WO2009093787A1

WO2009093787A1 - Production method of white clover with mutation in leaf number

Info

Publication number: WO2009093787A1
Application number: PCT/KR2008/004533
Authority: WO
Inventors: In Ja Song; Tae Woong Bae; Pyung Ok Lim; Hyo Yeon Lee; Si Yong Kang
Original assignee: Cheju National University Industry-Academic Cooperation Foundation
Priority date: 2008-01-25
Filing date: 2008-08-05
Publication date: 2009-07-30
Also published as: KR20090081784A; KR101248367B1

Abstract

Disclosed is a production method of a white clover with induced mutation in leaf number. The production method of a white clover comprises: treating flowers of a white clover with a mutagen; cultivating the mutagen-treated white clover to gather seeds of the white clover; and cultivating the seeds of the mutagen-treated white clover to select a white clover with induced mutation in leaf number.

Description

[DESCRIPTION]

[invention Title]

PRODUCTION METHOD OF WHITE CLOVER WITH MUTATION IN LEAF NUMBER

[Technical Field]

The present invention relates to a production method of white clover with induced mutation in leaf number.

[Background Art]

Trifolium repense L. is a perennial plant belonging to the Fabaceae family in the Rosales order of dicotyledon, and native to Europe. Also, this clover is called a white clover since it has white flowers .

The white clover takes root from knobs of its stems which spread on the ground, thus propagating an individual population, and comes into bloom by June to July in Korea. The white clover has 3 leaves, or sometimes 4 leaves. It has also been known that if the leaves of the white clover are four in number they bring good luck.

Technologies regarding the white clover are mainly divided into three groups: the first group is a technology of fancy products using leaves of a white clover, particularly 4 leaves of a white clover which is an emblem of good luck, the second group is a technology using physiological activities of a white clover, and the third group is a technology regarding breeding improvements in order to produce a 4-leaf clover.

Korean Patent No. 0391893 discloses a fancy product that is prepared by processing a 4-leaved clover into a constant size and shape and interposing the processed 4- leaved clover between transparent synthetic resin films. Further, Korean Design Registration No. 0190633 discloses the use of a 4-leaved clover for a necklace or a ring after sealing the 4-leaved clover between transparent synthetic resin films. These technologies belong to the first group. On the other hand, Korean Patent No. 0545043 disclosing an antioxidant cosmetic composition containing a trifolium repense L. extract belongs to the second group, and Korean Patent No. 0330804 disclosing a method for breeding a new plant variety of clover comprising collecting a creeping stem of a clover having 4 leaves, treating the creeping stem with BAP, IBA and NH₄NO₃ and asexually propagating the clover belongs to the third group. The present invention discloses a technology that can mass-produce 4 or more-leaved white clover by mutating a white clover.

[Disclosure] [Technical Problem] An aspect of the present invention provides a production method of a white clover with induced mutation in leaf number.

Another aspect of the present invention provides a production method of seeds of a white clover which has an induced mutation in leaf number.

Still another aspect of the present invention provides a production method of leaves with 4 or more-leaved variation.

Hereinafter, specific technical problems and other exemplary embodiments of the present invention will be described in detail, as follows.

[Technical Solution]

According to an aspect of the present invention, there is provided a production method of a white clover with an induced mutation in leaf number.

The method of producing a white clover with an induced mutation in leaf number includes: (a) treating flowers of a white clover with a mutagen; (b) cultivating the mutagen- treated white clover to gather seeds thereof; and (c) cultivating the seeds of the white clover to select a white clover with induced mutation in leaf number.

As seen from the following Examples and Comparative

Examples, the present inventors have found that, when white clover flowers are treated with a mutagen such as gamma rays (⁶⁰Co) or ethylmethane sulfonate (hereinafter, referred to as "EMS"), and the mutagen-treated white clover is cultivated to gather its seeds , and the seeds are sowed and germinated to cultivate a white clover, white clovers with induced mutation in leaf number are obtained at a rate of approximately 3.5% from plants which have survived gamma ray treatment ( 4 white clovers out of the 113 survived plants) and obtained at a rate of approximately 1.1% from EMS-treated survived plants (4 white clovers out of the 363 survived plants).

In particular, when seeds (Mi) gathered from the gamma ray-treated white clover are cultivated to obtain a first- generation white clover with induced mutation m leaf number, and seeds (M₂) are gathered from the first-generation white clover and cultivated to obtain a second-generation white clover, it is observed that all of 130 survived white clovers germinated from the 230 sowed seeds have induced mutation in leaf number. In this case, it is observed that the 130 surviving white clovers with induced mutation in leaf number mainly have 4 or 5 leaves, and occasionally have 6 or 7 leaves, that is, have 4 leaves with one leaf being partially divided into two leaves, have 3 large leaves and 2 small leaves , have 5 leaves with 2 overlapped leaves being viewed as 4 leaves, have 5 leaves with a cup-shaped leaf, etc.

The present invention is based on the above-mentioned experimental results . In this specification, the expression "white clover with induced mutation in leaf number" means a white clover that has 4 or more leaves at a higher frequency of occurrence than a wild-type white clover. In general, it has been known that a wild-type white clover having 4 or more leaves occurs at a frequency of 0.1% or less. This percentage is the percentage of the number of leaves having 4 or more-leaved variation among the total number of leaves in one white clover individual. Therefore, the expression "white clover with induced mutation in leaf number," as used herein, is defined as a white clover having more than 0.2% of 4 or more- leaved variation. In consideration of the results from the following Examples, the white clover with induced mutation in leaf number is preferably defined as a white clover having more than 1%, 3%, 5%, 10%, 15% or 20% of 4 or more-leaved variation, and more preferably defined as a white clover having more than 30% of 4 or more-leaved variation.

Also, the expression "white clover with induced mutation in leaf number" has the meaning that includes white clovers having 4 or 5 leaves, even 6 or 7 leaves. As long as it has 4 or more leaves, any white clover may be included within the scope of the present invention whether or not the leaves are different in size and/or shape from each other.

Also in this specification, the expression "induced mutation" means a genetic mutation that allows a mutant character (S) to be expressed in the next generation. Therefore, it may be understood that the expression "white clover with induced mutation in leaf number" means a white clover that is mutated in leaf number to express its phenotype in the following generations .

Also in this specification, the expression "flowers of a white clover" means flowers including all the sexual organs of a flower such as a pistil, a stamen, a calyx and petals, and which are all present in a differentiated state. In general, flowering is performed by forming a flower bud, differentiating the flower bud into flower organs such as a pistil, a stamen, a petal and a calyx and maturing the differentiated flower organs. Therefore, it may be understood that the expression "flowers of a white clover" means flowers spanning from when the flower organs are differentiated after the formation of the flower bud to a time point when the flowers are gone. Timing of the flowering may be controlled by artificially adjusting the temperature, light intensity, etc. since it is generally known the flowering of a white clover is affected by temperature, light intensity, etc. Also, the timing of the flowering may be varied according to region since climactic changes differ by region. For example, it has been known that a naturally-growing white clover, that is, one which grows without artificial handling of light, temperature, etc., comes into bloom by June to July in Korea; therefore, a white clover growing in Korea may be desirably used when the white clover is in full bloom by June to July.

Meanwhile, as seen from the following Comparative examples, it is revealed that a white clover is not mutated in leaf number when its seeds are treated with a mutagen. However, as seen from the following Examples, it is confirmed that a white clover is mutated in leaf number when its flowers are treated with a mutagen. Therefore, it may be understood that the feature of the present invention is to treat white clover flowers with a mutagen.

One reason for the present inventors to treat the white clover flowers with a mutagen is based on the fact that, when the flowering season is coincident with fertilization/insemination period and white clover flowers are treated with a mutagen in the flowering season, mutation rate of the white clover may be enhanced.

Also in this specification, the term "mutagen" means an agent that includes both physical mutagens and chemical mutagens. In general, the physical mutagens such as ⁶⁰Co, X- rays, γ-rays, β-rays (³²P, ³⁵S, etc.) and a neutron beam have been widely used to produce mutant plants (Hasegawa et al . 1995; Ling et al. 1991; Wang et al. 1988; Honda et al . 2006; Naito et al. 2005), and the chemical mutagens such as ethylmethane sulfonate (EMS), ethylethane sulfonate (EES), ethylene oxide (EO), N-methylcarbazole (NMC), and nitroso- methylurea have been widely used (Abe et al. 1995; Lee et al . 1998; Wu et al. 2005, Hohmann et al. 2005). In particular, EMS and ⁶⁰Co have been most widely used. Therefore, although the above-mentioned physical and chemical mutagens may all be used as the mutagen of the present invention, there is a preference for the use of EMS and ⁶⁰Co.

The treatment of the mutagens may be carried out according to any of the methods known in the art, depending on the kind of mutagen. Generally, the treatment of the physical mutagens will be carried out by irradiation, and the treatment of the chemical mutagens will be carried out by dipping organs of a plant to be treated ( flowers of a white clover in the present invention) in a chemical mutagen solution.

Meanwhile, the irradiation time of the physical mutagens , or the irradiation time and concentration of the chemical mutagens may be suitably determined by those skilled in the art, depending on the kind of mutagen used. In general, the more potent physical mutagens may induce a desired mutation of a white clover even when the white clover is irradiated with the physical mutagen for a shorter time period, compared to the relatively weak physical mutagens. Also, a high concentration of the chemical mutagens may induce a desired mutation of a white clover even when a white clover is dipped for a shorter time period, compared to a relatively low concentration of the chemical mutagens .

Therefore, though a white clover with induced mutation in leaf number may be obtained by treating white clover flowers with the physical or chemical mutagens, it should be understood that the kind of the mutagen used, the treatment methods of the mutagens, and the like are not intended to limit the scope of the invention.

Referring to the following Examples, however, a physical mutagen is preferably treated at a dose range of 15 to 100(Gy), and more preferably at a dose range of 20 to 80

(Gy), regardless of the kind of physical mutagens. In particular, ⁵⁰Co is preferably used as the physical mutagen.

The treatment of the chemical mutagen is preferably carried out by dipping a white clover in a 0.01% to 5% chemical mutagen solution for approximately 10 to 48 hours. On the basis of the present inventors' experience, when EMS is used as the chemical mutagen, the chemical mutagen is particularly preferably treated by dipping a white clover in a 0.01% to 2% chemical mutagen solution for approximately 24 hours, and more preferably 20 to 28 hours, as described later in the following Examples .

Meanwhile, the present inventors have found that the mutation in leaf number may be observed from all of surviving white clovers subjected to: treating flowers of a white clover with a mutagen, gathering first-generation seeds (Mi) from the white clover, cultivating the first-generation seeds (M₁) to select a first-generation white clover with the highest mutation in leaf number and cultivating a second- generation seeds (M₂) gathered from the first-generation white clover to grow into a second-generation white clover, as described above . Therefore, the production method of a white clover with induced mutation in leaf number according to one exemplary embodiment of the present invention may further comprise, after the step (c): gathering seeds from the selected white clover and cultivating the seeds of the selected white clover to select a white clover with induced mutation in leaf number. In this case, the selected white clover more preferably has the highest mutation rate in the leaf number; that is , the seeds are preferably gathered from the white clover having the highest mutation rate in leaf number.

Meanwhile, although not directly confirmed by the present inventors, it is expected from the above-mentioned facts that the mutation rate in leaf number may be increased across a series of generations. Therefore, it is preferred to repeat the above-mentioned steps of the production method.

The mutation rate in leaf number may be calculated as a rate of the number of leaves with 4 or more-leaved variation to the total number of leaves in a certain white clover individual. In this case, it is considered that the mutation rate in leaf number becomes higher as the number of the leaves with 4 or more-leaved variation increases with respect to the total number of the leaves of a white clover.

According to another aspect of the present invention, there is provided a production method of seeds of a white clover with induced mutation in leaf number.

The production method of seeds of a white clover with induced mutation in leaf number may comprise (a) treating flowers of a white clover with a mutagen and (b) cultivating the mutagen-treated white clover to gather seeds of the mutagen-treated white clover. Like the production method of a white clover with induced mutation in leaf number, the production method of seeds of a white clover with induced mutation in leaf number according to one exemplary embodiment of the present invention preferably includes cultivating the seeds gathered in the step (b) to grow into a white clover and gathering seeds from the grown white clover. Here, the seeds are preferably gathered from the white clover having the highest induced mutation rate in leaf number. Also, it is preferred that the above-mentioned steps be repeated for the same reasons as described above.

The definitions and descriptions of the production method of a white clover with induced mutation in leaf number explained in the above and other embodiments of the present invention are also effective as pertaining to the production method of seeds of a white clover with induced mutation in leaf number.

According to still another aspect of the present invention, there is provided a production method of white clover leaves with 4 or more-leaved variation. The production method of white clover leaves with 4 or more-leaved variation according to one exemplary embodiment of the present invention includes (a) treating flowers of a white clover with a mutagen; (b) cultivating the mutagen- treated white clover to gather seeds of the white clover; (c) cultivating the seeds of the white clover to grow into a mature white clover; and (d) collecting leaves with 4 or more-leaved variation from the grown white clover.

According to the production method of the present invention, it is possible to mass-produce white clover leaves with 4 or more-leaved variation. As seen from the following Examples, when first- generation seeds (Mi) are sowed to select a white clover having the highest mutation rate in leaf number from the white clovers showing mutation in leaf number, and a creeping stem is separated from the selected white clover to propagate a white clover, 4-leaf white clovers and 5-leaf white clovers occur at a significantly increased freguency of approximately 20% and approximately 60%, respectively, compared to the wild-type white clover.

Therefore, the production method of the present invention preferably comprises, after the step (c), selecting a white clover having the highest mutation rate in leaf number from the mature white clover, separating a creeping stem from the selected white clover and propagating the creeping stem of the white clover. Meanwhile, like the production method of a white clover with induced mutation in leaf number according to one exemplary embodiment of the present invention or the production method of seeds of a white clover with induced mutation in leaf number according to one exemplary embodiment of the present invention, the production method of white clover leaves with 4 or more-leaved variation of the present invention preferably further includes, after the step (c), gathering seeds from the grown white clover and cultivating the seeds of the white clover to grow into a white clover. Here, the seeds are preferably gathered from the white clover having the highest mutation rate in leaf number. Also, it is preferred to repeat the above-mentioned steps on the same ground as described above.

The definitions and descriptions of the production method of a white clover with induced mutation in leaf number explained in the above and other embodiments of the present invention are effective as pertaining to the production method of white clover leaves with 4 or more-leaved variation.

According to yet another aspect of the present invention, there is provided a method of propagating a creeping stem of 4 or more-leaved white clover.

The method of propagating a creeping stem of 4 or more- leaved white clover according to the present invention comprises: (a) treating flowers of a white clover with a mutagen; (b) cultivating the mutagen-treated white clover to gather seeds of the white clover; (c) cultivating the seeds of the white clover to select a white clover with induced mutation in leaf number; and (d) separating a creeping stem from the selected white clover and transplanting the creeping stem into the ground to propagate the separated creeping stem.

In this case, the creeping stem is preferably a creeping stem having the highest mutation rate in leaf number. Also, the method of propagating a creeping stem of 4 or more-leaved white clover preferably further includes, after the step (c): gathering seeds of the selected white clover and cultivating the seeds of the white clover to select a white clover with induced mutation in leaf number. In this case, the seeds are preferably gathered from the white clover having the highest mutation rate in leaf number Also, it will be preferred that the above-mentioned steps of the production method be repeated. The reason for the above is the same as described above, and the mutation rate in leaf number is the same as described above .

Also, the ground may be an outdoor soil or a flowerpot soil when the creeping stem is transplanted into the ground as described above.

Meanwhile, the definitions and descriptions of the production method of a white clover with induced mutation in leaf number according to one exemplary embodiment (including the other exemplary embodiments) of the present invention are also effective as pertaining to the method of propagating a creeping stem of 4 or more-leaved white clover.

[Advantageous Effects]

One aspect of the present invention may be useful to provide a production method of a white clover with induced mutation in leaf number.

Another aspect of the present invention may be useful to provide a production method of seeds of a white clover with induced mutation in leaf number.

Still another aspect of the present invention may be useful to provide a production method of white clover leaves with 4 or more-leaved variation.

It is expected that the white clover having 4 or more leaves produced according to the present invention may be effectively used as a raw material in fancy products, etc.

[Description of Drawings]

FIG. 1 is a photograph taken from a white clover after M₁ seeds of a white clover have been sowed and germinated into a plantlet and the plantlet is transplanted into flowerpots and cultivated in a greenhouse for 3-4 months. FIG. 2 is a photograph taken from a white clover after seeds of a mutagen (gamma rays ⁶⁰Co) -untreated white clover are sowed to cultivate a white clover.

FIG. 3 is a photograph taken from a white clover after a white clover having the highest mutation rate is selected from Mi-generation white clovers and grown.

FIG. 4 is a photograph taken from a white clover after a creeping stem of a white clover having the highest mutation in leaf number (i.e. 4 and 5 leaves) is selected from a population of Mi-generation white clovers having the highest mutation rate, and propagated. FIG. 4A is a photograph taken from a white clover after propagating a creeping stem of a white clover having a lot of 4 leaves, and FIG. 4B is a photograph taken from a white clover after propagating a creeping stem of a white clover having a lot of 5 leaves. Black stars represent 4-leaf clovers, and white stars represent 5-leaf clovers.

FIG. 5 is a photograph taken from a white clover after a creeping stem of a white clover having 3 leaves is separated and propagated.

FIG. 6 is a photograph taken from a white clover after M₂ seeds gathered from an Mχ-generation white clover are cultivated and propagated.

FIG. 7 is a photograph taken from a 7-leaf white clover when M₂ seeds are cultivated and propagated.

FIG. 8 is a photograph taken from a white clover having 4 leaves albeit one partially divided leaf when M₂ seeds are being cultivated and propagated.

FIG. 9 is a photograph taken from a white clover having 3 large leaves and 2 small leaves when M₂ seeds are being cultivated and propagated. FIG. 10 is a photograph taken from a white clover having 5 leaves with 2 overlapped leaves being viewed as 4 leaves when M₂ seeds are cultivated and propagated.

FIG. 11 is a photograph taken from a white clover having 5 cup-shaped leaves when M₂ seeds are being cultivated and propagated.

FIG. 12 is a photograph taken from a white clover obtained by cultivating seeds of a white clover treated with the mutagen "EMS." FIG. 13 is a photograph taken from a white clover obtained by cultivating seeds of a white clover treated with the mutagen "gamma rays (⁶⁰Co)."

FIG. 14 is a photograph taken from a white clover obtained by cultivating seeds of a white clover treated with the mutagen "EMS."

[Best Mode]

Hereinafter, specific Examples and Comparative examples of the present invention will be described in more detail.

However, it is understood that the specific Examples and Comparative examples are not intended to limit the scope of the present invention.

Examples : Production of white clover with induced mutation in leaf number Example 1 : Production of white clover with induced mutation in leaf number by irradiation of gamma rays (⁶⁰Co)

Example 1-1; Irradiation of gamma rays (⁶⁰Co) A white clover used in this experiment was Trifolium repens L., and flowers of a white clover whose flower stalk has a length of approximately 20cm were selected in June to July when the white clover came into bloom, and treated with gamma rays (⁵⁰Co). The irradiation of a white clover with gamma rays (⁶⁰Co) was carried out at doses of 25 and 50 (Gy) for approximately 1 hour and 2 hours, respectively, using an irradiation facility, which is owned by Institute of Radiology of the Cheju National University ( Jeju-do, Korea) . 48 hours after gamma ray irradiation, each of the plants was cultivated in a greenhouse of the Cheju National University under natural light condition, and seeds (Mi) were gathered from each of the plants after 1 and 2 months . The gathered seeds were treated at a low temperature in a 4 ^°C refrigerator, and then sowed to select plants with mutation in leaf number.

Example 1-2; Examination of Mi-generation white clover on germination rate and mutation rate in leaf number In order to select an Mi-generation white clover with mutation in leaf number, a wild-type white clover was treated with radioactive rays to obtain Mi white clover seeds, and a wild-type white clover was wound with a sandpaper to enhance germination rate, and sowed on a filter paper to germinate the seeds. The germinated seeds were cultivated at 28^°C±2 under a continuous light condition. 100 gamma ray-untreated seeds were used as the control, and 120 seeds treated with gamma rays were allotted to each experimental group. Four weeks after being sowed, the seeds were examined for germination rate. In the case of the control, 98 seeds were germinated with a germination rate of 98%. As for the experimental group, germination was observed in 84 seeds of the 25 (Gy)- treated group with a germination rate of 70%, and in 86 seeds of the 50 (Gy)-treated group with a germination rate of 71.7% (see Table 1) .

In each of the treated groups, the germinated plants were transplanted into flowerpots 4 weeks after the germination of the seeds, cultivated at 28±2^°C for 3 to 4 months under a natural light condition in a greenhouse while being fed water (see FIG. 1), and a population of white clovers with mutation in leaf number, leaf colors and leaf shapes was then observed.

The total of 170 seeds were germinated in the two treated groups except for the control, but 113 populations were grown into plants. Among them, the total 7 populations were observed as mutant white clovers, including 4 mutant white clovers with mutation in leaf number and 3 mutant white clovers with mutation in leaf colors and leaf shapes (see Table 1 ) .

No mutant white clover was observed in the control (see Table 1 and FIG. 2) .

TABLE 1

Germination rate and mutation rate in leaf number of white clover according to gamma ray-irradiation

Example 1-3; Selection of Mi-generation white clover with mutation in leaf number and analysis of mutation rate in leaf number in M₂-generation white clover

Among 7 populations with mutations in leaf out of the Mx-generation white clovers obtained by sowing seeds of a white clover irradiated with gamma rays, 4 populations with mutations m leaf number were selected, transplanted to a greenhouse, and propagated at 28^°C±2 for 6 months under a natural light condition while removing branches having 3 leaves and feeding water. In this case, the reason for propagating a white clover while removing branches having 3 leaves is to fix a leaf number of a white clover at 4 or 5 leaves . Among the primarily selected plants with mutation in leaf number, one population having the highest mutation in leaf number was selected and propagated at 28^°C±2 for approximately 1 year under a natural light condition (see FIG. 3), creeping stems of the white clovers having the highest mutation in each leaf number (i.e. 4 or 5 leaves) were selected by 4s, and the selected creeping stems were transplanted to a greenhouse of the Cheju National University, and then propagated at 28^°C±2 for 1 year under a natural light condition (see FIG. 4). Then, the total leaf number of the 8 creeping stems was summed up to analyze the mutation rate in leaf number in each population. The mutation rate in leaf number was determined by observing only fully spread leaves that were grown 2cm or more from the ground.

The experimental results are listed in the following Table 2.

TABLE 2

Selection of M₂-generation plant with mutation in leaf number and analysis of mutation rate in M₂-generation plants

As listed in the Table 2, creeping stems of the 3-leaf white clovers as the control were propagated at 28^°C±2 for 1 year under a natural light condition in a greenhouse ( see FIG. 5). As can be seen in Table 2, 99.9% of the white clovers have 3 leaves, 0.1% of the white clovers have 4 leaves, and no white clover has 5 leaves m the case of the control, but that 20.3% of the white clovers have 3 leaves, 19.9% of the white clovers have 4 leaves, and 59.9% of the white clover have 5 leaves in the case of M₂-generation white clovers. Therefore, it was confirmed that the 4-leaf white clovers and the 5-leaf white clovers occur at significantly increased frequencies in the M₂-generation white clovers, compared to the control .

Example 1-4: Analysis of germination rate and mutation rate m leaf number in M₂-generation white clover

Seeds (M₂) were gathered from the M_x-generat i_on white clovers cultivated and propagated in the Example 1-3, dried, treated at a low temperature in a 4^°C refrigerator, and then sowed on a filter paper to germinate white clover seeds. The germinated white clover seeds were cultivated at 28^°C±2 under a continuous light condition. The germinated plantlets were transplanted in flowerpots 4 weeks after the germination of the white clover seeds, cultivated for 1 month under a natural light condition in a greenhouse, transplanted into the ground, and then propagated at 28^°C±2 for 1 year under a natural light condition (see FIG. 6).

TABLE 3 Germination rate and mutation in leaf number of M₂ seeds

As listed in the Table 3, most of the 130 populations having the mutation in leaf number were mutant white clovers having 4 and 5 leaves, and populations having 6 and 7 leaves were sometimes observed (see FIG. 7). Also, populations were also observed which have 4 leaves with one leaf being partially divided into two leaves (see FIG. 8), have 3 large leaves and 2 small leaves (see FIG. 9), have 5 leaves with 2 overlapped leaves being viewed as 4 leaves (see FIG. 10), and have 5 leaves with a cup-shaped leaf (see FIG. 11).

Meanwhile, one population (M₂-3) having 3 leaves, one population (M₂-4) having 4 leaves and one population (M₂-5) having 5 leaves were selected from the 130 populations to count leaves. The results are listed in the following Table 4.

TABLE 4 Mutation in leaf number in M₂-generation plants

Total No. of 3-leaf No. of 4-leaf No. of 5-leaf leaf No. clovers ( ^s0 clovers (^s3) clovers )

M₂-3 382 172 (45) 155 (40 .6 ) 55 (14. 4)

M₂-4 466 151 (32.4 ) 184 (39 .5 ) 131 (28 .1 )

M₂-5 466 81 (17.4 101 (22 .7 ) 284 (60 .9 )

Example 2 : Production of white clover with induced mutation in leaf number by treatment of EMS

Flowers of a white clover were treated with EMS in the same manner as in Example 1. The EMS was used at an increasing concentration of 0.01%, 0.2% and 2%, and the EMS treatment was carried out by dipping a white clover for 24 hours in an EMS solution. Here, the white clover was cultivated in a dark room during the EMS treatment. After the EMS treatment, the white clover was cultivated under a natural light condition in a greenhouse of the Cheju National University, and its seeds were gathered 1 to 2 months after the cultivation of the white clover. The seeds were germinated in the same manner as in Example 1-2, and grown into plants. The number of the seeds was 200 per each EMS- treated group.

TABLE 5

Among the total 8 populations with mutation in leaf, 4 populations with mutations in leaf number were selected and transplanted to a greenhouse, and propagated at 28^°C±2 for 6 months under a natural light condition in the greenhouse while removing branches having 3 leaves and feeding water. In this case, the reason for propagating a white clover while removing branches having 3 leaves is to fix a leaf number of a white clover to 4 or 5 leaves, as described above. Among the primarily selected plants with a mutation in leaf number, only one population having the highest mutation in leaf number was selected and propagated at 28^°C±2 for approximately 1 year under a natural light condition. As a result, it was revealed that the creeping stems of the white clovers having lots of 4 and 5 leaves were plentifully observed at a level similar to that of the Example 1 (see FIG. 12).

Comparative examples : Mutation of white clover seeds by treatment of gamma rays or EMS

Comparative example 1 : Treatment of seeds with gamma rays (⁶⁰Co)

Seeds of a white clover were irradiated, respectively, with 25, 50 and 100 (Gy) of gamma rays (⁶⁰Co), and sowed and germinated in the same manner as in Example 1-2 to cultivate a white clover, but no white clover with mutation in leaf number was obtained (see FIG. 13).

Comparative example 2 : Treatment of seeds with EMS Seeds of a white clover were treated with EMS in the same manner as in Example 2, and sowed and germinated to cultivate a white clover, but no white clover with mutation in leaf number was obtained (see FIG. 14).

Claims

[CLAIMS]

[Claim l]

A method of producing a white clover with induced mutation in leaf number, comprising:

(a) treating flowers of a white clover with a mutagen;

(b) cultivating the mutagen-treated white clover to gather seeds of the white clover; and

(C) cultivating the seeds of the white clover to select a white clover with induced mutation in leaf number.

[Claim 2]

A method of producing seeds of a white clover with induced mutation in leaf number, comprising: (a) treating flowers of a white clover with a mutagen; and

(b) cultivating the mutagen-treated white clover to gather seeds of the white clover.

[Claim 3]

A method of producing white clover leaves with 4 or more-leaved variation, comprising:

(a) treating flowers of a white clover with a mutagen;

(b) cultivating the mutagen-treated white clover to gather seeds of the white clover;

(C) cultivating the seeds of the white clover to grow into a white clover; and

(d) collecting leaves with 4 or more-leaved variation from the grown white clover.

[Claim 4]

A method for propagating a creeping stem of 4 or more- leaved white clover, comprising:

(a) treating flowers of a white clover with a mutagen;

(c) cultivating the seeds of the white clover to select a white clover with induced mutation in leaf number; and

(d) separating a creeping stem from the selected white clover and transplanting the creeping stem into the ground to propagate the separated creeping stem.

[Claim 5]

The method according to any one of claims 1 to 4, wherein the mutagen is a physical mutagen selected from the group consisting of an X-ray, a γ-ray, a β-ray (³²P, ³⁵S, etc.) and a neutron beam.

[Claim 6]

The method according to claim 5, wherein the physical mutagen is irradiated at a dose range of 15 to 100 (Gy) . [Claim 7 ]

The method according to claim 5, wherein the physical mutagen is irradiated at a dose range of 20 to 80 (Gy) .

[Claim 8]

The method according to any one of claims 1 to 4, wherein the mutagen is a chemical mutagen selected from the group consisting of ethylmethane sulfonate (EMS), ethylethane sulfonate (EES), ethylene oxide (EO), and N-methylcarbazole (NMC).

[Claim 9]

The method according to any one of claims 1 to 4, wherein the mutagen is a chemical mutagen selected from the group consisting of EMS, EES, EO, and NMC, and the treatment of the chemical mutagen is performed by dipping flowers of the white clover in a 0.01% to 5% chemical mutagen solution for 10 to 48 hours.

[Claim lθ]

The method according to any one of claims 1 to 4, wherein the mutagen is ⁶⁰Co.

[Claim ll] The method according to any one of claims 1 to 4 , wherein the mutagen is EMS . [Claim 12]

The method according to claim 10, wherein the ⁶⁰Co is administered at a dose range of 15 to 100 (Gy) .

[Claim 13]

The method according to claim 10, wherein the ⁶⁰Co is administered at a dose range of 20 to 80 (Gy) .

[Claim 14]

The method according to claim 11, wherein the treatment with EMS is performed by dipping flowers of the white clover in a 0.01% to 2% EMS solution for 20 to 28 hours.

[Claim 15]

The method according to claim 1, further comprising, after the step (c): gathering seeds of the selected white clover and cultivating the seeds of the white clover to select a white clover with induced mutation in leaf number.

[Claim 16]

The method according to claim 15, wherein the selected white clover has the highest mutation rate in leaf number.

[Claim 17]

The method according to claim 2, further comprising: cultivating the seeds gathered in the step (b) to grow into a white clover and gathering seeds from the grown white clover.

[Claim 18] The method according to claim 17, wherein the white clover into which the seeds gathered in the step (b) are cultivated to grow has the highest mutation rate in leaf number.

[Claim 19]

The method according to claim 3, further comprising, after the step (c): gathering seeds from the grown white clover and cultivating the seeds of the white clover to grow into a white clover.

[Claim 20]

The method according to claim 19, wherein the grown white clover from which the seeds are gathered has the highest mutation rate in leaf number.

[Claim 2l]

The method according to claim 3, further comprising, after the step (c): selecting a white clover having the highest mutation rate in leaf number from the grown white clover, separating a creeping stem from the white clover having the highest mutation rate in leaf number and propagating the creeping stem of the white clover.

[Claim 22]

The method according to claim 3, further comprising, after the step (c): gathering seeds from the selected white clover with induced mutation in leaf number and cultivating the seeds of the selected white clover to select a white clover with induced mutation in leaf number.

[Claim 23]

The method according to claim 22, wherein the grown white clover from which the seeds are gathered has the highest mutation rate in leaf number.

[Claim 24]

The method according to claim 4 , wherein the creeping stem has the highest mutation rate in leaf number.