METHODE POUR AMELIORER LES RECOLTES

METHOD FOR INCREASING THE YIELD OF CROPS

Abrégé anglais

ABSTRACT OF THE DISCLOSURE
The yield of crops (such as rice, wheat, corn, potato, soybean
and the like main crops) can be increased according to a method
wherein plants capable of yielding such crops are treated at a
specific stage in their growing period with brassinolide, i.e.
(2.alpha.,3.alpha.,22R,23R)-tetrahydroxy-24S-methyl-B-homo-7-oxa-5.alpha.-
cholestan-6-one of the formula:
<IMG>

Revendications

Claims:
1. A method for increasing the yield of crops in
production of grains and vegetables selected from the
group consisting of wheat, rice, corn, soybean and potato,
characterized in that the seeds and/or plants of the grains
and vegetables are treated at a specific stage in the
growing period with (2.alpha., 3.alpha., 22R, 23R)-tetrahydroxy-24S-
methyl-B-homo-7-oxa-5.alpha.-cholestan-6-one of the formula (l):
<IMG> (I)
wherein,
(1) when the grain is wheat, the treatment is
carried out by dipping seeds of wheat prior to germination
in an aqueous solution of brassinolide of the formula (I)
having a concentration of from 10-3 to 10-1 ppm or by
applying an aqueous solution of brassinolide having a con-
centration of from 10-4 to 1 ppm to plant of wheat at
the flowering-ripening stage;
(2) when the grain is rice, the treatment is carried
out by dipping seeds of rice prior to germination in an
aqueous solution of brassinolide having a concentration of
10-2 ppm or by applying said aqueous solution either at
a concentration of from 10-4 to 10-2 ppm or at a dose
of 1.5 x 10-2 - 1.5 mg of brassinolide per 10 are, to

plant of rice at the maximum tillering stage;
(3) when the grain is corn, the treatment is carried
out by applying an aqueous solution of brassinolide having
a concentration of from 10-4 to 1 ppm to plant of corn
at the silking stage;
(4) when the grain is soybean, the treatment is
carried out by applying an aqueous solution of
brassinolide having a concentration of from 10-2 to 1
ppm to plant of soybean in the period from unfolding of
the 3rd trifoliolate leaf to flowering; and
(5) when the vegetable is potato, the treatment is
carried out by dipping the divided tuber of potato in an
aqueous solution of brassinolide having a concentration of
from 10-5 to 10-3 ppm just before incorporation of the
divided tuber into soil.
56

Description

lZt~S~8~)
Description of the P~i~r Arts:
Hitherto, a number of compounds have been found as
substances capable of controlling growth and propagation of
plants. srassinolide, one of such compounds, is a steroidal
plant'growth regulator isolated in 1979 from pollen of
Brassica napus L. and determined as (2a,3a,22R,23R)-tetra-
hydroxy-24S-methyl-B-homo-7-oxa-5~-cholestan-6-one having
the following structure [Nature, Vol. 281, pp.216-217(1979)]:
HO~ ~ I i
A great number of steroidal compounds are already known
as hormonesfor animals and insects, but brassinolide is a
steroidal compound found for the first time as a s~bstance
exhibiting physiological activities to plants. Thus, bras-
sinolide is considered to be the 6th plant hormone subsequent
to ethylene, auxin, gibberellin, cytokinin and abscicic acid,
and is still being studied for its distribution in plants
and its specific functions from the academic point of view.
Concerning the physiological effects of brassinolide to
plants, various kinds of bioassay including the second inter-
node elongation bioassay for kidney bean ~Phaseolus vulgaris),rice lamina inclination bioassay and raphanus bioassay for
radish ~Raphanus sativus~ have been made heretofore for
comparison with other plant hormones [The Society for Chemical
Regulation of Plants, Ja~an, 18(No.l), 38-54(1983~]. As a
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12~S680
result of ga-thering the previous reports, brassinolide is now
being watched as possessing unique activit~es different from
those seen in other plant hormones. Further, brassinolide
exhibits a strong synergistic effect with auxin to various
elongation bioassays for plants and also a synergistic effect
with cytokinin to a propagation test for callus, thus proving
significantly unique effects which will hardly be observed in
using such known hormone alone. With respect to distribution of
brassinolide in the plant kingdom, more than ten kinds of
brassinolide analogues are already discovered widely in
various plants in addition to ~rassinolide itself, and it is
confirmed experimentally that these brassinolide compounds
are contained widely in the higher plants such as rice
(Oryza sativa), kidney bean (Phaseolus vulgaris), ~hinese
cabbage (Brassica pekinensis), tea (Thea_sinensis~, chestnut
~ (Castanea spp.), hyacinth bean (Dolichos lablab), pine (Pinus
; thunbergii), cattail (Typha latifolia) and Distylium racemosum.
In the past, the effect of brassln~lide to plants is
known in the case of kidney bean wherein brassins as a crude
extract from pollen of Rape (Brassica napus L.) is used
[J.W. Mitchel and L.E. Gregory, Nature New Biology, 239, 253
(1972)] and in the case of radish, lettuce, kidney bean,
pepper (Piper nigrum) and potato (Solanum_tuberosum) wherein
brassinosteroids as synthetic analogous compounds are used
~Science, Vol. 212 (1981), 33-34]~ The treatment for plants
disclosed in these literatures comprises applying a lanolin
paste to seedlings of kidney bean in case of the brassins
and spraying an aqueous solution over seedlings in case
of the brass~ona~ter~ids.
In Japanese Laid-open Pàtent Appln. No. Sho. 57-118503,
~:
~ - 3--

~ L2~5680
there is. disclosed 2~, 3~ 22~ 23S tetrah~dr~xy-24S~ethyl-
22S, 23s~5a~cholestan ~6-one(22S, 23S-homobrassinolide,,
and their derivatives, one of the synthetic brassinolide
analogues, which are explained therein as a substance ef~ec-
tive ~or accelerating the growth of tomato (Lycopersiconesculentum), carrot (Daucus carota), mung bean ~Phaseolus
aureus), radish (Raphanus sativus), cucumber (Cucumis sativus)
and azuki bean (Phaseolus angularis) by dipping seeds or
seedlings of these plants in a solution of this 22S, 23S-
homobrassinolide prior to soil culture. This referencefurther discloses that when tubers of potato, sweet potato
seedlings, cuttings of branches of tea plant and seeds of
tabacco are dipped in a solution of the 22S, 23S-homobras-
sinolide prior to cultivation, the 22S, 23S-homobrassinolide
exhibits a growth-accelerating effect and that when the 22S,
23S-homobrassinolide is sprayed over fruit trees at the stage
of anthesis, the dlameter and weight of the fruits become
larger. In Japanese Laid-open Patent Appln. No. Sho.58-90578,
there are also disclosed new synthetic 2~, 3a, 22R, 23R~
tetrahydroxy-24S-ethyl-5~-cholestan ~6-one and their deriva-
tives (22R, 23R-homobrassinolide derivatives) and their use
for accelerating the growth of various plants and improving
the quality of agricultural products. In this reference,
an elongation test for azuki bean is carried out by treating
the seed with the 22R, 23R-homobrassinolide derivatives just
before or after germination, but no concrete disclosure is
given therein how the yield of crops is increased.
In general, plant-growth regulating agents are utilized
by artificially controlling the growth of plants to achieve
inc~eased yields of crops,re~ulation of the amount of the

~L265680
agr~cultural p~oduc~ p~oye~en~ in ~u~lit~ saving of work
time and power, and regulation of harvest time. These plant-
growth regulating agents are comprised chiefly of plant
hormones, synthétic compounds possessing activlties equivalent
to such plant hormones an~ chemical substances ha~ing
antagonistic effects to
- 4-~ -

~2656~30
these. Recognized now as plant hormones are auxin, gibberellin,
cytokinin, abscicic acid and ethylene. Many of the chemical
substances actually employed for agricultural use as plant-
growth regulating agents possess activity similar to those five
plant hormones.
On the other hand, a great number of compounds are known
which show physiological activity in vitro bioassay to plants,
but the number of the compounds actually utilized for practical
use are rather small. In ordinary in vitro bioassay, its
experimental system is simplified with a view to detecting only
a particular reaction sharply, minimizing the mutual effect with
other organs and tissues. However, a plant body
contains in its body a plurality of organs which are different
in age and function and grow under their mutual actions to
keep coordination. Thus, it is rather rare that the activity
observed in vitro bioassay is repraduced exactly in whole
plants. Further, strength of in vitro bioassay does not respond
to importance in practical use. It is often observed that sub-
stances which exhibit similar equivalent activity in in vitro
bioassay may exhibit quite different activity to cultivated
plants. In addition, it is also known that a quite different
activity is often exhibited according to the concentration of
the compound used. Furthermore, it is usual that the growing
phenomena observed in applying the growth-accelerating agent
to plants are differentaccording to the sort and age of the
plants. Thus, the growth reaction of plants to foreign substance
varies according to the sort of plantsand to the stage of their
growth. In the extreme case, plants may often show a counter-
reaction to such growth-increasing agent. It is quite impossible,
therefore, to estimate growth-regulating activity on the basis

~265~80
of its in vitro bioassay. Thus, development of a new plant-
growth regulating agent always encounters a yreat difficulty
and is only possible by repeating "trial and error" tests to
check i-tspractical usefulness.
All of the prior art methods hitherto known relate to
testswherein a crude extract containing brassinolide or a
synthetic brassinolide analogue is exclusively used for accelera-
ting the growth of plants, but fail to disclose the effect of
pure brassinolide itself on the growth of plants. Under these
circumstances, therefore, there was still a great demand for
developing a new method of using brassinolide for accelerating
the growth of plants, especially for increasing the yield of
crops.
BRIEF SUMMARY OF THE INVENTION
. . ~
Accordingly, it is an object of the present invention to
provide a method for increasing the yields of crops wherein
brassinolide isused under specific conditions. ;
It is another object of the present invention toprovide a
yield-increasing agent for crops which contains brassinolideO
It is still another object of the present invention to
provide the use of brassinolide for treating a plant capable
of yielding a crop at a specific stage in its growing period.
Other and further objects, features and advantages of the
present invention will be apparent more fully from the following
description.
Since the discovery of brassinolide, a number of synthetic
brassino]ide analogues having structures similar to brassinolide
have been developed and tested for various plants. Even if
such synthetically similar compound exhibits a high plant-growth
increasing activit:y at the level of in vitro bioassay, such

~2656~30
compound would not be always effective for whole plants and
would usually exhibit somewhat different activity according
to the sort and age of plants, methods for treatment and the
concentrations used. This will just apply to the case o~
brassinolide itself; in order to utilize only a specific
activity among the various plant-physiological activities of
brassinolide, a method for the treatment of plants with bras-
sinolide has to be investigated for every plant. Standing on
the above viewpoint, the present inventors have carefully paid
their attention to the yield-increasing effect among the various
activities exhibited by brassinolide and studied a method for
the treatment with brassinolide for individual plants to examine
the strength of the effect in connections with other related
factors such as the time of application, concentration, etc.
As a result of such extensive study it has now been found that
the yield of main crops such as wheat, rice, corn, soybean and
potato can be increased remarkably by applying a specific treat-
ment with brassinolide to plants capable of yielding these main
crops at a specific stage in their growing period~ The con-
~0 ditions for treating these plants with brassinolide are strictly
i critical. Thus, the conditions for the treatment are ~uiteinherent to the individual plants so that the yield-increasing
effect of brassinolide cannot be expected at all and, in the
extreme case, adverse effects may be incurred if the conditions
inherent to particularplants are changed. Such conditions in-
volve a combination of the concentration of brassinolide, the
; method of treatment and the time of application of brassinolide
to particular plants and would not be anticipated at all even
`~ by those skilled in the art.
In accordance with one aspect of the present invention,
.
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1265680
therefore, there is provided a method for increasing the
yield of crops in production of grains and vegetables
selected from the group consisting of wheat, rice, corn,
soybean and potato, characterized in that the seeds and/or
plants of the grains and vegetables are treated at a
specific stage in the growing period with (2~, 3~, 22R,
23R)-tetrahydroxy-24S-methyl-B-homo-7--oxa-5~-cholestan-
6-one of the formula tI):
0~ _
¢~
f~
HO~
~10 ""~
o
wherein, (l) when the grain is wheat, the treatment is
carried out by dipping seeds of wheat prior to germination
in an aqueous solution of brassinolide of the formula (I)
having a concentration of from lO 3 to lO l ppm or by
applying an aqueous solution of brassinolide having a con-
centration ~f from lO 4 to l ppm to plant of wheat at
the flowering-ripening stage; (2) when the grain is rice,
the treatment is carried out by dipping seeds of rice prior
to germination in an aqueous solution of brassinolide
having a concentration of lO 2 ppm or by applying said
aqueous solution either at a concentration of from lO 4
to lO 2 ppm or at a dose of l.5 x lO 2 _ l.5 mg of
brassinolide per lO are, to plant of rice at the maximum
tillering stage; (3) when the grain is cornr the treatment
~t~
~ 8

iX~56~0
is carried out by applying an aqueous solution of brassino-
lide having a concentration of from 10 4 to 1 ppm to
plant of corn at the silking stage; (4) when the grain is
soybean, the treatment is carried out by applying an
aqueous solution of brassinolide having a concentration of
from 10 2 to 1 ppm to plant of soybea~ in the period
from unfolding of the 3rd tri~oliolate leaf to flowering;
and (5) when the vegetable is potato, the treatment is
carried out by dipping the divided tuber o~ potato in an
aqueous sol~ltion of brassinolide having a concen~ration of
from 10 5 to 10 3 ppm just before incorporation of the
divided tuber into soil.
In accordance with another aspect of the present invention,
there is provided a yield-increasing agent for crops which
contains the compound of the formula I above as an active
ingredient in admixture with a carrier or diluent.
In accordance with still another aspect of the present
invention, there is provided the use of the compound of the
formula I for increasing the yield of crops.
DETAILED DESCRIPTION OF THE INVENTION
In the method of the present invention, it is important
that plants capable of yielding crops are treated with bras-
sinolide at a specific concentration and at a specific stage in
their growing period~ This specific stage in growing period is
inherent to indiuidual plants. By the term "growing period"
referred to herein is meant a period normally from germination
to ripening of crops in the form of grains, seeds, fruits or
tubers. In certain cases, the term "growing period" involves a
plant in the state of seed, grain or tuber. According to the
method of this invention, therefore, a plant capable of yielding
~,
- 8a -

~ ~65~80
a crop is treated with brassinolide at a specific concentration
at a certain stage in the growing period of the plant from the
state of seed (grain) or tuber prior to germination to the state
of ripened seed tgrain), fruit or tuber. Thus, for example,
the state of germination, seedling, tillering or branching,
emergence of ear and flowering are involved as intermediate
stages of the above growing period.
Brassinolide used in the present invention is a crystalline
substance soluble in organic solvents such asethanol and acetone
but sparingly soluble in water and can be synthesized according
to a method improving the method of Mori et al. [Mori et al.,
Agric. Biol. Chem. ~7(3), 663-664 (1983)] Brassinolide is
usually employed in the form of a liquid formulation as a stock
solution, an emulsifiable concentrate or a solid (powder or
granular) formulation as a water-dispersible agent. On actual
use, these formulations are diluted with a sufficient amount
of water to have a given concentration of brassinolide. It is
also possible to prepare a brassinolide paste based on lanolin
to apply brassinolide directly to a specific part of plant.
In the preparation of such liquid formulation, brassinoiide
is dissolved in or homogeneously mixed with an organic solvent
containing, if necessary, an auxiliary agent such as a wetting
agent. Illustrative as the organic solvent are, for example,
alcohols such as methanol, ethanol or propanol, ketones such as
acetone, mèthyl ethyl ketone or methyl isobutyl ketone, glycols
such as ethylene glycol, propylene glycol or diglyme, esters
such as ethyl acetate or butyl acetate, hemiethers such as
ethylene glycol monGmethyl ether, amides such as dimethylform-
amide or hexamethylphosphoramide, sulfones such as diethylsulfone,
ethers such as dioxane or tetrahydrofuran and allphatic or
.
_ g _

1265~j80
aromatic hydrocarbons such as hexane, toluene or xylene, and a
mixture of these.
In the preparation of the solid preparation in the form
of powder, tablets or granules, brassinolide is thoroughly mixed
with a solid carrier such as clay, acid actived clay, bentonite,
talc, diatomaceous earth or the like mineral material together
with an auxiliary agent such as a wetting agent, a dispersing
agent or an emulsifier.
In the preparation of the paste~ brassinolide is mixed with
vaselin, lanolin, petroleum jelly or the like waxy substance,
if necessary, together with an auxiliary agent such as an
extending agent or dispersing agent.
Examples of theauxiliary agent usually employed for im-
pro~ing the extending, permeating, dispersing, binding, wetting
and suspending properties of the above various formulations
included non-ionic surfactants such as polyoxyethylene alkyl-
phenyl ethers, polyoxyethylene dodecyl ethers, polyalkylene-
glycol alkyl ethers and polyoxyethylene resin acid esters;
anionic surfactants such as sodium dinaphthylmethanedisulfonate,
sodium ligninesulfonate and sodium dodecylbenzenesulfonate;
and other additives such as paraffin and D-sorbit.
The amount and concentration of brassinolide in these for-
mulation can be adjusted according to the sort of plants to be
treated, the sort of formulations, and the method for the
treatment. In general, brassinolide is allowed to be present
in such liquid or solid formulation in an amount of about
10-1000 ppm and the content of brassinolide in the formulation
is finally adjusted on actual useto~ concentration of about 10
ppm to 10 4 ppm by dilution of the formulation with water or
an extending agent. It is a matter of course, however, that
.,
- 10 -

~ 265680
the final concentration of brassinolide may be varied within
a wide range according to the purpose of treatment, the sort
of plants to be treated, the method of treatments and other
various factors.
The treatment of plants with brassinolide can be carried
out in various manners known per se. In case plants are to be
treated with brassinolide at the stage of their seeds or
tubers, for example, they axe normally dipped in a diluted
aqueous solution, dispersion or emulsion of the bras-
sinolide formulationsfor a given period of time at a given
concentration. Alternatively, a paste formulation of bras-
sinolide may be applied to the seeds or tubers by painting or
the like means. When plants are to be treated with brassinolide
at any stage of germination, seedling, branching or tillering,
emergence of ears, flowering and ripening, the plants are
preferably sprayed with an aqueous solution, dispersion or
emulsion of brassinolide at a given concentration according to
a usual spraying method. In this case, one or more of the above
mentioned various auxiliary agents may advantageously be in-
corporated into the brassinolide solution, dispersion or emulsionused with a view to improving a part or all of the wetting and
spreading property,penetrating ability, dispersibility, depo-
siting property, sticking property, suspensibility of the treat-
ing agent. The spraying operation itself can be carried out by
using any type of spray devices, but an aerial low volume spray
by an aircraft may preferably be adopted when the plants in a
broader area are treated within a short period of time. The
treatment with brassinolide can be carried out singly or in
combination with one or mcre of other treatmentssuch as
herbicidal or pesticidal treatments. In such combination treat-

i2~56~30
ment, one or more of the other plant hormones, fertilizers,herbicides, sterilizing agents, insecticides, etc. may be added
to the brassinolide formulations.
The present invention can more fully be understood from
the foilowing description in conjunction with the accompanying
drawings in which:
Figs. l(A)-l(C) are graphs showing results of tests wherein
wheat was treatedwith an aqueous solution of brassinolide
having a given concentration at various growing stages of wheat.
Figs. 2(A)-2(C) are graphs showing results of tests
wherein wheat was treated with the solution of brassinolide
having a given concentration after anthesis to check the
weight per grain and the relation between the concentration
of brassinolide on the grain-setting of a specific part of the
wheat ear.
Fig. 3 is a graph showing a result of test wherein
wheat was treated with the solution of brassinolide after
anthesis to check the grain weight per spikelet.
Fig. 4 is a graph showing a result of test wherein wheat
was treated with the solution of brassinolide after anthesis
to check the grain weight per spikelet.
Fig. 5 is a graph showing a result of tests wherein
wheat was treated with the solution of brassinolide after
authesis to check the relation between the total grain weight
of the specific spikelets and the concentration of brassinolide.
Figs. 6(A)and6~B)are graphs showing results of test
wherein soybean was treated with~an aqueous solution of
brassinolide having a given concentration to check the effect
of brassinolide on the growth of soybean at the early stages.
Figs. 7(A) and 7(B) are graphs showing results of tests
12 -

~ 1265~j~0
wherein soybean was treated with the solution o brassinolide
at certain growing stages to check the effect of brassinolide
on photosynthesis and the related factors.
Figs 8(A) and 8(B) are graphs showing results of tests
wherein soybean was treated with the solution of brassinolide
at certain growing stages to check the effect of brassinolide
on the seed-setting.
Figs 9(A) and 9(B) are graphs showing results of tests
wherein soybean was treated with the solution of brassinolide
to check its effect on the yield of soybean.
The method of the present invention can be carried out
for increasing the yield of wheat as one of the gramineous
crops. In this case, the yield-increasing efect of brassino-
lide is obviously exhibited when the wheat is treated with
brassinolide at a specific concentration at the stage of seed
prior to germination or in the flowering-ripening period. It
has been found that when wheat is -treated at its various
growing stages with brassinolide to check its influence on the
growing process of wheat, the ear weight of tillers and the ear
weight of the main stem axe significantly reduced in a plot
where brassinolide has been sprayed from the beginning of
tillering, as the concentration of brassinolide becomes higher.
It has also been found that in a plot where brassinolide has
been sprayed from the middle stage of spikelet differentiation,
the ear weight of tillers i5 reduced but no influence is found
in the ear weight of the main stem. In contrast, the ear
weights of both tillers and the main stem were obviously
increased as compared with the untreated control in a plot
where brassinolide has been sprayed from the beginning of
anthesis, thus resulting in increase in the percentage of

~x~s~o
grain-setting of floxets located in the upper part of a spike-
let. It has further been found that the treatment with bras-
sinolide increases the weight per grain in each setting posi-
tion of a particular spikelet sampled, the grain weight per
spikelet and the total weight of grains of each spikelet and
that the total grain weight becomes maximum by the treatment
with brassinolide at a concentration of 10 2 ppm, showing in-
crease by 10~ in comparison with the untreated control. The
treatment with brassinolide increases grain weight significantly
especially in spikelets and grains located in the upper part of
the ear. Thus, the treatment of wheat with brassinolide before
anthesis rather gives bad influence on the ripening of grains
and the yield of grains but the treatment of wheat with bras-
sinolide during the period from anthesis to ripening serves to
increase the percentage in grain-setting of florets located in
the upper part of a spikelet and the weight of grains located
in the upper part of a spikelet, thus bringing about an in-
creased yield of grains. It is quite unforeseeable therefore
that the percentage of grain-setting and the grain weight can
be remarkably increased by treating wheat during the period
from anthesis to ripening with brassinolide especially in the
upper part of a spikelet where the percentage of grain-setting
and the ~rain weight are usually low. It is also interesting
that as will be evident from Example 2 the yield-increasing
effect of prassinolide is exhibited also by treating the seed
of wheat with brassinolide. The concentration of brassinolide
varies according to the method of treatment and the sort of
~lants. In case of wheat, brassinolide is preferably sprayed
over the whole plant and the concentration of brassinolide
in this case is recommended to be about 10 1 _ 10 3 ppm. The
number of the treatment with brassinolide varies according to
.
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~S~i80
the method of treatment, the sort of plants and the concentration
used, but the treatment is usually carried out 1-5 times in case
of spraying brassinolide at a concentration of 10 2 ppm.
An exact reason for the development of this yield-increa-
sing effect is stillunknown at the present stage, but it has beenfound, according to the present inventors' study, that the pho-
tosynthetic rate, stomatal conductance (gs) and mesophyll con-
ductance (gm) are slightly promoted in young leaves but are
rather inhibited in senescent leaves. It is considered that
both of gs and gm partici.pate in the change in photosynthetic
rate by the treatment with brassinolide.
.In case corn is treated with brassinolide, a significant
yield-increasing effect is observed when an aqueous solution of
brassinolide is sprayed from the early silking stage to ear and
silk of the plant. As a result of the present inventors' study
on the length and diameter of ear, kernel weight, column number
of ear, kernel number per column and number of vacant kernels
by spraying a solution of brassinolide:having a given concentra-
tion onto ear and silk of corn at the early silking stage, it
2~ has been found that the treatment with brassinolide gave a
significant influence especially on the length of unfertile tip
portion of ear and the number of vacant kerners and increased
the yield by 18-33% by weight as compared with the untreated
control. As in the case of wheat, it is evident that the treat-
ment with brassinolide contributes to increasingthe rate of grain-
: setting of the floret located in the upper part of the ear where
grain -setting is usually extremely poor.
In case of treating paddy rice with brassinolide, the yield-
: increasing effect is observed when the seed is dipped in an
aqueous solution of brassinolide or when the paddy rice is
,. _ l,C; _

1.265~'30
treated with brassinolide at the maximum tillering stage.As a result of treating paddy rice at its several growing
stages with brassinolide at several levels of concentration,
it has been found that in a plot where the seed ~7as dipped
in the solution of brassinolide at a concentration of 10 2
ppm, the numbers of tillers and ears were increased by
7~ and 15~, respectively, as compared with the untreated
control so that the weight of unhulled rice was increased
by 17% as shown in Table 4 alt]hough the 1000-kernel weight
was almost unchanged and that in a plot where the paddy
rice was treated at the maximum tillering stage with brassino-
lide at concentrations of 10 2 ppm and 10 4 ppm by a foliage
treatment, the nu~er of grains per ear was increased by
11-13% as compared with the untreated control so that an
average ear weight was increased by 5-7~ (Table 5~O However,
a significant increase in the yield is not expected in case :
of treating the paddy rice with brassinolide at the young
panicle formation stage and anthesis. Concerning the rate .
of increasing the number of grains in each part of the ear
in the foliage treatment at the maximum tillering stage~ the
rate of increase in the upper lst-4th rachises is within
the range of 7-~% but that in the lower rachises is surprisingly
15-17~, thus showing the fact that brassinolide gives a
strong influence on the grains located in the l~wer rachises
25 where grainsare usually not fully grown. This tendency is :
seen in the foliage treatment at the young panicle formation
stage but th~e rate of increase is not so great as compared
with the above case. Thus, the yield-increasing effect
brought al ut by brassinolide sprayed at the marimum
. ~ - 16 -

~ s~
~illering stage results apparently from increase in th~ rate
of grain-setting in the lower part o~ ears.
The method of the present invention can al50 be carried
out for increasing the yield of soybean as one of pulse crops.
As a r,esult of treating soybean at its several growing stages
with brassinolide at several levels of concentration, it has
been found that brassinolide exhibits an accelerating effect
on the elongation of epicotyls, internodes, petioles and
branches. However, such effect varies according to the sort
of organs and the concentration of brassinolide; the growth of
leaf area was inhibited at a higher concentration of brassino-
lide. On the other hand, no significant influence is found on
the node order of the first branching,number of branGhing,number
of nodes on branches and node number of the main stem so that no
accelerating effect is observed on the ~ormation of organs. The
treatmentof soybean with brassinolide at the stage of unfold-
ing of the 4th trifoliolate leaf and after fully expanding of
the 4th trifoliolate leaf increase the photosynthetic rate.
The amount of chlorophyll in the leaf of each leaf position on
the stem is increased also by a consecutive treatment with bras- ¦
sinolide before or after flowering, thus showing a senescence-
preventing activity to leaves. As a result of tests for
examining the influence of brassinolide on the seed-setting
and yield of soybean, a consecutive treatment with brassinolide
before flowering increases the pod number of branch and the
seed number per pod so that the tot~lseed weight per plant can
be increased. Contrary to this a consecutive treatment with
brassinolide after flowering increases elongation of the
branches and petioles to allow them to curve and rather de-
3Q crea~e~ the Xate o~ seed~sett~n~ and the ~ield~ In case ofsoybean, therefore, the treatment in the period from the
- 17 -

~ 80
unfolding of the t~ird tri~oliol~te leaf to the beginning of
Elowering incre~ses the number of pods on branches and the seed
number per pod, to show the yield-increasing effect for soybean.
Consequently, the method for increasing the yield of pulse
crops according to the present invention comprises treating
a part of the plant such as flowers, leaves, stems or roots or
the whole part of the plant with brassinolide in the period
from the unfolding of the third trifollolate leaf to the begin-
ning of flowering. A proper device such as dipping, spraying,
applying or the like means can be used for treating plants with
brassinolide in the form of an aqueous solution, emulsion or
dispersion to apply brassinolide to a particular part of the
plants. The conaentration of brassinolide varies according to
the method of treatment or the sort of plants. In case of soy-
bean, for example, it is preferable to spray an aqueous solu-
¦ tion, dispersion or emulsion of brassinolide wholly over theplant at a concentration of about 10 1 _ 10 4 ppm. The number
of the treatment varies according to the method of treatment,
the sort of plants and the concentration of brassinolide used
but is usually within the range of 1-5 times.
In case of the method of the present invention is applied
to increase the yield of potato, it is preferable to dip divid-
ed tubers in an aqueous solution, dispersion or emulsion of
¦ brassinolide at a concentration of 10 3 to 10 5 ppm for 10-30
25 ¦ hours, thereby increasing the yield of tubers per plant by
¦ 10-20% as compared with the untreated control. An optimum
¦ concentration o~ brassinolide in this case is about 10 4 ppm.
¦ In a plot where an aqueous solution of brassinolide having a
¦ concentration o~ 10 4 ppm is sprayed two times over the whole
30 ¦ plants, the number and weight of tubers per plant was almost
e~l to t.ho~e o~ t~e un~treated~txol~thus ~ ng ~a ~ncXe~se
- 18 -

~26s~ao
in the yield of tubers. Thus, it is evidenk that the treat-
ment of tubers with brassinolide prior to incorporation into
soil is suitable for increasing tha yield of potato.
As brassinolide is a natural substance which is contained
widely in naturally occurring edible vegetables, it is a matter
of course that no problem arises in connection with the safety
and biodegradative property of brassinolide.
The present invention will now be illustrated in more
detailbyway of Formulation Examples and Examples wherein the
term "BR treatment" meaning the treatment with brassinolide
is used in Tables and in the descriptions relating to the
various examination items.

iZ656~(~
For~ul~tion Ex~le 1 (~ ui~ f~r~ul~tion~
A liquld for~ulation is pr~pared by dissolv~ng 100 mg
of br~ssinolide and 10 ml of Neoesterin*(a wetting agent
marketed by Kumiai Chemical Industry Co., Ltd., ~apan) in
990 ml of ethyl alcohol and homogeneously mixing the solution.
On actual use, the liquid formul~tion is dîluted w;th water
to a volume of 1,000-1,000,000 times.
Formulation Example 2 (an emulsifiable concentrate)
An emulsifiable concentrate is prepared by homogeneously
mixing the follow~ng ingredients:
Brassinolide 0.01 Part by weight
Dimethylformamide 60 Parts by weight
Xylene 30 Parts by weight .
Nitten (a wetting agent 10 Parts by weight
marketed by Nissan
Chemical Industries,Ltd.)
Formulation Example 3 (a water-dispersible powder)
A water-dispersible powder is prepared by thoroughly
mixing and pulverizing the following ingredients: .
20 Brassinolide 0.1 Part by weight
Diatomaceous Earth85 Parts by weight
Polyvinyl alcohol 5 Parts by weight
Sodium dodecylbenzene- 9.9 Parts by weight
sulfonate
Pormulation Example 4 (a paste)
. A paste is prepared by homogeneously mixing the following
ingredients:
Brassinolide 0.001 Part by weight
Ethyl alcohol 10 Parts by weight
.~: 30 Lanolin 90 Parts by weight
*Tradc mark - 20 -

12~S680
Example 1
. Using Asakaze wheat, a combination oE tests was carried
out according to the methods as shown in Table 1 to examine
the effect of brassinolide applied to the wheat at various
growing stages thereof.
A solution of 100 ppm brassinolide in ethyl alcohol
was diluted with a 1 : 5000 aqueous solution of Neoesterin and
the dilutedsolution waswidely sprayed at aconcentration
of 10 4, 10 2 or 10 ppm in a sufficient amount all over
the wheat. The cultivation of the wheat was performed in a
usual mannerand a yield survey was made in Test Runs II, III
and IV after harvest.
A survey was made for the five examination items, i.e.
(1) the effect on germination and seedling growth, (2) the
effect on tillering, ~3) the effect on heading, (4) the
effect on ear weight, and (5) the effect ongrain-setting of
ear on the main stem. Described below are results ofthe
tests made according to the individual examination items.
In addition, the results of the tests obtained in the test
runs II, III and IV are shown in Figs. l(A), l(B) and l(C),
respectively, and the results obtained in the test run
IV are shown especially in Figs. 2(A), 2(B) and 2(C) and
Figs. 3-5.
Results of the tests:
(1) The effect on germination and seedling growth
At a concentration of 10 or 10 ppm, elongation of
leaf sheath and roots was strongly inhibited but the number
of roots was somewhat increased.
(2) The effect on tillering
- 21 -

1~5680
Table l Testing method
Test Method for Method for
run cultivation BR treatment
(A) on a petri dish dipping the seeds for 24
hours in the aqueous
I (B) in a 2-liter beaker solution of BR at a given
charged with soil concentration
Seeding depth: 5 cm
sown a pot ~ith the seeds treated 7 times with the
on January 12th, 1984, aqueous solution of BR during
II the pot having been the period from March 3rd
placed in a green (the beginning of tillering)
house until March 3rd to April 20th (the beginning
and then placed outdoors of heading: April 29th)
sown a pot with the seeds treated 7 times with the
on November ~th, 1983, aqueous solution of BR during
III the pot being placed the period from March 3rd
outdoors (the middle stage of spikelet
defferentiation) to April
20th (the beginning of
headiny: April 27th)
; same as above treated 5 times with the
IV aqueous solution of BR during t~ le
period from May 2nd (the
beginning of anthesis) to
: May 30th
Remarks: (l) "sR" means brassinolide
. ~(2) In case of the test ~uns II, III and IV, the
wheat was harvested on June 8th.

12656~30
In the te~ ~u~s ~ ,a,~d I~- the ~lle~ nu~bex wa5 in-
creased as the cancentration o~ brass~nollde became higher but,
on the other hand, the percentage of productive stems was de-
creased so that the number of productive stems ~as decreased.
(3~ The effect on heading
In the test run II the BR treatment showed the effect of
retarding the heading time.
(4) The effect on ear weight
In the test run II, both ear weight of tillers and ear
weight of the main stem were significantly decreased as the
concentration of brassinolide became higher. In the test run
III the ear weight of tillers was decreased by the BR treatment
but no change was observed in the ear weight of the main stem.
In the test run IV both ear weight of tillers and ear weight
of the main stem were obviously increased by the BR treatment. ,
~5~ The effect on the grain-setting of ear on the main stem
In the test run IV, the BR treatment served to increase
the percentage of grain-setting of florets located in the upper
part of a spikelet. Further, the BR treatment increased the
weight per grain in each setting position of a particular
spikelet sampled, the grain weight per spikelet in the uppex,
middle and lower parts of the ear, respectively, and the total
weight of grains of each spikelet. The total grain weight
became maximum with the BR treatment as a concentration of 10
ppm and was increased by 1~% as compared with the untreated
control~ Increase in grain weight with the BR treatment was
found significant especially in spikelets and grains located
in the upper part of the ear.
In the ~raphs of Figs. l~A~, l(B) and l~C~ showing results
of the test runs II, III and IV, respectively~ the left-hand
- 23 -

~265~i8~1 ~
ordinAte st~nds ~O~ th~ ~ot~l eaX ~e~ht pe~ pot ~in terms of
g/pot~ while the r~ght-hand ordinate ~or the ear weight of
tillers or the ear we~ght of the main stem per pot (in terms
of g/pot). In e~ch graph, the abscissa stands ~or the concen-
tration of brassinolide used for the treatment (in terms ofppm), a solid line with black spots ~ stands for the
total ear weight, a broken line with white tria~gles (-
~for the ear weight of the main stem, a broken line with white
circles (--o--o--) for the ear weight of tillers, a vertical
bar represents the least significant difference between means
(p - 0.05), and values in parentheses are relative values in
case of the value in the untreated control being 100. Figs.
l(A~ and l(B) apparently show that the total weight of the
ears was rather decreased-as the concentration of brassinolide
became higher in the test runs II and III.
Among the graphs of Figs. 2(A), 2(B), 2(C~, 3, 4 and 5
showing results of the test for evaluating the effect of
brassinolide on grain-setting of ear on the main stem in the
test run IV, Figs. 2(A~, 2(B) and 2(C) show the weight per
grain and the relation between the concentration of bras-
sinolide in the treatment and the percentage of grain-setting
of the topmost fertile floret in the spikelets located in the
upper, middle and lower parts of the ear, respectively. ~,
Throughout Figs. 2(A), 2(B), 2(C), 3 and 4, the spikelet
located in the upper part of the ear (the upper spikelet) means
the third spikelet downwardly from the terminal spikelet while
the sp~elet located in the lower part of the ear (the low~r
spikelet~ means the third spikelet upwardly from the basal
spikelet. In Figs. 2(A), 2(B~ and 2(C), the position of
3~ grains in the spikelet was ar~itrarily de~ined as follows:

~ 2656~0
The bottommost position is defined as "a" and the positions
"b", "c" and "d" are then successively defined upwardl~ from
the bottommost position. In each of Figs. 2(A), 2(B) and 2(C),
the left-hand ordinate stands for the weight per grain (G.W.
in terms of mg) and the right-hand ordinate for the percentage
of ~ratn^setting of the topmost fertile floret (G.S.) while the
abscissa stands fox the concentration of brassinolide in the
treatment (in terms of ppm). In Fig. 2(~), the s~lid line
a (- o-~ - ) connects the plots standing for results in the
grain located in the bottommost position of the upper spikelet
treated at given concentrations of brassinolide, the broken
line b (--o--o--) connects the plots standing for results in
the grain in the position "b" of the upper spikelet treated
at given concentrations of brassinolide and the line c
(- ~ ~ ) with the notation "G.S." connects the plots standing
for "G.S." of the grain in the position "c" in the upper
spikelet treated at given concentrations of brassinolide, and
the line c (~ ) with the notation "G.W." connects the
plots standing for "G.W." of the grain in the position "c"
in the upper spikelet treated at given concentrations of bras-
sinolide. In Figs. 2(B) and 2(C), the lines a and b have the
same meanings as given in Figs. 2(A), the line c (-
~connects the plots standing for results in the grain located
in the position "c" in the middle or lower spiXelet treated
at given concentrations of brassinolide, the line d (~
with the notation "G.S." connects the plots standing for "G.S."
of the grain in the position "d" in the middle or lower
spikelet treated at given concentrations of brassinolide,
and the line cl (-{]---{C}-) with the notation "G.W." connects
the plots standing for "G.W." of the grain in the position "d"
- 25 ~

12~5~
in the middle QX lo~e~ ~p~ke~et txe~ted ~t ~i~en concentra-
tions of br~ssinolide, In case of the upper spikelet, onl~
3 grains were involved there~n and so the line d does not
exist in Fig. 2(~) in contrast to Fig. 2(B) or 2(C) showing
the result of 4 grains in the middle or lower spikelet.
Fig. 3 is a graph showing the grain weight per spikelet
at gi~en concentrations of brassinolide wherein the ordinate
stands for the grain weight per spikelet (in terms of mg)
while the abscissa for the concentration of brassinol~de
~in terms o~ ppm~ and wherein the line ~ stands for
the middle spikelet, the line (~ for the lower spikelet
and the line (-o-~-o-) for the upper spikelet.
Fig. 4 is a graph showing the grain weight per spikelet
(in terms of percentage) at given concentrations of bras-
sinolide wherein the ordinate stands for the grain weight per
spikelet(in terms of percentage) while the abscissa for the
concentration of brassinolide (in terms of ppm2 and wherein
the lines (-9--~ - and -o---o-~ have the same meanings
as given in Fig. 3.
Fig. 5 is a graph showing the rPlation between the total
grain weight of spikelets located in the upper, middle and
lower parts of the ear and the concentration of brassinolide
wherein the ordinate stands for the total grain weight (mg)
of spikelets located in the upper, middle and lower parts
(the upper~, middle and lower spikelets) while the abscissa for
the concentration of brassinolide (ppm). In Fig. 5, a vertical
bar represents the least significant difference between means
(p - 0.05~, and the values in parentheses axe relative values
in case of the value in the untreated control being 100.
Example 2
Wheat seeds ~Variety:Norin 61~ were treated with brass~ide
- 26 -

1~6568()
according to a dipping method wherein the seeds are dipped prior
to germination in aqueous solutions of brassinolide having
various concentrations (10 1, 10 3 and 10 5 ppm) for 12 hours.
A seed-disinfectant was added to the aqueous solutions at the
same time. The seeds thus treated were washed with water, dried
for 2 days at room temperature and used for the following test:
A cubic concrete pot having a size of 60cm in length,
60cm in width and 40cm in depth was filled with a diluvial soil
and sown with the wheat seeds in 3 rows with 30 seeds being
sown in each row. The sown seeds were covered with the diluvial
soil of 3cm in depth. Just 7 months after the seeding, the
total weight of ears and the total numberof ears were measured
and the weight per ear was calculated. A result of the test is
shown in Table 2 below.
Table 2
Agent Concentration Total Total Weight
weight number per ear
(ppm) of ears of ears
Brassinolide 10 116 101 115
Brassinolide 10-3 112 100 112
Control 100 100 100
Remarks: The results in the table are shown by relative values
in case of the value in the untreated control
being 100.
As is evident from Table 2, a significant increase in the
total w~ight of ears and the weight per ear is found in case
of the seeds treated with brassinolide.

~ 5~i80
Example 3
To ear and silk of corn (Zea mays) cultivated in a
green ho~lse an aqueous solution of brassinolide having a
concentration of 10 4, 10 2 or 10 was sprayed in a sufficient
amount 1-3 timss from the early silking stage. The test
conditions were as follows:
Kind of corn : Honey Bantam
Test scale : 3 plants/plot
Soil : sandy loam (alluvial soil)
Interval of spraying : 1-3 times at intervals of a week
from the early silking stage
Day of investigation : harvested and surveyed after the
lapse of one week from the day of the
third sprinkling
Examination items : length, diameter and weight of corn
ear, length of an unertile tip portion of
ear, kernel number per column, column
number of ear, number of vacant kernels
and total kernel number
Table 3 shows a result of the test. Below is a detailed
information on the individual examination items.
(1) The effect on the ear length:
The treatment with brassinolide showed a tendency of
increaslng the ear length somewhat within the range of 0-15%.
(2~ The effect on the diameter:
In each test area, the treatment with brassinolide showed
a tendency of increasing the diameter of ear somewhat within
the range of 2-14%.
(3) The effect on the length of an unfertile tipportion of ear:
In the plot where spraying of the aqueous brassinolide

~2~680
solution was carried out 2 times or 3 times, the length of
an unfertile tip portion of ear was significantly shortened.
In particular, in the plot where brassinolide was applied
at a concentration of 10 ppm, the length o~ an unfertile tip
portion of the ear was shortened to 39-46~ of the case
observed in the untreated control. In the plot where bras-
sinolide was applied at a concentration of 10 4 ppm, the
length was shortened to 25-36%. Thus, the treatment with
brassinolide obviously enhanced the ripening of the ear.
(4) The effect on the weight of ear:
In each plot, the weight of ear became greater than that
in the untreated control. The treatment with brassinolide at
concentrations of 10 ppm, 10 2 ppm and 10 4 ppm gave 25%,
33~ and 18~ increases (an average value in case of the
spraying by 1-3 times) respectively, in the weight of ear.
(5) The effect on the kernel number per column:
The treatment with brassinolide showed a tendency of
increasing the kernel number per column (the number of kernels
aligned vertically) somewhat within the range of 0-13~.
(6) The effect on the column number of the ear:
The treatment with brassinolide gave no influence on the
column number (the number of kernels aligned horizontally)
of the ear.
(7) The effect on the number of vacant kernels:
A remarkable result was observed for the number of vacant
kernels. In any of the test areas where brassinolide was
applied at a concentration of 10 ppm, the number of vacant
kernels was less than 10~ as compared with that in the
untreated control area. In the test areas where the concent-
3Q ration of brassinolide was 10 ppm or 10 4 ppm, the number

1~ti51i80
of vacant kernels was 11-24% or 15-35%, respectively.
(8) The effec-t on the total kernel number:
The treatment with brassinolide showed a tendency of
increasing the total kernel number within the range of 10-25%.
The foregoing results apparently show that the treatment
with brassinolide at the silking stage exhibited a tendency of
increasing the yield in various items represented by this
except the column number of the ear. In general, the effect
on increasing the yield of corn is determined by increase or
decrease in the length of an unfertile tip portion of the ear
and in the number of vacant kernels. The treatment with bras-
sinolide gave the most significant influence on these two
examinatlon items as compared with the untreated control.
It is considered therefore that the effect on increasing the
yield of corn by the treatment with brassinolide i5 exhibited
by enhancing the ripening of kernels at the top portion which
are usually hard to be ripened and decreasing the number of
vacant kernels.

iSg.i80
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-- 31 --

~656~0
Example 4
This example illustrates the effect of brassinolide on
increasing the yield of paddy rice in direct incorporation of
paddy field with rice seeds. Seeds of paddy rice (variety:
Musashikogane) were dipped before hastening of germination
in an aqueous solution of brassinolide having a concentration
of 10 ppm for 24 hours, germinated at 30C, and then coated
with Calper (Hodogaya Chemical Co., Ltd., Japan) in a ratio
of 10:8 and directly inccrporated into flooded paddy field.
The test conditions used were as follows:
Day of sowing : May 11th 1984
Type of sowing : incorporation of the flood paddy
field with the seeds at an interval
of 1.5 cm in rows (an interval between
the adjacent rows being 30 cm)
Depth of the incorporated seeds : 1 cm (covered with
diluvial soil)
Quantity of the incorporated seeds : 50a g/are
Scale : 20 m2/plot (duplication/test)
Day of harvest : October 2nd
Examination items : plant height and number of tillers
(on July 24th), weight of unhulled rice,
1000-kernel weight, and number of ears
(after harvest).
Table 4 shows a result of the tes~. Below is a general
evaluation on the individual examination items.
An a~erage plant height was not so influenced by the
treatment with brassinolide but the number of tillers was
increased by 7% as compared with the case of the untreated
control. The nu~ber of ears was increased in the treated plot
- 32 -

~56~
by 15% as compared with that in the untreated con~rol, but no
change was observed in 1000-kernel weight. Thus, the weight
of unhulled rice per unit test area was 17% greater thanthatin
the untreated control. .
Table 4
The yield-increasing effect of brassinolide on paddy
rice in case of treating the seeds before hastening
of germination
. Concentration Average Number weight 1000- N.umber of
10of plant of of Kernel ears
brassinolide height tillers unrhuiceed weight 2
ppm cm g/3.3m gNumber/3.3m
: . 2 ~O (%) (%) (%) (%)
10- 67.8 1275 2018 21.3910
. (101) (107) (117) (99)(115)
Control 67.1 1192 1725 21.52 791
. (100) (100) (100) (100)
Remarks: The values in parentheses are relative numbers
(in percentage) in case of the values in the
untreated control being 100.
Example S
Using a paddy rice (variety: Musashikogane), an aqueous
solution of brassinolide having a concentration of 10 ~ or
10 4 ppm was sprayed by a foliage treatment at certain growing
; stages, i.e. the maximum tillering, the young panicle forma-
tion stage and the anthesis stage, to check the influence of
brassinolide and its application time on increase of yield.

12~àS6~0
The test conditions employed were as follows:
Day of transplantation : May 17th 1984
Day of the beginning of heading : August 12th
Day of harvest : October 8th
Location of the test: Paddy field
Test scale : 10 m2/plot (duplication/test)
Spraying time : the maximum tillering stage
(on June 27th)
the young panicle formakion stage
(on July 20th)
the anthesis (on August 14th)
Amount of the solution sprayed : 15 liters/are
Examination items : yield, number of ears, average ear
weight, 1000~ernel weight and number
of grains per ear and rachis
Twenty hills per plot were harvested to check the yield,
the number of ears,the average ear weight, the 1000-kernel
weight and the number of grains per ear. The number of
grains was checked as per whole ear and as per the lst-4th
rachises counting downwardly from the ear top (the upper lst-
4th rachises) and rachises located in positions lower than
the 4th rachis (the lower rachises), respectively.
A result of the test is shown in Tables 5 and 6 and
briefly summarized below.
In the plot where the aqueous solution of brassinolide
was sprayed wholly over the paddy rice at the maximum tiller-
ing stage~ no change was observed in the number of ears but
the number of grains per ear was increased by 11-13% at each
concentration as compared with that in the untreated control
so that an average ear weight of the hills in the treated plot

12~5680
was increased by 5-7%. Consequently, a yield-increasing
effect as high as 10% by ear weight per 20 hills could be
recognized at any concentration of brassinolide. In this case,
the number of grains in the lst-4th rachises counting down-
wardly from the ear top was 7-9% greater than that in the
untreated control. Contrary to this, the number o~ grains in
the lower rachises was 15-17% greater than that in the un-
treated control, thus showing a tendency that the increasing
rate became higher as the grains were located in the lower
part of ears. In the test area where the spraying was carried
out at the young panicle formation stage, the number of grains
was increased by 9% in the rachis lower than the 4th rachis
irrespective of the concentration of brassinolide as in the
case of the plot where the spraying was carried out at the
maximum tillering stage, but the number of grains was
increased only by 2-3% in the upper lst-4th rachises so that
the number of grains per ear was increased by 5-6% as
compared with that in the untreated control. As the 1000-
kernel weight was slightly decreased to 98% of that in the
untreated control area, however, the ear weight per 20 hills
was almost equal to that in the untreated control. In the
; treated plot where the spraying was carried out at the
anthesis, an average ear weight, the 1000-kernel weight and
the number of grains were all equivalent to those in the
untreated control, and no difference was found in the ear
weight per 20 hills in both plots.

5ti~0
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- 36-

1~ti568~:)
Table 6
Distribution of the ear weights of paddy rice
treated with brassinolide
(Spraying at the maximum tillering stage)
trationl ga~ wei ght (g) ¦
of BR 0~0.75~1.0^-1.25,^-1.5~1.75-~2.0~ 2.25--
(ppm) 0.75 1.0 1.25 1.5 1.75 2.0 2.25 2.5 2.5
_ ,
1o~2 3.35.111.416.6 21.1 21.1 14.7 5.7 1.0
10-4 2.36.59.0 19.7 21.4 23.6 ~2.6 4.6 0.7
________ _____ ._____ _____ _____ ,_____ _____ _____ _____ _____
Control 3 S a 115 . 4 20 . 4 24 . 313. 4 6 .5 2 . 7 1. 2
(B)
Consen- _
tration Ea Weig] It (~
Of BR o ~ 0 ~ o ~ 'o ~ o ~ o _ 0 ~ o _
(ppm) 0.751.0 1.25 1.5 1.75 2.0 2.25 2.5
. . _
lo~2 3.38.4 19.8 36.4 57.5 78.6 93.399.0
10-4 2.38.8 17.8 37.5 58.9 82.1 g4.799.3
_______ _ ____ ___ ______ _____ _____ _____. ._____ ______
Con t ro 1 3.3 11.6 2 7 . 0 4 7 . 4 7 1 . 7 9 0 . 1 9 6.6 99.3
Remarks: (:L) "BR" means brassinolide.
(2) In Table (A), each range is-sectioned by 0.25g.
(3) In Table (A), each value stands for "total ear
weight" in terms of~percentage belonging to a
relevant weigh't range.
_ 37 -

~.265680
(4) In Table (B), each value stands for a total
sum of the values belonging up to the relevant
range in Table (A) in terms of percentage.
Example 6
A plurality of pots charged with soil were incorporated
with seeds of Soybean (variety: Enrei) and 2 or 3 plants of
the soybean were allowed to exist per pot and subjected to
a test under field conditions according to the testing
method as shown in Table 7. A solution of brassinolide in
an amount of 100 ppm in ethyl alcohol was diluted with a
1:5000 aqueous solution of Nitten and the diluted solution of
brassinolide was sprayed at a concentration of 10 2 or 10
ppm in a sufficient amount all over the soybean plants. The
cultivation of the soybe~n was carried out in a usual manner
and the location of each pot was changed every week.
A survey was made for each examination item in the test
runs I-IV as described in Table 7. Shown below are results of
the tests accordlng to the individual examination items. In
addition, the results of the tests obtained in the test runs
20 ¦ I-IV aee ~ho~n n ~a~les 8 and 9 an~ ~igs. 6-9.
'
- 38 -

~.2~5ti81)
Table 7 Testing method
Test
run Method ~or BR treatment Examination items
I After germination, 3 plants Two weeks after the B~
per pot treated with BR over treatment, the growth of
the consecutive two days at the plants were checked.
the stage of the primary leaf
expanding.
II Seeding was made on June 15th In the test area where
and, after germination, 2 the plants were treated
plants per pot of 1/5000 a. with BR at unfolding of
(are), were supplied with 2g the 4th trifoliolate leaf,
of a compound fertilizer (N, the plants were examined
P2O5 and K2O in a ratio of 10 days after the treat-
12:15:15) as a basic manure ment for photosynthesis inand treated with BR over the internal factors within
consecutive two days at
unfolding of the 4th tri- the leaf and 20 days after
foliolate leaf and after the treatment for the
the full expansion of the growth of thearPea where the
4th trifollolate leaf
plants were treated wlth
BR after the full expansion
of the 4th trifoliolate
leaf, the plants were
examined on the next day
of the treatment for photo-
synthesis in-the leaf and
. the relative factors
within the leaf.
III Seeding was made on June 16th The plants were harvested
and 3 plants per pot of on October 3rd and a
1/2000 a.(are), were sup- surve~ was made for
plied with 8g of a compound dimension of plant part,
fertilizer (N, P2O5 and K2O yield components and seed
in a ratio of 12:15:15) and weight.
4g of potassium magnesium
sulfate (55% in ratio of
K to Mg) and treated 4 times
with BR at àn interval of
6 days in the period.f~om
unfolding of the 3rd trifolio-
late leaf to flowering.
IV Seeded and fertilized in the Same as in the test run III.
: same manner as in the ~est
run III, and treated 7 times
with BR at an interval of 6
days in the period from
flowering to ripening.
..
j~ P~emarks "bR" means brass:i~nol ~de
.. - 39 -

1265680
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- 40 -

~lX6568~)
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Results of the tests:
1. The influence of the BR treatment on the growth
of soybean:
According to the methods of the test runs I-IV, soybean
was treated at several stages of growing with brassinolide
to ascertain the effect of brassinolide at each stage of
the growth of soybean.
(1) The BR treatment at the young seedling stage (during the
period of the primary leaf expanding)(the test run I):
At a concentration of 10 ppm, elongation of the epicotyl
was accelerated by 43% as compared with the case of the un-
treated control, but expansion of the primary leaf and the
first trifoliolate leaf was inhibited by 11-14~. At a
concentration of 10 2 ppm, a sl~ghtly accelerating effect
was observed in the growth of each organ but no significant
influence was considered in this respect~
(2~ The BR treatment at the seedling stage (at the stage of
unfolding the fourth trifoliolate leaf)(the test run II):
(a) Growth of stem : At a concentration of 10 2 ppm,
elongation of the third internode (at the middle of the
rapid elongation stage) and the fourth internode (at the
beginning of the rapid elongation ~tage) was accelerated
by 45% and 65%, respectively, as compared with the case
of the untreated control. By the BR treatment, elonga-
tion of the second internode (at the end of elongation)
was promptly stopped. At a concentration of 10 ppm,
an accelerating effect on elongation of internodes at
the middle of the elongation stage became weaker as
compared with the case at a concentration of 10 2 ppm,
and an inhibitory effect on elongation of internodes at

~L2~56~0
the end of the elongation stage hecame rather strong.
(b) Growth of leaves : In case of the BR treatment at
a concentration of 10 2 ppm, no significant influence
was observed on the leaf area, dry weight and petiole
length of the fourth trifoliolate leaf, but the BR treat-
ment at a concentration of 10 ppm gave a significant
influence on these factoxs. At the concentration of 10
ppm, the leaf area and the dry weight of the fourth
trifoliolate leaf were both decreased by 19% as compared
with the untreated control, but the petiole length became
longer by 12%.
(3) The concecutive BR treatment during the period from the
unfolding of the third trifoliolate ~eaf to the beginning of
l flowering stage (the test run III):
15¦ In case of a consecutive BR treatment at a concentration
of 10 ppm, the leaf area became smaller while the petiole
length became longer as shown in ~able 8. The test run III
was similar in this respect to the test runs I and II. No
l significant influence of the BR treatment was found on the
20¦ other dimensions of plant parts as shown in Table 9. The B~
treatment scarcely gave influence on the node order of first
¦ branching, the number of branches, the number of nodes on
branches and the number of nodes on the main stem.
(4) The consecutive BR treatment during the period from the
5 beginning of the flowering to the ripening (the test run IV):
A result of this test was almost same as that in the test
run III.
2. The influence of the BR treatment on photos~nthesis
and matt~r production:
~ ~11 The BR treatment before unfolding of the fouxth trifolio-

~ ;~656~0late leaf:
When the BR treatment was carried out at the stage of
unfolding the fourth trifoliolate leaf and the measurement
was made 10 days after the treatment, the photosynthetic
rate (CER) was somewhat increased. Both chlorophyll content
(ChQ a~b) and mesophyll conductance (gm) were also increased
slightly, but the stomataLl conductance (gs) was not changed by
the BR treatment at a concentration of 10 2ppm and was reduced
by 12~ by the BR treatment at a concentration of 10 ppm as

iL;~6~i80
compared with the untreated control.
(2) The BR treatment after fully expansion of the fourth tri-
foliolate lea~:
When the BR treatment was carried out af~er full
expansion of the four~h trifoliolate leaf and the measure-
ment was made on the next day of the treatment, the photo-
synthetic rate was enhanced by 10% at a concentration of 10 2
ppm and by 13% at a concentration of 10 ppm as compared
with the untreated control. In case of "gm" and "gs", the
values were also slightly greater than those of the untreated
control. No change was found in the chlorophyll content
measured 10 days after the BR treatment.
When the BR treatment was continued before or after
flowering, the chlorophyll content in leaves was slightly
increased in all trea~ed plots, thus exhibiting the effect of
preventing the senescene Qfleaves as shown in Table 9.
3. The influence of the BR treatment on pod-setting, seed-
setting and yield:
~1~ The influence on pod-setting and seed-setting:
In case the soybean plants were treated consecutively
with brassinolide before flowering (the test run III), no
difference was found in total pod number between the treated
plants and the untreated control, but the ratio in pod number
of the branches to the main stem was significantly changed.
By the BR treatment the pod number of the main stem was
decreased by 16-18% as compared with the untreated control,
but contrary to this, the pod number of the branches was in-
creased by 12~. It was also recognized that the seed number
per pod was increased by 6-9% by the BR treatment.
3Q In the test run IV wherein the soybean plants were treated

~s~
consecutively with brassinolide aftex flowering, the same
tendency as in the treat~ent before flo~ering (the test run
III) was seen with respect to the total pod number per plant
and the ratio in pod number of the branches to the main stem
in the treated plot at a concentration of lO 2 ppm. However,
the difference in the ratio in pod number of the branches to
the main stem became smaller. In the treated plot at a
concentration of 10 ppm, the pod numbers in the branches and
the main stem per plant were decreased as compared with those
of the untreated control, but the BR treatment gave no
influence on the seed number per pod.
(2) The influence on yield:
In the test run III, the seed weight in the main stem per
pot was decreased by 15% by the BR treatment as compared with
15¦ the case of the untreated control, but the seed weight in
l branches was increased by 18-21%, thus increasing the total
¦ seed weight per pot by 8%. The weight per seed in the main
stem was decreased by 3-5% by the BR treatment, but no
l influence on that was observed in the branches by the BR
2a ¦ treatment.
In the test run IV, no change was found in the seed
weight in branches per pot by the BR treatment but the seed
weight in the main stem was lightened by 9% at a concentra-
tion of lO ppm and by 20% at a concentration of la ppm as
compaxed with the cases of the untreated control so that the
total seed weight per pot was decreased by 3% and 8%/
respectively. No significant difference was found in the
weight per seed in the seeds either produced in the branches
or in the main stem, as compared with the untreated control.
3Q Of the c~r~phs of Figs. 6(~) and 6(B2 showing results o~

1~656~30
the test runs I and II, respectively, the graph of Fig. 6(A~
shows a result, as average graphical data, of the test wherein
soybean seedlings were treated twice with brassinolide during
the period of the primary lea expanding and 3 plants per
pot (5 pots per treatment) were checked 2 weeks after the
treatment and the graph of Fig. 6(B) shows a result, as average
graphical data, of the test wherein soybean seedlings were
treated twice with brassinolide at the stage of unfolding the
4th trifoliolate lea~ and 2 plants per pot ~5 pots per treat-
ment) were checked 3 weeks after the treatment. In Fig. 6(A),the left-hand ordinate ~tands for leaf area (cm2) while the
right-hand ordinate for the length (cm~ of various organs other
than leaf. In the graph, lines with white circles stand for
leaves while lines with black spots stand for organs other
than leaf. The line a stands for the first trifoliolate leaf,
the line b for the primary leaf and the line c for the second
trifoliolate leaf. On the other hand, the line _ stands for
epicotyl, the line e for hypocotyl, the line f for the first
internode and the line ~ for the second internode. In Fig.
6(B~, the upper part of the left-hand ordinate stands for the
leaf area in terms of cm2 and the lower part for the petiole
length in terms of cm while the upper part of the right-hand
ordinate stands for the leaf weight (dry) in terms of g and
the lower part for the internode length in terms of cm. The
line a relates to the leaf area, the line b to the leaf weight
~dry) and the line c to the petiole length. Regarding the
internode length, the numerals attached to the bold solid
lines with b:Lack spots stand for the corresponding internodes;
e.g~ the line 2 stands for the second internode and the line 5
3~ for the ~ifth inte~node. ~n e~ch graph, the ~bscissa stands
.
- ~8 -

12656~30
for the concentration of brassinolide used for the treatment
(in terms of ppm). Fiy7 6(~) apparently shows that expansion
of the leaves was strongly inhibited and the elongation o~
epicotyl was strongly influenced by the treatment with bras-
sinolide but no influence was observed in the growth of otherorgans. Fig. 6(B) shows that the area and dry weight of the
leaf was decreased by the treatment with brassinolide but the
petiole length was increased and a significant influence was
found on the internode length.
In Figs. 7(A) and 7(B), the upper part of the left-hand
ordinate stands for the photosynthetic rate (CER) in terms of
C2 mg/dm /hr and the lower part for the mesophyll conductance
[gm~ in terms of cm/sec, while the upper part of the right-
hand ordinate stands for the stomatal conductance (gs) in
terms of cm/sec and the lower part for the chlorophyll content
~Chl~ in terms of mg/dm . The abscissa in each graph stand
for the concentration of brassinolide used for the treatment
in terms of ppm. The graphs of Figs.7(A) and 71B) show the
in~luence of the treatment with brassinolide on the photo-
synthesis and its related factors of the fourth trifoliolate
leaf 7 The graph of Fig. 7~) shows a result, as average
graphical data, of the plot wherein the soybean plant was
treated twice with brassinolide at unf~lding of the 4th tri-
foliolate leaf and 4 leaves per-plot were checked 10 days
after the treatment, The graph of Fig. 7(B) shows a result,
as average graphical data, of the test wherein the soybean
plant was treated twice with brassinolide after the full
expansion of the 4th trifoliolate leaf and 4 leaves per plot
were checked on the next day of the treatment.
Figs. 8~A~ and 8~Bl are graphs showing the influence of
- 49 -

~2~i~;680
the treatment with brassinolide on pod-~setting and seed-
setting of soybean. In case of Fig. 8(A), the graph shows
a result, as average graphical data, of the test run III
wherein the soybean plants were repeatedly treated with
brassinolide (up to 4 times) be~fore flowering and 3 plants
per pot (8 pots per treatment) were checked after harvest.
In case of Fig. 8(B), the graph shows a result, as average
graphical data, of the test run IV wherein the soybean plants
were treated up to 7 times with brassinolide after flowering
and 3 plants per pot (8 pots per treatment) were checked
after harvest. In Figs~ 8(A) and 8(B), the upper part of the
left-hand ordinate stands for the total pod number per plant
and the lower part for the pod number of branches per plant
while the upper part of the right-hand ordinate stands for the
pod number in the main stem per plant and the lower part for
the seed number per pod. In each graph, the abscissa stands
for the concentration of brassinolide used for the treatment
(in terms of ppm2 and the values in parentheses are relative
values in case of the value in the untreated control beiny
100. For convenience's sake, the data on branches are shown by
broken lines with black spot and the data on the main stem are
shown by solid lines with white circles.
Figs. 9~Al and 9(B2 are graphs showing the influence of
the treatment with brassinolide on the yield of soybean in
the test runs III and IV, respectively. In graph of Figs.
9~AI and 9(B2, the upper part of the left-hand ordinate stands
for the total seed weight (in terms of g/pot), the middle part
for the seed weight of branches (in terms of g/pot2 and the
lower part for the weight per seed of branches ~in terms of
g~seedl while the upper part of the right-hand ordin~te stands

~ lZlj56~30
for the seed weight of the maln stem ~in terms o~ ~/pot~ and
the lower part for the weight per seed of the main stPm (in
terms of g/seed). The results shown in Figs. 9(A) and 9(B)
are average data from 3 plants per pot (8 pots per treatment)
and the values in parentheses are relati~e values in case of
the value in the untreated control being 100. As in the case
of Figs. 8(A) and 8(B), the data on branches axe shown by
broken lines with black spots and the data on the main stem by
solid lines with white circles. Vertical bar represents the
least significant difference between means (p = 0.05).
Example 7
A tuber of potato ~variety: Danshaku) was divided into
3 portions and each portion was dipped in an aqueous solution
of brassinolide at a given concentration for 24 hours and
incorporated into soil. In a plot whexe an aqueous solution
o~ brassinolide was sprayed by foliage treatment, the solution
at a concentration of 10 4 ppm was sprayed twice all over the
plant at the stage of flowering. After harvestt the number of
tubers per plant and the weight of tubers were investigated
2Q to evaluate the yield-increasing effect of brassinolide. The
test conditions employed were as follows:
Day of incorporation: March 8th 1985
Test place : crop field
Test scale : 60 cm x 4 m/plot (triplication
Soil : Volcanic ash earth
Treatment : ~Dipping treatmentl
One day before the incorporation, a tuber of
potato divided into 3 portions ~as dipped in
an aqueous solution of brassinolide at a
3Q concentxation o~ 10 3, 10 4 OX lQ 5 ppm for
- 51 -

1~5680
24 hours.
(Foliage treatment)
An aqueous solution of brassinolide having
a concentration of 10 4 ppm was sprayed
twice over the whole plant at the stage of
flowering in a volume of 20 ml per plant.
Examination items : the number of tubers per plant and
the weight of tubers (tubers in a size smal-
ler than a table-tennis ball are omitted.)
A result of the test is shown in Table 10 below. Below
is a general evaluation on the individual examination items.
In the plot where the tuber was subjected to the dipping
treatment, the number of tubers was increased at each concen-
¦ tration by 17-20% as compared with the untreated control.
On the other hand, an average weight of tuber was slightly
¦ increased in the plot where the concentration of brassinolide
was 10 4 ppm, but the weight was slightly d creased in the
plots where the concentration was 10 3 and 10 5 ppm. Thus~
l the yield per plant was increased in each of the plots where
20¦ the concentration was 10 3, 10 4 and 10 5 ppm in comparison
¦ with the untreated control, showing increase by 16%, 20~ and
¦ 10%, respectively. It is considered therefore that the increase
in the yield is chiefly ascribable to increase in the number
of tubers`per plant.
~5 Contrary to the plots where the dipping treatment was made,
an average weight of tuber was somewhat decreased in the plot
where the foliage treatment was made although a tendency of
slightly increasing the number of tubers per plant was observed.
Consequently, the yield per plant in the plotswhere the foliage
treatment was made was almost same as in the untreated control.
- 52 -

~L2~i56~0
It is understood that the preceding representative
examples may be varied within the scope of the present
specification both as to the sorts of plants and the treating
conditions by one skilled in the art to achieve essentially
the same results.
As many widely different embodiments of this invention
may be made without departing from the spirit and scope
thereof, it is to be construed that this invention is not
limited to the specific embodiment:s thereof except as defined
10 ~ in the pen~ed claims

~ 6568l)
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