Method for carrying out genetic transformation on transgenic rice T0 generation by anther culture and ultra-low temperature preservation technology
By using anther culture and cryopreservation technology, the problems of low anther culture efficiency and material waste in transgenic rice T0 generation plants have been solved, achieving efficient genetic transformation and haploid plant regeneration, and shortening the breeding cycle.
Patent Information
- Application Number
- CN202511579544.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-31
- Publication Date
- 2026-02-06
AI Technical Summary
In the anther culture of transgenic rice T0 generation plants, the number of homozygous positive plants is limited, the anther culture efficiency is greatly affected by genotype, and the amount of callus tissue is difficult to guarantee, which makes polyploid transformation difficult and the traditional method has a long breeding cycle.
Anther culture combined with cryopreservation technology was used, including cryotreatment, callus induction, subculture, cryopreservation and thawing recovery. Genetic transformation was carried out using Agrobacterium infection, and haploid plants were obtained through resistance screening and differentiation culture.
It shortens the research cycle, improves material utilization and transformation success rate, avoids material waste, is easy to operate, and is suitable for the preservation and regeneration of plants with specific functional gene positivity.
Smart Images
Figure CN121472319A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of anther culture and crop genetic breeding technology, and in particular to a method for genetic transformation of transgenic rice T0 generation using anther culture and cryopreservation technology. Background Technology
[0002] Rice is one of the world's most important food crops. Transgenic technology provides an efficient means of genetic improvement in rice, allowing for the targeted improvement of agronomic traits (such as disease resistance, insect resistance, stress resistance, and quality) by introducing exogenous genes. With the rapid development of biotechnology, using CRISPR gene editing technology for gene function research has become a trend. For example, intervening in key genes involved in mitosis to create polyploid plants is a current research hotspot in crop genetics and breeding. In transgenic rice research, the T0 generation plants are the initial generation after the integration of exogenous genes. Conventional transgenic rice offspring selection requires self-pollination to obtain genetically stable homozygous lines. However, taking the creation of polyploids as an example, by editing specific genes, non-disjunction of chromosomes in plant cells is induced, producing polyploid plants with doubled chromosomes. This raises a problem: further functional verification of transgenic plants is difficult due to the challenges posed by the resulting polyploid transgenic offspring for transformation.
[0003] Anther culture is an effective method for obtaining haploids and homozygous diploids. By culturing anthers in vitro, microspores can be induced to develop into haploid plants, which can then be rapidly converted to homozygous lines through chromosome doubling, significantly shortening the breeding cycle. Therefore, selecting anthers from T0 generation transgenic rice plants for callus induction and using them as genetic transformation recipients is a means to solve the dilemmas in gene function research. However, the number of homozygous positive T0 generation plants is usually limited, anther culture efficiency is greatly affected by factors such as genotype, and problems such as albino seedlings have always plagued the efficiency of anther culture, and the amount of callus tissue is difficult to guarantee for multiple transformations. Therefore, it is necessary to provide a method for anther culture and genetic transformation of T0 generation transgenic rice. Summary of the Invention
[0004] In view of this, the present invention provides a method for genetic transformation of transgenic rice T0 generation using anther culture and cryopreservation technology to solve the above problems.
[0005] To achieve the above-mentioned objectives, this invention provides the following technical solution: a method for genetic transformation of transgenic rice T0 generation using anther culture and cryopreservation technology, comprising the following steps: S1. Selection of explants: T0 generation transgenic rice plants were cultured to the booting stage, and after cold treatment, rice panicles at the edge of the uninucleate stage were harvested. S2 induction culture: After disinfecting the harvested rice panicles, the glumes were cut off, and the anthers were inoculated in M8 medium and cultured in the dark at 24~26℃ for 3~4 weeks to obtain callus tissue; S3. Subculture: Transfer the callus tissue to the subculture medium and subculture 2-3 times; S4. Cryopreservation: Inoculate the callus tissue into a high-glucose culture medium for 7-8 days, collect the callus tissue, dry it, and then aliquot it into cryovials. Place the cryovials into liquid nitrogen for preservation. S5. Thawing and recovery culture: Remove the cryovials from liquid nitrogen, thaw them in a water bath, and then inoculate the thawed callus tissue into the recovery culture medium; S6. Genetic transformation and screening: Callus tissue was infected with Agrobacterium and co-cultured for 45-50 h after infection; After co-culture, the callus tissue was screened for resistance and then cultured in the dark at 24-26℃ for 3-4 weeks to obtain resistant callus tissue. S7. Differentiation of resistant callus and plant regeneration: The resistant callus was transferred to a differentiation medium and cultured under light. Rice seedlings differentiated after 3-4 weeks of culture.
[0006] By adopting the above technical solution, the present invention has the following beneficial effects: (1) Shorten the research cycle: This invention uses T0 plants for anther culture, which shortens the breeding and generation cycle compared with the traditional method and accelerates the research process; (2) Improve material utilization: This invention utilizes ultra-low temperature preservation technology to achieve long-term preservation of transgenic T0 generation rice callus materials, which can be thawed and cultured as needed, avoiding material waste, and is especially suitable for specific functional gene positive plants; (3) Easy to operate and high conversion success rate: This invention uses direct freezing for ultra-low temperature preservation, avoiding complicated operation processes, and can preserve large quantities of callus materials. Through extreme low temperature, the activity of transgenic flower culture callus is screened, which improves the differentiation rate of callus tissue into seedlings in the later stage of screening. Attached Figure Description
[0007] Figure 1 This is a diagram of spikelets in the uninucleate marginal stage.
[0008] Figure 2 Images of newly formed callus after induced culture.
[0009] Figure 3 Diagram of anther callus tissue for restoration culture.
[0010] Figure 4 These are plants that grow from the differentiation and culture of callus tissue from resistant anthers. Detailed Implementation
[0011] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0012] Example 1 Rice variety: Chunyou 84 Step 1: Induction of callus from anthers of transgenic rice T0 generation plants Transgenic T0 rice plants that have been sequenced and validated are planted and grown to the booting stage (approximately 7-10 days before heading). The main panicle is selected and placed in a 10°C freezer for 5 days before harvesting the spikelets. If the number of plants is small, panicles can be harvested in batches. Spikelets at the edge of the uninucleate stage are selected (see...). Figure 1 After disinfecting with 75% alcohol for 1 min and 1% sodium hypochlorite for 10 min, the glumes were cut off and the anthers were inoculated in M8 medium and cultured in the dark at 26°C for 4 weeks. New callus tissue was observed after induction culture (see...). Figure 2 Due to the effects of target gene mutations on plants, the induction rate of transgenic T0 generations varies considerably. Knockout of chromosome-related genes has a certain impact on the seed setting rate of plants. Figure 2 It can be seen that although the number of induced calluses was limited, callus tissue was still successfully induced.
[0013] Step 2: Subculturing and cryopreservation of anther callus (1) After obtaining primary callus, the callus tissue should be transferred to subculture in a timely manner. Use N6 subculture medium (N6 medium + 2,4-D 2 mg / L + sucrose 30 g / L + agar 7 g / L, pH=6.0) for subculture three times to expand the callus tissue. The callus tissue cannot be expanded indefinitely. As the number of subcultures increases, its activity decreases. Callus cultured from transgenic anthers cannot be subcultured for a long time.
[0014] (2) The flower callus was inoculated into N6 high sugar medium (N6 medium + 2,4-D 2 mg / L + maltose 100 g / L + sorbitol 30 g / L + agar 7 g / L, pH = 6.0) for 7 days. The callus was collected into a large petri dish with filter paper, dried at 19℃ for 12 h, and then dispensed into 5 mL cryovials and stored in liquid nitrogen.
[0015] Step 3: Thawing and regeneration culture of callus preserved at ultra-low temperatures Remove the cryovials from liquid nitrogen and quickly place them in a 40°C water bath. Shake rapidly for 2 minutes. Inoculate the thawed callus onto recovery medium (N6 + 2,4-D 2 mg / L + KT 3 mg / L + sorbitol 30 g / L + maltose 30 g / L + agar 7 g / L, pH = 6.0). Callus regrowth can be observed. Figure 3The callus recovery rate was statistically analyzed (approximately 34%), and the callus was subcultured twice after recovery to obtain highly active callus for genetic transformation.
[0016] Step 4: Genetic transformation and screening of anther callus tissue (1) Agrobacterium-mediated transformation: Following the conventional rice genetic transformation process, Agrobacterium needs to avoid the resistance of the original transgene. The T0 generation of Chunyou 84 is Hpt resistant, so it is necessary to select an Agrobacterium vector carrying resistance other than Hpt. In this example, the Bar resistant Agrobacterium EHA105 strain was used and co-cultured for 2 days after infecting the callus.
[0017] (2) Screening culture: The co-cultured callus was washed three times with sterile water containing 300 mg / L carbenicillin, dried, and then inoculated into screening medium (N6+2,4-D 2 mg / L+Bar 30 mg / L+CB 500 mg / L+sucrose 30 g / L+agar 7 g / L pH 5.8) and cultured in the dark at 25℃ for 4 weeks, during which time it was transferred to screening medium once to obtain resistant callus.
[0018] Step 5: Differentiation of resistant callus and plant regeneration The resistant callus was transferred to differentiation medium (MS medium + 6-BA 2 mg / L + KT 2 mg / L + NAA 0.2 mg / L + IAA 0.2 mg / L + maltose 30 g / L + plant gel 4 g / L, pH = 6.0), and then transferred to a light culture chamber (16 h / d light intensity 2500 lx). After 3 weeks of culture, green seedlings differentiated. Figure 4 The regeneration rate is approximately 15%.
[0019] As can be seen from the above embodiments, the present invention provides a method for genetic transformation of transgenic rice T0 generation using anther culture and cryopreservation technology. The present invention improves the callus induction rate, callus recovery rate and plant regeneration rate of transgenic T0 generation rice.
[0020] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for genetic transformation of transgenic rice generation T0 using anther culture and cryopreservation technology, characterized in that, Includes the following steps: S1. Selection of explants: T0 generation transgenic rice plants were cultured to the booting stage, and after cold treatment, rice panicles at the edge of the uninucleate stage were harvested. S2 induction culture: After disinfecting the harvested rice panicles, the glumes were cut off, and the anthers were inoculated in M8 medium and cultured in the dark at 24~26℃ for 3~4 weeks to obtain callus tissue; S3. Subculture: Transfer the callus tissue to the subculture medium and subculture 2-3 times; S4. Cryopreservation: Inoculate the callus tissue into a high-glucose culture medium for 7-8 days, collect the callus tissue, dry it, and then aliquot it into cryovials. Place the cryovials into liquid nitrogen for preservation. S5. Thawing and recovery culture: Remove the cryovials from liquid nitrogen, thaw them in a water bath, and then inoculate the thawed callus tissue into the recovery culture medium; S6. Genetic transformation and screening: Callus tissue was infected with Agrobacterium and co-cultured for 45-50 h after infection; After co-culture, the callus tissue was screened for resistance and then cultured in the dark at 24-26℃ for 3-4 weeks to obtain resistant callus tissue. S7. Differentiation of resistant callus and plant regeneration: The resistant callus was transferred to a differentiation medium and cultured under light. Rice seedlings differentiated after 3-4 weeks of culture.
2. The method according to claim 1, characterized in that, The disinfection method described in S2 is as follows: disinfect with 75% alcohol for 60 seconds, and then disinfect with 1.5% sodium hypochlorite solution for 15-20 minutes.
3. The method according to claim 1, characterized in that, The subculture medium described in S3 is N6+2,4-D 2 mg / L + sucrose 30 g / L + agar 7 g / L, pH=6.
0.
4. The method according to claim 1, characterized in that, The high-glucose culture medium described in S4 is: N6 + 2,4-D 2 mg / L + maltose 100 g / L + sorbitol 30 g / L + agar 7 g / L, pH = 6.
0.
5. The method according to claim 1, characterized in that, The drying temperature described in S4 is 19°C, and the drying time is 12 hours.
6. The method according to claim 1, characterized in that, The recovery medium described in S5 is N6+2,4-D 2 mg / L+KT 3 mg / L+sorbitol 30 g / L+maltose 30 g / L+agar 7 g / L, pH=6.
0.
7. The method according to claim 1, characterized in that, The temperature of the water bath in S5 is 40°C, and the water bath time is 2 minutes; the Agrobacterium in S6 is Agrobacterium that does not carry the original transgenic resistance.
8. The method according to claim 1, characterized in that, The resistance screening described in S6 is as follows: wash the co-cultured callus tissue 3-5 times with sterile water containing 300 mg / L carbenicillin.
9. The method according to claim 1, characterized in that, The photoculture in S7 has a photoperiod of 16 h / d and a photointensity of 2400~2600 lx.
10. The method according to claim 1, characterized in that, The incubation period described in S1 is 7 to 10 days before heading.