A method of inducing plant callus under non-tissue culture conditions

Abstract

The application discloses a method for inducing plant callus under non-tissue culture conditions, and belongs to the field of plant tissue regeneration. The method comprises the following steps: selecting target plants, and growing rape and potato to the five-leaf stage, and growing Arabidopsis thaliana to 2-3 weeks after germination; applying 50-70 muA direct current to the main vein of the plant leaf for electric stimulation; the electric stimulation treatment mode is continuously applying current for 3-10 seconds, then interval 3-10 seconds, and then continuously applying current for 3-10 seconds; the treatment is performed once a day, and the treatment is continuously performed for 4-6 days; and then the whole plant is subjected to bagging culture for 36-60 hours, so that callus is formed in a local region. The method does not need in-vitro tissue culture, exogenous hormones and sterile conditions, can directly induce the dedifferentiation of local tissue of the whole plant in a natural or artificial environment, is simple in operation, high in induction efficiency, suitable for various plant types, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant callus induction, specifically relating to a method for inducing plant callus under non-tissue culture conditions. Background Technology

[0002] Callus is a highly vacuolated, amorphous mass of thin-walled cells formed by the dedifferentiation of plant organs, tissues, or cells under suitable conditions, either through in vitro culture or physical injury (such as cuts). Callus is fundamental to 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 generate callus tissue. Callus induction mainly involves regulating external conditions such as culture medium composition, exogenous hormone ratio, and environmental parameters to stimulate in vitro cultured plant explants to change their original 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 technology 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 still heavily relies on chemical induction with exogenous hormones (such as 2,4-D and NAA) and the use of in vitro cultured (tissue culture) plant explants. However, this method has drawbacks such as a long induction period (usually ≥14 days), significant genotype dependence (induction success rate of woody plants <40%), and a high risk of epigenetic interference.

[0005] Therefore, finding a method to 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 purpose of this invention is to overcome at least one deficiency of the prior art and to provide a method for inducing plant callus under non-tissue culture conditions.

[0007] The technical solution adopted in this 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) Select plants of dicotyledonous plants that have grown to the seedling stage;

[0010] (2) Apply 50-70μA DC current to the main vein of the plant leaves for electrical stimulation. The electrical stimulation method is to apply the current continuously for 3-10s, then apply the current continuously for 3-10s after an interval of 3-10s; treat once a day for 4-6 days.

[0011] (3) After the electrical stimulation treatment, the above-ground parts of the plant were covered by bagging for 36-60 hours to induce the formation of callus tissue.

[0012] Preferably, the dicotyledonous plants mentioned in step (1) include those from the Brassicaceae or Solanaceae families.

[0013] More preferably, the dicotyledonous plant mentioned 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. 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 leaf in step (2) includes contacting the contact parts of two electrodes with the main vein of the same leaf and placing them on both sides of the midpoint of the main vein, wherein the diameter of the electrode contact parts is less than or equal to the diameter of the main vein, and connecting the positive and negative terminals of a DC power supply to form a circuit.

[0018] More preferably, the contact portion of the two electrodes in step (2) is a flat-tipped needle made of stainless steel. The flat-tipped needle prevents the leaf from being pierced when it contacts the leaf. The outer diameter of the needle is preferably 0.5-0.7 mm. If the outer diameter is too small, it may easily damage the plant; if it is too large, it is inconvenient for precise operation. Therefore, an outer diameter of 0.5-0.7 mm is preferred.

[0019] And / or; the contact points of the two electrodes in step (2) are 1–3 cm apart from the contact points of the same leaf on the main vein. There are no requirements for the connection of the positive and negative electrodes; positive or negative contact is acceptable.

[0020] If the distance between the electrodes on the main pulse is less than 1 cm, the positive and negative electrodes are easily accidentally touched, causing a short circuit; if it exceeds 3 cm, the distance is too far, resulting in a large resistance, which may cause the stimulation current to be lower than the instrument's set 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), a DC current of 58-62 μA is applied for electrical stimulation; and / or, in step (2), the electrical stimulation treatment is to continuously apply current for 4-6 s, with an interval of 4-6 s, and then continuously apply current for another 4-6 s.

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

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

[0026] Preferably, in step (3), the relative humidity inside the bag is maintained at 85%-90%. In the example, the bagged plants are cultured in an artificial incubator with a humidity of 40%-45%, and the humidity inside the bag is 85%-90% after bagging. Generally, in temperate or drier areas, the outdoor relative humidity is usually 40%-60%, and this standard can also be achieved after bagging. Avoid outdoor environments with large fluctuations.

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

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

[0029] (a1) Application in the preparation of new plants;

[0030] (a2) Application 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 direct CRISPR-Cas9 editing on living leaf callus tissue (e.g., via Agrobacterium or nanocarriers), utilizing callus differentiation into complete plants carrying the target trait, avoiding the contamination risks that may occur with traditional tissue culture, and potentially shortening the genotype transformation cycle.

[0034] The crop breeding method includes inducing callus in living crops according to the above method, simultaneously applying drought / salt stress electric field signals, screening for resistant plants that regenerate from mutated callus, and constructing a rapid evolutionary system for stress resistance.

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

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

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

[0038] (3) Dependence on in vitro: A method for inducing plant callus under non-tissue culture conditions is proposed for the first time. The operation is performed directly on the whole plant without the need to establish a cell line, which greatly improves the applicable scenarios and efficiency.

[0039] (4) No exogenous hormones required: Completely eliminates the induction of hormones such as 2,4-D, avoiding hormone toxicity and epigenetic risks.

[0040] (5) Targeted and precise induction: Electrical signals are transmitted through the vascular bundle path to activate the local regeneration process and achieve efficient callus formation. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of an electrical stimulation induction experiment on rapeseed plants; where, Figure 1 A represents the high-humidity microenvironment culture of rapeseed after bagging. Figure 1 B and Figure 1 C represents callus induced by electrical stimulation of rapeseed.

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

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

[0044] Figure 4 This is a schematic diagram illustrating the use of a handheld needle electrode. Detailed Implementation

[0045] Current plant callus induction techniques still primarily rely on in vitro tissue culture of explants, requiring aseptic conditions and high concentrations of exogenous hormones (such as 2,4-D and NAA) for chemical induction. This method suffers from core drawbacks, including cumbersome procedures, high risk of contamination, long hormone induction cycles (typically ≥14 days), and efficiency significantly influenced by plant species. Particularly in woody plants, callus induction success rates are often less than 40%, limiting large-scale application.

[0046] Furthermore, existing technologies that promote plant callus proliferation through electrical stimulation still rely on established callus systems, require in vitro plant cell lines, cannot achieve in situ induction, and have long treatment cycles, making it difficult to meet the needs of efficient field applications.

[0047] To address the aforementioned bottlenecks, this invention provides a novel solution: in situ induction of plant callus tissue under non-tissue culture conditions through a synergistic technology of in vivo targeted electrical stimulation and high-humidity microenvironment.

[0048] In some specific embodiments, this application found that for rapeseed and potato plants at the five-leaf stage, two electrodes were precisely placed on the midrib of the second, third, or fourth leaf, positioned on either side of the midpoint of the midrib, with a distance of 1–3 cm between the contact points of the two electrodes, and a 60 μA DC current was applied. The stimulation method involved continuously applying the current for 5 seconds, stopping, pausing for 5 seconds, and then continuously applying the current for another 5 seconds. Continuous treatment for 5 days effectively activated cell reprogramming signals. Subsequently, bagging the entire plant to maintain a high-humidity microenvironment (relative humidity of 85%–90%) for 48 hours promoted the formation of light-colored or white granular callus tissue near the electrically stimulated area. Experimental results showed that callus tissue could be observed visually 48 hours after bagging (7 days after the first electrical stimulation). This method is also applicable to various plants such as Arabidopsis thaliana, demonstrating good applicability and reproducibility.

[0049] In some specific embodiments, the electrodes used are preferably handheld flat-tipped stainless steel needle electrodes. The contact portion of the electrode is a flat-tipped needle, and the outer diameter of the needle is preferably 0.5-0.7 mm (smaller than the diameter of the midrib). To facilitate flexible electrotreatment of the plant, the electrodes are held in hand to contact the midrib of the plant leaf, positioned on either side of the midpoint of the midrib. The distance between the contact portions of the two electrodes and the contact points on the same leaf on the midrib is preferably 1-3 cm, ensuring good conductive contact between the electrodes and the plant tissue without damaging the tissue.

[0050] The power supply uses a sourcemeter unit (SMU), which is a four-quadrant voltage / current source that can simultaneously and accurately measure current / voltage values. Preferably, the sourcemeter is set to constant current output with an output current of 60 microamps. Feedback from the sourcemeter indicates that the output current fluctuation is within ±1 nanoamp. The compliance voltage is selected appropriately based on the characteristics of the plant leaves. For most plants such as Arabidopsis, rapeseed, and potato, a compliance voltage of 12 volts ensures the output current reaches the set value (60 microamps); for crops such as corn and rice, a compliance voltage of 42 volts ensures the output current reaches the set value. Two electrodes are connected to the positive and negative terminals of the sourcemeter, respectively, forming a circuit after contacting the conductive surface of the plant. When applying electrical stimulation, attention should be paid to the output feedback of the sourcemeter to ensure a stable current flow through the plant tissue.

[0051] In this invention, callus formation is typically determined using morphological criteria. Specifically, this involves observing whether irregular, fragile, and typically light-colored / white / pale yellow / green cell clumps form on the explant surface, appearing as irregular, disordered clumps or nodules. This morphological characteristic is currently a commonly used and effective criterion for identifying callus formation.

[0052] In this invention, the term "non-tissue culture conditions" refers to culture conditions in which whole plants are treated directly in a natural or artificially controlled environment (such as a greenhouse or field) without relying on aseptic techniques, explant harvesting, and culture medium induction, unlike traditional tissue culture systems. This environment allows for the presence of microorganisms and includes both natural and conventional artificial culture environments, unlike the strictly aseptic conditions required for tissue culture.

[0053] In this invention, the term "targeted electrical stimulation" refers to the use of electrodes placed close to the main vein of a leaf to conduct electrical signals through the vascular bundles of the leaf vein, thereby 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 procedures, this invention enables in situ callus generation directly on the whole plant without removing tissue or performing aseptic operations.

[0055] "Five-leaf stage" and "six-leaf stage" are agricultural terms used to describe the growth and development stages of plants. Five-leaf stage: refers to the growth stage where the fifth leaf on the main stem (stem) of the plant has fully unfolded. Six-leaf stage: refers to the growth stage where the sixth leaf on the main stem (stem) of the plant has fully unfolded.

[0056] In a preferred embodiment of the present invention, such as Figure 4 As shown, each electrical stimulation operation uses only one pair of electrodes and is performed only on a single leaf, thus avoiding current shunting or plant damage. The contact points of the two electrodes on the same leaf's midrib are located on either side of the midrib's midpoint. The spacing between the electrode contacts can be adjusted flexibly according to the leaf size, only needing to be controlled within 1–3 cm. This ensures effective current conduction within the midrib tissue, and the contact points are made of stainless steel flat-tipped needles (0.5–0.7 mm outer diameter), ensuring no damage to plant tissue. There are no restrictions on the connection of the positive and negative electrodes; either orientation is 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 various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials used in this invention. In fact, the sources of biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted and used according to the suggestions in the embodiments.

[0059] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0060] Example 1

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

[0062] (1) Plant pretreatment: Rapeseed plants that had reached the five-leaf stage and were cultured in an artificial incubator at 22℃ with a photoperiod of 16h light / 8h darkness were selected as experimental materials. The rapeseed variety used in this embodiment was Zhongshuangshiyi. Those skilled in the art can select rapeseed plants that have been cultured to the five-leaf stage under field conditions; or they can select rapeseed plants of other varieties.

[0063] (2) Electrode positioning and stimulation: The electrodes are precisely placed in contact with the midrib of the second leaf, located on both sides of the midrib, and the distance between the contact parts of the two electrodes is controlled to be 1–3 cm; a 60 μA current is started for electrical stimulation. The stimulation method is to continuously apply the current for 5 seconds, stop, wait 5 seconds, and then continuously apply the current for 5 seconds.

[0064] To verify whether there are differences in the effects of electrical stimulation among different leaves, the third and fourth leaves were treated according to the above conditions.

[0065] 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 16 hours of light and 8 hours of darkness.

[0066] The electrodes used were handheld, flat-tipped stainless steel needle electrodes, with a needle outer diameter preferably of 0.5-0.7 mm. For easy handling of the plants, the electrodes were handheld and mounted on the midrib of the leaf, positioned on either side of the midpoint of the midrib. The distance between the two electrodes was preferably 1-3 cm to ensure good conductive contact between the electrodes and the plant tissue without damaging the tissue. Experimental results showed that, on the same leaf, there was no significant difference in the induction effect when the distance between the two electrodes on the midrib was adjusted between 1 cm and 3 cm.

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

[0068] (3) High-humidity microenvironment control: After the electrical stimulation treatment on the 5th day, a bagging treatment was performed. This involved using a transparent polyethylene film bag (e.g., Figure 1 (As shown in A) Completely cover the entire above-ground part of the plant from the top, gently tapping the bag opening against the edge of the pot or the base of the culture medium to create a relatively sealed, high-humidity environment. Place the bagged plant in an artificial incubator at 22℃ with a photoperiod of 16h light / 8h darkness. The covering should be maintained for 48 hours to maintain a microenvironment with a relative humidity of no less than 85% (85%-90%).

[0069] (4) Observation of callus induction: After the bagging treatment was completed (i.e., the 8th day of electrical stimulation treatment), light-colored nodular protrusions could be observed with the naked eye in the electrode treatment area. These light-colored nodules were callus tissue. Further observation using a stereomicroscope yielded the following results: Figure 1 As shown.

[0070] like Figure 1 As shown, on day 8 of electrical stimulation treatment, obvious light-colored nodular callus tissue masses were visible to the naked eye at the electrode contact area (leaf veins). Figure 1 The areas indicated by the red arrows (B and 1C) exhibit a texture that contrasts sharply with the surrounding green stems, initially identified as callus tissue. Microscopic verification further confirmed this. Figure 1 Image B is a magnified view of a section of the leaf vein, clearly showing light-colored particles attached to the leaf vein epidermis in dense clusters, with a diameter of approximately 0.5-1 mm. Figure 1C is a close-up at a higher magnification (stereoscopic microscope), further confirming 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 containing 24 rapeseed samples. The results are shown below and summarized in Table 1:

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

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

[0074] Third attempt: 21 plants were successfully induced (induction rate: 87.50%).

[0075] The overall induction rate remained stable between 83.3% and 91.7% (average: 87.5%), with small data variation (standard deviation of approximately 3.4%), demonstrating high reproducibility of the results.

[0076] Example 2

[0077] This embodiment 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 with Arabidopsis thaliana, the other experimental steps are the same as in Example 1. The specific steps are as follows:

[0078] (1) Plant pretreatment: Arabidopsis thaliana Col-0 plants that had grown for 2 weeks in an artificial culture chamber at 22℃ with a photoperiod of 16h light / 8h darkness were selected;

[0079] (2) Electrode positioning and stimulation: Precisely contact the electrodes with the midrib of the second leaf, positioning them on either side of the midrib's midpoint, controlling the distance between the contact points of the two electrodes to be 1–3 cm; initiate 60 μA current stimulation, applying the current continuously for 5 seconds, stopping, pausing for 5 seconds, and then applying the current continuously for 5 seconds again. A schematic diagram of the use of the handheld needle electrode is shown below. Figure 4 As shown.

[0080] To verify whether there are differences in the effects of electrical stimulation among different leaves, the third and fourth leaves were treated 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 16 hours of light / 8 hours of darkness.

[0082] (3) High humidity microenvironment control: 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 16h light / 8h darkness to maintain a high humidity environment for 48 hours.

[0083] (4) Observation of callus induction: After the bagging treatment was completed (i.e., the 8th day of electrical stimulation treatment), punctate yellow-green clumps could be observed with the naked eye in the electrode treatment area. These punctate yellow-green clumps were callus tissue. Further observation using a stereomicroscope yielded the following results: Figure 2 As shown.

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

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

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

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

[0088] Third attempt: 71 plants were successfully induced (induction rate: 98.61%).

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

[0090] Example 3

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

[0092] (1) Plant pretreatment: Select potato A056 plants that have grown to the five-leaf stage in an artificial culture box with a photoperiod of 16h light / 8h darkness at 22℃;

[0093] (2) Electrode positioning and stimulation: The electrodes are precisely placed on the midrib of the second leaf, on both sides of the midrib, and the distance between the contact parts of the two electrodes is controlled to be 1–3 cm; 60 μA current stimulation is initiated. The stimulation method is to apply the current continuously for 5 seconds, stop, wait 5 seconds, and then apply the current continuously for 5 seconds.

[0094] To verify whether there are differences in the effects of electrical stimulation among different leaves, the third and fourth leaves were treated 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 darkness.

[0096] (3) High humidity microenvironment control: 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 16h light / 8h darkness to maintain a high humidity environment for 48 hours.

[0097] (4) Observation of callus induction: After the bagging treatment was completed (i.e., the 8th day of electrical stimulation treatment), white granular protrusions could be observed with the naked eye in the electrode treatment area. These white granules were callus tissue. Further observation using a stereomicroscope yielded the following results: Figure 3 As shown.

[0098] like Figure 3 A and Figure 3 As shown in Figure B, on the 8th day of electrical stimulation treatment, dense white granular protrusions were observed on the surface of potato leaves. The white granules were clustered 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, this example further conducted two parallel replicate experiments, each containing 6 potato samples. The results are shown below and summarized in Table 1:

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

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

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

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

[0104]

[0105]

[0106] Comparative Example 1

[0107] Compared with Example 1, this comparative example is identical in all experimental steps except for the electrode positioning in step (2), including steps (1) and (3). Specific step (2) is as follows:

[0108] (2) Electrode positioning and stimulation: The electrodes were precisely placed in contact with the non-midrib area of ​​the leaf (i.e., the mesophyll area outside the vein area), and the distance between the contact points of the two electrodes was controlled to be 1–3 cm. A 60 μA current stimulation was initiated. The stimulation method was to apply the current continuously for 5 seconds, stop, wait 5 seconds, and then apply the current continuously for 5 seconds. The entire electrical stimulation treatment lasted for five days.

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

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

[0111] Comparative Example 1 shows that positioning the electrodes on both sides of the midpoint of the leaf midrib at a certain distance plays a key role in the effective induction of callus tissue, while positioning them on non-midrib sites fails to induce callus tissue formation.

[0112] Comparative Example 2

[0113] Compared with Example 1, this comparative example is identical in all experimental steps except that polyethylene bags were not used to cover the plants in step (3). Specifically, step (3) is as follows:

[0114] (3) Microenvironment control: After the electrical stimulation treatment ended on the 5th day, the plants were placed directly in an artificial incubator at 22℃ with a photoperiod of 16h light / 8h darkness. The humidity of the artificial incubator was 40%-45%.

[0115] The experimental materials included 12 rapeseed plants, 12 Arabidopsis thaliana 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 maintained its original morphology. No signs of swelling or callus formation were observed by visual inspection and microscopic examination, and no light-colored / white / pale yellow / green cell clumps were produced.

[0117] Comparative Example 2 results indicate that culturing in a high-humidity environment with a bag for 48 hours after electrical stimulation is a key step in inducing callus formation. The lack of this microenvironment regulation will lead to the inhibition of cell dedifferentiation and the inability of electrical stimulation to be effectively converted into a driving force for cell reprogramming. At the same time, a low-humidity environment may cause the wound to dry and harden, preventing the dynamic balance of extracellular matrix degradation and resynthesis required for loose callus.

[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 without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A method for inducing plant callus under non-tissue culture conditions, characterized in that, The method includes the following steps: (1) Select dicotyledonous plants that have grown to the seedling stage; (2) Apply 50-70 μA DC current to the main vein of the plant leaves for electrical stimulation. The electrical stimulation method is to apply the current continuously for 3-10s, then apply the current continuously for 3-10s after an interval of 3-10s. Treat once a day for 4-6 days. (3) After the electrical stimulation treatment, the above-ground parts of the plant were covered with bags for 36-60 hours to induce callus formation; The dicotyledonous plants mentioned in step (1) include those from the Brassicaceae or Solanaceae families; The cruciferous dicotyledonous plants mentioned in step (1) are selected from rapeseed and Arabidopsis thaliana, and the solanaceous dicotyledonous plants are selected from potato, tobacco and tomato.

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

3. The method according to claim 1, characterized in that, 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 placing them on both sides of the midpoint of the main vein, wherein the diameter of the electrode contact parts is less than or equal to the diameter of the main vein, and connecting the positive and negative terminals of a DC power supply to form a circuit.

4. The method according to claim 3, characterized in that, In step (2), the contact part of the two electrodes is a flat-headed needle tube made of stainless steel.

5. The method according to claim 3, characterized in that, In step (2), the contact points of the two electrodes are 1–3 cm apart from the contact points of the same leaf blade on the main vein.

6. The method according to claim 4, characterized in that, The outer diameter of the flat-headed 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 leaf from 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), a direct current of 58-62 μA is applied for electrical stimulation; And / or, in step (2), the electrical stimulation treatment is to continuously apply current for 4-6 seconds, with an interval of 4-6 seconds, and then continuously apply current for another 4-6 seconds.

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

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

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