JP7345760B2 - How to introduce substances into pollen - Google Patents

Description

The present invention relates to a method for introducing a substance into pollen, and more particularly, to a method for introducing a substance into pollen using a particle bombardment method.

Introduction of substances such as nucleic acids and proteins into cells is of great significance not only in basic research but also in various industrial applications. However, plants have hard cell walls, making it difficult to introduce substances from the outside compared to animals. Regarding the introduction of substances into plant cells, there are methods that utilize the phagocytosis of protoplasts whose cell walls have been removed (polyethylene glycol method), methods that introduce substances into cells using a fine syringe (microinjection method), Attempts have been made to create a hole in the cell membrane electrically and allow a substance to flow into the cell (electroporation method), and to cover microparticles with a substance and shoot it into the cell (particle bombardment method). Among these, the particle bombardment method is generally used as a method for introducing substances into plant cells because it is simple and applicable to a wide range of plants (Non-Patent Document 1). Furthermore, it has been reported that this method is particularly effective for various angiosperms, such as mature pollen having a hard cell wall (Non-Patent Document 2).

In the particle bombardment method, mature pollen is usually suspended in a liquid medium for pollen tube elongation (cultivation), and with the pollen absorbing water, it is placed on a support such as filter paper or nylon membrane while being suctioned with a vacuum pump. A method of collecting and introducing pollen is mainly used (Non-Patent Document 3, Non-Patent Document 4). Additionally, a method using an agar medium as a support has also been used (Non-Patent Documents 5 and 6).

However, the particle bombardment method using these as a support has the problem that the efficiency of introducing substances into pollen is low, and many pollen do not germinate pollen tubes normally (that is, the survival rate after introduction treatment is low). there were.

J. Finer, J. et al., Curr Top Microbiol Immunol. , 1999, volume 240, pages 59-80. Schreiber, D. N. et al., Plant Molecular Biology Reporter, 2003, Volume 1, Pages 31-41. Seguin, A. et al., Plant Tissue Culture Manual, 1996, pp. 417-462. Hao Wang et al., Nature protocols, 2011, vol. 6, no. 4, pages 419-426. Boavida, L. C. et al., Plant J. , 2007, Volume 52, Pages 570-582. Mizuta Y. et al., Plant J. , 2014, volume 78, pages 516-526

The present invention has been made in view of the problems of the prior art, and aims to provide a particle bombardment method capable of introducing substances into pollen with high introduction efficiency and high survival rate.

In order to achieve the above object, the present inventors used various artificial membranes as supports on which pollen was placed, and introduced substances into the pollen by a particle bombardment method. As a result, when hydrophilic polytetrafluoroethylene (hydrophilic PTFE) or hydrophilic mixed cellulose esters (hydrophilic MCE, i.e., a mixture of nitrocellulose and cellulose acetate) are used as supports, substances are introduced; It was also revealed that the number of pollen grains that survived and germinated into pollen tubes was significantly higher than on other supports.

On the other hand, even when using PTFE, when hydrophobic PTFE was used as a support, the number of pollen particles was smaller than that of hydrophilic PTFE. Furthermore, when only nitrocellulose or cellulose acetate was used as a support, the number of pollen grains was significantly lower than that of hydrophilic MCE composed of them. In addition, compared to hydrophilic PTFE or hydrophilic MCE, filter paper, nylon, and agar media, which have been used as supports, have a higher number of pollen particles that have been introduced with substances and have survived and germinated pollen tubes. There were few. Furthermore, we have found that when hydrophilic PTFE is used as a support, it is possible to introduce substances into the ostogen cells in pollen with high efficiency and survival rate.

That is, the present invention relates to a method for introducing a substance into pollen, and more specifically provides a method for introducing a substance into pollen using a particle bombardment method, as described below.
<1> A method of introducing a substance into pollen,
the step of bombarding pollen on a support with fine particles coated with a substance by a particle bombardment method, and the support is a polymer of at least one of hydrophilic polytetrafluoroethylene and hydrophilic mixed cellulose ester. A method comprising a support comprising:
<2> The method according to <1>, wherein the substance is a nucleotide and/or a peptide.
<3> The method according to <1> or <2>, wherein the fine particles are metal fine particles.
<4> A pollen support material for particle bombardment, comprising at least one polymer selected from hydrophilic polytetrafluoroethylene and hydrophilic mixed cellulose ester.
<5> A device for particle bombardment, comprising the pollen supporting material for particle bombardment according to <4>.

According to the present invention, it is possible to introduce substances into pollen with high introduction efficiency and high survival rate.

FIG. 2 is a diagram showing an overview of the particle bombardment method (before bombarding pollen with a substance) of the present invention. FIG. 2 is a diagram showing an overview of the particle bombardment method (when a substance is bombarded with pollen) of the present invention. It is a graph showing the supports used to place pollen and the number of surviving pollen particles into which a substance (plasmid DNA) was introduced by particle bombardment using these various supports. It is a graph showing the supports used to place pollen and the number of surviving oogen cells into which a substance (plasmid DNA) was introduced by the particle bombardment method using these various supports. It is a photograph showing the results of introducing a GFP expression vector into tetrads and microspores by the particle bombardment method of the present invention and observing them with a fluorescence microscope. In the figure, the lower photograph shows the result of fluorescence observation, and the upper photograph shows the result of superimposing the fluorescence observation result and bright field observation. The scale bar in the figure represents 100 μm. The area surrounded by a dotted circle indicates that GFP fluorescence was detected in the tetrad. It is a photograph showing the results of introducing a GFP expression vector into microspores by the particle bombardment method of the present invention and observing them with a fluorescence microscope. The notation in the figure is the same as in FIG. 4A. It is a photograph showing the results of introducing a GFP expression vector into immature pollen (two nuclei) by the particle bombardment method of the present invention and observing it with a fluorescence microscope. The notation in the figure is the same as in FIG. 4A. FIG. 2 is a photograph showing the results of introducing an RNA-protein complex containing carboxyfluorescein (FAM)-labeled RNA into mature pollen using the particle bombardment method of the present invention and observing it with a fluorescence microscope. FIG.

The method of introducing a substance into pollen of the present invention includes the step of bombarding pollen covered with a substance by a particle bombardment method, and wherein the support is made of hydrophilic polytetrafluoroethylene and hydrophilic polytetrafluoroethylene. A support comprising at least one polymer of mixed cellulose esters.

In the present invention, "pollen" means male gametes of plants, and includes not only mature pollen but also immature pollen. Immature pollen also includes pollen mother cells, tetrads (pollen tetrads), microspores, and 2-nucleated immature pollen. In addition, cells constituting pollen (androgen cells, sperm cells, pollen tube cells (vegetative cells), etc.) are also included in the pollen to which the substance of the present invention is introduced. The "plant" from which the pollen is derived is not particularly limited, and includes, for example, angiosperms including dicotyledons and monocots, gymnosperms, herbaceous plants, and woody plants. More specific examples of plants include tomatoes, tobacco, green peppers, chili peppers, eggplants such as eggplants; cucurbits such as cucumbers, pumpkins, and watermelons; vegetables such as cabbage, broccoli, Chinese cabbage, and Arabidopsis; celery and parsley. 1. Raw vegetables and spicy vegetables such as lettuce; Alliums such as green onions, onions, and garlic; Legumes such as soybeans, groundnuts, green beans, peas, azuki beans, mung beans, cowpeas, and fava beans; Other fruit vegetables such as strawberries and melons; Japanese radish , turnips, carrots, burdock, and other taproots; taro, cassava, potato, sweet potato, and other potatoes; rice, corn, wheat, sorghum, barley, rye, minatomojigusa, buckwheat, and other cereals; asparagus, spinach, etc. Petals such as lilies, lisianthus, stock, carnations, chrysanthemums; Lawns such as bentgrass and orifolia; Oil crops such as rapeseed, peanuts, rapeseed, oilseed rape; Japanese rushes, rushes, etc. Fiber crops; Forage crops such as clover, dent corn, and alfalfa; Deciduous fruit trees such as apples, pears, grapes, peaches, and kiwifruit; Citrus fruits such as mandarin oranges, oranges, lemons, and grapefruit; Satsuki and azaleas , cedar, poplar, para rubber tree, ginkgo, pine, and other woody plants.

The "substance" introduced into the pollen is not particularly limited, and includes, for example, biopolymers such as nucleotides (DNA, RNA), peptides, sugars, and lipids, and low-molecular compounds such as fluorescent dyes. Nucleotides include oligonucleotides, polynucleotides, and nucleic acids, peptides include oligopeptides, polypeptides, and proteins, and sugars include oligosaccharides and sugar chains. Furthermore, the "biopolymer" according to the present invention includes not only naturally occurring ones, but also their derivatives (e.g., cross-linked nucleotides, non-natural amino acids), and complexes thereof (e.g., Also included are glycoproteins, glycolipids, RNA-protein complexes).

In the method of introducing a substance into pollen of the present invention, as described above, a method is used in which fine particles coated with the substance are shot into an object (in the present invention, pollen) placed on a support by the particle bombardment method. The "particle bombardment method" is also known as the particle gun method, gene gun method, microparticle gun method, microparticle injection method, or particle delivery method, and is a method in which microparticles coated with a substance such as DNA are injected at high speed. This refers to a method of introducing into target cells. Hereinafter, an example of an embodiment of the particle bombardment method of the present invention will be described with reference to FIGS. 1A and 1B.

In the particle bombardment method, for example, before injecting a substance, the inside of the particle bombardment device 1 is made negative pressure, and then gas is supplied to the gas acceleration tube 2 until the rupture disk 3 reaches a bursting pressure. Next, as shown in FIG. 1B, due to the gas impact pressure generated by the rupture of the rupture disk 3 (the rupture disks 3a and 3b after rupture), the macrocarrier 4 has the fine particles 5 coated with the substance attached to its lower surface. will move in the direction of the object (pollen 7). Then, the macro carrier 4 stops at the stopping screen 6, and only the attached fine particles 5 are shot into the object (pollen 7) placed on the support 8.

In the particle bombardment method of the present invention, the support on which pollen is placed (also referred to as "pollen support material for particle bombardment") is designed to From the viewpoint of high pollen survival rate, the support contains at least one polymer selected from hydrophilic polytetrafluoroethylene and hydrophilic mixed cellulose ester.

In the present invention, "hydrophilic" means that the water contact angle is less than 90 degrees (the water contact angle is the static contact angle with respect to water droplets in the atmosphere at room temperature). "Hydrophilic polytetrafluoroethylene" is prepared by subjecting hydrophobic polytetrafluoroethylene (PTFE) to hydrophilic treatment. There are no particular restrictions on the "hydrophilic treatment," and examples include a method of introducing a substituent such as a hydroxyl group, a method of impregnating a hydrophilic resin such as polyvinyl alcohol (PVA) and then crosslinking it, and a method of crosslinking the membrane surface. Methods of surface treatment with hydroxypropyl acrylic acid, tetraethylene glycol acrylic acid, polyethylene glycol diacrylate, etc., methods of immersion in alcohols such as methanol, ethanol, 2-propanol, or other liquids with low surface tension, methods of surface treatment with electron beams, etc. Examples include a method of modifying by irradiating energy rays and a method of surface modification using an alkali metal material. "Hydrophilic mixed cellulose ester (hydrophilic MCE)" is a mixture of nitrocellulose and cellulose acetate, and is inherently hydrophilic.

These hydrophilic polymers can be appropriately prepared by those skilled in the art using known manufacturing methods. Moreover, since many are commercially available, they can also be prepared by purchasing them. For example, hydrophilic PTFE is commercially available as "Product name: Omnipore (registered trademark)" from MERCK, and "Product name: Hydrophilic PTFE membrane filter" is available from ADVANTEC. Hydrophilic MCE is commercially available as "Product name: MF-Millipore (registered trademark)" from MERCK, "Product name: Cellulose mixed ester type membrane filter" is commercially available from ADVANTEC, and Cytiva (formerly "Product name: Cellulose mixed ester membrane" is commercially available from GE Healthcare Corporation.

The "support" according to the present invention may be dense or porous, but from the viewpoint of stably maintaining a moisture content suitable for pollen germination, it is preferably porous. preferable. The shape of the "support" is not particularly limited and may be in the form of a membrane, film, sheet, or plate, but from the viewpoint of ease of placing pollen and culturing after bombardment, Preferably, the shape is Although it also depends on its shape, the pollen is usually placed on one surface of the support, as illustrated in FIG. 1A. As for the "support" according to the present invention, it is desirable that at least a part of the surface on which pollen is placed is made of hydrophilic PTFE and/or hydrophilic MCE, and may contain other materials. Here, "part" means 50% or more, preferably 70% or more, and more preferably 85% or more of the entire surface of one side of the support. In addition, such other materials include a plastic plate (material: polystyrene, etc.) for lining hydrophilic PTFE and/or hydrophilic MCE, a membrane made of a polymer other than hydrophilic MCE and hydrophilic PTFE (for example, Examples include membranes listed in Tables 1 to 3), filter paper, agar medium, glass plates, metal plates (aluminum plates, stainless steel plates, etc.), silicone resins (PDMS, etc.), and the like.

When the support contains a porous membrane of hydrophilic PTFE and/or hydrophilic MCE, the pore size of the hydrophilic PTFE and/or hydrophilic MCE is usually 0.1 to 100 μm, preferably 0.2 μm. ~50 μm, more preferably 0.8 ~ 10 μm. Further, the thickness of the hydrophilic PTFE and/or hydrophilic MCE in the support is usually 10 to 500 μm, preferably 15 to 300 μm, and more preferably 19 to 150 μm.

Furthermore, from the viewpoint of stably maintaining a moisture content suitable for the survival of pollen, it is desirable that the hydrophilic PTFE and/or hydrophilic MCE in the support contain a liquid. Examples of such liquids include pollen maintenance media (pollen tube elongation media, male gamete initial media, etc.), physiological saline, and buffers such as phosphate buffered saline (PBS). In addition, the content rate of the liquid can be appropriately adjusted by those skilled in the art depending on the type of target (pollen), etc., and is not particularly limited, but for example, 0.01 to 10 μL/mm ³ (1 to 3 μL/mm ³ etc.) can be mentioned.

In the particle bombardment method of the present invention, the material of the "fine particles" coated with the substance is not particularly limited, and examples thereof include fine particles (metal fine particles). Among these, gold particles are preferred. The particle diameter of the fine particles is not particularly limited, but is, for example, 0.05 to 5 μm, preferably 0.1 to 3 μm, and more preferably 0.2 to 1.1 μm. It is 5 μm, more preferably 0.4 to 0.8 μm.

"Coating" according to the present invention does not mean that the entire surface of the fine particle is covered with the substance, but also that a part of the surface is covered (the substance is attached to a part of the surface of the fine particle). It also includes the state. Here, "part" means 50% or more of the surface of the fine particles, preferably 85% or more, more preferably 90% or more, and still more preferably almost the entire surface (for example, 95% or more). Furthermore, "fine particles coated with a substance" may be in the form of a mixture of a substance and fine particles.

The method for coating the fine particles with the substance is not particularly limited, and any known method may be employed. For example, coating can be achieved by bringing the fine particles into contact with the introduced material. At this time, positively charged substances (e.g., polyamines such as spermidine) are used to dissolve the three-dimensional structure of plasmid vectors, and substances that promote adhesion of introduced substances to fine particles or precipitation of fine particles (e.g., calcium chloride, etc.) are used. By adding salt), more efficient coating can be achieved.

The fine particles coated with the substance are usually applied to the macrocarrier as uniformly as possible using a micropipette, etc., and then dried in a sterile environment such as a clean bench, so that the fine particles coated with the substance adhere to the bottom surface. A macrocarrier is prepared.

The method of injecting the fine particles coated with the substance is not particularly limited, but as described above, a method of injecting the particles with a gas set at a constant pressure is usually adopted. As the gas, an inert gas is preferably used, and for example, helium gas is used.

The gas pressure is preferably 30 to 2200 psi, more preferably 450 to 1200 psi. Note that the optimal gas pressure can be appropriately determined by a transient expression experiment or the like, depending on the type of fine particles, the type of target (pollen), the distance to the target, and the like.

The distance between the rupture disk and the macro carrier is preferably 2 to 12 cm, more preferably 2 to 4 cm. Further, the distance between the macrocarrier and the object (pollen) is preferably 2 to 12 cm, more preferably 3 to 6 cm. Note that the optimum pressure for these distances can be determined as appropriate by a transient expression experiment or the like, depending on the type of fine particles, the type of object (pollen), etc.

The number of times fine particles are shot into pollen may be at least once (for example, once or twice). Note that the optimum number of shots can be appropriately determined by a transient expression experiment or the like.

The preferred embodiment of the particle bombardment method of the present invention has been described above with reference to the schematic diagrams shown in FIGS. Any device (so-called gene gun, particle delivery system) that can eject fine particles coated with a substance at high speed without causing any damage can be used regardless of its form or injection method. More specifically, it is not limited to chamber-type, stationary-type, box-type, or installation-type gene guns (for example, BIO-RAD PDS-1000/He system), but also handheld gene guns, etc. ( For example, even with the Helios Gene Gun system manufactured by BIO-RAD, those skilled in the art can implement the present invention by appropriately setting conditions suitable for the device.

EXAMPLES Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to the following Examples.

(Example 1)
In order to find a suitable pollen support for the introduction of substances into pollen using the particle bombardment method, plasmid DNA was coated on the surface of fine gold particles and shot into N. benthamiana pollen placed on various supports. The plasmid DNA was then introduced and the number of viable pollen cells was counted. Specifically, it was performed as follows.

<Plasmid DNA>
The plasmid DNA to be introduced into the pollen contains a sequence (AtUBQ10p::sGFP) that connects the sGFP gene downstream of the Arabidopsis UBQ10 (polyubiquitin 10; AT4G05320) gene promoter, and a binary vector for Nicotiana benthamiana (Addgene, pGreen0029). and prepared. Fluorescent protein (GFP) will be expressed in the cytoplasm (pollen and pollen tube) of the pollen into which the plasmid DNA has been introduced, so using this as an indicator, pollen into which the plasmid DNA has been introduced can be identified.

<Coating of gold particles with plasmid DNA>
Gold particles with a diameter of 0.6 μm (BIO-RAD, #165-2262) were washed with 100% ethanol and suspended in sterile water to prepare water containing 30 mg/mL gold particles. 10 μL of DNA-coated gold particle-containing ethanol (described below) was used for each injection, and the plasmid DNA in the ethanol was mixed and added to a concentration of 500 ng per injection, followed by stirring. To this mixed solution of gold particles and plasmid DNA, 4 μL of 0.1 M spermidine and 10 μL of 2.5 M calcium chloride were added with stirring, and the gold particles coated with plasmid DNA were precipitated by spindown. Thereafter, the supernatant was removed, washed with 20 μL of 70% ethanol, and then resuspended in 10 μL of 100% ethanol to prepare DNA-coated gold particle-containing ethanol.

<Particle bombardment method>
10 μL of the DNA-coated gold particle-containing ethanol was used for each macrocarrier used for introduction by particle bombardment. After dropping the ethanol onto the macrocarrier and air-drying it, place the introduction device (PDS-1000/He, BIO-RAD, #165-2257 J1) at the top of the main body, that is, at a position 3 cm away from the rupture disk. Macro carriers were placed so that Collect mature pollen (3 anthers, pollen with male progenitor cells collected from anthers after anther opening) from a growing N. benthamiana flower, scatter the pollen on various supports, and use it as a macrocarrier. It was placed at a distance of 3 cm or 6 cm from the

As supports, various artificial membranes and filter papers moistened with pollen tube elongation medium and pollen tube elongation medium supplemented with 1% agarose were used as shown in Tables 1 to 3 below.

In addition, for the artificial membrane and filter paper, a plastic plate was laid to back them. In addition, the amount of culture medium dropped per unit volume onto various artificial membranes was 1.35 μL/mm ³ . Furthermore, the composition of the pollen tube elongation medium is as follows.
Composition of pollen tube elongation medium: 0.01w/v% boric acid, 1mM calcium chloride, 1mM calcium nitrate, 1mM magnesium sulfate, 10w/v% sucrose (adjusted to pH 6.5 with potassium hydroxide) (Hao Wang & Liwen Jiang, Nature protocols, 2011, vol. 6, pp. 419-426).

Using a rupture disk for 1,100 psi, each shot was performed once at a helium gas pressure of 1,100 psi and a vacuum of -25 to 27 inHg. Thereafter, for those that were injected onto the artificial membrane, the pollen together with the artificial membrane was transferred onto a pollen tube elongation medium containing 1% agarose, and cultured at room temperature for about 20 hours under moisturizing conditions and shielding from light.

Then, the expression of GFP fluorescence in pollen and pollen tubes was observed under a fluorescence microscope. Pollen tubes expressing the fluorescent protein were counted as pollen into which plasmid DNA had been introduced and were surviving and germinating pollen tubes, and the numbers were compared between the supports. The results obtained are shown in FIG. 2.

As is clear from the results shown in Figure 2, when hydrophilic polytetrafluoroethylene (hydrophilic PTFE) or hydrophilic mixed cellulose ester (hydrophilic MCE) is used as a support, plasmid DNA is introduced. Moreover, the number of pollen grains that survived and germinated into pollen tubes was significantly higher than that of other supports (see 1., 2., and 5. in FIG. 2).

On the other hand, even when using PTFE, the number of pollen particles was smaller in hydrophobic PTFE than in hydrophilic PTFE (see 3. and 4. in FIG. 2). Furthermore, when using only nitrocellulose or cellulose acetate, the number of pollen particles was significantly lower than when using mixed cellulose ester composed of them (see 6. to 9. in FIG. 2). Furthermore, compared to hydrophilic PTFE or mixed cellulose ester, filter paper, nylon, and agar medium (1% agarose), which have been used as supports for a long time, allow plasmid DNA to be introduced and survive to form pollen tubes. The number of germinated pollen grains was small.

(Example 2)
Using hydrophilic PTFE, which had the highest number of introductions into pollen (cytoplasm) in Example 1, we investigated whether the substance could be efficiently introduced into the ostogen cells within the pollen using AtUBQ10p::sGFP. Instead of plasmid DNA, plasmid DNA having the following 35Sp::H2B-tdTomato that can label pollen nuclei was used.

<Plasmid DNA>
A sequence (35Sp::H2B-tdTomato) that connects a gene encoding a fusion protein consisting of a nuclear export signal derived from Arabidopsis histone H2B (AT1G07790) and tdTomato downstream of the 35S promoter of cauliflower mosaic virus (CaMV) is inserted into a SpUC vector. Plasmid DNA to be inserted and introduced into pollen was prepared. A fluorescent protein (tdTomato) is expressed in the nucleus of the pollen (including the nucleus of the androgen cell) into which the plasmid DNA has been introduced.

Furthermore, particle bombardment was carried out in the same manner as in Example 1, except that the distance between the support (hydrophilic PTFE or 1% agarose pollen tube elongation medium) and the macrocarrier was set to 3 cm, and fluorescent proteins were expressed. The number of androgen cells in the pollen tube was counted and compared between the cases where hydrophilic PTFE was used as a support and the case where a 1% agarose pollen tube elongation medium was used. The results obtained are shown in FIG.

As is clear from the results shown in Figure 3, when hydrophilic PTFE was used as a support, the number of oogen cells in pollen tubes expressing fluorescent proteins was higher than when using 1% agarose. There were many.

(Example 3)
In order to confirm that the method of the present invention does not target a specific plant species, an attempt was made to introduce a substance into lisianthus pollen using a method similar to that described in Example 1.

Specifically, in the particle bombardment method, mature pollen from Lisianthus flowers (one anther, and rogen cells collected from the anther after anther opening) is used instead of the mature pollen from Nicotiana benthamiana flowers. pollen) was used. Further, the distance between the macrocarrier and the support was unified to 3 cm. Furthermore, the composition of the pollen tube elongation medium added to various artificial membranes was changed as follows.
1.6mM boric acid, 1.8mM calcium nitrate, 2mM acetic acid, 15w/v% sucrose (adjusted to pH 5.7 with potassium hydroxide) (Park Hee Sung, Journal of Life Science, 2004, Vol. 14, No. 6, Pages 1018-1022 (please refer).

Then, an attempt was made to introduce substances into lisianthus pollen in the same manner as in Example 1 except for the above changes. The results obtained are shown in Table 4.

As is clear from the results shown in Table 4, when using hydrophilic PTFE or hydrophilic MCE as a support, even for lisianthus pollen, agar medium (which has traditionally been used as a support) 1% agarose), the number of pollen particles into which plasmid DNA had been introduced and which survived and germinated pollen tubes was large (see the "Fluorescence" item in Table 4).

Therefore, it was confirmed that according to the method of the present invention, substances can be introduced into pollen with high survival rate and high introduction efficiency, regardless of the type of plant.

(Example 4)
In order to confirm that the method of the present invention does not target only mature pollen, we introduced substances into male gametes at an immature stage (tetrads, microspores, immature pollen with two nuclei). , I tried using the method shown below.

<Particle bombardment method>
A macrocarrier having the DNA-coated gold particles attached to its lower surface was prepared in the same manner as described in Example 1, and placed in an introduction device at a distance of 3 cm from the rupture disk.

The stages of male gametes to be introduced are as follows, and they were prepared from growing N. benthamiana flowers using the size of their buds as an index.
(1) Stage with a mixture of tetrads and microspores (2) Stage consisting of microspores (3) Stage consisting of immature pollen with two nuclei In addition, in (1) and (2), the male gametes of 20 anthers are The child was taken. In (3), male gametes from 15 anthers were collected. After collection, a portion of each male gamete was taken out and stained with DAPI to observe the number of nuclei within the male gamete and confirm that it was at each stage.

Next, each of the male gametes thus prepared was placed on a hydrophilic PTFE support (manufactured by MERCK, product name: Omnipore) to which a male gamete initial medium having the following composition was added at a concentration of 1.35 μL/ ^mm3 . ) and placed at a distance of 3 cm from the macrocarrier.
Composition of male gamete initial medium: 0.016mM boric acid, 1mM calcium nitrate, 1mM magnesium sulfate, 10mM potassium nitrate, 1w/v% peptone, 3mM glutamine, 1mM uridine, 0.5mM cytidine, 0.5M sucrose (20mM phosphate) (adjusted with buffer solution (pH 7.0)) (Jaroslav Tupy and Ludmila Rihova, Sexual Plant Reproduction, 1991, Vol. 4, pp. 284-287. See).

Then, using a rupture disk for 1,100 psi, each shot was performed once at a helium gas pressure of 1,100 psi and a reduced pressure of -25 to 27 inHg. Next, the pollen on the hydrophilic PTFE support was collected by washing with a male gamete initial medium, and cultured using the medium at room temperature in the dark for about 20 hours. Thereafter, the expression of GFP fluorescence in each male gamete was observed under a fluorescence microscope.

As a result, as shown in Figures 4A to 4C, GFP fluorescence was detected in male gametes at all stages, confirming that according to the present invention, substances can be introduced into pollen regardless of whether it is mature or immature. was completed.

(Example 5)
In order to confirm that the method of the present invention does not target only nucleotides, attempts were made to introduce other substances (RNA-protein complexes) into pollen using the method described below.

<Particle bombardment method>
In order to introduce an RNA protein complex (hereinafter also referred to as "RNP") into pollen (mature pollen), a macrocarrier for particle bombardment loaded with RNP was prepared by the following procedure.

RNP was prepared by mixing 3500 ng of commercially available Cas9 protein, 467 ng of tracrRNA, and 280 ng of crRNA in a 30 μl solution (20 mM Tris-HCl, 200 mM KCl, 2% sucrose). In order to detect the introduction of RNP, the tracrRNA used was a commercially available product (manufactured by Fasmac Co., Ltd.) that was fluorescently labeled with carboxyfluorescein (FAM: green fluorescence).

Next, by ultrafiltration, the RNP solution was replaced with a solution consisting of 20 mM Tris and 2% sucrose, and then replaced with 20 mM Tris. On the other hand, gold particles with a diameter of 0.6 μm (manufactured by BIO-RAD) were washed with 100% ethanol and suspended in sterile water to prepare a 30 mg/ml gold solution. Then, 10 μl of the gold solution was added per shot to 20 μl of the RNP solution after the buffer exchange, and 30 μl of the gold solution was added to one particle bombardment macro carrier (PDS-1000/He macro carrier manufactured by BioRad). was loaded. The loaded macrocarrier was degassed and dried at 25° C. for about 20 minutes under a reduced pressure of about 100 hPa (about 1/10 of 1 atm) to produce a macrocarrier for particle bombardment loaded with RNP.

Next, the macrocarrier was placed at the top of the introduction device (PDS-1000/He, BIO-RAD, #165-2257 J1), ie, at a distance of 3 cm from the rupture disk. Collect mature pollen (3 anthers, pollen with male progenitor cells collected from anthers after anther opening) from a growing N. benthamiana flower, and add 1.35 μL/mm of the above male gamete initial medium. ³ on a hydrophilic PTFE support (manufactured by MERCK, product name: Omnipore) and placed at a distance of 3 cm from the macrocarrier.

Then, using a rupture disk for 1,100 psi, each shot was performed once at a helium gas pressure of 1,100 psi and a reduced pressure of -25 to 27 inHg. Next, the expression of FAM fluorescence in the pollen on the support was observed under a fluorescence microscope.

As a result, as shown in FIG. 5, FAM fluorescence was detected in pollen, confirming that the present invention can be introduced into pollen regardless of the type of substance.

As explained above, according to the present invention, it is possible to introduce substances into pollen with high survival rate and high introduction efficiency.

For example, in the production of plants through genetic recombination of somatic cells, long-term cultivation is required. In contrast, in the present invention, since pollen is targeted, progeny can be obtained more easily and in a shorter period of time by pollinating pollen into which a substance such as a genetic material has been introduced. Therefore, it is possible to introduce substances such as genes into pollen with high survival rate and introduction efficiency, and furthermore, the ability to easily and quickly produce progeny using pollination methods will greatly contribute to the agricultural field.

DESCRIPTION OF SYMBOLS 1...Particle bombardment device, 2...Gas acceleration tube, 3...Rupture disk, 3a...Rupture disk after rupture, 3b...Rupture disk after rupture, 4...Macro carrier, 5...Fine particles coated with substance, 6...Speed Topping screen, 7...Pollen, 8...Support.

Claims (1)

A method of introducing a substance into pollen, the method comprising:
The particle bombardment method involves the process of bombarding pollen particles coated with a substance onto pollen particles placed on a support.
The support is a support containing at least one polymer selected from hydrophilic polytetrafluoroethylene and hydrophilic mixed cellulose ester,
the substance is a nucleotide and/or a peptide, and
The method , wherein the fine particles are fine particles made of gold or tungsten .