阅读说明：本技术 一种菰米遗传转化体系建立的方法 (Method for establishing wild rice genetic transformation system ) 是由 闫宁 吴丹丹 李亚丽 张晶 于秀婷 纪春 杨婷 郑凯丽 张宇 许敏敏 杜咏梅 于 2021-09-10 设计创作，主要内容包括：本发明属于植物基因工程领域。受限于菰米胚性愈伤的培养以及再生效率的低下,菰米的遗传转化体系迟迟未能建立。本发明一方面通过优化菰米胚性愈伤的培养条件和培养方式以及优化愈伤再生的培养基,极大程度地提高菰米愈伤的再生效率,为农杆菌侵染提供优质的胚性愈伤受体材料,为转化奠定坚实的基础；另一方面通过实验探究优化转化过程中的处理方式和培养条件,得到转化阳性株系,顺利建立稳定的农杆菌介导的菰米遗传转化体系。(The invention belongs to the field of plant genetic engineering. Limited by the low culture and regeneration efficiency of the rice embryogenic callus, the genetic transformation system of the rice cannot be established in late stage. On one hand, the culture conditions and the culture mode of the wild rice embryonic callus and the culture medium for callus regeneration are optimized, so that the regeneration efficiency of the wild rice callus is greatly improved, a high-quality embryonic callus receptor material is provided for agrobacterium infection, and a solid foundation is laid for transformation; on the other hand, a treatment mode and culture conditions in the optimized transformation process are explored through experiments to obtain a transformation positive strain, and a stable agrobacterium-mediated wild rice genetic transformation system is smoothly established.)

1. A method for establishing a wild rice genetic transformation system is characterized by comprising the following steps:

1) and (3) induction culture: performing callus induction culture on mature wild rice seeds on an induction culture medium, performing subculture on the callus induction culture medium, and selecting callus with good state and transferring the callus to the callus induction culture medium for pre-culture;

2) preparation of an agrobacterium liquid: inoculating agrobacterium to an agrobacterium activation culture medium for activation, and suspending and diluting the agrobacterium by using a heavy suspension to obtain an agrobacterium liquid with an OD660 value of 0.2-0.4;

3) infection of agrobacterium liquid: soaking the pre-cultured callus in the step 1) in the agrobacterium liquid obtained in the step 2) for infection for 15-25 min;

4) and (3) recovery culture and screening of callus: co-culturing the infected callus particles in the step 3), wherein the co-culturing conditions are as follows: filling three aseptic filter papers on a disposable culture dish, adding acetosyringone liquid culture medium with final concentration of 15-25mg/L, sucking agrobacterium liquid on the surface of the callus by the aseptic filter papers, uniformly dispersing the agrobacterium liquid on a co-culture medium, and culturing for 2-4 days in dark at 20-28 ℃; after the co-culture is finished, uniformly and sparsely spreading the co-cultured callus in a recovery culture medium, and culturing for 3-6 days at the temperature of 25-35 ℃ under illumination with the illumination period of 16h illumination/8 h darkness; after the recovery culture is finished, transferring the callus onto a screening culture medium, wherein the culture conditions are as follows: culturing at 25-35 deg.C under illumination with illumination period of 16h illumination/8 h darkness;

5) regeneration of callus tissue: screening the callus particles obtained in the step 4) on a culture medium for 3-4 weeks, transferring to a regeneration culture medium, and culturing under illumination at 25-35 ℃ with illumination cycle of 16h illumination/8 h darkness; the seedling grows to 2-5cm in length, is transferred to a rooting culture medium, is cultured under illumination at 25-35 ℃, and the illumination period is 16h illumination/8 h darkness.

2. The method for establishing a wild rice genetic transformation system according to claim 1, wherein the method comprises the following steps: the callus induction culture conditions in the step 1) are as follows: culturing with light at 4000Lux intensity and 16h light/8 h dark at 25-35 deg.C for 3 weeks to obtain embryogenic callus.

3. The method for establishing a wild rice genetic transformation system according to claim 1, wherein the method comprises the following steps: the OD660 value of the agrobacterium liquid during infection is 0.3, and the infection time of the callus particles is 20 min.

4. The method for establishing a wild rice genetic transformation system according to claim 3, wherein the method comprises the following steps: the final concentration of acetosyringone in the co-culture medium is 20mg/L, and the co-culture medium is cultured in the dark at 23 ℃ for 3 days.

5. The method for establishing a wild rice genetic transformation system according to claim 3, wherein the method comprises the following steps: the conditions of recovery culture, screening culture, regeneration culture and rooting culture are as follows: culturing at 30 deg.C under light with a light cycle of 16h light/8 h dark.

6. The method for establishing a wild rice genetic transformation system according to claim 1, wherein the method comprises the following steps: the formula of the callus induction culture medium is as follows: the culture medium contains N6 in a large amount of 20X50mL per liter; trace B5 1, 100X10 mL; trace B5 2, 1000X1 mL; vitamin B5, 100X10 mL; iron salt 100X10 mL; 0.5g of glutamine; proline 0.5 g; 0.3g of hydrolyzed casein; 2,4-D, 1mg/mL, 2 mL; 30g of cane sugar; 3g of plant gel.

7. The method for establishing a wild rice genetic transformation system according to claim 1, wherein the method comprises the following steps: the formula of the agrobacterium activating culture medium is as follows: the culture medium contains yeast extract 10g per liter; 10g of peptone; 50g of NaCl; kanamycin, 50mg/mL, 1 mL; rifampicin, 10mg/mL, 1.5 mL; acetosyringone, 20mg/mL, 1 mL; agar 12 g.

8. The method for establishing a wild rice genetic transformation system according to claim 1, wherein the method comprises the following steps: the formula of the recovery culture medium is as follows: the culture medium contains N6 in a large amount of 20X50mL per liter; trace B5 1, 100X10 mL; trace B5 2, 1000X1 mL; b5 vitamin 100X10 mL; iron salt 100X10 mL; 0.5g of glutamine; proline 0.5 g; 0.3g of hydrolyzed casein; 2,4-D, 1mg/mL, 2 mL; carbenicillin, 250mg/mL, 1 mL; 30g of cane sugar; 3g of plant gel.

9. The method for establishing a wild rice genetic transformation system according to claim 1, wherein the method comprises the following steps: the formula of the screening culture medium is as follows: the culture medium contains N6 in a large amount of 20X50mL per liter; trace B5 1, 100X10 mL; trace B5 2, 1000X1 mL; b5 vitamin 100X10 mL; iron salt 100X10 mL; 0.5g of glutamine; proline 0.5 g; 0.3g of hydrolyzed casein; 30g of cane sugar; 2,4-D, 1mg/mL, 2 mL; carbenicillin, 250mg/mL, 1 mL; Hyg-B, 50mg/mL, 1.4 mL; 3g of plant gel.

10. The method for establishing a wild rice genetic transformation system according to claim 1, wherein the method comprises the following steps: the formula of the regeneration medium is as follows: the culture medium contains N6 in a large amount of 20X50mL per liter; trace B5 1, 100X10 mL; trace B5 2, 1000X1 mL; b5 vitamin 100X10 mL; iron salt 100X10 mL; CuSO₄5mg/mL, 0.5 mL; 0.5g of MES; hydrolyzing 1g of casein; 30g of cane sugar; 30g of sorbitol; carbenicillin, 250mg/mL, 0.5 mL; Hyg-B, 50mg/mL, 0.4 mL; NAA, 1mg/mL, 0.5 mL; 6-BA, 1mg/mL, 0.5 mL; TDZ, 1mg/mL, 1 mL; amino acid 10X100 mL; 2.8g of plant gel.

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to a method for establishing a wild rice genetic transformation system.

Background

Chinese wild rice (Zizania latifolia) originated in China and is distributed in Japan, Korea, India and southeast Asia, Central and south China peninsula. Except for Tibet, there are wild rice distributions around rivers, lakes, ditches, ponds and paddy fields in China, especially in Yangtze river watershed. Wild rice (wild rice) is a caryopsis of wild rice in China, has been eaten as grain for over 3000 years in China, and is one of important 'six cereals' (rice, millet, sorghum, wheat and wild rice) for emperor to eat in ancient times. After the Tang dynasty, with the great increase of population, the increase of various farming activities such as lake-surrounding field building and the like, the popularization of rice planting technology, and the difficulty in harvesting and shelling the wild rice, the wild rice gradually fades out of sight of people. Subsequently, zizania rice is gradually used as a traditional Chinese medicinal material, and the records of the use of zizania rice in the treatment of diabetes (diabetes) and gastrointestinal diseases such as the Ming dynasty Li Shizhen Ben Cao gang mu and the like are recorded. Due to high nutritive value and unique flavor, the wild rice is widely appeared in dining tables and shops and is popular with more and more people.

Currently, the biological activity and health care effect of the wild rice in China are widely concerned by east Asian scientists such as China and Korea. The biological activity and health care value of the wild rice are proved to comprise antioxidant activity, improvement of insulin resistance, improvement of lipid toxicity, prevention of cardiovascular diseases and the like. The research on the nutritional ingredients of the zizania plants mainly focuses on the seeds of the zizania rice, and the zizania rice not only contains rich nutritional ingredients such as protein, amino acid, vitamin and mineral substances, but also contains a large amount of bioactive substances such as resistant starch, dietary fiber, phytosterol, anthocyanin and procyanidine. The zizania esculenta rice has smaller length and diameter compared to zizania esculenta rice. Compared with rice, the wild rice is a high-protein and low-fat health food.

The wild rice itself has some limitations, for example, the wild rice contains the shattering gene, which causes the mature seeds to be easy to shatter, and is not suitable for large-scale production and application. Therefore, the function research of the zizania latifolia related genes is particularly important, and the technologies and researches related to the zizania latifolia gene engineering are in urgent need of breakthrough. However, the low efficiency of culture and regeneration of the embryogenic callus of the wild rice has been limited, and the genetic transformation system of the wild rice has not been established in time.

Disclosure of Invention

Aiming at the problems that the wild rice genetic transformation system mediated by agrobacterium at present is not established yet, the invention provides a method for establishing the wild rice genetic transformation system. According to the invention, on one hand, the culture condition and the culture mode of the wild rice embryonic callus are optimized, and the culture medium for callus regeneration is optimized, so that the regeneration efficiency of the wild rice callus is greatly improved, a high-quality embryonic callus receptor material is provided for agrobacterium infection, and a solid foundation is laid for transformation; on the other hand, a treatment mode and culture conditions in the optimized transformation process are explored through experiments to obtain a transformation positive strain, and a stable agrobacterium-mediated wild rice genetic transformation system is smoothly established.

The invention is realized in such a way that the method for establishing the wild rice genetic transformation system comprises the following steps:

1) and (3) induction culture: performing callus induction culture on mature wild rice seeds on an induction culture medium, performing subculture on the callus induction culture medium, and selecting callus with good state and transferring the callus to the callus induction culture medium for pre-culture;

2) preparation of an agrobacterium liquid: inoculating agrobacterium to an agrobacterium activation culture medium for activation, and suspending and diluting the agrobacterium by using a heavy suspension to obtain an agrobacterium liquid with an OD660 value of 0.2-0.4;

3) infection of agrobacterium liquid: soaking the pre-cultured callus in the step 1) in the agrobacterium liquid obtained in the step 2) for infection for 15-25 min;

4) and (3) recovery culture and screening of callus: co-culturing the infected callus particles in the step 3), wherein the co-culturing conditions are as follows: filling three pieces of sterile filter paper on a disposable culture dish, adding the agrobacterium heavy suspension of acetosyringone with the final concentration of 15-25mg/L, sucking the agrobacterium liquid on the surface of the callus by using the sterile filter paper, uniformly dispersing the agrobacterium liquid on a co-culture medium, and culturing for 2-4 days in the dark at the temperature of 20-28 ℃; after the co-culture is finished, uniformly and sparsely spreading the co-cultured callus in a recovery culture medium, and culturing for 3-6 days at the temperature of 25-35 ℃ under illumination with the illumination period of 16h illumination/8 h darkness; after the recovery culture is finished, transferring the callus onto a screening culture medium, wherein the culture conditions are as follows: culturing at 25-35 deg.C under illumination with illumination period of 16h illumination/8 h darkness;

5) regeneration of callus tissue: screening the callus particles obtained in the step 4) on a culture medium for 3-4 weeks, transferring to a regeneration culture medium, and culturing under illumination at 25-35 ℃ with illumination cycle of 16h illumination/8 h darkness; the seedling grows to 2-5cm in length, is transferred to a rooting culture medium, is cultured under illumination at 25-35 ℃, and the illumination period is 16h illumination/8 h darkness.

The conditions for callus induction culture in the step 1) are preferably as follows: culturing with light at 4000Lux intensity and 16h light/8 h dark at 25-35 deg.C for 3 weeks to obtain embryogenic callus.

The final concentration of 2,4-D in the induction medium was 2 mg/L.

The agrobacterium species is preferably EHA 105; preferably, the first activation is carried out by dark culture at 28 ℃ for 24h, then the activated Agrobacterium is inoculated on a fresh Agrobacterium activation medium, and the second activation is carried out by dark culture at 28 ℃ overnight. Preferably, 1mL of acetosyringone stock solution with a final concentration of 20mg/mL is added to each liter of the inoculation culture medium, and the mixture is subpackaged in 50mL of sterilized centrifuge tubes, and 25mL of each tube is used as a heavy suspension.

The optimal OD660 value of the agrobacterium liquid during infection is 0.3, and the optimal infection time of the callus particles is 20 min. The concentration of the bacterial liquid with OD660 of 0.3 is suitable for screening subsequent transformants, the resistant callus rate is higher, and browning is reduced. The bacteria liquid with too high or too low concentration is not beneficial to the subsequent screening of transformants, the transformation efficiency is reduced due to too low concentration, and the overgrowth of agrobacterium is caused due to too high concentration, so that the callus browning is caused.

The co-cultured medium contains acetosyringone with a final concentration of 20mg/L, and is co-cultured in the dark at 23 ℃ for 3 d. The drying treatment in the co-culture process can reduce the water content in the co-culture process, and has the effects of increasing cell contact and inhibiting bacterial growth, but cannot be too dry, and can inhibit the growth of callus.

The conditions for resuming the culture are preferably: culturing at 30 deg.C for 5 days with illumination cycle of 16h light/8 h dark.

The conditions for the selection culture are preferably: culturing at 30 deg.C under light with a light cycle of 16h light/8 h dark.

The conditions for the regeneration culture are preferably: culturing at 30 deg.C under light with a light cycle of 16h light/8 h dark.

The conditions for rooting culture are preferably as follows: culturing at 30 deg.C under light with a light cycle of 16h light/8 h dark.

The formula of the callus induction culture medium is as follows: a large amount of 20X50mL per liter of medium containing N6; b5 minor 1, 100X10 mL; b5 minor 2, 1000X1 mL; vitamin B5, 100X10 mL; iron salt 100X10 mL; 0.5g of glutamine; proline 0.5 g; 0.3g of hydrolyzed casein; the final concentration of 2,4-D is 1mg/mL and 2 mL; 30g of cane sugar; 3g of plant gel.

YP, the formula of the acetosyringone agrobacterium activation medium is as follows: 10g of yeast extract; 10g of peptone; 50g of NaCl; kanamycin has a final concentration of 50mg/mL and 1 mL; the final concentration of rifampicin is 10mg/mL and 1.5 mL; the final concentration of acetosyringone is 20mg/mL and 1 mL; agar 12 g.

The formulation of the agrobacterium tumefaciens resuspension is as follows: a large amount of 20X50mL per liter of N6; b5 minor 1, 100X10 mL; b5 minor 2, 1000X1 mL; vitamin B5, 100X10 mL; iron salt 100X10 mL; proline 0.5 g; 0.5g of hydrolyzed casein; 2,4-D, 1mg/mL 2 mL; 20g of cane sugar; 10g of glucose; the final concentration of acetosyringone is 20mg/mL, 1 mL.

The formula of the recovery culture medium is as follows: a large amount of 20X50mL per liter of medium containing N6; b5 minor 1, 100X10 mL; b5 minor 2, 1000X1 mL; vitamin B5, 100X10 mL; iron salt 100X10 mL; 0.5g of glutamine; proline 0.5 g; 0.3g of hydrolyzed casein; the final concentration of 2,4-D is 1mg/mL and 2 mL; the final concentration of carbenicillin is 250mg/mL and 1 mL; 30g of cane sugar; 3g of plant gel.

The formula of the screening culture medium is as follows: a large amount of 20X50mL per liter of medium containing N6; b5 minor 1, 100X10 mL; b5 minor 2, 1000X1 mL; vitamin B5, 100X10 mL; iron salt 100X10 mL; 0.5g of glutamine; proline 0.5 g; 0.3g of hydrolyzed casein; 30g of cane sugar; the final concentration of 2,4-D is 1mg/mL and 2 mL; the final concentration of carbenicillin is 250mg/mL and 1 mL; the final concentration of Hyg-B is 50mg/mL, 1.4 mL; 3g of plant gel.

The formula of the differentiation medium is as follows: a large amount of 20X50mL per liter of medium containing N6; b5 minor 1, 100X10 mL; b5 minor 2, 1000X1 mL; vitamin B5, 100X10 mL; iron salt 100X10 mL; CuSO₄5mg/mL, 0.5 mL; 0.5g of MES; hydrolyzing 1g of casein; 30g of cane sugar; 30g of sorbitol; the final concentration of carbenicillin is 250mg/mL and 0.5 mL; the final concentration of Hyg-B is 50mg/mL, 0.4 mL; the final concentration of NAA is 1mg/mL, 0.5 mL; the final concentration of 6-BA is 1mg/mL, 0.5 mL; TDZ final concentration is 1mg/mL, 1 mL; amino acid 10X100 mL; 2.8g of plant gel.

The formula of the rooting culture medium is as follows: each liter of culture medium contains 2.165g of MS salt; vitamin B5, 100X10 mL; 20g of cane sugar; the final concentration of carbenicillin is 250mg/mL and 0.5 mL; the final concentration of NAA is 1mg/mL, 0.4 mL; 3.5g of plant gel.

Taking the extracted and transformed plant leaf DNA sample as a template, taking the screening marker HPT gene as a primer to carry out PCR amplification, running agarose gel electrophoresis on a PCR product to determine a transformed positive plant, and sequencing and comparing the product.

The invention has the following beneficial effects:

the structure and the state of the callus are crucial to the transformation efficiency, the callus growth condition is reasonably controlled, 16h light/8 h dark culture is favorable for formation of vascular tissues, and the vascular tissues are favorable for conduction of nutrients, so that the callus state is fuller, and in addition, 1-2 times of subculture in the callus culture process is favorable for growth and multiplication of the callus; the invention optimizes the callus regeneration culture medium, improves the regeneration efficiency of the callus and lays a solid foundation for transformation; the quantity and the state of the agrobacterium are important factors influencing transformation, and the method adopts the bacterial liquid with OD660 of 0.3, so that the transformation is favorably carried out; three pieces of sterile filter paper are laid on a disposable culture dish, and then an agrobacterium tumefaciens heavy suspension culture medium containing acetosyringone is added to co-culture infected callus particles, so that the transformation frequency is effectively improved; the explant used in the invention is convenient to obtain materials, and the stored mature seeds can be used at any time and are not limited by seasons.

Drawings

FIG. 1 is a diagram showing the callus state at different 2,4-D concentrations in the examples;

FIG. 2 is a diagram of the recovery of the wild rice callus in the examples;

FIG. 3 is a graph of callus proliferation of wild rice in the examples;

FIG. 4 is a diagram of the differentiation and shoot growth of the wild rice callus of the examples;

FIG. 5 is a diagram showing the growth state of the callus differentiation buds of the wild rice callus particles under different hormone combinations in the example;

FIG. 6 is a diagram of the effect of rooting and strengthening seedlings of black rice in the examples;

FIG. 7 is a gel electrophoresis diagram of PCR products of the black rice regenerated plant of the example, M-D2000 Marker; 1-a plasmid; 2-12-regenerating the single plant after transformation; 13-ddH₂O；

FIG. 8 is the alignment analysis of the sequencing results of the wild rice PCR products in the examples.

Detailed Description

The present invention will be described in further detail with reference to the following embodiments and the accompanying drawings. The equipment and reagents used in the examples and test examples were commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.

For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.

The normal temperature in the following embodiments of the present invention refers to a natural room temperature condition in four seasons, and is not subjected to additional cooling or heating treatment, and is generally controlled at a normal temperature of 10-30 ℃, preferably 15-25 ℃.

The invention discloses a method for establishing a wild rice genetic transformation system and application thereof, wherein mature wild rice seeds are selected as a tissue culture explant, 2,4-D NBCIM induction culture medium with the concentration of 2mg/L is selected to induce callus, the callus is subjected to subculture proliferation through the subculture medium, and the callus is pre-cultured, and the callus with good state and vigorous division is taken as a receptor to be subjected to genetic transformation by adopting an agrobacterium-mediated method to obtain a transgenic plant. The details are shown in the following examples.

Examples

1. Callus induction and proliferation

Selecting clean and mildew-free mature wild rice seeds, firstly disinfecting the seeds for 90s by using 75% ethanol, then disinfecting the seeds for 15min by using 0.1% mercury solution, placing the seeds in a constant-temperature shaking incubator during the disinfection, shaking and culturing the seeds at 30 ℃ and 180r/min, and after the disinfection treatment is finished, washing the seeds for more than 10 times by using sterile water until the seeds are clarified and soaked overnight;

peeling off the embryo of the disinfected seed by using a scalpel, inoculating the embryo on an inducing culture medium NBCIM (N-bromosuccinimide) with the concentration of 2mg/L, 4-D and the pH value of 5.8 under the culture condition of the temperature (28 +/-2 ℃), the illumination intensity of 1500-2000 Lux, the photoperiod of 16h illumination/8 h darkness and the callus can be obtained after 40D;

separating the obtained callus from embryo, removing redundant endosperm, subculturing on fresh callus induction culture medium, and culturing under the following conditions: the temperature is 30 ℃, and the illumination period is 16h of illumination/8 h of darkness.

Selecting the callus with good state, transferring the callus to an NBCIM culture medium for pre-culture for 3-5 days, collecting the small particles with good state and vigorous division into a 50mL sterile centrifuge tube by using a small spoon for agrobacterium infection, and co-culturing two dishes for each 10mL callus.

TABLE 12 influence of 4-D concentration on callus development rate and callus status

2. Genetic transformation of explants

Inoculating Agrobacterium EHA105 with plant expression vector PHUN411-sg2.0-BADH2 on YP acetosyringone culture medium, culturing in dark at 28 deg.C for 24 hr, inoculating on fresh YP acetosyringone culture medium, and culturing in dark at 28 deg.C overnight; scraping off the agrobacterium by using an inoculating loop, adding a resuspension solution (adding 1mL of acetosyringone storage solution (20 mg/mL)) into each liter of inoculation culture medium, subpackaging the suspension into 50mL of sterilized centrifuge tubes, taking 25mL of each tube as the resuspension solution to resuspend the agrobacterium, wherein OD660 values are 0.1, 0.3 and 0.5 respectively, and infecting the callus treated in the step 1 for 20min while gently shaking occasionally.

Pouring out the heavy suspension, placing on sterile filter paper until no excess bacteria liquid exists on the surface of the callus, and then carrying out co-culture. Setting three co-culture conditions, namely a common culture medium; secondly, three pieces of sterile filter paper are placed on a disposable culture dish pad with the thickness of 90 multiplied by 25 mm; thirdly, adding 2.5mL of inoculation culture medium (containing acetosyringone with the final concentration of 20 mg/L) after three sterile filter papers are placed on a disposable culture dish pad with the diameter of 90 multiplied by 25 mm; the infected callus with OD value of 0.3 is evenly dispersed on three different co-culture media after being sucked dry, and the dispersion degree of agrobacterium is observed after dark culture at 23 ℃ for 3d, the result shows that the drying treatment in the co-culture process can reduce the moisture content in the co-culture process, and the effects of increasing cell contact and inhibiting the growth of bacteria are achieved, but the drying treatment cannot be too much to inhibit the growth of callus, so the filter paper wetted by adding acetosyringone is most suitable for co-culture of infected callus particles; then uniformly spreading the co-cultured callus in a recovery culture medium by a small spoon (each dish is divided into at least two dishes in the recovery culture medium), wherein the culture conditions are temperature (28 +/-2 ℃), the illumination intensity is 1500-2000 Lux, the photoperiod is 16h illumination/8 h darkness, after 5d, transferring the callus to a screening culture medium (the callus which grows and drops from the original callus is regarded as an independent transformant), each culture dish is inoculated with 25 calli, after one round of screening, the calli with good states are selected according to the conditions, two or three rounds of screening are continued, each screening period is 14d-20d, the culture conditions are 30 ℃ illumination culture, the photoperiod is 16h illumination/8 h darkness, after 14d, the resistant callus rate is counted, and the result shows that the bacteria liquid with over-high or over-low concentration is not beneficial to the subsequent screening of transformants, and the low concentration can cause the reduction of transformation efficiency, and the concentration of the bacillus subtilis is too high, the agrobacterium can grow excessively to cause callus browning, so that the concentration of the bacterium liquid with the OD660 of 0.3 is more suitable for screening subsequent transformants, the callus resistance rate is higher, and the browning is reduced.

TABLE 2 Effect of different coculture conditions on transformation

TABLE 3 influence of the bacterial liquid concentration on the screening of transformants

3. Regeneration and transplantation of transgenic plants

After screening for 3-4 weeks on a culture medium, selecting 3-5 fresh resistant callus small particles with good growth state from each independent transformant, transferring to a regeneration culture medium (each culture dish is connected with 8 independent transformants), and culturing under the conditions of 30 ℃ illumination and 16h illumination/8 h darkness in a photoperiod; until the length of the seedling grows to be about 2-5cm, only one well-grown seedling is taken from each independent transformant and transferred to a rooting medium (callus from which the seedling has been taken is discarded and marked at the bottom of the culture dish to prevent confusion with callus from other independent sources, which are undifferentiated or not taken). Each root finger vessel is connected with a seedling. Culturing at 30 deg.C under illumination with photoperiod of 16h light/8 h dark.

TABLE 4 Effect of regeneration Medium with different hormone ratios on callus differentiation

4. DNA extraction of transgenic plant, PCR amplification of screening marker gene and product sequencing

And (3) putting a proper amount of wild rice leaves of 11 seedlings obtained by rooting into a 2mL centrifuge tube, adding 1 particle of 5mm grinding steel balls, putting the sample into a high-throughput tissue grinder, and grinding after fixation is completed, wherein the grinding time is set to be 2min, and the rotation speed is 1500 rpm. And taking out the ground sample, adding 800 mu L CTAB extraction liquid, reversing, uniformly mixing, placing in a 65 ℃ water bath kettle, and carrying out warm bath for 45min, wherein the centrifuge tube is gently shaken every 15 min. After cooling, an equal volume of chloroform was added: isoamyl alcohol (24:1), mixing well, laying on shaking table, shaking for 5min (frequency is to fully mix the extract and chloroform), 12000rpm, centrifuging at 20 deg.C for 10 min. And (3) sucking the supernatant into another clean 1.5mL centrifuge tube, adding precooled isopropanol with the same volume as the supernatant, reversing, uniformly mixing, and freezing for 30min in a refrigerator at the temperature of-20 ℃. Centrifuging at 12000rpm at 20 deg.C for 10min, and removing supernatant by pipette. Rinsing and precipitating twice with 75% ethanol, and standing for 5min each time; the ethanol was removed as much as possible by pipette and the tube was placed upside down on absorbent paper and dried until the precipitate appeared translucent. The precipitate was dissolved in 100. mu.L of sterile water. 2 mu.L of DNA was electrophoresed to check the quality of DNA and stored at-20 ℃.

The obtained DNA samples of 11 independent individuals were used as templates, and PCR amplification was performed using primers for screening marker HPT genes, and the amplification system was as follows:

TABLE 5 PCR amplification System

An amplification primer: HPT-F: CGCCGATGGTTTCTACAA (SEQ NO:1)

HPT-R:GGCGTCGGTTTCCACTAT(SEQ NO:2)

Amplification conditions: pre-denaturation at 95 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 56 deg.C for 30s, extension at 72 deg.C for 60s, and final extension at 72 deg.C for 7min under 32-33 circulation.

Target fragment: 839 BP.

The PCR product (SEQ NO:3) was run on an agarose gel for electrophoresis, and the gel image is shown in FIG. 7. As can be seen from the electrophoresis gel chart, the individual 2 was a transformation-positive individual, and the transformation-positive rate was 1/11.

The result of the PCR product sequencing is shown in FIG. 8, and it can be seen that the sequencing result of the PCR product completely matches the sequence of hyg gene.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.

Sequence listing

<110> tobacco institute of Chinese academy of agricultural sciences

HEFEI JIANGU BIOTECHNOLOGY Co.,Ltd.

<120> method for establishing wild rice genetic transformation system

<141> 2021-09-10

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 1

cgccgatggt ttctacaa 18

<210> 2

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 2

ggcgtcggtt tccactat 18

<210> 3

<211> 815

<212> DNA

<213> Artificial Sequence

<400> 3

gggtttcatc gggcactttg catcggccgc gctccgattc cggaagtgct tgacattggg 60

gagtttagcg agagcctgac ctattgcatc tcccgccgtt cacagggtgt cacgttgcaa 120

gacctgcctg aaaccgaact gcccgctgtt ctacaaccgg tcgcggaggc tatggatgcg 180

atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc 240

ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac 300

tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg 360

atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc 420

aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg 480

ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt 540

atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccacga 600

ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc 660

aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc 720

gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt 780

gtagaagtac tcgccgatag tggaaaccga cgcca 815