JP2773062B2 - Mass Rapid Propagation of Citrus Plants - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for rapidly mass-producing citrus plants, and more particularly, to stable and healthy somatic cells from undifferentiated cultivated cells (embryogenic callus) capable of forming somatic embryos of citrus plants in liquid culture. The present invention relates to a method for producing a large amount of embryos in a short time.

[0002]

2. Description of the Related Art Conventionally, as a method of inducing somatic embryos from undifferentiated culture cells of a plant, for example, undifferentiated culture of a carrot of the Apiaceae family or an eggplant of the Solanaceae family in a liquid medium or a solid medium is known. A method for inducing somatic embryos by culturing cells in a liquid medium having another composition [Hirada Hiroshi et al., 1st edition, "Plant Cell Tissue Culture", 1st edition, pp. 91-96, 19
In 1979, published by Rigaku Kogyo Co., Ltd., JP-A-62-232312], or in citrus family, callus derived from the nucleus obtained by culturing ovules on a solid medium was used as a protoplast, and this was cultured on a soft agar medium. Method to induce somatic embryos (A.
Vardi et al. Theor. Appl. Genet. 62, 171-176 (198
2)] has been reported.

[0003] However, when the purpose is to grow a large amount of plants, in the former method, since somatic embryos are induced by liquid culture, the somatic embryos are often in a water-immersion state.
(vit-rification), the plant regeneration rate is low, and the latter method is an experimental proof that the plant is simply regenerated from single-cell protoplasts. Also, it takes about 6 months to obtain a plant, and it cannot be adopted as a mass propagation method at all.

[0004] In order to solve these drawbacks, undifferentiated cells grown in liquid culture recently using asparagus of the lily family are cultured in a solid medium to induce somatic embryos more efficiently than before. [Saito et al.,
Breeding 39 (Annex 2), pp. 72-73 (1989)].

[0005]

DISCLOSURE OF THE INVENTION The present invention provides a method for inducing a stable and healthy somatic embryo from a undifferentiated cultured cell of a Citrus family plant more efficiently in a quantitative and timely manner. It is intended to establish a system for reproducing a large amount and in a short time.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above-mentioned object, and as a result, undifferentiated cultured cells of Rutaceae plants obtained by culturing in a liquid medium were sterilized on a solid medium. After being dried by forced forced drying, by culturing it, we have obtained a new finding that stable and healthy somatic embryos can be induced extremely efficiently more than ever before, and based on this finding, we have completed the present invention. Reached.

[0007] That is, a first invention of the present invention relates to a method for growing a Rutaceae plant, which comprises culturing undifferentiated proliferating cultured cells having the ability to form somatic embryos of Rutaceae plants to induce somatic embryos, Undifferentiated cultured cells of the Citrus family obtained by culturing in a solid medium are dried by aseptic forced drying, and then cultured to induce somatic embryos. It is a rapid growth method.

[0008] Further, a second aspect of the present invention, which includes a preferred embodiment of the first aspect, comprises culturing undifferentiated proliferating cells having the ability to form somatic embryos of Rutaceae plants. In a method for growing Rutaceae plants that induces somatic embryos, the cells having the ability to form somatic embryos obtained by culturing cells having the ability to form somatic embryos of Rutaceae plants in a liquid medium containing a plant hormone that inhibits somatic embryogenesis The undifferentiated growth cultured cells are washed with a liquid that does not contain the plant hormone, suspended in the liquid, and diffused on a solid medium that does not contain the phytohormone. This is a method for mass-proliferation of a Citrus family plant, which comprises aseptically drying the surface of a grown culture cell and the solid medium, and then culturing it to induce a somatic embryo.

In the second invention, sterile water is particularly preferable as the liquid containing no plant hormone. Hereinafter, the present invention will be described in detail. First, the Citrus family plant used in the present invention is not particularly limited, but among them, for example, sweet oranges, grapefruit, lemon, Sudachi, citrus such as Satsuma mandarin, and the like are preferred, and sweet oranges are particularly preferred. . For orchards that cannot reproduce seeds, grafts, cuttings,
Propagation is carried out by breeding methods such as harvesting, but it takes a long time, such as several years, to obtain a large amount of seedlings, for example, to grow them to tens of thousands. The present invention is particularly effectively applied to such a plant.

The citrus plants include not only varieties but also mutants at the cell (including protoplast) level and plant level, hybrids by crossing, somatic hybrids by cell fusion [eg, Plants such as somatic hybrids obtained by cell fusion of cells and cells having no somatic embryogenic ability (such as mesophyll cells) (see JP-A-61-192283); It may be your body. Next, the cells having the ability to form somatic embryos of these Rutaceae plants may be any as long as they have somatic embryogenic ability. For example, those obtained by collecting and culturing ovules (cultures derived from pearl hearts) Cells), cultured cells via protoplasts, cultured cells accompanying (by-produced) somatic embryos obtained by cell fusion using protoplasts (see, for example, the above-mentioned JP-A-61-192283),
Cultured cells in which a foreign gene has been introduced into cells, as well as virus-free ones thereof, may be mentioned.

[0011] Then, by culturing the cells having the ability to form somatic embryos in a liquid medium containing a plant hormone that suppresses somatic embryo formation, a large number of undifferentiated and expanded cultured cells having the ability to form somatic embryos can be obtained. Can proliferate. One example of this is, for example, 8 days from the flowering date of Rutaceae plants such as sweet orange, grapefruit and lemon.
Aseptically take out the ovule from the flowers until about a week,
A medium containing no plant hormones, for example, a medium containing 5% (w / v) sucrose and 0.8 to 1.0% (w / v) agar (eg, an MT agar medium, etc., MT medium: Murashige and Tucker) Medium (Proc. First Int. Citrus Symp. 3 , 1155
1161161 (1969)), a medium containing no plant hormones and agar is referred to as MT medium or MT] or a medium containing benzylaminopurine, naphthaleneacetic acid, maltoextract, adenine sulfate, etc. By culturing under low light for about 2 to 5 months, first, cultured cells derived from the nucleus are obtained. The obtained cultured cells are maintained and cultured in a solid medium by a conventional method.

Next, a liquid medium containing a plant hormone which suppresses the ability to form somatic embryos, for example, an MT liquid medium containing 1 to 20 mg / l of one or more plant hormones such as benzyladenine and kinetin. For example, by subculturing 2 to 10 times about every two weeks, somatic embryogenesis is suppressed, and undifferentiated cultured cells that have grown in large quantities can be obtained. In this case, the culture was carried out at
It is preferable to carry out rotation shaking culture at 0 to 150 rpm.

The identification of the ability to form somatic embryos in cells of Rutaceae plants may be performed according to a conventional method. For example, an MT solid medium containing a plant hormone such as benzyladenine or kinetin that suppresses somatic embryo formation is used. The cells in the maintenance culture are transplanted to the MT solid medium not containing the plant hormone, and cultured at 20 to 30 ° C. under low light for 1 to 2 months, during which green somatic embryo formation is confirmed. For example, it is identified as having somatic embryogenesis ability.

Subsequently, the undifferentiated proliferating cultured cells having the ability to form somatic embryos thus obtained are washed with a liquid containing no plant hormone which suppresses somatic embryo formation. The purpose of this is to remove plant hormones that suppress somatic embryogenesis as described above when somatic embryos are induced in a subsequent step. As the liquid containing no plant hormone to be used, various media may be used. However, as a result of various studies on this point, the present inventors have found that the use of sterilized water is simple and economically advantageous. Not only that, the germination rate of the induced somatic embryos is equal to or higher than that in the case where the medium is used, and thus it has been found that the germination rate is more preferable. According to the washing with sterilized water, one of the features of the method of the present invention is aseptic forced drying in the next step, that is, drying of the surface of the undifferentiated growth cultured cells and the solid medium. It is considered that there was no increase in the component concentration on the cell surface and the medium surface observed.

This washing is carried out by, for example, taking a suspension of undifferentiated cultured cells in a centrifuge tube, adding the above liquid, and allowing the suspension to stand or centrifugal separation, and removing the supernatant, for example, two to three times. Done. This is then centrifuged and the amount of compressed cells
(packed cell volume: PCV), and using the same liquid as the washing liquid, low density, for example, 50-200 for PCV.
Suspend by a factor of two. Then, this suspension is diffused on the surface of a solid medium containing no plant hormones for suppressing somatic embryo formation for inducing somatic embryos.

The composition of the solid medium used at this time may be appropriately selected so as to be suitable for somatic embryo formation. Instead of sucrose, maltose is 2 to 10% (w / v) and malt extract is 100 to 100%. A preferred example is an MT medium containing 1000 mg / l and adenine sulfate of 10 to 100 mg / l. In addition, as sugar, for example, maltose,
Lactose, galactose, sucrose, glucose and the like can be mentioned, but maltose is particularly preferred, followed by galactose and lactose.

As the solid medium, for example, 0.2 to 1.0%
(w / v) gel light [GELRITE = Trademark of Merck Co., Ltd. (trade name, manufactured by Sanei Chemical Industry Co., Ltd., GELLAN GUM)) P
A polysaccharide produced outside the cells by se-udomonas elodea is purified by deacetylation and then purified to a dry powder, which contains glucose, rhamnose, uronic acid, etc.] Medium, 0.5 to 2% (w / v) agar medium, agarose medium and the like. Particularly, a high concentration of about 1% (w / v) gellite medium prevents water immersion and obtains a healthy somatic embryo. Is preferred. The diffusion amount of the suspension in the solid medium is about 1 to 10% (v / v).

Next, the solid culture medium in which the suspension of the undifferentiated cultured cells has been spread on the surface is forcibly dried aseptically. This drying step is the most characteristic feature of the present invention. In other words, the aseptic forced drying causes a rapid decrease in the surface water of the undifferentiated cultured cells and the solid medium, thereby reducing the induced water-immersion state of the somatic embryo, which is one of the drawbacks of the conventional method. It is eliminated at once, and a stable and healthy somatic embryo can be obtained as shown in Examples described later, and effects such as improvement of the regeneration efficiency of the plant can be obtained. Aseptic forced drying is different from natural drying that occurs gradually only in a state of being exposed to the outside air, and forcibly or artificially promotes drying by aseptic blowing of air with adjusted temperature and humidity. And preferably at a temperature of 20 to 30 ° C. and a humidity of 1
Perform for 5 to 20 hours under the condition of 0 to 50%. The means for aseptic forced drying is not particularly limited. For example, a clean bench (sterile ventilation laboratory bench) installed in a room where the temperature and humidity can be adjusted, an aseptic blast drying device whose temperature and humidity can be adjusted, and A culturing facility equipped with the aseptic blast drying device may be appropriately selected and used.

Next, culture for inducing somatic embryos is performed at 20 to 30 ° C., preferably 23 to 28 ° C., for 3 to 6 weeks, preferably for about 4 weeks. This culture is preferably performed under aseptic aeration conditions from the viewpoint of homogenization of somatic embryos, efficiency of acclimation, and the like. The aseptic ventilation conditions include, for example, a membrane having aseptic ventilation (for example, Milli-seal, Milliwrap (trade name, manufactured by Millipore) of a Teflon-made sterile ventilation membrane)
And a stopper [for example, New Silicosen (trade name, manufactured by Shin-Etsu Polymer Co., Ltd.)] and the like. The culture is 1,000-4,000 lux, preferably 2,000-3,000.
The operation is carried out at 000 lux under continuous or intermittent, preferably continuous, illumination.

By culturing in this manner, a cotyledon-like somatic embryo can be obtained. The somatic embryo of the Citrus family plant obtained as described above can be turned into artificial seeds, and further cultured to regenerate the plant.
Culture conditions and the like in this case may be appropriately selected depending on the type of the plant. For example, cotyledon-like somatic embryos are placed on an MT solid medium containing 1 to 20 mg / l of gibberellin and 2 to 8% (w / v) of sucrose, and intermittently illuminated under aseptic aeration using Milliseal or the like. [Approx. 16 hours light (3,000
~ 5,000lux), dark for 8 hours]
By culturing for 4 weeks [regeneration culture [I]], a plant with particularly elongated shoots (buds) is regenerated. The buds usually start to appear about one week after the culture.

Still another medium, for example, 0.01 to 0.1 mg / l naphthalene acetic acid, 1 to 5% (w / v) sucrose
And placed on an MT agar medium containing
Culture for 4 to 6 weeks under the same conditions as in [Regeneration culture [II]]
After the rooting, the plant can be obtained by grafting the stem apex or raising the pot. In addition, the somatic embryo of the obtained Rutaceae plant can be obtained immediately by omitting the regeneration culture [I] and immediately cultivating the regeneration culture [II] to obtain a plant body.

In the case of Rutaceous plants, it takes several years to grow in conventional grafting methods such as grafting and cuttings. However, according to the method of the present invention, for example, in the case of sweet orange, 1 g of undifferentiated Approximately 15.0
00 somatic embryos were obtained, and at a high rate of 70%, that is, 10,000 plants, the period from the start of solid culture of undifferentiated proliferating cultured cells to potting was as short as 2 to 4 months. Obtained between.

[0023]

Examples are shown below. Example 1 An ovule was aseptically taken out of a flower of a sweet orange flower on the day of flowering with tweezers, and this was extracted with malt extract 10
Cultured cells derived from pearls were obtained by placing on an MT medium containing 0 mg / l and adenine sulfate 20 mg / l and containing no plant hormone, and culturing for 150 days. The cells having the ability to form somatic embryos were treated with the plant hormone kinetin (10 mg).
/ L of MT liquid medium containing 50 ml / l three times every two weeks, and transfer the obtained undifferentiated expanded cultured cells to a graduated centrifuge tube.
0.5 ml was taken, and the suspension was added with 15 ml of sterile water and suspended. After standing, the operation of removing the supernatant was repeated twice to wash the undifferentiated cells. This compressed cell volume (PCV) of 100
2 ml of this suspension in a 200 ml plant culture flask, MT1% (w / v) gellite medium [maltose 5% (w / v) instead of sucrose, maltoextra 500 mg / l, adenine sulfate 40 mg / l
l containing] (50 ml). Without capping the flask, the undifferentiated proliferating cultured cells were forcibly dried at a temperature of 26 ° C. and a humidity of 30 to 40% for 15 hours on a clean bench (TYPE PCV manufactured by Hitachi, Ltd.), which is a sterile ventilation test bench. And after aseptically drying the surface of the solid medium,
The flask was covered with aluminum foil to which a sterile air-permeable membrane Milli-seal (trade name, manufactured by Millipore) was attached, and
The cells were cultured under continuous lighting at 3,000 lux at ℃. One month later,
Somatic embryos were induced in most cell masses, and about 200 cotyledon-like somatic embryos were obtained per flask. This means that about 15,000 somatic embryos were obtained per gram of undifferentiated cultured cells.

Next, 100 obtained somatic embryos were subjected to MT0.5 containing gibberellin 10 mg / l and sucrose 2% (w / v).
% (W / v) gellite medium (referred to as regeneration medium (I) containing GA in Example 2) (50 ml), covered with aluminum foil covered with milliseal, and placed at 26 ° C., 4,000
0 lux long day conditions (16 hours light, 8 hours dark)
For one month. 72 germinated during this period were immediately 0.05 mg / l naphthaleneacetic acid and 2% sucrose.
(w / v) MT 0.2% (w / v) Gelrite medium (50 ml)
Then, the plate was covered with aluminum foil to which a milliseal was attached, and cultured for about one month at 4,000 lux at 26 ° C. for about one month.

That is, plants were obtained from about 70% of the somatic embryos within three months from the start of the solid culture of the undifferentiated expanded cultured cells. 1 g of sweet orange
Approximately 10,000 plants per undifferentiated cultured cell of the present invention can be obtained. Example 2 Induction of somatic embryos and germination of somatic embryos were carried out in the same manner as in Example 1 except that the step of aseptic forced drying in the induction stage of somatic embryos and the presence or absence of the application of a milliseal were as shown in the following table. I let you. The following table shows the number of individuals that germinated within one month after culture in the regeneration (I) medium containing GA per 100 somatic embryos obtained at this time.

The result of No. 1 in the table is the same as that of Example 1.
belongs to.

[0027]

[Table 1]

From the table, prior to the induction of somatic embryos, the surfaces of the undifferentiated expanded cultured cells and the solid medium were aseptically forcedly dried (N
o. 1 and No. 2 = method of the present invention), it was found that the germination rate of the induced somatic embryos can be significantly improved by drastically drying, and the obtained plants are extremely healthy. Met. Example 3 Mass Rapid Propagation of Somatic Hybrids (1) Method for Production of Somatic Hybrids
Protoplasts were isolated from the cultured cells of Sudachi subcultured on a 0.3% (w / v) gellite medium containing mg / l and the mesophyll tissue of Mexican lime by the method of Vardi et al. After mixing these, 1 MH
z, an alternating current of 500 V / cm for 15 seconds, then 1.5 KV / cm
The operation of giving a DC pulse having a cm pulse width of 40 μsec was repeated twice to fuse the protoplasts. The fused protoplasts were then sucrose 2
The cells were cultured in MT agarose medium containing 0.5% (w / v) to obtain somatic embryos derived from hybrid cells.

[0029] These somatic embryos were prepared using MT 0.2 containing malt extract 500 mg / l and adenine sulfate 40 mg / l.
A regenerated plant was obtained by transplanting into a% (w / v) gellite medium and subcultured (the plant was confirmed to be a somatic hybrid by chromosome number and nuclear ribosomal DNA analysis). . Further, a part of the somatic embryo was transferred to an MT0.2% (w / v) gellite medium containing 5 mg / l of benzyladenine, and subcultured to induce undifferentiated cultured cells.

(2) Maintenance of undifferentiated cultured cells of somatic cell hybrids and assay of somatic embryogenesis ability Undifferentiated cultured cells of somatic cell hybrids of Sudachi and Mexican lime are undifferentiated from ordinary citrus pearl MT0.2 containing 5 mg / l of benzyladenine as well as growing cultured cells.
% (W / v) gellite medium. For assay of somatic embryogenesis, sucrose 5% (w / v)
Was transplanted to a 0.2% (w / v) MT gel-free medium containing plant hormones that inhibit somatic embryogenesis, with maltose 5% (w / v). Formed green somatic embryos. From this, it was confirmed that the undifferentiated expanded cultured cells have somatic embryogenesis ability.

(3) Mass Rapid Propagation of Somatic Hybrids The cells having somatic embryogenic ability of the somatic hybrids obtained as described above were placed every two weeks in 50 ml of MT liquid medium containing 5 mg / l of benzyladenine. By culturing the cells three times, undifferentiated cells grown in large quantities were obtained. About 0.5 ml of the undifferentiated cultured cells thus obtained was placed in a graduated centrifuge tube, and 15 ml of sterile water was added thereto to suspend the cells. After standing, the operation of removing the supernatant was repeated twice. The growth culture cells were washed. The suspension was suspended in sterilized water 100 times the volume of the compressed cells (PCV), and 2 ml of the suspension was placed in a 200 ml plant culture flask with MT1% (w / v) gellite medium [maltose 5 instead of sucrose. % (W / v), containing 500 mg / l of malt extract and 40 mg / l of adenine sulfate] (50 m
l) spread on the surface. Without capping the flask,
A clean bench [TYPE PCV manufactured by Hitachi, Ltd.] which is an aseptic ventilation experiment table, temperature 26 ° C, humidity 15-25%,
After forcibly drying for 8 hours, the surfaces of the undifferentiated proliferating cultured cells and the solid medium are aseptically dried, and then the flask is covered with a sterile and air-permeable stopper New Silicocene (trade name of Shin-Etsu Polymer Co., Ltd.). , 26 ° C, 3000 lux continuous light. Six weeks later, cotyledon-like somatic embryos were induced in many cell masses, and about 50,000 somatic embryos were obtained per gram of undifferentiated expanded cultured cells.

Next, the obtained somatic embryo was subjected to naphthaleneacetic acid 0.0
5 mg / l, MT 0.2% (w / v) sucrose 2% (w / v)
v) Place on a gellite medium (50 ml), cover with new silicocene, and incubate for about 5 weeks at 4,000 lux at 26 ° C for about 5 weeks. I raised the pot. That is, plants were obtained from about 50% of the somatic embryos in about three months after the start of solid culture of the undifferentiated cultured cells.

Thus, in the case of a somatic hybrid of Sudachi and Mexican lime, about 2.5
00 plants will be obtained.

[0034]

As described above, according to the method of the present invention, a large amount of stable and healthy somatic embryos can be obtained from undifferentiated proliferating cultured cells having the ability to form somatic embryos of Rutaceae plants in a short time. The plant can be efficiently regenerated by further culturing it. Thus, the method of the present invention is industrially extremely significant.

Continuation of the front page (72) Inventor Junichi Shimizu 27-5 Tsurubon, Noda City, Chiba Prefecture (72) Inventor Shigetaka Ishii 101, Miyazaki, Noda City, Chiba Prefecture (56) References JP-A-1-218519 (JP, A) Kaihei 1-218520 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) A01H 4/00 BIOSIS (DIALOG) JICST file (JOIS)

Claims (3)

(57) [Claims] 1. A citrus plant obtained by culturing in a liquid medium in a method for growing a citrus plant to induce a somatic embryo by culturing undifferentiated cultured cells capable of forming somatic embryos of a citrus plant. Undifferentiated cultured cells having the ability to form somatic embryos are dried on a solid medium by aseptic forced drying, and then cultured to induce somatic embryos. Law. 2. A method for growing a Rutaceae plant, which comprises culturing undifferentiated proliferating cells having the ability to form somatic embryos of Rutaceae plants to induce somatic embryos. After culturing cells in a liquid medium containing a plant hormone that suppresses somatic embryogenesis, undifferentiated proliferating cultured cells having somatic embryogenesis ability are washed with a liquid containing no plant hormone, and then suspended in the liquid. After turbidity and spreading on a solid medium not containing the plant hormone, the undifferentiated growth cultured cells and the surface of the solid medium are aseptically dried by aseptic forced drying, and then cultured. A method for rapidly mass-producing citrus plants, comprising inducing somatic embryos. 3. The method according to claim 2, wherein the liquid containing no plant hormone is sterilized water.