Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INV NE TION
COMPOSITION FOR ACCELERATING SEED GERMINATION AND PLANT GROWTH
FtFt p OF THE INVENTION
The present invention relates to agriculture. More
specifically, the invention relates to plant seed germination, seedling
emergence,
quiescence-breakage and plant growth. Even more specifically, the present
invention relates to compositions which accelerate plant seed germination,
seedling emergence and plant growth of numerous types of crops and to
methods using same.
_BACKGROUND OF THE INVENTION
Symbiotic microorganisms can promote the growth of
legumes by way of biological fixation of nitrogen. More specfically,
fiizobiaceae
are gram-negative soil bacteria which fix nitrogen and are involved in
symbiotic
association with these legumes. This symbiotic association between the
bacteria and the legume enables the latter to grow in soils having low
assimilable nitrogen levels. In return, through photosynthesis, the legume
provides the bacteria with the energy it requires to reduce the atmospheric
nitrogen into ammonia. This ammonia can then be used by the legume and
enters into the nitrogen metabolism. The legume, of the Fabaceae family, forms
nodules in which the rhizobia proliferate. The Rhizobiaceae family is in a
state
of taxonomic flux. It has been reported to comprise four main genera:
Rhizobium, Bradyrhizobium, Sinorhizobium and Azorhizobium (U.S. 5,549,718).
The symbiotic relationship between nitrogen-fixing bacteria or rhizobia and
plants of the Fabaceae family enables the growth of the latter in soils having
low
levels of available nitrogen, thus reducing the need for nitrogen fertilizers.
Since
nitrogen fertilizers can significantly increase the cost of crops, and are
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associated with a number of polluting effects, biological means to stimulate
this
symbiotic relationship and/or to decrease the use of nitrogen fertilizers is
of
great importance.
Initial recognition between 8. japonicum and soybean
involves exchange of molecular signals (Stacey et al, 1995). Legume roots
secrete phenolic compounds (Dakora 8~ Philips, 1996; Peters & Verma, 1990),
largely from the area of root hair emergence, which act as chemo-attractants
to
(brady)rhizobia (Nap & Bisseling, 1990), and activate the nod genes. Flavones,
isoflavones, and flavanones have been identified as the inducing molecules for
(brady)rhizobial chemotaxis and for expression of nod genes, e.g. genistein,
daidzein and several related compounds in soybean (Peters & Verma, 1990).
These plant-to-bacteria signal compounds cause expression of the bacterial nod
(also nol and noe) genes very rapidly (only a few minutes after exposure) and
at very low concentrations (10' to 10-e M) (Peters et al., 1986). Generally
this
is through an interaction with nodD, which activates the common nod genes,
although the situation may be more complex, as is the case in 8. japonicum,
where nodD,, nodD2 and nodVW are involved (Gillette & Elkan 1996; Stacey
1995). Nod genes have been identified in the rhizobia that form nitrogen
fixing
relationships with numbers of the Fabiaceae family (see 5,549,718 and
references therein). Recently, the plant-to-bacteria signal molecules have
been
shown to promote soybean nodulation and nitrogen fixation under cool soil
temperatures (CA 2,179,879) and increase the final soybean grain yield on
average of 10% in the field and up to 40% under certain conditions.
Among the products of the nod genes induced by the plant
phenolic signal molecules are various enzymes involved in the synthesis of a
series of lipo chitooligosaccharides (LCOs) (Spaink, 1995; Stacey, 1995).
These newly synthesized LCOs act as bacterium-to-plant signals, inducing
expression of many of the early nodulin genes (Long, 1989). This results in
root
hair deformation (including curling), cortical cell division leading to
initiation of
nodule meristems, secretion of additional nod gene inducers, and initiation of
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infection threads (Verma, 1992). These bacterium-to-plant signals exert a
powerful influence over the plant genome and, when added in the absence of
the bacteria, can induce the formation of root nodules (Truchet et al., 1991).
Thus, the bacteria-to-plant signals can, without the bacteria, induce all the
gene
activity for nodule organogenesis (Denarie et al., 1996; Heidstra & Bisseling,
1996). Moreover, the above-mentioned activities induced by LCOs can be
produced. by concentrations as low as 10-'° M (Stokkermans et al.
1995). The
mutual exchange of signals between the bacteria and the plant are essential
for
the symbiotic interaction. Rhizobia mutants unable to synthesize LCOs will not
form nodules. Analysis of the B. japonicum nod genes indicates that ability to
induce soybean nodulation requires at least: 1 ) a basic tetrameric Nod factor
requiring only nodABC genes or 2) a pentameric LCO (C18:1, C16:0 or C16:
fatty acid and a methyl-fucose at the reducing end, sometimes acetylated)
requiring nodABCZ genes (Stokkermans ef al. 1995).
When added to the appropriate legume, LCOs can cause the
induction of nodule meristems (Denarie et al., 1996), and therefore cell
division
activity. One previous publication has shown that LCOs can induce cell cycle
activities in a carrot embryogenesis system at levels as low as 10-'° M
(De Jong
ef al. 1993).
A chemical structure of lipo chitooligosaccharides, also
termed "symbiotic Nod signals" or "Nod factor", has been described in U.S.
Patents 5,549,718 and 5,175,149. These Nod factors have the properties of a
lectin ligand or lipo-oligosaccharide substances which can be purified from
bacteria or synthesized or produced by genetic engineering.
The relationship between environmental variables, such as
low root zone temperature (RZT) and pH, and the interplay of molecular signals
has only recently become a subject of investigation. For example, some
soybean genotypes have less synthesis abilities for isoflavones under cool
soil
temperature, whereas a higher isoflavone concentration is needed to turn on
the
nod genes of B. japonicum (Zhang and Smith 1995 and 1997). The plant-to-
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bacteria signal molecules (i.e. isoflavones) have been shown, among other
things, to overcome the negative effect of low temperature on the early events
of symbiotic nitrogen fixation (Canadian application number 2,179,879).
While the effects of plant-to-bacteria signal molecules (i.e.
isoflavones) on modulation, nitrogen fixation, growth and protein yield of
legumes, such as soybean, and on bacteria-to-plant signal molecules (LCOs)
on modulation and nitrogen fixation in legumes have been described under
certain conditions, the effect of the bacteria-to-plant signal molecules on
the
growth of non-legumes is unknown. In fact, the role of such bacteria-to-plaint
signal molecules on non-legumes has never been assessed. In addition, the
effect of LCOs on processes other than modulation of legumes has yet to be
studied.
There thus remains a need to assess the effect of LCOs on
seed germination, seedling emergence andlor growth of plants in general and
especially of non-legume plants.
The present invention seeks to meet these and other needs.
SUMMARY OF THE INVENTION
The invention concerns a composition for enhancing seed
germination, seedling emergence and growth of plants and especially of crop
plants. More specifically, the present invention relates to a composition
comprising an t_CO which can increase seed germination and/or seedling
emergence andlor growth of a legume, in addition to acting as a trigger to
initiate
legume symbiotic nitrogen fixation. More particularly, the invention relates
to
increased seed germination and/or seedling emergence andlor growth of
soybean, pea and red clover.
Surprisingly, the compositions of the present invention act not
only on legumes such as soybean but on plants in general as exemplified with
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non-legume crops from different plant families Poaceae, Cucurbitaceae,
Malvaceae. Asteraceae, Chenopodiaceae and Solonaceae. More specifically,
the non-legume crops exemplified herein include corn, cotton, cantaloupe,
cucumber. canola, lettuce, potato and beet. The present invention thus also
5 refers to compositions for enhancing seed germination and/or seedling
emergence andlor growth of non-legumes. More particularly, the invention
relates to compositions comprising an LCO for enhancing seed germination,
seedling emergence and growth of non-legumes. Non-limiting examples of such
non-legumes include cotton, corn, canola, potato, cucumber, cantaloupe,
lettuce
and beet. Broadly, the present invention relates to compositions comprising an
LCO for promoting growth of a crop. Non-limiting examples of crop plants
include monocot, dicot, members of the grass family (containing the cereals),
and legumes.
Thus, the present invention relates to agricultural
compositions comprising at least one LCO (and methods of using same) for
promoting seed germination, and/or early development of seedlings, andlor
emergence of sprouts from tubers, andlor rapid development of new plants from
higher plant perinating structures.
In a particular set of experiments in the field, a composition
of the present invention comprising an LCO was shown to significantly enhance
early plant growth.
The invention in addition relates to methods for enhancing
seed germination and/or seedling emergence and/or growth of plants and/or for
breaking the dormancy thereof comprising a treatment in the vicinity of a seed
or seedling or plant with an effective amount of an agricultural composition
comprising an LCO and an agriculturally suitable carrier for a sufficient time
and
under conditions which enable an increased germination of the seed and/or an
increased emergence of the seedling andlor an increased growth of the plant
and/or a triggering of the growth of a dormant plant.
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The invention also relates to compositions and methods for
breaking the dormancy of a plant and initiating the growth thereof. In a
particular embodiment, the invention relates to the breaking of dormancy of
potato.
The Applicant is the first to show that a composition
comprising an LCO can have a significant effect on seed germination, and/or
seedling emergence of legumes. Moreover, the Applicant is the first to show
the
surprising effect of signal molecules involved in bacteria-legume signalling
on
the growth of non-legume plants. In addition, the Applicant is the first to
show
that a composition comprising an LCO had an effect on non-legume seed
germination and/or seedling emergence and/or plant growth of the non-legume.
Also, the Applicant is the first to show that an LCO can not only act as a
dormancy breaker but that it can also significantly increase the yield of a
dormant plant following the dormancy breakage, when compared to known
dormancy breakers.
While the seed germination andlor seedling emergence
and/or plant growth enhancing capabilities of the compositions of the instant
invention are demonstrated with corn, cotton, canoia, potato, cantaloupe,
lettuce, beet, cucumber, soybean, pea and red clover, they are applicable to
plants in general and more especially to crop plants. Indeed, the plants
chosen
for the experiments presented herein are crops from significantly divergent
plants in eight distinct families: (1 ) corn, the only monocot tested herein,
in the
family of grasses (Poaceae), which also contains the cereals; (2) cucumber and
cantaloupe, the latter being a plant used horticulturally, and being slow to
germinate at low temperature [its base temperature is about 14°CJ
(Cucurbitaceae); (3) cotton, one of the most important fibre crops on the
planet
(Malvaceae); (4) lettuce (Asteraceae); (5) beet (Chenopodiaceae); (6) potato,
a very important crop (Solonaceae, which also includes tobacco, peppers and
tomato); and two families of legumes (7) canoia, representing the mustard
group
(Brassicaceae) and (8) soybean (representative of oil seed crop), bean
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(representative of a crop for human consumption) and red clover and alfalfa
(forage legumes) (all of the Fabaceae family).
In view of the diversity of the plants tested, and of the similar
results obtained with these different crop plants, it can be predicted that
such
results will apply to crop plants in general. It follows that a person skilled
in the
art can adapt the teachings of the present invention to other crops. Non-
limiting
examples thereof include tobacco, tomato, wheat, barley, rice, sunflower and
plants grown for flower production (daisy, carnation, pansy, gladiola, lilies
and
the like). It will be understood that the compositions can be adapted to
specific
crops, to meet particular needs.
In accordance with the present invention, there is thus
provided an agricultural composition for enhancing plant crop seed germination
and/or seedling emergence andlor growth of a plant crop comprising a growth-
promoting amount of at least one lipo chitooligosaccharide (LCO) together with
an agriculturally suitable carrier. There is also provided a composition for
breaking the dormancy andlor quiescence of a plant, comprising a growth-
promoting amount of at least one lipo chitooligosaccharide (LCO) together with
an agriculturally suitable carrier. Furthermore, there is provided a method
for
enhancing seed germination and/or seedling emergence and/or growth of a
plant, comprising a treatment in the vicinity of one of a seed, root or plant
with
a composition comprising an agriculturally effective amount of a lipo
chitooligosaccharide (LCO) in admixture with an agriculturally suitable
carrier
medium, wherein the effective amount enhances seed germination andlor
seedling emergence and/or growth of the plant in comparison to an untreated
plant. There is further provided a method for enhancing seed germination
andlor seedling emergence and/or growth of a plant crop comprising incubating
a rhizobial strain which expresses a lipo chitooligosaccharide (LCO) in the
vicinity of one of a seed andlor root of the plant such that the LCO enhances
seed germination and/or seedling emergence andlor growth of the plant crop.
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As used herein, the term "rhizobia" is used broadly to refer to
bacterial strains which are involved in a nitrogen fixing symbiotic
relationship
with a legume.
As used herein, the term "LCO" refers broadly to a Nod factor
which is under the control of at least one nodulation gene (nod gene), common
to rhizobia. LCO therefore relates to a bacteria-to-plant signal molecule
which
induces the formation of nodules in legumes and enables the symbiotic bacteria
to colonize same. Broadly, LCOs are lipo chitooiigosaccharide signal
molecules,
acting as phytohormones, comprising an oligosaccharide moiety having a fatty
acid condensed at one of its end. An example of an LCO is presented below as
formula i
CHaORt CHtORs
0
O G
NH-R~
2o NH-CO-x,
in which:
G is a hexosamine which can be substituted, for example, by an acetyl group on
the nitrogen, a sulfate group, an acetyl group andlor an ether group on an
oxygen,
R,, R2, R3, R5, R6 and R,, which may be identical or different, represent H,
CH3C0-, CxHyCO- where x is an integer between 0 and 17, and y is an integer
between 1 and 35, or any other acyi group such as for example a carbamyl,
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R4 represents a mono-, di- or triunsaturated aliphatic chain containing at
least
12 carbon atoms, and
n is an integer between 1 and 4.
More specific LCOs from R. melilofi have also been described
in 5,549,718 as having the formula II
OR
/
~ ~ ~spH
O O
HO
0 ~~~ii~ 0
N 'I ' ~H
HO ~ HO IM OH
CO
a~, j
H _ i GIs
(~~s
CH
II
cx
I
(~sh
in which R represents H or CH3C0- and n is equal to 2 or 3.
Even more speck LCOs include NodRM, NodRM-1, NodRM-
3. When acetylated (the R = CH3C0-), they become AcNodRM-1, and
AcNodRM-3, respectively (U.S. 5,545,718).
LCOs from B. japonicum have also been characterized in U.S.
5,175,149 and 5,321,011. Broadly, they are pentasaccharide phytohormones
comprising methylfucose. A number of these B. japonicum-derived LCOs are
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described : BjNod-V (C,B;,); BjNod-V (A~, C,B:,), BjNod-V (C,s;,); and BjNod-V
(A~, C,s:o), with "V" indicating the presence of five N-acetylglucosamines;
"Ac"
an acetylation; the number following the "C" indicating the number of carbons
in the fatty acid side chain; and the number following the ":" the number of
5 double bonds.
it shall also be understood that compositions comprising
different LCOs, are encompassed within the scope of the present invention.
Indeed, while the present invention is exemplified with NodBj-V(C,e:,e)11 also
known as BjNod-V(C,B;,MeFuc); NodRM-V(C,6_2, S); and NodRl, any LCO
10 produced by a rhizobia which is capable of entering into a nitrogen
fixation
relationship with a legume (i.e. a member of the Fabiaceae family) is expected
to have the potential to show the same properties as those described herein.
1t wilt be clear to the person of ordinary skill that the selection of a
rhizobia
known to be expressing LCOs at high levels, or known to express an LCO
having an effect on a broader spectrum of legumes could be advantageous.
It will also be clear that the LCO compositions of the present
invention could also comprise more than one signal molecule. Non-limiting
examples of such compositions include agricultural compositions comprising in
addition to one LCO: (1) at least one additional LCO; (2) at least one plant-
to-
bacteria signal molecule; (3) gibberellic acid or other agents or compounds
known to promote growth or fitness of plants; and mixtures of such
compositions
(1), (2) or{ 3).
It shall be clear that having ident~ed new uses for LCO,
bacteria could be genetically engineered to express nod genes and used for
producing LCOs or for direct administration to the plants and/or seeds.
Thus, while the instant invention is demonstrated in particular
with LCOs from Bradyrhizobium japonicum, Rhizobium meliloti and R.
ieguminosarum and selected legumes and non-legume crops, the invention is
not so limited. Other legume crops, non-legume crops and rhizobial strains may
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be used using the same principles taught herein. Preferred matching of
rhizobia
with legume crop groups include, for example:
rhizobial species Legume crow ra oun
R. meliloti alfalfa, sweet clover
R. leguminosarum peas, lentils
R. phaesolii beans
Bradyrhizobium japonicum soybeans
R. trifolii red clover
As will be apparent to the person of ordinary skill to which the
present invention is directed, the growth-stimulating compositions of the
present
invention can be applied to other crop plants and especially to other warm
climate adapted crop plants (plants or crops having evolved under warm
conditions (i.e. tropical, subtropical or warm temperature zones] and whose
metabolism is optimized for such climates). It should be understood that the
growth-enhancing compositions of the present invention should find utility
whenever a particular crop is grown in a condition which limits its growth.
More
particularly, whenever a particular plant crop is grown at a temperature which
is
below its optimum temperature for seed germination, seedling emergence,
growth and the like. Such temperatures are known in the art. For example,
optimum temperatures for germination of corn, soybean, rice and cotton are
30°C, 34-36°C, 30-32°C, and 34°C, respectively.
The minimum germination
temperatures (or base temperatures) for these crops are 9°C,
4°C, 8 to 10°C,
and 14°C, respectively, while the maximum germination temperatures are
40°C,
42-44°C, 44°C and 37°C, respectively. The compositions of
the present
invention therefore find utility, among other things, in enhancing germination
of
warm climate adapted crops when grown at temperatures between their base
temperature for seed germination, andlor seedling emergence and/or growth
and their optimum temperature for germination. The compositions of the present
invention find utility in general in enhancing seed germination andlor
seedling
emergence andlor growth of crop plants when grown under conditions which
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delay or inhibit seed germination and/or seedling emergence thereof. Non-
limiting examples of such inhibiting conditions (as known from their
signalling
inhibition in bacteria-legume interactions, their inhibition or delay of the
bacteria-
plant symbiotic relationship) include pH stress, heat-stress, and water
stress.
It will be nevertheless recognized that the compositions and
methods of the present invention enhance growth of plants grown under optimal
conditions.
Thus, the compositions and methods of the present invention
should not be limited to plants growing under sub-optimal conditions.
The term "environmental conditions which inhibit or delay the
bacterial-plant symbiotic relationship" should be interpreted herein as
designating environmental conditions which postpone or inhibit the production
and exchange of signal molecules between same and include, without being
limited thereto: conditions that stress the plant, such as temperature stress,
water stress, pH stress as well as inhibitory soil nitrogen concentrations or
fixed
nitrogen.
"An agriculturally effective amount of a composition" for
increasing the growth of crop plants in accordance with the present invention
refers to a quantity which is sufficient to result in a statistically
significant
enhancement of growth andlor of protein yield and/or of grain yield of the
plant
crop as compared to the growth, protein yield and grain yield of the control-
treated plant crop. As will be seen below, the growth promoting activity of
the
LCOs are observable over a broad range of concentrations. Indeed, LCO
growth-promoting activities can be observed at an applied concentration of
about 10-5 to 10-'4 M, preferably about 10'6 to about 1 Q~'2 M and more
preferably
about 10-' to about 10''° M.
The term "immediate vicinity of a seed or roots" refers to any
location of a seed or roots wherein if any soluble material or composition is
so
placed, any exhibit of the plant or of the bacteria, or bacterial cells will
be in
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actual contact with the seed as it germinates or the roots as they grow and
develop.
Direct or indirect methods of inoculation with the composition
of the present invention can be employed. During direct inoculation the
composition is applied directly to the seed prior to sowing. This can most
simply
be accomplished by spraying the seed with or dipping the seed into a liquid
culture containing the desired components.
The recitation "short season condition" refers herein broadly
to temperatures of the middle and temperate zones and shorter. Typically, the
active growing season is around 1/2 to 2/3 of the year. Short season
conditions
broadly refers to a frost-free period of less than half the year, often on the
order
of 100 frost-free days.
By "nodulation gene-inducing" or "nod gene-inducing" is
meant bacterial genes involved in nodule establishment and function.
By "seed germination" is meant a clear evidence of root
growth developing from the embryo on the seed. When referring to an
"increased seed germination", the Applicant refers to a significant difference
in
seed germination between the treated versus the control seed.
"Seedling emergence" is meant to refer to growth of the plant
which is observable above the rooting medium surface. When referring to an
"enhanced seedling emergence", the Applicant refers to a significant
observable
difference between the growth of the seedling in the treated versus the
control.
BRIEF DESCRIPTION OF THE DRAWING
Having thus generally described the invention, reference will
now be made to the accompanying drawing, showing by way of illustration a
preferred embodiment thereof, and in which:
Figure 1 shows the seed germination enhancing effect of a
composition according to the present invention on corn.
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Other objects, advantages and features of the present
invention will become more apparent upon reading of the following
non-restrictive description of preferred embodiments with reference to the
accompanying drawing which is exemplary and should not be interpreted as
limiting the scope of the present invention.
~~ESw,°.IPTION OF THE PREFERRED EMBODIMENT
While the effects on nodulation were detected upon treatment
of soybean with SoyaSignal~ (a composition comprising both the plant-to
bacteria and bacteria-to-plant signal molecules), it was also noted that in
many
of the field experiments the plants that received some sort of genistein
treatment
emerged from the soil sooner. Thus, an experiment, in which genistein alone,
B. japonicum alone, and genistein plus 8. japonicum were added to soybean
seeds under controlled environment conditions, was conducted. Because slow
germination of corn (and other plants, as well) is a serious agricultural
problem
in eastern Canada because the weather conditions limit the growth thereof, com
was also treated in a similar fashion. The experiment showed that the seed
germination and seedling emergence promoting effect was present with the
combination of genistein plus B. japonicum, leading to the conclusion that the
enhancing effects were due to the LCOs produced by genistein exposed B.
japonicum. Purification (HPLC and otherwise) of the LCO most abundantly
produced by genistein-stimulated B. japonicum (NodBj-V(C,e:,e11 )) was carried
out. This was aided by the gracious gift of enough LCO material to standardize
the assay (G. Stacey, University of Tennessee at Knoxville; U.S. 5,175,149 and
U.S. 5,321,011) which allowed both isolation and quantification. With isolated
NodBj-V(C,8;,,11 ), research on the ability of this compound to stimulate seed
germination, seedling emergence and growth of leguminous and non-
leguminous plants could be conducted.
These experiments surprisingly demonstrated that the
addition of SoyaSignal~ (which comprises both an isoflavone and an LCO; the
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latter at a concentration of about 10-5M) accelerates the germination of com
seeds, whereas isoflavone solutions alone do not. Presumably this effect was
due to the LCOs produced by B. japonicum cells and induced by the presence
of isoflavones: When the seedlings were harvested (still at the mesocotyl
stage)
5 they were 44% longer and 33% as heavier in the genistein-B. japonicum
treated
versus non-treated plants (Figure 1 ). In addition, not only did seedling
emergence increase, but the rate of cotton seed germination was also
accelerated by the application of SoyaSignal~. The germination rate of cotton
seeds treated with SoyaSignal~ (0.66 mllkg seed) increased by 145% compared
10 to those control seeds that were treated with pure water. Both the corn and
cotton experiments were conducted at low temperatures, 15°C and
17°C for
corn seeds and for cotton seeds, respectively.
The field trial showed that the time of tasselling of sweet com
treated with SoyaSignal~ (planted on May 6 on the Experimental Farm of McGill
15 University, Quebec) was 1 to 2 days earlier compared to that of untreated
plants. Soybean seeds that received SoyaSignal~ (planted on June 22 in
Martinsville, Illinois) emerged 8 hours earlier compared to control seeds
while
the first trifoliar fully expanded 1 day earlier. At the agronomy farm of
Purdue
University, Indiana, soybeans planted in early June and observed in early July
were already one stage further in their development (V6) compared to the
control plants (V5). In a farmer trial (in Jackson, Illinois), plants that
received
SoyaSignal~ had many more nodules on the secondary roots and were 10%
taller than untreated plants.
Thus, an LCO (a bacteria-to-plant signal molecule involved
in the establishment of the symbiotic relationship between a rhizobia and a
legume) can promote growth of corn, a monocot distantly related to legumes.
Based on the evolutionary divergence of corn from legumes and the significant
response thereof to the LCO treatments, corn was used as a model plant
system in follow-up experiments. These experiments demonstrated that the
results obtained with corn were also observable with all other crop plants
tested.
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Taken together, the laboratory data and field trials presented
herein show that an LCO can increase seed germination, seedling emergence
and plant growth of legumes and non-legume plants under controlled
environment and field conditions.
The signal molecules are also shown to break the dormancy
of potato tubers. Of note, the dormancy experiments showed that the signal
solution was better at increasing the yield of potato tubers as compared to
other
dormancy breakers (i.e. giberellic acid).
The precise mechanism of action of LCOs on seed
germination, seedling emergence, dormancy and plant growth of legumes and
non-legumes is not fully understood. The general understanding of the role of
LCOs in signalling during the establishment of the legume-rhizobia symbiosis
was described above. When added to the appropriate legume, LCOs can cause
the induction of nodule meristems. Thus, it is possible that LCOs might be
normal signal molecules in higher plants, so that exogenously supplying them
simply increases their levels and, therefore, the activity of the things they
would
normally regulate. Alternatively, there may be an endogenous class of signal
molecules which play important roles in plant development, and have a
conformation similar to those of LCOs. One possible candidate for this is the
oligosaccharins (Fry ef al. 1993), some of which do stimulate meristem
activity
(Pavlova et al. 1992). LCOs are somewhat similar in structure and chemistry to
the oligosaccharins (Fry et al. 1993) and can, in the broadest sense, be
included
in that group (Stokkermans et al. 1995). However, the signal molecules with a
similar conformation need not be chemically similar, as demonstrated by the
ability of opiates (plant alkaloids) to fit into receptor sites normally
occupied by
endorphrins (oligo-peptides). Nothing is known regarding the mechanisms by
which LCOs cause this activity. Without being limited to a particular theory,
the
present invention is nevertheless the first to have identified a seed
germination,
and/or seedling emergence andlor dormancy breaking andlor plant growth
promoting effect of a composition comprising LCOs on non-legume plants.
SUBSTITUTE SHEET (RULE 26)
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Crops, such as soybean, corn and cotton evolved in relatively
warm climates and, as a result, have high base temperatures for germination,
being of about 5°C for soybean,10°C for corn and 14°C for
cotton. These high
base germination temperatures lead to slow emergence after planting, resulting
in slow leaf ground cover early in the season (when the temperature is sub-
optimal), which in turn leads to poorer early season light interception,
poorer
competition with weeds (and therefore greater need for herbicide application)
and increased soil erosion during heavy rainfall events. To simplify, these
crops
are often grown under conditions which limit their seed germination, andlor
seedling emergence andlor growth. Hence, the use of a growth-promoting
factor which is in limiting amount can compensate for a deficiency or stress
in
the growth conditions. Using SoyaSignal~ as a plant growth regulator could
thus
partially overcome the negative effects of environmental stress conditions,
such
as low soil temperature on crop seed germination, seedling emergence and
plant development. Thus, the present invention provides the means to improve
the production of crops of tropical and subtropical origin in the temperate
zones
and may extend their production into shorter season areas. In addition, the
present invention provides the means to improve production of crops growing
under stress conditions.
The present invention is illustrated in further detail by the
following non-limiting examples.
EXAMPLE 1
Induction of LCO production by Bradyrhizobium japonicum
The first culture containing Bradyrhizobium japonicum (strain
532C) was grown at 28°C in 100 -125 mL of sterile yeast mannitol media
(YEM)
with pH 6.8, shaken at 150 rpm until the ODszo reaches 0.4-0.6 (4-6 days).
Thereafter, a 2L bacterial subculture was started by inoculating with material
from the first culture (5 mL of first culture per 250 mL of YEM media), for 5-
7
days (ODszo - 0.8-1.0), as above. At this stage, 0.25 L of 50 ~cM genistein
(in
SUBSTITUTE SHEET (RULE 26)
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18
methanol) were added to each 250 mL of bacterial subculture (genistein
concentration of 5 ~cM ) and the culture was incubated for 48-98 hours, the
flavone thereby inducing LCO production in the bacterial cells.
EXAMPLE 2
Induction of LCO production by Rhizoblum meliloti
or Rhlzoblum legtrminosarum
The first culture of Rhizobium meliloti strain RCR2011 was
grown at 28°C in 100-125 mL of sterile yeast mannitol media (YEM) with
pH 6.8,
shaken at 150 rpm until the ODsp reaches 0.4-0.6 (2-3 days). Thereafter, a 2L
bacterial subculture was started by inoculating first culture (5 mL of first
culture
per 250 mL of YEM media), for 2-3 days (ODD - 0.8-1.0), as above. At this
stage, 0.25 mL of 50 uM luteolin (in methanol) was added to each 250 mL of
bacteria! subculture (luteoiin concentration of 5 ~cM ) and the culture was
incubated for 48 hours, the fiavone thereby inducing LCO production in the
bacterial cells.
For LCO production by Rhizobium leguminosarum, the
rhizobia was grown similarly as above. The flavone (naringenin) was added to
the subculture of R. ieguminosarum (10 NM) and the procedure carried out as
above.
EXAMPLE 3
Extraction and purification of LCOs
Two liters of bacterial subculture were phase-partitioned
~ against 0.8 L of HPLC-grade 1-butanol by shaking overnight. The upper
butanol layer was then transferred to a 1 L evaporation flask and evaporated
at
80°C to 2-3 mL of light brown, viscose material with a Yamato RE500
Rotary
Evaporator, which was resuspended in 4 mL of 1896 aoetonitrile and kept in the
dark at 4 °C in a sealed glass vial.
* Trade-mark
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PCT/CA99/00666
HPLC analysis was conducted with a Vydac C18 reversed-
phase column with flow rate 1.0 mUmin and a Vydac guard column. As a
baseline, acetonitrile (AcNI HzO; wlw) was run through the system for at least
min. When the sample was loaded, an isocratic elution was started by 18%
5 of AcN for 45 min. This step aims at removing all non-polar contaminant
light
fractions. Thereafter, a gradient elution for 90 min. with 18-82% AcN was
performed. LCOs began to elute after 94-96 min. of HPLC run time.
For the purification of LCOs from R. leguminosarum (which
nodulates numerous legumes), the HPLC peaks were identified and compared
10 to those obtained with B. japonicum and R. meliloti. LCO peaks which were
different from those of these two other rhizobia were identified collected.
Thus,
it is strongly suggested that the R. leguminosarum LCOs used herein are
different from that of B. japonicum or R. meJiloti.
EXAMPLE 4
Effect of LCO on emergence of some plant species
Plastic pots (7.5 cm dia) were filled with 15g of autoclaved
vermiculite. Seeds of corn (Zea mays - Poaceae), bean (Phaseolus vulgaris -
Fabaceae), canola (Brassica napus - Brassicaceae), cucumber (Cucumis
sativus - Cucurbitaceae), cantaloupe (Cucumis melo - Cucurbitaceae), cotton
(Gossypium sp.- Malvaceae), lettuce (Lactuca sativa - Asteraceae), beet (Beta
vulgaris - Chenopodiaceae), and soybean (Glycine max - Fabaceae), were
placed at 2.5 cm deep at the rate of 5 or 10 seeds per pot. Pots were
irrigated
with either 25 mL of LCO solution at different concentrations (10'6 - 10'"M)
or
aqueous acetonitrile or water, as controls. Acetonitrile was included as one
of
the controls since LCO was purified in this solvent (see Example 3), after 4
days
the pots were irrigated with 10 mL of water once every two days. Each
treatment
had 4 replications in a randomized block design. Pots were placed on a green
house bench maintained at 25 t 2°C with a day/night cycle 16/8h and
relative
humidity of 70%, or in a growth chamber set at 15°C with a 16:8
daylnight cycle.
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As defined above, seed germination has occurred when clear
evidence of root growth developping from the embryo on the seed is observed.
As the time required for seedling emergence of the species
used in the experiment varied considerably, observation on seedling emergence
5 was recorded when the emergence was observed for at least 50% in most of the
treatments. As defined above, seedling emergence has occurred when growth
of the plant can.be observed above the rooting medium surface. The percent
emergence was calculated. The data were analyzed with Statistical Analysis
System, version 6.12 (SAS institute inc. Cary, NC, USA).
10 LCO treatment reduced the time required from sowing to
emergence of a number of economically important plant species tested. Among
the species tested, Z. mays, L. sativa, B. vulgaris, P. vulgaris, and G. max
showed significant increases in emergence when treated with LCO at 25°C
(Table 1 ), while, C. sativus and B. napes showed similar effects at
15°C (Table
15 2).
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TABLE 1
Effect of iipo chitooligosaccharide on
seedling emergence (%) at 25°C
TreatmentZea Beta Glycine Gossy- Cucumis Letuca Phaseolus
mays vulgarismax pium meio sativa vulgaris
sp.
Control 40 cW 33 a 40 a 55 b 80 a 5 d 44 abc
10-sM 76 ab NT 65 d 88 a NT 45 a 67 abc
10-'M 68 abc 66.6 80 be 66 ab 100 b 35 ab 89 a
10$M 84 a NT 90 ab 88 a NT 10 do 78 ab
10-9M 88 a 66 b 100 a 88 a 100 b 20 bcd 67 abc
10-'M 84 a NT 70 cd 88 a NT 25 abcd 44 abc
10-"M 68 abc 86 b 50 a NT 100 b 26 abcd 22 c
10''2M 48 abc NT 80 be NT NT 30 abc 33 be
10''3M 40 c 80 b 70 cd NT 100 b 5 d 33 be
10''"M 40 c NT 70 cd NT NT 10 d 33 be
'~ means with in the same column, followed by the same letter are not
significantly
different (p s 0.05) by ANOVA protected LSD test.
NT- Not tested
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TAB
Effect of lipo chitooligosaccharide
on seedling emergence at 15°C
Treatment Cucumis sativus Brassica napus
Control 60 c'~ 32.5 c
Lipo chitooligosaccharide
10-6M 65 abc 35 be
10-'M 85 ab 32 c
10-8M 80 ab 35 be
10'9M 70 abc 52 ab
10-'M 50 c 62 a
10'"M 80 ab 47 be
10-'ZM 80 ab 45 be
10-'3M 70 abc 37 be
10''"M 70 abc 30 c
Tmeans with in the same column, followed by the same letter are not
significantly
different (p s 0.05) by ANOVA protected LSD test.
SUBSTITUTE SHEET (RULE 26)
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23
In some plants, germination/emergence promoting effects of
LCOs is seen at all temperatures suitable for growth, while in others, it is
only
observed under temperature-limiting conditions.
EXAMPLE 5
Effects of LCO on early growth of corn
The percent seedling emergence was recorded at 4 days after
sowing (DAS). Plant height was recorded from 4 DAS to 15 DAS. Plants were
harvested at 15 DAS and leaf area, root length and the number of roots per
plant
were recorded. The plants were then dissected and placed in paper covers and
dried at 90°C for 24h and the dry weights of roots, shoots and the
spent seeds
recorded. The data were analyzed with the Statistical Analysis System version
6.12
(SAS institute Inc. Cary, NC, USA).
!n LCO treatments, seedling emergence started 2-3 days after
seeding while in the control it was 3-4 days. LCO treatments significantly
increased
leaf area, root length, number of roots, shoot dry weight and root dry weight,
while
the weight of spent seed recorded significant decreases as compared to the
control
(Table 3). The optimum effect was observed at an LCO concentration of 10~BM.
The decrease in spent seed weight is attributed to the rapid transfocation of
stored
reserve from the seed endosperm to the embryo. Of interest, a dramatic
increase
of a-amylase activity was observed in the treated seeds.
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24
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SUBSTITUTE SHEET (RULE 26)
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Taken together, Examples 4 and 5 and Tables 1-3 show that
LCOs can stimulate seedling emergence in all tested plants. In addition, a
significant growth stimulation of corn was observed. Furthermore, the spent
seed
weight results suggested that LCOs also had an effect on seed germination for
all
5 tested plants. The growth-promoting effect of LCOs on corn, a plant quite
distantly
related to legumes (i.e. com is a monocot), strongly suggests that plants in
general
should show the same growth-responses to LCO treatment.
EXAMPLE 6
10 Dormancy breaking activity of LCO on potato mini tubers
Signal solution is a bacterial fermentation tank product,
comprising approximately 10'6 M LCO from 8. japonicum. More specifically,
signal
solution is the supernatant from a culture of B. japonicum in which genestein
(a
flavone) had been introduced to promote LCO expression. Following the
15 subculture of B. japonicum, the bacteria was removed. While the stimulatory
effect
of Signal solution in the soybean-Bradyrhizobium japonicum complex has been
described {Zhang and Smith, 1995; Zhang ef al. 1996), the effects of these
plant
substances in other plant species and their associated rhizospheres' organisms
have not been investigated.
20 Gibberellic acid (GA) and kinetin affect both the germination rate
and the percent germination of crop seeds.
Some studies have indicated that plant growth regulators
(PGRs), such as gibberellic acids (GAs), stimulate seed germination at low
temperatures. Durrant and Mash (1991) reported that adding gibberellins (GA4n)
25 to sugar-beet seeds (Beta vulgaris L. Var. altissima) was beneficial to
seed
germination under cold, wet conditions.
Kepczynski and Bialecka reported that Methyl jasmonate (JA-
MB) inhibited or retarded germination of Amaranfhus caudatus seeds in darkness
at 24°C. Ethephon, ACC (1-aminocyclopropane-1-carboxylic acid) and
gibberellins
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(GA3 or GA4+,) partially or completely reversed this inhibition depending on
the
concentration of JA-Me applied.
Indeed, gibberellic acid, as well as bromoethane, are used
commercially to break dormancy and to stimulate sprout formation.
Treatments were carried out on microtubers (200-400 mg) that
had been cold-stored for 8 wk to determine their effect on breaking dormancy.
Signal solution was used at full strength (100%) or diluted to 20% (as for
soybean),
12%, or 6% of full strength. GA3 (500 mg I-'), water soaking, and control
treatments
were performed for comparison purposes. Microtuber soaking treatments lasted
24
h and then incubation occurred either in the light (40 p mol m-2s-' coot-white
fluorescent) or in the dark. Five microtubers were used in each treatment.
Observations for sprouted microtubers were made at 1 and 2 wk.
One hundred % signal solution was as effective as GA3 (500 mg
f') when evaluated after 1 wk with respect to the number of sprouted
microtubers.
Table 4 shows the effect of signal solution (SS) on dormancy breaking of
potato
microtubers as compared to the known dormancy breaker gibberellic acid (GA3)
(200-450 mg) that had been cold-stored at 5°C for 8 weeks and evaluated
after
treatment and incubation with or without light for 1 and 2 weeks, for number
of
sprouts and for number with multiple sprouts ( r 1 ) at 2 weeks. One hundred
signal solution induced multiple sprouts and dark incubation favoured
sprouting as
compared with the light regime after 1 wk of incubation. The exact cause of
dark
incubation favouring 100% signal solution is not understood. One hundred %
signal .
solution was more effective than diluted signal solution when numbers of
sprouted
microtubers were counted after 1 wk. After 2 wk of incubation all treatments
were
equally effective in causing sprouting but the signal solution and GA3
solutions were
most promotive of multiple sprouting which did not occur in the water soaking
treatment and only in the control treatment incubated in the dark.
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TA L 4
PCT/CA99I00666
Effects of signal solution (SS) on dormancy breaking
of potato microtubers as compared to the known
dormancy breaker gibberellic acid (GA3)
Number of Number of Mean
sprouted
Treatments microtubers multiple number
of
sprouted sprouts
microtuberst
1wk 2wk (2wk) SE
GA3 500 mgl-' ght 115 5I5 2/5 2.5 t
+ li 0.5
GA3 500 mgi-' 5I5 4I5 2.5 t
- light 5/5 0.3
100 % SS + 0/5 5/5 215 2.510.5
light
100 % SS - 5/5 5/5 3/5 2.310.3
light
20 % SS + light0/5 415 0 0
20 % SS - light215 515 2/5 2.0 t
0
12%SS+light 015 4/5 1/4 2.010
12%SS-light 215 5/5 2l5 2.010
6 % SS + light0/5 4/5 214 2.0 t
0
6 % SS - light1/5 5/5 2/5 2.O t
0
Water + light OI5 4I5 0 0
Water - light 2I5 5/5 0 0
Control + light015 515 0 0
Control - light1I5 5/5 1/5 2.0 t
0
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Tuber sprouting of potatoes is somewhat comparable to seed
germination in the sense that the plant meristems are activated and the plant
is
beginning to grow, following quiescence. A signal molecule involved in
bacteria-
legume signalling was shown to be effective in breaking the dormancy of a
plant
{potato) that is distantly related to the legumes. LCOs therefore seem to have
a
broad effect on breaking the dormancy or quiescence of plants.
EXAMPLE 7
Effects of combinations of GA3 and 100% signal solution in breaking
dormancy of potato tubers
The data presented in Example 6 suggested that the 100%
signal solution (and thus the LCO purified from 8. japonicum) was effective in
breaking microtuber dormancy. However, the microtubers used in the trial had
been cold-stored for 8 wk. In this trial, the effects of signal solution were
evaluated
in combination with GA3 on minitubers with only 3 wk of cold storage. It was
also
investigated whether the effect of 100% signal solution might be synergistic
if used
with GA3 (500 mg f').
Minitubers (20-30 g) with 3 wk cold storage treatment were
soaked for 24 h in 500 mg I-' GA3, 100% signal solution, or a mixture of the
two.
Another treatment involved successive soaking for 12 h each, in first GA3, and
then
signal solution. A control treatment without soaking was also performed. Eight
minitubers were used per treatment which were applied at room temperature
(20°C). Microtubers were observed after 2 wk and the number of sprouted
minitubers and the number with multiple sprouts were counted.
All treatments were able to break minituber dormancy except the
control (Table 5). The 500 mg f' GA3 treatment alone or together with 100%
signal
solution, for 24 h, caused 100% sprouting, and significantly more multiple
sprout
formation than the other treatments. The 100% signal solution alone or in
combination with 500 mg f' GA3 were as effective as 500 mgl-' GA3 alone for
breaking dormancy within 2 wk. However, less multiple sprouting occurred with
100% signal solution alone, or following 12 h GA3 treatment, compared with the
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PCT/CA99/00666
GA3 treatment alone or the combined GA3 and Signal treatments. When working
under the conditions tested, there were no clear synergistic effects of 100%
signal
solution in combinations with 500 mg 1-' GA3 on the number of sprouted tubers
or
multiple sprouts.
TABLE 55
Individual and combined effects of GA, and 100%
signal solution (SS) on dormancy breaking of minitubers
that had been cold-stored for 3 weeks
Treatments Number Number of Mean number
of
sprouted minituber withof sprouts t SE
minituber multiple sprouts
GA3 500 mg f' 8l8 6I8 3.37 t 0.62
24 h
100% SS 7I8 2I7 1.33 t 0.42
24 h
GA3 500 mg I-' 8/8 6/8 2.37 t 0.41
+
100% SS
12h+12h
GA3 500 mg I-' 8/8 6/8 3.75 t 0.67
+
100% SS
Combination
24 h
Control 0/8 0/8 0/8
These results suggest that bacteria-legume signal molecules are
effective in breaking the dormancy of potatoes. Taken together with the
results
5 presented above (i.e. Example 4 and Table 1 ) showing the effects of a pure
LCO
in breaking the quiescence of seeds and promoting growth of a variety of
distantly
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related plants, strongly supports the contention that LCOs are effective at
breaking
the dormancy of potatoes and promoting the activity of plant meristems in
general.
EXAMPLE 8
5 Effectiveness of GAS and signal solution, as compared to bromoethane
and mechanical injuries, in breaking dormancy
Bromoethane (BE) was reported to break potato tuber dormancy
when applied as a fumigant and it was found that BE at a concentration of 0.2
ml
10 I-' was the most effective (Coleman, 1983). Conventionally, large potato
tubers are
cut into small pieces, each containing an eye, to be used as seed pieces. To
obtain
quick and uniform sprout emergence, potato tubers should be cut at least 2 wk
before planting (Slomnicki and Rylski, 1964). Mechanical injury is also shown
to
contribute to sprout induction. The objective of this experiment was to
compare the
15 effects of known dormancy-breaking treatments on microtubers and minitubers
1)
BE; 2) GA3; and 3) mechanical injury; with the newly identified dormancy
breaker:
LCOs.
Microtubers (200-500 mg) cold-stored for 8 wk and minitubers
(20-35 g) cold-stored for 0, 2, or 8 wk were used for these experiments. BE
(0.2 ml
20 I-') and mechanical injury (cutting in half, microwaving at full power for
10 sec)
were compared with GA3 (500 mgl-'), 100% signal solution, water soaking, and
control treatments. Six microtubers or minitubers were used per treatment.
Observations were made at 1, 2, 3, and 4 wk intervals and the number of
sprouted
tubers were counted. The evaluation period was extended because tubers with
little
25 or no cold storage treatment took longer to sprout.
GA3 was the only agent which was able to break dormancy of
minitubers that had not been cold-stored; 0/6 at 2 wk but 4I6 by 4 wk
(minituber, 0
wk storage; Table 6). Minitubers with 2 wk cold storage that were treated with
GA3
also broke-dormancy; OI6 at 2 wk but 5/6 by 4 wk with 216 showing multiple
shoots.
30 Signal solution treatment of minitubers cold-stored for 2 wk caused 1/6
(with
multiple shoots) minitubers to break dormancy after 4 wk. For minitubers cold-
stored for 8 wk, all treatments (including water soaking and control), except
the
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microwaving, showed some sprouting the first week and multiple sprouting was
evident in the GA3 (6/6), 100% signal solution {4/6), and BE (1/6) treatments
by 2
wk. Cutting caused sprouting in 9/12 cut halves by 2 wk but only a few
multiple
shoots (2/12) were evident by 3 wk.
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PCT/CA99/00666
With microtubers cold-stored for 8 wk, dormancy-breaking
occurred within the first week in all treatments except microwaving. After 2
wk,
sprouting had progressed in all treatments; water (416) was similar to GA3
solution (5/6), BE (4/6), signal solution (4/6), and cutting (8112). Only GA3
solution (3/6 at 2 wk, 616 at 4 wk) and signal solution (116 at 4 wk) caused
multiple sprout formation.
Signal solution (100%) was effective in causing sprouting in
minitubers with 2 or 8 wk cold storage and microtubers with 8 wk cold storage
but it was ineffective on minitubers that had not been cold-stored. GA3 and
100
% signal solution induced multiple sprouts from different eye-points unlike
the
control BE, or cutting treatments, that induced single sprouts only from the
rose
end. BE worked well in inducing single sprouts in minitubers and microtubers
with 8 wk cold-storage but was not as effective as GA3 for minitubers that had
not been cold-stored.
Cutting minitubers or microtubers in half after 8 wk cold
storage induced single sprouts on each cut half. This occurred quite
efficiently
in minitubers (9112 in 2 wk, 12/12 in 4 wk) and somewhat less efficiently with
microtubers (8/12 in 2 wk, 10112 in 4 wk). Cutting was not effective in
breaking
dormancy in minituber without cold storage and worked poorly in minitubers
that
had been cold stored for only 2 wk (OI12 after 2 wk, 1112 after 4wk).
Sprouting
from two halves was good, in the sense that by cutting minitubers or
microtubers
in half, two propagules, each with one sprout were derived although a very
insignificant number of minituber halves (2/12) showed multiple sprouting.
However, cutting was risky in that this sometimes provided opportunities for
fungal or bacterial infection. Microwaving induced limited sprouting but only
in
minitubers or microtubers that had been cold-stored for 8 wk, and not in the
minitubers with 0 or 2 wk cold storage. Microwaving caused some tuber
damage that may account for the reduced sprouting observed.
In short, signal solution was effective in promoting both
sprouting and multiple sprouting of mini- and micro-tubers and, of the tested
treatments, only GA3 was better.
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EXAMPLE 9
Effectiveness of anti-ABA compared with other dormancy breaking
treatments
Anti-Abscisic acid (anti-ABA), the acetyienic analog of ABA,
has never been used to induce sprouting in dormant potato tubers since it was
first shown to be an ABA antagonist (Wilen et al., 1993). However, anti-ABA
has
been used to terminate dormancy in canola seeds (PBI Bulletin, 1995). The
objective of this experiment was to test anti-ABA for breaking dormancy in
potato minitubers and compare its efficacy with other dormancy-breaking
treatments.
Microtubers (200-600 mg) were cold-stored for 3 wk prior to
the experiment. Seven microtubers were used per treatment. Treatments
included 24 h soaks in anti-ABA or GA3 (500 and 250 mg f', respectively)
applied alone or in combination, GA3 (500 mg f' in combination with 100%
signal
solution, and water. Bromoethane (0.2 ml I-') and control treatments were also
performed. Observations were made after 2 wk in the dark at room temperature
(20°C). Data included number of sprouted microtubers and number of
muftipie
sprouts. Means of sprout number were calculated only from microtubers that
had sprouted
2p Anti-ABA alone and in successive treatments or in
combination with GA3 was effective in breaking microtuber dormancy (Table 7).
Among the different treatments using anti-ABA and GA3 the greatest mean
number of sprouts (1.8 * 0.48) occurred when microtubers were soaked in a
mixed solution of 500 mg I-' GA3 and 500 mg I-' anti-ABA for 24 h but it was
not
significantly different from the 500 mg f' GA3 treatment (1.711 0.28). The
combined signal solution and GA3 was not more effective than GA3 alone and
was less effective than any GA3 and anti-ABA treatment in breaking dormancy.
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TABLE 7
Sprouting performance on microtubers cold-stored
for 3 weeks and evaluated at 2 weeks after
exposure to dormancy-breaking agents.
Treatments No. sprouted Mean No.
microtubers sprouts
SE
GA3 500 mg f' 7I7 1.71 t
0.28
24 h
GA3 500 mg I-' + 100% SS 3/7 1.33 t
0.29
24 h
Anti-ABA 500 mg I-' 5/7 1.4 t 0.24
24 h
Anti-ABA 250 mg I-' 5/7 1.2 t 0.20
24 h
GA3 500 mg I-' 12 h 6/7 1.33 t
0.21
Anti-ABA 500 mg I-' 12 h
GA3 250 mg I-' 12 h 7I7 1.57 t
0.29
Anti-ABA 250 mg I-' 12 h
GAa 500 mg i-' + Anti-ABA 500 mg I-' S/7 1.8 t 0.48
Combination 24 h
GA3 250 mg I-' + Anti-ABA 250 mg I-' 5/7 1.6 t 0.24
Combination 24 h
Bromoethane 1/7 1.0 t 0
Water 1/7 1.0 t 0
Control (no treatment) 0/7 0
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The overall results with anti-ABA underline its importance as
a potential dormancy-releasing agent, as much so as GA3 Anti-ABA and GA3
both induced multiple sprouts but sprouts were longer after GA3 than anti-ABA
treatment. Both agents caused sprouts to emerge at various eyes over the tuber
surface. However, the GA3-induced multiple sprouts were profusely branched;
a group of sprouts protruded from each eye, while the anti-ABA-induced sprouts
were singles. The mechanism of dormancy breaking by anti-ABA and GA3
therefore was similar, but GA3 appeared stronger. These agents should be
tested on an equimolar basis in the future.
EXAMPLE 10
Harvests from minitubers sprouted using a range of
dormancy-breaking treatments
There is only limited information on the relative yield
performance of potato tubers that were treated with dormancy-breaking agents
(Choudhury and Ghose, 1960; Slomnicki and Rylski, 1964). Yields from potato
tubers that were treated with GA3 at 25-100 mg f' (Choudhuri and Ghose, 1960)
or 5-40 mg I~' (Slomnicki and Rylski, 1964) were reduced compared with
untreated controls. The objective of this experiment was to evaluate the
effect
of dormancy-breaking agents on subsequent yield in greenhouse pot trials.
Minitubers (20-35 g) that were cold-stored for 8 wk were given
dormancy-releasing treatments including 24 h soaking in GA3 (500 mg i-'), 100%
signal solution, or water. Other treatments included BE (0.2m I-'), cutting in
half,
and the control. All minitubers were observed at 3 wk following treatment and
the number of sprouts were noted at the time of planting. Five minitubers per
treatment were individually planted into 11 x 12 cm plastic pots in the
greenhouse. The potting mixture was 2:1 peat:perlite without fertilizer added.
The pots were arranged in a complete randomized design and watered equally
every alternate day. Harvest occurred after 60 d and tuber yields (number and
fresh weight) were recorded.
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GA3 caused significantly more sprouts per minituber (4.2 t
0.37) than the other treatments, with 100% signal solution (2.0 t 0.31 ) and
BE
(1.8 t 0.2) giving intermediate values, and water-soaking and cutting similar
to
the control (Table 8). The average number of tubers per plant was greatest in
the GA3 treatment (3.6); almost double that of other treatments that were not
different from the control. Suprisingly, however, the mean fresh weight of
tubers
(per replicate i.e. pot basis) harvested from minitubers exposed to the 100%
signal solution treatment was the greatest (34.97 g); greater than the control
fresh weight and three times more than the GA3treatment The size and shape
of tubers harvested from the GA3 treatment were small and more elongated than
that of the control and other treatments. Yields from BE treated minitubers
were
significantly lower compared with controls. The cut halves each yielded almost
the same as uncut controls and had similar fresh weight to control (28.41 vs
27.16). Two cut halves of each minituber together would effectively double
control yield and bring the mean number of tubers into the GA3 treatment
range.
However, cutting into halves posed a problem of infection and decomposition at
the cut surfaces.
Thus, although signal solution is not as efficacious as GA3 in
breaking dormancy {as evaluated by the number of sprouts), it however is
significantly more efficient than GA3 in increasing the tuber yield. LCOs
therefore appear as the best agents to promote dormancy breaking and yield
increases in potato.
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TABL 8
Harvests after 60 d from minitubers that were forced to break
dormancy by different methods.
Treatments Mean number Mean number of Mean fresh
sprouts at tubers produced weight
planting per replicate (g)
SE
GA3 4.2 + 0.37 3.fi"' ' 11.13
d
Cutting into 1.2 0.20 2.0 28.41 b
halves
(1J2 minituber)
Bromoethane 1.8 0.20 2.0 14.01 d
100% Signal 2.0 0.31 1.8 34.97 a
soln.
Water 1.2 0.20 1.0 17.41 c
Control 1.0 0 2.0 27.16 b
Numbers represented by the same letter are not significantly different at the
0.05
level.
It shall be recognized therefore that agricultural compositions
comprising at feast one LCO and gibberellic acid (GA3 and others known in the
art) could be advantageously used in accordance with the methods of the
present invention to break dormancy and/or quiescence of crop plants.
EXAMPLE 11
Other LCOs
Following the methods described above, the LCO most
abundantly produced by R. meliloti (Nod Rm-V(C,6.2, S)) was isolated and
tested
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on alfalfa (Medicago saliva) seeds. Briefly, 10 seeds were placed in a disk of
filter paper on a petri plate. The filter paper was wetted with 5 ml of the
appropriate LCO solution. Data were taken at 12 hour intervals upon the
radicle
(an embryonic root). The number of seed with an emerged radicle were
counted. Each treatment was repeated four times. The data presented in table
9 indicate a clear acceleration of growth. In this case no standard for HPLC
calibration was available, so a relative dilution series was used. in
addition, a
cluster of peaks specifically induced by the specific flavone of Rhizobium
leguminosarum (bv phaseoli (strain 127K105]) were collected and tested on corn
(Zea mat's), red clover (TrifoGum repens, Fabaceae) and pea (Pisum sativum,
Fabaceae) (Table 10). In each case, a stimulation of seed germination was
observed. Of note, Rhizobium leguminosarum produces a large number of
LCOs. A subset of these LCOs was selected from a range of the HPLC profile
where the LCOs from 8. japonicum and R. meliloti did not occur. Taken
together, these results clearly demonstrate that the promoting effects of LCOs
on plant growth disclosed herein are observable with LCOs from different
bacterial strains involved in bacteria-legume signalling. Consequently, the
presented data strongly suggests that LCOs in general should demonstrate the
same effects on seed germination, seedling emergence, growth, dormancy
breakage and the like.
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TABLE 9
PCT/CA99100666
Effect of LCO isolated from Rhizobium meliloti (RCR 2011 )
on germination of alfalfa after 24 h of treatment
Treatment Percent
Germination
Control 16.7b
10'' dilution 26.6ab
10'2 dilution 26.6ab
10'3 dilution 36.6a
LSD (p<0.05) 19.2
In column numbers followed by same letter do not differ significantly by an
ANOVA protected LSD test at p < 0.05
TABLE 10
Effect of LCOs of Rhizobium leguminosarum by phaseoli (strain
127K105) on seed germination (%) of corn (after 48h), red clover
(after 12h) and pea (after 48h) at 25°C
Treatment Corn Red Clover Pea
Control 20a 43.3bc 26.6b
10'' dilution 26.6a 26.6c 26.6b
10-Z dilution 60.Ob 63.3ab 20.Ob
10'3 dilution 20.Oa 66.6a 73.3a
LSD (p < 0.05) 19.9 23.3 29.7
1n column numbers followed by same letter do not differ significantly by
anANOVA protected LSD test at p < 0.05
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EXAMPLE 12
Germination versus emergence
Seeds of corn (cv Pioneer 3921 ) were surface sterilized in
2% sodium hypochlorite solution for 2 minutes and placed in 9 cm diameter
Petri
plates containing a sheet of filter paper soaked in 10 ml of the required test
solution (LCO 10-5 - 10''3). Water served as the control. Observations on
germination, length of root primodia and shoot were taken after 72 h of
incubation at 25°C. The data was analyzed for significance by an ANOVA
protected LSD test using SAS system Version 6.1 (SAS Inc., Cary, NC, USA).
TABLE 11
Effect of lipo chitooligosaccharide [Bj Nod-V (C,e:, MeFuc)J on
germination of corn (Zea mays L.) after 72 h of treatment
Treatment Percent Length of root Length of shoot
germination primodia (mm) primodia {mm)
Control 46.6 a 32.3 a 4.6 a
LCO 10'SM 80 be 53.0 ab 12.3 ab
LCO 10''M 73.3 b 57.6 be 15.0 ab
LCO 10'9M 73.3 b 48.0 ab 9.6 a
LCO 10'"M 100 c 78.6 c 21.0 b
LCO 10'"M 80 be 43.0 be 8.3 a
LSD (p<0.05)22.6 24.3 11.0
In column numbers followed by same letter do not differ significantly by an
ANOVA protected LSD test at p < 0.05.
Table 11 shows that incubation of corn seeds with LCO
solution significantly improved the germination of corn and increased the
length of both shoot and roots.
5
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EXAMPLE 13
Seedling emergence-promoting effects of LCOs
under field conditions
PCT/CA99/00666
Seeds of corn, cotton, beet, and soybean which showed
promising results under laboratory conditions were tested for seedling
emergence under field condition. Seeds were surface sterilized with 2% sodium
hypochlorite and soaked in different concentrations (10-5, 10-', 10-9M) of LCO
solution for 12 h. Water served as the control. The study was conducted at the
experimental field of the Macdonald campus of McGill University, Ste-Anne-de-
Belleveue. Quebec, Canada. The field was ploughed to a fine tilth. seeds were
hand planted in 1 m rows at 2.5 to 3 cm deep with three replications per
treatment. The percent seedling emergence was observed at six days after
planting during which time at least 50% of the seeds emerged in the
treatments.
The data was analyzed for significance by an ANOVA protected LSD test using
SAS system Version 6.1 (SAS Inc., Cary, NC, USA).
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TABLE 12
PCT/CA99/00666
Effect of lipo chitooligosaccharide [Bj Nod-V (C,e:, MeFuc)~ on seedling
emergence under field condition
Treatment Corn Cotton Beet Soybean
Control 41.6 6.6 a 26.6 a 16.6 a
a
LCO 10-SM 80.0 16.6 28.3 a 26.6 ab
b a
LCO 10-'M 60.0 60.0 46.6 c 33.3 b
b b
LCO 10-9M 53.3 23.3 38.3 ab 63.3 c
b a
LSD (P< 0.05)35.7 28.0 16.3 16.6
In column numbers followed by same letter do not differ significantly by an
ANOVA protected LSD test at p < 0.05
Table 12 shows that LCO [Bj Nod-V (C,B:, MeFuc)] treatment
enhanced the seedling emergence under field conditions of all the crop species
studied. The best effect was observed in cotton where LCO at 10''M improved
the emergence by more than 9 times as compared to the control. The effective
concentration of LCO varied with the species.
Table 12 also validates the laboratory results presented
herein by demonstrating that the stimulatory effects of LCOs are operating on
four different crops under field conditions.
Thus the present invention provides agricultural compositions
and methods by which LCO could be used to enhance the germination, seedling
emergence, root growth and improve early growth of crops under laboratory or
field conditions.
CONCLUSION
The present invention therefore provides evidence that,
among other things: (1 ) lipo chitooligosaccharide (LCO) treatment enhances
the
seedling emergence of higher plant seeds (egs. Z. mays, L. sativa, 8.
vulgaris,
P. vulgaris, G. max C. sativus, 8. napes and M. safiva); (2) lipo
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PCT/CA99/00666
chitooligosaccharide breaks the dormancy of potato (Solanum tuberosum)
minitubers and increases their yield; (3) lipo chitooligosaccharide improves
emergence and early growth, including root growth, of Z. mays giving a
competitive advantage over non treated ones; (4) lipo chitooligosaccharide
enhances the translocation of stored seed reserve; and (5) lipo
chitooligosaccharide enhances seed germination.
Although the present invention has been described
hereinabove by way of preferred embodiments thereof, it can be modified,
without departing from the spirit and nature of the subject invention as
defined
in the appended claims.
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PCT/CA99/00666
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