Anthurium andraeanum genetic transformation method

Abstract

The invention discloses a genetic transformation method of anthurium andraeanum, and belongs to the field of biological breeding, the method comprises the steps of callus induction, callus propagation, callus infection and the like, in the method, through treatment of a specific resting culture medium, growth of positive cells is recovered; and screening the calluses through two rounds of propagation, so that the positive calluses can be propagated. The efficiency of anthurium callus infection is improved by improving an infection solution, a co-culture medium, a resting culture medium, a screening culture medium and the like. Besides, in the method, specific pre-culture is carried out on the agrobacterium dip dyeing liquid, expression of related protein infected by agrobacterium is induced in advance, and the infection efficiency of the agrobacterium on the callus can be improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological breeding, and is suitable for a genetic transformation method for transgene of all varieties of Anthurium. BACKGROUND

[0002] Anthurium andraeanum Linden, as a perennial herb of Anthurium in Araceae, becomes an important ornamental flower in the world with unique spathes and bright colors, and is favored in the cut flower and potted plant markets.

[0003] The traditional propagation method of Anthurium andraeanum Linden cannot meet the market demand, and the genetic transformation technology opens up a new path for Anthurium breeding, which can break through the limitations of traditional breeding and improve the varieties in a targeted manner. The Agrobacterium-mediated method is a common genetic transformation method for Anthurium, but has problems of strong genotype dependence and low transformation frequency. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a genetic transformation method for Anthurium andraeanum Linden, and improve the transformation efficiency.

[0005] The technical scheme of the present application is a genetic transformation method for Anthurium andraeanum Linden, comprising the following steps:

[0006] (1) Callus induction: placing the cleaned and disinfected leaf petioles of Anthurium andraeanum Linden on an induction medium, and inducing callus at 25°C in the dark; the induction medium comprises 1 / 2MS, 30g / L sucrose, 0.2mg / L 2,4-D, 1mg / L 6-BA, and pH5.8;

[0007] (2) Propagation of callus: transferring the callus obtained in step (1) to a propagation medium, and culturing at 25°C in the dark to propagate the callus; the propagation medium comprises MS, 30g / L sucrose, 0.2mg / L 2,4-D, 1mg / L 6-BA, and pH5.8;

[0008] (3) Callus dipping:

[0009] a. cutting the propagation callus obtained in step (2) into small pieces, soaking with Agrobacterium dipping liquid, constantly shaking the liquid during the soaking, pouring out the liquid after 15 minutes, and transferring the callus to sterile filter paper and air-drying;

[0010] b. then transferring the callus to a co-culture medium and culturing at 21°C in the dark for 3 days;

[0011] c. then transferring the callus to a resting medium and culturing in the dark for 10 days;

[0012] d. screening the callus after resting culture on a screening medium;

[0013] e、then the screened resistant callus is transferred to the pre-differentiation medium for growth for 1 round;

[0014] f、then the resistant callus is transferred to the differentiation medium for differentiation;

[0015] g、finally the obtained seedlings are transferred to the rooting medium for rooting;

[0016] the co-culture medium is composed of MS, 30 g / L sucrose, 0.85 mg / L silver nitrate, 200 μM AS, 1 mg / L 6-BA, pH 5.8;

[0017] the resting medium is composed of MS, 30 g / L sucrose, 300 mg / L Tim, 0.2 mg / L 2,4-D, 1 mg / L 6-BA, pH 5.8;

[0018] the screening medium is composed of MS, 30 g / L sucrose, 300 mg / L Tim, 20 mg / L Hgy, 0.2 mg / L 2,4-D, 1 mg / L 6-BA, pH 5.8;

[0019] the pre-differentiation medium is composed of MS, 30 g / L sucrose, 300 mg / L Tim, 20 mg / L Hgy, 1 mg / L 6-BA, pH 5.8;

[0020] the differentiation medium is composed of MS, 30 g / L sucrose, 300 mg / L Tim, 20 mg / L Hgy, 2 mg / L 6-BA, pH 5.8.

[0021] Further, the Agrobacterium dipping solution is prepared by the following method:

[0022] (1) the Agrobacterium tumefaciens containing the target plasmid is streaked on a YEB solid plate and cultured at 28℃ for 2 days;

[0023] (2) a single colony is picked from the plate and inoculated into YEB liquid medium, and cultured at 28℃ overnight with shaking;

[0024] (3) inoculated into new YEB liquid medium at a ratio of 1:100, and cultured at 28℃ with shaking until OD 600 0.6-0.8, centrifuged at 3000 rpm for 10 minutes to collect the bacterial cells;

[0025] (4) resuspend the bacterial cells with the infection solution, and incubate at 25℃ with shaking at 60 rpm for 2-3 hours;

[0026] The infection solution is composed of MS, 68.4 g / L sucrose, 36 g / L glucose, 200 μM AS, 1 mg / L 6-BA, pH 5.2.

[0027] Further, the number of times of screening the callus subjected to the resting culture on the screening medium is two rounds.

[0028] Compared with the prior art, the present application has the following beneficial effects:

[0029] 1. In the present application, the positive cells are recovered by resting culture, and then the positive callus is expanded by two rounds of expansion screening, so that the positive callus is expanded. Through this optimization step, the positive callus is improved each time, and only a small amount of callus is browned and dead. If the callus is not subjected to resting culture, but is directly cultured in the pre-differentiation medium or the screening medium, most of the callus will be dead. Therefore, by improving the infection solution, the co-culture medium, the resting culture medium and the screening medium, the efficiency of the infection of the callus of the red palm is improved.

[0030] 2. In the present application, the Agrobacterium dipping solution is subjected to specific pre-culture, the expression of the Agrobacterium infection-related protein is induced in advance, and the infection efficiency of the Agrobacterium on the callus is improved. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Red palm leaves;

[0032] Figure 2 Inducing callus;

[0033] Figure 3 Expanding callus;

[0034] Figure 4 Callus differentiation;

[0035] Figure 5 PCR identification, M: DNA marker DL2000; 1: control; 2-8: transformed seedlings. DETAILED DESCRIPTION

[0036] In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the experimental materials are purchased from commercial channels unless otherwise specified.

[0037] 2,4-D: 2,4-dichlorophenoxyacetic acid; 6-BA: 6-benzyladenine; AS: acetyl-syringone; Tim: thidiazuron; Hgy: hygromycin; MS: Phyto Technology M519.

[0038] Example 1

[0039] (1) Induction of callus tissue in Anthurium

[0040] Take tender leaves of Anthurium and wash them with clean water. Figure 1 Then, sterilize the leaves in a clean bench with a 2% sodium hypochlorite solution (with a few drops of Tween 20 added) for 15 minutes, stirring occasionally. Next, rinse five times with sterile water, place the leaves on sterile filter paper, and blot away any remaining water from the leaf surface.

[0041] Use a scalpel to remove the leaf margins, then cut the leaf blade and petiole into pieces about 1 cm long, and place them separately on different induction culture media:

[0042] Sample 1: 1 / 2 MS, 30 g / L sucrose, 0.2 mg / L 2,4-D, 1 mg / L 6-BA, pH 5.8;

[0043] Sample 2: 1 / 2 MS, 30 g / L sucrose, 0.2 mg / L 2,4-D, 1.5 mg / L 6-BA, pH 5.8;

[0044] No. 3: 1 / 2 MS, 30 g / L sucrose, 0.3 mg / L 2,4-D, 1 mg / L 6-BA, pH 5.8.

[0045] Callus induction was performed at 25℃ in the dark. The results of explant culture on different callus induction media showed that callus grew rapidly on medium No. 1. Figure 2 Callus induction was performed on Anthurium leaves and petioles using culture medium No. 1. Callus could be induced in just over a month under dark culture conditions. The callus induction rate of petioles was higher than that of leaves, indicating that petioles are suitable for callus induction (Table 1).

[0046] Table 1. Callus induction from different explants

[0047] Explant Callus induction rate Leaf blade 89 Petiole 96

[0048] (2) Proliferate callus tissue

[0049] The induced callus tissues were transferred to different propagation media to propagate the callus tissues:

[0050] No. 1: MS, 30 g / L sucrose, 0.2 mg / L 2,4-D, 1 mg / L 6-BA, pH 5.8;

[0051] Item 2: MS, 30 g / L sucrose, 1 mg / L 6-BA, pH 5.8.

[0052] Incubation at 25°C in the dark revealed that callus tissue in medium 1 grew faster than that in medium 2. Figure 3 Culture medium No. 1 was selected as the culture medium for proliferating callus tissue.

[0053] (3) Preparation of Agrobacterium infection solution

[0054] LBA4404 bacteria containing the pCAMBIA1300 plasmid were streaked onto YEB solid plates and incubated at 28°C for 2 days. Single colonies were picked from the plates and inoculated into YEB liquid medium, and cultured overnight at 28°C with shaking. The culture was then inoculated into fresh YEB liquid medium at a 1:100 ratio and cultured at 28°C with shaking until OD (dose elapsed). 600 The bacterial cells were collected by centrifugation at 3000 rpm for 10 minutes, with a pH of 0.6-0.8. The cells were then resuspended in infection buffer (MS, 68.4 g / L sucrose, 36 g / L glucose, 200 μM AS, 1 mg / L 6-BA, pH 5.2) and incubated at 25°C with shaking at 60 rpm for 2-3 hours. Pre-culturing Agrobacterium allows for the early induction of infection-related protein expression, which can improve the infection efficiency of Agrobacterium on callus tissue.

[0055] (4) Infection of callus tissue in Anthurium

[0056] Cut the anthurium callus into small pieces and soak them in the prepared Agrobacterium infection solution for 15 minutes, shaking the solution continuously. Pour out the solution and transfer the callus to sterile filter paper to air dry. Then transfer the callus to a co-culture medium (MS, 30 g / L sucrose, 0.85 mg / L silver nitrate, 200 μM AS, 1 mg / L 6-BA, pH 5.8) and incubate in the dark at 21°C for 3 days. Finally, transfer the callus to two different culture media:

[0057] Resting medium: MS, 30 g / L sucrose, 300 mg / L Tim, 0.2 mg / L 2,4-D, 1 mg / L 6-BA, pH 5.8

[0058] Screening medium: MS, 30 g / L sucrose, 300 mg / L Tim, 20 mg / L Hgy, 0.2 mg / L 2,4-D, 1 mg / L 6-BA, pH 5.8

[0059] The callus was cultured in the dark for 10 days. Results showed that most of the callus died when cultured on selection medium after infection had ceased; however, callus on resting medium did not show browning and grew normally. Because Anthurium callus grows slowly, transferring it to selection medium might prevent positive cells from having sufficient time to develop, and the death of negative cells could lead to browning and death of the entire callus. Therefore, resting culture of the infected callus was chosen to allow positive cells to proliferate.

[0060] Callus tissues that had undergone resting culture were screened twice on selection medium. Then, resistant callus tissues were transferred to pre-differentiation medium (MS, 30 g / L sucrose, 300 mg / L Tim, 20 mg / L Hgy, 1 mg / L 6-BA, pH 5.8) for one round of growth, followed by transfer of resistant callus tissues to differentiation medium (MS, 30 g / L sucrose, 300 mg / L Tim, 20 mg / L Hgy, 2 mg / L 6-BA, pH 5.8) to differentiate into seedlings. Figure 4 Finally, the obtained seedlings are transferred to a rooting medium to root.

[0061] During callus infection, the callus tissue is cut into pieces approximately 0.5-8 cm in size and allowed to recover the growth of positive cells through resting culture. Two rounds of callus propagation and selection further increase the number of positive callus. This optimized process increases the number of positive callus with each infection, resulting in only a small amount of callus turning brown and dying. If callus is cultured directly on pre-differentiation or selection media without resting culture, most of the callus will die. Therefore, improvements to the infection solution, co-culture medium, resting medium, and selection medium have enhanced the efficiency of Anthurium callus infection.

[0062] One leaf was cut from a resistant seedling, and genomic DNA was extracted using the CTAB method. Primers were designed based on the vector, and PCR was used to identify whether the T-DNA region of pCAMBIA1300 was inserted into the genome of the resistant seedling. Electrophoresis results showed that the target fragment was amplified. Figure 5 This indicates that the resistant seedlings obtained were all transgenic plants.

Claims

1. A method for genetic transformation of Anthurium, characterized in that, Includes the following steps: (1) Callus induction: The cleaned and disinfected petioles of Anthurium were placed on the induction medium and inducing callus in the dark at 25°C; the composition of the induction medium was: 1 / 2 MS, 30 g / L sucrose, 0.2 mg / L 2,4-D, 1 mg / L 6-BA, pH 5.8; (2) Callus propagation: The callus obtained in step (1) was transferred to a propagation medium and cultured in the dark at 25°C to propagate the callus; the propagation medium consisted of MS, 30 g / L sucrose, 0.2 mg / L 2,4-D, 1 mg / L 6-BA, and pH 5.

8. (3) Callus infiltration: a. Cut the proliferated callus obtained in step (2) into small pieces, soak them in Agrobacterium tumefaciens infusion solution, and shake the solution continuously during the process. After 15 minutes, pour out the solution, transfer the callus to sterile filter paper, and air dry. b. Then transfer the callus tissue to a co-culture medium and incubate in the dark at 21°C for 3 days; c. Then transfer the callus tissue to resting culture medium and incubate in the dark for 10 days; d. Screen the callus tissue after resting culture on screening medium; e. Then, the selected resistant callus tissues are transferred to predifferentiation medium for one round of growth; f. Then transfer the resistant callus to a differentiation medium to differentiate into seedlings; g. Finally, the obtained seedlings are transferred to a rooting medium to root. The co-culture medium consisted of: MS, 30 g / L sucrose, 0.85 mg / L silver nitrate, 200 μM AS, 1 mg / L 6-BA, pH 5.8; The resting culture medium consisted of: MS, 30 g / L sucrose, 300 mg / L Tim, 0.2 mg / L 2,4-D, 1 mg / L 6-BA, pH 5.8; The screening medium consisted of: MS, 30 g / L sucrose, 300 mg / L Tim, 20 mg / L Hgy, 0.2 mg / L 2,4-D, 1 mg / L 6-BA, pH 5.8; The predifferentiation medium consisted of: MS, 30 g / L sucrose, 300 mg / L Tim, 20 mg / L Hgy, 1 mg / L 6-BA, pH 5.8; The differentiation medium consisted of: MS, 30 g / L sucrose, 300 mg / L Tim, 20 mg / L Hgy, 2 mg / L 6-BA, pH 5.

8.

2. The method according to claim 1, characterized in that, The Agrobacterium-containing infusion solution was prepared by the following method: (1) Streak Agrobacterium tumefaciens containing the target plasmid on YEB solid plates and incubate at 28°C for 2 days; (2) Pick a single colony from the plate and inoculate it into YEB liquid medium, and incubate overnight at 28°C with shaking; (3) Inoculate into fresh YEB liquid medium at a ratio of 1:100 and incubate at 28°C with shaking until OD. 600 The bacterial cells were collected by centrifugation at 3000 rpm for 10 minutes to a value of 0.6-0.

8. (4) Resuspend the bacterial cells in the infection solution and incubate at 25°C with shaking at 60 rpm for 2-3 hours. The composition of the infection solution was: MS, 68.4 g / L sucrose, 36 g / L glucose, 200 μM AS, 1 mg / L 6-BA, pH 5.

2.

3. The method according to claim 1, characterized in that, The number of times the callus tissue that has undergone resting culture is screened on the screening medium is two rounds.

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