Note: Descriptions are shown in the official language in which they were submitted.
l 1 '7 3 f~
MET~OD FOR PROMOTING PL~T GROW rH
BACKGROUNI:) OF IrHE IN VENTION
1. Field o The Invention
This invention relates to a me~hod of promo~cing
plant growth and more particularly to the use of
certain synthetic brassinosteroids to pro~ote plant
cell elongation and cell division and th~reby increase
~he vegetative growth of pLants. , ;
2. Description of Ar~ -
Plant growth substanoes occupy an important
place in the growth and developme~tal processes of all
plant s~ecies. The pioneers in plant growth sub3tances,
Charles Darwin, Boycen-Jensen, and o~hers, recognized
that plant grow~h phenomenon was under control of
~ome chemical ~ubstances produced by the ~;lan~s an~
in 1928 F. W. Went successfully demonstrated the
existence of growth-regulating substances in plants.
- The plan~ growth hormones, au~ins f~om oat seedlin~s,
a~d gibberellins from a fwngus, and several secondary
pLant produc~s such as phenolics, l~pids, s~eroids and
terpenoids also were shown to be r~sponsible or plan~
growth and de~elopment. Some of the lat~er elici~
growth responses in conjunction with the endogenous
growth hormones. Cer~ain syn~hetic compo~nds, although
diferent than the na~u;~al growth subs~ances, also
~nduce ~i~ilar biologiGal responses.
BrassinoLide (2~,3~,22~,23~-teerahydroxy-24~-~ethyl-
B-homs-7~oxa-5a-cholestan-6-one) isolated rom pollen of
.. . . .
rape, Brassica na~us L., produces a nov~l growth~pro~oting
efect when applied to young pinto bean plants. 1173659
SUMMARY OF THE INVENTION
An obJect of this invention i5 to provide synthecic
brassinosteroids for increasing vegetati~e plan~ growth,
biomass production, and crop yield.
~ nother obj ect is to provide suitable methods by
which the synthetic brassinosteroid~ may be applied -
to optimize their effects.
S~ill another object ls to prov~de ~Ra~S ~or
determining appropriate conditions ~or applying ~he
synthetic brassinosteroids.
! A further obiect is to provide suitable dosages
of the synthetic brassinosteroids to obtain the desired
enhancement o plant growth.
A still further object is to provide synthetic
brassinosteroids tha~ are economically feasible to make
and us e .
According to ~his invention the above objects are
accomplished by a method wherein plant cell elongation
and cell div~sion and thereby plant growth is promo~ed
by applying in a ~uitable manner to a pla~t an effectivc
growth promoting amount of a polyhydroxylated
steroidal Lactone having the general for~ula
. .
F~
~a~
.O . O~ 2
~173659
in which R is selected from ~he group consisting of
H~
'~f~ I HO~
`~W VI
HO~ I t .
`'~' II H~ VII
ÇH~ - ;
III H~ 1~, ~t 2
` ~ VIII
- H~
. IV ~nd
~- V ~ IX
Hereinafter ~ the compounds will be ref rred to
by the R substituen~. For example, ~he compound of
the general formula in which R is substituent IV
1~ will be referred to as compourld IV.
_ _ _ . _ . _ .. . .. . . . . . . . . . . . . .. .
~ 3 fi -~ ~
. ~ESCRIPTXON OF THE INVENTLON
Synthetic brassinos~eroids promote plant cell
elongation and cell division which are essen~ial for
the normal growth o~ higher plants. In accordance
with this invention, brassinosteroids when applied to
seedlings under greenhouse and ~ield conditions
increased the vegetative growth of all plants thus far
examined as well as the cxop yield of several varieties
. of vegetables, including le~tuce, radishes, bush beans,
and barley. Ex~erimental evidence indicates that the
growth promoting effects caused by brassinosteroids
work in conjunction with the intact plant endogenous
hormones, such as auxins, gibberellins and cytokinins.
Thus, the mode and me~hod of applica~ion, conditions
for application, and dosage, are vital parameters
for the brassinosteroids to be effective.
The brassinosteroids were first tes~ed in the
pinto bean firs~ internode test system which has come
to be used as one of several model biological systems
~or evaluating plant growth promoting activi~ies o~
brassinosteroids and o~her plant growth regulators.
In this test the biologically ac~ive brassinosteroids
enhance the growth of isolated bean first internode
sections. This response is dependent upon the presence
of an external auxin supply and the dosage o the
chemicals.
Known quantities of the brassinosteroids wexe
firs~ dissolved in a specific volume of ethanol, then
ten microliters were transferred ~o a s~all filter
~ 6 lj 9
. paper di~c, ~he disc dried, ~nd then ap~lied ~o one
side and nea~ ~he base of the internode section that
had been pre~reated in a similar manner with 10 ng
of auxin. Both the filter paper disc and the plant
sections are in close con~act wi~h a water satura~ed
sponge r~hich serves as a continuous source of
moisture that is essential for sustaining the growth
of the plan~ section.
The rela~ive biological aetivity of the various
brassinosteroids was determined from a series of
regression lines which, as seen in Table 1, yields
a good estimation of the activities of the brassino-
steroids. For exam~le, for every ten fold încrease
in the amount of compound IV, ~he xelative ra~e of
growth increases by 2.947 mm/hrtng over that of the
` control. In other words, one nanogram o compound IV
enhances the auxin induced cell enlargement almost
three fold, thus resulting in increased grow~h of the
plan~.
- 20 Interestingly, similar compounds that contain
only the cholesterol side chain or the cholesterol
side chain with an u-orien~ed methyl at C~24 caused
no growth response. Thus substituent~ at C-22, C-23,
and C-24 that are present in the side chain o ~he
brassinos~eroids of this invention are also necessary
reauiremen~s for the plant growth promoting effects
of brassinosteroids.
Com~olmds tha~ were active in the first internode
test system were further examined for their plan~
growth~promo~ing efects on young see~lings grown under
.1. ~.7:3fi~
. . ~ . .
greenhouse conditions. Brassinosteroits a~ concentra-
tions of 1. 0 or 10. 0 ~g per 10 mg of lanolin were
applied to the axils of ~he first p ir of leaves of
10 da~ old bean seedlings. The trea~ed plan~s
produced more foliage and yielded a greater weight of
be~s ~han the control plarlts (Table 2).
The brassinos~eroids successfully promoted growth
of barley when applied to the ~lants by repeated
spray or leaf-dip methods. ~oncentrations of the
.la stexoids at Q. Q01, 0. Ql, and 0.1 mg/li~er were prepared
by initially dissol~in~ thP steroid in 1.0 ml of
ethanol and then diluting with water to a final volume
of one liter. Four drops of Tween 20 was added to each
stock solu~ion to increase wetting caDabilities. Any
surfactant that does not harm plant material may be
used in place of Tween 20. The barley plants were
sprayed lO days after planting and again 2 and 4 days
after the first application. S;milarly prepared
solu~ions were used in the leaf-dip application method.
In this case 10 ml of each stock solution was transferret
to a scintillation vial and placed into a 4-inch pot
that contained five plants. The oldest leaf of each
plan~ was placed into the solution and the leaf was
held in place with a cotton plug. Plants were measused
two weeks after initia~ion of treatments. Compound III
caused an increased grow~h in barley when applied by
bo~h the repeated spray and the leaf-dip method.
In rield plots, lettuce in 20 f~ot rows of 20-25
plants per row with 4 rows per plot and radishes in
--6--
~1736 A~ ~
5 foot rows and one oot apart were trea~ed as young
I emerging seedlings by spraying wi~h a solution of
i brassinosteroid III at concentration of 0.01 mg/li~er.
Spray applications were repea~ed twice at ~wo day
intervals to assure ~hat all seedlings received
treatment as the plants emerged from the ground.
Normal cultural ~ractices and recommended insecticide
treatments were followed and continued weeding by
hand were conducted. The cro~s were harvested at the
end of their growing season and weighed. The yield
of lettuce and radishes were enhanced significan~ly
by treatment with brassinosteroid III. These field
resul~s witbi. lettuce and radishes are summarized in
Table 4 along with the summary of the results of
other untreated and brassinos~eroid-treated crops.
Table 1 .~.17~365
. .,
Enhancement o~ auxin activi~y by brassinosteroid~
in ~he bean first-inte~node bioassay
Increase of growth over
- Compound the auxin treated control
%
_____.
III 244.0
IV 295.0
VI 145.0
Table 2
The ef~ect of the brassinosteroid I~I on foliage,
plan~ weight and fruit production of bush
beans four weeks after treatment.
~g of III per 10.0 mg
lanolin per plant
0.0 1.0 10.0
Total l~af area -/ .
(cm2)/plant 493.00 734.00 692.00
Leaf area (cm2)/ 1/
lea~ 43. sa 56.80 59~60
Total fresh weightl/
(gm)/plant 17.02 25.03 24,27
Fresh weight of 1/
fruit (gm)/plant 13.24 20.02 19~18
1/ Averages from 10 plants per treatment.
_ . _ _ .. , _ . .. . , . .. _ . .. ... . . ...
Table 3 ~ ~ 73fi5(3
.
The er~fects of brasslnosterold III
applica~ions on ~he growth o~ barley
Coneen~ra~ion Spray ap~lications Lea-dip
of III 2X 4X anplication
( continuous
~eeding)
Average le,~gth in mm 1/
Control 252 . 70243 . 90 253 . 30
0.001 mg/liter 271.30Z96.90 285.30
0 . 01 mg/li~er 279 . 80 270 . 20 289 . 30
O .1 mg/Liter 280 . 6~` 276 . ~60 2~4 .~
/ 10 plants/treatment
Table 4
The yield of harvestable products from
untreated and brassirlo~teroid III
treat~ crops
Crop Untrea~edl/ Treatedl/ (~rowth
S timulation
gm. gm. %
Lettuce (var. )
Salad BowlJ171. 38 215 .11 25 . 2
Salad Bowl3/189 . 45 221. 75 14. 6
GrPat Lakes2/727 .10 906 . 30 24. 7
Great Lakes3/942 . 94 1220 . 87 29 . 5
Radish 20 . 8 24. 9 19 . 8
Pepper 72 . 4 77 . 6 7 .1
Bean 15 . 3 16 . 2 5 .
Fresh weight ~er plant
~ Early Crop
--I Lace Crop 9
