Instantaneous transformation method for living grape leaves

Abstract

The invention provides a live grape leaf instantaneous transformation method, and belongs to the technical field of breeding. The method comprises the following steps: (1) culturing grape seedlings; (2) instantaneous transformation of grape leaves: taking hydroponic grape seedlings in a suitable transformation stage, and wrapping grape root systems with wet cotton; completely intruding grape leaves into the infection penetrating fluid, and vacuumizing; taking out the grape seedlings after preset time, continuously placing the grape seedlings in the Hoagland culture solution, and recovering culture. According to the method, the root system of the grape is wrapped by the wet cotton, so that the root system is prevented from being damaged by vacuum water loss, the condition that the leaves are mechanically damaged due to transformation in a traditional syringe or centrifugal tube is avoided, and the grape leaves are completely intruded into the infection penetrating fluid, so that the uniformity and stability of transformation of the living leaves can be ensured; according to the method, the transformation efficiency and stability are effectively improved, the transformation period can be remarkably shortened, more efficient and reliable technical support is provided for grape gene function research, and the method has wide application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of breeding, and in particular to a method for instantaneous transformation of living grape leaves. Background Art

[0002] As an important economic crop, grapes have always been an important object of plant breeding and genetic research due to their significant economic value. Conventional hybrid breeding is inefficient and time-consuming, which limits the development of new germplasm and the progress of variety improvement. With the continuous development and improvement of molecular breeding technology, the breeding of new grape varieties will be more accurate and efficient. In order to improve the quality, stress resistance and improve the characteristics of grape varieties, although a stable grape genetic transformation system is the best choice, it faces some challenges in actual operation, such as low transformation efficiency and long transformation cycle, which greatly limits the research progress. Therefore, the development of a fast and efficient grape leaf transient transformation technology is of great significance to promote gene function verification.

[0003] Although some transient transformation methods have been proposed and applied, these technologies still face some significant limitations. For example, when using a syringe or centrifuge tube for transformation, mechanical damage to the leaves often occurs, which not only affects the transformation effect, but also has a negative impact on plant health; the diffusion effect of the injected liquid is usually poor, resulting in uneven distribution of the transformation liquid on the leaves, thus affecting the transformation efficiency and stability; in addition, the in vitro transient transformation method often causes irreversible damage to the leaves. This damage not only causes the leaves to rot and die quickly after treatment, but also affects the reliability of subsequent experimental data. These problems have brought many difficulties in actual operation, making the balance between efficient transformation and plant health maintenance in existing transient transformation technologies more complicated. Summary of the invention

[0004] In view of this, in order to solve the deficiencies in the above-mentioned background technology, the present invention provides a method for instantaneous transformation of living grape leaves, which wraps the grape roots with wet cotton to avoid vacuum dehydration damage to the roots, avoids the mechanical damage to the leaves caused by the traditional transformation using a syringe or a centrifuge tube, and completely immerses the grape leaves in the infection infiltration liquid to ensure the uniformity and stability of the transformation of the living leaves, effectively improves the transformation efficiency and stability, and can significantly shorten the transformation cycle, providing more efficient and reliable technical support for the study of grape gene function, and has broad application prospects.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for instantaneous transformation of living grape leaves comprises the following steps:

[0007] Step (1), cultivation of grape seedlings:

[0008] Select healthy grape seedlings from their own roots, clean the substrate on the roots and place them in Hogland nutrient solution for hydroponics until the number and state of their leaves reach an appropriate transformation stage;

[0009] Step (2), instantaneous transformation of grape leaves:

[0010] Take the hydroponic grape seedlings at a suitable transformation stage and wrap the grape roots with wet cotton; completely immerse the grape leaves in the infiltration solution and evacuate the solution; take out the grape seedlings after a preset time, continue to place them in the Hoagland culture solution, and resume culture.

[0011] Preferably, in step (2), the preparation method of the infection penetrant is as follows:

[0012] Pick the target gene monoclonal colony and place it in LB culture medium containing resistance for expansion culture until the bacterial solution turns orange-yellow; collect the bacteria after centrifugation, resuspend the bacteria with the prepared infiltration solution, and adjust the OD 600 At 0.5-0.6, the infection penetrant is obtained.

[0013] Preferably, the permeate is:

[0014] Each liter of distilled water contains 2.1325 g of MES, 2.033 g of MgCl2, and 750 ul of acetosyringone.

[0015] Preferably, the prepared infection penetrant is allowed to stand at 28° C. for 2-3 hours.

[0016] Preferably, in step (2), vacuum is drawn at 88 kPa for 15-20 minutes.

[0017] Preferably, in step (1), the Hoagland culture solution is: 1.26 g of Hoagland powder and 0.945 g of calcium salt are added to each liter of distilled water.

[0018] Preferably, in step (1), the hydroponic conditions are: hydroponic culture at 25° C. with light for 4-6 days to allow the grape seedlings to complete the water transfer.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The instantaneous transformation method of living grape leaves provided by the present invention wraps the grape root system with wet cotton to avoid vacuum water loss and damage to the root system, avoids the situation where the traditional transformation is carried out in a syringe or a centrifuge tube and causes mechanical damage to the leaves, and completely infiltrates the grape leaves into the infection infiltration liquid to ensure uniform and stable transformation of the living leaves, effectively improves the transformation efficiency and stability, and can significantly shorten the transformation cycle, providing more efficient and reliable technical support for grape gene function research, and has broad application prospects.

[0021] The instantaneous transformation method of living grape leaves proposed for the first time in the present invention simplifies the experimental operation steps and reduces the demand for complex equipment compared with traditional transformation methods; the whole living seedling infection and transformation significantly reduces the mechanical damage to the leaves and avoids rot and death; the use of improved infection methods and vacuum treatment steps greatly improves the transformation efficiency of exogenous genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the instantaneous transformation process of living grape leaves. Figure A shows the hydroponic culture of self-rooted grape seedlings; Figure B shows the root system of grape seedlings wrapped in wet cotton; Figure C shows the device for infecting grape leaves with bacterial solution; Figure D shows the vacuum extraction stage; Figure E is a partial enlarged view of Figure D (the stage when bacterial solution penetrates the leaves).

[0023] Figure 2 Comparison diagram of in vivo and in vitro transformation of grape leaves. AB is the observation of grape phenotypes on the 0th and 15th days after in vivo bacterial infection and transformation; CD is a partial enlargement of grape leaves in B; EF is the observation of grape leaf phenotypes on the 0th and 5th days after in vitro bacterial infection and transformation; GH is a partial enlargement of F.

[0024] Figure 3 This is the identification of transiently expressed proteins and their functional verification. Figure A shows the continuous protein expression after VvNAC33 transient transformation; Figure B shows the drought resistance function of VvNAC33 verified by transient transformation. DETAILED DESCRIPTION

[0025] The present invention provides a method for instantaneous transformation of living grape leaves, comprising the following steps:

[0026] Step (1), cultivation of grape seedlings:

[0027] Select healthy grape self-rooted seedlings, clean the substrate on the roots and place them in Hoagland nutrient solution for hydroponics until the number and state of their leaves reach a suitable transformation stage (preferably culture for 3-5 days until the number and state of their leaves reach a suitable transformation stage).

[0028] The preferred Hoagland culture medium is: 1.26 g of Hoagland powder and 0.945 g of calcium salt are added to each liter of distilled water.

[0029] The hydroponic conditions are preferably: 25°C light hydroponic culture for 4-6 days to allow the grape seedlings to complete the water transfer.

[0030] Among them, strong self-rooted grape seedlings can be selected from strong 5-8 leaf self-rooted grape seedlings. The appropriate transformation stage refers to the prevention period from the emergence of grape seedlings to before germination.

[0031] Step (2), instantaneous transformation of grape leaves:

[0032] Take hydroponic grape seedlings at a suitable transformation stage, wrap the grape roots with wet cotton (to avoid vacuum water loss and damage to the roots, as well as mechanical damage and rot to the grape seedlings caused by traditional centrifuge tubes); completely immerse the grape leaves in the infection infiltration solution (to ensure uniform and stable transformation of living leaves), and evacuate (preferably 88kpa vacuum for 15-20 minutes and then release); take out the grape seedlings after a preset time, continue to place them in the Hoagland culture solution, and resume culture.

[0033] In the present invention, in step (2), the preparation method of the infection penetrant is as follows:

[0034] Pick the target gene monoclonal colony and place it in LB culture medium containing resistance for expansion culture until the bacterial solution turns orange-yellow; collect the bacteria after centrifugation, resuspend the bacteria with the prepared infiltration solution, and adjust the OD 600 At 0.5-0.6, the infection penetrant is obtained. Preferably, the prepared infection penetrant is allowed to stand at 28° C. for 2-3 hours.

[0035] Wherein, the permeate is preferably:

[0036] Each liter of distilled water contains 2.1325 g of 2-(N-morpholinyl)ethanesulfonic acid hydrate (MES); 2.033 g of MgCl2; and 750 ul of acetosyringone.

[0037] The technical solution of the present invention is clearly described in detail below in conjunction with specific embodiments.

[0038] Example 1

[0039] 1. Cultivation of grape seedlings

[0040] (1) Prepare Hoagland nutrient solution by adding 1.26 g of Hoagland powder and 0.945 g of calcium salt to 1 L of distilled water;

[0041] (2) Select healthy grape seedlings with 5 to 8 leaves, gently separate the seedlings from the soil, clean the substrate attached to the roots, and then place them in Hoagland nutrient solution for hydroponics;

[0042] (3) inserting the separated grape seedlings into Hoagland's nutrient solution and culturing them hydroponically at 25°C for 5 days to allow the grape seedlings to complete the water transfer;

[0043] (4) Cultivate for another 4 days until the number and condition of the leaves reach an appropriate transformation stage.

[0044] 2. Preparation of infection penetrant

[0045] (1) Pick a monoclonal colony of the target gene (taking VvNAC33, a gene that has been confirmed to be involved in drought resistance regulation, as an example) and place it in LB culture medium containing resistance for expansion culture until the bacterial solution turns orange-yellow;

[0046] (2) Prepare the infection infiltration solution: 1L of distilled water contains 2.1325g of MES, 2.033g of MgCl2, and 750ul of acetosyringone;

[0047] (3) Pour the VvNAC33 bacterial suspension into 50 mL centrifuge tubes, centrifuge at 5000 g for 10 minutes at room temperature, collect the bacterial cells, resuspend the cells in the prepared permeabilization solution, and adjust the OD 600 is 0.6;

[0048] (4) Let the prepared infection solution stand at 28°C for 3 hours before use.

[0049] 3. Instantaneous transformation of grape leaves

[0050] (1) Take a hydroponic grape seedling at an appropriate transformation stage and wrap the grape root system with wet cotton;

[0051] (2) The grape leaves are completely immersed in a beaker containing the infection penetrant, with the roots placed outside the beaker to ensure that the root system is not damaged and prevent rot;

[0052] (3) Close the lid, connect the vacuum pump to the vacuum chamber, draw a vacuum at 88 kPa, a large number of bubbles on the leaf surface are removed, and the infected penetrant begins to boil;

[0053] (4) The pressure remains constant and the gas is released after 15-20 minutes;

[0054] (5) Take out the grape seedlings, rinse them with clean water, and place them back in the Hoagland culture medium for cultivation at 25°C under light.

[0055] Comparative Example 1

[0056] The grape leaves were collected and subjected to instantaneous transformation treatment using the same treatment method as in Example 1. The leaves were taken out after vacuuming, and the excess bacterial solution on the surface of the leaves was gently wiped off. The grape petioles were wrapped with wet cotton and sealed with plastic wrap to prevent excessive water loss. The leaves were cultured at 25°C under light.

[0057] After testing and verification, according to Figure 1 The in vivo grape leaf transient transformation method performed according to the steps shown is superior to the in vitro grape leaf transformation method. Figure 2 As shown, the in vivo transformation method of the present invention significantly prolongs the subsequent experimental operation time, while avoiding interference factors such as rot and mildew. In the study of stress such as drought treatment, inoculation of diseases and fungi, this method provides great convenience.

[0058] Example 2

[0059] 1. Detection of exogenous genes in transient transformation in vivo

[0060] (1) Collecting grape seedling leaves at 1, 5, 10, 15, and 20 days after in vivo transient transformation;

[0061] (2) After grinding with liquid nitrogen, extract leaf protein;

[0062] (3) Western-blot was used to detect the expression duration of VvNAC33 protein.

[0063] 2. Verifying the feasibility of transient transformation in vivo using known functional genes

[0064] (1) Construct an overexpression vector of the drought resistance regulatory gene VvNAC33, which has been confirmed to be involved in drought resistance, and transform Agrobacterium;

[0065] (2) Three grape seedlings were taken and VvNAC33 was transiently overexpressed in grape leaves;

[0066] (3) Another three grape seedlings were vacuum-infected with only the infiltration solution (without the VvNAC33 gene) as a control group;

[0067] (4) After recovery, 4% PEG 6000 was added to both the control and experimental groups to simulate drought treatment;

[0068] (5) Treat with drought for 3 days, and observe and record the phenotype.

[0069] After testing and verification, Figure 3 As shown, the instantaneous gene transformation experiment verifies that the present invention can indeed be effectively used for gene function identification. Since grape leaves can maintain their natural physiological environment in the living state, the gene expression effect after transformation can last for about 16 days, which is more durable and stable than the traditional method. This method greatly improves the efficiency of gene function research and allows gene function evaluation to be performed directly in the leaves.

Claims

1. A method for instantaneous transformation of living grape leaves, characterized in that: The steps include: Step (1), cultivation of grape seedlings: Select healthy grape seedlings from their own roots, clean the substrate on the roots and place them in Hogland nutrient solution for hydroponics until the number and state of their leaves reach the appropriate transformation stage; Step (2), instantaneous transformation of grape leaves: Take the hydroponic grape seedlings at a suitable transformation stage and wrap the grape roots with wet cotton; completely immerse the grape leaves in the infiltration solution and evacuate the solution; take out the grape seedlings after a preset time, continue to place them in the Hoagland culture solution, and resume culture.

2. The method for instantaneous transformation of living grape leaves according to claim 1, characterized in that: In step (2), the preparation method of the infection penetrant is as follows: Pick the target gene monoclonal colony and place it in LB culture medium containing resistance for expansion culture until the bacterial solution turns orange-yellow; collect the bacteria after centrifugation, resuspend the bacteria with the prepared infiltration solution, and adjust the OD 600 At 0.5-0.6, the infection penetrant is obtained.

3. A method for instantaneous transformation of living grape leaves according to claim 2, characterized in that: The permeate is: Each liter of distilled water contains 2.1325 g of MES, 2.033 g of MgCl2, and 750 ul of acetosyringone.

4. The method for instantaneous transformation of living grape leaves according to claim 2, characterized in that: Let the prepared infection penetrant stand at 28°C for 2-3 hours.

5. The method for instantaneous transformation of living grape leaves according to claim 1, characterized in that: In step (2), vacuum is applied at 88 kPa for 15-20 minutes.

6. The method for instantaneous transformation of living grape leaves according to claim 1, characterized in that: In step (1), the Hoagland culture solution is prepared by adding 1.26 g of Hoagland powder and 0.945 g of calcium salt to each liter of distilled water.

7. The method for instantaneous transformation of living grape leaves according to claim 1, characterized in that: In step (1), the hydroponic conditions are: 25° C. light hydroponic culture for 4-6 days to allow the grape seedlings to complete the water transfer.

Images