U.S. patent application number 14/569141 was filed with the patent office on 2015-04-30 for composition and method for stress mitigation in plants.
This patent application is currently assigned to STOLLER ENTERPRISES, INC.. The applicant listed for this patent is Stoller Enterprises, Inc.. Invention is credited to Albert Liptay, Ronald Salzman, Jerry H. Stoller.
Application Number | 20150119247 14/569141 |
Document ID | / |
Family ID | 46927999 |
Filed Date | 2015-04-30 |
United States Patent
Application |
20150119247 |
Kind Code |
A1 |
Stoller; Jerry H. ; et
al. |
April 30, 2015 |
Composition and Method for Stress Mitigation in Plants
Abstract
A composition and method to mitigate plant autophagy and/or
apoptosis of newly developing cells in plants grown under
environmentally stressful growing conditions, such as high
temperature. Exogenous application of a cytokinin, preferably
kinetin, to either the roots or the foliage (i.e., flowers and
leaves) of plants has been discovered to overcome, or at least
substantially mitigate, autophagy when applied during or just prior
to flowering. Experimental results indicate that high
temperature-induced autophagy, and subsequent new cell apoptosis,
is the result of a deficiency of cytokinin in the plant tissues.
The application of low concentrations of potassium together with
the cytokinin appears to provide a synergistic effect by amplifying
the effect of the cytokinin to lessen autophagy and increase crop
productivity.
Inventors: |
Stoller; Jerry H.; (Houston,
TX) ; Liptay; Albert; (Houston, TX) ; Salzman;
Ronald; (College Station, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Stoller Enterprises, Inc. |
Houston |
TX |
US |
|
|
Assignee: |
STOLLER ENTERPRISES, INC.
Houston
TX
|
Family ID: |
46927999 |
Appl. No.: |
14/569141 |
Filed: |
December 12, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13433050 |
Mar 28, 2012 |
8932987 |
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14569141 |
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13429014 |
Mar 23, 2012 |
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13433050 |
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61469044 |
Mar 29, 2011 |
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Current U.S.
Class: |
504/124 |
Current CPC
Class: |
C05F 11/10 20130101;
A01N 43/90 20130101; A01N 43/90 20130101; A01N 59/00 20130101; A01N
2300/00 20130101; A01N 59/00 20130101; A01N 47/36 20130101 |
Class at
Publication: |
504/124 |
International
Class: |
A01N 43/90 20060101
A01N043/90; A01N 59/00 20060101 A01N059/00 |
Claims
1-7. (canceled)
8. A method of farming comprising the steps of: readying an
autophagy-inhibiting agent for application to growing plants, said
autophagy inhibiting agent consisting of a plant hormone, said
plant hormone including a cytokinin, readying a potassium salt for
application to said growing plants, and applying said
autophagy-inhibiting agent and said potassium salt in an aqueous
solution to the foliage or flowers of said growing plants, or to
the soil in which said plants are growing, during or just prior to
flowering, said aqueous solution applied to said growing plants
such that between about 0.09 grams to about 0.76 grams of cytokinin
are applied per acre of growing plants wherein, said potassium salt
is applied to the foliage or flowers of said growing plants, or to
the soil in which said plants are growing, at the rate of about
0.25 lb. to about 0.50 lbs. potassium salt per acre.
9. (canceled)
10. A method of growing plants comprising the steps of: readying a
plant hormone and potassium for application to foliage or flowers
of plants that grow in an average daytime temperature above about
20 degrees Celsius, said plant hormone having cytokinin as a
majority component, and applying an aqueous solution of said plant
hormone and said potassium to the foliage or flowers of said
plants, or to the soil in which said plants are growing, during or
just prior to flowering, said cytokinin applied to said plants at a
rate between about 0.09 grams per acre to about 0.76 grams per acre
wherein, said potassium is applied to the foliage or flowers of
said plants, or to the soil in which said plants are growing, at a
rate of between about 0.25 lb. to about 0.50 lbs. per acre.
11. (canceled)
12. The method of claim 10 wherein, said average daytime
temperature above about 20 degrees Celsius occurs during or just
prior to flowering.
13. The method of claim 10 wherein, said average daytime
temperature exceeds about 30 degrees Celsius.
14. A composition for mitigating environmental stress in growing
plants, said composition comprising: a plant hormone including
primarily a cytokinin, said cytokinin being present in an aqueous
solution of said composition at a concentration of between about
0.1 ppm to about 3.4 ppm cytokinin, said composition being an
amount effective to lessen plant autophagy under an average daytime
growing temperature above 30 degrees Celsius and a potassium salt
present in said composition at a concentration of between about 500
ppm to about 4,000 ppm potassium salt.
15. (canceled)
16. A composition for lessening environmental stress in growing
plants, said composition comprising: a plant hormone having
cytokinin as a majority component, said cytokinin being present in
an aqueous solution of said composition at a concentration such
that between about 0.09 grams to about 0.76 grams of cytokinin may
be applied per acre of said growing plants when said composition is
so applied, said composition being an amount effective to lessen
plant autophagy under an average daytime growing temperature above
30 degrees Celsius and a potassium salt being present in said
aqueous solution of said composition at a concentration such that
between about 0.25 lb. to about 0.50 lbs. of potassium salt may be
applied per acre of said growing plants when said composition is so
applied.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 13/433,050, filed Mar. 28, 2012 and allowed on
Nov. 17, 2014, which is a continuation-in-part of co-pending U.S.
patent application Ser. No. 13/429,014, filed Mar. 23, 2012, which
itself claims priority to U.S. Provisional Application Ser. No.
61/469,044, filed Mar. 29, 2011.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates generally to a method and composition
for mitigating plant autophagy or the degradation of older plant
cells to supply nutrients to deficient newly-forming plant cells,
which can occur during development (e.g., flowering) of new plant
cells under stressful conditions, such as high temperatures.
Moreover, this invention relates to the enhanced development of
seeds, and consequently, the increased yield of harvestable grains
from plants, including crop plants, that experience environmentally
stressful growing conditions during development and growth.
[0004] 2. Description of the Related Art
[0005] Traditionally, mineral fertilizers have been predominately
applied to growing crop plants. Difficulties arise, however, when
external stresses impede successful plant development, especially
of grain or seed crops and/or other crops. Physical stresses, such
as those inflicted by environmental temperatures being either too
low or too high, and in particular high temperatures, are
especially problematic. Moreover, the state-of-the-art agronomic
practice does not employ plant growth regulators to overcome a
plant's difficulty, due to such stresses, in producing sufficient
amounts of nutrients, e.g., sugars, to prevent autophagy (i.e.,
cannibalization of previously-formed plant cells by newly-forming
cells to compensate for a dearth of cell nutrients). It is well
known that mineral fertilizers provide sixteen minerals that are
necessary for crop growth and development. Signaling molecules,
such as plant growth regulators or other molecules, are known to
enhance crop productivity through the expression of certain genes.
Furthermore, much research has been conducted into the use of plant
growth regulators and their effects on plant growth and
development. However, until disclosure of the invention herein, it
has not been known that the application of certain "signaling
molecules" improves plant productivity by mitigating plant
autophagy caused by environmental stresses, such as high growing
temperatures.
[0006] Considering the sheer amount of research into techniques and
compositions to improve food production as well as the continual
need for greater food production to feed an exponential human
population growth, there is a long felt and unfulfilled need for
improved methods and compositions to improve plant productivities,
especially in view of higher environmental temperatures and other
harsher growing conditions.
[0007] 3. Identification of Objects of the Invention
[0008] An object of the invention is to accomplish one or more of
the following:
[0009] Provide a method and composition to enhance the productivity
and growth of crop plants;
[0010] Provide a method and composition to enhance the productivity
and growth of crop plants grown under harsh environmental
stresses;
[0011] Provide a method and composition to enhance the productivity
and growth of plants grown under high temperature conditions;
[0012] Provide a method and composition to increase the synthesis
of nutrients by plants;
[0013] Provide a method and composition to mitigate plant autophagy
and/or apoptosis;
[0014] Provide a method and composition to enhance productivity and
crop growth during the vegetative stages of crop growth, prior to
the reproductive stages of crop growth;
[0015] Provide a method and composition to enhance the seed size of
grain crops, and therefore crop yield, under high temperature
stress by increasing the availability of water for grain sizing;
and
[0016] Provide a method and composition to enhance the seed size of
grain crops and therefore crop yield under high temperature stress
by increasing the availability of water for grain sizing.
[0017] Other objects, features, and advantages of the invention
will be apparent from the following specification and drawings to
one skilled in the art.
SUMMARY OF THE INVENTION
[0018] The objects identified above, along with other features and
advantages of the invention are incorporated into a method and
composition for growing plants, especially crop plants, to be more
productive and/or resilient to stressful growing conditions, such
as high temperature. When growing temperatures are too high, the
development of flowers and subsequent embryos (seeds) is known to
be compromised, with the concomitant result that productivity of
crop grains or other types of agronomic harvest is impaired and
crop yields can be drastically decreased. Under extreme growing
conditions, such as high temperatures, plants experience an
inability to produce the nutrients, such as sugars, necessary for
conducting normal anabolic processes (i.e., flower and embryo/seed
development). To compensate for this dearth of nutrients, plants
growing under these stress conditions typically undergo autophagy,
or self-cannibalization, to secure the necessary nutrients to the
newly formed cells.
[0019] Exogenous application to the plant canopy (i.e. leaves and
flowers) of the plant growth regulator/hormone cytokinin has been
discovered to prevent such autophagy by inducing the necessary
production of sufficient nutrients (i.e., sugars) for the growth of
new plant cells (i.e., successful and complete seed development).
It is thought that genes controlling for increased levels of
photosynthates (i.e., plant sugar/energy producers) are triggered
by exogenous application of cytokinin. Additionally, the
application of low concentrations of potassium along with the
cytokinin has been found to substantially increase the effect of
the cytokinin. Such results are unexpected with such low potassium
concentrations, because they differ from the physiological effects
normally attributed to higher application rates of fertilizer-grade
potassium. It is thought that the application of low concentrations
of potassium act much like other signaling molecules (e.g.,
hormones) in stimulating transcription of particular genes, such as
the genes that express cytokinin effects, or provide an enhanced
level of energy or enhance other hormones that have an effect on
increasing yields such as abscisic acid or responsiveness to same.
The synergy from the application of low concentrations of potassium
and cytokinin to growing plants may also be realized under lower
stress growing conditions.
[0020] The disclosed composition and its method of application
represents a practical approach to mitigating plant autophagy, and
any ensuing apoptosis, that results from stressful plant growing
conditions, such as high temperatures. The method preferably
includes the application of a plant hormone, primarily a cytokinin,
to the foliage and/or flowers of plants at or about the time of the
beginning of plant flowering (e.g., during meiosis and when pollen
is about to enter dehiscence). This autophagy-inhibiting agent is
preferably the cytokinin, kinetin, however, other forms of
cytokinin may be used singularly or in combination, such as zeatin,
various forms of zeatin, N6-benzyl adenine, N6-(delta-2-isopentyl)
adenine, 1,3-diphenyl urea, thidiazuron, CPPU (forchlorfenuron) or
other chemical formulations with cytokinin-like activity.
Preferably, but optionally, a low concentration of potassium is
also applied together with the plant hormone to enhance the effects
of the plant hormone as previously described.
[0021] In a first step, the cytokinin plant hormone is readied for
application to the plants to be treated. The cytokinin plant
hormone is preferably applied to the plants as an aqueous solution.
Therefore, readying the cytokinin plant hormone may include one or
more of the following activities: diluting the cytokinin plant
hormone in sufficient water to create the desired concentration of
cytokinin in the applied mixture/composition, adding low
concentrations of potassium to the cytokinin plant hormone
mixture/composition to enhance the effects of the applied
cytokinin, loading the cytokinin plant hormone with or without
potassium (or an aqueous mixture thereof) into a sprayer or tank
for subsequent application to the plants to be treated, calibrating
the sprayer or dosing applicator to meter the desired amount of the
cytokinin plant hormone mixture to the plants to be treated and
transporting the cytokinin plant hormone with or without potassium
(or an aqueous mixture thereof) to the location of the plants to be
treated.
[0022] Preferably, the cytokinin concentration in an undiluted
aqueous solution ranges from about 0.01% to about 0.10%. A
commercially-available, undiluted cytokinin solution, X-Cyte (a
product of Stoller USA, Houston, Tex.), supplies the preferred
cytokinin concentration of about 0.04%. At the preferred cytokinin
concentration, the undiluted aqueous solution of cytokinin is
applied in a second step to plants to be treated at the rate of
between about 1/4 to 4 pints solution per acre of growing plants
and more preferably between 1 to 2 pints solution per acre of
growing plants. Such application equates to a rate of between about
0.09 to about 0.76 grams cytokinin per acre of growing plants
(diluted in 60 gallons of water per acre), and more preferably, at
a rate of between about 0.19 to about 0.38 grams cytokinin per acre
of growing plants (diluted in 60 gallons of water per acre).
Potassium, if applied with the cytokinin, is preferably applied at
very low concentrations. The potassium application rates are
preferably between about 1/4 lb. to about 2 lbs. per acre, more
preferably between about 1/2 lb. to about 11/2 lbs. per acre, and
most preferably about 1 lb. per acre. The cytokinin and/or
potassium can be applied either to the leaves, as stated above, or
to the soil at the same concentrations. It may be applied to the
soil in any appropriate fashion, such as, for example, in an opened
furrow near the plant roots, which furrow may subsequently be
closed. It may also be applied with various forms of irrigation,
such as overhead or drip tape, or furrow irrigation, among
others.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] By way of illustration and not limitation, the invention is
described in detail hereinafter on the basis of the accompanying
figures, in which:
[0024] FIG. 1 is a histogram of experimental results testing
whether impaired seed/silique development under high temperature
growth conditions is caused by a nutrient/sugar insufficiency,
which may incite apoptosis of newly-forming plant cells via
autophagy; and
[0025] FIG. 2 is a histogram of experimental data that verifies the
results obtained in FIG. 1, namely that high temperature yield
reduction, caused by autophagy, is mainly due to an insufficiency
of the plant growth regulator/hormone, cytokinin.
DESCRIPTION OF THE PREFERRED IMPLEMENTATIONS OF THE INVENTION
[0026] A preferred implementation of the invention addresses one or
more of the deficiencies of the prior art and incorporates at least
one of the objects previously identified. The invention employs a
plant growth regulator, preferably a cytokinin, which when
appropriately applied to plants has been discovered to enhance the
synthesis and transfer of sufficient nutrients, such as sugars, for
the growth and development of the reproductive parts (e.g., in
particular, the pollen) of plants grown under stressful conditions,
such as high temperatures. For the purposes of this invention, high
growing temperatures include growing temperatures above about 25
degrees Celsius (77 degrees Fahrenheit), but more commonly growing
temperatures above about 30 degrees Celsius (86 degrees
Fahrenheit). Even a temperature greater than about 20 degrees
Celsius (68 degrees Fahrenheit) may be considered a "high"
temperature, depending on the plant type (e.g., wheat barley and
rye) and/or locality (e.g., distance from the earth's poles). Such
high temperatures have been found to compromise crop plant
productivity. This is thought to be the result of the reduction of
cytokinin plant hormones in the plant due to the high
temperatures.
[0027] A reduction in the level of cytokinin in the plant tissues
incites autophagy--self-cannibalization--of healthy plant tissues
to provide the required nutrients for reproductive development. The
stress of autophagy can compromise seed formation (Cheikh et al.
1994), structural strength and/or physical integrity of the
reproductive organs (and thus successful egg fertilization) (Liptay
et al. 1994), cell arrangement and organ functionality (Lolle et
al. 1998), cell replication (Takahshi et al. 2008) and cell growth
(Szekeres et al. 1996). These stress effects are due to autophagy
of pre-formed tissues in the various processes of plant growth and
development mentioned previously. Furthermore, this autophagy
results in apoptosis of potential crop products, thereby
significantly reducing crop yield.
[0028] Exogenous applications of cytokinin to the flowers and
leaves (i.e., foliage) of plants provides the spatially-required,
growth regulator signaling effect needed for enhanced synthesis of
nutrients/sugars for use by tender new cells. The cytokinin and/or
potassium can also be applied to the soil in which plants are
growing at the same concentrations. It may be applied to the soil
in any appropriate fashion, such as, for example, in an opened
furrow near the plant roots, which furrow may subsequently be
closed. It may also be applied with various forms of irrigation,
such as overhead or drip tape, or furrow irrigation, among others.
Enhancement of nutrient synthesis, via cytokinin application, is
believed to result in a more complete development of the biological
tissues for plant reproduction. Specifically, the availability of
an adequate supply of nutrients/energy leads to the successful
development of the male sperm, including the various tissues and
biological signals responsible for its development. An adequate
energy source also aids in the various stages of development of the
pollen in which the sperm are protected by encasement. Also,
adequate nutrients/energy are available to assist the male sperm in
its journey from the pollen grain, through the developing pollen
tube and into the female ovary for fertilization of the egg. Thus,
cytokinin application results in the successful formation of seed
embryos and associated tissues of the crop plant, thereby
overcoming autophagy and any resultant apoptosis.
[0029] Additionally, the application of low concentrations of
potassium along with the cytokinin has been found to substantially
increase the effect of the cytokinin on plant tissues. Such results
are unexpected and differ from the physiological effects normally
attributed to higher application rates of typical fertilizer-grade
potassium. The physiological effects of higher applied potassium
concentrations include: maintaining turgidity in the plants and
thus ensuring a water supply, neutralizing anions helping to
stabilize pH of the cytoplasm, and general metabolic processes. To
induce these physiological effects, the concentration of applied
potassium must be on the order of typical fertilizers. The low
concentrations of potassium, disclosed herein, employed for
signaling effect are at least ten percent lower than typical
potassium fertilizer applications, such as those described in U.S.
Pat. No. 4,581,056 issued to Nooden et al. or in A. A. Csizinszky,
Foliar and Soil-Applied Biostimulant Studies with Microirrigated
Pepper and Tomato, 103 PROC. FLA. STATE HORT. SOC. 113-17 (1990).
It is thought that potassium, applied in low concentrations, acts
much like other signaling molecules (e.g., hormones) in aiding
transcription of particular genes, such as the genes that are
expressed in response to applied cytokinin. Potassium, if applied
with the cytokinin, is preferably applied at very low
concentrations between about 1/4 lb. to about 2 lbs. per acre, more
preferably between about 1/2 lb. to about 11/2 lbs. per acre, and
most preferably about 1 lb. per acre. The signaling effect of the
potassium has been found to be increasingly diminished for
potassium application rates greater than about 2 lbs. per acre.
[0030] A preferred implementation of the invention facilitates the
successful synthesis of nutrients/sugars, such that these
nutrients/sugars (i.e., chemical energy) may be transferred to the
developing male plant organs. The method preferably includes the
application of a plant hormone, primarily a cytokinin, to the
foliage and/or flowers of plants at or about the time of plant
flowering. The period of potential effectiveness of cytokinin
application may range from several weeks prior to flower emergence,
up to and including flower emergence, pollination, and during
subsequent embryo development. A preferred time is during the
process of meiosis, when pollen mother cells my fail to
differentiate, or fail to divide. This time of meiosis occurs in
dicot plants during an interval after flower bud differentiation,
but before the flowers open. In monocot plants, such as grains, it
corresponds approximately to the "early boot" stage (Feekes stage
10.0) in grain development. Another preferred time is during pollen
release from the tapetum and pollen sacs, when pollen is about to
enter dehiscence (or shed). In dicot plants, this generally falls
within the period ranging from immediately before the opening of
the flower up until the flower dies and falls off the plant. In
monocot plants, pollen release corresponds approximately to Feekes
stage (10.5.1) in grain development. The application of cytokinin
after flowering may also enhance the development of the embryo
under high temperature growing conditions and thereby increase
yield.
[0031] The autophagy-inhibiting agent is preferably the cytokinin,
kinetin, however, other forms of cytokinin may be used singularly
or in combination, such as zeatin, various forms of zeatin,
N6-benzyl adenine, N6-(delta-2-isopentyl) adenine, 1,3-diphenyl
urea, thidiazuron, CPPU (forchlorfenuron) or other chemical
formulations with cytokinin-like activity. Preferably, but
optionally, a low concentration of potassium is also applied
together with the plant hormones to enhance the effects of the
plant hormone, cytokinin.
[0032] In a first step, the cytokinin plant hormone is readied for
application to the plants to be treated. The cytokinin plant
hormone is preferably applied to the plants as an aqueous solution.
Application of agricultural chemicals may be accomplished in any of
several ways well known to those skilled in the art, including but
not limited to, spraying, drip lines, side dressing, etc.
Therefore, readying the cytokinin plant hormone may include one or
more of the following activities: diluting the cytokinin plant
hormone in sufficient water to create the desired concentration of
cytokinin in the applied mixture/composition, adding a low
concentration of potassium to the cytokinin plant hormone
mixture/composition to enhance the effect of the applied cytokinin,
loading the cytokinin plant hormone with or with out potassium (or
an aqueous mixture thereof) into a sprayer or tank for subsequent
application to the plants to be treated, calibrating the sprayer or
dosing applicator to meter the desired amount of the cytokinin
plant hormone to the plants to be treated and transporting the
cytokinin plant hormone with or without potassium (or an aqueous
mixture thereof) to the location of the plants to be treated.
[0033] Preferably, the cytokinin concentration in an undiluted
aqueous solution ranges from about 0.01% to about 0.10%. A
commercially-available, undiluted cytokinin product, X-Cyte (a
product of Stoller USA, Houston, Tex.), supplies the preferred
cytokinin concentration of about 0.04%. At the preferred cytokinin
concentration, the undiluted aqueous solution of cytokinin is
applied in a second step to plants to be treated at the rate of
between about 1/4 to 4 pints solution per acre of growing plants
and more preferably between 1 to 2 pints solution per acre of
growing plants. Such application equates to a rate of between about
0.09 grams to about 0.76 grams of cytokinin per acre of growing
plants (diluted in 60 gallons of water per acre for a
sprayed/applied solution), and more preferably, at a rate of
between about 0.19 to about 0.38 grams cytokinin per acre of
growing plants (diluted in 60 gallons of water per acre for a
sprayed/applied solution), depending on the specific plant/crop
species. Thus, as disclosed above, the amount of cytokinin applied
to the growing plants (1 to 2 pints of undiluted cytokinin solution
per acre, which is equivalent to 0.1 ppm to 1.66 ppm cytokinin of
the sprayed/applied solution per acre) is much lower than previous
cytokinin applications to growing plants. In fact, the preferred
application rates are more than ten fold lower than other reported
ranges of cytokinin application, which are from about 20 ppm to
about 400 ppm as suggested by N. G. Denny, User Guide of Plant
Growth Regulators. If the duration of flower development is
lengthy, the application may need to be repeated for the newly
developing flowers.
[0034] The cytokinin and/or potassium (discussed in further detail
below) can be applied either to the leaves of the plants, or to the
soil at the same concentrations. It may be applied to the soil in
any appropriate fashion, such as, for example, in an opened furrow
near the plant roots, which furrow may subsequently be closed. It
may also be applied with various forms of irrigation, such as
overhead or drip tape, or furrow irrigation, among others.
[0035] Potassium, if applied together with the cytokinin, is also
preferably applied at low concentrations. (Alternatively, the
potassium may be applied prior to or after a separate application
of cytokinin. However, such separate applications are not optimal,
because they are more energy and time intensive.) The potassium is
preferably applied as a potassium salt, such as that found in
potash, however other forms of potassium known to those skilled in
the art may be equally employed. The potassium application rates
are preferably between about 1/4 lb. to about 2 lbs. per acre
(equivalent to about 500 ppm to about 4,000 ppm potassium of the
sprayed solution per acre), more preferably between about 1/2 lb.
to about 11/2 lbs. per acre, and most preferably about 1 lb. per
acre. The synergistic effects of applying low concentrations of
cytokinin along with low concentrations of potassium to growing
plants may not be limited to high stress growing conditions but may
also be realized under lower stress growing conditions.
[0036] Preferred implementations of the invention are further
described in the following several examples. However, these
examples are not meant in any way, and should not be interpreted,
to limit the scope of the invention disclosed herein.
Example 1
[0037] Experiments were conducted to determine the extent to which
the plant growth regulator, cytokinin, when appropriately applied
to plants growing in high temperature environments, enhanced sugar
levels and increased the transfer of sugars to energy deficient
portions of the plant. These experiments were also designed to
determine whether the plant tissues under high temperature stress
suffered from one or both of two potential problems: First, whether
under high temperature stress, there is a deficiency of cytokinin
in the plant tissue. Second, whether under high temperature stress,
there is degradation of the cytokinin (which is a normal process to
regulate the balance of plant growth regulators in plant tissues,
in this case by reducing the amount of active cytokinin in the
plant tissue).
[0038] The data from these experiments, as shown in FIGS. 1 and 2,
clearly indicate that the problem suffered by plants subjected to
high temperature stresses during their flowering phase is an
inadequacy of cytokinin in the reproductive tissues of the plants.
The results further show that appropriate, exogenous cytokinin
application, as disclosed herein, increases plant development and
crop yields in high temperature growing environments (i.e.,
increases the number and quality of seeds that are properly
developed, thus increasing yield at harvest). FIG. 1 is a histogram
of experimental results testing whether impaired seed/silique
development under high temperature growth conditions is caused by a
nutrient/sugar insufficiency, which may incite apoptosis of
newly-forming plant cells by autophagy and FIG. 2 is a histogram of
experimental data that verifies the results obtained in FIG. 1,
namely that high temperature yield reduction, caused by autophagy,
is mainly due to an insufficiency of the plant growth
regulator/hormone, cytokinin.
[0039] These experiments employed the model crop plant, Arabidopsis
thaliana, whose genome was mapped as early as the year 2000. To
ensure uniformity, the tested plants were selected such that they
were in the beginning stages of flowering and had balanced numbers
of flowers across groups. The control agent as well as all tested
agents were applied in a water solution containing 0.01%
Silwet.RTM. super spreader (obtained from General Electric) to the
leaves and flowers (i.e., foliage) of the tested plants. As shown
in FIG. 1, a control agent (Ctl) consisting of an aqueous solution
of 0.01% Silwet.RTM. super spreader was applied to a control plant
group. A second tested agent consisting of
N-(2-chloro-pyridin-4-yl)-N-phenyl-urea (CPPU) at 100 .mu.g/l (0.1
ppm) was applied to a second plant grouping to test whether the
difficulty in seed formation is due to cytokinin insufficiency or
overactive enzymes that break down cytokinin in the plant. The
third and fourth agents tested consisted of 6-benzyl adenine (6-BA)
and kinetin (KIN)--both cytokinins--each applied at the rate of 100
.mu.g/1 (0.1 ppm). Finally, the fifth and sixth agents tested
consisted of sucrose sugars applied in solutions of 20 mM (SUC 20)
and 100 mM (SUC 100), respectively, applied as readily-available
energy sources.
[0040] Aqueous solutions of the tested agents prepared to the
stated concentrations/doses were sprayed onto test groups of
flowers and leaves (i.e., foliage) of Arabidopsis thaliana until
runoff at one day prior to heat exposure and again at four days
after the beginning of heat exposure. The total duration of heat
exposure was fourteen (14) days. The growing temperatures were
maintained at between 33-36 degrees Celsius during sixteen (16)
hour days, and at about 25 degrees Celsius during eight (8) hour
nights. The experiments were conducted on poly(methyl
methacrylate)-enclosed shelves, i.e., Plexiglass.RTM.-enclosed
shelves, using four dual fluorescent lamps and a
thermostat-controlled exhaust fan. The seed pods (i.e. siliques),
filled with developed seeds (siliques >7 mm long and/or >1 mm
wide and/or bearing seeds), were counted just prior to the
beginning of the experiment, and again after fourteen (14) days of
heat exposure treatments.
[0041] As shown in FIG. 1, the plant growth regulator, cytokinin,
can mitigate the sugar deficiency in the plant tissues caused by
high growing temperatures, thus preventing autophagy. This result
is indicated by the increased number of seeds (i.e., siliques) in
the cytokinin tested agents-6-benzyl adenine (6-BA) and kinetin
(KIN). The slightly negative results encountered with the
N-(2-chloro-pyridin-4-yl)-N-phenyl-urea (CPPU) tested agent (i.e.,
a cytokinin oxidase inhibitor) combined with the positive results
encountered with the cytokinin tested agents clearly indicate that
the problem of high temperature induced autophagy is due to a
cytokinin insufficiency (i.e., lack of energy synthesis for the
newly forming cells). It should be noted that the two sucrose
tested agents also alleviated the sugar/photosynthate deficiency
thereby preventing plant autophagy. However, sucrose testing agents
provide expensive, temporary relief, because the sucrose breaks
down rapidly in situ.
[0042] FIG. 2 presents an additional set of experimental results
that reinforce the results obtained for the previous experiment
(FIG. 1). The second experiment was conducted in the same manner
and using the same control and tested agent concentrations as the
first experiment, with the exception of CPPU application. Thus, the
respective aqueous solutions as used in the first experiment, with
the exception of CPPU, were sprayed onto test groups of Arabidopsis
thaliana foliage until runoff at one day prior to heat exposure and
again at four days after the beginning of heat exposure. The total
duration of heat exposure was fourteen (14) days. The growing
temperatures were maintained at between 33-36 degrees Celsius
during sixteen (16) hour days, and at about 25 degrees Celsius
during eight (8) hour nights. The experiments were conducted on
poly(methyl methacrylate)-enclosed shelves, i.e.,
Plexiglass.RTM.-enclosed shelves, using four dual fluorescent lamps
and a thermostat-controlled exhaust fan. The seed pods (i.e.
siliques), filled with developed seeds (siliques >7 mm long
and/or >1 mm wide and/or bearing seeds), were counted just prior
to the beginning of the experiment, and again after fourteen (14)
days of heat exposure treatments. The results of the second
experiment may be similarly interpreted as the results of the first
experiment, thereby confirming that the plant growth regulator,
cytokinin, can mitigate the sugar deficiency in the plant tissues
caused by high growing temperatures, thus reducing autophagy.
Example 2
[0043] In this example, the effect of the plant growth regulator
cytokinin, specifically kinetin, applied at the beginning of
flowering and two to four weeks thereafter to field-grown beans
(i.e., lima beans) in Gustine, Calif. was observed. Growing
temperatures up to approximately 35 degrees Celsius were recorded.
Table 2 (below) provides the results of this replicated, randomized
experiment. The lima bean yields were increased significantly
(i.e., less seeds succumbed to autophagy and seed death or
collapse) when the aqueous solution of kinetin was applied just
prior to flowering to the lima bean foliage at either a rate of one
pint per acre or two pints per acre. The difference of "t" test of
5% is significant.
[0044] TABLE 1: Crop Yield for Kinetin Solution Application at
Flowering for Rates of 0 pt/acre, 1/2 pt/acre, 1 pt/acre and 2
pt/acre
TABLE-US-00001 Lima Bean Yield Gustine, CA Year 2010 Average
Average Average Average Average Yield Yield Yield Yield (lb/plot)
(lb/plot) (lb/plot) (lb/plot) X-Cyte pt/acre 0 0.5 1 2 Average
yield (lb/plot) 6.86 6.915 7.845 8.11375 t test vs. control
0.416532 0.000115 0.000307 t test vs. 1/2 pt/acre 0.003132 0.006917
t test vs. 1 pt/acre 0.09629 rep 1 7.13 6.47 8.13 8.61 rep 2 6.84
8.34 8.02 8.02 rep 3 6.59 7.11 7.65 7.83 rep 4 6.8 6.68 8.07 8.37
rep 5 7.17 6.45 7.38 8.51 rep 6 7 7.17 7.55 6.85 rep 7 6.7 6.49
7.91 8.53 rep 8 6.65 6.61 8.05 8.19
Example 3
[0045] In this example, the effect of the plant growth
regulator/hormone, cytokinin, applied together with low
concentrations of potassium, was observed. The cytokinin that was
field-applied was X-Cyte, as previously disclosed. In these
unreplicated field trials, conducted in Ohio over a three year
period, potassium at 1/2 lb. to 1 lb. per acre and cytokinin at 1
pint per acre were applied to field corn. The average increase in
yield attained by applying potassium, in addition to cytokinin, was
approximately fifteen (15) bushels per acre.
Example 4
[0046] In this example, the effect of the plant growth regulator
cytokinin, specifically kinetin, and applied at various times
before the reproductive stages of growth, was observed. In Table 2,
V7, V10, V13, and V16 refer to stages of growth of the corn plant.
V7 refers to the growth stage where the collar of the seventh leaf
is visible, V10 refers to the growth stage where the collar of the
tenth leaf is visible, V13 refers to the growth stage where the
collar of the thirteenth leaf is visible, and V16 refers to the
growth stage where the collar of the sixteenth leaf is visible. V16
is also just prior to the reproductive stage of growth. The corn
crops were grown in Weslaco, Tex. The water levels were either none
(i.e., simply rain referred to as dryland), or drip through a "drip
tape" (i.e., drip irrigation whereby sufficient water was applied
for more optimal growth of the crop). Results are given for yield
(measured in bushels per acre), and the weight of the seed
(measured in grams per 1,000 kernels). T tests indicate whether
there were differences. All treatments with exogenous cytokinin (in
this case kinetin) enhanced yield in a highly significant fashion.
Seed size was generally increased about the same with more optimal
watering both for the untreated control and the exogenously applied
cytokinin. However, under dryland conditions (no added water or
irrigation) the cytokinin treatment increased seed size in a highly
significant fashion over the untreated control.
TABLE-US-00002 TABLE 2 Treatment of field-grown corn crops before
the reproductive stage of growth with cytokinin, in this case
kinetin, on the effect of the treatment on crop yield (measured in
bushels of grain per acre), and seed size (measured in grams per
1,000 kernels of seed). Yield Kernel wt Sowing T test 1,000 SD T
test Harvest Yield SD vs con Kernel Kernel vs con EXPT Stage
Variety Water Dates Bu/acre Yield P= wt g wt P= 1 control B25DC25
Drip Oct. 1, 2011 174 11.6 296 7.1 R80 Jan. 7, 2012 1 V10 B25DC25
'' Oct. 1, 2011 259 7.0 0.01% 298 2.2 NS R80 Jan. 7, 2012 1 V16
B25DC25 '' Oct. 1, 2011 311 8.8 0.01% 298 1.0 NS R80 Jan. 7, 2012
----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- 2
control Asgrow Dry Mar. 15, 2011 91 7.4 227 5.1 7573 land Jul. 15,
2011 2 V10 Asgrow Dry Mar. 15, 2011 149 3.3 0.01% 244 5.0 0.01%
7573 land Jul. 15, 2011 ----- ----- ----- ----- ----- ----- -----
----- ----- ----- ----- 3 control H2684162 Dry Feb. 15, 2011 101
2.2 260 15.1 land Jun. 10, 2011 3 V16 '' Dry Feb. 15, 2011 207 4.9
0.015 296 3.6 0.01% land Jun. 10, 2011 3 V13 '' Dry Feb. 15, 2011
208 9.1 0.01% 299 10.1 0.01% land Jun. 10, 2011 3 V10 '' Dry Feb.
15, 2011 211 4.6 0.01% 296 6.6 0.01% land Jun. 10, 2011 3 V7 '' Dry
Feb. 15, 2011 212 6.4 0.01% 295 9.6 0.015 land Jun. 10, 2011
[0047] The Abstract of the disclosure is written solely for
providing the United States Patent and Trademark Office and the
public at large with a means by which to determine quickly from a
cursory inspection the nature and gist of the technical disclosure,
and it represents one preferred implementation and is not
indicative of the nature of the invention as a whole.
[0048] While some implementations of the invention have been
illustrated in detail, the invention is not limited to the
implementations shown; modifications and adaptations of the
disclosed implementations may occur to those skilled in the art.
Such modifications and adaptations are in the spirit and scope of
the invention as set forth in the claims hereinafter:
* * * * *