U.S. patent application number 17/480616 was filed with the patent office on 2022-06-16 for chemicals which alter the production of metabolites in cultivated plants.
The applicant listed for this patent is Impello Biosciences, Inc. Invention is credited to Michael KEY.
Application Number | 20220183290 17/480616 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-16 |
United States Patent
Application |
20220183290 |
Kind Code |
A1 |
KEY; Michael |
June 16, 2022 |
CHEMICALS WHICH ALTER THE PRODUCTION OF METABOLITES IN CULTIVATED
PLANTS
Abstract
Elicitors are applied to cultivated crops in order to improve
plant productivity and/or harvestable crop value in agricultural
and horticultural operations providing increased production of
metabolites in cultivated plants.
Inventors: |
KEY; Michael; (Fort Collins,
CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Impello Biosciences, Inc |
Fort Collins |
CO |
US |
|
|
Appl. No.: |
17/480616 |
Filed: |
September 21, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16118415 |
Aug 30, 2018 |
11147271 |
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17480616 |
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62552084 |
Aug 30, 2017 |
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International
Class: |
A01N 37/42 20060101
A01N037/42; A01N 59/00 20060101 A01N059/00; A01N 37/40 20060101
A01N037/40; C05F 11/10 20060101 C05F011/10 |
Claims
1) At least one elicitor for increasing production of one or more
selected inherent plant chemicals which have value in plant
cultivation comprising applying an effective amount of at least one
elicitor to plant cells being cultivated after which the selected
inherent plant chemical production is increased.
2) The at least one elicitor of claim 1 wherein the elicitor
comprises Methyl Jasmonate.
3) The at least one elicitor of claim 1 wherein the at least one
elicitor comprises a composition consisting of Methyl Jasmonate and
Jasmonic Acid including homologues, isomers or derivatives of the
listed elicitors.
4) The composition of claim 3 wherein the composition further
consists of Methyl salicylate including homologues, isomers or
derivatives of the listed elicitors.
5) The composition of claim 4 wherein the composition further
consists of Salicylic Acid including homologues, isomers or
derivatives of the listed elicitors.
6) The composition of claim 5 wherein the composition further
consists of Silicic acid including homologues, isomers or
derivatives of the listed elicitors.
7) The at least one elicitor of claim 1 wherein the at least one
elicitor comprises a composition of two or more elicitors selected
from the group consisting of Methyl Jasmonate, Jasmonic Acid,
Methyl salicylate, Salicylic Acid, Silicic acid and their
homologues or isomers or derivatives.
8) The composition of claim 2 wherein the composition is applied to
plant cells in the form of seed, clone stock, flowers, roots,
leaves, or other plant tissue.
9) The inherent plant chemicals of claim 1 wherein the inherent
plant chemicals consist of metabolites.
10) The inherent plant chemicals of claim 9 wherein the inherent
plant chemicals are selected from the group consisting of secondary
metabolites, phytochemicals, colors, aromas, flavors, acids and
phenolics.
11) A method of increasing production of one or more selected
inherent plant chemicals which have value in plant cultivation by
using the composition of claim 7 comprising applying the
composition at selected time points resulting in increased
production of selected inherent plant chemicals.
12) The method of claim 11 wherein selected inherent plant
chemicals include secondary metabolites, phytochemicals, colors,
aromas, flavors, acids and phenolics.
13) The method of claim 11 wherein applying the composition is
selected from a group consisting of a foliar spray, a root drench
and a gas.
14) The method of claim 11 wherein applying the composition is
directed to subterranean plant cells, aerial plant cells or
both.
15) The method of claim 11 wherein the selected time points consist
of a time point during plant growth cycle.
16) The method of claim 15 wherein the selected time points consist
of a time point selected from the group consisting of vegetative
stage and reproductive stage.
Description
[0001] This application claims benefit of U.S. Provisional Patent
Application Ser. No. 62/552,084 filed Aug. 30, 2017 and is hereby
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention involves adding chemical and
biochemical compounds to cultivated crops in order to improve plant
productivity and/or harvestable crop value in agricultural and
horticultural operations. More specifically, the invention
describes isolations, compositions and combinations of chemical
elicitors wherein increased activity is provided and the
compositions and combinations provide increased production of
metabolites in cultivated plants.
BACKGROUND
[0003] Cells and tissues of all organisms must respond and adapt to
changes in external environmental conditions. In many cases, cells
contain specific receptors for particular chemicals. When such
chemicals come into contact with cell surfaces, they bind
specifically to particular receptors. This binding then triggers a
cascade of events within the cells, including up- and
down-regulation of genes and activation or repression of specific
pathways within the cells. Those processes result in substantial
changes in cellular physiology. Thus, these elicitors are triggers
of dramatic physiological responses. Moreover, a very small
quantity of the elicitor molecule is often sufficient to cause
major changes in cellular physiology. Such compounds generally are
effective at micromolar concentrations. An understanding of such
elicitors has a major impact on cellular physiology and permits
metabolic engineering to achieve beneficial changes in organismal
activity.
[0004] Plants produce both primary (essential) and secondary
(non-essential) metabolites during growth. Secondary metabolites
are not necessary for the plant's survival but are small molecules
(MW<1,000 g/mol) that contribute to plant growth, development,
defense, and reproductive capabilities. Numerous secondary
metabolites, including alkaloids, terpenoids and isoprenoids, and
phenolics, among others, have commercial value in industries
ranging from nutraceuticals to pharmaceuticals to agrochemicals.
Previously, elicitation has been utilized on cell suspensions and
in vitro plant cultures to induce the production of some
plant-derived secondary metabolites, but these applications have
generally been limited to the large-scale production of plant
products that are not adequately produced in planta. Such products
are suitable for in vitro production because both the organic
synthesis and the extraction yield of such natural products from in
planta applications is impractical from cost standpoints. Elicitors
have been used in agricultural applications to promote systemic
acquired resistance for crop protection but have not been used to
increase crop value.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] FIG. 1 is a first graph comparing MeJA vs. non-MeJA
treatment measuring total Cannabinoid (mg/g) content and a second
graph comparing MeJA vs. non-MeJA treatment measuring total CBD
(mg/g) content at 28 days.
[0006] FIG. 2 is a first graph comparing MeJA vs. non-MeJA
treatment measuring total THC (mg/g) content and a second graph
comparing elicitors MeJA and/or SA and/or Si treatment vs. Control
measuring total Terpene (mg/g) content at 28 days.
[0007] FIG. 3 is a first graph comparing MeJA vs. non-MeJA
treatment measuring total Cannabinoid (%/w) content and a second
graph comparing MeJA vs. non-MeJA treatment measuring total CBDA
(%/w) content at 42 days.
[0008] FIG. 4 is a first graph comparing MeJA+SiA+SA treatment vs.
non-elicitor control measuring total Cannabinoid (%/w) content; a
second graph comparing MeJA or MeJA+SiA or MeJA+SiA+SA vs.
non-elicitor control measuring total Cannabinoid (%/w) content; and
a third graph comparing MeJA vs. non-MeJA treatment measuring total
THCA (%/w) content at 42 days.
[0009] FIG. 5 is a first graph comparing MeJA+SiA+SA treatment vs.
non-elicitor control measuring total Cannabinoid (%/w) content; a
second graph comparing MeJA or MeJA+SiA or MeJA+SiA+SA vs.
non-elicitor control measuring total CBD (%/w) content; and, a
third graph comparing MeJA+SiA+SA treatment vs. non-elicitor
control measuring total CBDA (%/w) content at 63 days.
[0010] FIG. 6 is a first graph comparing MeJA+SiA+SA treatment vs.
non-elicitor control measuring total Cannabinoid (mg/g) content; a
second graph comparing MeJA+SiA+SA treatment vs. non-elicitor
control measuring total CBD (mg/g) content; and, a third graph
comparing MeJA+SiA+SA treatment vs. non-elicitor control measuring
total Myrcene (mg/g) content.
[0011] FIG. 7 is a first graph comparing MeJA+SiA+SA treatment vs.
non-elicitor control measuring total Linalool (mg/g) content; a
second graph comparing MeJA+SiA+SA treatment vs. non-elicitor
control measuring total B-caryophyllene (mg/g) content; and, a
third graph comparing MeJA+SiA+SA treatment vs. non-elicitor
control measuring total Humulene (mg/g) content.
[0012] FIG. 8 is a first graph comparing MeJA+SiA+SA treatment vs.
non-elicitor control measuring total B-pinene (mg/g) content; a
second graph comparing MeJA+SiA+SA treatment vs. non-elicitor
control measuring total a-pinene (mg/g) content; and, a third graph
comparing MeJA+SiA+SA treatment vs. non-elicitor control measuring
total Limonene (mg/g) content.
[0013] FIG. 9 is a first graph comparing MeJA+SiA+SA treatment vs.
non-elicitor control measuring max CBD (mg/g) content; a second
graph comparing MeJA+SiA+SA treatment vs. non-elicitor control
measuring max THC (mg/g) content.
[0014] FIG. 10 is a first graph comparing MeJA+SiA+SA treatment vs.
non-elicitor control measuring total CBDA (mg/g) content; a second
graph comparing MeJA+SiA+SA treatment vs. non-elicitor control
measuring total Cannabinoid (mg/g) content.
[0015] FIG. 11 is a first graph comparing MeJA+SiA+SA treatment vs.
non-elicitor control measuring total THCA (mg/g) content; a second
graph comparing MeJA+SiA+SA treatment vs. non-elicitor control
measuring total CBD (mg/g) content.
[0016] FIG. 12 is a first graph comparing MeJA+SiA+SA treatment vs.
non-elicitor control measuring total CBGA (mg/g) content; a second
graph comparing MeJA+SiA+SA treatment vs. non-elicitor control
measuring total CBC (mg/g) content.
[0017] FIG. 13 is a graph comparing MeJA+SiA+SA treatment vs.
non-elicitor control measuring 49 THC (mg/g) content.
[0018] FIG. 14 is a first graph comparing MeJA+SiA+SA treatment vs.
non-elicitor control measuring A-bisabolol (mg/g) content; a second
graph comparing MeJA+SiA+SA treatment vs. non-elicitor control
measuring myrcene (mg/g) content.
[0019] FIG. 15 is a first graph comparing MeJA+SiA+SA treatment vs.
non-elicitor control measuring Nerolidol-trans (mg/g) content; a
second graph comparing MeJA+SiA+SA treatment vs. non-elicitor
control measuring B-caryophyllene (mg/g) content.
[0020] FIG. 16 is a first graph comparing MeJA+SiA+SA treatment vs.
non-elicitor control measuring B-pinene (mg/g) content; a second
graph comparing MeJA+SiA+SA treatment vs. non-elicitor control
measuring a-pinene (mg/g) content.
[0021] FIG. 17 is a first graph comparing MeJA+SiA+SA treatment vs.
non-elicitor control measuring limonene (mg/g) content; a second
graph comparing MeJA+SiA+SA treatment vs. non-elicitor control
measuring total terpene (mg/g) content.
SUMMARY
[0022] Chemical and/or biochemical isolates and mixtures are used
herein to stimulate the biosynthesis of plant metabolites in
plants, altering the content of phytochemicals in harvestable plant
parts, ultimately increasing crop value. Applications include but
are not limited to Cannabis crop varieties known as hemp and
marijuana varieties.
[0023] In a preferred embodiment at least one elicitor is required
for increasing production of one or more selected inherent plant
chemicals which have value in plant cultivation comprising applying
an effective amount of at least one elicitor to plant cells being
cultivated after which the selected inherent plant chemical
production is increased. The at least one elicitor may be a
composition of two or more elicitors selected from the group
consisting of Methyl Jasmonate, Jasmonic Acid, Methyl salicylate,
Salicylic Acid, Silicic acid and their homologues or isomers or
derivatives. The composition may be applied to plant cells in the
form of seed, clone stock, flowers, roots, leaves, or other plant
tissue. The inherent plant chemicals may be selected from the group
consisting of secondary metabolites, phytochemicals, colors,
aromas, flavors, acids and phenolics. The selected time points are
determined such that increased production of selected inherent
plant chemicals occurs. The composition may be applied using a
foliar spray, a root drench or a gas to subterranean plant cells
and/or aerial plant cells.
[0024] In preferred embodiments, compositions will include
components such as additives, auxiliaries, and excipients in
addition to the primary chemical ingredients. Additional components
may act to improve the stability of the composition, improve the
homogeneity of the composition, improve the function of the
composition in planta, or provide other qualities to the
composition and/or to the methodology of the present disclosure.
Possible additional components include amino acids, minerals,
salts, solvents, stabilizers, surfactants, hormones, enzymes,
vitamins, chitin, chitosan, carboxylic acids, carboxylic acid
derivatives, linoleic acid and other fatty acids, volatile organic
compounds (VOCs), microbial consortia or isolates, bioregulators,
biostimulants, and other additives known in the art to elicit a
biological, biochemical, physiological, and/or physiochemical
response from the plant, or to stabilize the composition, or to
elicit specific metabolite production in the plant.
[0025] The compositions disclosed herein include liquid and/or dry
forms and include dry stock components that are added to water or
other liquids prior to application to the plant in an aqueous form.
Liquid compositions include aqueous, polar, or non-polar solutions.
The compositions may be provided in concentrated or diluted forms.
In a preferred embodiment, the composition is diluted. In another
preferred embodiment, the composition is concentrated. In yet
another preferred embodiment the composition is aqueous.
[0026] Methods include using the disclosed composition to increase
the value of cultivated plants. The composition can be applied to
seed, seedling, clone stock, vegetative tissues, root tissues,
flowering tissues, and mature plant parts. The composition may be
applied in liquid or dry form. The composition may be applied to
the soil, to the plant, or to both the soil and the plant. The
composition may be applied to plant parts using methods known in
the art, such as foliar spray, atomization, fumigation, or
chemigation. The composition may be applied to the soil using
methods known in the art such as irrigation, chemigation,
fertigation, or injection. The composition may be applied to a soil
or a water or a carbon dioxide or a fertilizer source, including
hydroponic and aeroponic and carbon dioxide injection systems,
which is delivered to the plant in a liquid, dry, or gaseous form.
In preferred embodiments, the plant may be grown indoors or
outdoors, in a controlled or uncontrolled environment, in fields or
in containers. The plant may be grown in soil-based media,
soil-less media, or a media containing both soil-less and
soil-based components. The plant may be grown in coco, rockwool,
peat moss, or other acceptable medias well-known in the art. The
plant may be grown in indoor or outdoor environments with organic
(Carbon-based), inorganic (synthetic), or a combination of both,
fertilizers, amendments, adjuvants, pesticides, and supplements. In
preferred embodiments where the composition is applied to immature
plants, seeds, or seedlings, the composition may also be applied to
the soil or plant later in the plant's life prior to harvest, or
applications of the composition may be repeated prior to harvest.
When the composition is applied to growing plant parts or flowers,
the same composition may be reapplied at a later stage of growth,
or a different composition may be applied at the same time or at a
later stage of growth, or both.
[0027] Methods further include application to living or dormant
plant tissues, both above-ground and below-ground, to alter the
biosynthesis and/or overall yield of valuable plant products to
increase the value of the crop relative to its market.
DETAILED DESCRIPTION
Elicitors as Biostimulants to Alter Secondary Metabolite
Production
[0028] Chemical compounds known as elicitors are plant signaling
molecules that influence plant health, growth, and stress
management. Elicitors may act on two primary plant signaling
pathways--the salicylate and jasmonate pathways--that are
responsible for modulating plant responses to abiotic and biotic
stimuli. Salicylates (SA) are derivatives of salicylic acid that
occur naturally in plants and serve as a natural immune hormone and
preservative, protecting the plants against diseases, insects,
fungi, and harmful bacteria. Jasmonates (JA) are lipid-based plant
hormones that regulate a wide range of processes in plants, ranging
from growth and photosynthesis to reproductive development. In
particular, JAs are critical for plant defense against herbivory
and plant responses to poor environmental conditions and other
kinds of abiotic and biotic challenges. Plants direct significant
energetic resources to the production of compounds along these
pathways during times of stress, herbivory, and pathogen invasion
in order to better defend themselves from the stressor. The
presence of these compounds in planta dictate numerous plant
responses and alter gene expression, influencing biochemical
synthesis pathways and inducing the production of secondary
metabolites such as phenolics, stilbenoids, terpenes, and
highly-specific compounds such as cannabinoids, whose endogenous
production is generally recognized as limited to plant species
within the Cannabis genera.
[0029] In preferred embodiments, Methyl Jasmonate (MeJA), Salicylic
acid (SA), and Silicic Acid (SiA) are considered for use
independently or as a mixture to alter the production of valuable
secondary metabolites by contacting some part of the plant or its
environment. One or more elicitors may be mixed with one or more
auxiliaries, adjuvants, excipients, surfactants, or other
chemicals. Elicitors may be applied simultaneously but separately
from other plant growth products, such as nutrients and pesticides,
for improved performance or facility.
[0030] The disclosed compositions and methods may be used to
increase crop value by contacting young plants, seeds, clones or
scions, vegetative plants, or other non-reproductive plant parts,
or reproductive plant parts, to induce some desired response. The
value of the crop may be determined by quantifying the
concentration of secondary metabolites in plant parts with mass
spectrometry, or by weight or volume measurements, or yield
(concentration, weight, density, or relative abundance) of
structures or organs, or by other physical or chemical means.
[0031] The disclosed compositions and methods are used to increase
the value of Cannabis crops relative to crops not treated with the
compositions and methods, or to increase the value of Cannabis
crops relative to crops treated with other variations of the
present disclosure and relative to the perceived market value of
each plant component. In more than one iteration, the compositions
and methods can be used to increase the production of valuable
metabolites by weight, or to decrease the production of undesirable
metabolites by weight, as determined by chemical analysis of plant
or flower parts during the reproductive (i.e., flowering) phase of
plant growth.
[0032] The profile of secondary metabolites in Cannabis plants can
be the primary determinant of the crop's value. In hemp crops,
low-THC varieties are not only mandated by law but coveted by
consumers and cultivators. Hemp crops are generally utilized for
their secondary metabolites produced in planta in flower organs,
which are extracted and refined using various techniques such as
lipid or hydrocarbon extractions. In high THC varieties, which are
restricted to the marijuana markets, less emphasis is placed on the
variety of secondary metabolites and greater emphasis is placed on
the concentration of THCA and its derivatives. In both instances,
the secondary metabolites of greatest interest are cannabinoids and
terpenes.
EXAMPLES
[0033] Example 1: Assessment of MeJA, SiA, and SA Eliciting
Properties. Applicant tested the performance, both efficacy and
effectiveness, of the eliciting compositions and methods to alter
secondary metabolite production in a controlled environment with a
commercial hemp varietal (Happy Camper, CBDRx LLC, Boulder, Colo.).
The compositions demonstrate bioactivity in planta beyond what was
hypothesized. Notably, the compositions applied demonstrated the
ability to both increase and decrease the production of dominant
secondary metabolites such as cannabidiol (CBD), cannabidiolic acid
(CBDA), tetrahydrol cannabidiol (THC), tetrahydrol cannabidiolic
acid (THCA), and common terpenes such as humulene,
beta-caryophyllene, and myrcene. Our results show that positive
results can be obtained from the application of both elicitors,
depending on which secondary metabolites are of greatest interest
to the applicator.
[0034] Applicant performed an indoor cultivation experiment with 30
identical Cannabis sp. hemp varietal clones (Happy Camper, CBDRx
LLC, Colorado). Each of the thirty plants was randomly assigned to
one of six treatment groups to test the efficacy and effectiveness
of each elicitor compound in isolation or in combination. Plants
were maintained in an isolated indoor growing environment under
eight, 600-watt double-ended electronic high pressure sodium lamps
(Gavita International B.V) under a photoperiod of 12 hours of light
per 24-hour day and potted in 1-gallon black plastic pots with a
55% coco coir/45% perlite media (Miller Soils LLC, Longmont, Colo.,
USA), then transplanted into 10-gallon fabric pots (Smart Pot, High
Caliper Growing, USA) filled with a mixture of 75% coco coir and
25% cork (DNA-Mills Ltd, London, England), irrigated as needed with
tap water and fertilized consistently to maintain good health with
a 4-20-39 base nutrient supplemented with 15-0-0 Calcium Nitrate
and commercially available magnesium sulfate (Hydro Gardens
International, Colorado Springs, Colo.) at a rate of 300-500 ppm
(TDS) as needed. A microbial inoculant (Tribus Original, Impello
Biosciences, Ft. Collins, Colo.) was applied at a rate of 1
mL/gallon of nutrient solution every watering to improve plant
health. Drip emitters and an automated watering system supplied
water and nutrients equally to each plant. Temperatures in the
growing environment were maintained at 75.degree. F..+-.15.degree.
F. with a relative humidity range of 30-60%. Every 14 days of
growth, all plants were supplemented with a fish protein
hydrolysate at a rate of 5 ml/gallon of the previously mentioned
nutrient solution to improve nutrition (Innate Bloom 4-2-1, Impello
Biosciences, Colorado).
[0035] Treatment groups included (1) applications of MeJA as a
foliar spray at a concentration of 7.5 mM, (2) SA as a foliar spray
at a concentration of 0.5 mM, (3) SiA as a root drench at a
concentration of 3 mM, (4) MeJA as a foliar spray at a
concentration of 7.5 mM and a root drench of SiA at a concentration
of 3 mM, and (5) MeJA as a foliar spray at a concentration of 7.5
mM and a root drench of SiA at a concentration of 3 mM and foliar
applications of SA at a concentration of 0.5 mM. The control group
was not treated with any applications aside from normal
fertilization. Foliar applications of MeJA were performed in the
morning hours, within 30 minutes of daybreak, with applications
covering plant leaves until dripping. Applications of SA for all SA
treatment groups were performed in the morning hours, within 30
minutes of daybreak, with applications covering plant leaves until
dripping. Applications of SiA were performed by hand additions of
SiA mixtures at the desired concentration to the plant roots of
treatment plants; control plants were treated with plain water in
the same volume at the same time without the addition of SiA.
[0036] 7.5 mM solutions of MeJA (mixture of isomers) were prepared
in 1-liter volumes using 93% purity MeJA stock solution (TCI
America, CAS #1101843-02-0) added to reverse osmosis water dropwise
with a pipettor while stirring. The pH of the final solution was
not adjusted. 0.5 mM solutions of SA were prepared from a 0.5M
stock solution, which was prepared using Salicylic Acid powder
(ThermoFisher Scientific, CAS #69-72-7), dissolved in 200-proof
anhydrous EtOH, denatured (VWR Life Sciences) and stored in a
sealed glass bottle. Plant-useable solution was prepared by
diluting 1000 .mu.L, of the stock SA solution in 999 mL of reverse
osmosis water. Foliar applications of MeJA and SA were made with a
1-L volume low-pressure hand-pump sprayer (Mondi Products LTD,
Canada). Silicic Acid solutions were prepared as needed using
Silicic Acid stock powder (extra pure light DAB, Millipore Sigma,
CAS #7699-41-4), dissolved in reverse osmosis water to the desired
final concentration.
[0037] Plants were maintained under identical conditions in the
same growing environment. All plants were randomly arranged in the
room using an online experiment randomizer tool (randomizer.com).
Foliar treatments of SA and MeJA were performed with all
environmental ventilation (exhaust and circulation fans) turned off
to minimize overspray onto non-treatment plants, and plants were
spatially separated. Treatments were applied according to a defined
schedule. For all treatment groups receiving SA, applications
occurred every 10 days, starting on day 0 of the reproductive cycle
when the photoperiod was changed from 18 hours of light per day to
12 hours of light per day and applied again on days 10, 20, and 30.
MeJA applications, for all treatment groups receiving MeJA, were
applied every 14 days, starting on the 7th day of the reproductive
cycle and applied again on days 21, 35, and 49. SiA applications,
for all treatment groups receiving SiA, were applied weekly
starting on day 1 of the reproductive cycle and continued through
harvest.
[0038] Quantitative observations were made three times throughout
the experiment. On day 28 of the reproductive cycle, approximately
2 grams of flowers (buds) of similar size and from similar
locations on each plant were harvested and dried for analysis.
Samples were taken from each plant, air dried at approximately
100.degree. F. in a Nesco American Harvest food dehydrator (Metal
Ware Corp, WI, USA), then combined and homogenized into a single
representative sample for each treatment group. These samples were
submitted to a third-party lab (ProVerde, Milford, Mass.) for
chemical analysis via mass spectrometry. Statistical analysis was
performed on the results group-wise, and differences between
treatment groups was observed as shown in FIG. 1 & FIG. 2. This
quantitative procedure was performed again on day 42 of the
reproductive cycle, when similar sized flower sections were
harvested from each plant, dried at .about.100.degree. F., and
submitted for chemical analysis via mass spectrometry (Botanacor,
Denver, Colo.). In this case, three samples from each treatment
group were submitted for chemical analysis via HPLC-MS.
Statistically significant differences were observed in the
concentration of secondary metabolites between treatment groups as
shown in FIG. 3 & FIG. 4. On day 62, plants were harvested and
hung to dry. Five samples of each group were taken for chemical
analysis, again selecting similar top flower buds from each plant
for consistency. These samples were dried at 95.degree. F. for 85
hours prior to chemical analysis via HPLC-MS (Botanacore, CO).
Again, statistically significant differences in secondary
metabolite production were observed as shown in FIG. 5.
[0039] Statistical analysis for all quantitative data and
observations were performed via two-sample Student's t-Tests
assuming unequal variance using Microsoft Excel software. Data was
graphed using box-and-whisker plots in Microsoft Excel software.
Statistical significance was defined by a level of significance of
p.ltoreq.0.05 in the t-Test analyses.
[0040] Day 28 Assay Summary (FIGS. 1&2): Total cannabinoids
(+61.96%), total CBD (+61.49%), and total THC (+61.30%)
concentrations were all significantly increased with applications
of methyl jasmonate either alone or in conjunction with other
elicitors at a p.ltoreq.0.05 level of significance. Total terpene
production was significantly increased (+35%) by applications of
either MeJA and/or SA compared to control or si only at a
p.ltoreq.0.05 level of significance.
[0041] Day 42 Assay Summary (FIG. 3&4): Total cannabinoids
(THC, THCA, CBD, CBDA) were significantly increased (+54.67%) at
the p.ltoreq.0.001 level of significance in treatment groups that
received applications of MeJA either alone or in combination
compared to the treatment groups that did not receive MeJA
applications. Total CBDA was increased significantly (+54.99%) in
plants that received MeJA treatments relative to plants that did
not receive MeJA treatments at a p.ltoreq.0.0005 level of
significance. Total THC-A was increased significantly (+56.82%) in
plants that received MeJA treatments relative to plants that did
not receive MeJA treatments at a level of significance less than
0.0005. Plants that received all treatments showed significantly
increased total cannabinoid concentrations (+56.31%) relative to
the plants in the control group at a p.ltoreq.0.01 level of
significance. Plants that received MeJA treatments showed
significantly increased (+41.49%) total cannabinoid concentration
relative to plants in the control group at a p.ltoreq.0.005 level
of significance.
[0042] Day 63 Assay Summary (FIG. 5): Total cannabinoids (THC,
THCA, CBD, CBDA) were increased significantly (+41.48%) in plants
that received ALL elicitor treatments compared to control plants
(+41.48%, p.ltoreq.0.05). CBD content was significantly increased
in plants treated with only MeJA (+23.27%, p.ltoreq.0.05), MeJA and
SiA (+18.96%, p.ltoreq.0.05), and ALL elicitor treatments (+33.62%,
p.ltoreq.0.01) compared to control plants. CBDA content was
significantly increased in plants treated with ALL elicitors
compared to control plants (+42.67%, p.ltoreq.0.05). Plants
receiving some sort of MeJA treatment (MeJA only, MeJA with SiA, or
ALL treatments) produced significantly higher (+58.64%,
p.ltoreq.0.000001) total cannabinoid contents than plants that did
not receive any MeJA treatments (control, SA only, SiA only).
Plants receiving some sort of MeJA treatment (MeJA only, MeJA with
SiA, or ALL treatments) produced significantly higher (+33.11%,
p.ltoreq.0.05) total cannabinoid contents than plants in the
control group. THCA was significantly decreased (-45.67%,
p.ltoreq.0.05) in SA treatment plants versus control plants.
[0043] MeJA treatments consistently increased cannabinoid
production throughout the plant's flowering cycle, regardless of
whether it was applied alone or in conjunction with other
treatments. MeJA increased terpene production regardless of whether
it was applied alone or in conjunction with other elicitor
treatments. SA increased terpene production regardless of whether
it was applied alone or in conjunction with other elicitor
treatments, but decreased THCA production when it was applied
independently without supplemental MeJA applications which could be
valuable to hemp producers. The former findings could be valuable
to both marijuana and hemp producers.
[0044] Example 2 Assessment of Secondary Metabolite Profile
Alteration with Foliar Applications of the Elicitors MeJA and SA
and Elicitor Primer SiA. Sixteen (16) identical clones from a
single mother plant (Happy Camper, CBDRx, USA) were rooted into 1''
rockwool cubes (Grodan B.V.) prior to subsequent transplanting into
4'' rockwool cubes (Grodan B.V). All sixteen plants were randomly
divided into two groups, either a treatment group or a control
group. All sixteen plants were maintained under identical
conditions prior to and during the experiment. Plants were
maintained in an isolated indoor growing environment on two
elevated black plastic 4'.times.8' grow tables (Botanicare,
Chandler, Ariz., USA) with eight plants per table; treatment plants
were kept on one table and control plants on the other. Light was
supplied via eight, 600-watt double-ended electronic high-pressure
sodium lamps (Gavita International B.V) under a photoperiod of 12
hours of light per 24-hour day. Plants were irrigated as needed
with tap water and fertilized consistently to maintain good health
with a 4-20-39 base nutrient supplemented with 15-0-0 Calcium
Nitrate and commercially available magnesium sulfate (Hydro Gardens
International, Colorado Springs, Colo.) at a rate of 300-500 ppm
(TDS) as needed. A microbial inoculant (Tribus Original, Impello
Biosciences, Ft. Collins, Colo.) was applied at a rate of 1
mL/gallon of nutrient solution every watering to improve plant
health. Temperatures in the growing environment were maintained at
75.degree. F..+-.15.degree. F. with a relative humidity range of
30-60%. Every 14 days of growth, all plants were supplemented with
a fish protein hydrolysate at a rate of 5 ml/gallon of the
previously mentioned nutrient solution to improve nutrition (Innate
Bloom 4-2-1, Impello Biosciences, Colorado). Drip emitters and an
automated watering system supplied water and nutrients equally to
each plant.
[0045] 7.5 mM solutions of MeJA (mixture of isomers) were prepared
in 1-liter volumes using 93% purity MeJA stock solution (TCI
America, CAS #1101843-02-0) added dropwise with a pipettor to
reverse osmosis water in a 1-L Erlenmeyer flask while stirring. pH
was not adjusted, and 100 .mu.L of Tween-20 (VWR, CAS #9005-64-5)
were added to the 1 L solution to improve homogenization. 0.5 mM
solutions of SA were prepared from a 5 mM stock solution, which was
prepared using Salicylic Acid powder (ThermoFisher Scientific, CAS
#69-72-7), dissolved in reverse osmosis water and stored in a glass
bottle. The plant-useable 0.5 mM solution was prepared by diluting
100 mL of the stock SA solution in 900 mL of reverse osmosis water
and mixing with 100 .mu.L of Tween-20. The plant-useable 0.1 mM
solution was prepared by diluting 20 mL of the stock SA solution in
980 mL of reverse osmosis water and mixing with 100 .mu.L of
Tween-20. The 3 mM plant-useable Silicic Acid (SiA) solutions were
prepared as needed from a 0.25M Silicic Acid stock solution, which
was prepared with Silicic Acid powder (extra pure light DAB,
Millipore Sigma, CAS #7699-41-4), dissolved in reverse osmosis
water and stored in a sealed glass bottle. The stock solution was
heated and stirred to homogenize prior to use and diluted in water
to the desired final concentration of 3 mM. 100 .mu.L/L of Tween-20
was added to the solution as a surfactant. Control plants received
foliar applications of 100 .mu.L/L of Tween-20 in reverse osmosis
water only at the same volume and application timings as the
treatment groups. Foliar spray treatments were performed with all
environmental ventilation (exhaust and circulation fans) turned off
to minimize overspray onto other treatment groups.
[0046] All eight (8) plants in the treatment group were subjected
to applications of MeJA (7.5 mM), SA (0.1-0.5 mM) and SiA (3 mM) as
foliar sprays. Treatments were applied individually on designated
days. MeJA applications occurred every 14 days, starting on the
seventh (7th) day of the reproductive cycle. SA applications
occurred every 10 days, starting 17 days prior to initiation of the
reproductive cycle and continuing every 10 days. The final two
applications of SA were applied at a concentration of 0.1 mM to
minimize the potential negative interactions between SA and MeJA
applications; all applications prior to that were applied at a
concentration of 0.5 mM SA. SiA applications were performed weekly,
beginning 6 days after the initiation of the flowering cycle.
Plants in the control group were sprayed with a solution of
distilled water and 0.01% v/v Tween-20 on the days that treatment
plants received applications and in the same fashion. Foliar
applications of MeJA, SA, SiA, and control solutions were made with
a 1-L volume low-pressure hand-pump sprayer (Mondi Products LTD,
Canada). After 7 weeks, plants were harvested, and samples from
each plant were taken from similar locations on the plant's
flowering stalks, labeled, and air dried in a Nesco American
Harvest food dehydrator (Metal Ware Corp, WI, USA) at 95.degree. F.
for 72 hours until fully dry.
[0047] Plant samples were submitted to a third-party lab (ProVerde,
Milford, Mass.) for analysis. Cannabinoid contents were quantified
via Convergence Chromatography (CC) and terpenes were analyzed via
Gas Chromatography-Mass Spectrometry (GC-MS). All data was analyzed
for statistical significance using two-sample Student's t-Test
assuming unequal variance and plotted on box-and-whisker plots
using Microsoft Excel software. Statistical significance was
defined by a level of significance of p.ltoreq.0.05 in the t-Test
analyses.
[0048] Total THC, total CBD, and total cannabinoids were all
significantly increased (+32.75%, +27.98%, +28.14%) in the plants
in the treatment group at a significance level of p.ltoreq.0.00005
relative to the control group (FIG. 6). Total terpene
concentrations were increased significantly in the treatment plants
relative to the control plants at the following percentage
increases and significance levels: Myrcene (+58.23%, p.ltoreq.0.02)
(FIG. 6), B-caryophyllene (+30.73%, p.ltoreq.0.02) (FIG. 7),
Linalool (+66.66, p.ltoreq.0.005) (FIG. 7), Humulene (+29.65,
p.ltoreq.0.05) (FIG. 7), B-pinene (+43.13%, p.ltoreq.0.05),
a-pinene (+71.15%, p.ltoreq.0.01), and Limonene (+82.46%,
p.ltoreq.0.02) (FIG. 8).
[0049] Example 3: Assessment of Secondary Metabolite Profiles in
plants treated with MeJA, SA, SiA simultaneously. Based on the
results of our initial experiments, an experiment was designed to
test the efficacy and effectiveness of applying all three eliciting
compounds as a single treatment. A single-treatment elicitor may
prove more economically viable and applicable in a real-life
cultivation setting. A six-plant experiment was designed, using
identical clone stock from a single variety (WHY, CBDRx, USA),
where three plants were assigned to the treatment group and three
plants were assigned to the control group.
[0050] All plants were rooted in 1-inch rockwool cubes (Grodan
B.V.) under identical conditions prior to transplant into 1-gallon
black plastic pots filled with a 55% coco coir, 45% perlite media
(Miller Soils LLC, CO, USA). Treatment plants were treated with a
foliar application of 7.5 mM MeJA, 0.05 mM SA, and 3 mM SiA in the
same solution. The solution was prepared by first adding 6 mL of a
0.25M SiA stock solution, which was prepared by dissolving Silicic
Acid powder (extra pure light DAB, Millipore Sigma, CAS #7699-41-4)
in reverse osmosis water, to approximately 488 mL reverse osmosis
water on a stir plate. Then, 5 mL of a 0.5 mM SA stock solution
(prepared using Salicylic Acid powder (ThermoFisher Scientific, CAS
#69-72-7), dissolved in reverse osmosis water and stored in a glass
bottle) was added to the solution while stirring. Next, 886.5 .mu.L
of 93% stock MeJA (TCI America, CAS #1101843-02-0) was added to the
mixture dropwise while stirring. Finally, 100 .mu.L/0.5 L Tween-20
(VWR, CAS #9005-64-5) was added to the mixture to improve
homogenization of the mixture and improve functionality on the
plant. Control plants were treated with an aqueous water solution
supplemented with 100 .mu.L/0.5 L Tween-20 without pH adjustment on
the same days as the treatment group. Treatment plants were removed
from the growing environment prior to applications to avoid
contamination of the control group. Treatments were made in the
early morning hours within 30 minutes of daybreak. Treatments were
made every 10 days, beginning on the 14th day of the reproductive
(flowering) cycle and continuing until the 44th day of flower.
Plants were harvested on day 51 of the flowering cycle.
[0051] Plants were maintained in an isolated indoor growing
environment on an elevated black plastic 4'.times.8' grow table
(Botanicare, Chandler, Ariz., USA). Light was supplied via eight,
600-watt double-ended electronic high-pressure sodium lamps (Gavita
International B.V) under a photoperiod of 12 hours of light per
24-hour day. Plants were irrigated as needed with tap water and
fertilized consistently to maintain good health with a 4-20-39 base
nutrient supplemented with 15-0-0 Calcium Nitrate and commercially
available magnesium sulfate (Hydro Gardens International, Colorado
Springs, Colo.) at a rate of 300-500 ppm (TDS) as needed. A
microbial inoculant (Tribus Original, Impello Biosciences, Ft.
Collins, Colo.) was applied at a rate of 1 mL/gallon of nutrient
solution every watering to improve plant health. Temperatures in
the growing environment were maintained at 75.degree.
F..+-.15.degree. F. with a relative humidity range of 30-60%. All
plants were hand watered with 1 L of nutrient solution
as-needed.
[0052] At the time of harvest, plants were cut at the stalk
immediately below the first node. Plants were immediately weighed,
and their weights were recorded. Contrary to the visual
observations, treatment plants demonstrated an approximately 14%
increase in weight, although this difference was not significant at
the p.ltoreq.0.05 level. Samples were analyzed for cannabinoids via
HPLC and for terpenes via GC-MS (ProVerde Labs, Milford, Mass.,
USA). Plants were hang-dried at approximately 80.degree. F. until
moisture was removed, at which point each plant was submitted for
chemical analysis. Plants in the treatment group showed an
approximately 25% increase in dry weight, but this difference was
also not significant at the p.ltoreq.0.05 level of
significance.
[0053] Statistical analysis for all quantitative data and
observations were performed via two-sample Student's t-Tests
assuming unequal variance using Microsoft Excel software. Data was
graphed using box-and-whisker plots in Microsoft Excel software.
Statistical significance was defined by a level of significance of
p.ltoreq.0.05 in the t-Test analyses.
[0054] Cannabinoid Summary: Chemical analysis of the flower
material revealed significant differences in cannabinoid production
between plants in the treatment and control groups. Plants in the
treatment group showed significantly increased levels of the
following cannabinoids at a confidence of p.ltoreq.0.02: Max CBD
(+12.84% increase), Max THC (+12.21% increase) (FIG. 9), CBDA
(+12.56% increase), and Total Cannabinoids (+13.07% increase) (FIG.
10). Plants in the treatment group also demonstrated significantly
increased levels of THCA (+12.92% increase) and CBD (+17.46%
increase) (FIG. 11) at a confidence level of p.ltoreq.0.01 and
significantly increased levels of CBGA (+20.89% increase) at a
confidence of p.ltoreq.0.001 and CBC (+21.38% increase) (FIG. 12)
at a confidence of p.ltoreq.0.005. Plants in the treatment group
did show an increase in .DELTA.9-THC content (+9.09% increase)
(FIG. 13) compared to the control group, but this difference was
not significant. It should be noted that CBC and CBGA production
increases are of significant importance and demonstrate that the
effect of the elicitor treatments occurs upstream of the
cannabinoid biosynthesis step, meaning that the effects of the
treatments are not limited to cannabinoid production, which is in
line with our previous findings. Moreover, the significant
increases we saw in CBGA production in the treatment group is
crucial to the production of other cannabinoids that may be
expressed in different Cannabis sativa varieties, since CBGA is the
necessary precursor to all other cannabinoids derived by the
olivetolic acid pathway.
[0055] Terpene Summary: Chemical analysis of the flower material
revealed significant increases (+20.62%) in the production of the
terpene A-bisabolol (FIG. 14) in plants in the treatment group
relative to plants in the control group at the p.ltoreq.0.05 level
of significance. Plants in the treatment group expressed increased
concentrations of other terpenes including myrcene (+320.09%) (FIG.
14), Nerolidol-trans (+22.57%) (FIG. 15), B-caryophyllene
(+14.38%), B-pinene (+45.60%) (FIG. 15), a-pinene (+37.73%) (FIG.
16), and limonene (+135.57%) (FIG. 16), and total terpene content
(+93.75%) (FIG. 17) at p.ltoreq.0.05.
[0056] Example 4: Assessment of Flower Yield in plants treated with
varying concentrations of MeJA. In our previous experiments,
applications of MeJA demonstrated remarkable bioactivity and
biostimulation in regard to the production of highly-valuable
secondary metabolites such as cannabinoids. In turn, we designed an
experiment to evaluate the efficacy and effectiveness of applying
MeJA as a foliar spray to plants in different concentrations.
[0057] Fourteen (14) identical plants from clone (Charlotte x
Tangie, CBDRx) were cultivated in 1-gallon black plastic pots in a
media mixture of 55% coco coir, 45% perlite (Miller Soils LLC,
Colorado). Plants were rooted in 1-inch A-OK rockwool cubes
(Grodan, B.V.) uniformly prior to transplant into the 1-gallon
pots. At the time of transplant, plants were also transitioned from
a photoperiod of 18 hours of light per day to a photoperiod of 12
hours of light per day to induce flowering. Each plant was
maintained with a standard, commercially available 3-part (4-20-39,
CaNO3, MgSO4) nutrient solution at a recommended rate of 350 ppm
(Hydro Gardens International, CO), hand watered as needed with 1 L
per plant. Plants were randomly assigned to one of four groups,
including three treatment groups and one control group: 0 mM MeJA
(control), 5 mM MeJA (low), 7.5 mM MeJA (med), and 10 mM MeJA
(high). Plants were maintained under identical environmental
conditions on a 4'.times.8' black plastic table (Botanicare, USA)
under 1200 w of light (Gavita Inc, Ne). A microbial inoculant
(Tribus Original, Impello Biosciences, Ft. Collins, Colo.) was
applied at a rate of 1 mL/gallon of nutrient solution every
watering to improve plant health. Temperatures in the growing
environment were maintained at 75.degree. F..+-.15.degree. F. with
a relative humidity range of 30-60%.
[0058] Treatment groups were treated with the appropriate
concentration of MeJA, prepared prior to application from a 93%
MeJA stock solution (TCI America, CAS #1101843-02-0) diluted in
reverse osmosis water and homogenized by mixing on a stir plate
with the addition of 0.01% (100 .mu.L/L) Tween-20 (VWR, CAS
#9005-64-5) to aid in homogenization and to act as a surfactant,
improving functionality when applied to the plant surfaces. MeJA
mixtures were prepared in 250 mL Erlenmeyer flasks, capped with
rubber stoppers while stored at room temperature to prevent
volatilization of the MeJA out of solution. The mixture was fully
homogenized and appear opaque white in color. For each use, the
prepared flasks were shaken vigorously to ensure homogenization,
then poured into micro-spray bottles with a capacity of 50 mL that
produced a fine mist. A separate bottle was used for each treatment
group and the control group sprays. Control groups were treated
with a solution of water and 0.01% (100 .mu.L/L) Tween-20 without
MeJA. Neither the control nor treatment compositions were pH
adjusted. Beginning on day 7 of the reproductive cycle, plants were
treated in the early morning hours within 30 minutes of daybreak
with a foliar spray until dripping from foliage, with treatments
recurring every 14 days. Each plant was removed from the growing
area for the spray treatment to reduce potential contamination
between treatment groups and allowed to dry for less than 1 hour
prior to returning to the growing environment.
[0059] No significant differences in weights between the treatment
groups were observed. The highest recorded plant weight was in the
10 mM MeJA treatment group. This suggests that in planta exogenous
foliar applications of MeJA to cultivated Cannabis sativa in our
desired concentrations did not negatively impact plant
performance.
[0060] The foregoing is considered as illustrative only of the
principles of the invention. Furthermore, since numerous
modifications and changes will readily occur to those skilled in
the art, it is not desired to limit the invention to the exact
construction and operation shown and described. Therefore, all
suitable modifications and equivalents fall within the scope of the
invention.
* * * * *