Method for inducing plant callus under non-tissue culture condition

Abstract

The invention discloses a method for inducing plant callus under a non-tissue culture condition, and belongs to the field of plant tissue regeneration. The method comprises the steps that target plants are selected, rape and potatoes grow to a five-leaf stage, and arabidopsis thaliana grows to germinate for 2-3 weeks; applying 50-70 [mu] A direct current to main veins of plant leaves for electrical stimulation; the electrical stimulation treatment mode is that the current is continuously applied for 3-10 seconds, and then the current is continuously applied for 3-10 seconds after 3-10 seconds; treating once every day, and continuously treating for 4-6 days; and then carrying out bagging culture on the whole plant for 36-60 hours, and inducing a local area to form calluses. The method does not need in-vitro tissue culture, exogenous hormones and sterile conditions, can directly induce the local tissue dedifferentiation of the whole plant in a natural or artificial environment, is simple and convenient to operate, has high induction efficiency, is suitable for various plant types, and has wide application prospects.

Description

Technical Field

[0001] The invention belongs to the field of plant callus induction, and particularly relates to a method for inducing plant callus under non-tissue culture conditions. Background Art

[0002] Callus is a highly vacuolated, amorphous mass of thin-walled cells formed by plant organs, tissues, or cells in vitro, through dedifferentiation under suitable conditions, stimulated by in vitro culture or physical injury (such as a cut). Callus is the foundation of gene transformation, tissue culture, and plant regeneration, and is widely used in breeding, natural product extraction, and other fields.

[0003] However, not all plant wounds can produce callus. Callus induction primarily involves manipulating external conditions such as culture medium composition, exogenous hormone ratios, and environmental parameters to stimulate in vitro plant explants to alter their metabolic patterns, enhance the synthesis and metabolism of substances such as proteins and nucleic acids, and initiate the process of cell dedifferentiation to form amorphous cell clusters. This technique plays a fundamental role in the study of somatic embryogenesis, the construction of plant regeneration systems, and metabolic regulation mechanisms.

[0004] Currently, plant callus induction is still highly dependent on chemical induction with exogenous hormones (such as 2,4-D and NAA) and uses plant explants cultured in vitro (tissue culture). This method has defects such as a long induction period (usually ≥14 days), significant genotype dependence (the induction success rate of woody plants is <40%), and a high risk of epigenetic interference.

[0005] Therefore, finding a method that can achieve rapid and stable induction of plant callus under non-in vitro conditions is an urgent problem to be solved in the field of plant tissue culture and regeneration. Summary of the Invention

[0006] The object of the present invention is to overcome at least one deficiency of the prior art and provide a method for inducing plant callus under non-tissue culture conditions.

[0007] The technical solution adopted by the present invention is:

[0008] A first aspect of the present invention provides a method for inducing plant callus under non-tissue culture conditions, the method comprising the following steps:

[0009] (1) Selecting dicotyledonous plants grown to the seedling stage;

[0010] (2) Applying 50-70 μA direct current to the main veins of the plant leaves for electrical stimulation; the electrical stimulation treatment method is to continuously apply the current for 3-10 seconds, then wait for 3-10 seconds and then continue to apply the current for 3-10 seconds; the treatment is performed once a day for 4-6 days;

[0011] (3) After the electrical stimulation treatment, the aboveground parts of the plants were covered with bags for 36-60 hours to induce callus formation.

[0012] Preferably, the dicotyledonous plant in step (1) includes Cruciferae or Solanaceae.

[0013] More preferably, the dicotyledonous plant in step (1) is selected from one of rapeseed, potato, tobacco, tomato or Arabidopsis thaliana.

[0014] Preferably, the seedling stage in step (1) includes the five-leaf stage, the six-leaf stage, or 2-3 weeks after germination, wherein 2-3 weeks after germination is approximately equal to the five-leaf stage or the six-leaf stage.

[0015] Preferably, when the plant in step (1) is selected from rapeseed or potato, the seedling stage is the five-leaf stage;

[0016] And / or; when the plant in step (1) is selected from Arabidopsis thaliana, the seedling stage is 2-3 weeks after germination.

[0017] Preferably, the method of applying electrical stimulation to the main vein area of ​​the plant leaves in step (2) includes contacting the contact parts of two electrodes with the main vein of the same leaf, and being located on both sides of the midpoint of the main vein, wherein the diameter of the electrode contact part is less than or equal to the diameter of the main vein, and connecting the positive and negative poles of the DC power supply to form a loop.

[0018] More preferably, the contact portion of the two electrodes in step (2) is a stainless steel flat-end needle. When the flat-end needle contacts the leaf, it can prevent the leaf from being pierced. The outer diameter of the needle is preferably 0.5-0.7 mm. A needle with a diameter that is too small can easily injure the plant, while a needle with a diameter that is too large can make precise operation difficult. Therefore, the outer diameter is preferably 0.5-0.7 mm.

[0019] and / or; in step (2), the contact portion of the two electrodes is 1–3 cm away from the contact position on the main vein of the same leaf. There are no requirements for the positive and negative electrode connections; both positive and negative contacts are acceptable.

[0020] If the distance between the electrodes on the main vein is less than 1 cm, the positive and negative poles may be easily touched by mistake and cause a short circuit if the distance is too close; if it exceeds 3 cm, the distance is too far and the resistance is large, which may easily cause the stimulation current to be lower than the instrument setting value.

[0021] Preferably, the two electrodes in step (2) are handheld electrodes.

[0022] Preferably, in step (2), the plant leaf is at least one of the second, third, and fourth leaves.

[0023] Preferably, in step (2), 58-62 μA direct current is applied for electrical stimulation; and / or, in step (2), the electrical stimulation treatment method is to continuously apply current for 4-6 seconds, take an interval of 4-6 seconds, and then continuously apply current for 4-6 seconds.

[0024] More preferably, in step (2), 60 μA direct current is applied for electrical stimulation, and the electrical stimulation treatment method is to continuously apply the current for 5 seconds, take a 5-second interval, and then continuously apply the current for 5 seconds.

[0025] Preferably, in step (3), the bagging is a transparent plastic film bag; the plastic includes polyethylene (PE), polypropylene (PP), etc.

[0026] Preferably, in step (3), the relative humidity in the bag is maintained at 85%-90%. In the embodiment, the bagged plants are cultured in an artificial incubator with a humidity of 40%-45%. After bagging, the humidity in the bag is 85%-90%. Generally, in temperate or dry areas, the outdoor relative humidity is usually 40%-60%, and this standard can be achieved after bagging. It is sufficient to avoid a fluctuating outdoor environment.

[0027] Preferably, the culture conditions of the plants in step (2) and / or step (3) are a temperature of 20-25° C. and 16 h light / 8 h dark.

[0028] The second aspect of the present invention provides an application of the plant callus prepared by the above method, wherein the application is any one of the following (a1) to (a4):

[0029] (a1) Use in preparing new plants;

[0030] (a2) Use in the preparation of genetic transformation systems;

[0031] (a3) Application in plant cell culture;

[0032] (a4) Application in crop breeding.

[0033] Methods for genetic transformation of callus tissue include directly performing CRISPR-Cas9 editing on living leaf callus tissue (such as through Agrobacterium or nanocarrier delivery), using callus to differentiate into complete plants carrying target traits, avoiding the contamination risks that may occur in traditional tissue culture, and potentially shortening the genotype transformation cycle.

[0034] The crop breeding method includes inducing callus in a targeted manner on a living crop according to the above method, simultaneously applying drought / salt stress electric field signals, screening resistant plants regenerated from the mutant callus, and constructing a rapid evolution system for stress resistance.

[0035] The beneficial effects of the present invention include at least:

[0036] (1) Improved efficiency: The time for rapeseed leaf vein callus formation is shortened to 7 days (the traditional in vitro method requires 14 days).

[0037] (2) Simple operation and low cost: No clean bench, sterile environment or chemical additives are required, suitable for on-site processing, and more convenient to operate.

[0038] (3) Independence from in vitro culture: For the first time, a method for inducing plant callus tissue under non-tissue culture conditions was proposed. The operation was completed directly on the whole plant without the need to establish a cell line, which greatly improved the application scenarios and efficiency.

[0039] (4) No need for exogenous hormones: completely abandon the induction of hormones such as 2,4-D to avoid hormone toxic side effects and epigenetic risks.

[0040] (5) Targeted and precise induction: Electrical signals are transmitted through the vascular pathway to activate local regeneration programs and achieve efficient wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The diagram is a schematic diagram of the electrical stimulation induction experiment of rapeseed plants; Figure 1 A is the high humidity microenvironment culture after rapeseed is bagged. Figure 1 B and Figure 1 C is the callus tissue induced by electrical stimulation of rapeseed.

[0042] Figure 2 This is the callus tissue induced by electrical stimulation of Arabidopsis thaliana.

[0043] Figure 3 This is callus tissue induced by electrical stimulation of potato.

[0044] Figure 4 Schematic diagram of the use of handheld needle tube electrode. DETAILED DESCRIPTION

[0045] Existing plant callus induction techniques still primarily rely on ex vivo tissue culture of explants, requiring chemical induction in a sterile environment with high concentrations of exogenous hormones (such as 2,4-D and NAA). This method suffers from core drawbacks, including cumbersome procedures, high contamination risks, a long hormone induction cycle (typically ≥14 days), and significant influence of plant species on its efficiency. In woody plants in particular, the success rate of callus induction is often less than 40%, hindering its large-scale application.

[0046] In addition, the use of electrical stimulation to promote the proliferation of plant callus reported in the prior art still relies on an established callus system and requires an in vitro plant cell line, which cannot achieve in situ induction and has a long processing cycle, making it difficult to meet the needs of efficient field application.

[0047] In response to the above bottlenecks, the present invention provides a novel solution, namely, to achieve in situ induction of plant callus under non-tissue culture conditions through the synergistic technology of in vivo targeted electrical stimulation and high humidity microenvironment.

[0048] In some specific embodiments, the present application found that for rapeseed and potato plants in the five-leaf stage, two electrodes were precisely placed in contact with the main vein of the second, third, or fourth leaf, and located on both sides of the midpoint of the main vein. The contact parts of the two electrodes were 1-3 cm apart, and a 60μA DC current was applied. The stimulation method was to apply the current continuously for 5 seconds and then stop, and then apply the current continuously for 5 seconds after an interval of 5 seconds. Continuous treatment for 5 days can effectively activate cell reprogramming signals. The entire plant was then bagged to maintain a high humidity (relative humidity of 85%-90%) microenvironment for 48 hours, which can promote the formation of light-colored or white granular callus tissue near the electrical stimulation area. The experimental results showed that callus tissue could be observed with the naked eye 48 hours after bagging (7 days after the first electrical stimulation). This method is also applicable to a variety of plants such as Arabidopsis, showing good applicability and repeatability.

[0049] In some specific embodiments, the electrodes used are preferably handheld flat-end needle-tube stainless steel electrodes, the contact portion of which is a flat-end needle tube, and the outer diameter of the needle tube is preferably 0.5-0.7 mm (smaller than the diameter of the main vein). To facilitate flexible electrical treatment of plants, the electrodes are handheld to contact the main vein of the plant leaves and are located on both sides of the midpoint of the main vein. The distance between the contact portions of the two electrodes and the contact points on the main vein of the same leaf is preferably 1-3 cm, ensuring good conductive contact between the electrodes and the plant tissue without crushing the tissue.

[0050] The power supply device adopts a source meter unit (SMU), which is a four-quadrant voltage source / current source and a measuring instrument that can accurately measure the current value / voltage value simultaneously. Preferably, the source meter operation mode is set to: constant current output, the output current is set to 60 microamperes, and the feedback of the source meter on the output current shows that the output current fluctuates at the level of ±1 nanoampere. The compliance voltage is reasonably selected according to the characteristics of the plant leaves themselves. For most plants such as Arabidopsis, rapeseed, and potatoes, the compliance voltage is set to 12 volts to ensure that the output current reaches the set value (i.e., 60 microamperes); for crops such as corn and rice, the compliance voltage is set to 42 volts to ensure that the output current reaches the set value. The two electrodes are respectively connected to the positive and negative poles of the source meter, and a circuit is formed after the electrodes contact the conductive surface of the plant; when applying electrical stimulation, attention should be paid to the output feedback of the source meter to ensure that the current flows stably through the plant tissue.

[0051] In the present invention, callus formation is typically determined based on morphological criteria. Specifically, this involves observing the formation of amorphous, fragile, usually light-colored / white / yellowish / green cell clumps on the explant surface, which appear as irregular, disordered masses or tumors. This morphological characteristic is currently a common and effective criterion for identifying callus formation.

[0052] In the present invention, the term "non-tissue culture conditions" refers to culture conditions that directly manipulate whole plants in a natural or artificially controlled environment (such as a greenhouse or field) without relying on traditional tissue culture systems that require aseptic manipulation, explant excision, and culture medium induction. These environments allow the presence of microorganisms, including natural environments or conventional artificial culture environments, and are distinguished from the strict aseptic conditions required for tissue culture.

[0053] In the present invention, the term "targeted electrical stimulation" refers to the process of placing electrodes close to the main veins of leaves, utilizing the vascular bundles of the leaf veins to conduct electrical signals, activating the dedifferentiation pathways of adjacent parenchyma cells, and achieving targeted electrical signal delivery and local induction.

[0054] Compared with existing in vitro tissue culture operations, the present invention can directly generate callus tissue in situ on the entire plant without cutting tissue or performing sterile operations.

[0055] "Five-leaf stage" and "six-leaf stage" are agricultural terms used to describe the stages of plant growth and development. The five-leaf stage refers to the stage when the fifth leaf on the plant's main stem (main stalk) has fully expanded. The six-leaf stage refers to the stage when the sixth leaf on the plant's main stem (main stalk) has fully expanded.

[0056] In a preferred embodiment of the present invention, Figure 4 As shown, only one pair of electrodes is used for each electrical stimulation operation, and electrical stimulation is only performed on a single leaf, thereby avoiding current shunting or plant damage. The contact positions of the two electrodes and the main vein of the same leaf are located on both sides of the midpoint of the main vein. The spacing between the electrode contact parts can be flexibly adjusted according to the size of the leaf, and the spacing only needs to be controlled at 1-3 cm. That is, close to the middle area of ​​the leaf to ensure that the current is effectively conducted in the main vein tissue. At the same time, the contact part is a flat-headed needle tube made of stainless steel (outer diameter of 0.5-0.7 mm), which can ensure that the plant tissue is not damaged. There is no requirement for the connection of positive and negative poles, and both positive and negative contacts are acceptable.

[0057] Unless otherwise specified, the methods used in the following examples are conventional methods. For specific steps, please refer to: Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0058] The methods for obtaining the various biological materials described in the examples merely provide experimental methods for achieving the disclosed objectives and should not be construed as limiting the sources of the biological materials used in the present invention. In fact, the sources of biological materials used are diverse, and any legally and ethically accessible biological material may be substituted for and used as indicated in the examples.

[0059] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0060] Example 1

[0061] This example provides a method for inducing plant callus under non-tissue culture conditions, using rapeseed plants as experimental materials. The specific steps are as follows:

[0062] (1) Plant Pretreatment: Rapeseed plants grown in an artificial incubator at 22°C with a photoperiod of 16 h light / 8 h dark and reaching the five-leaf stage were selected as experimental materials. The rapeseed variety used in this example was Zhongshuangyi. Those skilled in the art may select rapeseed plants grown in the field at the five-leaf stage or other varieties of rapeseed plants.

[0063] (2) Electrode positioning and stimulation: The electrodes were placed precisely on the main vein of the second lobe, on both sides of the midpoint of the main vein, with the contact points of the two electrodes kept 1–3 cm apart. A 60 μA current was initiated for electrical stimulation. The stimulation was performed by applying the current continuously for 5 s, stopping the current, and then applying the current continuously for another 5 s after a 5-s interval.

[0064] In order to verify whether there are differences in the electrical stimulation effects between different leaves, the third and fourth leaves were treated separately according to the above conditions.

[0065] The electrical stimulation treatment lasted for five days. During the treatment period, the plants were placed in an artificial incubator at 22°C with a photoperiod of 16 hours light and 8 hours dark without bagging.

[0066] The electrodes used are handheld, flat-tipped stainless steel needle electrodes, preferably with an outer diameter of 0.5-0.7 mm. To facilitate flexible plant treatment, the electrodes are handheld and mounted on the main vein of the plant leaf, on either side of the midpoint of the main vein. The distance between the two electrodes is preferably 1-3 cm, ensuring good conductive contact with the plant tissue without crushing it. Experimental results show that on the same leaf, there is no significant difference in the induction effect when the spacing between the two electrodes on the main vein is adjusted between 1 cm and 3 cm.

[0067] The power supply unit uses a Sourcemeter Unit (SMU), a four-quadrant voltage / current source that can simultaneously and accurately measure current and voltage. Preferably, a Keithley 2401 or 2450 series SMU is used as the power supply unit. The operating mode is set to constant current output, with the output current set to 60 microamperes. Feedback from the SMU indicates that the output current fluctuation is within ±1 nanoampere.

[0068] (3) High humidity microenvironment regulation: After the electrical stimulation treatment on the 5th day, the cells were bagged. That is, a transparent polyethylene film bag (such as Figure 1 (As shown in A), completely cover the entire aerial part of the plant from the top, gently snapping the bag onto the edge of the pot or the base of the culture medium to create a relatively enclosed, high-humidity environment. Place the bagged plants in an artificial incubator at 22°C with a photoperiod of 16 hours light / 8 hours dark. Maintain the cover for 48 hours to maintain a microenvironmental condition with a relative humidity of at least 85% (85%-90%).

[0069] (4) Observation of callus induction: After the bagging treatment (i.e., the 8th day of electrical stimulation treatment), light-colored tumor-like protrusions can be observed with the naked eye in the electrode-treated area. The light-colored tumors are callus tissue. Further observation using a stereo microscope showed the following results: Figure 1 shown.

[0070] like Figure 1 As shown, on the 8th day after the electrical stimulation, obvious light-colored tumor-like callus blocks were visible to the naked eye in the electrode contact area (leaf veins) ( Figure 1 The texture of the tissue (indicated by the red arrows in B and 1C) is in sharp contrast to the surrounding green stems and is initially identified as callus tissue. Figure 1 B is a partial magnified image of the leaf vein, which clearly shows that light-colored particles are densely clustered and attached to the leaf vein epidermis, with a diameter of about 0.5-1mm. Figure 1C is a close-up at a higher magnification (stereo microscope), which further confirms that the structure is irregularly differentiated callus tissue with a typical granular and loose structure, which is significantly different from the compact texture of healthy leaf tissue.

[0071] To evaluate the callus induction effect of the method provided in Example 1 in rapeseed, this example further conducted three parallel replicate experiments, each including 24 rapeseed samples. The results are shown below and summarized in Table 1:

[0072] First time: 20 strains were successfully induced (induction rate: 83.33%);

[0073] Second time: 22 strains were successfully induced (induction rate: 91.67%);

[0074] The third time: 21 strains were successfully induced (induction rate: 87.50%).

[0075] The overall induction rate was stable between 83.3% and 91.7% (average: 87.5%), and the data variation was small (standard deviation of about 3.4%), proving that the results were highly reproducible.

[0076] Example 2

[0077] This example provides a method for inducing plant callus under non-tissue culture conditions, using Arabidopsis thaliana as the experimental material. Compared with Example 1, except that rapeseed is replaced by Arabidopsis thaliana, the remaining experimental steps are the same as those in Example 1. The specific steps are as follows:

[0078] (1) Plant pretreatment: Select Arabidopsis Col-0 plants grown for 2 weeks in an artificial incubator at 22°C with a photoperiod of 16 h light / 8 h dark;

[0079] (2) Electrode positioning and stimulation: The electrodes are precisely placed in contact with the main vein of the second leaf, and are located on both sides of the midpoint of the main vein, with the contact parts of the two electrodes kept 1–3 cm apart; 60 μA current stimulation is initiated, with the current applied continuously for 5 seconds, then stopped, followed by a 5-second interval, and then continued for another 5 seconds. Figure 4 shown.

[0080] In order to verify whether there are differences in the electrical stimulation effects between different leaves, the third and fourth leaves were treated separately according to the above conditions.

[0081] The entire stimulation treatment lasted for five days; during the treatment period, the plants did not need to be bagged and were cultured in an artificial incubator at 22°C with a photoperiod of 16h light / 8h dark.

[0082] (3) High humidity microenvironment regulation: After the fifth day of treatment, the plants were covered with transparent polyethylene bags and placed in an artificial incubator at 22°C with a photoperiod of 16 h light / 8 h dark, maintaining a high humidity environment for 48 h.

[0083] (4) Observation of callus induction: After the bagging treatment (i.e., the 8th day of electrical stimulation treatment), yellow-green punctate protrusions can be observed with the naked eye in the electrode treatment area. The yellow-green punctate protrusions are callus tissue. Further observation using a stereo microscope showed the following results: Figure 2 shown.

[0084] like Figure 2 A. Figure 2 As shown in Figure B, on the 8th day after electrical stimulation, densely distributed yellow-green dot-like callus masses were observed at the leaf veins of Arabidopsis thaliana. These masses were unevenly clustered and had a diameter of about 0.2-0.5 mm, and were clearly raised on the leaf surface.

[0085] To evaluate the callus induction effect of the method provided in Example 2 in Arabidopsis, this example further conducted three parallel replicate experiments, each including 72 Arabidopsis samples. The results are shown below and summarized in Table 1:

[0086] First round: 70 strains were successfully induced (induction rate: 97.22%);

[0087] Second round: 69 strains were successfully induced (induction rate: 95.83%);

[0088] The third time: 71 strains were successfully induced (induction rate: 98.61%).

[0089] The overall induction rate was as high as 97.2% (average), with a variation of only ±1.4%, indicating excellent data stability.

[0090] Example 3

[0091] This example provides a method for inducing plant callus under non-tissue culture conditions, using potato as the experimental material. Compared with Example 1, except that rapeseed is replaced by potato, the remaining experimental steps are the same as those in Example 1. The specific steps are as follows:

[0092] (1) Plant pretreatment: Potato A056 plants grown to the five-leaf stage in an artificial incubator at 22°C with a photoperiod of 16 h light / 8 h dark were selected;

[0093] (2) Electrode positioning and stimulation: The electrodes were placed precisely on the main vein of the second leaf, on either side of the midpoint of the main vein, with the contact points of the two electrodes kept 1–3 cm apart. A 60 μA current stimulation was initiated. The stimulation was performed by applying the current continuously for 5 s, stopping the current, and then applying the current continuously for another 5 s after a 5-s interval.

[0094] In order to verify whether there are differences in the electrical stimulation effects between different leaves, the third and fourth leaves were treated separately according to the above conditions.

[0095] The entire electrical stimulation treatment lasted for five days; during the treatment, the plants did not need to be bagged and were cultured in an artificial incubator at 22°C with a photoperiod of 16h light / 8h dark.

[0096] (3) High humidity microenvironment regulation: After the fifth day of treatment, the plants were covered with transparent polyethylene bags and placed in an artificial incubator at 22°C with a photoperiod of 16 h light / 8 h dark, maintaining a high humidity environment for 48 h.

[0097] (4) Observation of callus induction: After the bagging treatment (i.e., the 8th day of electrical stimulation treatment), white granular protrusions can be observed with the naked eye in the electrode treatment area. The white particles are callus tissue. Further observation using a stereo microscope showed the following results: Figure 3 shown.

[0098] like Figure 3 A and Figure 3 As shown in B, on the 8th day after electrical stimulation, dense white granular protrusions were observed on the surface of potato leaves. The white granules gathered in clusters on the leaf surface, and the callus tissue was concentrated in the electrode contact area.

[0099] To evaluate the callus induction effect of the method provided in Example 3 in potatoes, two parallel replicate experiments were conducted in this example, each including 6 potato samples. The results are shown below and summarized in Table 1:

[0100] First time: 5 strains were successfully induced (induction rate: 83.33%);

[0101] Second time: 6 strains were successfully induced (induction rate: 100%).

[0102] The overall induction rate reached 91.7% (11 / 12), and the induction rate of the second experiment was 100%, proving that the technology was extremely stable.

[0103] Table 1 Statistical results of plant callus induction experiment

[0104]

[0105]

[0106] Comparative Example 1

[0107] Compared with Example 1, except for the different electrode positioning in step (2), the other experimental steps including steps (1) and (3) are the same. The specific step (2) is as follows:

[0108] (2) Electrode Positioning and Stimulation: Electrodes were precisely placed in contact with the non-vein region of the leaf (i.e., the mesophyll region outside the vein region), with the contact points of the two electrodes kept 1–3 cm apart. A 60 μA current stimulation was initiated. The stimulation was performed by applying the current continuously for 5 seconds, stopping, then stopping for 5 seconds, and then applying the current continuously for another 5 seconds. The entire electrical stimulation treatment lasted for five days.

[0109] The experimental materials included 12 rapeseed plants, 12 Arabidopsis plants and 6 potato plants.

[0110] The experimental results showed that no light-colored / white / light yellow / green cell clusters were observed in the electrode contact area in all treated plants.

[0111] The results of Comparative Example 1 showed that positioning electrodes on both sides of the midpoint of the main vein of the leaf and at a certain distance played a key role in the effective induction of callus tissue, while positioning them at non-main vein locations failed to induce the formation of callus tissue.

[0112] Comparative Example 2

[0113] Compared with Example 1, this comparative example is the same as that of Example 1, except that the polyethylene bag is not used to cover the plants in step (3). The other experimental steps, including steps (1) and (2), are the same. The specific step (3) is as follows:

[0114] (3) Microenvironmental control: After the electrical stimulation treatment on the fifth day, the plants were directly placed in an artificial incubator at 22°C with a photoperiod of 16 h light / 8 h dark. The humidity of the artificial incubator was 40%-45%.

[0115] The experimental materials included 12 rapeseed plants, 12 Arabidopsis plants and 6 potato plants.

[0116] The experimental results showed that in all treated plants (i.e., those not covered with polyethylene bags), the tissue in the electrode contact area always maintained its original morphology. Visual observation and microscopic examination revealed no signs of swelling or callus, and no light-colored / white / light yellow / green cell clumps were produced.

[0117] The results of Comparative Example 2 showed that 48 hours of high-humidity environment culture in bags after electrical stimulation treatment is a key step in inducing callus formation. The lack of this microenvironmental regulation will lead to the obstruction of the cell dedifferentiation process, and electrical stimulation cannot be effectively converted into the driving force for cell reprogramming. At the same time, the low humidity environment may cause the wound surface to dry and harden, preventing the dynamic balance of extracellular matrix degradation and resynthesis required for loose callus tissue.

[0118] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions that do not depart from the concept of the present invention are within the scope of protection of the present invention.

Claims

1. A method for inducing plant callus under non-tissue culture conditions, characterized in that: The method comprises the following steps: (1) Selecting dicotyledonous plants grown to the seedling stage; (2) Applying 50-70 μA direct current to the main veins of the plant leaves for electrical stimulation; the electrical stimulation treatment method is to continuously apply the current for 3-10 seconds, then wait for 3-10 seconds and then continue to apply the current for 3-10 seconds; the treatment is performed once a day for 4-6 days; (3) After the electrical stimulation treatment, the aboveground parts of the plants were covered with bags for 36-60 hours to induce callus formation.

2. The method according to claim 1, characterized in that The dicotyledonous plant in step (1) includes Cruciferae or Solanaceae; Preferably, the dicotyledonous plant is selected from one of rapeseed, potato, tobacco, tomato or Arabidopsis thaliana.

3. The method according to claim 1, characterized in that The seedling stage in step (1) includes the five-leaf stage, the six-leaf stage, or 2-3 weeks after germination.

4. The method according to claim 1, wherein The method of applying electrical stimulation to the main vein area of ​​the plant leaf in step (2) includes contacting the contact parts of two electrodes with the main vein of the same leaf, and being located on both sides of the midpoint of the main vein, wherein the diameter of the electrode contact part is less than or equal to the diameter of the main vein, and connecting the positive and negative poles of the DC power supply to form a loop.

5. The method according to claim 4, characterized in that The contact portion of the two electrodes in step (2) is a flat-end needle tube made of stainless steel; and / or; in step (2), the contact portions of the two electrodes are 1–3 cm away from the contact position on the main vein of the same leaf.

6. The method according to claim 5, characterized in that The outer diameter of the flat-head needle tube in step (2) is 0.5-0.7 mm.

7. The method according to claim 1, characterized in that In step (2), the plant leaf is selected from at least one of the second, third, and fourth leaves of the plant during its growth and development.

8. The method according to claim 1, characterized in that In step (2), 58-62 μA direct current is applied for electrical stimulation; And / or, in step (2), the electrical stimulation treatment method is to continuously apply current for 4-6 seconds, take an interval of 4-6 seconds, and then continuously apply current for 4-6 seconds.

9. The method according to claim 1, characterized in that In step (3), the bagging is a transparent plastic film bag.

10. Use of the plant callus prepared by the method according to any one of claims 1 to 9, characterized in that: The application is any one of the following (a1) to (a4): (a1) Use in preparing new plants; (a2) Use in the preparation of genetic transformation systems; (a3) Application in plant cell culture; (a4) Application in crop breeding.

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