Method for producing plant cells into which biological function control factors have been introduced

Abstract

To provide a method for producing a plant cell in which a biofunction controlling factor is introduced into an alive plant cell highly efficiently even when the factor is a biofunction controlling factor having low permeability of a biological membrane or the like in the plant cell.SOLUTION: A method for producing a plant cell into which a biofunction controlling factor is introduced, where by performing humidity control treatment so as to make the relative moisture content of a plant cell when the moisture content of the plant cell before humidity control treatment is 100% becomes 90 to 40%, and by immersing the plant cell after the humidity control treatment into solution containing the biofunction controlling factor, a plant cell into which the biofunction controlling factor is introduced is obtained.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing a plant cell into which a biological function control factor has been introduced. [Background technology]

[0002] In research and development in the field of plant science, including plant cell engineering, various staining treatments, enzyme reaction treatments, genetic recombination, etc. are performed on plant cells as materials. Compared to the commonly performed staining procedures involving freezing and chemical fixation, and extraction and reaction procedures using physically crushed plant cells, in research and development that maintains the morphology and function of living cells, it was necessary to consider what kind of treatment should be performed on the cell walls and cell membranes (biomembranes) unique to plant cells. However, the cell walls and cell membranes (biomembranes) of plant cells have various transport mechanisms for compounds (e.g., changes in the composition and extension of the cell wall, partial decomposition and repair, semipermeability of the biomembrane, various transporters, cytosis, etc.), and their regulatory mechanisms are diverse and complex. Up to now, methods have been considered that select and use cells of model plants that are easy to handle in a limited manner, methods that perform chemical treatments such as surfactant treatment, and methods that elucidate one of the various transport mechanisms to develop new compounds, but these require the development of technology specialized for the target plant cells and strict condition adjustments, and a simpler method was desired.

[0003] Patent Document 1 describes a method for cryopreserving plant cells, comprising the steps of: a) pretreating plant cells with a cryopreservative and a stabilizer; b) acclimating the pretreated plant cells to low temperature; c) loading the plant cells with a loading agent; d) vitrifying the plant cells with a vitrification solution; and e) freezing the vitrified plant cells at a cryopreservation temperature. It is said that this method provides a method for cryopreserving plant cells and a method for recovering living plant cells from long-term or short-term cryopreservation.

[0004] Patent Document 2 describes a method for producing artificial seeds using plant somatic embryos, characterized in that the somatic embryos are dried and stored. It is said that a method for producing artificial seeds that can be stored for long periods of time can be provided by placing the somatic embryos in a dry state and regenerating a plant body from the dried somatic embryo in the same manner as before drying.

[0005] However, Patent Documents 1 and 2 do not describe or suggest the introduction of biofunction control factors into plant cells, and there is a need for a method for producing plant cells that can introduce biofunction control factors into living plant cells with high efficiency. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2008-5846 A [Patent Document 2] Japanese Patent Application Publication No. 2-31624 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made to solve the above-mentioned problems, and aims to provide a method for producing plant cells in which a biofunction control factor has been introduced into living plant cells with high efficiency, even if the biofunction control factor has low permeability through biological membranes in plant cells. [Means for solving the problem]

[0008] The above problem is solved by providing a method for producing plant cells into which a biofunction control factor has been introduced, the method being characterized in that a humidity control treatment is performed so that the relative moisture content of the plant cells is 90 to 40%, when the moisture content of the plant cells before the humidity control treatment is taken as 100%, and the plant cells after the humidity control treatment are immersed in a solution containing the biofunction control factor to obtain plant cells into which a biofunction control factor has been introduced.

[0009] In this case, a preferred embodiment is to carry out the humidity conditioning treatment using a porous carrier, and a preferred embodiment is to carry out the humidity conditioning treatment after processing the mass of plant cells into a sheet-like form.

[0010] In a preferred embodiment, the humidity conditioning time for conditioning to a desired relative moisture content in the humidity conditioning treatment is determined by the following formula (1) and / or formula (2). (Number 1) y=-ax+b (1) [In formula (1), y is the relative moisture content (%), x is the time (minutes), and a and b are positive real numbers.] (Number 2) y=ce -dx (2) [In formula (2), y is the relative water content (%), x is the time (minutes), and c and d are positive real numbers.] Effect of the Invention

[0011] The method for producing plant cells of the present invention makes it possible to introduce a biofunction control factor into living plant cells with high efficiency, even if the biofunction control factor has low permeability through the biomembrane of the plant cell. The plant cells thus obtained into which the biofunction control factor has been introduced are suitable for use in the production of various raw materials, the development of new materials, and the like. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram showing an outline of a method for producing a plant cell into which a biofunction control factor has been introduced according to the present invention. [Diagram 2] This is a graph showing the results of humidity conditioning treatment of lemon callus using silica gel and the approximate formula obtained from the treatment. [Diagram 3] These are photographs showing the appearance of lemon callus when the relative moisture content was changed by 10% increments using silica gel. [Figure 4] This is a photograph showing the appearance of lemon callus after re-culture, which was re-cultured with different relative moisture contents after humidity conditioning treatment. [Diagram 5]This is a graph showing the proliferation of lemon callus that was re-cultured with different relative moisture contents after humidity conditioning treatment. [Figure 6] FIG. 1 shows micrographs of the tissue morphology of each plant cell (callus). [Figure 7] The photographs show the results of each plant cell (callus) being re-cultured together with untreated plant cells (callus) after being subjected to humidity adjustment so that the relative moisture content was 50%. [Figure 8] This is a graph showing the results of humidity conditioning treatment of lemon callus by changing the type of porous carrier. [Figure 9] This is a graph showing the results of humidity conditioning treatment of lemon callus by changing the type of porous carrier and the approximate formula obtained from the results. [Figure 10] This is a graph showing the results of humidity conditioning treatment using different amounts of silica gel and coffee (nEC) callus. [Figure 11] FIG. 1 shows fluorescence micrographs of plant cells (callus) into which a nuclear-staining fluorescent probe was introduced. [Figure 12] This figure shows the results of GUS staining of untreated lemon callus as a control, and of untreated lemon callus into which the GUS (β-glucuronidase) gene had been introduced and after adjustment to 50% humidity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention is a method for producing plant cells into which a biofunction control factor has been introduced, characterized in that the plant cells are subjected to humidity control treatment so that the relative moisture content of the plant cells is 90-40% when the moisture content of the plant cells before the humidity control treatment is taken as 100% (hereinafter, sometimes referred to as "first step"), and the plant cells after the humidity control treatment are immersed in a solution containing a biofunction control factor (hereinafter, sometimes referred to as "second step") to obtain plant cells into which a biofunction control factor has been introduced. By performing humidity control treatment as in the present invention, it has become possible to introduce a biofunction control factor into living plant cells with high efficiency. As is clear from the examples described below, when a DNA-binding nuclear staining fluorescent probe reagent used for detecting dead cells was introduced as a biofunction control factor into each plant cell subjected to humidity control treatment so that the relative moisture content is 50%, the introduction efficiency was at least several dozen times higher than that of each untreated plant cell (not subjected to humidity control treatment). It is difficult to introduce the nuclear staining fluorescent probe reagent for detecting dead cells into plant cells that retain their biological activity, so it is surprising that a biofunction control factor can be introduced into living plant cells with high efficiency. Thus, even if a biofunction control factor has low permeability through biomembranes in plant cells, the present invention can be introduced into living plant cells with high efficiency, and therefore the present invention is of great significance.

[0014] In the present invention, the humidity control treatment is performed so that the relative moisture content of the plant cells is 90 to 40% when the moisture content of the plant cells before the humidity control treatment is 100%. The inventors have studied that when the relative moisture content exceeds 90%, the biological activity of the plant cells is maintained but the efficiency of introducing the biological function control factor is low, while when the relative moisture content is less than 40%, the biological function control factor can be introduced but it is difficult to maintain the biological activity of the plant cells. Therefore, it is important to perform the humidity control treatment so that the relative moisture content of the plant cells is 90 to 40%. From the viewpoint of increasing the efficiency of introducing the biological function control factor while maintaining the biological activity of the plant cells, the upper limit of the relative moisture content in the first step is preferably 80% or less, more preferably 70% or less, even more preferably 65% ​​or less, and particularly preferably 60% or less. On the other hand, the lower limit of the relative moisture content in the first step is preferably 45% or more, more preferably 50% or more.

[0015] In the present invention, the plant cells to be subjected to the humidity control treatment include not only plant cells constituting general plant tissues but also plant cultured cells, and the plant cultured cells include callus cultured in a solid medium and suspension cultured cells cultured in a liquid medium. The plant cultured cells have a primary wall mainly composed of cellulose, hemicellulose and pectin, and in addition to the primary wall, they have a secondary wall containing hydrophobic lignin, have a higher water affinity than the plant cells constituting general plant tissues, and have a moisture content of about 90 to 98%. As the plant cells to be subjected to the humidity control treatment, plant cultured cells are preferably used from the viewpoint of high proliferation, and among them, callus is more preferably used.

[0016] The plant cells to be subjected to the humidity conditioning treatment are preferably cells of at least one plant selected from the group consisting of conifers, broad-leaved trees, fruit trees, vegetables, grains, and other plants. Conifers include cedar, cypress, red pine, larch, sawara, Chinese fir, etc. Broad-leaved trees include poplar, maple, cherry, amplexica, umbellata, Japanese cinnamon, Japanese cinnamon, eucalyptus, etc. Fruit trees include lemon, yuzu, orange, pomegranate, akebi, peach, fig, kiwi fruit, etc. Vegetables include eggplant, tomato, pepper, strawberry, shiso, dokudami, etc. Grains include rice, wheat, barley, corn, oat, etc. Other plants include bamboo, mangrove, coffee, tea, vanilla, etc.

[0017] In the first step, the method of performing humidity control treatment on the plant cells is not particularly limited, and examples thereof include a method of performing humidity control treatment using equipment having a temperature control function such as a dryer, a constant temperature incubator, a natural convection dryer, a constant temperature and humidity chamber for blowing air, a heat block, a dehumidifier, etc.; a method of performing humidity control treatment by drying under reduced pressure, etc.; a method of performing humidity control treatment using a saturated aqueous solution of inorganic salts, etc.; a method of performing humidity control treatment using a porous carrier such as silica gel, zeolite, charcoal, and fired ceramics, etc. From the viewpoint of being sterilizable and being able to be handled at room temperature, a method of performing humidity control treatment using a saturated aqueous solution of inorganic salts, etc. and / or a method of performing humidity control treatment using a porous carrier is preferably adopted, and among them, from the viewpoint of being able to easily perform humidity control treatment in a sterile environment and being able to be repeatedly recycled, a method of performing humidity control treatment using a porous carrier is more preferably adopted. Among the porous carriers, from the viewpoint of being excellent in humidity control and being able to be repeatedly recycled, at least one selected from the group consisting of silica gel, charcoal, and zeolite is preferably used, at least one selected from the group consisting of silica gel and charcoal is more preferably used, and silica gel is particularly preferably used.

[0018] The amount of the porous carrier used when performing humidity control treatment on plant cells using a porous carrier is appropriately determined according to the weight of the plant cells, but it is preferable to use 0.1 to 100 g of the porous carrier per 100 mg of plant cells. If the amount of the porous carrier used per 100 mg of plant cells is less than 0.1 g, the humidity control time may be too long, so it is more preferable that the amount is 0.5 g or more, even more preferable that the amount is 1 g or more, and particularly preferable that the amount is 3 g or more. On the other hand, if the amount of the porous carrier used per 100 mg of plant cells exceeds 100 g, the humidity control time cannot be shortened according to the amount used, and the cost may be high, so it is more preferable that the amount is 80 g or less, even more preferable that the amount is 50 g or less, and particularly preferable that the amount is 30 g or less.

[0019] In a preferred embodiment, in the first step, the plant cell mass is processed into a sheet before the humidity conditioning treatment. Also, it is preferred to divide the plant cell mass into a plurality of pieces before the humidity conditioning treatment. This makes it possible to shorten the humidity conditioning time required to adjust the humidity to a desired relative moisture content, compared to a plant cell mass of the same weight.

[0020] In the first step, the temperature at which the humidity conditioning treatment is carried out is not particularly limited, but is preferably 5 to 50° C. If the temperature at which the humidity conditioning treatment is carried out is less than 5° C., condensation may occur, reducing the efficiency of the humidity conditioning treatment and the biological activity of the plant cells may not be sufficiently maintained, so the temperature is more preferably 10° C. or higher, even more preferably 15° C. or higher, and particularly preferably 20° C. or higher. On the other hand, if the temperature at which the humidity conditioning treatment is carried out exceeds 50° C., the biological activity of the plant cells may not be sufficiently maintained, so the temperature is more preferably 40° C. or lower, even more preferably 35° C. or lower, and particularly preferably 30° C. or lower.

[0021] In the first step, the time for performing the moisture conditioning process is appropriately adjusted depending on the extent to which the relative moisture content is to be adjusted, but in a preferred embodiment, the moisture conditioning time for adjusting the moisture to the desired relative moisture content is determined by the following formula (1) and / or formula (2).

[0022] (Number 3) y=-ax+b (1) [In formula (1), y is the relative moisture content (%), x is the time (minutes), and a and b are positive real numbers.]

[0023] (Number 4) y=ce -dx (2) [In formula (2), y is the relative water content (%), x is the time (minutes), and c and d are positive real numbers.]

[0024] As a result of the study by the present inventor in the examples described below, it has become clear that the time required to condition the moisture to the same relative moisture content varies depending on the shape of the plant cells and the type of porous carrier used in the moisture conditioning treatment, and that there are cases where the linear approximation formula of formula (1) is applicable and cases where the exponential approximation formula of formula (2) is applicable. When the moisture conditioning treatment is performed slowly, a method of determining the moisture conditioning time when conditioning the moisture to a desired relative moisture content using the exponential approximation formula of formula (2) is preferably adopted. On the other hand, when the moisture conditioning treatment is performed in a short time, such as when a porous carrier such as silica gel with excellent moisture conditioning properties is used, a method of determining the moisture conditioning time when conditioning the moisture to a desired relative moisture content using the linear approximation formula of formula (1) is preferably adopted. That is, according to the present invention, it is possible to simply and accurately determine the moisture conditioning time when conditioning the moisture to a desired relative moisture content.

[0025] In the present invention, the plant cells after the humidity control treatment are immersed in a solution containing a biofunction control factor in the second step, thereby obtaining plant cells into which a biofunction control factor has been introduced. The plant cells after the humidity control treatment have a relative water content controlled to be within a certain range while maintaining high biological activity, and when immersed in a solution containing a biofunction control factor, the plant cells absorb water and the biofunction control factor is rapidly taken up into the plant cells. The inventors speculate that by controlling the relative water content of the plant cells to be within a certain range, some modification is made to a state in which the cell wall and part of the cell membrane in the plant cells can be introduced with the biofunction control factor. According to the present invention, even if the biofunction control factor has low permeability to the biomembrane in the plant cells, the biofunction control factor can be introduced into the living plant cells with high efficiency.

[0026] The biofunction control factor introduced into the plant cell in the second step means a foreign substance to the plant cell to be introduced, which functions as a biofunction control factor in the plant cell, and the foreign substance may be isolated in nature or artificially synthesized. As the biofunction control factor, at least one selected from the group consisting of fluorescent probe reagents such as nuclear staining reagents, mitochondrial staining reagents, Golgi apparatus staining reagents, endoplasmic reticulum staining reagents, and live cell staining reagents, amino acids, peptides, proteins, nucleic acids, plasmids, and vectors is preferably used. As is clear from the examples described below, when a DNA-binding nuclear staining fluorescent probe reagent used for detecting dead cells was introduced as a biofunction control factor into each plant cell that had been subjected to humidity control treatment so that the relative moisture content was 50%, the introduction efficiency was at least 10 times higher than that of each untreated plant cell. In addition, when a recombinant Agrobacterium with a plasmid introduced as a biofunction control factor was introduced into lemon callus that had been subjected to humidity control treatment so that the relative moisture content was 50%, the expression efficiency was at least 3 times higher than that of untreated lemon callus (not subjected to humidity control treatment). Thus, in the field of plant science including plant cell engineering, when performing various vital staining, enzyme reactions, transient gene introduction, genetic recombination, genome editing, etc. of plant cells, it is necessary to introduce a biofunction control factor into the plant cell. However, even if the biofunction control factor has low permeability to the biological membrane of the plant cell, it is possible to introduce the biofunction control factor into the living plant cell with high efficiency, and it can be applied to genome editing in addition to genetic recombination. The present invention can be used as a plant growth and metabolic function modification technology in the field of plant science including plant cell engineering. The plant cell into which the biofunction control factor obtained by the present invention has been introduced can be suitably used for various raw material production such as food, pharmaceuticals, and breeding, and new material development. EXAMPLES

[0027] The present invention will now be described in more detail with reference to examples.

[0028] (1) Dry heat sterilization of porous carrier A certain amount of porous carrier silica gel (Nacalai Tesque, Inc., Silica gel (blue) (medium grain) "17091-55 (CAS RN 7631-86-9)") was placed in a 200 mL glass bottle, the bottle was covered with aluminum foil, and then sterilized with dry heat at 160°C for 2 hours in a natural convection dryer (EYELA Corp., "NDO-450"), and then stored in a natural convection dryer (EYELA Corp., "NDO-450") at 60°C.

[0029] (2) Humidity control treatment of lemon callus Using the porous carrier (1) above, 100 mg of lemon callus was placed in an aluminum cup (10 mm length x 10 mm width x 10 mm height), and the aluminum cup was placed in a 200 mL glass bottle at room temperature (25°C) and the lid was placed on the bottle to perform humidity conditioning. The lemon callus that had not been subjected to humidity conditioning was considered to have a relative moisture content of 100%, and the weight change of the lemon callus was measured every 30 minutes up to 120 minutes using an electronic balance (A&D Corp. "HR-250AZ", minimum display 0.1 mg) to calculate the relative moisture content. As shown in Figure 2, when 100 mg of lemon callus was subjected to humidity conditioning every 30 minutes up to 120 minutes using 5 g of silica gel, the approximate formula y = -0.0053x + 0.971, R 2 = 0.99 was obtained. In other words, it became clear that the relative moisture content of the humidity conditioning treatment could be calculated by measuring the weight change of the callus at regular intervals according to the approximate equation of the humidity conditioning-drying curve, y = -ax + b (y is the relative moisture content (%), x is the time (minutes), and a and b are positive real numbers).

[0030] (3) Appearance of lemon callus after humidity conditioning The moisture conditioning treatment of 100 mg of lemon callus using 5 g of silica gel was carried out in the same manner as in (2) above, and the appearance of the lemon callus was observed when the relative moisture content was changed by 10% at a time. Photographs of the obtained appearance are shown in Figure 3. When the relative moisture content was less than 40%, the lemon callus tended to shrink significantly.

[0031] (4) Proliferation of lemon callus after humidity conditioning The lemon calli treated with humidity control in (3) above were recultured for 4 weeks at 25°C in the dark on MT medium (Murashige T & Tucker DPH (1969) Growth factor requirements of citrus tissue culture. Proc. 1st. Citrus Symp. 3: 1155-1161) supplemented with 10 μM of auxin 2,4-D and 3 μM of cytokinin BA as plant hormones. The photograph of the appearance obtained is shown in Figure 4. In addition, the weight of each lemon callus after reculture for 4 weeks was measured to confirm the proliferation of the lemon callus after humidity control treatment. The results are shown in Figure 5. Good proliferation was observed when the relative moisture content was between 100% and 40%, and high biological activity was maintained. On the other hand, a decrease in proliferation was confirmed when the relative moisture content was less than 40%.

[0032] (5) Proliferation of each plant cell (callus) after humidity control treatment Using 5 g of silica gel, 100 mg of plant cells (callus) from Akebia, fig, coffee (nEC: non embryogenic callus), coffee (EC: embryogenic cells), bamboo (Bm), rice, and peach were subjected to humidity conditioning treatment in the same manner as in (2) above, so that the relative moisture content of each was 50%. Microscopic photographs of the tissue morphology of each plant cell (callus) used are shown in Figure 6. 100 mg of each untreated plant cell (callus) and each plant cell (callus) after the above-mentioned 50% humidity conditioning were re-cultured in the following media at 25°C for 4 weeks. The media used are as follows: Bamboo (Bn) and rice Modified MS medium: MS (Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473-497) with four times the amount of KH2PO4 (680mg / l) (MSp680) was used, with the addition of 10μM of the auxin Picloram (Ogita, S., Kikuchi, N., Nomura, T. and Kato, Y. (2011) A Practical Protocol for Particle Bombardment-mediated Transformation of Phyllostachys Bamboo Suspension Cells. Plant Biotechnology, 28, 43-50.). Coffee (nEC) and Coffee (EC) For coffee (nEC), a medium containing the above MSp680 plus 10 μM 2,4-D was used, and for coffee (EC), a medium containing the above MSp680 plus 10 μM 2,4-D and 3 μM of the cytokinin Kinetin was used. Akebia The medium used was the above MS supplemented with 3 μM 2,4-D and 3 μM BA. Figs and peaches The medium used was the above MS supplemented with 10 μM 2,4-D and 3 μM BA. Photographs comparing the appearance before and after reculture are shown in Figure 7. In addition, the surface area of ​​each plant cell (callus) was analyzed every week from the start of reculture to four weeks later using the open source image analysis software "ImageJ," and a relative value was calculated with the surface area at the start of reculture set at 1. The results are shown in Table 1.

[0033] [Table 1]

[0034] (6) Comparison of humidity control treatments based on the shape of plant cells (callus) and the type of porous carrier Using 5 g of each porous carrier (silica gel (Nacalai Tesque, silica gel (blue) (medium grain) "17091-55 (CAS RN 7631-86-9)"), bamboo charcoal (Moso bamboo, 5 mm square), zeolite (Gex, "Best Sand", 3-5 mm square)), moisture conditioning treatment was performed in the same manner as in (2) above until the relative moisture content of 100 mg of lemon callus became 50%. Note that each porous carrier was sterilized by dry heat in the same manner as in (1) above and stored in a natural convection dryer at 60°C. Two types of lemon callus were used: one in the form of a lump (diameter 5-7 mm) and one processed into a sheet-like form from the lump (12-15 mm square, 1.5-2.5 mm thick). The results are shown in Figures 8 and 9. As shown in Figure 9, when silica gel was used, the linear approximation equation for the humidity control treatment of lemon callus processed into a sheet was y = -0.0031x + 0.9845, R 2 =0.9954 and the exponential approximation equation y=1.0069e -0.004x , R 2 = 0.9958 was obtained, and when zeolite was used, the linear approximation equation was y = -0.0015x + 0.9517, R 2 =0.9584 and the exponential approximation equation y=0.9658e -0.002x , R 2 = 0.9895 was obtained. Comparing the linear approximation formula and the exponential approximation formula, there was almost no difference between the two for silica gel, but it is considered that the method of calculating the relative moisture content of the moisture conditioning treatment by measuring the weight change of the callus at regular intervals according to the linear approximation formula y = -ax + b (y is the relative moisture content (%), x is the time (minutes), and a and b are positive real numbers) is more suitable. On the other hand, for zeolite, moisture is conditioned slowly, so the exponential approximation formula y = ce -dx It is considered that a more appropriate method would be to calculate the relative moisture content of the humidity-conditioning treatment by measuring the change in weight of the callus at regular intervals in accordance with (y is the relative moisture content (%), x is the time (minutes), and c and d are positive real numbers).

[0035] (7) Comparison of humidity control treatments using different amounts of plant cells (callus) and porous carriers Using 5g or 50g of silica gel, 100mg and 1g of coffee (nEC) callus were subjected to humidity conditioning treatment in the same manner as described above in (2). The humidity conditioning treatment was continued until the callus was almost completely dry. Two types of 1g of coffee nEC callus were used: a 1g lump and one divided into 10 pieces of 100mg each. The results are shown in Figure 10.

[0036] Example 1 (8) Introduction of nuclear staining fluorescent probe Using 5 g of silica gel, 100 mg of each plant cell (callus) of lemon, rice, coffee (nEC), and bamboo (Bm) was subjected to humidity control treatment in the same manner as in (2) above, so that the relative moisture content of each plant cell (callus) was 50%. 100 mg of each untreated plant cell (callus) and each plant cell (callus) after the 50% humidity control treatment were placed in a 1.5 mL Eppendorf tube, and about 100 μL of an aqueous solution (5 nM) of a DNA-binding nuclear staining fluorescent probe reagent ("SYTOX Green" manufactured by Thermo Fisher Scientific Co., Ltd.) used to detect dead cells, which was diluted 1 / 1000 with phosphate buffer solution (PBS), was added as a biological function control factor, and water absorption treatment was performed for about 5 minutes. Photographs observed using an inverted fluorescent microscope ("CKX53" manufactured by Olympus Corporation) with a B excitation filter are shown in Figure 11. Note that when observing, each sample after water absorption treatment was placed on a slide glass, and phosphate buffer solution (PBS) was dropped on it to prevent drying, and then the inverted fluorescent microscope was operated. In the untreated plant cells (callus), the biomembrane permeability was low and almost no staining was observed. In contrast, in the plant cells (callus) after 50% humidity adjustment, high-intensity staining was observed while maintaining sufficient viability. In addition, the open source image analysis software "ImageJ" was used to analyze the stained areas of the untreated and 50% humidity-adjusted plant cells (callus) in Figure 11, and the introduction efficiency (fold) was calculated. The results are shown in Table 2. In this example, the aqueous solution containing the biofunction control factor was prepared with PBS that had been sterilized with high-pressure steam at 120°C for 15 minutes in an autoclave (TOMY's "FLS-1000"), or sterilized with Millipore's sterilization filter "Millex GP 0.22μm".

[0037] [Table 2]

[0038] Example 2 (9) Introduction of the GUS (β-glucuronidase) gene A vector (pIG121Hm) incorporating the GUS gene was introduced into Agrobacterium (EHA105 strain) by electroporation according to a standard method to obtain recombinant Agrobacterium. The recombinant Agrobacterium solution (adjusted to about 0.2 in terms of OD600) was added to untreated lemon callus and lemon callus obtained in the same manner as in (2) above after adjusting the humidity to 50%, and the callus was cultured in a medium (MT medium described above) at 25°C for 48 hours, after which GUS staining was performed according to a standard method. In GUS staining, the area where the GUS gene was expressed is stained blue with X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide cyclohexylammonium salt) reagent. The appearance and color gradation peak photographs obtained after 3 hours are shown in Figure 12. In addition, the stained areas of the untreated lemon callus (control), the untreated lemon callus into which the GUS gene had been introduced, and the lemon callus into which the GUS gene had been introduced after adjusting the humidity to 50% in the external photograph of Figure 12 were analyzed using the open source image analysis software "ImageJ," and the stained ratio (%) relative to the total area of ​​each lemon callus was calculated. The results are shown in Table 3. The GUS gene incorporated into the pIG121Hm vector contains an intron in its sequence, and is expressed when the inserted gene is properly incorporated into the nucleus of the plant cell and spliced. Therefore, the GUS staining results show that transient gene expression was efficiently induced.

[0039] [Table 3]

Claims

1. A method for producing a plant cell into which a biofunction control factor has been introduced, comprising the steps of: the biological function control factor is at least one selected from the group consisting of a fluorescent probe reagent, a peptide, a nucleic acid, and a plasmid; the plant cells are cultured plant cells including callus or suspension culture cells, The method is characterized in that a humidity control treatment is performed using 3 to 100 g of a porous carrier per 100 mg of plant cells so that the relative moisture content of the plant cells is 90 to 40%, when the moisture content of the plant cells before the humidity control treatment is taken as 100%, and the plant cells after the humidity control treatment are immersed in a solution containing the biofunction control factor to obtain plant cells into which the biofunction control factor has been introduced.

2. The method according to claim 1, wherein the plant cell mass is processed into a sheet-like form and then the humidity conditioning treatment is carried out.

3. 3. The method according to claim 1, wherein the humidity conditioning time for conditioning the moisture to a desired relative moisture content in the humidity conditioning treatment is determined by the following formula (1) and / or formula (2): (Equation 1) y=-ax+b (1) [In formula (1), y is the relative water content (%), x is the time (minutes), and a and b are positive real numbers.] (Equation 2) y=ce －ｄｘ (2) [In formula (2), y is the relative water content (%), x is the time (minutes), and c and d are positive real numbers.]

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