JP5738545B2 - Liquid medium for suspension culture of endosperm cells of plants belonging to the genus Brassica or genus Brassica, and method for culturing the suspension of endosperm cells of the plants of genus Brassica or genus Brassica using the same - Google Patents

Description

The present invention relates to a liquid medium for culturing endosperm cells of a plant of the genus Brassica or genus Brassica and an endosperm cell suspension culture method of the plant of the genus Brassica or genus Brassica using the same. More specifically, the present invention relates to a liquid medium for culturing endosperm cells of a plant of the genus Brassicum or genus Brassica suitable for reducing the size of a cell mass formed by suspension culture in a liquid medium. Relates to a method for culturing an endosperm cell suspension of a Brassica plant or a Brassica plant utilizing the above.

  In the present specification, “suspension culture” means a method of culturing plant cells in a liquid medium while applying physical stimulation such as shaking, vibration, swirling, and bubbling.

  Plant cells have a strong cell wall structure and thus have strong adhesion between cells. Generally, plant cell masses (such as callus) formed on a solid medium are composed of a large number of cells and tend to be large. In the enlarged cell mass, the environmental conditions are different between the inside of the mass and the surface layer, so that homogeneous cells could not be obtained, which was insufficient for passage or use as an experimental sample.

  In contrast, some model cells, such as tobacco BY-2 cells, Arabidopsis T87 cells, Deep cells, MM1 cells, MM2d cells, and Alex cells, have physical properties in suspension culture performed in a liquid medium with shaking or stirring. It is known that adhesion between cells is weakened by mechanical stimulation. As a result, the cultured cell mass is composed of a relatively small number of 10 or more cells, and it is easy to obtain homogeneous cells, and an excellent experimental sample can be obtained (see Patent Document 1).

JP-A-9-205918

  However, since plant cells that can reduce the size of the cell mass only by physical stimulation in suspension culture are limited to some of the model cells described above, most of the other plant cells are cultured in a solid medium. As in the case of suspension culture, the cell mass becomes large even in suspension culture. For this reason, since a homogeneous cell cannot be obtained, it is desired to reduce the cell mass by suspension culture.

  By the way, in recent years, with the growing need to use plant biomass as an alternative energy for fossil fuels, oil plant plants that can be applied to biodiesel fuel production have been rapidly planted. Euphorbiaceae genus Brassica and Brassica are attracting attention as new plantation plants because they contain about 30% by weight of oil in their seeds. However, breeding research has not been conducted, and varieties having good characteristics such as high fat content have not been sufficiently selected or produced. At present, there are many unclear points in the generation of oil storage organs and the mechanism of oil synthesis and accumulation, and no early evaluation method for productivity and definitive productivity-enhancing genes have been found. The cultivation of Brassica plants requires a large-scale greenhouse and fields, and at the same time, restrictions such as a period of several months from germination to seed harvesting hinder research promotion, and the genes and environment involved in oil production Techniques for quickly and comprehensively evaluating the factors are not well established. In order to quickly and comprehensively evaluate and study the functions and effects of genes and environmental factors involved in oil and fat productivity, it is necessary to develop tools that can be used for the evaluation of oil and fat productivity.

  In the seeds, fats and oils are stored in the seeds as energy sources (nutrients) during germination, as are starches and sugars. The genus Brassica and Brassica store oils and fats in highly developed endosperm that occupies 60-70% by volume of the seed volume. Therefore, endosperm is the subject of research when the oil and fat component and the modification of the content are targeted for breeding in the genus Brassica or Brassica.

  Since the cells that make up the endosperm are special cells generated by double fertilization, research using endogeneous somatic cells (immature embryos, embryos, cotyledons, hypocotyls, roots, stems, leaves, etc.) It cannot properly simulate or evaluate events that occur in cells. Therefore, a method for preparing cell protoplasts from endosperm has been developed for a long time, and has been used for enzymological studies and molecular biology studies. However, as a characteristic of endosperm cell protoplasts, it has been reported that in cells with advanced developmental stages (cells with accumulated storage substances and enlarged), physical damage during preparation is large and the survival rate is significantly reduced. To prepare high-quality endosperm protoplasts, it is necessary to collect pre-mature and mature endosperm, but a large amount of plant samples are required, and cell preparation at the same development stage is extremely difficult.

  In general, in research on specific tissues and cells, the use of cultured cells is also effective for preparing homogeneous plant samples. That is, by developing cultured cells derived from endosperm, the time required for sample preparation can be drastically shortened, and the labor for cell preparation can be omitted. Furthermore, cultured cells are effective for analyzing metabolites such as proteins, carbohydrates and lipids, and at the same time have many advantages in analyzing enzyme activity and the like by transiently expressing genes or proteins. For these reasons, it has been desired to establish a technique for culturing endosperm cells of the plants of the genus Brassica or Brassica.

  However, the technology for culturing endosperm cells of the genus Brassica or the genus Brassica has not been established so far, and the above analysis and the like cannot be performed satisfactorily. In order to obtain homogeneous cells, it is desirable to reduce the cell mass by suspension culture. Therefore, it is desired to establish a technique for culturing endosperm cells and to reduce the size of the cell mass without increasing the size by suspension culture.

The present invention has been made in view of such a demand, and is capable of realizing effective cell culture for endosperm cells of plants of the genus Brassica and Brassica, and further, a cell mass at the time of suspension culture is obtained. It is an object of the present invention to provide a liquid medium for suspension culture of endosperm cells of a Brassica plant or Angiobula plant that can prevent enlargement, and an endosperm cell suspension culture method of an Brassica plant or an Brassica plant using the same. To do.

  In order to achieve such an object, the present inventors have conducted intensive research, and as a result, by adding cytokinin and auxin to a basic medium for culturing plant cells, the endosperm cells of the Brassica plant and the Brassica plant can be obtained. It has been found that growth can be drastically improved and the growth of endosperm cells can be drastically improved by making these addition amounts within a specific range.

However, it was found that the cell mass becomes large and homogeneous cells cannot be obtained only by suspending and culturing the endosperm cells of the Brassica plant or the Brassica plant in a liquid medium supplemented with cytokinin and auxin. Therefore, the present inventors have conducted intensive studies and found that the addition of cytokinin and auxin simultaneously with the addition of gibberellin can reduce the cell mass while promoting proliferation. As a result, gibberellin can reduce the cell walls of plant cells and weaken the adhesion of cell walls between cells. As a result, the cell mass formed in suspension culture can be reduced in size. As a result , the present invention was completed.

That is, the liquid medium for endosperm cell suspension culture of the Brassica plant or the Brassica plant of the invention of claim 1 is added with gibberellin in an amount of 0.5 to 1.0 μmol / L , and further added with cytokinin and auxin. It is characterized by having. Gibberellin promotes the growth of plant tissues by loosening the cell walls of plant cells when it acts on plant tissues, but it weakens cell-cell adhesion by loosening the cell walls when acting on cell clusters that are not tissues. The mass can be subdivided into small cell masses consisting of few cells. In addition, the addition of cytokinin and auxin promotes the growth of endosperm cells of the plant belonging to the genus Brassica or the plant belonging to the genus Brassica. For this reason, the cell mass is reduced in size while maintaining the cell growth ability of the endosperm cells of the Brassica plant or the Brassica plant .

In this case, as in the invention of claim 2, wherein the di Bererin _(sometimes referred to as GA ₃ in the following description.) Gibberellin A ₃ is preferably a. These further promote the downsizing of the cell mass.

In this case, as in the invention described in claim 3 , it is preferable that cytokinin is 5 to 20 μmol / L and auxin is 5 to 20 μmol / L. Furthermore, as in the invention described in claim 4 , it is preferable that cytokinin is benzyladenine and auxin is 3-indoleacetic acid. By these, the cell proliferation of the endosperm cells of the Brassica plant or the Brassica plant is further improved.

On the other hand, the endosperm cell suspension culture method of the Brassica plant or the Brassica plant of the invention according to claim 5 is the endosperm cell of the Brassica plant or the Brassica plant of any one of claims 1 to 4. By using a liquid medium for suspension culture, the endosperm cells of the Brassica plant or the Brassica plant are suspended and cultured . Therefore, endosperm cells while being accelerated growth, reduction in size of the cell mass is achieved.

According to the first aspect of the present invention, the cell mass can be miniaturized while maintaining the cell growth ability of the endosperm cells of the Brassica plant or the Brassica plant. Therefore, since it becomes easy to obtain homogeneous cells by suspension culture, excellent cultured cells can be obtained as experimental samples.

In addition, according to the invention described in claim 2 , since it is possible to further reduce the size of the cell mass, it becomes easier to obtain homogeneous cells by suspension culture.

Moreover, according to the invention of Claim 3 and 4 , a cell mass can be reduced in size, further improving the cell growth of the endosperm cell of a Brassica genus plant or a Brassica genus plant.

Further, according to the invention described in claim 5, since it becomes easy to obtain a homogeneous endosperm cell for the plant of the genus Brassica or the genus Brassica, it is possible to obtain an excellent endosperm cell as an experimental sample.

It is a graph showing the relationship between the amount of gibberellin added and the number of cells / lumps in the plant cell suspension culture method using the plant cell culture liquid medium of the present invention, and there is no significant difference between the same alphabets (Multiple comparison of Tukey, p <0.05). It is a microscope picture which shows the cell cultured by the plant cell suspension culture method of this invention, (A) (B) does not add gibberellin, (C) (D) adds gibberellin 1.5 micromol / L, (E) (F) shows the case where gibberellin is added at 3.0 μmol / L. It is a graph which shows the relationship between the addition amount of gibberellin and the cell diameter in the plant cell suspension culture method of the present invention, and there is no significant difference between the same alphabets (Tukey's multiple comparison, p <0.05). It is a graph which shows the relationship between a culture | cultivation period when subcultured by suspension culture, and a cell volume.

  Hereinafter, the configuration of the present invention will be described in detail based on an example of an embodiment shown in the drawings.

  In the present invention, suspension culture of plant cells is performed using a liquid medium in which gibberellin is added to a liquid medium for suspension culture of plant cells.

  The present invention can target all plant cells whose cell mass can be enlarged by suspension culture. Specific examples include endosperm cells of plants belonging to the genus Brassica or genus Brassica, but are not limited thereto, and for example, snapdragons, ice plants and the like can also be used as culture targets.

  As a liquid medium for suspension culture of plant cells, for example, Murashige Skog (MS) medium or B5 medium can be used, but is not limited thereto. Specifically, Murashige Skog (MS) medium (WAKO) having a pH of 5.8 containing 3% by weight of sucrose can be preferably used, but is not limited thereto.

As gibberellin, gibberellin A _{1 to} A ₁₃₆ or a mixture of two or more of these can be used, and gibberellin A ₃ can be suitably used. However, gibberellin that can be used in the medium of the present invention includes these. It is not limited.

  The amount of gibberellin added is preferably 0.5 to 1.5 μmol / L, more preferably 0.5 to 1.0 μmol / L, and 1.0 μmol / L. Further preferred. If the amount of gibberellin added is too high, the cells are enlarged due to the cell elongation promoting effect of gibberellin, and physical damage is likely to occur during swirling and shaking in suspension culture. If the amount of gibberellin added is too small, it is difficult to reduce the size of the cell mass by gibberellin.

  Here, when the endosperm cells of the Brassica plant or the Brassica plant are to be cultured, cytokinin and auxin are further added to the liquid medium. Thereby, the proliferation of the endosperm cells of the plants of the genus Brassica and the plants of the genus Brassica is dramatically promoted.

  Cytokinin, whether natural or synthetic, can be used in the medium of the present invention. Specifically, for example, benzyladenine, 6-methylaminopurine (MAP), 2-isopentenylaminopurine (2-iP), kinetin, zeatin, thiadiazulone, or a mixture of two or more thereof may be used. Although it is preferable to use benzyladenine, cytokinins that can be used in the medium of the present invention are not limited to these.

  Auxin can be used in the medium of the present invention, whether natural or synthetic. Specifically, for example, 3-indoleacetic acid, dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA), indolebutyric acid (IBA), 4-CPA, 1-naphthylacetamide, ethiclozete, cloxiphonac , Dichloroprop, or a mixture of two or more of these, preferably using 3-indoleacetic acid, dichlorophenoxyacetic acid (2,4-D), and using 3-indoleacetic acid Is more suitable, but the auxin that can be used in the medium of the present invention is not limited thereto.

  Here, the amount of cytokinin added is preferably 5 to 20 μmol / L, more preferably 10 to 20 μmol / L, and even more preferably 10 μmol / L. The amount of auxin added is preferably 5 to 20 μmol / L. However, if the amount of auxin added is too large, the rate of formation of proliferating cell mass may decrease. It is more preferable to make it less than L, and it is further more preferable to set it as 5 micromol / L. It is most preferable that the amount of cytokinin added is 10 μmol / L, and the amount of auxin added is 5 μmol / L.

  The endosperm cells of the Brassica plant and the Brassica plant are cultured in suspension using, for example, the medium of the present invention as follows.

  Prior to culturing endosperm cells using the liquid medium of the present invention, passage cells are established. In order to establish this passage cell, immature seeds are collected from immature fruits of the plants of the genus Brassica and the plant of the genus Brassica, the surface of the immature seeds is sterilized, and then the shell is taken, and a certain size (for example, It is obtained by selecting seeds having a constant minor axis size), sterilizing the ovule end portion (without cotyledons) from which the pericarp has been removed, for example, with antiformin and washing with distilled water or the like. This plant piece is composed of nacre, nacre, and endosperm. The plant piece is sliced in a direction perpendicular to the cotyledon, and sucrose is added to a plant cell culture medium containing cytokinin and auxin, for example, Murashige Skog (MS) medium (WAKO) having a pH of 5.8 to a final concentration of 3% by weight. Place on the added medium.

  For example, the case of culturing endosperm cells of Sina brachigill will be described in detail. For example, immature seeds are collected from immature fruits estimated to be 1 to 4 months after ripening of Sina brabili, The seeds are soaked in 70% by volume ethanol for 10 minutes, the seed surface is sterilized, and the shells are removed. Then, seeds having a minor axis of 8 to 12 mm are selected, and the ovule end portion (excluding cotyledons) from which the skin has been removed is immersed in antiformin (effective chlorine concentration 1% by weight) for 5 minutes, and then washed with distilled water three times. To obtain plant pieces composed of nacre, nacre and endosperm. Then, this plant piece is sliced in a direction perpendicular to the cotyledon, cut into a thickness of 1.0 to 2.0 mm, and placed on a plant cell culture medium containing cytokinin and auxin.

  The culture is performed at 16 ° C. to 27 ° C., preferably 20 ° C. to 25 ° C., more preferably 23 ° C., and is performed in a culture room having a fixed day length (for example, 16 hour day length). After the month culture), the proliferated cells are transplanted to a new medium under the same conditions, and then the passage is repeated every certain period (for example, 2 weeks). Thereby, the passage cells of the endosperm cells of the plants belonging to the genus Brassica and the plants belonging to the genus Brassica can be obtained.

  Here, by collecting the endosperm from the seeds of cinnabra brasil with a short axis of 8 to 12 mm, it becomes possible to efficiently collect undifferentiated endosperm cells. That is, when the short axis of the seed is 12 mm or less, undifferentiated endosperm cells can be obtained, and when the short diameter of the seed is 8 mm or more, the collection operation is facilitated and only the endosperm cells are obtained. Can be collected reliably.

  Then, the passage cells are transplanted into the liquid medium of the present invention, and suspension culture is performed at 16 ° C to 27 ° C, preferably 20 ° C to 25 ° C, more preferably 23 ° C. Suspension culture is preferably performed by, for example, swirl culture (120 rpm), but is not limited to this method.

The above-described embodiment is an example of a preferred embodiment of the present invention, but the present invention is not limited to this, and various modifications can be made without departing from the gist of the present invention. For example, in the above-described embodiment, the case where cinnamon braggili is used as a specific example of the plant of the genus Brassica and the genus Brassica has been described. The present invention can also be applied to coral brachigiri, maple coral braggili, western squirrel, nanyo cherry, red basilica, salmon bayotropha, and the like.

  In addition, when other than endosperm cells of plants belonging to the genus Brassica and Brassica genus are to be cultured, addition of cytokinin and auxin to the liquid medium is not essential, and suspension culture can be performed without adding these. .

Hereinafter, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way.
(A) Examination of callus induction conditions (1) Examination of plant hormone addition effect Aleurites fordii that grows naturally in Shimada City, Shizuoka Prefecture was used as an experimental material. An immature seed having a minor axis of 8 to 12 mm, estimated to be within 3 months after fruit set, was immersed in 70% by volume ethanol for 10 minutes to sterilize the seed surface, and then the shell was removed. The exposed endosperm was immersed in antiformin (effective chlorine concentration 1% by weight) for 5 minutes, washed 3 times with distilled water, and used for cell culture on an agar medium. This plant piece is composed of nacre, nacre, and endosperm.

  The basic medium was Murashige Skog (MS) medium (WAKO) having a pH of 5.8 containing 3% by weight of sucrose, and the support material was 0.8% by weight of agar. The plant hormone to be added is 0-20 μmol / L of synthetic cytokinin (benzyladenine: BA), synthetic auxin (2,4-dichlorophenoxyacetic acid: 2,4-D) or natural auxin (3-indoleacetic acid: IAA). Used at a concentration of 0-20 μmol / L.

  The above-mentioned plant pieces were sliced in a direction perpendicular to the cotyledons, cut into sections having a thickness of 1.0 to 2.0 mm, and placed on the above-mentioned medium containing plant hormones under various conditions.

  Upon callus induction, the concentration conditions of two plant hormones (auxin and cytokinin) were examined. The culture was performed in a culture room at 23 ° C. for 16 hours, and after one month of culture, the presence or absence of callus formation was determined under a stereomicroscope.

  From the results shown in Table 1, by using BA and 2,4-D together, the callus formation rate is dramatically improved, BA is 5-20 μmol / L, 2,4-D is 5-20 μmol / L. As a result, it was clarified that the callus formation rate was dramatically improved, and that BA was 10 μmol / L and 2,4-D was 10 μmol / L, and the callus formation rate was further improved dramatically. .

  In addition, from the results shown in Table 2, by using BA and IAA in combination, the callus formation rate is drastically improved. By setting BA to 5 to 20 μmol / L and IAA to 5 to 20 μmol / L, callus formation is achieved. It was revealed that the rate of callus formation was further improved by dramatically increasing the rate and setting BA to 10 μmol / L and IAA to 10 μmol / L.

  From the above results, the callus formation rate is drastically improved by adding cytokinin and auxin to the medium, and the callus formation rate is further increased by making cytokinin 5-20 μmol / L and auxin 5-20 μmol / L. It was clarified that the callus formation rate was dramatically improved by setting cytokinin to 10 μmol / L and auxin to 10 μmol / L.

(2) Influence of culture temperature The suitable culture temperature in the subculture callus was examined.

  After subculture of the callus mass, the cells were cultured at various temperatures for 2 weeks (Table 3). As a result, callus formation was observed at a temperature of 16 to 27 ° C., and in particular, at a temperature of 23 ° C., the callus formation rate reached a maximum of 100%.

  From the above results, it was considered that the culture temperature was preferably 16 to 27 ° C, and most preferably 23 ° C.

(B) Examination of suspension culture conditions

(1) Passage cells to be used Aleurites fordii, which is native to Shimada City, Shizuoka Prefecture, was used as an experimental material. An immature seed having a minor axis of 8 to 12 mm, estimated to be within 3 months after fruit set, was immersed in 70% by volume ethanol for 10 minutes to sterilize the seed surface, and then the shell was removed. The exposed endosperm was immersed in antiformin (effective chlorine concentration 1% by weight) for 5 minutes, washed 3 times with distilled water, and used for cell culture on an agar medium.

  As for the medium, sucrose was added to Murashige Skog (MS) medium (WAKO) having a pH of 5.8 to a final concentration of 3% by weight, and synthetic cytokinin (benzyladenine: BA) was added at a final concentration of 10 μmol / L, natural auxin (3 -Indoleacetic acid: IAA) was added to a final concentration of 10 μmol / L. As a supporting material, 0.8% by weight of agar was contained.

  As a culture method, an endosperm slice sliced to a thickness of 1.0 to 2.0 mm was placed on a medium. One month later, the grown cells were transplanted to a fresh medium under the same conditions. Thereafter, the passage was repeated every 2 weeks, and this was used as the passage cell used in this Example.

(2) Concentration of cytokinin and auxin Prior to the examples, in order to confirm the effect of gibberellin on the proliferation of Sinabragi endosperm cells, the cells obtained in the above passage were treated in an agar medium supplemented with gibberellin (GA ₃ ). Cultured. The medium used here is a basic medium obtained by adding sucrose to a final concentration of 3% by weight to Murashige Skog (MS) medium (WAKO) having a pH of 5.8 containing 0.8% by weight of agar as a support material. BA was added at a final concentration of 10 μmol / L, IAA was added at a final concentration of 10 μmol / L, and gibberellin (GA ₃ ) was added at a final concentration of 0 (negative control), 1.5, and ₃ μmol / L (Table 4). Bottom). As a result, the growth rate of the proliferating cell mass, which was 88.2% when GA ₃ was not added, slightly decreased to 70.6-77.8% by the addition of GA ₃ .

Therefore, BA was added to the above basic medium so that the final concentration was 10 μmol / L, IAA was final concentration 5 μmol / L, and GA ₃ was final concentration 0 (negative control), 1.5 μm, 3 mol / L (Table 4). Top). As a result, the growth rate of the proliferating cell mass, which was 88.2% when GA ₃ was not added, increased to 94.4% by the addition of 1.5 μmol / L GA ₃ .

  Based on this result, BA was determined to have a final concentration of 10 μmol / L and IAA was determined to have a final concentration of 5 μmol / L in cell separation studies for cell culture in suspension.

(3) Concentration of gibberellin The endosperm-derived cells, which are the above-described passage cells, were transplanted into a liquid medium not containing a support material (agar), and swirl culture was performed at 23 ° C. and 120 rpm. Here, the liquid medium was obtained by adding sucrose to a final concentration of 3% by weight to Murashige Skog (MS) medium (WAKO) having a pH of 5.8. After culturing for 2 weeks, whole cells were collected after removing masses with a diameter of 3 mm or more. The cells that were allowed to stand for 10 minutes and naturally settled were washed twice with a liquid medium (without a plant growth regulator).

  Then, in order to examine the influence of the gibberellin concentration, the following experiment was performed with different gibberellin addition concentrations.

(Examples 1-3)
The cells after washing were treated with BA: 10 μmol / L, IAA: 5 μmol / L, GA ₃ : 0.5 (Example 1), 1.0 (Example 2), 1.5 (Example 3) μmol / L. It transplanted to each liquid medium of addition, and swirl culture was performed at 23 degreeC and 120 rpm, respectively. After 2 weeks of culture, the number of cells constituting the mass and the size of the cells were measured.

  The results are shown in FIGS. As shown in FIG. 1, the average number of cells per lump was 24.3 ± 2.2 (average value ± standard error, n = 30, the same applies hereinafter) in Example 1, and 16.9 ± in Example 2. The number was 1.7 and the number in Example 3 was 17.6 ± 1.6. Moreover, as shown in FIG. 2, in the culture medium after 2 weeks of culture, the number of cells per mass was small and a relatively small cell mass appeared. Furthermore, as shown in FIG. 3, the cell diameter was 43.2 ± 1.3 (mean value ± standard error, n = 60, the same applies hereinafter) μm in Example 1, and 38.7 ± 0.00 in Example 2. 9 μm and 46.7 ± 1.3 μm in Example 3. Therefore, compared with the case where the gibberellin is not added (Comparative Example 1), almost no cell hypertrophy was observed.

(Comparative Example 1)
The washed cells were transplanted to a liquid medium containing BA: 10 μmol / L, IAA: 5 μmol / L but not GA ₃ , and swirling culture was performed at 23 ° C. and 120 rpm. After 2 weeks of culture, the number of cells constituting the mass and the size of the cells were measured.

  The results are shown in FIGS. As shown in FIG. 1, the average number of cells per mass is 55.7 ± 7.4 (average value ± standard error, n = 30), and the number of cells per cell mass is higher than that of Examples 1-3. There were many. Moreover, as shown in FIG. 2, a large cell mass was observed in the medium after 2 weeks of culture. Furthermore, as shown in FIG. 3, the cell diameter was 40.3 ± 0.9 (mean value ± standard error, n = 60) μm, which was substantially the same as in Examples 1 to 3.

(Comparative Example 2)
The cells after washing were transplanted into a liquid medium supplemented with BA: 10 μmol / L, IAA: 5 μmol / L, GA ₃ : 3.0 μmol / L, and swirl culture was performed at 23 ° C. and 120 rpm. After 2 weeks of culture, the number of cells constituting the mass and the size of the cells were measured.

  The results are shown in FIGS. As shown in FIG. 1, the average number of cells per lump was 18.8 ± 2.1 (average value ± standard error, n = 30), which was the same as in Examples 1 to 3. Moreover, as shown in FIG. 2, in the culture medium after 2 weeks of culture, the number of cells per mass was small and a relatively small cell mass appeared. Furthermore, as shown in FIG. 3, the cell diameter was 69.1 ± 2.3 (mean value ± standard error, n = 60) μm, and larger cells were observed compared to Examples 1-3. In this comparative example, it is considered that the effect of gibberellin to loosen the cell wall became stronger and the cells were enlarged.

  In all of Examples 1 to 3 and Comparative Examples 1 and 2, the total number of cells in the medium was almost the same.

  Therefore, it was confirmed that by adding gibberellin in a concentration range of 0.5 to 1.5 μmol / L, cell separability can be improved without enlarging the cells while maintaining the cell proliferation ability. In particular, it was found that the effect was maximum when gibberellin was 1.0 μmol / L.

(4) Passage 1.8 mL of the suspension cells obtained in Example 3 were transplanted to 30 mL of liquid medium (CIM) supplemented with BA: 10 μmol / L, IAA: 5 μmol / L, GA ₃ : 1.5 μmol / L. Cell volume was measured after 3 weeks of culture. After the measurement, the suspension cultured cells were divided into two, transplanted to a new liquid medium having the same composition, and further measured after culturing for 3 weeks, and this was repeated 3 times every 3 weeks. The result is shown in FIG. As shown in the figure, the cell volume increased about twice in 3 weeks. In addition, the proliferation ability was maintained after at least 4 passages (after 84 days). For this reason, it was proved that the suspension cells had sufficient passage ability.

  Moreover, although the present Example is performed about the Brassica genus Sinabraagiri, it is estimated that the same result can be obtained also about the Brassica genus plant and the Astragalus genus plant which have the same property.

  The liquid medium for plant cell suspension culture and the plant cell suspension culture method using the same are used as a technique for miniaturizing a cell mass formed by suspension culture in a liquid medium. In particular, it is suitably used as a technique for reducing the cell mass while maintaining the cell proliferation ability.

Claims (5)

A liquid medium for endosperm cell suspension culture of a plant belonging to the genus Brassicella or Nanobula plant , characterized in that gibberellin is added in an amount of 0.5 to 1.0 μmol / L , and cytokinin and auxin are further added . Liquid medium of claim 1 wherein the gibberellin is gibberellin A _3. The liquid medium according to claim 1 or 2, wherein the cytokinin is 5 to 20 µmol / L and the auxin is 5 to 20 µmol / L. The liquid medium according to any one of claims 1 to 3, wherein the cytokinin is benzyladenine and the auxin is 3-indoleacetic acid. An endosperm of a Brassica plant or a Brassica plant characterized by suspending and culturing an endosperm cell of a Brassica plant or a Brassica plant using the liquid medium according to any one of claims 1 to 4. Cell suspension culture method.