Agrobacterium tumefaciens-mediated phalaenopsis genetic transformation method

Abstract

The invention discloses an agrobacterium tumefaciens-mediated phalaenopsis genetic transformation method, a complete plant is obtained through protocorm-like body induction, protocorm-like body proliferation, genetic transformation and protocorm-like body differentiation seedling, the method has the characteristics of simple and feasible process, short cultivation period, high transformation efficiency and the like, the obtained protocorm-like body can be stored for a long time, and the method is suitable for large-scale popularization and application. And the material is a good acceptor material, and can meet the requirements of genetic transformation research and annual production of seedlings of phalaenopsis amabilis.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant transgenic technology, and specifically relates to a method for genetically transforming Phalaenopsis '0908' by using young leaves of sterile seedlings obtained by culturing Phalaenopsis orchid stem segments as explants and induced protocorms as transformation receptor materials through Agrobacterium-mediated genetic transformation of Phalaenopsis '0908'. Background Art

[0002] Phalaenopsis is a perennial herbaceous plant of the genus Phalaenopsis in the family Orchidaceae. It is native to China, Thailand, the Philippines, etc. and is most widely planted in the area south of the Yangtze River in my country. Phalaenopsis is named because it resembles a butterfly. Phalaenopsis has a variety of flower shapes and colors, and some varieties have a charming fragrance. It is loved by people all over the world and is one of the most ornamental and economical orchid flowers. Phalaenopsis is a monopodial epiphytic orchid, and the plant rarely develops side branches, so it cannot be propagated by division. And its seeds contain almost no endosperm, which usually cannot provide nutrition for its germination. Traditional hybrid breeding of Phalaenopsis needs to follow the natural laws of plant growth, and the breeding cycle is relatively long. With the continuous development of biotechnology, genetic engineering technology has become more and more mature. The use of exogenous genes introduced into Phalaenopsis can improve the traits that need to be controlled and keep other traits relatively stable at the same time, which speeds up the breeding process and improves the efficiency of selecting new varieties of Phalaenopsis.

[0003] At present, most Phalaenopsis transgenic projects use Agrobacterium-mediated genetic transformation, but the transformation efficiency is low or it is difficult to obtain transgenic plants. Therefore, the present invention provides a high-efficiency transformation method using protocorm-like bodies as transformation receptor materials, which greatly improves the efficiency of Agrobacterium-mediated genetic transformation. Summary of the invention

[0004] The purpose of the present invention is to provide a method for genetic transformation of Phalaenopsis mediated by Agrobacterium, specifically a method for genetic transformation of Phalaenopsis '0908' mediated by Agrobacterium using protocorm-like bodies as receptors, which lays a foundation for further genetic improvement thereof by genetic engineering means.

[0005] The present invention is implemented by the following technical solutions:

[0006] The invention discloses an Agrobacterium-mediated genetic transformation method for Phalaenopsis, comprising four steps: protocorm-like body induction, proliferation and cultivation process of transformed receptor materials, genetic transformation process, and differentiation of resistant protocorm-like bodies into seedlings.

[0007] 1. Protocorm-like body induction process: The young leaves of sterile seedlings obtained from the primary culture of peduncle axillary buds are used as explants, cut and inoculated into induction medium, and induced into protocorms-like bodies at 25±2℃, light intensity of 1500-2000lx, and light time of 14h light / 10h dark;

[0008] 2. Proliferation and culture process of transformation receptor materials: the induced protocorm-like bodies are inoculated into a proliferation culture medium for proliferation culture to provide transformation receptor materials for genetic transformation;

[0009] 3. Genetic transformation process: Agrobacterium carrying the target gene is streaked and cultured on a solid or liquid LB medium containing 100 mg / L Kana, cultured in the dark at 28°C for 26-48 hours, and a single colony is picked and placed in a 10 ml LB liquid medium containing 100 mg / L Kana for culture, and cultured at 26-28°C with shaking (220 rpm) overnight. 0.8-1 ml of bacterial solution is inoculated into 50 ml LB liquid medium (containing 100 mg / L Kana), and cultured on a shaking table at 28°C and 220 rpm for 10 hours to 14 hours, and the concentration of the bacterial solution is detected by a spectrophotometer until the bacterial solution OD600 = 0.6-0.8. The cultured bacterial solution is divided into 50 ml centrifuge tubes in a clean bench, and centrifuged at 4500 rpm for 10 minutes at room temperature using a high-speed refrigerated centrifuge, and the supernatant is discarded to collect the bacteria. Add 50 ml of transformation medium to suspend the bacteria. Specifically, put the mixture of transformation medium and bacteria into a shaker at 28°C and 150rpm to resuspend for 30-40min to obtain an Agrobacterium suspension for later use. Select the protocorm-like body in good condition obtained by proliferation culture in step 2 as the receptor material, place it in the prepared Agrobacterium suspension for infection (the infection condition is shaking on a shaker at 100-110rpm for 30min), inoculate the infected protocorm-like body on the co-culture medium for co-culture, wash it three times with sterile water containing 100mg / LCef after 3 days of co-culture, wash it three times with sterile water, place it on filter paper for air drying, and then transfer it to the selective proliferation medium for selective proliferation culture;

[0010] 4. The process of differentiating the resistant protocorms into seedlings: The resistant protocorms obtained by selective culture and screening in step 3 are transferred into a seedling differentiation medium to differentiate into seedlings, and finally the transgenic plants of Phalaenopsis '0908' are obtained.

[0011] In the above technical scheme, further, the components of the protocorm-like induction medium in step 1 include: 1 / 2MS basic medium, 6-BA 3-4 mg / L, TDZ 0.5-0.8 mg / L, sucrose 30 g / L, plant gel 3 g / L, peptone 1-1.5 g / L, CW 15%, add purified water to 1 L, pH 5.5-5.8.

[0012] Furthermore, the proliferation culture process of the protocorm-like bodies in step 2 is carried out under the conditions of 25±2°C and a light intensity of 1500-2000lx; the components of the proliferation culture medium include: 1 / 2MS basic culture medium, 6-BA 4mg / L, Ad 3mg / L, AC1g / L, sucrose 15g / L, plant gel 3g / L, corn flour 10g / L, CW 15%-20%, and distilled water is added to make the volume 1L, pH 5.5-5.8; the proliferation culture medium is updated every 4 weeks, and the protocorm-like bodies that have been cultured for 10-15d in the last updated proliferation culture medium are selected as the recipient material for genetic transformation.

[0013] Furthermore, in the step 3, the co-cultivation is carried out in the dark at 25±2°C for 3 days, and then the culture medium is transferred to the protocorm-like body selective proliferation culture medium for selective proliferation culture for 8-10 weeks, the culture medium is replaced every 2-3 weeks, and the selective proliferation culture is carried out at 25±2°C and a light intensity of 1500-2000lx.

[0014] Further, the culture medium of Agrobacterium in step 3 is solid or liquid LB culture medium (containing 100 mg / L Kana), and its main formula is: peptone 10 g / L + yeast extract 5 g / L + 10 g / L sodium chloride, agar powder 7.5 g / L is added to the solid culture medium, and the pH value is 6.0; the transformation medium of Agrobacterium is liquid MS + 100 μmol / L AS; the co-cultivation culture medium components include: MS basic culture medium, 6-BA 3 mg / L, AS 100 μmol / L, sucrose 30 g / L, Phytagel 3 mg / L, pH 5.8; the selective proliferation culture medium components include: 1 / 2MS basic culture medium, 6-BA 4 mg / L, Hyg 10 mg / L, Cef 200-300 mg / L, CW15%-20%, sucrose 15 g / L, Phytagel 3 g / L, pH 5.5-5.8;

[0015] Furthermore, the composition of the culture medium for differentiation of protocorms-like bodies into seedlings in step 4 is: 1 mg / L of Huabao No. 1 + 1 mg / L of Huabao No. 2 as the basic culture medium, and also includes 2 mg / L of 6-BA, 10 mg / L of Hyg, 200-300 mg / L of Cef, 30 g / L of sucrose, 15%-20% of CW, 3 g / L of Phytagel, pH 6.0. The differentiation into seedlings is carried out at 25±2°C and a light intensity of 1000-1500 lx.

[0016] Further, the Hyg is hygromycin: it is prepared into a 100 mg / L mother solution with sterile purified water; Cef is cefotaxime: it is prepared into a 200 mg / L mother solution with sterile purified water; AS is acetosyringone: AS powder is weighed, first dissolved with an organic solvent dimethyl sulfoxide, and finally fixed to volume with sterile distilled water to prepare a 10 μmol / ml mother solution. The above three biochemical reagents are all sterilized by filtration with a 0.22 μm filter membrane and stored at -20°C;

[0017] The 1 / 2MS, MS, Huabao No. 1 and Huabao No. 2 are basic culture media, 6-BA is 6-benzyladenine, Phytagel is plant gel, CW is coconut milk, AS is acetosyringone, and Ad is adenine sulfate.

[0018] In order to facilitate the understanding of the present invention, the applicant has explained the three key points in the above steps as follows: 1. Infection conditions: The infection conditions play a key role in whether resistant protocorms can be obtained. The applicant has found that for the '0908' variety of Phalaenopsis, the infection needs to be carried out on a shaker at 100-110rpm for 30 minutes. The shaking speed of the shaker cannot be too fast, otherwise the receptor material may be damaged and the infection efficiency may be reduced. The time must be strictly controlled within 30 minutes. If the time is too long, the Agrobacterium will overflow seriously during the later culture and it will be difficult to remove the bacteria. If the infection time is too short, the Agrobacterium will not be able to adsorb on the wound part or surface of the receptor material, resulting in a reduction in the infection efficiency.

[0019] 2. Co-cultivation time: The co-cultivation stage is an important stage for the transfer of Agrobacterium T-DNA to plant cells. The co-cultivation time has an important impact on the transformation rate. Studies have shown that the most suitable co-cultivation time for Phalaenopsis '0908' variety is 2 days. If the co-cultivation time is too short, the efficiency of genetic transformation will be reduced and no transformed plants can be obtained; if the co-cultivation time is too long, Agrobacterium will overgrow and the plant cells will die due to poisoning;

[0020] 3. Selection of screening pressure: The control of screening pressure (hygromycin content) in the corresponding culture medium during the selection and proliferation culture in step 3 and the differentiation and seedling culture in step 4 also has a very important impact on improving the transformation efficiency. Only by finding the correct screening pressure can the pressure for the subsequent identification of transgenic materials be reduced. Studies have shown that for the '0908' variety of Phalaenopsis, the hygromycin concentration is 10-12 mg / L. The Hyg concentration should be appropriately reduced during the selection and proliferation process, because the vitality of the receptor material will be reduced to a certain extent after infection. If the selection and screening concentration is too high, it will be unfavorable for its screening. The concentration can be appropriately increased in the subsequent selection, differentiation, and seedling stages;

[0021] The present invention utilizes the protocorm-like bodies that have been induced and proliferated as the transformation receptor material, and can directly carry out selective proliferative culture after infection, thereby shortening the culture period and successfully obtaining the Phalaenopsis '0908' transgenic plants.

[0022] The beneficial effects of the present invention are:

[0023] 1. The method of the present invention successfully achieved the transformation of Phalaenopsis '0908' protocorms as the receptor material, and obtained transgenic plants containing GUS reporter gene and DhGA2ox through Agrobacterium-mediated and further culture, which laid a good foundation for the transformation of the target gene.

[0024] 2. The operation method is simple and the cultivation cycle is short.

[0025] 3. The explants used in the present invention are relatively easy to obtain, and the tissue culture of Phalaenopsis '0908' can be carried out year-round, and protocorms-like bodies can be obtained as receptor materials through proliferation culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Schematic diagram of the genetic transformation process of the present invention;

[0027] Figure 2 : PCR detection gel image of transgenic plants of the present invention;

[0028] Figure 3 : GUS staining diagram of the transgenic plant of the present invention;

[0029] Figure 4 : GUS activity diagram of the transgenic plants of the present invention. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. The described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0031] Example 1

[0032] Experimental material selection, culture medium design and genetic transformation process

[0033] 1. Source of test materials and their processing:

[0034] 1) Receptor material: The protocorms induced from the leaves of Phalaenopsis '0908' sterile seedlings grown for 30-45 days were cultured for proliferation, and the vigorously growing protocorms were selected as the receptor material;

[0035] 2) Strains and plasmids: The Agrobacterium strain used in the experiment was EHA105, and the plant expression vector was pCAMBIA1301 (containing the GUS gene and the hygromycin resistance gene hpt). The GUS gene contains an intron to ensure that it is only expressed in plant cells and not in Agrobacterium.

[0036] 2. Culture medium design

[0037] Table 1 lists the components and amounts of various culture media of the present invention.

[0038] Table 1 Phalaenopsis in vitro culture medium design

[0039]

[0040]

[0041] Hygromycin in Table 1: prepared into a 100 mg / L mother solution with sterile purified water; Cef is cefotaxime: prepared into a 200 mg / L mother solution with sterile purified water; AS is acetosyringone: AS powder is weighed, first dissolved with an organic solvent dimethyl sulfoxide, and finally fixed to volume with sterile purified water to prepare a μmol / ml mother solution. The above three biochemical reagents are all sterilized by filtration with a 0.22 μm filter membrane and stored at -20°C;

[0042] The 1 / 2MS, MS, Huabao No. 1 and Huabao No. 2 are basic culture media, 6-BA is 6-benzyladenine, Phytagel is plant gel, CW is coconut milk, AS is acetosyringone, and Ad is adenine sulfate.

[0043] 3. Preparation of GUS dye solution

[0044] Table 2 shows the reagents and dosage of GUS histochemical staining solution.

[0045] Table 2 GUS histochemical staining solution

[0046]

[0047]

[0048] Among them, X-Gluc solution should be prepared and used immediately and added last, and GUS dye solution should be kept away from light.

[0049] 4. Genetic transformation process

[0050] 1) Preparation of receptor materials: Using young leaves of Phalaenopsis grown for 30-45 days obtained by the sterile seedlings obtained by primary culture of peduncle axillary buds as explants, cutting and inoculating them into protocorm-like body induction medium to directly induce protocorm-like bodies; transferring the induced protocorm-like bodies into proliferation medium for proliferation culture, updating the proliferation medium every 4 weeks, and selecting the protocorm-like bodies that have been proliferated and cultured for 10-15 days in the last updated proliferation medium as the receptor materials for genetic transformation;

[0051] 2) Preparation of Agrobacterium suspension: Agrobacterium carrying the target gene was streaked and cultured on LB solid medium containing 100 mg / L Kana, cultured in the dark at 28°C for 26-48 hours, and a single colony was picked and placed in 10 ml LB liquid medium containing 100 mg / L Kana for culture, and cultured at 26-28°C with shaking (220 r / min) overnight. 0.8-1 ml of bacterial solution was pipetted into 50 ml LB liquid medium (containing 100 mg / L Kana), and cultured on a shaking table at 28°C and 220 rpm for 10-14 hours. When the bacterial solution was observed to be orange-yellow, the concentration of the bacterial solution was detected with a spectrophotometer, and the culture was terminated when the bacterial solution OD600 = 0.6-0.8; the cultured bacterial solution was dispensed into 50 ml centrifuge tubes in a clean bench, and centrifuged at 4500 rpm for 10 minutes at room temperature using a high-speed refrigerated centrifuge, and the supernatant was discarded to collect the bacterial bodies. Add 50 ml of transformation medium to suspend the bacteria. Specifically, put the mixture of transformation medium and bacteria into a shaker at 28°C and 150 rpm for 30-40 minutes to obtain Agrobacterium suspension for later use.

[0052] 3) Infection: directly put the protocorm-like body prepared in step 1) into the Agrobacterium suspension prepared in step 2) and infect on a shaker at 100-110 rpm for 30 min;

[0053] 4) Co-cultivation: Place the infected protocorm-like bodies on sterile filter paper to absorb the excess bacterial liquid on the surface, transfer to co-cultivation medium, and culture at 25±2℃ in the dark for 3 days;

[0054] 5) Selection culture and differentiation into seedling culture: The protocorm-like bodies after 3 days of co-culture were first washed three times with sterile water containing 100 mg / L Cef, then washed three times with sterile water and placed on filter paper for air drying, and then transferred to the selection proliferation medium, and resistant protocorm-like bodies were screened at 25±2°C and a light intensity of 1500-2000lx. The medium was renewed every two weeks and cultured for 8-10 weeks; the resistant protocorm-like bodies that survived and grew well after screening were inoculated into the differentiation into seedling culture medium, and cultured at 25±2°C and a light intensity of 1000-1500lx. Fresh culture medium was renewed every 2-3 weeks until complete plants were obtained;

[0055] 6) Transgenic plant detection: The obtained transgenic plants and untransformed plants were placed in GUS dye solution for GUS activity detection, with untransformed plants as negative controls; leaves of transformed plants were used as materials, total DNA was extracted by CTAB method, and PCR detection was performed.

[0056] 5. Test results

[0057] After GUS activity detection and PCR detection, the GUS transformation efficiency was 9.91%, and the PCR transformation efficiency was 19.24%.

[0058] Figure 2 The results of PCR detection of transgenic plants, where M: Marker DL 5000; 1-9: Phalaenopsis transgenic experimental group; 12-15: Phalaenopsis control group; P: positive control; W: water. PCR detection of 9 strains found that strain 1 was a non-transgenic strain, and strains 2-9 were transgenic strains.

[0059] Figure 3 GUS staining of transgenic plants. Figure 3 It can be seen that blue spots appeared in the transgenic strains, proving that the GUS gene has been successfully introduced into the Phalaenopsis.

[0060] Figure 4 is the GUS activity diagram of transgenic plants. Figure 4 It can be seen that the GUS activity of the three transgenic lines is significantly higher than that of the control.

Claims

1. A method for genetic transformation of Phalaenopsis mediated by Agrobacterium, characterized in that: The following steps are involved: 1) Protocorm-like body induction culture: The young leaves of sterile seedlings obtained from the primary culture of peduncle axillary buds were cut and inoculated into induction medium at 25±2°C, with a light intensity of 1500-2000lx and a lighting time of 14h light / 10h dark to induce protocorms-like bodies; 2) Proliferation culture of transformed receptor materials: the protocorm-like bodies induced in step 1) are placed in a proliferation culture medium and cultured at 25±2°C and a light intensity of 1500-2000 lx; ​​the proliferation culture medium is renewed every 4 weeks, and the protocorm-like bodies that have been cultured for 10-15 days in the last renewed proliferation culture medium are selected as the receptor materials for genetic transformation; 3) Genetic transformation: The protocorm-like body obtained by the proliferation culture in step 2) is used as a receptor material, and is placed in an Agrobacterium suspension containing the target gene for infection, and the infection conditions are a shaking table speed of 100-110 rpm and shaking for 30 minutes; the infected protocorm-like body is inoculated on a co-culture medium for co-culture, and after co-culture at 25±2°C in the dark for 3 days, it is first washed 3 times with sterile water containing 100 mg / L Cef, and then washed 3 times with sterile water, and then placed on filter paper to dry, and then transferred to a selective proliferation medium for selective proliferation culture; 4) Cultivation of resistant protocorms-like differentiation into seedlings: The resistant protocorms-like differentiation obtained by selection and culture in step 3) are transferred to the seedling differentiation medium for cultivation. The seedling differentiation culture is carried out at 25±2°C and a light intensity of 1000-1500lx to finally obtain the transgenic plants of Phalaenopsis '0908'.

2. The method for genetic transformation of Phalaenopsis mediated by Agrobacterium according to claim 1, characterized in that: In step 1), the induction medium comprises: 1 / 2MS basic medium, 6-BA 3-4 mg / L, TDZ 0.5-0.8 mg / L, sucrose 30 g / L, plant gel 3 g / L, peptone 1-1.5 g / L, CW 15%, and distilled water to 1 L, pH 5.5-5.

8.

3. The method for genetic transformation of Phalaenopsis mediated by Agrobacterium according to claim 1, characterized in that: In step 2), the components of the proliferation medium include: 1 / 2MS basic medium, 6-BA 4mg / L, Ad3mg / L, AC 1g / L, sucrose 15g / L, plant gel 3g / L, corn flour 10g / L, CW 15%-20%, distilled water is added to make up to 1L, pH 5.5-5.

8.

4. The method for genetic transformation of Phalaenopsis mediated by Agrobacterium according to claim 1, characterized in that: In step 3), the method for obtaining the Agrobacterium suspension containing the target gene is as follows: streak the Agrobacterium carrying the target gene on a solid or liquid LB medium containing 100 mg / L Kana, culture it in the dark at 28°C for 26-48 hours, pick a single colony and place it in 10 ml of LB liquid medium containing 100 mg / L Kana, and culture it at 26-28°C and 220 rpm overnight; draw 0.8-1 ml of the bacterial solution and inoculate it into 50 ml of LB liquid medium containing 100 mg / L Kana, and culture it on a shaker at 28°C and 220 rpm for 10 hours to 14 hours, until the bacterial solution OD 600 =0.6-0.8; the bacterial solution was dispensed into 50 ml centrifuge tubes in a clean bench, centrifuged at 4500 rpm for 10 min at room temperature, the supernatant was discarded, and the bacteria were collected; 50 ml of transformation medium was added to suspend the bacteria, specifically, the mixture of the transformation medium and the bacteria was placed in a shaker at 28°C and resuspended at 150 rpm for 30-40 min to obtain the Agrobacterium suspension containing the target gene.

5. The method for genetic transformation of Phalaenopsis mediated by Agrobacterium according to claim 4, characterized in that: The solid or liquid LB culture medium includes: 10 g / L peptone + 5 g / L yeast extract + 10 g / L sodium chloride, 7.5 g / L agar powder is added to the solid culture medium, and the pH value is 6.0; the transformation medium is liquid MS + 100 μmol / L AS.

6. The method for genetic transformation of Phalaenopsis mediated by Agrobacterium according to claim 1, characterized in that: In step 3), the components of the co-culture medium include: MS basic medium, 6-BA 3mg / L, AS 100μmol / L, sucrose 30g / L, Phytagel 3mg / L, pH 5.8; the components of the selective proliferation medium include: 1 / 2MS basic medium, 6-BA 4mg / L, Hyg 10mg / L, Cef 200-300mg / L, CW15%-20%, sucrose 15g / L, Phytagel 3g / L, pH 5.5-5.

8.

7. The method for genetic transformation of Phalaenopsis mediated by Agrobacterium according to claim 1, characterized in that: In step 3), the selective proliferation culture is carried out for 8-10 weeks, and the selective proliferation culture medium is replaced every 2-3 weeks. The selective proliferation culture is carried out at 25±2°C and a light intensity of 1500-2000lx.

8. The method for genetic transformation of Phalaenopsis mediated by Agrobacterium according to claim 1, characterized in that: In the step 4), the differentiation seedling culture medium includes 1 mg / L Huabao No. 1 and 1 mg / L Huabao No. 2, 2 mg / L 6-BA, 10 mg / L Hyg, 200-300 mg / L Cef, 30 g / L sucrose, 15%-20% CW, 3 g / L Phytagel, and pH 6.

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