JPH06209666A - Cyclamen - Google Patents

Abstract

PURPOSE:To establish a technique of clone mass proliferation for an elite individual of a cyclamen. CONSTITUTION:An embryo taken out of a full-ripe seed, sterilized and subjected to water absorption and germination hastening treatment is bedded on a Linsmaier-Skoog (LS) solid culture medium containing 2,4-D, cytokinin and sucrose to induce an adventitious bud of the cyclamen so as not to have a great difference in differentiation degree between the species and strains. The adventitious bud of the cyclamen is differentiated and grown to a rodlike embryo, which is then cultured at 10-25 deg.C in the dark. Thereby, the rooting (germination) ratio is improved. The adventitious bud of the cyclamen is cultured on a solid culture medium by using the 2,4-D and kinetin to create a cyclamen breeding mother plant capable of regenerating a plant body. As a result, the efficiency for creation of an F1 hybrid parent derived from the adventitious bud is improved.

Description

Detailed Description of the Invention [Industrial applications]

The present invention relates to a method for regenerating cyclamen plants.

[0002]

2. Description of the Related Art Up to now, little research has been reported on the mass multiplication of excellent cyclamen horticultural varieties, but Otani et al.
We have reported a method of inducing callus from the expanded leaves of the plant one year after sowing of "Rose" to obtain adventitious embryos from the callus (Journal of the Horticultural Society of Japan, Vol. 58, Supplement 1, 1989).
574). According to this, the adventitious embryo induction procedure of Otani et al. Is as follows.

First, after sterilizing the developed leaves, 2,4-dichlorophenoxyacetic acid (hereinafter referred to as 2,4-D), kinetin, gellan gum, and 5% sucrose are added to adjust the pH to 5.0.
Two types of callus are obtained by culturing in the dark in the LS medium prepared as above. One is flyable callus and the other is dense and hard callus. The flyable callus was added to α-naphthalene acetic acid (hereinafter referred to as NAA), kinetin, etc. in an LS medium to continue callus proliferation and adventitious embryo formation at the same time,
Subculture is performed every 40 to 50 days.

Somatic embryos formed in the subculture medium are NA
When cultivated in an LS medium of 1/2 concentration containing A, kinetin, gellan gum, 5% sucrose and the like under illumination, a young plant is obtained via a tuber-like body. The seedlings are cultured in a ½ concentration LS hormone-free medium (containing 1.5% sucrose and 0.2% gellan gum) under illumination to obtain plants. Then, when a plant body having 5 to 10 expanded leaves is potted, it blooms. In addition, it is reported that indoleacetic acid (IAA) is added to the somatic embryo at the time of inducing a seedling (G. WICART et al., Protoplasma 11).
9: 159-167, 1984).

Also, Ohashi et al. Have cultivar No. It has been reported that plantlets could be obtained from tissues (roots) of 12 cultured strains via soft callus and adventitious embryo (Horticological Society Magazine, Volume 60, Supplement 1 (1991)). According to this report
6% sucrose, pH 5.6, gellite 0.2% of tissues in buds (styles, ovary walls, petals, anthers, placenta) or tissues of cultures of the same strain (leaf blades, petioles, tubers, roots) As a basic medium containing MS medium containing 2,4-D 1.0 mg / liter and 2,4-D 1.0 mg / liter, BA
The cells are placed in two types of medium supplemented with 0.1 mg / liter, and after 30 days of placement, transplanted to an MS medium containing 6% sucrose, pH 5.6, and 0.2% gellite. All the cultures during this period were performed at 25 ° C. in the dark, and soft callus was formed in the placenta and the root, and the root was 2,4-D1.0 mg.
Adventitious embryos were formed only when placed in a medium containing 0.1 mg / liter of BA and 0.1 mg / liter of BA.
5%) plantlets were differentiated.

[0006]

According to the above-mentioned callus and adventitious embryo induction method of cyclamen by Otani et al., 1 mg / liter of 2,4-D (auxin) and 0.5 mg / liter of kinetin (cytokinin) are Although 100% callus is induced, no flyable callus forming somatic embryos was obtained from this callus. Fryable callus is a hormone of 1 mg / liter 2,4
-D alone with medium plus 1 mg / l 2,4
-D and 0.1 mg / liter of kinetin from each medium 14.3% (3% when including dense and hard callus)
5.7%) and 46.7%. Such a low rate of formation of fryable callus is not suitable for liquid culture, which is advantageous for large-scale culture of somatic embryos from callus. In the induction method of Ohashi et al., The induction of cyclamen embryoid body callus showed a large difference in the degree of differentiation between varieties and strains,
It has been reported that the optimum concentrations of plant regulators differ among varieties and lines that are only differentiated.

The present inventors have already cultivated explants of cyclamen, which are fixed species of Persicum, in a medium containing 2,4-D and kinetin to induce callus, and the callus is somatic embryo in a medium containing no hormone. Of the somatic embryo by culturing the somatic embryo with shaking in a liquid medium, and succeeding in isolating the somatic embryo, and applying a patent for the method (Japanese Patent Application No. 2-41).
6880).

The present inventors have completed the following liquid culture technology and applied for a patent in order to establish an efficient method for inducing cyclamen somatic embryos in a liquid culture system suitable for large-scale culture ( Japanese Patent Application No. 3-349126). That is, the method is to culture explants of cyclamen in a solid medium containing 2,4-D and kinetin to generate callus, and then in a solid medium containing no hormones, a nodule-shaped somatic embryo Induce
The somatic embryo is cultivated in a liquid medium containing 2,4-D and kinetin, and from the obtained culture, only a cell mass having a size of 30 μm or more and 2 mm or less is subcultured to be proliferated while being subcultured. This is a somatic embryo induction method. But,
This method also succeeded only when the petiole was used as an explant, and in cyclamen, there were large differences among varieties and individual differences, and a means to solve this was needed. Therefore, a first object of the present invention is to establish a method for inducing cyclamen so that the induction of cyclamen embryoid body callus does not significantly differ between cultivars and strains.

The optimum temperature for germination of cyclamen seeds is 20.
Approximately ℃, but about rooting of somatic embryo, germination was carried out at 26 ℃ in the report of Otani et al.
There are only cases where germination was carried out at 25 ° C and 25 ° C, and detailed examination on the rooting condition of adventitious embryos derived from cyclamen seeds has not been made. Therefore, a second object of the present invention is to establish rooting conditions for rod-shaped embryos differentiated and developed from adventitious embryos such as cyclamen seeds.

Furthermore, the regenerated plant derived from the somatic embryo of rice has a fertility rate of -85.9% to -27% depending on the type of culture system.
However, Matsuno et al. Reported that this was due to male sterility (Breeding Journal Vol. 41,
Separate volume 2, 312 pages, 1991). On the other hand, in cyclamen, Otani et al. Reported in the above-mentioned magazine that plants regenerated from somatic embryos were normal. On the other hand, the present inventors have conducted repeated studies to regenerate a plant from the somatic embryo of cyclamen developed by the present inventors. Therefore, a third object of the present invention is to improve the efficiency of producing F ₁ hybrid parents of cyclamen derived from somatic embryos of cyclamen. As described above, the present invention aims to establish a technique for mass-producing a clone of excellent cyclamen individuals.

[0011]

The first object of the present invention described above is achieved by the following constitution of the first invention. That is,
This is a method for inducing cyclamen embryoid body callus in which embryos taken out from matured seeds that have been sterilized and subjected to water-emergent germination are placed on an LS solid medium containing 2,4-D, cytokinin and sucrose and cultured. The amount of 2,4-D and cytokinin added to the medium is not particularly limited, but each is 0.1-10 pp.
m, preferably 0.001 to 10 ppm.
As the cytokinin, kinetin, BA (benzylaminopurine) or zeatin can be used. Further, it is desirable for the induction of embryoid body callus that the medium contains 1 to 12% or more of sucrose. As the medium for culturing the embryos taken out from the mature seeds used in the present invention, a medium generally used for culturing plants can be used as long as it is a solid medium. Further, the sterilization treatment carried out in the present invention can be carried out by a generally used sterilization treatment method. In addition, water sprouting treatment can also be carried out by a generally used sterilization treatment method.

According to the present invention, embryoid body callus grows while producing spherical embryos or rod-like embryos. The present inventors have previously proposed a large-scale culture method of a cyclamen somatic embryo in a liquid medium (Japanese Patent Application No. 3-349126), which uses a petiole as an explant. As mentioned above, this Japanese Patent Application No. 3
According to the method described in No. 349126, adventitious embryos that can be mass-cultured in liquid are selected with a mesh of 2 mm or less and 30 μm or more. According to the present invention, embryos in ripe seeds are rod-shaped embryos having a length of about 2 mm and a diameter of about 0.2 mm. Callus derived from the rod-shaped embryos has a rod-shaped embryo of a length of about 2 mm or less. It is composed of a spherical embryo having a size of 3 and a small cell mass smaller than that, and can be a callus that meets the purpose of liquid culture disclosed in Japanese Patent Application No. 3-349126. In this way, it became possible to mass-cultivate somatic embryos from callus with no significant difference in the degree of differentiation between varieties and strains, expanding the possibility of developing an efficient mass-propagation method using somatic embryos in liquid culture of cyclamen. .

The second object of the present invention is achieved by the constitution of the following second invention. That is, after differentiation and development of somatic embryos of cyclamen into rod-shaped embryos, they were developed in the dark at 10 to 25 ° C.
It is a method of rooting somatic embryos of cyclamen. The rooting temperature of the somatic embryo is 10 to 25 ° C, preferably 1
It is 5-23 degreeC. At this time, if the gelling concentration of the solid medium for rooting the rod-shaped embryos is set to 0.2 to 0.4%, the germination (tubering, rooting, germination) of adventitious embryos is improved. Also,
The rooting rate of adventitious embryos is also improved by setting the concentration of inorganic salts in the solid medium for rooting the rod-shaped embryos to 1/2 or less of the basal medium in which differentiation and development have been performed. The somatic embryo can also be obtained from the root tip. However, the cytokinin used for root tip culture is limited to kinetin.

The third object of the present invention is achieved by the constitution of the following third invention. That is, it is a method for producing a cyclamen breeding mother book in which a somatic embryo of cyclamen is cultured on a solid medium using 2,4-D and kinetin to regenerate a plant. Further, at this time, by selecting a mutant strain of male sterility having a normal chromosome number among the regenerated plants, it becomes possible to produce an F ₁ hybrid which does not require skillful emasculation.

[0015]

The first aspect of the present invention focuses on the fact that the seeds have a strong seed coat, and even if the seeds having a strong seed coat are subjected to severe sterilization treatment, the embryos in the inside are hardly affected. It was completed based on the finding that it is easy to sterilize an explant (embryo).

In general, it is very difficult to sterilize leaf pieces and petioles, and therefore, yellowing treatment (treatment of making leaves yellow by irradiating light (Introduction to illustrated tissue culture, edited by Yoshiro Furukawa, Seibundou Shinkosha, 72) (See page, etc.) and then sterilized.In addition, a powerful and harmful sterilizing agent called mercury chloride is also used.On the other hand, according to the present invention, ripe seeds are sterilized and subjected to water-absorption germination treatment. Since the removed embryos are used as explants, embryoid body callus can be obtained for most varieties.

According to the second invention, at 25 ° C., the overall average is 1
Only 20% of the roots were rooted, but at 20 ° C., 38% of the roots were rooted on average, and the differentiation rate was improved by about 40 times (excluding the callus). Thus, by improving the differentiation rate (rooting rate) of the somatic embryo, it is possible to reduce the seedling production cost in the somatic embryo culture system.

Generally, three strains, a male sterile strain, a maintenance strain, and a recovery strain, are required to produce an F ₁ hybrid.
Selection by crossing has a long cultivation period (about 1 for cyclamen)
However, according to the third invention, it is possible to induce mutations by culturing at the flask level to create a male sterile system, and thus the efficiency is improved. Once a male-sterile culture system is created, clones can be propagated in culture, so a maintenance system becomes unnecessary.
A hybrid system can be produced by the two-method method of male-sterile system and recovery system. Male sterile cyclamen with normal chromosome number can be obtained from regenerated plant derived from somatic embryo.

[0019]

EXAMPLES Examples of the present invention will be described. Example 1 7 ripe seeds of cyclamen variety "Finlandia"
40% in 0% ethanol for 10 seconds, 1% antiformin
After soaking for 1 minute and sterilizing to sterilize, water-absorbing germination treatment is performed, and then LS hormone-free solid medium is used for 25 minutes.
It is allowed to stand for 1 to 30 days in the dark at 0 ° C. for water absorption and germination treatment.
Then, the embryo is taken out, and this is 2, 4-D at 1 ppm,
Embryoid callus is obtained by allowing it to stand in an LS solid medium containing 0.1 ppm of kinetin and 5% of sucrose at 25 ° C. in the dark for 2 months. The photograph of the obtained embryoid body callus is shown in FIG. Further, since the present invention is a method which does not select a variety,
Other varieties such as "Saga", "Rosamunde", "Large White", etc. can be similarly guided.

As described above, this Japanese Patent Application No. 3-3491.
According to the method described in No. 26, there are 2 somatic embryos that can be mass-cultured in liquid.
The mesh is selected with a mesh of 30 mm or less and 30 mm or less. The embryos in the ripe seeds described in this example have a length of about 2
mm, a rod-shaped embryo with a diameter of about 0.2 mm, but the callus derived from the rod-shaped embryo has a rod-shaped embryo with a length of about 2 mm,
It is composed of a spherical embryo having a size smaller than that and a small cell mass having a size smaller than that, and can be a callus that meets the purpose of liquid culture disclosed in Japanese Patent Application No. 3-349126. In this way, it becomes possible to carry out mass culture of adventitious embryos from callus, which has no great difference in the degree of differentiation between varieties and strains.

Example 2 This example is a method of rooting from a somatic embryo of cyclamen. The procedure for inducing callus and adventitious embryo from petiole of cyclamen and rooting from adventitious embryo will be explained. First, 1,4-ppm of 2,4-D from petiole of variety "Large White",
Callus is induced by standing still in the dark at 25 ° C. for 30 days in an LS solid medium containing 0.5 ppm of kinetin and 5% of sucrose. Add this callus to MS solid medium containing 3% sucrose for 25
The somatic embryo is induced by allowing it to stand in the dark at 30 ° C. for 30 days. The obtained somatic embryo was treated with 2,4-D at 1 ppm and kinetin at 0.
Liquid culture is carried out for 21 days in the dark at 25 ° C. in an LS liquid medium containing 5 ppm and 5% sucrose. After 21 days, the somatic embryos obtained after 21 days are sorted by a filter into those having a size of less than 500 μm and 106 μm or more and those having a size of 500 μm or more, and those having a size of less than 500 μm and 106 μm or more are returned to the somatic embryo liquid culture system again, Differentiate and develop a rod-shaped embryo with a size of 500 μm or more in MS liquid medium. At this time, the somatic embryo density in MS liquid medium was 1
Do not exceed 00 / ml. The rod-shaped embryo thus obtained by differentiating and developing the somatic embryo was rooted under the following conditions.
Rooting data at 25 ° C and 20 ° C are shown in Tables 1 and 2.

(Table 1) Effect of environmental conditions on rooting rate of somatic embryos of cyclamen (temperature x gellite concentration x inorganic salt concentration) gellite concentration (%) 0.2 0.25 0.3 0.4 none 1 0 0 0 6 machine Concentration 1/2 0 6 0 0 Salinity 1/5 − 0 0 0 (%) 1/10 − − 0 0 Overall average rooting rate: 1% (25 ° C.)

(Table 2) Effect of environmental conditions on rooting rate of somatic embryos of cyclamen (temperature x gellite concentration x inorganic salt concentration) gellite concentration (%) 0.2 0.25 0.3 0.4 none 1 19 13 38 19 machine Concentration 1/2 19 44 38 38 38 Salinity 1 / 5-50 50 50 (%) 1 / 10-69 56 56 Overall average rooting rate: 38% (20 ° C) Both of the experiments shown were conducted under the following conditions. (1) Excludes callus from the rooted population (2) The number of adventitious embryos tested in each section is 16 (3) Both under 25 ° C and 20 ° C under dark conditions for 2 months, the basic medium is MS medium (Same as basic medium for differentiation and development) In the table, (-) indicates that the experiment was impossible because the medium did not solidify.

In addition, in FIG. 2, the inorganic salt concentration × 1, 25 ° C.,
Gelrite concentration of 0.2% in the dark for 2 months
(1-1 in the upper left of the figure), 0.25% (1- in the upper right of the figure)
2), 0.3% (1-3 in the lower left of the figure) and 0.4% (1-4 in the lower right of the figure). Further, in FIG. 3, gellite concentration is 0.2% (1-1 in the upper left of the figure), 0.25 (1-2 in the upper right of the figure), 0.3 with respect to the inorganic salt concentration × 1 at 20 ° C. % (1-3 in the lower left of the figure), 0.4%
(1-4 in the lower right of the figure) shows the state of each rooting. In addition, in FIG. 4, the concentration of inorganic salt × 1/2, 20 ° C., the gellite concentration of 0.2% in the culture in the dark for 2 months (the upper left in the figure 2-
1), 0.25 (upper right 2-2 in the figure), 0.3% (lower left 2-3 in the figure), 0.4% (lower right 2-4 in the figure) Show. In FIG. 5, the inorganic salt concentration × 1/5, 20 ° C.,
Gelrite concentration of 0.25% in culture in the dark for 2 months
(3-2 in the upper part of the figure), 0.3% (3-3 in the lower left part of the figure),
The state of each rooting of 0.4% (3-4 in the lower right of the figure) is shown.
In addition, in FIG. 6, the concentration of inorganic salt × 1/10, 20 ° C., in the dark, for 2 months, the gellite concentration was 0.3% (4-3 on the left in the figure), 0.4 (right on the figure) 4-4) of each rooting is shown. As shown in Tables 1 and 2, root averages only 1% at 25 ° C, but averages 38% at 20 ° C.
The roots were rooted and the differentiation rate was improved by about 40 times (except for callus). The rooting rate was 44% at maximum when the gellite concentration was changed from 0.2 to 0.4%. 44/38 against the average rooting rate of 38% when the temperature was set to 20 ° C
≈1.2 times higher rooting rate. Further, when the concentration of the inorganic salt was ⅕ or less, the rooting rate was 50% or more, the maximum was 69%, which was 69 / 44≈1.6 times improved. When the inorganic salt concentration is 1/10 or less, the medium does not solidify and root growth is suppressed.

Further, in connection with this embodiment, the root can be rooted from the somatic embryo of cyclamen by the following method. This method involves inducing callus from a petiole of cyclamen in a solid medium containing 2,4-D and kinetin, and then inducing an adventitious embryo in a solid medium without hormones.
Shake culture in a liquid medium containing -D and kinetin, and
Spherical embryos of 00 μm or larger were differentiated and developed into rod-shaped embryos in a hormone-free liquid medium, and the rod-shaped embryos were hormone-free and the ratio of the nitrogen source ammonia nitrogen to nitrate nitrogen was set to 1/10 or less. This is a method of culturing in a solid medium and rooting.

More specifically, the rooting method will be described. "Large white" petioles of 2,4-D at 1 ppm, kinetin at 0.5 ppm and sucrose at 5% in an LS solid medium at 25 ° C in the dark. Incubate for 30 days to induce callus. Then, in order to induce somatic embryos from this callus, MS liquid medium containing 3% sucrose was added at 25 ° C. in the dark for 30 days.
The somatic embryos obtained are placed for 2 days and then 2,4-D
It is cultured in an LS liquid medium containing pm and kinetin at 0.01 ppm and sucrose 5% at 25 ° C. in the dark for 21 days.
In this way, from the somatic embryos obtained, smaller than 500 μm 1
The size of 106 μm or more and the size of 500 μm or more are selected and 106 μm or less is selected.
Somatic embryos with a size of m or more are returned to the LS liquid medium and liquid-cultured again. On the other hand, somatic embryos with a size of 500 μm or more are differentiated and developed in MS liquid medium at an somatic embryo density of 100 / ml or less. After 21 days in the dark at 20 ° C., germination (tubering, rooting, germination) was performed. The state of germination is shown in FIG. 7, gellite concentration 0.2%).

According to the above method for differentiating and developing spherical embryos of 500 μm or more into rod-like embryos in a hormone-free liquid medium, the rooting rate was 44/50 × 100 = 88%, and the MS medium of Example 2 was used. 88/69 = 1.28 times better than when I did it. In addition, in the MS medium of Example 3, about 5 to 30% of callus is transformed from rod-shaped embryos, whereas callus is completely suppressed by this method.

Furthermore, the seedling production cost in the somatic embryo culture system can be reduced by improving the differentiation rate (rooting rate). Generally, when a somatic embryo is transformed into a plug seedling, an ungerminated part becomes a missing strain, and a great amount of labor is required to replace a germinated part of this part. It is important to improve the germination rate, and the method has a great effect on the omission of this step. Furthermore, the rooting rate when rooted in an MS medium in which the ratio (molar ratio) of ammonia nitrogen to nitrate nitrogen was 1/2 was a maximum of 69% (Example 2), and the period until rooting was Although it takes 2 months, this method can be done in 1 month and the period can be shortened to 1/2.

The rod-shaped embryo can also be induced by the following method. That is, cyclamen explants are cultured in a solid medium containing 2,4-D and kinetin to produce callus, and then a nodule-shaped adventitious embryo is induced in the hormone-free solid medium, Adventitious embryos were cultured in a liquid medium containing 2,4-D and kinetin, and 100 μm or more was obtained from the obtained culture.
Preferably, a somatic embryo having a size of 500 μm or more is shake-cultured in a hormone-free liquid medium, and when the somatic embryo is differentiated and developed, the somatic embryo density is set to 100 / ml or less. is there.

It is known that the density of carrots affects the differentiation and development of somatic embryos, and 80 to 90% of them differentiate (differentiate and develop) when the density is 150 ng / ml or less. However, up to now, no example has been known in which the optimum conditions for the differentiation and development of globular embryos into rod-shaped embryos have been examined in cyclamen. According to the above-mentioned differentiation and development method, somatic embryos of cyclamen do not differentiate and develop unless the somatic embryo density is 100 / ml or less.
The present inventors have found that the differentiation rate at 0 / ml is about 45%. Since the differentiation and development can be controlled by the liquid medium, it is possible to scale up to tank culture. Further, since the globular embryos can be differentiated and developed synchronously, the seedling production cost can be reduced.

A specific example will be described. First, "Large White" petioles are cultured in LS solid medium (2,4-D 1 ppm, kinetin 0.5 ppm, sucrose content 5%) at 25 ° C for 30 days in the dark to induce callus. The adventitious embryo at 25 ° C. in MS solid medium containing 3% sucrose from the obtained callus,
Incubate in the dark for 30 days. Then, this somatic embryo 2,
4-D, 1 ppm of kinetin, and 5% of sucrose are cultivated in an LS liquid medium at 25 ° C. in the dark for 21 days. Then, an adventitious embryo having a size of less than 500 μm and a size of 106 μm or more and an adventitious embryo having a size of 500 μm or more are selected, and the former is cultivated in the dark at 25 ° C. in the LS liquid medium to obtain a size of 500 μm or more. Sano adventitious embryos in hormone-free MS liquid medium at 25 ° C for 2
After treatment for 1 day, the embryo differentiates into a rod-shaped embryo and develops. Table 3 shows the effect of culture density on the differentiation and development of somatic embryos.

(Table 3) (Effect of Culture Density on Differentiation and Development of Adventitious Embryo) Initial density of culture Differentiation and development of somatic embryo (stick embryo) Differentiation and development rate (ke / ml) Density (ke / ml) (% ) 655 0 0 321 0 0 91 6 6.6 47 21 44.7 Moreover, a photograph of an adventitious embryo differentiated and developed at a low density is shown in FIG.
Fig. 9 shows a photograph of an adventitious embryo that does not differentiate and develop at high density.

Two methods for inducing somatic embryos of cyclamen will be described in connection with this embodiment. One of them is that the root end of the germinated plant of cyclamen is 2,4-
L containing D and kinetin and containing 3% or more of sucrose
It is a method for inducing cyclamen embryoid body callus, which comprises culturing by placing on S solid medium. The callus grows while generating spherical embryos or rod-like embryos.

This method will be described in detail. A cyclamen variety "Saga" was immersed in 70% ethanol for 10 seconds and 1% antiformin for 40 minutes, and placed in an LS hormone-free medium at 20 ° C. in the dark and still. Aseptically seeded by stationary culture to form seedlings, and their root tips are 2, 4
Incubate in an LS solid medium containing 1 ppm of -D, 0.1 ppm of kinetin and 9% of sucrose at 20 ° C in the dark for 2 months. The obtained embryoid body callus is shown in FIG. In addition, in FIG. 11, L was the same as above except that sucrose was 1%.
Callus was cultured in S solid medium under the same conditions, but no embryoid body was generated (FIG. 11). At this time, LS
When the sucrose concentration in the solid medium was 1%, no embryoid callus was obtained.

The method for inducing cyclamen embryoid body callus using this root tip is the same as the method of Otani et al. Except that the root is used for the explant, and the method of Ohashi et al. Is the same method except that BA of cytokinin was changed to kinetin and the basic medium was changed from MS medium to LS medium.

A large number of explants, including the lateral roots, were obtained from one individual at the root end of cyclamen, and the root end was a tissue with vigorous division / proliferation similar to the growth point. Also has a strong advantage. Furthermore, similarly, even if embryoid body callus cannot be obtained in most tissues depending on the variety and individual, it can be obtained by using the root as an explant. Furthermore, the reproducibility of embryoid callus induction per explant was 92% (12 /
13 units x 100), 17% in Finlandia (2
Co / 12 co × 100).

The following method was to use MS liquid medium containing picloram to reduce the phosphate concentration of the MS liquid medium containing picloram and zeatin to the liquid culture cells of cyclamen that were kept growing. The somatic embryo induction method is characterized by culturing in a medium to induce somatic embryos.
The phosphoric acid concentration in this method is preferably reduced to 1/20 to 1/5. In this method, the method of inducing somatic embryos in liquid culture previously filed by the present inventors (Japanese Patent Application No. 3-84733).
No.) inducing somatic embryos (tuber-like bodies) has an efficiency of 12 per 1 ml.
However, the somatic embryo induction efficiency was 20 / ml, which was 1.67 times that of the conventional case. For example, 1 p of picloram
Liquid culture (25 ° C, dark) in MS medium containing pm, then 2 ppm picloram and 0.2 ppm zeatin
And is cultured in an MS medium containing 1/10 of phosphoric acid. Two
By the week, embryoid bodies at an early stage of spherical embryos were obtained at 20 / ml. A photograph of the obtained embryoid body (adventitious embryo) is shown in FIG.

In general, plants usually have worse environmental conditions (cold,
When it comes to dryness, it blooms, bears fruit, and takes the form of seeds that can maintain life even under adverse conditions, allowing the next generation to survive. In this method, it is considered that the stress of decreasing the phosphate essential for life support brought about the seed-forming (adventitious embryo induction) function potentially possessed by the cells. Since this method is a liquid culture system, it can be scaled up to tank culture. Industrial production such as tank culture becomes possible, and planned seedling production becomes possible.

Example 3 This example relates to a method for producing a breeding mother of cyclamen, which was carried out by the following method. First, 1 part of "Large White" petiole of 2,4-D, 1 ppm of kinetin and 5% of sucrose was added to LS solid medium.
Incubate at 25 ° C in the dark for a month. Then, the somatic embryo is induced by allowing it to stand in an MS solid medium containing 3% sucrose at 25 ° C. in the dark for 1 month. The adventitious embryos were further transferred to hormone-free MS solid medium for 2 months in the dark for 2 months.
Incubate at 0 ° C. The seedlings were placed in pots and cultivated. When male sterility was investigated in the 7 flowering strains, all of the 7 strains were sterile. The sterile strains are shown in FIG.

So far, no male sterile sterile cyclamen regenerated from somatic embryos have been known. According to this example, it is possible to omit the mating work for producing F ₁ hybrids that do not require emasculation. For example, F ₁ seeds can be obtained by placing male-sterile strains and recovery-type strains in a net chamber and releasing bees, flies, and the like. Although emasculation requires skill, the method of the present example is possible even if the skill level of those skilled in the art is not high.

[0041]

EFFECTS OF THE INVENTION According to the first aspect of the present invention, an embryoid body callus can be induced regardless of differences between varieties and individuals. Further, according to the second invention, the rate of differentiation from somatic embryos is improved. According to the third aspect of the present invention, once a male sterile culture system is created, clonal expansion can be performed in culture, and F ₁ hybrid production that does not require emasculating mating can be achieved, thus simplifying the mating work.

[Brief description of drawings]

FIG. 1 is a photograph showing the morphology of the organism relating to the embryoid body callus obtained in Example 1 of the present invention.

FIG. 2 is a photograph of the morphology of an organism showing the effect of gellite concentration on the rooting of somatic embryos of Example 2 of the present invention.

FIG. 3 is a photograph of the morphology of an organism showing the effect of gellite concentration on the rooting of somatic embryos of Example 2 of the present invention.

FIG. 4 is a photograph of the morphology of an organism showing the effect of gellite concentration on the rooting of somatic embryos of Example 2 of the present invention.

FIG. 5 is a photograph of the morphology of an organism showing the effect of inorganic salt concentration on the rooting of somatic embryos of Example 2 of the present invention.

FIG. 6 is a photograph of the morphology of an organism showing the effect of inorganic salt concentration on the rooting of somatic embryos of Example 2 of the present invention.

FIG. 7 is a photograph of the morphology of an organism showing the appearance of rooting from a rod-shaped embryo related to Example 2 of the present invention.

FIG. 8 is a photograph of the morphology of an organism showing an adventitious embryo differentiated and developed at a low initial culture density related to Example 2 of the present invention.

FIG. 9 is a photograph of the morphology of an organism showing an adventitious embryo that does not differentiate and develop at a high initial culture density related to Example 2 of the present invention.

FIG. 10 is a photograph showing the morphology of the organism relating to the embryoid body callus related to Example 2 of the present invention.

FIG. 11 is a photograph showing the morphology of the organism relating to the embryoid body callus obtained in the comparative example related to Example 2 of the present invention.

FIG. 12 is a photograph showing the morphology of organisms related to embryoid bodies induced at 2 weeks of phosphate starvation culture related to Example 2 of the present invention.

FIG. 13 is a photograph showing the morphology of organisms related to the somatic embryo-derived male sterile strain of Example 3 of the present invention.

Claims (3)

[Claims] 1. A cyclamen embryoid callus characterized by culturing by culturing an embryo taken out from a sterilized and water-absorptive germinated mature seed on an LS solid medium containing 2,4-D, cytokinin and sucrose. Induction method. 2. Differentiating a cyclamen somatic embryo into a rod-shaped embryo,
A method for rooting an adventitious cyclamen embryo, which comprises rooting at 10 to 25 ° C. in the dark after development. 3. A method for producing a cyclamen breeding mother book, which comprises culturing a somatic embryo of cyclamen in a solid medium containing 2,4-D and kinetin to regenerate a plant.