the infection solution used in this example has the following composition: 1 / 4 MS (containing 10% sucrose and pH 5.8), 100µM~200µM acetylsuccinone, and 150mg / L dithiothreitol.

[0094] Example 7 A non-tissue culture-dependent genetic transformation method for promoting effective potato transformation using growth factors is disclosed. Compared with Example 6, the only difference is the composition of the sprout selection solution used in the potato transformation process. Specifically, the sprout selection solution used in this example consists of: plant hormone H solution (containing 4 mg / L Zeatin, 1.5 mg / L indole-3-acetic acid, 0.2 mg / L gibberellin and 0.5 mg / L 6-benzylaminopurine) and 6 mg / L PPT.

[0095] Example 8 A non-tissue culture-dependent genetic transformation method for promoting efficient potato transformation using growth factors is disclosed. Compared with Example 6, the only difference is the composition of the sprout selection solution used in the potato transformation process. Specifically, the sprout selection solution used in this example consists of: plant hormone H solution (containing 4 mg / L Zeatin, 1.5 mg / L indole-3-acetic acid, 0.2 mg / L gibberellin and 0.5 mg / L 6-benzylaminopurine), 6 mg / L PPT and 100 mM StREF1.

[0096] Example 9 A non-tissue culture-dependent genetic transformation method for promoting effective potato transformation using growth factors is disclosed. Compared with Example 6, the only difference is the composition of the sprout selection solution used in the potato transformation process. Specifically, the sprout selection solution used in this example consists of: plant hormone H solution (containing 4 mg / L Zeatin, 1.5 mg / L indole-3-acetic acid, 0.2 mg / L gibberellin and 0.5 mg / L 6-benzylaminopurine) and 6 mg / L PPT, 50 mg / L Gln and 50 mg / L Asn.

[0097] Effect Example The methods described in Examples 2 through 9 were used to perform tissue culture-independent genetic transformation of potatoes, resulting in different transgenic seedlings. PCR was performed on these seedlings to assess the genetic transformation efficacy of different methods in tissue culture-independent potato transformation. The PCR detection process is as follows: Genomic DNA was extracted from plants using the NuClean Plant Genomic DNA Kit (Kangwei Century Biotechnology Co., Ltd., China). Then, according to the manufacturer's instructions, PCR amplification of the plant genome was performed using a 2×Taq PCR StarMix (Dye) (GenSta, China). The primers used are shown in Table 1. The fragment size amplified using BarGFPuv_T_F and BarGFPuv_T_R was 571 bp, while the fragment amplified using dsRED_T_F and dsRED_T_R was 815 bp.

[0098] Table 1 Primer sequences used for PCR detection

[0099] PCR detection was performed on transgenic seedlings obtained using different transformation methods. The results showed that all resistant seedlings possessed the resistance gene bar (571 bp) and the target gene dsRED fragment (815 bp) from the vector. Figure 4 Therefore, the resistance screening used in this invention has a very good effect. Representative results in Table 2 show that in different infection solutions, when the infection solution formulation is only 1 / 4 MS + AS + DTT (i.e., Examples 6 to 9), transgenic seedlings cannot be obtained. However, adding zeatin to the infection solution helps in obtaining transgenic seedlings. Furthermore, in different induction and screening solutions, adding 100 mM StREF1 effectively induces the production of transgenic seedlings, while the addition of Gln and Asn has little effect on the genetic transformation of potatoes.

[0100] The transgenic seedlings obtained using the non-tissue culture-dependent transformation methods of Examples 2 and 4 were observed for any deformities. The results showed that no deformities were observed in the transgenic seedlings obtained using the non-tissue culture-dependent transformation methods of Examples 2 and 4. The expression of GFP and dsRED in the roots was detected using a Leica M205FA microscope (Leica, Germany). The wavelength ranges of the excitation filters used for GFP and dsRED were 450–490 nm and 540–580 nm, respectively. In potato roots, both GFPuv and dsRED were expressed well. Figure 5 As can be seen, the reporter gene can be effectively expressed in some transgenic lines obtained using the methods of Examples 2 and 4.

[0101] Further propagation of the various transgenic plants ( Figure 3 (c) Potatoes from all lines were harvested. One potato tuber from each line was germinated, and the germinated seedlings were subjected to PCR testing. The results showed that the asexually reproduced offspring of the above lines all carried the transgenic fragment ( Figure 4 (and Table 2). Therefore, the transgenic plants obtained by transformation using the methods of Example 2 and Example 4 can be propagated asexually.

[0102] Table 2. Representative results obtained from different conversion methods

[0103] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A reagent composition comprising an infection solution and a shoot induction screening solution; wherein, the infection solution comprises acetyl-syringone, dithiothreitol and a plant hormone; the shoot induction screening solution comprises a plant hormone, glufosinate, a regeneration factor.

2. The reagent composition of claim 1, wherein, the acetyl-syringone has a final concentration of 50-300 µM in the infection solution; preferably, the dithiothreitol has a final concentration of 100-300 mg / L in the infection solution; preferably, the plant hormone has a final concentration of 1-8 mg / L in the infection solution; preferably, the infection solution further comprises a culture medium; preferably, the culture medium comprises MS medium; preferably, the culture medium contains sucrose.

3. The reagent composition according to claim 1 or 2, characterized in that, the plant hormone comprises at least one of zeatin, indole-3-acetic acid, gibberellin and 6-benzylaminopurine; preferably, the regeneration factor is StREF1, and the amino acid sequence of the StREF1 is shown in SEQ ID NO: 1; preferably, the shoot induction screening solution further comprises glutamine and asparagine.

4. The reagent composition according to claim 3, wherein the glufosinate has a final concentration of 1-20 mg / L in the shoot induction screening solution; preferably, the StREF1 has a final concentration of 80-200 mM in the shoot induction screening solution; preferably, the glutamine has a final concentration of 10-150 mg / L in the shoot induction screening solution; preferably, the asparagine has a final concentration of 10-150 mg / L in the shoot induction screening solution. 5.Use of the reagent composition of any one of claims 1-4 in plant genetic transformation or in the preparation of a product for plant genetic transformation. preferably, the plant comprises at least one of potato, sweet potato, taro and yam; preferably, the genetic transformation is non-tissue culture dependent genetic transformation. 6.A product comprising the reagent composition of any one of claims 1-4. 7.A potato genetic transformation method comprising the step of treating potato using the reagent composition of any one of claims 1-4 or the product of claim 6.

8. The method for genetic transformation of potato according to claim 7, characterized in that, the potato genetic transformation method comprises the following steps: (1) cutting and perforating a potato without dormant bud points and with epidermis to obtain explants; (2) treating the explants with vacuum infiltration in the infection solution of any one of claims 1-4, wherein the infection solution contains Agrobacterium capable of expressing a heterologous gene; (3) taking out the explants, drying and planting in a substrate for co-culture, followed by alternating culture under light / dark conditions, using the shoot induction screening solution of any one of claims 1-4 to induce germination, and obtaining transgenic potato.

9. The method for genetic transformation of potato according to claim 8, characterized in that, the vacuum infiltration treatment is performed by vacuumizing, maintaining at a pressure of 0.08-0.1 MPa for 3-10 min, and then slowly releasing, and repeating 2-4 times; preferably, after the vacuum infiltration treatment, the explants are further soaked in the infection solution for 5-30 min; preferably, the co-culture is performed in dark conditions at 20-28 ℃ for 3-6 days; preferably, the OD value of Agrobacterium in the infection solution is 0.2-1.

10. Use of the method for genetic transformation of potato according to any one of claims 7 to 9 in potato breeding.

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