Efficient anthurium andraeanum transgenosis method

Abstract

The invention discloses an efficient anthurium andraeanum transgenosis method. The method provided by the invention comprises the following steps: S1, inducing embryonic calluses by taking leaves of tissue culture seedlings of Anthurium fortunei with petioles as explants; s2, the embryogenic callus is scratched and then put into an agrobacterium engineering bacterium solution to be infected; s3, carrying out co-culture on the infected callus, washing and degerming, and then carrying out recovery culture; s4, carrying out screening culture on the callus subjected to recovery culture to obtain a transformant, and carrying out differentiation culture on the transformant to obtain a test-tube plantlet; and S5, transferring the test-tube plantlet into a rooting and seedling strengthening culture medium, and culturing to obtain a 'Furii' anthurium transgenic plant. The acceptor adopted by the method has relatively strong transformation capacity, the co-culture time is prolonged, effective infection of agrobacterium can be ensured, the recovery culture stage is beneficial to callus recovery and transformed cell proliferation after infection, the transfer and integration probability of T-DNA fragments is improved, efficient genetic transformation of 'Fuy' anthurium is realized, and the transformation rate reaches 51.61%.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering. More specifically, it relates to a highly efficient transgenic method for Anthurium andraeanum. Background Art

[0002] Anthurium andraeanum is a perennial evergreen herb of the genus Anthurium in the family Araceae. This plant is famous for the unique shape of its spathe, bright and diverse flower colors, and long ornamental period, so it is widely used in the production of potted flowers and cut flowers. Due to its popularity among consumers, the sales volume of Anthurium andraeanum ranks second only to orchids, and it has a very broad market prospect.

[0003] The breeding of new varieties is the basis for the efficient and sustainable development of the Anthurium andraeanum industry. Hybrid breeding is the main method for breeding new varieties of Anthurium andraeanum, but there are some disadvantages in Anthurium andraeanum hybrid breeding, such as long breeding cycle, narrow range of resource utilization, and inability to achieve directional improvement, which is not conducive to the rapid and directional cultivation of breakthrough new varieties and shortening the breeding gap with foreign countries as soon as possible. Genetically modified breeding (GMB) refers to a method of cultivating new varieties by adding one or more genes to a biological genome through modern molecular biology techniques. Through genetically modified breeding, the species boundary can be broken, the utilization range of genetic resources can be broadened, and the directional and efficient transfer of known functional genes can be realized. This breeding method with precise and directional gene manipulation has higher efficiency and stronger pertinence. Agrobacterium-mediated method is the earliest and most widely used transgenic method. Agrobacterium carries the Ti plasmid related to the induction of plant tumor formation. The target gene can be inserted into the transfer DNA to T-DNA, so as to realize the transfer and expression of foreign genes, and then change the genetic characteristics of plants; it has many advantages such as simple operation, low cost, good repeatability, high transformation rate, less gene silencing phenomenon, short transfer cycle, ability to insert large fragments of DNA, and no need for protoplast culture to regenerate plants.

[0004] Factors affecting the Agrobacterium-mediated transgenic effect include the genotype and type of receptor materials, the species of Agrobacterium strains, the concentration of engineered bacterial solution, the infection time, the co-culture time, the concentration of acetosyringone, etc. There have been many research reports on Agrobacterium-mediated transgenic anthurium (Yi Maosheng et al., 2015; Kong et al, 2017). The research results show that varieties, receptors, co-culture time, etc. have a great impact on the genetic transformation efficiency of anthurium. So far, the research on anthurium transgenic breeding still stays in the stage of method research, rather than variety breeding. No commercial new anthurium varieties have been selected through this method (Teixeira da Silva et al, 2015), and the genetic transformation efficiency of Agrobacterium-mediated anthurium is still relatively low. Due to the large number of anthurium varieties, including genotypes from different original variety parental materials, different varieties are suitable for different parts as genetic transformation receptors, and the effects of genetic transformation are also different. At present, for different anthurium varieties such as 'Yali Red', 'Telensa', 'Pink Champion', 'Alabama', '13100-A3', etc., the total conversion rate of transgenic using Agrobacterium still needs to be improved. And there is no genetic transformation method for the anthurium variety 'Furi', and there is a lack of transgenic breeding methods applicable to more different anthurium varieties to achieve efficient genetic transformation of anthurium.

[0005] Therefore, further carrying out research on anthurium transgenic breeding technology and establishing an efficient and repeatable genetic transformation technology system for anthurium are of great significance for further using transgenic breeding methods to cultivate new anthurium varieties, shortening the gap with foreign anthurium breeding, and promoting the innovative development and high-quality development of China's anthurium industry. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defects of the existing anthurium transgenic methods, such as low transformation efficiency and poor stability, and the lack of a genetic transformation method for the new anthurium variety 'Furi', and to provide an efficient anthurium transgenic method.

[0007] The above object of the present invention is achieved by the following technical solutions:

[0008] The present invention provides an efficient anthurium transgenic method, including the following steps:

[0009] S1. Using the leaves of 'Furi' anthurium tissue culture seedlings with petioles as explants to induce callus;

[0010] S2. Scratching the induced embryogenic callus and then putting it into the engineered Agrobacterium solution for infection;

[0011] S3. First co-culturing the infected callus, washing and removing bacteria, and then performing recovery culture;

[0012] S4. The callus after recovery culture is screened to obtain transformants, and the transformants are differentiated to obtain test-tube plantlets.

[0013] S5. The test-tube plantlets are transferred to a rooting and strong-seedling medium for cultivation to obtain transgenic Anthurium andraeanum plants of 'Furui'.

[0014] In the present invention, the callus induced from the leaves of tissue-cultured seedlings of Anthurium andraeanum 'Furui' with petioles of 1 - 3 mm is used as the receptor. Through the Agrobacterium-mediated method, it is co-cultured with Agrobacterium for 2 d. After co-culture, recovery culture is carried out, and then it is screened on a medium containing 30 mg / L hygromycin for 2 rounds. Finally, through differentiation and PCR detection, transgenic seedlings are obtained. The present invention uses the embryogenic callus of Anthurium andraeanum 'Furui', which has strong transformation ability; at the same time, the co-culture time is extended to ensure the effective infection of Agrobacterium; a recovery culture stage under light conditions is added, which is beneficial to the recovery of callus after infection and the proliferation of transformed cells, and improves the probability of transfer and integration of T-DNA fragments; then 2 rounds of screening culture are carried out to improve the screening efficiency and effect of transformants, realizing the efficient genetic transformation of Anthurium andraeanum 'Furui' with a transformation rate of 51.61%, laying a reliable technical foundation for cultivating new varieties of Anthurium andraeanum using transgenic technology.

[0015] Preferably, in S1, the leaves of tissue-cultured seedlings of Anthurium andraeanum 'Furui' with petioles of 1 - 3 mm are used as explants to induce callus.

[0016] Preferably, the induction culture conditions in S1 are: 25 ± 2 °C, cultured for 50 - 70 d under dark conditions; the induction medium is MS + 0.5 mg / L 6-BA + 1.0 mg / L 2,4-D + 30 g / L sugar + 8 g / L carrageenan, and the pH value is 5.8 ± 0.2.

[0017] Preferably, in S2, the induced embryogenic callus is scratched and then pre-cultured. The pre-culture conditions are: pre-cultured in the dark at 24 - 26 °C for 4 - 8 d; the pre-culture medium is MS + 0.5 mg / L 6-BA + 1.0 mg / L 2,4-D + 0.1 mg / L PVP + 30 g / L sugar + 8 g / L carrageenan, and the pH value is 5.8 ± 0.2.

[0018] Preferably, the Agrobacterium engineering bacterial solution in S2 contains 100 μmol / L AS and MS liquid medium, and the OD 600 value is not less than 0.2.

[0019] Preferably, the Agrobacterium is EHA105 strain.

[0020] Preferably, the Agrobacterium engineering bacterial solution in S2 is shaken twice in an LB medium containing 0.1% kanamycin until the OD 600It reaches 0.6 - 0.8 and is resuspended with the prepared suspension medium (MS liquid medium) added with 100 μmol / L acetosyringone.

[0021] Preferably, the infection method in S2 is as follows: Immerse the callus in the Agrobacterium infection solution and shake it at 160 rpm in a shaker at 28°C for 30 min. Pour out the bacterial solution, take out the callus, and blot the excess bacterial solution on the surface with sterile filter paper.

[0022] Preferably, the co - culture conditions in S3 are: Co - culture in the dark at 24 - 26°C for 1 - 4 d; the co - culture medium is MS + 0.5 mg / L 6 - BA + 1.0 mg / L 2,4 - D + 0.1 mg / L PVP + 100 μmol / L AS + 30 g / L sugar + 8 g / L carrageenan, and the pH value is 5.8 ± 0.2.

[0023] More preferably, the co - culture time is 2 d.

[0024] Preferably, the washing and de - bacterization in S3 are as follows: Wash 3 times with sterile water, change to sterile water containing 500 mg / L Cef, wash at 160 rpm in a shaker at 28°C for 30 min, and then wash 3 times with sterile water containing 500 mg / L Cef, and then transfer it to the recovery medium containing 300 mg / L cephalosporin.

[0025] Preferably, the recovery culture conditions in S3 are: The culture temperature is 25 ± 2°C, and the light time is 12 h / d; the recovery medium is MS + 0.5 mg / L 6 - BA + 1.0 mg / L 2,4 - D + 0.1 mg / L PVP + 300 mg / L Cef + 30 g / L sugar + 8 g / L carrageenan, and the pH value is 5.8 ± 0.2.

[0026] Preferably, the transformant screening method in S4 is as follows: When the callus grows to about 0.5 cm in length, transfer it to the screening medium for screening culture for 20 - 40 d, and then transfer the surviving tissue blocks to a new screening medium for continuous culture to obtain the transformant; the screening medium is MS + 0.5 mg / L 6 - BA + 1.0 mg / L 2,4 - D + 0.1 mg / L PVP + 300 mg / L Cef + 30 mg / L hygromycin + 30.0 g / L sucrose + 7.0 g / L carrageenan, and pH = 5.8 ± 0.2.

[0027] Preferably, the differentiation culture conditions in S4 are: The light intensity is 1500 ± 100 lx, the light time is 12 h / d, and the temperature is 25 ± 2°C; the differentiation medium is MS + 1.0 mg / L 6 - BA + 1.5 mg / L NAA + 300 mg / L Cef + 30.0 g / L sugar + 8.0 g / L carrageenan, and pH = 5.8 ± 0.2.

[0028] Preferably, the rooting and seedling strengthening culture conditions in S5 are as follows: light intensity is 2200±100 lx, light time is 12 h / d, and temperature is 25±2 °C; the rooting and seedling strengthening culture medium is: MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.2 g / L activated carbon + 300 mg / L Cef + 30.0 g / L sugar + 8.0 g / L carrageenan, pH = 5.8±0.2.

[0029] The present invention has the following beneficial effects:

[0030] The present invention provides an efficient genetic transformation method for Anthurium andraeanum. Using embryogenic callus induced from the leaves of 'Furi' Anthurium andraeanum tissue culture seedlings with petioles of 1-3 mm as the transgenic receptor, Agrobacterium infection is carried out, and co-cultivation is carried out for 2 d to ensure the effective infection of Agrobacterium; recovery culture is carried out under light of 12 h / d, which is beneficial to the recovery of callus after infection and the proliferation of transformed cells, and improves the probability of transfer and integration of T-DNA fragments; then two rounds of screening culture are carried out to improve the screening efficiency and effect of transformants; finally, after differentiation and seedling formation culture, transgenic plants are successfully obtained, and the transformation efficiency is 51.61%. The present invention optimizes the key links such as the genotype and type of receptor material, infection, transfer and integration of T-DNA fragments, and screening of transformants in Agrobacterium-mediated genetic transformation, significantly improves the genetic transformation efficiency of Anthurium andraeanum, has a simple operation process, is easy to master, saves time and effort, and is particularly suitable for the genetic transformation of 'Furi' Anthurium andraeanum. Description of the Drawings

[0031] Figure 1 Shows the effects of different hygromycin concentrations on the browning and death of Anthurium andraeanum callus (a. 0 mg / L; b. 10 mg / L; c. 20 mg / L; d. 30 mg / L; e. 40 mg / L).

[0032] Figure 2 Shows the genetic transformation process of Anthurium andraeanum AaMYB9 gene mediated by Agrobacterium (a. pre-culture of Anthurium andraeanum callus; b. Agrobacterium infection; c. co-culture of Anthurium andraeanum callus; d. recovery culture of Anthurium andraeanum callus; e. screening culture of Anthurium andraeanum callus; f. differentiation culture of Anthurium andraeanum callus and formation of resistant buds of Anthurium andraeanum).

[0033] Figure 3 Is the Super1300-GFP plasmid.

[0034] Figure 4 Is the Super1300-AaMYB9-GFP recombinant plasmid.

[0035] Figure 5Partial PCR detection electrophoresis diagram of the regenerated Anthurium andraeanum plants transformed with Super1300-AaMYB9-GFP (in the figure, M is DL2000 Marker; - is the blank control; + is the positive control plasmid (910bp); 1, 2, 3, 4, 5, 6, 7, 8, 10, 17, 18, 20, 21, 22, 25, 31 are positive plants). Detailed implementation manners

[0036] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0038] The callus of Anthurium andraeanum used in the present invention is derived from the induction of the leaves of tissue culture seedlings including petioles of 1-3 mm.

[0039] The MS medium used in the following examples is Murashige & Skoog medium (a commercial medium, in powder form, soluble in water), with relatively high inorganic salt and ion concentrations, which is a relatively stable ion balance solution. It has a high nitrate content, and the quantity and proportion of its nutrients are appropriate, which can meet the nutritional and physiological needs of plant cells. The formula of LB medium is tryptone 10 g / L, yeast 5 g / L, and NaCl 10 g / L.

[0040] Example 1 Establishment of the genetic transformation method of Anthurium andraeanum callus

[0041] 1. Agrobacterium liquid infection

[0042] (1) Embryogenic callus induction: Take the leaves of 'Furi' Anthurium andraeanum tissue culture seedlings with petioles of 1-3 mm as explants for callus induction. The induction medium is MS + 0.5 mg / L 6-BA + 1.0 mg / L 2,4-D + 30 g / L sugar + 8 g / L carrageenan, and the pH value is 5.8 ± 0.2. Cultivate under dark conditions at a temperature of 25 ± 2 °C, and use the embryogenic callus produced after 60 days of cultivation as the transgenic receptor. The embryogenic callus has strong transformation ability. Scratch the callus and transfer it into the pre-culture medium. The pre-culture medium is MS + 0.5 mg / L 6-BA + 1.0 mg / L 2,4-D + 0.1 mg / L PVP + 30 g / L sugar + 8 g / L carrageenan, and the pH value is 5.8 ± 0.2. Pre-culture in the dark at 25 °C for 6 days.

[0043] (2) Construction of expression vector: The EHA105 Agrobacterium was used to construct the expression vector. After the construction of the expression vector, shaking culture was carried out in two steps, first small-scale shaking and then large-scale shaking. For both shaking cultures, LB medium containing 0.1% kanamycin was used. For the first shaking, 500 μL of LB + 0.5 μL of Kan (50 mg / mL) + colony were used; at 28 °C, 200 rpm for 12 h. For the second shaking, 100 mL of LB + 100 μL of Kan (50 mg / mL) + 100 μL of seed liquid were used, and shaking was carried out at 28 °C, 200 rpm for 14 h until the OD 600 reached about 0.6 - 0.8. After the engineering bacterial liquid was shaken well, the bacteria were collected by centrifugation and the strain was resuspended. The shaken bacteria were poured into 50 mL tubes, with 40 mL - 50 mL aliquoted into each tube, and two tubes were filled. At room temperature, centrifugation was carried out at 5000 rpm for 10 min; the bacterial cells were collected and resuspended with the prepared suspension medium (MS liquid medium), and 100 μmol / L acetosyringone was added before use to resuspend to an Agrobacterium liquid with an OD 600 value of 0.2 for standby.

[0044] (3) Infection: The callus of the receptor material cut and cultured was infected in the engineering Agrobacterium liquid. It was shaken at 160 rpm in a 28 °C shaker for 30 min. The bacterial liquid was poured out, and the callus was taken out and the excess bacterial liquid on the surface was blotted dry with sterile filter paper.

[0045] 2. Co-culture and recovery culture

[0046] The embryogenic callus after infection is first co-cultured and then recovered. First, transfer it to the co-culture medium (containing 200 μmol / L acetosyringone AS): MS + 0.5 mg / L 6-BA + 1.0 mg / L 2,4-D + 0.1 mg / L PVP + 200 μmol / L AS + 30.0 g / L sugar + 8.0 g / L carrageenan, pH = 5.8 ± 0.2. Co-culture in the dark at 25 °C for 2 days, discard the dead materials, wash and remove bacteria, wash with sterile water until clear, and then wash 4 times with sterile water containing 500 mg / L cefotaxime. The first time, shake and wash in a shaker at 28 °C and 160 rpm for 30 min, and the remaining 3 times, let it stand for washing to remove the excess bacterial solution. Then spread the washed materials on filter paper to dry, and transfer them to the recovery medium (containing 300 mg / L cefotaxime Cef): MS + 0.5 mg / L 6-BA + 1.0 mg / L 2,4-D + 0.1 mg / L PVP + 300 mg / L Cef + 30 g / L sugar + 8 g / L carrageenan, pH value is 5.8 ± 0.2, and recover in the light for 12 h / d. The culture temperature is 25 ± 2 °C. Recover in the light for 12 h / d, change the medium every 15 days, and the culture temperature is 25 ± 2 °C, change the medium every 15 days; adding a recovery culture stage is beneficial to the recovery of callus after infection and the proliferation of transformed cells, and improves the probability of T-DNA fragment transfer and integration.

[0047] 3. Differentiation and seedling culture

[0048] The callus about 0.5 cm long that grew after the above-mentioned recovery culture is screened to obtain transformants, and then the transformants are differentiated to obtain test-tube seedlings. The specific method is as follows: If there is no obvious Agrobacterium in the medium, transfer it to the screening medium (MS + 0.5 mg / L 6-BA + 1.0 mg / L 2,4-D + 0.1 mg / L PVP + 300 mg / L Cef + 30 mg / L hygromycin + 30.0 g / L sucrose + 7.0 g / L carrageenan, pH = 5.8 ± 0.2), change the medium every 30 days, discard the dead tissue blocks, and transfer the surviving tissue blocks to a new screening medium for continuous screening. A total of 2 rounds of screening are carried out to improve the screening efficiency and effect of transformants.

[0049] Transfer the surviving materials after the above screening to the differentiation medium (MS + 1.0 mg / L 6-BA + 1.5 mg / L NAA + 300 mg / L Cef + 30.0 g / L sugar + 8.0 g / L carrageenan, pH = 5.8 ± 0.2), the light intensity is 1500 ± 100 lx, the light time is 12 h / d, the temperature is 25 ± 2 °C, change the medium every 30 days, and culture into small seedlings.

[0050] When the seedlings grow to 1.0 - 1.5 cm, transfer the seedlings into the rooting and strong seedling culture medium (MS + 1.0 mg / L 6 - BA + 0.1 mg / L NAA + 0.2 g / L activated carbon + 300 mg / L Cef + 30.0 g / L sugar + 8.0 g / L carrageenan, pH = 5.8 ± 0.2), and culture them under the conditions of light intensity of 2200 ± 100 lx, light time of 12 h / d, and temperature of 25 ± 2 °C. When the seedlings grow to 3 cm, transgenic plants are obtained.

[0051] Example 2 Influence Factors of Genetic Transformation of Anthurium Callus

[0052] 1. Influence of Different Concentrations of Hygromycin on the Survival Rate of Anthurium Callus

[0053] In order to determine the most suitable hygromycin screening concentration for Anthurium callus during the genetic transformation process, in this example, during the stage of differentiating into seedlings and culturing, Anthurium callus was respectively inoculated into the screening media containing 0 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, and 40 mg / L hygromycin and cultured for 40 d, and the medium was changed every 20 d. Other culture methods were the same as those in Example 1.

[0054] The results are as Figure 1 shown. It shows that in different media, Anthurium callus shows different degrees of browning. The statistical results of the survival rates of Anthurium callus with different hygromycin concentrations are shown in Table 1 below. When the hygromycin concentration is 40 mg / L, the survival rate of Anthurium callus is 23.0% ( Figure 1 e); when the hygromycin concentration is 30 mg / L, the survival rate of Anthurium callus is 57.5% ( Figure 1 d); when the hygromycin concentration is 20 mg / L, the survival rate of Anthurium callus is 60.3% ( Figure 1 c); when the hygromycin concentration is 10 mg / L, the survival rate of Anthurium callus is 75.0% ( Figure 1 b); when the hygromycin concentration is 0 mg / L, the survival rate of Anthurium callus is 97.0% ( Figure 1 a). It shows that as the hygromycin concentration increases, the survival rate of Anthurium callus becomes lower. A low concentration of hygromycin screening pressure is likely to produce chimeras and false - positive plants, and too high a concentration will make it difficult for the receptor materials after screening to recover and grow. Therefore, the most suitable hygromycin screening concentration for the genetic transformation process of Anthurium callus is 30 mg / L.

[0055] Table 1 Survival Rates of Anthurium Callus with Different Hygromycin Concentrations

[0056]

[0057] Example 3 A Method for Transgenic Anthurium with Embryogenic Callus as the Receptor

[0058] In this example, Agrobacterium-mediated genetic transformation of the Anthurium MYB9 gene was used. The specific method is as follows:

[0059] (1) Using the leaves of 'Furi' Anthurium tissue culture seedlings with petioles of 1 - 3 mm as explants for induction culture to obtain Anthurium embryogenic callus as the transgenic receptor. Scratch the surface of the callus with a knife and place the callus in the dark for pre-culture ( Figure 2 a);

[0060] (2) Construct the expression vector Super1300 - AaMYB9 - GFP to transform Anthurium callus. Use the Super1300 - GFP plasmid ( Figure 3 ) to construct the Super1300 - AaMYB9 - GFP overexpression vector by the homologous arm method ( Figure 4 ). After the plasmid construction, shake the bacteria in two batches. Both batches of shaking use LB medium containing 0.1% kanamycin. The first batch of shaking uses 500 μL LB + 0.5 μL Kan (50 mg / mL) + colony; at 28 °C, 200 rpm, for 12 h. The second batch of shaking uses 100 mL LB + 100 μL Kan (50 mg / mL) + 100 μL seed solution, and shake at 28 °C, 200 rpm for 14 h until the OD 600 reaches about 0.6 - 0.8; after the engineering bacteria solution is shaken well, pour the shaken bacteria into 50 mL tubes, aliquot 40 mL - 50 mL per tube, fill two tubes, at room temperature, centrifuge at 5000 rpm for 5 min; collect the bacterial cells, and use the prepared suspension medium (MS liquid medium), add 100 μmol / L acetosyringone before use to re-suspend to an Agrobacterium solution with an OD 600 value of 0.2 for standby;

[0061] (3) Infection: Transfer the pre-cultured callus into the Agrobacterium solution of Super1300 - AaMYB9 - GFP containing the target gene, and shake and infect at 160 rpm for 30 min ( Figure 2 b), pour out the bacterial solution, take out the callus, and blot the excess bacterial solution on the surface with sterile filter paper;

[0062] (4) Co-culture: Transfer the callus into the co-culture medium (MS + 0.5 mg / L 6 - BA + 1.0 mg / L 2,4 - D + 0.1 mg / L PVP + 200 μmol / L AS + 30.0 g / L sugar + 8.0 g / L carrageenan, pH = 5.8 ± 0.2), and co-culture in the dark at 25 °C for 2 d ( Figure 2 c);

[0063] After co-culture, discard the dead materials, wash them with sterilized water until clear, and then wash them 4 times with sterilized water containing 500 mg / L cefotaxime. The first wash is carried out by shaking at 160 rpm in a shaker at 28 °C for 30 min, and the remaining 3 washes are carried out by static washing to remove the excess bacterial liquid.

[0064] (5) Recovery culture: Spread the washed materials on filter paper to dry, and transfer them to the recovery medium (MS + 0.5 mg / L 6-BA + 1.0 mg / L 2,4-D + 0.1 mg / L PVP + 300 mg / L Cef + 30.0 g / L sugar + 8.0 g / L carrageenan, pH = 5.8 ± 0.2). The callus of Anthurium andraeanum gradually turns green and shows a large amount of proliferation and a small amount of differentiation ( Figure 2 d);

[0065] (6) Screening culture: If no obvious Agrobacterium appears in the medium, transfer it to the screening medium (MS + 0.5 mg / L 6-BA + 1.0 mg / L 2,4-D + 0.1 mg / L PVP + 300 mg / L Cef + 30 mg / L hygromycin + 30.0 g / L sucrose + 7.0 g / L carrageenan, pH = 5.8 ± 0.2), and change the medium every 30 d for a total of 2 rounds of screening ( Figure 2 e);

[0066] (7) Differentiation into seedlings: Transfer the surviving materials to the differentiation medium (MS + 1.0 mg / L 6-BA + 1.5 mg / L NAA + 300 mg / L Cef + 30.0 g / L sugar + 8.0 g / L carrageenan, pH = 5.8 ± 0.2) for culture, and change the medium every 30 d; when the seedlings grow to 1.0 - 1.5 cm ( Figure 2 f), transfer the seedlings to the rooting and strong seedling medium (MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.2 g / L AC + 300 mg / L Cef + 30.0 g / L sugar + 8.0 g / L carrageenan, pH = 5.8 ± 0.2) for culture. When the seedlings grow to 3 cm or more, the transgenic plants are obtained.

[0067] Extract the DNA of the regenerated plants and perform PCR detection. Calculate the conversion rate according to the following formula:

[0068] Conversion rate = number of positive plants / total number of plants detected × 100%.

[0069] According to the above method, a total of 31 regenerated plants were identified, and the detection results are as Figure 5 shown, among which there are 16 positive plants and the transformation efficiency is 51.61%.

[0070] In summary, the present invention provides an efficient genetic transformation method for Anthurium andraeanum. Using the embryogenic callus of 'Furi' Anthurium andraeanum induced from the leaves of tissue-cultured seedlings with petioles of 1-3 mm as the receptor, it is infected with Agrobacterium tumefaciens EHA105 and co-cultured for 2 days to ensure the effective infection of Agrobacterium. The recovery culture is carried out under a light duration of 12 h / d, which is beneficial to the recovery of callus after infection and the proliferation of transformed cells, and improves the probability of transfer and integration of T-DNA fragments. Two rounds of screening culture are carried out to improve the screening efficiency and effect of transformants. Finally, after differentiation and seedling formation culture, transgenic plants are successfully obtained, and the transformation efficiency is 51.61%. The present invention optimizes the key links in Agrobacterium-mediated genetic transformation, such as the genotype and type of receptor materials, infection, transfer and integration of T-DNA fragments, and screening of transformants, significantly improves the genetic transformation efficiency of Anthurium andraeanum, has a simple operation process, is easy to master, saves time and effort, and is particularly suitable for the genetic transformation of 'Furi' Anthurium andraeanum.

[0071] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A highly efficient transgenic method for Anthurium andraeanum, characterized in that: The following steps are involved: S1. Using petiole-bearing leaves of 'Furui' anthurium andraeanum tissue culture seedlings as explants, callus induction was performed; S2. The induced embryonic callus was scratched and then placed in the Agrobacterium engineering bacterial solution for infection; S3. co-cultivating the infected callus, washing and sterilizing it, and then recovering it; S4. The callus tissue after recovery culture is screened and cultured to obtain transformants, and the transformants are differentiated and cultured to obtain test tube plantlets; S5. The test tube seedlings were transferred into a rooting and seedling strengthening medium to obtain transgenic 'Furui' anthurium andraeanum plants.

2. The method according to claim 1, characterized in that: In S1, explants were induced using leaf materials from tissue culture seedlings with 1-3 mm petioles.

3. The method according to claim 1, characterized in that: The induction culture conditions in S1 are: 25±2°C, culture in the dark for 50 to 70 days; the induction culture medium is MS+0.5mg / L 6-BA+1.0mg / L 2,4-D+30g / L sugar+8g / L carrageenan, and the pH value is 5.8±0.

2.

4. The method according to claim 1, characterized in that: The induced embryonic callus tissue in S2 was scratched and then pre-cultured. The pre-culture conditions were: pre-culture at 24-26°C in the dark for 4-8 days. The pre-culture medium was MS+0.5mg / L 6-BA+1.0mg / L 2,4-D+0.1mg / L PVP+30g / L sugar+8g / L carrageenan, with a pH of 5.8±0.

2.

5. The method according to claim 1, characterized in that: The Agrobacterium engineering culture medium in S2 contained 100 μmol / L AS and MS liquid culture medium, and the culture OD 600 The value should not be less than 0.

2.

6. The method according to claim 5, characterized in that: The Agrobacterium was strain EHA105.

7. The method according to claim 1, characterized in that: The co-culture conditions of S3 were: co-culture in the dark at 24-26°C for 1-4 days; the co-culture medium was MS+0.5mg / L 6-BA+1.0mg / L 2,4-D+0.1mg / L PVP+100μmol / L AS+30g / L sugar+8g / L carrageenan, with a pH of 5.8±0.

2.

8. The method according to claim 1, characterized in that: The conditions for recovery culture in S3 are: culture temperature 25±2℃, light duration 12h / d; recovery culture medium is MS+0.5mg / L 6-BA+1.0mg / L2,4-D+0.1mg / L PVP+300mg / LCef+30g / L sugar+8g / L carrageenan, pH value is 5.8±0.

2.

9. The method according to claim 1, characterized in that: The method for screening transformants in S4 is as follows: when callus tissue with a length of about 0.5 cm is grown, it is inoculated into a screening medium and cultured for 20 to 40 days, and then the surviving tissue blocks are transferred to a new screening medium for continued culture to obtain transformants; the screening medium is MS+0.5 mg / L6-BA+1.0 mg / L 2,4-D+0.1 mg / LPVP+300 mg / L Cef+30 mg / L hygromycin+30.0 g / L sucrose+7.0 g / L carrageenan, pH=5.8±0.

2.

10. The method according to claim 1, characterized in that: The conditions for differentiation culture in S4 are: light intensity of 1500±100lx, light duration of 12h / d, temperature of 25±2°C; differentiation medium is MS+1.0mg / L 6-BA+1.5mg / L NAA+300mg / L Cef+30.0g / L sugar+8g / L carrageenan, pH=5.8±0.2.

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