Flower initiation inducer

An agent for inducing flower bud formation in plants comprising an unsaturated fatty acid having an oxo group and a hydroxy group, a hydroperoxy group, or an oxo group, a hydroxy group, and a hydroperoxy group; a method for preparing said agent for inducing flower bud formation; and, a method for inducing flower bud formation using said agent for inducing flower bud formation are provided.

TECHNICAL FIELD 
The present invention relates to agents for inducing flower bud formation, 
methods for the production thereof, and methods for inducing flower bud 
formation using said agents for inducing flower bud formation. 
BACKGROUND ART 
It is well known that flower formation of plants is controlled by day 
length. It has also been found that the part that responds to the day 
length is the leaf blade and flower formation begins at the meristem and 
that a certain signal is sent from the leaf blade via the petiole and the 
stem to the meristem where flower formation starts. The signal is called 
"florigen." It is obvious that the isolation and identification of 
florigen would enable the artificial control of the flowering timing of 
plants irrespective of day length, which would no doubt have enormous 
impacts on many plant-related fields. 
Thus, attempts have been made to artificially control the timing of 
flowering of plants by elucidating the mechanism of the process of flower 
formation. 
For example, it was found that gibberellin, a growth hormone of plants, 
when applied, causes flower bud formation of long-day plants even under 
short-day conditions and that pineapples start flower formation after the 
application of .alpha.-naphthalene, a synthetic auxin, which is currently 
used industrially. 
However, it is also known that these plant hormones are florigen-related 
substances, which are different from florigen itself. 
Therefore, it is often required to set various conditions such as the 
timing and the environments of applying these plant hormones to plants, 
etc. As a result, there is a need for further advancement of flowering 
methods, or more specifically, the establishment of flowering techniques 
through isolation and identification of substances which are directly 
involved in flower bud formation. 
It has also been reported that the phenomenon of flower bud formation based 
on photoperiodis is inhibited by a dry stress in the plants of the genus 
Pharbitis, the genus Xanthium, and the genus Lolium (for the genus 
Pharbitis and the genus Xanthium: Aspinall 1967; for the genus Lolium: 
King and Evans). Furthermore, it has also been reported that flower bud 
formation is induced by low temperature (Bernier et al. 1981; Hirai et al. 
1994), high illumination (Shinozaki 1972), poor nutrition (Hirai et al. 
1993), or shortage of nitrogen sources (Wada and Totuka 1982; Tanaka 1986; 
Tanaka et al. 1991). 
However, these reports are mere observations of phenomena and do not 
directly specify the above-mentioned florigen and there is still a need 
for the establishment of the flowering method based on the understanding 
from the material aspect. 
DISCLOSURE OF THE INVENTION 
Thus, the problem to be solved by the present invention is to locate an 
inducer of flower bud formation that is directly involved in flowering and 
thereby to provide an agent for inducing flower bud formation having said 
inducer of flower bud formation as an active ingredient. 
The present invention first provides an agent for inducing flower bud 
formation comprising a fatty acid of 4 to 24 carbon atoms having an oxo 
group and a hydroxy group and containing 0 to 6 double bonds. 
The present invention also provides an agent for inducing flower bud 
formation comprising a fatty acid of 4 to 24 carbon atoms having an oxo 
group, a hydroxy group and a hydroperoxy group and containing 0 to 6 
double bonds. 
The present invention further provides an agent for inducing flower bud 
formation comprising a fatty acid of 4 to 24 carbon atoms having a 
hydroperoxy group and containing 0 to 6 double bonds. 
The present invention further provides an agent for inducing flower bud 
formation which is obtained by incubating a yeast mass or a tissue of an 
angiospermal plant or an aqueous extract thereof with a fatty acid. 
The present invention further provides an agent for inducing flower bud 
formation comprising various agents for inducing flower bud formation 
mentioned above and norepinephrine. 
The present invention further provides a kit for inducing flower bud 
formation comprising an agent for inducing flower bud formation. 
The present invention further provides a method of inducing flower bud 
formation comprising applying said agent for inducing flower bud formation 
to a plant.

MODE FOR CARRYING OUT THE INVENTION 
Fatty Acids Having the Activity of Inducing Flower Bud Formation 
The first embodiment of the present invention of fatty acids having the 
activity of inducing flower bud formation is a fatty acid of 4 to 24 
carbon atoms having an oxo group and a hydroxy group and containing 0 to 6 
double bonds. Said oxo group and said hydroxy group preferably constitute 
an .alpha.-ketol structure or a .gamma.-ketol structure: 
##STR1## 
The number of double bonds is preferably two to five, more preferably two 
or three, and most preferably two. The number of carbon atoms is 
preferably 14 to 22, more preferably 16 to 22, and most preferably 18. The 
representative fatty acids having the .alpha.-ketol structure include 
9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid (sometimes referred to 
herein as Factor-C (FC)) and 12-oxo-13-hydroxy-9(Z),15(Z)-octadecadienoic 
acid: 
##STR2## 
9-hydroxy-10-oxo-12(z),15(Z)-octadecadienonic acid 
##STR3## 
12-oxo-13-hydroxy-9(z),15(Z)-octadecadienoic acid. 
Also, the fatty acids having the .gamma.-ketol structure include 
10-oxo-13-hydroxy-11(E),15(Z)-octadecadienoic acid and 
9-hydroxy-12-oxo-10(E),15(Z)-octadecadienoic acid: 
##STR4## 
10-oxo-13-hydroxy-11(E),15(Z)-octadecadienoic acid 
##STR5## 
9-hydroxy-12-oxo-10(E),15(Z)-octadecadienoic acid. 
The second embodiment of the present invention of the fatty acids having 
the activity of inducing flower bud formation is a fatty acid of 4 to 24 
carbon atoms having an oxo group, a hydroxy group, and a perhydroxy group 
(--O--OH) and containing 0 to 6 double bonds. Said oxo group and said 
hydroxy group constitute an .alpha.-ketol structure or a .gamma.-ketol 
structure and most preferably an .alpha.-ketol structure. The number of 
double bonds is preferably two to five, more preferably two or three, and 
most preferably two. The number of carbon atoms is preferably 14 to 22, 
more preferably 16 to 22, and most preferably 18. The representative fatty 
acids belonging to this embodiment include 
9-hydroperoxy-12-oxo-13-hydroxy-10(E),15(Z)-octadecadienoic acid and 
9-hydroxy-10-oxo-13-hydroperoxy-11(E),15(Z)-octadecadienoic acid: 
##STR6## 
9-hydroperoxy-12-oxo-13-hydroxy-10(E),15(Z)-octadecadienoic acid 
##STR7## 
9-hydroxy-10-oxo-13-hydroperoxy-11(E),15(Z)-octadecadienoic acid. 
The third embodiment of the present invention of the fatty acids having the 
activity of inducing flower bud formation is a fatty acid of 4 to 24 
carbon atoms having a hydroperoxy group and containing 0 to 6 double 
bonds. Said oxo group and said hydroxy group constitute an .alpha.-ketol 
structure or a .gamma.-ketol structure and most preferably an 
.alpha.-ketol structure. The number of double bonds is preferably two to 
five, more preferably two or three, and most preferably three. The number 
of carbon atoms is preferably 14 to 22, more preferably 16 to 22, and most 
preferably 18. The representative fatty acids belonging to this embodiment 
include 9-hydroperoxy-10(E),12(Z),15(Z)-octadecatrienoic acid and 
13-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid: 
##STR8## 
9-hydroperoxy-10(E),12(Z),15(Z)-octadecatrienoic acid 
##STR9## 
13-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid. 
Among the various fatty acids mentioned above, 
9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid, i.e. Factor C, may be 
prepared by the extraction methods from plants, chemical synthetic 
methods, and the enzymatic methods. Other fatty acids may be prepared by 
the chemical synthetic methods or the enzymatic methods. 
The Extraction Methods 
Lemna paucicostata used as a source material in this extraction method is a 
small water plant floating on the surface of a pond or a paddy field of 
which each thallus floating on the water produces one root in the water. 
Its flowers are formed on the side of the thallus in which two male 
flowers comprising only one stamen and a female flower comprising one 
pistil are enveloped in a small common bract. 
This Lemna paucicostata has a relatively fast growth rate (i.e., the rate 
of flower formation is rapid. The Lemna paucicostata 151 strain used for 
checking induction of flower bud formation in the assay system mentioned 
below conducts flower formation within only seven days), and has excellent 
properties as an assay system related to flower bud formation such as the 
ability of controlling induction of flower bud formation, etc. 
The ability of inducing flower bud formation has been at least found in the 
homogenates of this Lemna paucicostata. 
Furthermore, the fraction obtained by removing the supernatant from the 
mixture of the supernatant and the precipitate that was obtained by 
subjecting said homogenate to centrifuge (8000.times.g, ca. 10 minutes) 
may be used as a fraction containing Factor C. 
Thus, Factor C can be isolated and/or purified using the above-mentioned 
homogenate as the starting material. 
As a starting material preferred in terms of preparation efficiency there 
may be mentioned an aqueous solution obtained after floating or immersing 
Lemna paucicostata in the water. The aqueous solution is not specifically 
limited so long as the Lemna paucicostata is viable. 
The specific embodiments of the preparation of this aqueous solution will 
be described in the examples below. 
The immersing time may be, but is not limited to, two to three hours at 
room temperature. 
In preparing the starting material for Factor C by the method mentioned 
above, it is preferred to subject Lemna paucicostata to a specific stress 
in advance which enables induction of flower bud formation for better 
efficiency of Factor C production. 
Specific examples include dry stress, heat stress, osmotic stress, etc. as 
the above-mentioned stress. 
The dry stress may be imposed, for example, at a low humidity (preferably 
at a relative humidity of 50% or lower) at room temperature, preferably at 
24 to 25.degree. C. by leaving the Lemna paucicostata spread out on a dry 
filter paper. The drying time in this case is longer than about 20 
seconds, preferably 5 minutes or more, and more preferably 15 minutes or 
more. 
The heat stress may be imposed, for example by immersing Lemna paucicostata 
in a hot water. The temperature of the hot water in this case can be 
40.degree. C. to 65.degree. C., preferably 45.degree. C. to 60.degree. C., 
and more preferably 50.degree. C. to 55.degree. C. As the time required 
for treating in the hot water, about five minutes is sufficient, but at a 
relatively low temperature, for example the treatment of Lemna 
paucicostata in a hot water of about 40.degree. C., treatment for more 
than two hours is preferred. Furthermore, after said heat stress 
treatment, Lemna paucicostata is preferably returned to cold water as 
quickly as possible. 
The osmotic stress may be imposed, for example, by exposing Lemna 
paucicostata to a solution of high osmotic pressure such as a solution of 
a high sugar concentration and the like. The sugar concentration in this 
case is 0.3M or higher for mannitol, for example, and preferably 0.5M or 
higher. The treatment time is one minute or longer in the case of a 
solution of 0.5M mannitol, and preferably three minutes or longer. 
Thus, the starting material containing the desired Factor C may be 
obtained. 
The strains of Lemna paucicostata that constitute a basis for the various 
starting materials mentioned above are preferably, but not limited to, the 
strains that especially efficiently produce an inducer of flower bud 
formation (for example, Lemna paucicostata strain 441). Such a strain of 
Lemna paucicostata can be obtained by the conventional selection methods 
or by the gene engineering methods. 
Subsequently, the starting material thus prepared may be subjected to the 
following isolation and/of purification methods to produce the desired 
Factor C. 
It is to be understood that the separation methods as described herein are 
only illustrative and that these separation methods do not limit in any 
way the separation methods of Factor C from the above-mentioned starting 
materials. 
First the above-mentioned starting material is subjected to solvent 
extraction to extract a Factor C-containing component. The solvents used 
in such solvent extraction methods include, but not limited to, 
chloroform, ethyl acetate, ether, butanol, and the like. Among these 
solvents chloroform is preferred because it can remove impurities 
relatively easily. 
By washing and/or concentrating the oil layer fractions obtained by this 
solvent extraction by a commonly known method and then by subjecting to 
high performance liquid chromatography using a column for the 
reverse-phase partition column chromatography such as an ODS (octadodecyl 
silane) column etc. to isolate and/of purify the fractions having the 
ability of inducing flower bud formation, Factor C can be isolated. 
In addition, it is also possible to use combinations of other commonly 
known methods for separation such as ultra-filtration, gel filtration 
chromatography and the like. 
Chemical Synthetic Methods 
Next, chemical synthetic methods of fatty acids having the effect of 
inducing flower bud formation of the present invention will be explained. 
Factor C (i.e., 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid) can be 
synthesized according to the following scheme (A) (Method 1). 
Nonanedioic acid mono ethylester (I) used as the starting material is 
reacted with N,N'-carbonyldiimidazole to make an acid imidazolide, which 
is then reduced with LiAlH.sub.4 at a low temperature to convert to the 
aldehyde (3). On the other hand, cis-2-hexen-1-ol (4) is reacted with 
triphenyl phosphine and carbon tetrabromide. The thus obtained (5) is 
reacted with triphenyl phosphine and then reacted in the presence of 
n-BuLi with chloroacetaldehyde to construct a cis olefin which is 
converted to (7). Then, after reaction with methylthio methyl p-tolyl 
sulfone, it is reacted in the presence of NaH with the previously derived 
aldehyde (3). The derived secondary alcohol (9) is protected with 
tert-butyldiphenylsilylchloride, acid-hydrolyzed, and deprotected to 
convert to Factor C (9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid) 
(12). 
##STR10## 
Furthermore, as the second method for chemically synthesizing Factor C, 
there is mentioned a method in which 1,9-nonanediol (1') is used in place 
of nonanedionic acid mono ester (1) as the starting material, which is 
oxidized with MnO.sub.2 to give a dialdehyde (2') which is further 
oxidized with KMnO.sub.4 to give a monoaldehyde monocarboxylic acid (3') 
and then esterified to form an intermediate compound (3) in the above 
scheme (1). The subsequent reactions can be carried out according to the 
scheme (A) in the above method (1). The reaction route from 1,9-nonanediol 
(1') to the intermediate (3) is shown in scheme (B) 
##STR11## 
Also, 12-oxo-13-hydroxy-9(Z),15(Z)-octadecadienoic acid can be synthesized 
according to, for example, the synthetic scheme (C). That is, nonanedioic 
acid mono ethyl ester (1) as the starting material is reacted with thionyl 
chloride to give an acid chloride (2), which is then reduced with 
NaBH.sub.4 to give an acid alcohol (3). Then, after the free carboxylic 
acid is protected, it is reacted with triphenyl phosphine and carbon 
tetrabromide and the thus obtained (5) is reacted with triphenyl phosphine 
and then further reacted in the presence of n-BuLi to give 
chloroacetaldehyde to construct a cis olefin which is converted to (6). 
Then, after reaction with methylthio methyl p-tolyl sulfone, it is reacted 
in the presence of nBuLi with the aldehyde (3) which was separately 
derived from the PCC oxidation of cis-2-hexen-1-ol (8), and finally 
deprotected to give 12-oxo-13-hydroxy-9(Z),15(Z)-octadecadienoic acid 
1(10). 
Furthermore, 10-oxo-13-hydroxy-11(E),15(Z)-octadecadienoic acid can be 
synthesized according to, for example, the synthetic scheme (D). That is, 
methyl vinyl ketone (1) used as the starting material is reacted with 
trimethylsilyl chloride in the presence of LDA and DME, and to the silyl 
ether (2) thus obtained MCPBA and trimethylamine hydrofluoric acid are 
added at a low temperature (-70.degree. C.) to give a ketoalcohol (3). 
Then, after the carbonyl group is protected, triphenyl phosphine and 
trichloroacetone are used as the reaction reagents to give (5) without 
adding a chloride to the olefin. Then in the presence of tributylarsine 
and K.sub.2 CO.sub.3, formic acid is reacted to construct a trans olefin 
to give a chloride (7). Then, (7) and the aldehyde (8) obtained by the PCC 
oxidation of cis-2-hexen-1-ol are reacted to give (9). Furthermore, a 
binding reaction of (9) and 6-heptenoic acid (10) is conducted and finally 
deprotected to give 10-oxo-13-hydroxy-11(E),15(Z)-octadecadienoic acid 
(11). 
##STR12## 
The Enzymatic Method 
Next, the methods of enzymatic synthesis are explained. For example, Factor 
C of the present invention may be synthesized by the enzymatic method in 
the following manner. As the starting material for the enzymatic synthesis 
of Factor C, .alpha.-linolenic acid may be used. .alpha.-linolenic acid is 
an unsaturated fatty acid contained in plants and the like in a relative 
abundance. An .alpha.-linolenic acid that was isolated and/or purified 
using the commonly known methods from these animals and plants etc. may be 
used as the starting material for production of Factor C, or it is also 
possible to use commercial products. 
In this enzymatic method, .alpha.-linolenic acid as the substrate is 
brought to the action of lipoxygenase (LOX) to introduce a hydroperoxy 
group (--OOH) at position 9. Lipoxygenase is an oxidoreductase that 
introduces molecular oxygen as a hydroperoxy group into an unsaturated 
fatty acid having the cis,cis-1,4-pentadiene structure. Its presence in 
living organisms has been confirmed in animals and plants. 
In plants, for example, its presence has been recognized in soybeans, seeds 
of flaxes, alfalfa, barley, broad beans, lupines, lentils, field peas, 
rhizomes of potatoes, wheat, apples, baker's yeast, cotton, roots of 
cucumbers, gooseberries, grapes, pears, beans, rice bran, strawberries, 
sunflowers, tea leaves and the like. 
Lipoxygenase as used herein may be of any origin so long as it can 
introduce a hydroxyperoxy group into position 9 of .alpha.-linolenic acid. 
In conducting the above-mentioned lipoxygenase treatment using 
.alpha.-linolenic acid as the substrate it is of course preferred to let 
the enzymatic reaction proceed at an optimum temperature and an optimum pH 
of the lipoxygenase used. The lipoxygenase as used herein may be one that 
was extracted and/or purified from an above-mentioned plant and the like 
in a commonly known method, or it is possible to use a commercial product. 
In this manner, 9-hydroperoxy linolenic acid 
(9-hydroperoxy-cis-12,15-octadecadienoic acid) is prepared from 
.alpha.-linolenic acid. 
Subsequently, the 9-hydroperoxy linolenoic acid used as the substrate is 
brought to the action of hydroperoxy isomerase to prepare the desired 
Factor C. Hydroperoxy isomerase is an enzyme having the activity of 
converting a hydroperoxy group to a ketol body via epoxidization. It has 
been found in, for example, plants such as barley, wheat, corn, cotton, 
egg plants, seeds of flaxes, lettuce, oats, spinach, sunflowers, and the 
like. 
Hydroperoxy isomerase as used herein is not specifically limited so long as 
it can form an epoxy group by dehydrating a hydroperoxy group at position 
9 of 9-hydroperoxy linolenoic acid and it can thereby give the desired 
Factor C by a nucleophilic reaction of OH.sup.-. 
In conducting the above-mentioned hydroperoxide isomerase treatment using 
9-hydroperoxy linolenic acid as the substrate it is of course preferred to 
let the enzymatic reaction proceed at an optimum temperature and an 
optimum pH of the hydroperoxide isomerase used. 
The hydroperoxide isomerase as used herein may be one that was extracted 
and/or purified from a plant mentioned above in a known method, or it is 
also possible to used a commercial product. 
The above two-step reaction may be conducted in either a discreet manner or 
a continuous manner. Furthermore, it is possible to obtain Factor C by 
using the crude purified or purified product of the above-mentioned enzyme 
to proceed the above-mentioned enzymatic reaction. It is also possible to 
obtain Factor C by immobilizing the above-mentioned enzyme on a carrier to 
prepare these immobilized enzymes and then subjecting the substrate to a 
column treatment or a batch treatment. 
It is known that in obtaining Factor C by a nucleophilic reaction 
(mentioned above) of OH.sup.- after an epoxy group was formed, a 
.gamma.-ketol compound is formed as a byproduct in addition to an 
.alpha.-ketol unsaturated fatty acid depending on the manner of reaction 
in the neighborhood of the above epoxy group. 
The byproducts such as a .gamma.-ketol compound and the like can be readily 
removed by a commonly known separation method such as HPLC and the like. 
A synthetic route for synthesis of Factor C by the above-mentioned 
enzymatic method is described as scheme (E). 
##STR13## 
The preparation of Factor C by the enzymatic methods were explained in 
detail as above. Lipoxygenases or allene oxide synthase that convert a 
double bond in a fatty acid to an .alpha.-ketol structure occur widely in 
the yeast and angiosperms. Thus, according to the present invention the 
agent for inducing flower bud formation of the present invention can also 
be obtained by incubating a yeast mass, a vegetative body of an 
angiospermal plant, or a product containing the enzyme such as the 
homogenate, aqueous extract thereof, etc. with a fatty acid containing a 
double bond in a medium that is permissive for the enzymatic reaction such 
as an aqueous medium. 
As the yeast used in this case, for example, a yeast belonging to the genus 
Saccharomyces such as Saccharomyces cereviceae may be used. 
Also, as the angiosperms, as plants, for example, belonging to the subclass 
Archichlamydeae of the class Dictyledoneae, there are mentioned: 
the family Casuarinaceae of the order Verticillatae; the families 
Saururaceae, Piperaceae, and Chloranthaceae of the order Piperales; 
the family Salicaceae of the order Salicales; 
the family Myricaceae of the order Myricales; 
the family Juglandaceae of the order Juglandales; 
the families Betulaceae and Fagaceae of the order Fagales; 
the families Ulmaceae, Moraceae, and Urticaceae of the order Urticales; 
the family Podostemaceae of the order Podostemonales; 
the family Proteaceae of the order Preteales; 
the families Olacaceae, Santalaceae, and Loranthaceae of the order 
Santalales; 
the families Aristolochiaceae and Rafflesiaceae of the order 
Aristolochiales; 
the family Balanophoraceae of the order Balanophorales; 
the family Polygonaceae of the order Polygonales; 
the families Chenopodiaceae, Amaranthaceae, Nyctaginaceae, Cynocrambaceae, 
Phytolaccaceae, Aizoaceae, Portulacaceae, Basellaceae, and Caryophyllaceae 
of the order Centrospermae; 
the families Magnoliaceae, Trochodendraceae, Cercidiphyllaceae, 
Nymphaeaceae, Ceratophyllaceae, Ranunculaceae, Lardizabalaceae, 
Berberidaceae, Menispermaceae, Calycanthaceae, Myristicaceae, and 
Lauraceae of the order Ranales; 
the families Papaveraceae, Capparidaceae, Cruciferae, and Resedaceae of the 
order Rhoeadales; 
the families Droseraceae and Nepenthaceae of the order Sarraceniales; 
the families Crassulaceae, Saxifragaceae, Pittosporaceae, Hamamelidaceae, 
Platanaceae, Rosaceae, and Leguminosae of the order Rosales; 
the families Oxalidaceae, Geraniaceae, Tropaeolaceae, Linaceae, 
Erythroxylaceae, Zygophyllaceae, Rutaceae, Simaroubaceae, Bruseraceae, 
Meliaceae, Polygalaceae, Euphorbiaceae, and Callitrichaceae of the order 
Geraniales; 
the families Buxaceae, Empetraceae, Coriariaceae, Anacardiaceae, 
Aquifoliaceae, Celastraceae, Staphyleaceae, Icacinaceae, Aceraceae, 
Hippocastanaceae, Sapindaceae, Sabiaceae, and Balsaminaceae of the order 
Sapindales; 
the families Rhamnaceae and Vitaceae of the order Rhamnales; 
the families Elaeocarpaceae, Tiliaceae, Malvaceae, and Sterculiaceae of the 
order Malvales; 
the families Actinidiaceae, Theaceae, Guttiferae, Elatinaceae, 
Tamaricaceae, Violaceae, Flacourtiaceae, Stachyuraceae, Passifloraceae, 
and Begoniaceae of the order Parietales; 
the family Cactaceae of the order Opuntiales; 
the families Thymelaeaceae, Elaegnaceae, Lythraceae, Punicaceae, 
Rhizophoraceae, Alangiaceae, Combretaceae, Myrtaceae, Melastomataceae, 
Hydrocaryaceae, Oenotheraceae, Haloragaceae, and Hippuridaceae of the 
order Myrtiflorae; and 
the families Araliaceae, Umbelliferae, and Cornaceae of the order 
Umbellifloraea. 
Also, as plants belonging to the subclass Symperalea of the class 
Dictyledoneae, there are mentioned: 
the family Diapensiaceae of the order Diapensiales; 
the families Clethraceae, Pyrolaceae, and Ericaceae of the order Ericales; 
the families Myrsinaceae and Primulaceae of the order Primulales; 
the family Plumbaginaceae of the order Plumbaginales; 
the families Ebenaceae, Symplocaceae, and Styracaceae of the order 
Ebenales; 
the families Oleaceae, Loganiaceae, Gentianaceae, Apocynaceae, and 
Asclepiadaceae of the order Contoratae; 
the families Convolvulaceae, Polemoniaceae, Boraginaceae, Verbenaceae, 
Labiatae, Solanaceae, Scrophulariaceae, Bignoniaceae, Pedaliaceae, 
Martyniaceae, Orobanchaceae, Gesneriaceae, Lentibulariaceae, Acanthaceae, 
Myoporaceae, and Phrymaceae of the order Tubiflorae; 
the family Plantaginaceae of the order Plantaginales; 
the families Rubiaceae, Caprifoliaceae, Adoxaceae, Valerianaceae, and 
Dipsacaceae of the order Rubiales; 
the family Cucurbitaceae of the order Cucurbitales; and 
the families Campanulaceae and Compositae of the order Campanulatae. 
Furthermore, as plants belonging to the class Monocotyledoneae, there are 
mentioned: 
the families Typhaceae, Pandanaceae, and Sparganiaceae of the order 
Pandanales; 
the families Potamogetonaceae, Najadaceae, Scheuchzeriaceae, Alismataceae, 
and Hydrocharitaceae of the order Helobiae; 
the family Triuridaceae of the order Triuridales; 
the families Gramineae and Cyperaceae of the order Glumiflorae; 
the family Palmae of the order Plamales; 
the families Araceae and Lemnaceae of the order Arales; 
the families Eriocaulaceae, Bromeliaceae, Commelinaceae, Pontederiaceae, 
and Philydraceae of the order Commelinales; 
the families Juncaceae, Stemonaceae, Liliaceae, Amaryllidaceae, 
Dioscoreaceae, and Iridaceae of the order Liliiflorae; 
the families Musaceae, Zingiberaceae and Cannaceae of the order 
Scitamineae; and 
the families Burmanniaceae and Orchidaceae of the order Orchidales. 
These plants are used in the form of a vegetative body, seeds, and the 
treated products thereof that were treated in various ways without 
inactivating enzymes, such as a dried product, a homogenate, an aqueous 
extract, a pressed juice, and the like. Specifically, since chlorophill is 
known as an inhibitor of lipoxygenase, it is preferred to use the part 
(seeds) containing no chlorophyll such as wheat, rice, barley, soybeans, 
corn, beans, and the like. 
Furthermore, as in the case of the enzymatic production of Factor C, the 
enzyme products of lipoxygenase and hydroperoxide isomerases can be used 
in addition of the above-mentioned plants and the treated products 
thereof. 
Lipoxygenase introduces a hydroperoxy group using as the substrate a highly 
unsaturated fatty acid having a cis, cis-1,4-pentadiene structure in the 
following reaction. 
##STR14## 
Thus, any fatty acids having the above structure in their carbon chain may 
be used as a fatty acid for the enzymatic method of the present invention. 
Such fatty acids include, for example, cis-9,12-octadecadienoic acid 
(linolenic acid; C18:2, cis-9,12), trans-9,12-octadecadienoic acid 
(linolelaidic acid; C18:2, trans-9,12), 9,11-(10,12)-octadecadienoic acid 
(C18:2, .DELTA.9,11(10,12)), cis-6,9,12-octadecatrienoic acid 
(.gamma.-linolenic acid; C18:3, cis-6,9,12), cis-9,12,15-octadecadienoic 
acid (linolenic acid; C18:3, cis-9,12,15), trans-9,12,15-octadecatrienoic 
acid (linolenelaidic acid; C18:3, trans 9,12,15), 
cis-6,9,12,15-octadecatrienoic acid (C18:4, cis-6,9,12,15), 
cis-11,14-eicosadienoic acid (C20:2, cis-11,14), cis-5,8,11-eicosatrienoic 
acid (C20:3, cis-5,8,11), 5,8,11-eicosatrienoic acid (C20:3, 
5,8,11-ynoic), cis-8,11,14-eicosatrienoic acid (C20:3, cis-8,11,14), 
8,11,14-eicosatrienoic acid (C20:3, 8,11,14-ynoic), 
cis-11,14,17-eicosatrienoic acid (C20:3, cis-11,14,17), 
cis-5,8,11,14-eicosatetraenoic acid (arachidonic acid; C20:4, 
cis-5,8,11,14), cis-5,8,11,17-eicosapentaenoic acid (C20:5, 
cis-5,8,11,14), cis-13,16-docosadienoic acid (C22:2, cis-13,16), 
cis-13,16,19-docosatrienoic acid (C22:3, cis-13,16,19), 
cis-7,10,13,16-doxosatetraenoic acid (C22:4, cis-7,10,13,16), 
cis-7,10,13,16,19-doxosapentaenoic acid (C22:5, cis-7,10,13,16,19), 
cis-4,7,10,13,16,19-docosahexaenoic acid (C22:6, cis-4,7,10,13,16,19) and 
the like. 
Fatty acids can be selected depending on the kind of the fatty acid to be 
produced. For example, in order to obtain 
9-hydroperoxy-10(E),12(Z),15(Z)-octadecatrienoic acid and 
13-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid belonging to the 
third embodiment of the present invention by the enzymatic method, it is 
only required to bring linolenic acid to the action of lipoxygenase. When 
hydroperoxide isomerase is further used, as described above, Factor C can 
be obtained. 
By further bringing Factor C to the action of lipoxygenase, 
9-hydroxy-10-oxo-13-hydroperoxy-11(E),15(Z)-octadecadienoic acid can be 
obtained. By further bringing 13-hydroxy-12-oxo-9(Z),15(Z)-octadecadienoic 
acid to the action of lipoxygenase, 
9-hydroperoxy-12-oxo-13-hydroxy-10(E),15(Z)-octadecadienoic acid can be 
obtained. 
Incubation of an enzyme, a yeast mass or a vegetative body or the treated 
products thereof with a fatty acid is conducted as described above for the 
enzymatic production of Factor C. 
Also, Factor C can be obtained by the chemical synthetic reactions in 
addition to the above-mentioned extraction methods and the enzymatic 
methods. 
On the other hand, norepinephrine that exhibits the desired effect of 
inducing flower bud formation in combination with an unsaturated fatty 
acid of the present invention may be the one that was synthesized by a 
commonly known method, or it is of course possible to use a commercial 
product. 
According to the present invention, the (+) type norepinephrine in addition 
to the naturally occurring (-) type norepinephrine or mixtures thereof may 
be used. 
The agents for inducing flower bud formation of the present invention 
(hereinafter referred to as the invention agents for inducing flower bud 
formation) thus produced having as active ingredients an unsaturated fatty 
acid or an unsaturated fatty acid and norepinephrine are provided. 
Among the invention agents for inducing flower bud formation, those having 
an unsaturated fatty acid as the sole active ingredient are intended to 
exhibit the desired effect of inducing flower bud formation in combination 
with norepinephrine potentially present in plants, or to exhibit the 
desired effect of inducing flower bud formation by combining this form of 
the invention agent for inducing flower bud formation with a 
norepinephrine agent depending on the kind and state of the plants. 
Furthermore, among the invention agents for inducing flower bud formation, 
those forms having an unsaturated fatty acid and norepinephrine as the 
active ingredients can be conveniently used by blending the above active 
ingredients in such a ratio that exhibits the most intense effect of 
inducing flower bud formation of the invention agents for inducing flower 
bud formation. 
The ratio of blending an unsaturated fatty acid and norepinephrine in the 
invention agent for inducing flower bud formation can be adjusted as 
appropriate depending on, but not limited to, the above-mentioned purpose 
and furthermore the property of the plant used. When the presence of 
norepinephrine is not taken into consideration, as, For example, in the 
plants of the family Lemnaceae such as Lemna paucicostata, the equimolar 
blending of the two (an unsaturated fatty acid and norepinephrine) is 
preferred in that it can exhibit more effectively the desired effect of 
the present invention. When the two are not blended in equimolar amounts 
for Lemna plants, the effect exhibited tends to be almost equal to the 
effect that would be exhibited when the two are blended at a concentration 
of the ingredient contained in a smaller amount. 
The invention agents for inducing flower bud formation are in most cases 
more effective when administered while treating the subject plants 
depending on the property of the plants. For example, in the ratio of 
short-day plants such as Pharbitis nil, strain Violet described below in 
Examples etc., it is more effective to conduct a certain dark treatment 
prior to using the invention agents for inducing flower bud formation. 
The above active ingredients may be used as they are as the invention 
agents for inducing flower bud formation, but they may be blended as 
appropriate depending on the desired dosage form applicable to plants, for 
example a pharmaceutically applicable known carrier ingredient such as a 
liquid, a solid, a powder, an emulsion, a low-floor additive, etc. and an 
adjuvant etc. in the range that does not hinder the desired effect of 
inducing flower bud formation. For example, as the carrier ingredients 
when the invention agent for inducing flower bud formation is a low-floor 
additive or a solid, mostly inorganic materials such as talc, clay, 
vermiculite, diatomaceous earth, kaolin, calcium carbonate, calcium 
hydroxide, white clay, silica gel etc., and solid carriers such as wheat 
flour, starch, etc.: or when it is a liquid, mostly water, aromatic 
hydrocarbons such as xylene etc., alcohols such as ethanol, ethylene 
glycol, etc., ketones such as acetone, ethers such as dioxane, 
tetrahydrofuran, etc., dimethylformamide, dimethyl sulfoxide, 
acetonitrile, etc. are used as the above-mentioned carrier ingredient. As 
pharmaceutical adjuvants, for example, anionic surfactants such as alkyl 
sulfate esters, alkyl sulfonates, alkylaryl sulfonates, dialkylsulfo 
succinates, etc., cationic surfactants such as higher aliphatic amines 
etc., nonionic surfactants such as polyoxyethylene glycol alkyl ether, 
polyoxyethylene glycol acyl ester, polyoxyethylene glycol polyhydric 
alcohol acyl ester, cellulose derivatives, etc., thickeners such as 
gelatin, casein, gum Arabic, etc., bulking agents, binders, etc. can be 
blended as appropriate. 
Furthermore, as needed, plant growth-control agents such as benzoic acid, 
nicotinic acid, nicotinamide, pipecolic acid, or the like can be blended 
into the invention agents for inducing flower bud formation so long as it 
does not affect the above-mentioned desired effect of the present 
invention. 
The invention agents for inducing flower bud formation mentioned above may 
be used for a variety of plants in a manner suitable for the dosage form. 
According to the present invention, for example, it is possible to effect 
spraying, dropping, applying, and so on of the inducers as a liquid or an 
emulsion onto the meristem, the surface and/or the back surface of leaves 
of the plant to be flowered, the entire plant and the like, or to effect 
absorption from the soil by the root as a solid or a powder. When the 
plant to be flowered is a water plant such as Lemna paucicostata, it is 
also possible to effect absorption from the root as a low-floor additive 
or gradual dissolution of the solid in the water. 
According to the present invention, a kit for inducing flower bud formation 
that takes a form of a kit containing Factor C which is an active 
ingredient mentioned above, an unsaturated fatty acid, and norepinephrine 
is also provided. The purpose and effects of said kit for inducing flower 
bud formation are the same as those mentioned above for the invention 
agents for inducing flower bud formation. 
The types of plants to which the invention agents for inducing flower bud 
formation or the kit for inducing flower bud formation can be applied are 
not specifically limited and the invention agents for inducing flower bud 
formation are effective for both dicotyledons and monocotyledons. 
As the dicotyledons, there are mentioned the plants of, for example, the 
family Convolvulaceae including the genus Pharbitis (Pharbitis nil, strain 
Violet), the genus Calystegia (C. japonica, C. hederacea, C. soldanella), 
genus Ipomoea (I. pes-caprae, I. batatas), and the genus Cuscuta (C. 
japonica, C. australis), the family Casuarinaceae, the family Saururaceae, 
the family Piperaceae, the family Chloranthaceae, the family Salicaceae, 
the family Myricaceae, the family Juglandaceae, the family Betulaceae, the 
family Fagaceae, the family Ulmaceae, the family Moraceae, the family 
Urticaceae, the family Podostemaceae, the family Proteaceae, the family 
Olacaceae, the family Santalaceae, the family Loranthaceae, the family 
Aristolochiaceae, the family Rafflesiaceae, the family Balanophoraceae, 
the family Polygonaceae, the family Chenopodiaceae, the family 
Amaranthaceae, the family Nyctaginaceae, the family Cynocrambaceae, the 
family Phytolaccaceae, the family Aizoaceae, the family Portulacaceae, the 
family Basellaceae, the family Caryophyllaceae, the family Magnoliaceae, 
the family Trochodendraceae, the family Cercidiphyllaceae, the family 
Nymphaeaceae, the family Ceratophyllaceae, the family Ranunculaceae, the 
family Lardizabalaceae, the family Berberidaceae, the family 
Menispermaceae, the family Calycanthaceae, the family Lauraceae, the 
family Papaveraceae, the family Capparidaceae, the family Cruciferae, the 
family Droseraceae, the family Nepenthaceae, the family Crassulaceae, the 
family Saxifragaceae, the family Pittosporaceae, the family 
Hamamelidaceae, the family Platanaceae, the family Rosaceae, the family 
Leguminosae, the family Oxalidaceae, the family Geraniaceae, the family 
Linaceae, the family Zygophyllaceae, the family Rutaceae, the family 
Simaroubaceae, the family Bruseraceae, the family Meliaceae, the family 
Polygalaceae, the family Euphorbiaceae, the family Callitrichaceae, the 
family Buxaceae, the family Empetraceae, the family Coriariaceae, the 
family Anacardiaceae, the family Aquifoliaceae, the family Celastraceae, 
the family Staphyleaceae, the family Icacinaceae, the family Aceraceae, 
the family Hippocastanaceae, the family Sapindaceae, the family Sabiaceae, 
the family Balsaminaceae, the family Rhamnaceae, the family Vitaceae, the 
family Elaeocarpaceae, the family Tiliaceae, the family Malvaceae, the 
family Sterculiaceae, the family Actinidiaceae, the family Theaceae, the 
family Guttiferae, the family Elatinaceae, the family Tamaricaceae, the 
family Violaceae, the family Flacourtiaceae, the family Stachyuraceae, the 
family Passifloraceae, the family Begoniaceae, the family Cactaceae, the 
family Thymelaeaceae, the family Elaegnaceae, the family Lythraceae, the 
family Punicaceae, the family Rhizophoraceae, the family Alangiaceae, the 
family Melastomataceae, the family Hydrocaryaceae, the family 
Oenotheraceae, the family Haloragaceae, the family Hippuridaceae, the 
family Araliaceae, the family Umbelliferae, the family Cornaceae, the 
family Diapensiaceae, the family Clethraceae, the family Pyrolaceae, the 
family Ericaceae, the family Myrsinaceae, the family Primulaceae, the 
family Plumbaginaceae, the family Ebenaceae, the family Symplocaceae, the 
family Styracaceae, the family Oleaceae, the family Loganiaceae, the 
family Gentianaceae, the family Apocynaceae, the family Asclepiadaceae, 
the family Polemoniaceae, the family Boraginaceae, the family Verbenaceae, 
the family Labiatae, the family Solanaceae, the family Scrophulariaceae, 
the family Bignoniaceae, the family Pedaliaceae, the family Orobanchaceae, 
the family Gesneriaceae, the family Lentibulariaceae, the family 
Acanthaceae, the family Myoporaceae, the family Phrymaceae, the family 
Plantaginaceae, the family Rubiaceae, the family Caprifoliaceae, the 
family Adoxaceae, the family Valerianaceae, the family Dipsacaceae, the 
family Cucurbitaceae, the family Campanulaceae, the family Compositae, and 
the like. 
Furthermore, as monocotyledons there are mentioned the plants of the family 
Lemnaceae including the genus Spirodela (S. polyrhiza) and the genus Lemna 
(L. paucicostata, L. trisulca), the family Typhaceae, the family 
Sparganiaceae, the family Potamogetonaceae, the family Najadaceae, the 
family Scheuchzeriaceae, the family Alismataceae, the family 
Hydrocharitaceae, the family Triuridaceae, the family Gramineae, the 
family Cyperaceae, the family Palmae, the family Araceae, the family 
Eriocaulaceae, the family Commelinaceae, the family Pontederiaceae, the 
family Juncaceae, the family Stemonaceae, the family Liliaceae, the family 
Amaryllidaceae, the family Dioscoreaceae, the family Iridaceae, the family 
Musaceae, the family Zingiberaceae, the family Cannaceae, the family 
Burmanniaceae, the family Orchidaceae, and the like. 
EXAMPLES 
The present invention will now be explained more specifically with the 
following examples. It should be understood, however, that these examples 
do not limit the technical scope of the present invention in any way. 
Example 1 
Preparation of Factor C by the Extraction Method 
Lemna paucicosta strain 441 (hereinafter referred to as "P441"; this strain 
was obtained from Atushi Takimoto, professor emeritus of Kyoto University, 
Faculty of Agriculture, who is one of the inventors of the present 
invention; the strain is available on request) was aseptically subcultured 
in the 1/2 diluted Hutner's medium [Hutner 1953; the composition of the 
undiluted Hutner's medium is KH.sub.2 PO.sub.4 (400 mg), NH.sub.4 NO.sub.3 
(200 mg), EDTA.2K (690 mg), Ca(NO.sub.3).sub.2.4H.sub.2 O (354 mg), 
MgSO.sub.4.7H.sub.2 O (500 mg), FeSO.sub.4.7H.sub.2 O (24.9 mg), 
MnCl.sub.2.4H.sub.2 O (17.9 mg), ZnSO.sub.4.7H.sub.2 O (65.9 mg), 
CaSO.sub.4.5H.sub.2 O (3.95 mg), Na.sub.2 MoO.sub.4.2H.sub.2 O (14.2 mg), 
H.sub.3 BO.sub.3 (14.2 mg), Co(Mo.sub.3).sub.2.6H.sub.2 O (0.2 mg)/1000 ml 
distilled water, pH 6.2 to 6.4] containing 1% sucrose under continuous 
illumination by a daylight fluorescent lamp (illuminated to the plant at a 
rate of about 5 W/m.sup.2 using Hitachi FL20 SSD) at 24 to 25.degree. C. 
Then after washing the culture of P441 in distilled water it was 
transferred to 1/10 diluted E medium [Cleland and Briggs 1967; the 
composition of the 1/10 E medium is Ca(NO.sub.3).sub.2.4H.sub.2 O (118 
mg), MgSO.sub.4.7H.sub.2 O (40.2 mg), KH.sub.2 PO.sub.4 (68.0 mg), 
KNO.sub.3 (115 mg), FeCl.sub.3.6H.sub.2 O (0.54 mg), tertarate (0.30 mg), 
H.sub.3 BO.sub.3 (0.29 mg), ZnSO.sub.4.7H.sub.2 O (0.022 mg), Na.sub.2 
MoO.sub.4.2H.sub.2 O (0.013 mg), CuSO.sub.4.5H.sub.2 O (0.008 mg), 
MnCl.sub.2.4H.sub.2 O (0.36 mg), EDTA-2K (1.21 mg), EDTA.NaFe(III) salt 
(0.77 mg)/1000 ml distilled water], and was aseptically cultured under 
continuous illumination by a daylight fluorescent lamp (about 5 W/m.sup.2) 
at 24 to 25.degree. C. for 6 to 12 days. 
The thus prepared P441 culture was subjected to a drought stress by leaving 
it spread out on a dry filter paper at a low humidity (a relative humidity 
of 50% or less) at about 24 to 25.degree. C. for 15 minutes. 
The drought stress-treated P441 (75 g) was immersed in 1.5 liter of 
distilled water at 24 to 25.degree. C. for 2 hours. 
The P441 was then removed from the above immersing solution and 1.5 liter 
of chloroform was added in three portions to said immersing solution to 
extract. The chloroform layer obtained was washed with water, and then 0.1 
ml of acetic acid was added thereto followed by evaporation to dryness. To 
the residue 500 .mu.l of methanol was added to dissolve the residue. 
Subsequently the above methanol solution was subjected to high performance 
liquid chromatography [column: ODS (octadecylsilane) column (.PHI. 
10.times.250 mm, CAPCELLPAK C18: manufactured by Shiseido Co., Ltd.); 
solvent: 50% distilled water containing 0.1% trifluoroacetic acid and 50% 
acetonitrile containing 0.085% trifluoroacetic acid as the mobile phase at 
a flow rate of 4.00 ml/min to collect the active fractions (the activity 
was measured in the method similar to the one in the test example 
described below] and fractions at an elution time of about 15 minutes were 
collected. 
To the active fractions thus collected, ethyl acetate was added and the 
ethyl acetate layer was separated and then was washed with water, followed 
by evaporation of this ethyl acetate to dryness to obtain about 1 mg of 
the desired purified product as a dry solid. 
In order to determine the structure of the dry solid the chemical shifts 
thereof were measured using .sup.13 C-NMR (the above dry solid was 
dissolved in a mixture of one drop each of methyl alcohol-d4 and acetic 
acid-d4 to make a sample for measurement). 
As a result, the chemical shifts and the chemical structural formula 
characterized by the chemical shifts were determined as follows: 
1: 178.47(s), 2: 35.71(t), 3: 26.82*(t), 4: 31.11(t), 5: 26.92*(t), 6: 
35.36(t), 7: 78.61(d), 8: 213.78(s), 9: 38.38(t), 10: 122.95(d), 11: 
133.45(d), 12: 27.46(t), 13: 128.38(d), 14: 134.55(d), 15: 22.28(t), 16: 
15.39(q) (See FIG. 1 for the chart; each number at the head of the 
chemical shift corresponds the circled number representing each carbon 
atom in the chemical structural formula below). 
means that its assignment has not been known. 
##STR15## 
The result evidently revealed that the dry solid is the desired purified 
.alpha.-ketol unsaturated fatty acid (Factor C). 
Example 2 
The Effect of Factor C of Inducing Flower Bud Formation in Lemna 
paucicostata 
The effect of Factor C obtained in the preparation example above of 
inducing flower bud formation was evaluated using the P151 strain of Lemna 
paucicostata (hereinafter referred to as "P151"; this strain was obtained 
from Atushi Takimoto, professor emeritus of Kyoto University Faculty of 
Agriculture, who is one of the inventors of the present invention; the 
strain is available on request) as a model plant in terms of its rate of 
flower formation (%) (the number of thalluses for which flower formation 
was observed/the total number of thalluses.times.100). 
Thus, 0.155 mg of the above-mentioned Factor C was dissolved in 0.15 ml of 
water, to which were added 50 .mu.l of 10 mM norepinephrine and 25 .mu.l 
of 0.5M tris buffer, pH 8.0. The solution was incubated at 25.degree. C. 
for six hours. 
Then in order to obtain Factor C and norepinephrine in the concentrations 
shown in Table 1, the solution incubated under the condition mentioned 
above was added to 10 ml of the assay medium (1/10 E medium+1 .mu.m 
benzyladenine, but with no addition of sugar) in a 30 ml flask. The 
results are shown in Table 2. 
TABLE 1 
______________________________________ 
NEeq FCeq NEeq FCeq 
______________________________________ 
A 0.3 .mu.M 7.8 nM D 0.3 .mu.M 
2.9 nM 
A 3 .mu.M 78 nM D 3 .mu.M 29 nM 
A 10 .mu.M 260 nM D 10 .mu.M 98 nM 
B 0.3 .mu.M 78 nM E 0.3 .mu.M 29 nM 
B 3 .mu.M 780 nM E 3 .mu.M 290 nM 
B 10 .mu.M 2.6 mM E 10 .mu.M 980 nM 
C 30 nM 78 nM F 30 nM 29 nM 
C 100 nM 260 nM F 100 nM 98 nM 
C 0.3 .mu.M 780 nM F 0.3 .mu.M 29 nM 
C 3 .mu.M 7.8 .mu.M F 3 .mu.M 2.9 .mu.M 
C 10 .mu.M 26 .mu.M F 10 .mu.M 9.8 .mu.M 
______________________________________ 
One colony each of P151 was inoculated to the assay medium to which each 
concentration of Factor C was added and cultured under continuous 
illumination by a daylight fluorescent lamp (illuminated to the plant at a 
rate of about 5 W/m.sup.2 using Hitachi FL20 SSD) at 24 to 25.degree. C. 
for seven days to determine the rate of flower formation mentioned above 
(Table 2). 
The tests for the same system were conducted in three flasks and the test 
for the same system was conducted at least twice. The results shown in 
Table 2 are the mean of each test .+-.SE (standard error). 
TABLE 2 
______________________________________ 
30 nM NE 100 nM NE 0.3 .mu.M NE 
3.0 .mu.M NE 
10 .mu.M NE 
______________________________________ 
A -- -- 1.6 .+-. 1.6 
29.5 .+-. 3.5 
51.5 .+-. 2.4 
B -- -- 29.0 .+-. 10.3 34.7 .+-. 4.4 44.8 .+-. 0.7 
C 3.0 .+-. 3.0 40.6 .+-. 2.1 46.3 .+-. 3.6 56.2 .+-. 1.2 53.2 .+-. 1.1 
D -- -- -- 16.3 .+-. 6.4 50.6 
.+-. 7.0 
E -- -- 1.9 .+-. 1.1 61.6 .+-. 1.2 65.0 .+-. 0.4 
F 11.7 .+-. 2.7 39.2 .+-. 7.9 63.2 .+-. 1.2 60.8 .+-. 1.9 66.8 .+-. 
______________________________________ 
3.5 
The result evidently revealed that the activity of inducing flower bud 
formation increases largely in a dose-dependent manner, and that in the 
experiment groups C and F in which Factor C content was equimolar to that 
of norepinephrine or higher, the activity of inducing flower bud formation 
appears even at such an extremely low concentration of 30 nM of 
norepinephrine. 
In other words, it was revealed that when the Factor C content is equimolar 
to that of norepinephrine, the desired activity of inducing flower bud 
formation in Lemna paucicostata is most efficiently exhibited. 
Thus, the activity of inducing flower bud formation by the combined 
administration of Factor C and norepinephrine at the above concentrations 
was observed. 
Furthermore, as explained below, since the activity of inducing flower bud 
formation by Factor C is observed in Pharbitis nil, strain Violet that is 
a dicotyledon strain entirely different from that of monocotyledonous 
Lemna paucicostata, it is evident that the activity of inducing flower bud 
formation will be observed in the plants of the family Lemnaceae including 
the genus Spirodela and the genus Lemna. 
Example 3 
The Effect of Factor C of Inducing Flower Bud Formation in Pharbitis nil, 
Strain Violet 
Nine grams of the seeds of Pharbitis nil, strain Violet were treated with 
concentrated sulfuric acid for 20 minutes and then left under running 
water overnight. Then, the seeds were placed in the damp sea sand with the 
navel part of the seeds facing upward to radicate. These radicated seeds 
were planted in sea sands at a depth of about 1.5 to 2.0 cm and were 
cultured under continuous illumination (about 5 days). 
Pharbitis nil, strain Violet of which leaves were just unfolded by the 
cultivation was transferred to the culture liquid [KNO.sub.3 (250 mg), 
NH.sub.4 NO.sub.3 (250 mg), KH.sub.2 PO.sub.4 (250 mg), 
MgSO.sub.4.7H.sub.2 O (250 mg), MgSO.sub.4.4H.sub.2 O (1 mg), Fe-citrate 
n-hydrate (6 mg), H.sub.3 BO.sub.3 (2 mg), CuSO.sub.4.5H.sub.2 O (0.1 mg), 
ZeSO.sub.4.7H.sub.2 O (0.2 mg), Na.sub.2 MoO.sub.4.2H.sub.2 O (0.2 mg), 
Ca(H.sub.2 PO.sub.4).sub.2.2H.sub.2 O (250 mg)/1000 ml distilled water]. 
The culture system was subjected to the dark treatment with giving the test 
drugs such as Factor C obtained in the above preparation example directly 
to the hypocotyl of Pharbitis nil, strain Violet through cotton threads, 
and then was grown under continuous illumination at 28.degree. C. for 16 
days and on day 16 the number of flower buds were confirmed by observation 
using the stereomicroscope. 
The dark treatment was conducted overnight (16 hours of dark treatment) or 
for two nights (16 hours of dark treatment+8 hours of light treatment+16 
hours of dark treatment). 
The result of the overnight treatment is shown in FIG. 2, and the result of 
the two-night treatment is shown in FIG. 3. 
In both figures, the control group is the group that was given distilled 
water; 1 .mu.M (FC), 10 .mu.M (FC), and 100 .mu.M (FC) are the group that 
received each concentration of Factor C in the WS (water stressed); and 
Factor C+NE means that 10 .mu.M norepinephrine was incubated with the WS 
containing W.mu.M Factor C. The WS means the immersing water of the Lemna 
paucicostata P441 strain that was subjected to dry stress by the method 
described in the test example 1. 
Though the activity of Factor C of inducing flower bud formation was 
observed for the overnight treatment group as compared to the control 
group as is shown in FIG. 2, a stronger activity of inducing flower bud 
formation was observed when administered in combination with 
norepinephrine. 
In contrast, when attention is paid to the control group or the difference 
in the concentration of drugs, the average number of flower buds increased 
in a dose-dependent manner in the two-night dark treatment group shown in 
FIG. 3 indicating at least an enhanced activity of inducing flower bud 
formation. 
Thus, the activity of Factor C of inducing flower bud formation in 
Pharbitis nil, strain Violet was observed. 
As described above, since the activity of inducing flower bud formation is 
observed in Lemna paucicostata that is a strain entirely different from 
that of Pharbitis nil, strain Violet, it is evident that the activity of 
inducing flower bud formation will be observed in the plants of the family 
Convolvulaceae including the genus Pharbitis, the genus Calystegia, the 
genus Ipomoea and the genus Cuscuta. 
Furthermore, as described above, since the activity of inducing flower bud 
formation was observed for both monocotyledons and dicotyledons that are 
entirely different strains from each other by giving Factor C etc., it was 
strongly suggested that the activity of inducing flower bud formation will 
be observed for plants in general by the administration of Factor C. 
Example 4 
Pharbitis nil, strain Violet germinated as described in Example 3 was 
prepared. Separately, an aqueous solution of Factor C at a concentration 
of 1 .mu.M, 10 .mu.M or 50 .mu.M was prepared, which was sprayed onto both 
of the upper and the lower of the cotyledons immediately prior to the dark 
treatment and daily for 10 days after the dark treatment. The number of 
flower buds on day 14 is shown in FIG. 4. Thus, an activity of inducing 
flower bud was also evidently observed by the method of spraying Factor C 
on the leaves. 
Example 5 
Twenty pots of Dendrobium hybridum Hort. Redstar were cultivated by giving 
as appropriate oil cake and a liquid fertilizer (Hyponex) from April to 
July. After stopping fertilization, from August to December the plants 
were grouped into the experimental group and the control one. For the 
experimental group cultivation was continued by spraying an aqueous 
solution of 50 .mu.M Factor C on the entire plant daily from Monday 
through Friday every week. Dendrobium was kept in the greenhouse in order 
not to bring the lowest temperature fall under 10.degree. C. even in 
winter. The control group was treated similarly using water. 
The results are shown in Table 3. 
TABLE 3 
______________________________________ 
Relative value of 
number of flowerings 
Time of flowering per plant 
______________________________________ 
Experiment February (7 pots), 
132 
March (3 pots) 
Control (water) March (8 pots), 100 
April (2 pots) 
______________________________________ 
As described above, Factor C promoted the flower bud formation of 
Dendrobium hybridum Hort. Redstar. 
Example 6 
Twenty pots of Cymbidium hybridum Hort. Raspberry Mille-feuille were 
cultivated by giving as appropriate oil cake and a liquid fertilizer 
(Hyponex) from April to August. After stopping fertilization, from 
September to November the plants were grouped into the experimental group 
and the control group. For the experimental group, cultivation was 
continued by spraying an aqueous solution of 50 .mu.M Factor C on the 
entire plant daily from Monday through Friday every week. The control 
group was treated similarly using water. Cymbidium was kept in the 
greenhouse in order not to bring the lowest temperature fall under 
10.degree. C. even in winter. 
The results are shown in Table 4. 
TABLE 4 
______________________________________ 
Relative value of 
number of flowerings 
per plant 
______________________________________ 
Experiment January (1 pot), 
151 
February (7 pots), 
March (2 pots) 
Control (water) March (10 pots) 100 
______________________________________ 
As described above, Factor C promoted the flower bud formation of Cymbidium 
hybridum Hort. Raspberry Mille-feuille. 
Example 7 
Seeds of Dianthus caryophyllus L were planted in September and repotted in 
March. After repotting, an aqueous solution of 50 .mu.M Factor C was 
sprayed onto the entire plant daily from Monday through Friday every week. 
In July, the number of the flowered plants per 100 plants were counted. 
The results are shown in Table 5. 
TABLE 5 
______________________________________ 
Relative value of number of flowerings 
per plant 
______________________________________ 
Experiment 142 
Control (water) 100 
______________________________________ 
As described above, Factor C promoted the flower bud formation of Dianthus 
caryophyllus L. 
Example 8 
Pharbitis nil, strain Violet germinated as described in Example 3 was 
prepared. An aqueous solution of 
9-hydroperoxy-10(E),12(Z),15(Z)-octadecatrienoic acid in a concentration 
of 10 .mu.M, 50 .mu.M or 100 .mu.M was prepared, which was introduced into 
the hypocotyl through cotton threads similarly as described in Example 3. 
The number of flower buds on day 14 is shown in Table 6. 
TABLE 6 
______________________________________ 
The number of flower buds on day 14 
______________________________________ 
Control (water) 
0.892 
10 .mu.M 1.425 
50 .mu.M 1.623 
100 .mu.M 2.209 
______________________________________ 
Mean of n=16. 
Example 9 
By repeating the method of Example 8, 
9-hydroxy-10-oxo-13-hydroperoxy-11(E),15(Z)-octadecadienoic acid was used 
as the test substance. The number of flower buds on day 14 is shown in 
Table 7. 
TABLE 7 
______________________________________ 
The number of flower buds on day 14 
Control (water) 
1.203 
10 .mu.M 1.392 
50 .mu.M 1.572 
100 .mu.M 1.704 
______________________________________ 
Mean of n=16. 
Example 10 
Preparation of Factor C by the Enzymatic Method 
Non-heated wheat was ground to powder and 200 g of the powder (for 
soybeans, commercial products are available from Sigma) was dispersed in 
one liter of water. 0.5 g of linolenic acid was added thereto and 
incubated while stirring at 30.degree. C. After incubation for three days, 
it was extracted with chloroform. After evaporating chloroform in the 
evaporator it was fractionated using a silica gel column (carrier: Wakogel 
C-200, Wako Pure Chemical Industries, Co. Ltd., Solvent: 
ether-benzen-ethanol-acetic acid 40:50:2:0.2) and collected. 
Fractions of soybeans were similarly obtained. 
In a similar manner to those described in Examples 1 to 6, each of the 
above fractions was confirmed to promote flower bud formation. 
Example 11 
Ten grams of dry soybeans that were pulverized using a mixer was taken and 
suspended into 10 ml of deionized water. After adding 20 mg of linolenic 
acid it was reacted while stirring for 2 days keeping the temperature at 
30.degree. C. The soybean powder was removed by filtration and the aqueous 
layer was extracted with ethyl acetate. After evaporating ethyl acetate 
under reduced pressure, it was dissolved again in 25 ml of water (sample 
A). Some were not dissolved but were used as they are. 
To one ml of sample A were added 10 .mu.l of 10 mM norepinephrine (NE) and 
5 .mu.l of 0.5 M tris buffer, pH 8.0, and then incubated overnight at 
25.degree. C. It was added to a medium for the Lemna (strain 151) so that 
the concentration of NE becomes 0.3 .mu.M, 1 .mu.M, or 3 .mu.M. One colony 
of Lemna (strain 151) was planted and cultured under continuous 
illumination (Hitachi FL20SSD, 10 Wm.sup.-2) for one week (25.degree. C.). 
The rate of flower bud formation was evaluated by the percentage of flower 
formation. 
The results are shown in Table 8. 
TABLE 8 
______________________________________ 
Concentration of NE (.mu.M) 
0.3 1 3 
______________________________________ 
Linolenic acid 
0 0 0 
Sample A 44.3 .+-. 6.5 58.2 .+-. 2.3 56.9 .+-. 4.3 
______________________________________ 
The results are shown in the mean.+-.SD for three samples. 
As described above, it was confirmed that though linolenic acid itself has 
no activity of inducing flower bud formation it can be converted to a 
substance having the activity of promoting flower bud formation by the 
enzymatic treatment. The effect of using it in combination with 
norepinephrine was also confirmed. 
Example 12 
Using sample A prepared in Example 10 in a method described in Example 1, 
the induction of flower bud formation in Pharbitis nil, strain Violet was 
evaluated. The results are shown in Table 9 below. 
TABLE 9 
______________________________________ 
The number of flower buds of 
Treatment Pharbitis nil, strain Violet 
______________________________________ 
Water 1.3 
Linolenic acid 0.9 
Sample A 2.5 
______________________________________ 
Mean of n=24 
Sample A had the activity of promoting flower bud formation with a 
significant difference of P&lt;0.1 as compared to water or linolenic acid. 
Example 13 
Threshed dry wheat was ground to powder and treated by the method described 
in Example 11 to obtain sample B. 
Then, in a similar manner to that described in Example 11 its activity of 
inducing flower bud formation in Lemna paucicostata. The results are shown 
in Table 10. 
TABLE 10 
______________________________________ 
Concentration of NE (.mu.M) 
0.3 1 3 
______________________________________ 
Linolenic acid 
0 0 0 
sample B 55.4 .+-. 2.5 52.9 .+-. 5.4 58.4 .+-. 3.6 
______________________________________ 
As described above, the effect of the enzymatic treatment of linolenic acid 
and the effect of using in combination with neoepinephrine were confirmed. 
Example 14 
Replacing linolenic acid with arachidonic acid the method of Example 11 was 
repeated to obtain sample C. 
Using this sample C its activity of inducing flower bud formation in Lemna 
paucicostata was evaluated by the method described in Example 11. The 
results are shown in Table 11. 
TABLE 11 
______________________________________ 
Concentration of NE (.mu.M) 
0.3 1 3 
______________________________________ 
Arachidonic acid 
0 0 0 
Sample C 10.9 .+-. 2.3 27.5 .+-. 1.4 48.9 .+-. 2.7 
______________________________________ 
As described above, the effect of the enzymatic treatment of arachidonic 
acid and the effect of using in combination with neoepinephrine were 
confirmed. 
Example 15 
The method of Example 3 was repeated except that the time of the dark 
treatment was fixed at 16 days and the number to be observed n was set at 
50. The results are shown in FIG. 5. The results revealed that the number 
of flower buds significantly increased when the concentration of Factor C 
was 1 to 10 .mu.M. 
Example 16 
The method of Example 3 was repeated except that the time of the overnight 
dark treatment was set at 14, 16, or 18 hours and the concentration of 
Factor C was fixed at 10 .mu.M. The results are shown in FIG. 6. The 
results evidently revealed that the number of flower buds increased by 10 
mM of Factor C irrespective of the time of the dark treatment. 
INDUSTRIAL APPLICABILITY 
According to the present invention, the agents for inducing flower bud 
formation that directly affect the flower bud formation of plants and a 
kit for inducing flower bud formation are provided.