Method for efficiently inducing cultivated walnut hairy roots by agrobacterium rhizogenes

Abstract

The invention relates to a method for efficiently inducing hairy roots of cultivated species walnut by agrobacterium rhizogenes. The method comprises the following steps: selecting seeds, soaking the seeds, breaking shells, baking and accelerating germination to obtain seeds with good germination state and vigorous vitality; when radicles germinate to break through seed coats to form main roots, and the main roots extend to 2-6 cm and the diameter is 2-3 mm, agrobacterium rhizogenes are used for infecting the main roots; through co-culture and rooting culture, transgenic hairy roots can grow on main roots; the germinated main roots in the period are strong in cell division ability, vigorous in growth and extremely easy to be infected by agrobacterium rhizogenes, and the inductivity of positive transgenic hairy root plants can be greatly improved. Experiments show that by adopting the patent method, the experiment operation is simple and convenient, the materials do not need to be subjected to detoxification and degerming treatment, the proportion of positive hairy roots of cultivated species walnuts (warm 185) can reach 90%, the proportion of positive hairy root plants can reach 97% or above, and the method can be applied to work such as gene function verification on walnut indigenous species.

Description

Technical Field

[0001] The invention belongs to the technical field of cultivated walnut transgenic technology, and particularly relates to a method for efficiently inducing hairy roots of cultivated walnut using Agrobacterium rhizogenes. Background Art

[0002] At present, the commonly used genetic transformation technologies for walnuts mainly include the pollen tube channel method, microinjection method and Agrobacterium-mediated method. The explant materials used mainly include young leaves, petioles, stems, induced callus tissues and somatic embryos of walnuts. However, walnut genetic transformation still faces difficulties. First, the walnut material itself contains rich secondary metabolites such as phenols, which can easily cause the explant material to brown, contaminate and die during the culture process; second, the regeneration ability of walnut callus and somatic embryos is extremely weak, and the callus redifferentiation, cell embryo maturation, germination and plant regeneration technology require high standards; third, the existing walnut system is inefficient and unstable, and lacks an efficient genetic transformation system.

[0003] Due to the particularity of walnut materials, the application of Agrobacterium tumefaciens in walnut genetic transformation is cumbersome and time-consuming, and it is difficult to efficiently obtain complete regenerated plants or partial plant organs. At present, there is no report on an efficient hairy root induction system on walnut. By using the rooting characteristics of Agrobacterium rhizogenes, genetically stable transgenic hairy root organs can be obtained in a short period of time. Therefore, it can be used as a transgenic method for walnut genetic improvement and gene function analysis.

[0004] The invention adopts the Agrobacterium rhizogenes-mediated method and utilizes young roots germinated from walnut seeds to obtain a stable walnut transgenic hairy root system, thereby providing a simple and efficient walnut transgenic method. Summary of the invention

[0005] The purpose of the present invention is to provide a method for efficiently inducing hairy roots of cultivated walnuts by using Agrobacterium rhizogenes to solve the following technical problems.

[0006] The technical problems to be solved are as follows:

[0007] (1) To provide a simple walnut genetic modification operation process;

[0008] (2) Selecting technical means suitable for walnuts to significantly reduce time costs;

[0009] (3) Improve the efficiency of transgenic walnuts and establish a stable and efficient transgenic method.

[0010] The present invention adopts the following technical solutions:

[0011] A method for efficiently inducing hairy roots of cultivated walnuts by using Agrobacterium rhizogenes comprises the following steps:

[0012] Step 1, treating walnut seeds by low-temperature soaking, shelling and low-temperature germination methods, waiting for the radicle to germinate and break through the seed coat to form a taproot, and the taproot elongates to 2-6cm and 2-3mm in diameter, and then serving as an infection material for later use;

[0013] Step 2, preparing an Agrobacterium rhizogenes infection solution;

[0014] Step 3: Use the root-generating Agrobacterium infection solution to infect the main root of the infected material within 3 cm from the cotyledon connection point. After the infection is completed, co-cultivation and rooting culture are carried out.

[0015] Furthermore, the step 1 specifically includes the following steps:

[0016] Step 1.1, Seed selection: Select fruits of appropriate size, full kernels, no mechanical damage, and no mold contamination as materials for germination of seedlings;

[0017] Step 1.2, soaking seeds: Soak the seeds in water at 5-10℃, change the water 1-2 times a day, and soak for 7-14 days to allow the walnut seeds to fully absorb water;

[0018] Step 1.3, baking: After the soaked walnut seeds are drained, they are placed in an oven and baked at 30-35°C for 5-10 hours to keep the surface of the walnut shell dry;

[0019] Step 1.4, shell cracking: Apply force vertically along the suture line to gently squeeze the top of the walnut to crack it, and use a tool to remove part of the hard shell at the top to expose the seed germination point;

[0020] Step 1.5, germination: Place the walnut seeds on the surface of the pot vertically with the suture line, and place them layer by layer in an offset manner so that the radicle of each seed has enough space to grow downward when it germinates. After the seeds are placed in the pot layer by layer, cover them with plastic wrap to maintain a certain humidity in the pot. Place the plastic pot in an incubator and culture it in the dark at 20-23℃ for 10-20 days. Observe regularly during the period. When the radicle germinates and breaks through the seed coat to form a taproot, and the taproot elongates to 2-6cm and 2-3mm in diameter, it is ready for use.

[0021] Furthermore, the step 2 specifically includes the following steps:

[0022] Step 2.1, prepare an infection buffer containing 10 mM MES, 10 mM MgCl 2, 200 μM AS, and adjust the pH to 5.5-5.7 with 1 M KOH solution;

[0023] Step 2.2, melt the K599 Agrobacterium rhizogenes containing AtUBQ10::GFP stored at -80°C, take 10 μL and add it to 1 ml LB medium containing corresponding antibiotics, and activate the bacteria by shaking;

[0024] Step 2.3: Expand the culture of the activated Agrobacterium rhizogenes until OD 600 When the concentration reaches 1.0-1.2, the bacteria are collected by centrifugation at 5000 rpm, and then suspended and washed with infection buffer, and the bacteria are collected by centrifugation at 5000 rpm;

[0025] Step 2.4: Resuspend the cells in infection buffer and adjust the OD 600 To 0.6-1.0, stand in the dark at 25℃ for 2-4 hours to obtain the Agrobacterium rhizogenes infection solution.

[0026] Furthermore, the step 3 specifically includes the following steps:

[0027] Step 3.1, prepare peat soil and vermiculite in a ratio of 1:1, add purified water, and put the prepared substrate into a cultivation pot with a substrate thickness of 6-8 cm;

[0028] Step 3.2, infection and transformation: take out the walnut seeds with taproots obtained in step 1, place them in a collection basin, cover them with a wet cloth to keep them moist, use a sterile syringe to absorb the prepared infection solution, with the needle tip facing up, tilted at 45 degrees, and pierce holes around the taproot within 3 cm from the cotyledon connection point from bottom to top, with a depth of about 1.0-1.5 mm, and pierce 20-30 holes in total to obtain the infected material;

[0029] Step 3.3, transfer the infected material obtained in the previous step into a cultivation pot, place it for 20-30 minutes, then cover it with 2.0-3.0 cm of substrate, and cover it with plastic wrap to keep it moist;

[0030] Step 3.4, transfer to an incubator and culture in the dark at 21-23°C for 2-3 days;

[0031] Step 3.5: After the co-cultivation is completed, perform rooting culture at 23°C, dark for 8 hours and 25°C, light for 16 hours, and observe the germination of transgenic hairy roots after 2-3 weeks.

[0032] After adopting the above technical solution, the present invention has the following advantages:

[0033] 1. The present invention adopts low-temperature seed soaking, shell breaking and low-temperature germination technology to greatly reduce the massive reproduction of microorganisms during the germination of walnut seeds, especially significantly shorten the time required for seed germination and germination, greatly saving time cost.

[0034] 2. The seeds of cultivated walnut are relatively large in size, and the radicle germinates and breaks through the seed coat to form a taproot. The taproot extends to 2-6cm and has a diameter of 2-3mm. The radicle at this stage is of moderate size and has a very low degree of lignification, which is convenient for genetic engineering operations and suitable for Agrobacterium infection by syringe puncture method. This method has low requirements for instruments and equipment, is simple and convenient to operate, and has high efficiency.

[0035] 3. The seed materials in this period have good germination, strong vitality, and vigorous growth. The root cells within 3 cm from the cotyledon connection point on the taproot have strong division ability, rapid growth and expansion characteristics, and potential rooting ability, which are easily infected by Agrobacterium. It can greatly improve the positive hairy root induction rate and the positive hairy root plant rate. Experiments show that using our patented method, the proportion of positive hairy roots in cultivated walnut (temperature 185) can reach 90%, and the positive hairy root plants can reach more than 97%.

[0036] The present invention is described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Indicates the infection treatment method, where a is a walnut seed; b is a young walnut taproot damaged by a syringe; c is a small droplet of infection liquid formed at the wound when the taproot is damaged; d is a walnut taproot that has grown for too long, and it can be seen that young stems have grown, and the taproot has begun to swell and lignify, which is no longer suitable for infection operation;

[0038] Figure 2 The results of hairy root induction are shown in the handheld fluorescence detection results, where Figure a shows the positive callus at 14 days; Figure b shows the positive callus and the formed hairy roots at 21 days; Figure c shows the positive hairy roots cultured for 35 days; Figures de are the partial enlarged views of the growth of the positive hairy roots corresponding to Figures ac, respectively;

[0039] Figure 3 This is a technical operation flow chart. DETAILED DESCRIPTION

[0040] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0041] Preparation

[0042] (1) Required strains: Construction of K599 Agrobacterium carrying AtUBQ10::GFP vector

[0043] (2) Prepare reagents: 1M MES; 2M MgCl2; 0.5M AS (DMSO); 1M KOH

[0044] (3) Required instruments: light incubator, pH meter, 50 ml centrifuge tube, high-speed refrigerated centrifuge, UV spectrophotometer, blue-cap bottle, pipette, etc.

[0045] Example 1: Preparation of Wen 185 Infected Materials

[0046] (1) Seed selection: Generally, W185 walnuts harvested this year or the previous year are selected. Fruits of appropriate size, full kernels, no mechanical damage, and no mold contamination are selected as materials for germinating seedlings.

[0047] (2) Soaking seeds: Soak the seeds in water at a ambient temperature of 10°C, changing the water twice a day for 10 days to allow the walnut seeds to fully absorb water. During this process, you can use heavier objects to completely immerse the walnut seeds in water to prevent them from floating and incomplete soaking.

[0048] (3) Baking: After soaking, drain the walnut seeds and place them in an oven at 35°C for 8 hours to keep the surface of the walnut shell dry.

[0049] (4) Shell breaking: Apply force vertically along the suture line to gently squeeze the top of the walnut to crack it, and use a tool to remove part of the hard shell at the top to expose the seed germination point;

[0050] (5) Germination: Prepare a plastic pot of appropriate size (can be placed in an incubator), place walnut seeds on the pot surface with the suture line perpendicular to the suture line, and place them layer by layer in an offset manner so that the radicle of each seed has enough space to grow downward when it germinates. After the seeds are placed layer by layer, cover them with plastic wrap to maintain a certain humidity in the pot. Place the plastic pot in an incubator and culture it in the dark at 22°C for about 10-20 days. Observe it regularly during the period. When the radicle germinates and breaks through the seed coat to form a taproot, and the taproot elongates to 2-6 cm and has a diameter of 2-3 mm, it is ready for use.

[0051] Example 2: Activation of Agrobacterium and preparation of infection solution

[0052] (1) The expression vector carrying the AtUBQ10::GFP fragment was constructed into K599 Agrobacterium by electroporation, and the strain was preserved after PCR verification.

[0053] (2) The concentrations of the components in the infection buffer are: 10 mM MES, 10 mM MgCl2, 200 μM AS, and the pH is adjusted to 5.65 with 1 M KOH. Store away from light for future use. Note: The infection buffer must be prepared and used immediately to ensure a good infection effect.

[0054] (3) Thaw the K599 Agrobacterium rhizogenes containing AtUBQ10::GFP stored at -80°C on ice, take 10 μL and add it to 1 ml LB medium (containing the corresponding antibiotics), and activate the bacteria by shaking.

[0055] (4) The activated Agrobacterium was expanded and cultured until OD600 reached 1.0, and the cells were collected by centrifugation at 5000 rpm. The cells were suspended and washed with infection buffer and centrifuged at 5000 rpm to collect the cells.

[0056] (5) Resuspend the cells in infection buffer and adjust the OD600 to 0.8. Incubate at 25°C in the dark for 3 h.

[0057] Example 3: Infection and transformation

[0058] (1) Peat soil and vermiculite are prepared in a ratio of 1:1, and an appropriate amount of purified water is added. It is advisable to squeeze the substrate by hand until water hangs between the fingers without falling off. The prepared substrate is placed in a cultivation pot with a substrate thickness of about 6-8 cm;

[0059] (2) Figure 1 (b, c) Carefully remove the walnut seeds with the taproot, place them in a collection basin, and cover them with a damp cloth to keep them moist. Use a sterile syringe to absorb the prepared infection solution, with the needle tip facing up, tilted 45 degrees, and pierce holes around the taproot from bottom to top, with a depth of about 1.0-1.5mm. After each piercing, gently press the syringe push handle so that a small drop of liquid can hang on the hole. Generally, 20-30 holes are appropriate.

[0060] Example 4: Co-cultivation

[0061] (1) The infected material is promptly transferred to a cultivation pot and placed for 20 minutes. Then, a 2.0-3.0 cm thick substrate is placed on top and covered with plastic wrap to keep it moist.

[0062] (2) Transfer to an incubator and culture in the dark at 22°C for 3 days;

[0063] Example 5: Rooting culture

[0064] After the co-cultivation was completed, rooting culture was carried out at 23°C, dark for 8 hours; and 25°C, light for 16 hours. The germination of transgenic hairy roots was observed after 2-3 weeks.

[0065] like Figure 2 As shown, it can be seen that the method of the present invention can achieve a positive hairy root ratio of 90% for cultivated walnut (temperature 185), and a positive hairy root plant ratio of more than 97%.

[0066] The above is an example of the best implementation of the present invention, and the parts not described in detail are common knowledge of ordinary technicians in the field. The protection scope of the present invention shall be based on the content of the claims, and any equivalent transformation based on the technical enlightenment of the present invention is also within the protection scope of the present invention.

Claims

1. A method for efficiently inducing hairy roots of cultivated walnuts by Agrobacterium rhizogenes, characterized in that: The following steps are involved: Step 1, treating walnut seeds by low-temperature soaking, shelling and low-temperature germination methods, waiting for the radicle to germinate and break through the seed coat to form a taproot, and the taproot elongates to 2-6cm and 2-3mm in diameter, and then serving as an infection material for later use; Step 2, preparing an Agrobacterium rhizogenes infection solution; Step 3: Use the root-generating Agrobacterium infection solution to infect the main root of the infected material within 3 cm from the cotyledon connection point. After the infection is completed, co-cultivation and rooting culture are carried out.

2. The method for efficiently inducing hairy roots of cultivated walnuts by Agrobacterium rhizogenes according to claim 1, characterized in that: The step 1 specifically comprises the following steps: Step 1.1, Seed selection: Select fruits of appropriate size, full kernels, no mechanical damage, and no mold contamination as materials for germination of seedlings; Step 1.2, soaking seeds: Soak the seeds in water at 5-10℃, change the water 1-2 times a day, and soak for 7-14 days to allow the walnut seeds to fully absorb water; Step 1.3, baking: After the soaked walnut seeds are drained, they are placed in an oven and baked at 30-35°C for 5-10 hours to keep the surface of the walnut shell dry; Step 1.4, shell cracking: Apply force vertically along the suture line to gently squeeze the top of the walnut to crack it, and use a tool to remove part of the hard shell at the top to expose the seed germination point; Step 1.5, germination: Place the walnut seeds on the surface of the pot vertically with the suture line, and place them layer by layer in an offset manner so that the radicle of each seed has enough space to grow downward when it germinates. After the seeds are placed in the pot layer by layer, cover them with plastic wrap to maintain a certain humidity in the pot. Place the plastic pot in an incubator and culture it in the dark at 20-23℃ for 10-20 days. Observe regularly during the period. When the radicle germinates and breaks through the seed coat to form a taproot, and the taproot elongates to 2-6cm and 2-3mm in diameter, it is ready for use.

3. The method for efficiently inducing hairy roots of cultivated walnuts by Agrobacterium rhizogenes according to claim 1, characterized in that: The step 2 specifically includes the following steps: Step 2.1, prepare an infection buffer containing 10 mM MES, 10 mM MgCl 2, 200 μM AS, and adjust the pH to 5.5-5.7 with 1 M KOH solution; Step 2.2, melt the K599 Agrobacterium rhizogenes containing AtUBQ10::GFP stored at -80°C, take 10 μL and add it to 1 ml LB medium containing corresponding antibiotics, and activate the bacteria by shaking; Step 2.3: Expand the culture of the activated Agrobacterium rhizogenes until OD 600 When the concentration reaches 1.0-1.2, the bacteria are collected by centrifugation at 5000 rpm, and then suspended and washed with infection buffer, and the bacteria are collected by centrifugation at 5000 rpm; Step 2.4: Resuspend the cells in infection buffer and adjust the OD 600 To 0.6-1.0, stand in the dark at 25℃ for 2-4 hours to obtain the root-generating Agrobacterium infection solution.

4. The method for efficiently inducing hairy roots of cultivated walnuts by Agrobacterium rhizogenes according to claim 1, characterized in that: The step 3 specifically comprises the following steps: Step 3.1, prepare peat soil and vermiculite in a ratio of 1:1, add purified water, and put the prepared substrate into a cultivation pot with a substrate thickness of 6-8 cm; Step 3.2, infection and transformation: take out the walnut seeds with taproots obtained in step 1, place them in a collection basin, cover them with a wet cloth to keep them moist, use a sterile syringe to absorb the prepared infection solution, with the needle tip facing up, tilted at 45 degrees, and pierce holes around the taproot within 3 cm from the cotyledon connection point from bottom to top, with a depth of about 1.0-1.5 mm, and pierce 20-30 holes in total to obtain the infected material; Step 3.3, transfer the infected material obtained in the previous step into a cultivation pot, place it for 20-30 minutes, then cover it with 2.0-3.0 cm of substrate, and cover it with plastic wrap to keep it moist; Step 3.4, transfer to an incubator and culture in the dark at 21-23°C for 2-3 days; Step 3.5: After the co-cultivation is completed, perform rooting culture at 23°C in the dark for 8 hours and at 25°C in the light for 16 hours, and observe the germination of transgenic hairy roots after 2-3 weeks.

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