Agrobacterium tumefaciens-mediated transgenic method of paphiopedilum protocorm

Abstract

The invention discloses an agrobacterium tumefaciens-mediated transgenic method of a paphiopedilum protocorm. The method comprises the following steps: longitudinally cutting or needling a paphiopedilum protocorm, infecting the paphiopedilum protocorm in an agrobacterium solution, inoculating the protocorm into a co-culture medium for culture, then transferring the protocorm into a bacteriostatic culture medium for bacteriostatic treatment, and culturing the protocorm in a screening culture medium containing hygromycin to obtain the paphiopedilum protocorm. And finally, transferring the survival protocorm into a hygromycin-free H26 # culture medium for continuous culture until a complete plant is formed. According to the method, factors such as a paphiopedilum protocorm treatment mode, infection time, co-culture time, acetosyringone addition concentration, hygromycin selection pressure and the like are optimized, so that the conversion rate of an agrobacterium tumefaciens-mediated transgenic method of the paphiopedilum protocorm is remarkably improved, and a genetic transformation system taking the paphiopedilum 'Glory 90' protocorm as a receptor is established. The method disclosed by the invention has the advantages of simplicity in operation and high conversion rate, and is beneficial to molecular breeding of the paphiopedilum and acceleration of the breeding process of the paphiopedilum.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an Agrobacterium-mediated transgenic method for Paphiopedilum protocorms. Background Art

[0002] Paphiopedilum has a high ornamental value due to its unique flower shape, gorgeous flower color, and long-lasting ornamental flowering period. It is a very popular high-end flower in the international flower market. However, due to excessive excavation of Paphiopedilum and destruction of its habitat, Paphiopedilum has become one of the most endangered plant species in the world. All wild species are listed in Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) and are prohibited from trade. Various breeding methods can be used to select new Paphiopedilum varieties that are freely traded in the international market and have high ornamental value to meet market needs, which has broad market prospects. However, traditional methods such as hybrid breeding have problems such as long breeding cycle and low controllability of traits. Plant transgenic technology has been widely used in plant improvement and genetic breeding. Commonly used methods include Agrobacterium-mediated method, DNA direct insertion method, pollen tube channel method and plant virus-mediated method. Among them, Agrobacterium-mediated method is the most widely used transgenic method, which has the advantages of simple operation, high transformation efficiency, good repeatability, single copy, less gene silencing phenomenon, and the ability to transform larger fragments.

[0003] Methods for transgenic orchids include Agrobacterium-mediated method, ovary injection method and gene gun method, etc. Successful species include Dendrobium, Phalaenopsis, Oncidium and Orchidaceae, etc. Ovary injection method has also been successful in Paphiopedilum. However, since it is difficult to establish an effective regeneration system for Paphiopedilum, there is no report on transgenic Paphiopedilum using Paphiopedilum protocorms mediated by Agrobacterium. This patent uses Paphiopedilum 'Glorious 90', which is bred independently, as a material and establishes a genetic transformation system using its protocorm as a receptor, which has the advantages of high transformation efficiency and has important application prospects. At present, there is no report on Agrobacterium-mediated transgenic method based on Paphiopedilum protocorms. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for Agrobacterium-mediated transgenic of Paphiopedilum protocorms.

[0005] The Agrobacterium-mediated transgenic method of Paphiopedilum protocorms of the present invention comprises the following steps: longitudinally cutting or pricking the Paphiopedilum protocorms, placing them in Agrobacterium bacterial solution for infection, inoculating the protocorms into a co-culture medium for culture, then transferring them to an antibacterial culture medium for antibacterial treatment, placing them in a screening culture medium containing hygromycin (Hyg) for culture, and finally transferring the surviving protocorms to a hygromycin-free H26 culture medium for continued culture until a complete plant is formed.

[0006] Preferably, the method comprises the following steps: cutting or pricking the protocorm of Paphiopedilum longitudinally, placing it in an Agrobacterium bacterial solution for infection for 20 minutes, taking out the protocorm, absorbing the bacterial solution with sterile filter paper, inoculating it into an H26 medium containing acetosyringone and culturing it for 3 days, then transferring it to an H26 medium containing cephalosporin for antibacterial treatment, placing it in an H26 medium containing 15 mg / L hygromycin and culturing it for 15 days, then transferring the surviving protocorm to an H26 medium containing 30 mg / L hygromycin and continuing to culture it for 60 days, replacing the fresh medium every 15 days; finally, transferring the surviving protocorm to an H26 medium without hygromycin and continuing to culture it until a complete plant is formed.

[0007] The added concentration of acetosyringone (AS) is 200 μmol / L.

[0008] The added concentration of cephalosporin (Cef) is 300 mg / L.

[0009] The H26 culture medium is composed of: 1g / L Huabao No. 1 + 15g / L sucrose + 2g / L peptone + 5.0g / L agar + 1.5g / L activated carbon + 10mg / L vitamin B1 + 1mg / L vitamin B6 + 1mg / L niacin + 2mg / L glycine + 1mg / L NAA + 0.1g / L inositol + 50mL / L coconut juice, and the solvent is water.

[0010] The culture conditions are: culture temperature 26-28°C, illumination time 12-16h / d, and illumination intensity 1600-2000lx. Preferably, the culture is carried out in a culture room with a temperature of 28°C, illumination time 16h / d, and illumination intensity 1600-2000lx.

[0011] The induction step of the Paphiopedilum protocorm is as follows: soaking the Paphiopedilum 'Brilliant 90' fruit 150-210 days after pollination in 75% alcohol by volume for 30 seconds, then placing it in a 0.1% mercuric chloride solution for disinfection for 15 minutes, rinsing it with sterile water for 5 times, cutting the fruit, and inoculating the powdered embryo into a seed germination medium: 1 / 4MS medium + 50-100mL / L coconut juice + 0.1-0.5mg / L naphthaleneacetic acid + 1.0-2.0g / L activated carbon for cultivation.

[0012] The Agrobacterium is Agrobacterium tumefaciens EHA105, which contains a plasmid pCAMBIA1301 carrying a GUS gene and a hygromycin resistance gene hpt.

[0013] The present invention has the following beneficial effects:

[0014] The invention significantly improves the conversion rate of the Agrobacterium-mediated transgenic method of the Paphiopedilum protocorm by optimizing factors such as the treatment method of the Paphiopedilum protocorm, infection time, co-cultivation time, acetosyringone addition concentration, hygromycin selection pressure, etc., and establishes a genetic transformation system with the Paphiopedilum 'Brilliant 90' protocorm as the receptor. The method of the invention has the advantages of simple operation and high conversion rate, and is conducive to the molecular breeding of Paphiopedilum and accelerates the breeding process of Paphiopedilum. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The effect of different hygromycin concentrations on the survival rate of protocorms.

[0016] Figure 2 GUS staining of protocorms mediated by Agrobacterium. A: blank control, without any transformation treatment; B: protocorms mediated by Agrobacterium. The arrows indicate the blue color of the protocorms stained with GUS. DETAILED DESCRIPTION

[0017] The following are specific implementation examples of the present invention. It should be noted that these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Modifications and replacements made to the details and forms of the implementation scheme within the scope of the present invention fall within the protection scope of the present invention.

[0018] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The experimental reagents used in the following examples are all conventional biochemical reagents unless otherwise specified.

[0019] The formula of the H26 medium used in the following examples is: 1g / L Huabao No. 1 + 15g / L sucrose + 2g / L peptone + 5.0g / L agar + 1.5g / L activated carbon + 10mg / L vitamin B1 + 1mg / L vitamin B6 + 1mg / L nicotinic acid + 2mg / L glycine + 1mg / L NAA + 0.1g / L inositol + 50mL / L coconut juice, and the solvent is water. The preparation method is: dissolve the above ingredients in water, stir and mix, and sterilize to obtain.

[0020] Example 1

[0021] 1. Induction of protocorms:

[0022] The fruits of Paphiopedilum 'Glorious 90' 150 days after pollination were soaked in 75% alcohol by volume for 30 seconds and then placed in a 0.1% mercuric chloride solution for disinfection for 15 minutes. After rinsing with sterile water for 5 times, the fruits were cut open and the powdered embryos were inoculated into a seed germination medium (1 / 4MS medium + 50-100mL / L coconut juice + 0.1-0.5mg / L naphthaleneacetic acid + 1.0-2.0g / L activated carbon) for cultivation. The germination rate was 75-80% at 60 days.

[0023] 2. Agrobacterium-mediated transformation of protocorms:

[0024] (1) Determination of hygromycin selective pressure

[0025] The protocorms of Paphiopedilum modii with the same growth were selected and cultured on H26 medium containing Hyg (hygromycin) concentrations of 0mg / L, 10mg / L, 15mg / L, 20mg / L, 25mg / L, 30mg / L, 35mg / L, 40mg / L, and 45mg / L. It was found that the protocorms grew vigorously on H26 medium without selection pressure (Hyg concentration of 0mg / L), and the protocorms had formed seedlings after 2 months of growth. The growth of protocorms was obviously inhibited at other Hyg concentrations; with the increase of Hyg concentration in the culture medium, the protocorms gradually turned white and brown. When the Hyg concentration began to exceed 30mg / L, the survival rate of the protocorms was 20% ( Figure 1 ).

[0026] Therefore, the appropriate Hyg concentration should be 30 mg / L. Combined with the growth state of protocorms on H26 medium with different Hyg concentrations and the changing trend of their survival rate, in order to obtain resistant receptor materials to the maximum extent and avoid the existence of false positives as much as possible, it was determined that the gradient selection pressure method should be used for screening in the screening stage, that is, the protocorms were transferred to H26 medium with a Hyg concentration of 15 mg / L for 15 days, and then the surviving receptor materials were transferred to H26 medium with a Hyg concentration of 30 mg / L for continued 60 days, and fresh medium was replaced every 15 days; finally, the surviving receptor materials were transferred to H26 medium without Hyg for continued culturing until complete plants were formed.

[0027] (2) Effects of protocorm treatment methods on transformation

[0028] The protocorms of Paphiopedilum were subjected to Agrobacterium transformation under the conditions of longitudinal section, needle puncture and no treatment, and then transferred to H26 medium containing 300 mg / L cephalosporin for antibacterial treatment and selected culture on the screening medium for 75 days, and the survival rate of resistant receptor materials was counted. The experimental results show (Table 1) that the transformation efficiency of receptor materials is different when different treatment methods are used. When the protocorms were longitudinally sectioned, the transformation rate was 4.17%, and the protocorms were lightly punctured with a dissecting needle for about 5 times, and the transformation rate was 4.17%, while the protocorms were not subjected to any wound treatment and directly exposed to the Agrobacterium infection solution, and the transformation rate at this time was 0.

[0029] Table 1 Effects of protocorm treatment methods on transformation

[0030]

[0031] (3) Effect of infection time on transformation

[0032] The infection time of Agrobacterium affects its transformation efficiency. When the infection time of Agrobacterium is 10 minutes, the transformation rate is 0.83%, and when the infection time is 20 minutes, the transformation rate reaches the highest value, which is 5.00%. When the infection time is 30 minutes, the transformation efficiency drops to 4.17%, and then the transformation rate decreases significantly with the extension of the treatment time. When the infection time is 50 minutes, all the protocorms die and the transformation rate is 0. This shows that too short or too long infection time will cause a decrease in the transformation rate of the resistant receptor material. Therefore, the best infection time in this experiment is 20 minutes (Table 2).

[0033] Table 2 Effect of infection time on transformation

[0034]

[0035] (4) Effect of co-culture time on transformation

[0036] Table 3 shows that the conversion rate of the resistant receptor material was the highest at 3 days of co-culture, which was 4.17%. The growth of the receptor materials at 5 and 7 days of co-culture was observed, and it was found that the edges of the sterile filter paper in the co-culture medium were all Agrobacterium-grown, and there were visible Agrobacterium around a small number of receptor materials. Most of these protocorms with Agrobacterium would still grow bacteria even after washing, sterilization or antibacterial treatment in the later stage. Finally, the receptor materials were very likely to die due to the attachment of bacteria and competition for nutrients; one resistant material survived in the receptor materials at 7 days of co-culture, which may be because the conversion efficiency was improved after sufficient contact with Agrobacterium during the co-culture process, and the growth of Agrobacterium was successfully inhibited during the later sterilization process. Therefore, the optimal co-culture time determined in this experiment is 3 days.

[0037] Table 3 Effect of co-culture time on transformation

[0038]

[0039] (5) Effect of AS (acetosyringone) concentration on conversion

[0040] In the establishment of Agrobacterium-mediated genetic transformation system of monocotyledonous plants, phenolic compounds such as AS are often added at a certain concentration to improve transformation efficiency. In this experiment, different concentrations of AS were added to the co-culture medium. The results showed (Table 4) that the addition of AS was conducive to the acquisition of resistant receptor materials. With the increase of the concentration gradient of added AS, the transformation rate showed a trend of first increasing and then decreasing. The highest transformation rate was 5.83% at a concentration of 200 μmol / L.

[0041] Table 4 Effect of AS addition concentration on transformation

[0042]

[0043] Example 2

[0044] An Agrobacterium-mediated transgenic method for Paphiopedilum protocorm comprises the following steps:

[0045] 1. Induction of protocorms:

[0046] The fruits of Paphiopedilum 'Glorious 90' 210 days after pollination were soaked in 75% alcohol by volume for 30 seconds and then placed in a 0.1% mercuric chloride solution for disinfection for 15 minutes. After rinsing with sterile water for 5 times, the fruits were cut open and the powdered embryos were inoculated into a seed germination medium (1 / 4MS medium + 50-100mL / L coconut juice + 0.1-0.5mg / L naphthaleneacetic acid + 1.0-2.0g / L activated carbon) for cultivation. The germination rate was 75-80% at 60 days.

[0047] 2. Agrobacterium-mediated transformation of protocorms:

[0048] After longitudinal cutting or acupuncture of Paphiopedilum protocorms, they were placed in the bacterial solution of Agrobacterium tumefaciens EHA105 for infection for 20 minutes. The protocorms were taken out and the bacterial solution was dried with sterile filter paper. Then, they were inoculated into H26 medium containing 200 μmol / L acetosyringone and cultured for 3 days. Then, they were transferred to H26 medium containing 300 mg / L cephalosporin for antibacterial treatment. Then, they were placed in H26 medium containing 15 mg / L hygromycin and cultured at 28°C and 16 h of light exposure. / d, light intensity of 1600-2000lx culture room for 15d, then transfer the surviving protocorms to H26 medium containing 30mg / L hygromycin, and continue to culture for 60d in a culture room at 28℃, light time 16h / d, light intensity 1600-2000lx, and replace fresh culture medium every 15d; finally, transfer the surviving protocorms to H26 medium without hygromycin and continue to culture until a complete plant is formed.

[0049] 3. Detection of genetically modified materials

[0050] GUS histochemical staining was performed on the protocorms mediated by Agrobacterium tumefaciens. The results are shown in Figure 2 . Figure 2 The results showed that the transgenic protocorms showed clearly visible GUS-stained blue spots, while the protocorms in the blank control group that were not transformed with Agrobacterium did not have any blue spots, indicating that the plasmid containing the GUS reporter gene fragment was successfully transferred into the protocorms of Paphiopedilum.

[0051] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineering and technical personnel in the field should fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for Agrobacterium-mediated transgenic of Paphiopedilum protocorms, characterized in that: The following steps are involved: The protocorms of Paphiopedilum are cut longitudinally or punctured with a needle and then infected with Agrobacterium solution. The protocorms are inoculated into a co-culture medium for culture, then transferred to an antibacterial culture medium for antibacterial treatment, and then cultured in a screening culture medium containing hygromycin. Finally, the surviving protocorms are transferred to a hygromycin-free H26 culture medium for continued culture until a complete plant is formed.

2. The Agrobacterium-mediated transgenic method of Paphiopedilum protocorm according to claim 1, characterized in that: The following steps are involved: After longitudinal cutting or acupuncture of Paphiopedilum protocorm, it was placed in Agrobacterium bacterial solution for infection for 20 minutes, the protocorm was taken out and the bacterial solution was dried with sterile filter paper, and then inoculated into H26 medium containing acetosyringone and cultured for 3 days, and then transferred to H26 medium containing cephalosporin for antibacterial treatment, and then placed in H26 medium containing 15 mg / L hygromycin and cultured for 15 days, and then the surviving protocorm was transferred to H26 medium containing 30 mg / L hygromycin and continued to be cultured for 60 days, and fresh culture medium was replaced every 15 days; finally, the surviving protocorm was transferred to H26 medium without hygromycin and continued to be cultured until a complete plant was formed.

3. The Agrobacterium-mediated transgenic method of Paphiopedilum protocorm according to claim 2, characterized in that: The added concentration of the acetosyringone is 200 μmol / L.

4. The Agrobacterium-mediated transgenic method of Paphiopedilum protocorm according to claim 2, characterized in that: The added concentration of cephalosporin is 300 mg / L.

5. The Agrobacterium-mediated transgenic method of Paphiopedilum protocorm according to claim 2, characterized in that: The H26 culture medium is composed of: 1g / L Huabao No. 1 + 15g / L sucrose + 2g / L peptone + 5.0g / L agar + 1.5g / L activated carbon + 10mg / L vitamin B1 + 1mg / L vitamin B6 + 1mg / L niacin + 2mg / L glycine + 1mg / L NAA + 0.1g / L inositol + 50mL / L coconut juice, and the solvent is water.

6. The Agrobacterium-mediated transgenic method of Paphiopedilum protocorm according to claim 2, characterized in that: The culture conditions are: culture temperature 26-28°C, light exposure time 12-16h / d, and light intensity 1600-2000lx.

7. The Agrobacterium-mediated transgenic method of Paphiopedilum protocorm according to claim 2, characterized in that: The induction step of the Paphiopedilum protocorm is as follows: soaking the Paphiopedilum 'Brilliant 90' fruit 150-210 days after pollination in 75% alcohol by volume for 30 seconds, then placing it in a 0.1% mercuric chloride solution for disinfection for 15 minutes, rinsing it with sterile water for 5 times, cutting the fruit, and inoculating the powdered embryo into a seed germination medium: 1 / 4MS medium + 50-100mL / L coconut juice + 0.1-0.5mg / L naphthaleneacetic acid + 1.0-2.0g / L activated carbon for cultivation.

8. The Agrobacterium-mediated transgenic method of Paphiopedilum protocorm according to claim 2, characterized in that: The Agrobacterium is Agrobacterium tumefaciens EHA105.

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