Stable genetic transformation method for infecting lily scales without depending on tissue culture technology

Abstract

The invention relates to the technical field of lily genetic breeding, in particular to a stable genetic transformation method for infecting lily scales without depending on a tissue culture technology. According to the method, a stable genetic transformation system without tissue culture of the lily scales is established through an agrobacterium tumefaciens mediated method, and different agrobacterium tumefaciens strains, infection conditions, infection time and the infection efficiency of paclobutrazol (PBZ) on the lily scales are systematically explored. The defect that a genetic transformation system needs to use a sterile tissue culture technology to induce calluses is overcome. By optimizing key parameters of a tissue culture-free genetic transformation system of the lily scales, a reference basis is provided for subsequently establishing an efficient and stable genetic transformation system.

Description

Technical Field

[0001] The present invention relates to the technical field of lily genetic breeding, and in particular to a stable genetic transformation method that does not rely on tissue culture technology to infect lily scales. Background Art

[0002] At present, the genetic transformation system of lily is still immature, and it is necessary to induce callus tissue through tissue culture to study and verify gene function. However, this method requires not only mastering tissue culture skills, but also the establishment and possession of a sterile tissue culture environment. Moreover, the induction system of lily callus tissue has not been fully established, and the induction is difficult. The induction amount is difficult to meet the required amount for genetic transformation. This makes it impossible to carry out stable genetic transformation in lily plants, which seriously hinders research on lily genetic breeding and gene editing.

[0003] Lily genetic transformation mainly adopts transient transformation method, which is usually mediated by Agrobacterium, pollen magnetization, particle bombardment and PEG-Ca 2+ Agrobacterium-mediated method, etc. Agrobacterium-mediated method is the most commonly used method in the genetic transformation of lily. Agrobacterium-mediated method is relatively simple to operate, low cost, and has high transformation efficiency. However, its transformation efficiency will be affected by factors such as explant type, Agrobacterium strain and culture conditions. Studies have shown that the callus regeneration ability of lily decreases with age, and callus tissue from different sources varies significantly. In addition, during the transient transformation process, the regeneration frequency of lily is relatively low, and the growth state of the regenerated plants is also unstable. Although transient transformation technology can achieve rapid expression of plant genes, its integration efficiency is relatively low, which will lead to unstable gene expression and inability to stably inherit to offspring. Therefore, it greatly limits the application of gene editing technology in the genetic improvement and breeding of lily (variety).

[0004] In the existing technology, some studies have attempted to use callus tissue as a receptor material for genetic transformation. For example, the scale tissue of Lilium formosanum 'Sally' was successfully used to construct a genetic transformation system through Agrobacterium-mediated method (Zhang Huimin, Wei Yi, Li Lianlian, et al. Optimization of lily regeneration system and construction of genetic transformation system of Lilium formosanum 'Sally'. Bulbous Perennial Flower Branch of Chinese Horticultural Society. Research Progress of Chinese Bulbous Perennial Flowers, College of Agriculture, Yunnan University, 2023, 2.). However, it still has problems such as strong genotype dependence, poor genetic transformation stability and difficulty in regeneration after transformation.

[0005] The stable genetic transformation method for lilies through callus induction relies on tissue culture techniques and a sterile culture environment. Furthermore, the differentiation and regeneration efficiencies of callus induction in lilies are low, limiting the stable inheritance of exogenous genes in vivo and hindering functional research on lily genes and cultivar breeding and improvement efforts. Chinese invention patent publication number CN 117016092A discloses a non-tissue culture immature embryo generation system and efficient genetic transformation method for lilies, addressing the difficulty of obtaining immature embryos in tissue culture. However, there are no reports of a stable genetic transformation system for lily scales that does not require tissue culture. Summary of the Invention

[0006] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a stable genetic transformation method that does not rely on tissue culture technology to infect lily scales, overcomes the defect that the lily genetic transformation system requires the use of sterile tissue culture technology to induce callus tissue, and optimizes the key parameters of the genetic transformation system of lily scales that does not require tissue culture.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0008] A stable genetic transformation method is provided that does not rely on tissue culture technology to infect lily scales, comprising the following steps:

[0009] (1) Infect lily scales by shaking the fungus;

[0010] (2) scale differentiation into bulblets;

[0011] (3) PCR detection and identification of the number of successfully infected bulblets to determine the transformation rate.

[0012] Furthermore, during the process of shaking the lily scales, the modified MMA resuspension was prepared as follows: 4.43 g / LMS, 20 g / L sucrose, 1.95 g / L MES, 10 mM MgCl2, 100 μM acetosyringone and 10 μM paclobutrazol, and adjusted to pH = 5.6.

[0013] Furthermore, the best infection system for lily scales is to adjust the OD of the infection solution by mediating the EHA105 strain. 600 =1.0, and the bacteria were shaken at 200 rpm in a 28°C shaker for 15 min or 30 min to complete the infection, and the target gene could be successfully integrated into the lily genome.

[0014] The beneficial effects of the present invention are:

[0015] The present invention establishes a stable genetic transformation system for lily scales without tissue culture through the Agrobacterium-mediated method, systematically explores different Agrobacterium strains, infection conditions and infection times, as well as the infection efficiency of paclobutrazol (PBZ) on lily scales, providing technical support for lily gene function research and new variety breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a flow chart of the method of the present invention;

[0017] Figure 2 This is a schematic diagram of the root growth of differentiated bulbs in the GV3101 Agrobacterium-treated group;

[0018] Figure 3 This is a schematic diagram of the root growth of differentiated bulbs in the EHA105 Agrobacterium-treated group;

[0019] Figure 4 This is the result of mixed sample detection of differentiated bulblets of lily scales; LhERF4-F / LhERF4-R was used as the primer pair to detect the target sequence of PCR amplification of exogenous genes, with a length of 408bp, EH⑥-1 was the first group of the EH⑥ treatment group, EH⑤-7 was the seventh group of the EH⑤ treatment group, CK was the positive control, M was the marker, and the band sizes were 1500bp, 900bp, 700bp, 500bp, 400bp, 200bp and 100bp, respectively. DETAILED DESCRIPTION

[0020] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0021] Example

[0022] 1. Transformation of exogenous gene plasmid into Agrobacterium competent cells (GV3101 and EHA105)

[0023] (1) Thawing competent cells: Take Agrobacterium competent cells (GV3101 and EHA105) stored at -80°C and place them on ice until they melt (approximately ice-water mixture);

[0024] (2) Plasmid transformation: Add 5 μL of pCAMBIA3301-LhERF4 plasmid (0.1 μg / μL) to 100 μL of competent cells and mix thoroughly by inverting the tube. Avoid vigorous shaking. Then perform the following treatments in sequence: ice bath for 5 min, quick freeze in liquid nitrogen for 5 min (quickly insert liquid nitrogen), heat shock in a 37°C water bath for 5 min, and cool in an ice bath for 5 min.

[0025] (3) Recovery culture: Add 700 μL of antibiotic-free LB liquid medium, mix well, and place in a shaker at 28°C (200 rpm) for 2 h to allow the cells to recover and express resistance genes;

[0026] (4) Spreading screening: Take the revived bacterial solution, centrifuge it at 12,000 rpm for 1 min, discard most of the supernatant, retain about 100 μL, gently pipette to resuspend the bacteria, spread it evenly on a LB solid plate containing 100 mg / L Kan and 50 mg / L Rif, and culture it upside down in a 28°C incubator for 48 h. Observe the growth;

[0027] (5) Shake culture: Pick three single clones from different strains, add them into 300 mL of LB liquid medium containing 50 mg / L Kan + 50 mg / L Rif, and shake culture at 200 rpm in a 28°C incubator for 24 h;

[0028] (6) Agrobacterium detection: Take the cultured Agrobacterium liquid separately and perform PCR detection to determine whether it has been successfully transferred into the Agrobacterium strain.

[0029] 2. Agrobacterium infection

[0030] (1) Modified MMA resuspension solution: 4.43 g / L MS (without vitamins), 20 g / L sucrose, 1.95 g / L MES, 10 mM MgCl2, 100 μM acetosyringone (AS) and 10 μM paclobutrazol (PBZ), adjusted to pH = 5.6;

[0031] (2) Harvesting the bacteria: After shaking the bacteria, GV3101 and EHA105 strains were centrifuged separately to collect the bacteria;

[0032] (3) Resuspension of bacterial suspension: After centrifugation, discard the supernatant and resuspend the culture in MMA resuspension buffer. Adjust the OD600 to 1.0 or 1.5, respectively. After resuspension, add the culture to a 500 mL Erlenmeyer flask.

[0033] (4) Infection: Take healthy middle scales of Lilium truncatum without lesions, clean them, and add 50 scales of Lilium truncatum to each triangular flask. Shake the scales in a shaker at 28°C and 200 rpm for 15 min or 30 min. After shaking, remove the scales (use sterilized tweezers to avoid contamination) and place them in a tray for embedding. The scale treatment is shown in Table 1.

[0034] Table 1 Statistics of lily treatments infected with Agrobacterium

[0035]

[0036] 3. Inducing differentiation of bulblets

[0037] (1) Low-temperature vernalization: Seal the tray of embedded scales with plastic wrap and pierce it. The plastic wrap seal maintains humidity to prevent dehydration of the bulbs. The piercing ensures moderate gas exchange and avoids anaerobic respiration. Place in a constant temperature incubator at 4°C for 2 months. The low temperature of 4°C can effectively activate dormancy release-related genes in lily bulbs. Dark culture simulates the natural overwintering environment and inhibits germination inhibition mediated by photosensitive pigments.

[0038] (2) Vernalization removal and regeneration culture: Take out the lily bulbs after low temperature induction ( Figure 2 and Figure 3 ), tear off the plastic wrap, dig out the scales and re-insert them into the substrate at a 45° angle (buried 1 / 3 deep, with the tip facing upwards), and culture them in a moisturizing environment at 20℃-25℃ to induce differentiation and rooting.

[0039] Depend on Figure 1-2 It can be seen that the root growth of the GV3101 Agrobacterium-treated group treated for 15 minutes was better than that of the 30-minute treatment, and the short-time treatment was more suitable for the root growth of the GV3101 Agrobacterium-treated group; the root growth of the EHA105 Agrobacterium-treated group treated for 30 minutes was better than that of the 15-minute treatment, and the long-time treatment was more suitable for the root growth of the EHA105 Agrobacterium-treated group.

[0040] 4. Sample DNA Extraction and Detection

[0041] (1) The number and differentiation rate of bulblets differentiated from lily scales were counted. Bulblets were randomly selected from each treatment group for phenotypic observation and photography, and root growth was compared. The roots of the differentiated lily bulbs were then sampled. Ten bulblets were grouped and mixed for sampling (Tables 2 and 3) in each treatment group. The samples were stored in liquid nitrogen during sampling. After sampling, the samples were stored at -80°C for later use.

[0042] (2) The extraction steps of sample genomic DNA were referred to the instructions of the EasyPureUniversal Plant Genomic DNA Kit of Beijing Quanshijin Biotechnology Co., Ltd.

[0043] (3) Using the extracted differentiated bulblet DNA as a template, PCR was performed to detect the transformation efficiency of Agrobacterium infection. The results showed that among the ten bulblets infected with EHA105⑤ in group 7 and the ten bulblets infected with EHA105⑥ in group 1, there were 10 plants that were successfully transformed ( Figure 4 ).

[0044] Table 2 Statistics of lily scales and differentiated bulblets treated with GV3101 Agrobacterium

[0045]

[0046] Table 3 Statistics of lily scales and differentiated bulblets treated with EHA105 Agrobacterium

[0047]

[0048] Depend on Figure 4 It can be seen that the best infection system for lily scales is to adjust the OD of the infection solution by mediating the EHA105 strain. 600 =1.0, and the bacteria were shaken at 200 rpm in a 28°C shaker for 15 min or 30 min to complete the infection, and the target gene could be successfully integrated into the lily genome.

[0049] Chinese invention patent publication number CN 111197055 A discloses an Agrobacterium-mediated, efficient genetic transformation system for lily scales. Compared to that patent, the present invention establishes a stable genetic transformation system for lily scales that does not require tissue culture, using Agrobacterium-mediated infection, shake infection, and improved resuspension solutions. The system systematically explores the effects of different Agrobacterium strains, infection conditions, and infection times, as well as the infection efficiency of paclobutrazol (PBZ) on lily scales (adding PBZ to the resuspension solution can improve infection and transformation efficiency). In contrast, the non-tissue culture immature embryogenesis system and efficient genetic transformation method for lily disclosed in Chinese invention patent publication number CN 117016092 A involves sterilizing seeds with potassium permanganate, placing them in a sterile culture dish, wrapping them with sterile moist gauze, and culturing them in a lighted incubator. It can be seen from this that the method of the present invention is different from the above-mentioned patented methods, and it does not constitute a technical suggestion or inspiration for this application. A person skilled in the art cannot obtain the present invention by combining the above-mentioned patent documents without paying creative labor. The present invention is original.

[0050] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the present invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0051] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A stable genetic transformation method that does not rely on tissue culture technology to infect lily scales, characterized in that: The following steps are involved: (1) Infect lily scales by shaking the fungus; (2) scale differentiation into bulblets; (3) PCR detection and identification of the number of successfully infected bulblets to determine the transformation rate.

2. The method for stable genetic transformation of lily scales without relying on tissue culture technology according to claim 1, characterized in that: During the process of shaking infection of lily scales, the modified MMA resuspension was prepared as follows: 4.43 g / L MS, 20 g / L sucrose, 1.95 g / L MES, 10 mM MgCl2, 100 μM acetosyringone and 10 μM paclobutrazol, and adjusted to pH = 5.

6.

3. The method for stable genetic transformation of lily scales without relying on tissue culture technology according to claim 2, characterized in that: The best infection system for lily scales is to adjust the OD of the infection solution by mediating the EHA105 strain. 600 =1.0, and the bacteria were shaken at 200 rpm in a 28°C shaker for 15 min or 30 min to complete the infection, and the target gene could be successfully integrated into the lily genome.

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