JPH05500304A - A method of introducing molecules, especially genetic material, into plant cells - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] A method of introducing molecules, especially genetic material, into plant cellsTechnical field The present invention relates to a method for introducing molecules, particularly genetic material, into intact plant cells. .

Background technology Animal cells and protoplasts are injected with molecules such as plasmid DNA, RNA, There are several known ways to introduce genetic material, such as viruses or fragments of these materials. It is being

Such known methods include, inter alia, chemical methods, electroporation ( electroporation) and microinjection.

Such known methods are suitable for both transient and stable transformation. It can be implemented in In general, achieving transient expression All disclosed methods are useful for achieving stable transformation. I understand.

For the introduction of genetic material, both plant protoplasts and animal cells are have one important characteristic in common: they are isolated from their surroundings only by a membrane . On the other hand, in addition to the plasma membrane, intact plant cells have a cell wall that cannot be penetrated by polymeric compounds. the cell wall is composed of cellulose fibrils, pectin and often lignin It is a blinding network.

The similarity between plant protoplasts and animal cells is that plant protoplasts have genetic material Is it normal to know whether known methods for introducing cells can also be used in animal cells? This is further emphasized by the fruit.

For this purpose, the following method is used: The DNA is precipitated to obtain a crystalline product. Animal cells (Graham and van den Eb.

rVirology, 52.456-457.1973 J) and plant Rotoplus I□ (Hain et al., rMol, Gen, Genet, 1 99.161-168. 1985 J) is able to absorb the above-mentioned products. Wear.

Protoplasts (Deshayes et al., rEMBO, 4, 2731-2737 ．． 1985J) or animal cells (Felgner et al., Proc, Nat. l, Acad, Sci, USA.

84, 7413.1987), and the fusion was made with polyethylene glycol. It causes.

Use polyethylene glycol (PEG). In this method, plant protoplasts polyethylene glycol, usually 40% polyethylene glycol Add Cole 6000 (Krens et al., rNature, 296.72 ~74.1982J) oSimilarly, polyethyleneimine or polyL-ol You can use Nitin.

Perform electroporation. In this case, animal cells or plant protoplasts A suspension of microorganisms is exposed to short electrical pulses of high field strength in the presence of genetic material (N euman et al., rEMBOJ,,l.

841-845.1982 J; Fromm et al., rNature, 319 ．． 791-793°1986J).

Perform microinjection. In this case, the ultra-fine micropipera I~ and plant protoplasts (Crossway et al., rMol.

Gem, Genet, 20.179.1986) or animal cells (Cap pechi.

rcell, 22.479-488.1980).

International Publication No. 89102464 describes the transformation of animal cells with DNA fragments. to the animal cell, sufficient to damage the cell or without killing the cell. It is disclosed that this is carried out by applying ultrasonic treatment. animal cell and plant pro Because of the above-mentioned similarities with toplasts, this method also applies to plant protoplasts. It should be obvious that it could be used to introduce NA fragments. Up None of the methods described above are suitable for introducing plasmid DNA into intact plant cells. Therefore, a person skilled in the art who has read the above-mentioned international publication will understand that this method uses protoplasts. It seems that the conclusion can be reached that it can be implemented using However, if a person skilled in the art It is impossible to think of using the above-mentioned method, which is difficult to penetrate the cells. . Therefore, plant cell walls generally contain relatively large molecules such as DNA and proteins. Impermeability to children is widely accepted by those skilled in the art.

Therefore, in order to solve this problem, the cell walls of plant cells must first be hydrolyzed with enzymes. the need to remove by solution and then introduce genetic material into the generated protoplasts. There was. However, this is usually difficult. Is all plants protoplasts? Is it usually more difficult to regenerate from intact plant cells than from intact plant cells? Because there is. Therefore, protoplasts can be created by directly introducing genetic material into intact plant cells. issues related to the generation of protoplasts as well as issues related to regeneration from protoplasts. There is a great need to find ways to avoid it.

Recently, methods have been developed to introduce plasmid DNA into intact plant cells. this method is based on high-velocity bombardment with small particles coated with plasmid DNA. <　(Klein et al., rNature.

327, 70-73.1987''). However, this method requires very expensive equipment. and requires considerable specialized knowledge to operate properly.

Therefore, simpler and cheaper methods of introducing molecules, especially genetic material, into intact plant cells are needed. is necessary.

In the present invention, the method is completely effective, relatively inexpensive and easily accessible. by mild sonication using a method that requires only hand-held equipment. discovered that it is possible to achieve the introduction of molecules, particularly genetic material, into intact plant cells by .

Description of the invention The object of the invention is to introduce molecules, especially genetic material, into intact plant cells in a cheap and effective manner. The purpose is to provide a method for introducing

It is an object of the present invention to subject the medium containing said plant cells and said molecules to a mild ultrasound treatment. This is achieved by the method of the present invention, which is characterized in that it carries out the following steps.

The method of the present invention is a useful method different from conventionally known introduction methods. present invention The method provides a novel, low-cost method that is quick and easy to implement. . Based on the introductory experiments already carried out, the method of the present invention has proven that the known methods are insufficient. We predict that this method will be superior when used on a large number of other biological materials. I can do something.

In the present invention, cells are disrupted to produce a homogenate, or cells are lysed. subjecting plant cells to ultrasonication, which is significantly milder than the sonication used to . This sonication is reasonably mild so that a sufficient number of plant cells remain alive. Ru. To ensure viability, the plant cell suspension is heated between 5 and 300 W, preferably 3 Output power below 600W, such as 0-90W (i.e., the “output power” of the power supply unit) (output power read from the control unit and supplied to the sound wave generating means), 5 For ultrasonic waves in the frequency range of kHz to 10MHz, especially lO to 100kHz, 10100O such as 100-30QQms, preferably 400-10100O Advantageously, the exposure period is less than or equal to 0.

Examples of molecules that can be introduced into plant cells by the method of the present invention include DNA, plasmid DNA, A, RNA, virus, protein, lipid, pharmaceutical composition, small molecule, organelle or are fragments of these substances.

The method of the invention can be advantageously used to introduce molecules into sugar beet or tobacco plant cells. can be used.

Introduction of genetic material such as plasmid DNA into sugar beet or tobacco plant cells frequency range from 10 to 30 kHz at an output power of 75 to 100 W. By using ultrasonic waves at a temperature of 500 to 10,100 O, Good results can be obtained.

A particularly suitable medium for plant cells and molecules when sonicated is 21-28% It is a CPW containing sucrose.

When introducing plasmid DNA by the method of the present invention, plasmid DNA in the medium Particularly favorable results are obtained when the A8 degree is 10 μg/ml or more.

The gentle sonication of the method of the invention has an acute point. using a sound wave generating means, and immersing the sound wave generating means only in the upper part of the medium. It is advantageous to do so.

By the method of the present invention, plant cells of dicotyledonous plants such as sugar beet plants and tobacco plants can be obtained. It was found that molecules were introduced into the cells. The method of the present invention applies molecules to monocot plant cells. It is also perfectly suitable for introducing.

Further scope of applicability of the method according to the invention will become apparent from the detailed description given below. but Although the detailed description and specific examples indicate preferred embodiments of the invention, By way of example only, those skilled in the art will appreciate that various modifications within the spirit and scope of the invention can be made. Variations and modifications will be apparent from this detailed description.

The method of the present invention generates ultrasonic waves in the culture medium for a period of 10100O0 or less. Advantageously, this can be carried out by using a device equipped with sound wave generation means that can be able to. Such a device uses ultrasonic waves at frequencies ranging from lO to 100kHz. It is advantageous to provide the system so that it occurs.

The apparatus used to carry out the method of the invention uses acoustic waves to perform various tasks. The power supplied to the generating means can be adjusted to a predetermined value in the range of 5 to 300W. , so that the duration of sonication can be adjusted to the range of 100~10100O0. It can be attached to.

The sonic source of this device can be placed in a suitable container, such as an Eppendorf. It is advantageous to form it in such a way that it can be immersed into a tube. Sound wave generation means Conventional ultrasound, which destroys or dissolves cellular material, is an example of Known from the device.

Experiments have shown that such conventional ultrasound equipment can be used in the method of the present invention. . The present invention was developed using a cell lysis device, which is located in the United States and Branson, Eagle Road, Danbury, Cat. Commercially available from the company under the product name Sonifier B15. Available.

This device is capable of generating ultrasonic waves with a frequency of 20 kHz. including the scope of claims. The power values expressed in watts in this specification are based on the output control section of the power supply unit. is the output power as read from . During the experiment, the ultrasonic generation means were measured from the surface. 2 to 3 mm. From preliminary experiments using a calorimeter to experimental operations. The ultrasonic power applied to the liquid during operation is approximately 5-10% of the applied output power. I understand that. If other devices are used, those skilled in the art will be able to use them as shown in the examples below. The appropriate adjustment can be made by and effective molecular transfer is performed and sufficient viability is maintained. In other words, This adjustment corresponds to the sonication force in the experiments performed as described in the Examples. or occur. For comparison, the above-mentioned device Sonifier-815 was used, with no exceptions noted. Whenever possible, lyse the cells or disrupt the cells to produce a homogenate under the same conditions. When processing intact cells, adjust the output power to 80-100W. , adjust the processing period to 30000-250000ms.

As mentioned above, several methods are known for introducing genetic material into plant cells. In these methods, the plant cells are first transformed into plant protoplasts; Rotoplasts do not have a cell wall barrier, so they cannot be introduced into protoplasts. The amount of genetic material produced increases. On the other hand, some plant species, especially monocots, It is difficult to regenerate plastids into whole plants. using protoplasts of certain plant species. Favorable results were obtained when using intact plant cells instead of protoplasts. , a plant embryo or other morphogenetic material There are other plant species for which the use of al) is more suitable. From experiments, the method of the present invention Genetic material can be introduced directly into intact cells using milder sonication. This method of introduction is uncomplicated and can be used in plant species that can be successfully genetically engineered. This has been found to be advantageous as it allows for a significant increase in the number of

The relevant genetic material is in particular DNA or fragments thereof, such as plasmid DNA. . In addition, the method of the present invention can be used to inject RNA or fragments thereof into cells, as well as viruses, e.g. It is also suitable for introducing equipment for pathological tests. In addition, the method of the present invention can also be applied to proteins, Introduction of lipids, pharmaceutical compositions, small molecules, and organelles and virus particles It is also suitable for

The culture medium may be any suitable conventional culture medium for cells or protoplasts. can.

The method of the present invention uses technology that temporarily weakens both the cell membrane and the cell wall. This is demonstrated in intact plant cells in the Examples below.

These examples demonstrate that plasmid DNA can penetrate sonicated plant cells. and

Callus obtained from embryos was used to determine genotype Ml (Copenhagen, Denmark). Danisco (available from DAN [SCO>) sugar beet (Beta Bvlgari) Preparation for cell suspension culture of Betavulgaris L. do. This cell suspension was incubated at 25°C on a rotating shaking table in the dark. Cultivate. Cells were incubated with 5.7 μM indoleacetic acid and 4.4 μM benzyl acetate. Murashige and Skoog with addition of Denine (Physiol, Subculture in the medium of Plant, 15°473-479.1962) By doing so, the fiber CPW is based on Frearson et al. (Dev, Biol, 3 3. 130-137. As described by (1973), generally It is an aqueous solution of an inorganic salt mixture containing about 10mM (7) Ca"".

Ultrasonication The cell suspension used for ultrasonication is prepared by removing cells 3 to 4 days after subculturing. These cells were treated with CPW13S (i.e., CP containing 1396 sorbitol). W) and finally these cells were dissolved in 4 volumes of CPW1 in CPW13S. Prepared by suspending in a ratio of 1 part by volume to 3S. Next, Eppe 2193 sucrose and plant cells (500,000 cells/7nl) in a Lindorf tube. Plasmid DNA was added to the suspension in 0.35 ml of CPW containing . The plasmid DNA used was labeled with the labeling enzyme chloramphenicol-acetate. A plasmid encoding a transferase, in this case Sweden, Code available from Pharmacia LKB Biotechnology, Uprasa. 27-4909, CaMVCM.

Sonifier-815 (Eagle Road, Danbury, CT, USA) After rapid shaking, the microtip of the Immerse in the upper half of the cell suspension (i.e. 2-3 centimeters measured from the surface) . Ultrasonic pulses with a frequency of 20 kHz are supplied. of the power shown in the experimental results. The value is determined based on the output drawing department. In this case, 1 unit corresponds to 15W of power. do. At 10W power, the effective acoustic power is 0.22W/am2 . The duration of the process is expressed as a duty circle in %. ), and adjust by 10% at this time. ensures that each ultrasound pulse has a duration of 110 ms. 1 The number of such pulses of 10m5 can be set by an on/off switch. can.

The ultrasonicated cells were then placed in MS medium <Murashige and S Hoog (see above) in a Petri dish and incubated in a greenhouse for 2 days at 23°C. Ru. The presence of CAT activity indicates that 14c-labeled chloramphenicol can be obtained from treated cells. of the sample and then heating the sample to 60°C for 6 minutes. be done. After cooling, acetyl-coenzyme A was added to the sample to perform acetyl-cofermentation. The final concentration of element A is 0.71 mM. Plasmid introduction is performed using chloramphenicin. The evaluation was made by measuring the transformation rate (%) of call (CA). used The method is described by Gorman et al. (Mo1.Cel.

Biol., 2.1044-1051.1982). Ru.

Example 1 This example describes the introduction of plasmid DNA into intact sugar beet cells of genotype M1. I will clarify.

Suspension cultures of sugar beet cells were maintained by subculturing as described above and then The cells were sonicated, cultured, and analyzed. The measurement results of CAT activity are shown in Table 1.

Output time Transformed CA (Watt) (ms) (%) 000.03 60 800 0, 06 75 800 0, 21 90 800 0, 17 105 800 0, 07 Example 2 This example describes the introduction of plasmid DNA into intact tobacco cells.

Suspension cultures of tobacco cells were grown as described above for subculture of sugar beet cell suspensions. The cells were maintained by subculturing.

However, as a medium, Murashige and Skoog (Phsiol, Plant, 15.473-497.1962') 0.2mg / l of 2.4-dichlorophenoxyacetic acid, 0.1 mg / (! Added thiamine hydrochloride of 0.9 mg/(!) and KH2PO4 of 0.2 g/A. A medium with a pH of 6.0 was used.

Cells were removed 2-3 days after subculturing, and CPW13S (i.e., 1396 CPW containing sorbitol) and finally these cells were washed twice with It was suspended in CPW13S at a ratio of 4 parts by volume of CPW13S to 1 part by volume.

A sample of 0.357 nl was taken and each sample was injected with plasmid, CaMVCN. was added to give a final plasmid concentration of 100 μg/ml. Then these details The cells were subjected to ultrasonic treatment under the conditions shown in Table 2. After 2 days of culture in the medium described above. Then, the cells were taken out and the CAT activity of the cells was measured. The measurement results are shown in Table 2.

Table 2 Output time Transformed CA (Watt) (ms) (%) 0 0 0, 05 75 570 0, 06 75 800 0, 13 75 1000 0, 07 Clearly, the methods of the invention can be used to introduce molecules into intact plant cells.

Having thus described the invention, it will be obvious that the same may be modified in many ways. That's it. Such changes should not be construed as a departure from the spirit and spirit of the invention. All such modifications that are obvious to a person skilled in the art are included within the scope of the invention. included.

international search report international search report PCT/CK 90100166

Claims (10)

[Claims] 1. In introducing molecules, particularly genetic material, into an intact plant cell, said plant cell and to plant cells, characterized in that a medium containing the molecules is subjected to mild ultrasonication. How to introduce molecules. 2. The above-mentioned mild ultrasonic treatment is performed by applying a power of 600 W or less to the sound wave generating means at 10,000 watts. Claim 1 characterized in that this is carried out by supplying for a period of less than ms. Method described. 3. The above-mentioned mild ultrasonic treatment is performed by applying a power of 5 to 300 W to the sound wave generating means at 100 to 300 W. Claim 1 characterized in that it is carried out by supplying for a period of 00 ms. Method described. 4. The above-mentioned mild ultrasonic treatment was performed by applying a power of 30 to 90 W to the sound wave generating means at 400 to 1 000 ms and the frequency of the sound wave is between 10 and 100 kHz. 2. The method of claim 1, wherein the method is carried out by determining a range. 5. DNA, plasmid DNA, RNA, virus, protein, lipid, pharmaceutical composition , small molecules, organelles and fragments of these substances. 2. The method of claim 1. 6. Claim 1, wherein the plasmid DNA concentration in the medium is 10 μg/ml or more. How to write 7. The gentle ultrasonic treatment is carried out using a sound wave generating means having an acute point. 2. The method according to claim 1, wherein the sound wave generating means is immersed only in the upper portion of the medium. 8. 2. The method of claim 1, wherein the plant cell is a monocot. 9. 2. The method of claim 1, wherein the plant cell is a dicotyledonous plant. 10. The method according to claim 9, wherein the plant cell is a sugar beet or tobacco plant cell. Law.