Note: Descriptions are shown in the official language in which they were submitted.
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Improving the yield of plants
Technical field
The invention relates to the use of betaine to
improve the yield of plants. The invention relates
especially to the use of betaine to improve the yield
of grain legumes. According to the invention, the yield
can be ; _ ~ved both under normal and stress conditions,
10 i.e. when the conditions are poor due to e.g. drought,
high salinity, low temperatures, humidity or
environmental pollutants interfering with the growth.
The invention also relates to grain legumes treated with
betaine and to the parts thereof, especially seeds, and
15 to products prepared from these.
R:~-L~J~ d
The envi~ t and conditions for growth
considerably affect the yield of plants. Optimum growth
environment and conditions usually result in a yield
20 that is large in quantity and high in quality. Under
poor growth conditions both the quality and the quantity
naturally deteriorate.
The physiological properties of a plant are
preferably manipulated by means of br~; ng, both with
25 traditional br~A;ng methods and for example with
genetic manipulation.
Several different solutions concerning
cultivation t~hn;que have been developed to improve the
growth conditions and yield of plants. Selecting the
30 right plant for the right growth location is self-
evident for a person skilled in the art. During the
growing season plants may be protected with ~chAn;cal
means by utilizing for example different gauzes or
plastics or by cultivating the plants in greenhouses.
35 Irrigation and sprinkler irrigation, fertilizers and
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plant nutrients are generally used in order to ; ,~ove
the growth. Surfactants are often used in ~onn~ction
with applying pesticides, protective agents and
minerals. Surfactants ; _ ~ve the penetration of these
substA~ces into plant cells, thereby ~nh~nC; ng and
incre~;~ the effect of the aforementioned agents and
simultaneously reducing their harmful effects on the
envil. ?nt However, different methods of cultivation
t~hn;que are often laborious and impractical, their
effect is limited (the ecQn ~cal size of a greenhouse,
the limited protection provided by gauzes, etc.), and
they are also far too expensive on a global scale. No
econ~ ; cally acceptable chemical solutions for
protecting plants from stress conditions have been
described so far.
Water supply is more i _~L Lant than any other
envil~ -ntal factor for the productivity of a crop,
even though the sensitivity of plants to drought varies.
Irrigation is usually utilized to ensure sufficient
water supply. However, there are significant health and
envilc ntal problems related to irrigation, for
example a sharp decrease in water resources,
deterioration of water quality and deterioration of
agricultural lands. It has been calculated in the field
that about half of the artificially irrigated lands of
the world are damaged by waterlogging and salinization.
An indication of the significance and scope of the
problem is that there are 255 million hectares of
irrigated land in the world, and they account for 70%
of the total world water consumption. In the United
States alone, there are over 20 million hectares of
irrigated land mainly in the area of the 18 western
states and in the southeastern part of the country. They
use 83% of the total water consumption for irrigation
alone. It can also be noted that the use of irrigation
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water increases every year especi A 1 ly in industrial
countries. In addition to these problems, another
drawback of irrigation is the high cost.
Another serious stress factor is the s~l; ni ty
of soil. The salinity of soil can be defined in
different ways; according to the general definition,
soil is saline if it contains soluble salts in an amount
sufficient to interfere with the growth and yield of
several cultivated plant species. The most~ of the
salts is sodium chloride, but other salts also occur in
varying combinations AeF~nAl ng on the origin of the
saline water and on the solubility of the salts.
It is difficult for plants growing in saline
soil to obtain a sufficient amount of water from the
soil having a negative osmotic potential. High
co~ntrations of sodium and chloride ions are toxic to
plants. An additional problem is the lack of minerals,
which occurs when sodium ions compete with potassium
ions required, however, for cell growth, osmoregulation
and pH stabilization. This problem occurs especially
when the calcium ion ~o~c~ntration is low.
The productivity of plants and their
sensitivity to the salinity of soil also depend on the
plant species. Halophytes require relatively high sodium
chloride contents to ensure optimum growth, whereas
glycophytes have low salt tolerance or their growth is
considerably inhibited already at low salt
ron~ntrations, There are great differences even between
different cultivars of a cultivated plant species. The
salt tolerance of one and the same species or cultivar
may also vary depenAing for example on the stage of
growth. In the case of low or moderate salinity, the
slower growth of glycophytes cannot be detected in the
form of specific symptoms, such as chlorosis, but it is
shown in the stunted growth of the plants and in the
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colour of their leaves that is darker than normal.
Moreover, the total leaf area is reduced, carbon dioxide
assimilation decreases and protein synthesis is
inhibited.
Plants can adapt to some extent to growth and
stress conditions. This ability varies considerably
depending on the plant species. As a result of the
aforementioned stress conditions, certain plants begin
to produce a growth hormone called abscisic acid (ABA),
which helps the plants to close their stomata, thus
reducing the severity of stress. However, ABA also has
harmful side effects on the productivity of plants. ABA
causes for example leaf, flower and young fruit drop and
inhibits the formation of new leaves, which naturally
leads to reduction in yield.
Stress conditions and especially lack of water
have also been found to lead to a sharp decrease in the
activity of certain enzymes, such as nitrate reductase
and phenylalanine ammonium lyase. On the other hand, the
activity of alpha-amylase and ribonuclease increases.
No chemical solutions, based on these findings, to
protect plants have been described so far.
It has also been found that under stress
conditions certain nitrogen compounds and amino acids,
such as proline and betaine, are accumulated in the
regions of growth of certain plants. The literature of
the art discusses the function and meaning of these
accumulated products. On the one hand it has been
proposed that the products are by-products of stress and
thus harmful to the cells, on the other hand it has been
estimated that they may protect the cells (Wyn Jones,
R.G. and Storey, R.: The Physiology and Biochemistry of
Drought Resistance in Plants, Paleg, L.G. and Aspinall,
D. (Eds.), Academic Press, Sydney, Australia, 1981).
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Zhao et al. (in J. Plant Physiol. 140 (1992)
541 - 543) describe the effect of betaine on the cell
membranes of alfalfa. Alfalfa seedlings were sprayed
with 0. 2M glycinebetaine, whereafter the seedlings were
uprooted from the substrate, washed free of soil and
exposed to temperatures from -10~C to -2~C for one hour.
The seedlings were then thawed and planted in moist sand
for one week at which time regrowth was apparent on
those plants that had survived. Glycinebetaine clearly
improved the cold stability of alfalfa. The effect was
particularly apparent at -6~C for the cold treatment.
All controls held at -6~C for one hour died, whereas 67
of the seedlings treated with glycinebetaine survived.
Itai and Paleg (in Plant Science Letters 25
( 1982) 329 - 335) describe the effect of proline and
betaine on the recovery of water-stressed barley and
cucumber. The plants were grown in washed sand, and
polyethylene glycol (PEG, 4000 mol. wt.) was added to
the nutrient solution for four days in order to produce
water stress, whereafter the plants were allowed to
recover for four days before harvesting. Proline and/or
betaine (25 mM, p 6.2) was sprayed on the leaves of the
plant either on the first or third day of the stress or
~ iately before harvesting. As regards barley, it was
noted that betaine supplied either before or after the
stress had no effect, whereas betaine added in the end
of the stress was effective. Proline had no effect. No
positive effect was apparent for cucumber. On the
contrary, it was found out that both betaine and proline
had a negative effect.
Experiments aiming at clarifying the effects
of betaine and proline on plants have thus yielded
contradictory results. There are no commercial
applications based on these results.
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Brief description of the invention
The purpose of the present invention was to
find a way to partially replace irrigation so that the
amount and quality of the yield could be simultaneously
ensured. Another purpose of the invention was to find
a way to protect plants also under other stress
conditions, such as during high salinity often ~onnected
with drought, at low temperatures, etc. M~eover, a
further aim was to find a way to increase the yield
under normal conditions without utilizing methods that
would consume envi I -ntal resources or harm the
enviL~ -nt .
In conne~-tion with the present invention it has
been proved that the yield of grain legumes can be
considerably improved by means of exogenously applied
betaine. Betaine has been found to be effective in
improving the yield both under normal and stress
conditions, and it has no such detrimental effects as
the side effects of ABA. Betaine application makes it
possible to considerably reduce for example the need for
artificial irrigation, thus saving the envil~ -nt and
cutting down the costs to a great extent. An
advantageous feature of the invention is also the
decrease of the antinutrient concentration of plants as
a result of the betaine application. A good example of
this is the low alkaloid content of lupins treated with
betaine, i.e. about half of the normal level.
The invention thus relates to the exogenous use
of betaine to i~ ~ve the yield of grain legumes. The
invention relates especially to the use of betaine to
improve the seed yield of grain legumes.
According to the invention, betaine is used
exogenously to improve the yield of grain legumes both
under normal and stress conditions.
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The invention also relates to the exogenous use
of betaine to reduce the antinutrient content of grain
legumes, especially to reduce the alkaloid content of
lupin.
The invention further relates to grain legumes
treated exogenously with betaine and to the parts
thereof, particularly seeds, and to their use as such
and for example in food, ~n; ~1 feed and forage
industries.
The invention also relates to a method of
improving the yield of grain legumes, in which method
betaine is exogenously applied to growing grain legumes.
The invention further relates to a method of
reducing the antinutrient content of grain legumes, in
which method betaine is exogenously applied to growing
grain legumes. The invention especially relates to a
method of reducing the alkaloid content of lupins, in
which method betaine is exogenously applied to growing
lupins.
Betaine is applied to the plant in either one
or several dosages. The application may be performed for
example by spraying together with some other spraying
of for example a pesticide, if desired. Betaine used
according to the invention is transported to plant
cells, where it actively regulates the osmotic balance
of the cells and also participates in other processes
of cell metabolism. A plant cell treated with betaine
is more viable even when subjected to exogenous stress
factors.
The betaine treatment according to the
invention is economically advantageous, and the yield
increases in an amount that is economically profitable
and significant. The treatment does not produce
significantly more work since it may be performed
together with other sprayings, and it does not re~uire
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new investments in machinery, equipment or space. It
should also be noted that betaine is a non-toxic natural
product, which has no detrimental effects on the quality
of the yield. Betaine is also a stable substance that
"- ~;n~ in the plant cells and thereby has a long-
st~nAing effect.
Detailed description of the invention
Betaine refers to fully N-methylated amino
acids. BetA1nes are natural products that have an
important function in the metabolism of both plants and
Ani ~ One of the most common betAi n~ is a glycine
derivative wherein three methyl groups are attached to
the nitrogen atom of the glycine molecule. This betaine
compound is usually called betaine, glycinebetaine or
trimethylglycine, and its structural formula is
presented below:
CH3
I
CH3 - N~ - CH2COO-
I
CH3
Other bet~; n~s are for example alaninebetaine
and prolinebetaine, which has been reported to for
example prevent perosis in chicks. R.G. Wyn Jones and
R. Storey describe bet~inec in detail in The Physiology
and Biochemi.~try of Drought Resistance in Plants (Paleg,
L.G. and Aspinall, D. (Eds.), A~mic Press, Sydney,
Australia, 1981). The publication is included herein by
reference.
Betaine has a bipolar structure and it contains
several chemically reactive methyl groups which it can
donate in enzyme-catalyzed reactions. Most organisms can
synthesize small amounts of betaine for example for the
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methyl function, but they cannot react to stress by
substantially increasing the production and storage of
betaine. Best known organisms accumulating betaine are
plants belonging to the Chenopodiaceae family, for
example sugar beet, and some microbes and marine
invertebrates. The main reason for the betaine
accumulation in these orgAn;~ s is probably that betaine
acts as an osmolyte and thus protects the cells from the
effects of osmotic stress. One of the main functions of
betaine in these plants and microbes is to increase the
osmotic strength of the cells when the conditions
require this, for example in case of high salinity or
drought, thus preventing water loss. Unlike many salts,
betaine is highly compatible with enzymes, and the
betaine content in cells and cell organelles may
therefore be high without having any detrimental effect
on the metabolism. Betaine has also been found to have
a stab;~ ng effect on the operation of macromolecules;
it improves the heat resistance and ionic tolerance of
enzymes and cell membranes.
Betaine can be recovered for example from sugar
beet with chromatographic methods. Betaine is
c~ -rcially available from Cultor Oy, Finnsugar
Bioproducts as a product that is crystalline water-free
betaine. Other betaine products, such as betaine
monohydrate, betaine hydrochloride and raw betaine-
cont~;n;ng liquids, are also ~- -rcially available and
they can be used for the purposes of the present
invention.
According to the present invention, betaine is
thus used exogenously to improve the yield of grain
legumes, such as soybean, faba bean, green bean and
other beans, pea, lupin, etc. According to the
invention, betaine is used to improve the yield of grain
legumes both under normal and stress conditions, i.e.
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when the plants are subjected to periodic or continuous
exogenous stress. Such exogenous stress factors include
for example drought, h~gh temperatures, high soil
salinity, air pollution, such as ozone, nitrogen oxides,
sulphur dioxide and sulphuric acid (acid rain),
environmental poisons, herbicides, pesticides, etc.
Treating plants sub;ected to stress conditions
exogenously with betaine for example improves the
adaptation of the plants to the conditions and maintains
their growth potential longer, thereby improving the
yield-producing capacity of the plants. Betaine is also
a stable substance that L~ -i ns in the plant cells. The
positive effect of betaine is thereby long-standing and
~1 i ni shes only gradually due to dilution caused by the
growth.
Even though this reference and the claims use
the word 'betaine', it is clear that according to the
invention several different bet~i n~ can be used, if
desired. It should also be noted that betaine is used
here as a general term which thus covers different known
betaines.
Betaine is applied to the plants in either one
or several dosages. Application in a single dose is
considered preferable. The amount used varies dep~n~ing
on the grain legume species and cultivar, and on the
stage and conditions of growth. A useful amount may be
for example about 0.1 to 20 kg of betaine per hectare.
A preferable amount is thus for example about 1 to 6 kg
of betaine per hectare. The amounts given here are only
suggestive; the scope of the present invention thus
contains all amounts that work in the manner described
herein.
Any method suitable for the purpose may be used
for the application of betaine. Betaine can be applied
separately or together with other plant protectants,
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pesticides or nutrients, such as fungicides and urea or
micronutrients. Betaine can be applied easily for
example by ~laying. Foliar application of betaine and
possible other agents through spraying is a preferable
method which enables a more rapid response than methods
involving root application. However, there may be
different problems related to this method, such as low
penetration f~on,~ntrations in leaves with thick
cuticles, run-off from hydrophobic surfaces, w~ching off
by rain, rapid drying of the solution and leaf damage,
and therefore other methods may also be used to apply
betaine, if desired.
According to the invention, an aqueous solution
of betaine is preferably used.
The time of the treatment according to the
invention may also vary. If betaine is applied in a
single dosage, the treatment is usually performed at an
early stage of growth, for example on plants of about
5 to 20 cm, or when the leaves have just come out. If
betaine is applied in several dosages, a new spraying
is performed preferably in the beg; nn i ng of flowering
or when stress can be forecasted on the basis of the
weather.
The betaine treatment according to the
invention considerably improves the yield of grain
legumes, for example the amount and quality of the
yield. The treatment according to the invention can also
reduce the need for artificial irrigation. The treatment
according to the invention is e~on- ;cally advantageous
and the increase in yield is economically profitable and
significant. In connection with the invention it has
been shown that for example lupin yield can be increased
by over 28% with a suitable betaine dosage, for example
about 6 kg/ha. It should also be noted that even though
the amount of yield increases to a considerable extent,
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the quality does not deteriorate. On the contrary, in
connection with the present invention it has been shown
that the antinutrient content of plants, for example the
alkaloid content of lupins, considerably decreases as
a result of the betaine application according to the
invention.
High ro~ntrations of alkaloids are poisonous
to ~ni~-l cells, and therefore a low alkaloid content
in lupins is an important criterion of quality in view
of lupin applications. One of the quality requirements
for use in foodstuffs is that lupin seeds contain less
than 0.02% of alkaloids. There has generally been a
t~n~en~y to maintain the level of alkaloids as low as
possible by selecting cultivars with a low alkaloid
content. Since lupin cultivars with higher alkaloid
contents produce higher yields, this approach has not
been considered very advantageous. Eliminating alkaloids
or reducing their amount during the processing of lupins
has also been utilized, but this naturally increases the
number of processing steps and the costs.
Most of the lupin yield is used as forage or
in some other form as food for animals whereupon the
advantages of lupin are its high contents of protein,
amino acids and energy. The highest allowed alkaloid
content for these applications is 0.04%. However, even
with low concentrations alkaloids cause a bitter off-
taste, wherefore ~ni~l ~ tend to avoid eating forage or
other feed cont~; n; ng alkaloids. The use of lupins in
~ni ~1 food and forage applications is therefore
restricted due to their alkaloid content. It is also
known that the alkaloid content of lupins increases
under stress conditions.
The considerable decrease in the alkaloid
content of lupins achieved with the betaine treatment
according to the present invention is therefore a
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significant additional advantage which is even more
marked in view of the notable positive effect of betaine
on the stress resistance of plants.
According to the invention, the yield of grain
legumes can thus be improved both under normal and
stress conditions, which in addition to drought include
for example high salinity often ~onn~ted with drought,
high temperature, etc. Furthermore, the invention also
makes it possible to grow grain legumes on lands that
were previously considered unfit for cultivation.
The invention will be described in greater
detail by means of the following examples. The examples
are only provided to illustrate the invention, and they
should not be considered to limit the scope of the
invention in any way.
Example 1
Effect of betaine application on lupin yield
The effect of betaine application on lupin
yield was e~r;ned at Murdoch University, Perth,
Australia. The experiment was conducted under field
conditions during the winter of 1994, which was cooler
and rainier than usual, but during which water stress
did occur, however.
The experiment was conducted according to a
split-plot design utilizing plots of 8 m2. The plots
were divided into four sub-plots that were treated with
different betaine ~o~c~ntrations. The betaine
concentrations used were 0 (control), 2 kg/ha, 4 kg/ha
and 6 kg/ha. The soil was sandy (98% sand, 1% silt and
1% clay) with a low nitrogen, phosphorus and potassium
content and poor water and nutrient retention
properties. The amount of irrigation was normal. The
cultivar was Gungurru. The results are shown in Table
1.
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14
Table 1
Effect of betaine application on lupin yield
betaine yield of lupin seeds
concentration
(kg/ha) kg % of control
0 (control) 0.979 100
2 0.992 101
4 1. 048 107
6 1.253 128
The results show that the yield increased over
the control in all the experiments conducted. The best
results were obt~;ne~ with a betaine application rate
of 4 or 6 kg/ha.
Example 2
Effect of betaine application on lupin yield
under dry conditions
The effect of betaine application on lupins
growing under water stress was ~Y~ ;ned by repeating the
experiment described in Example l, but with a 50%
reduction in irrigation from the optimum amount. The
results are shown in Table 2.
Table 2
Effect of betaine application on lupin yield under water
stress
betaine yield of lupin seeds
concentration
(kg/ha) kg % of control
0 (control) 0.856 100
2 0.938 110
4 O. 912 107
6 1.031 120
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The yield also increased clearly in this
experiment compared with the control. It can also be
noted that utilizing the higher glycine betaine
concentration of 6 kg/ha provided similar results with
a low irrigation level (50~) as utilizing the lower
betaine rate of 2 to 4 kg/ha with optimum irrigation
(Example 1). This means that the same yield can be
achieved by decreasing irrigation if a higher betaine
application rate is used simultaneously.
Example 3
Effect of betaine application on the alkaloid
content of lupins
The alkaloid content of lupins grown and
treated according to Examples 1 and 2 was determined
with the method described by Priddis [Journal of
Chromatography, 261 (1983) 95 - 101]. The results are
shown in Table 3.
Table 3
Effect of betaine application on the alkaloid
content of lupin seeds
treatment alkaloid content (%)
control I 0.04
25 irrigation rate 50%
6 kg/ha betaine 0.02
irrigation rate 50%
30 control II 0.02
irrigation rate 100%
6 kg/ha betaine 0.01
irrigation rate 100%
The results show a clear decrease in the total
alkaloid content of lupins, which is a very surprising
and positive result from the betaine application
according to the invention.
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16
Example 4
Effect of betaine application on the early
development of green bean seeds
The effect of betaine on the germination
fre~uency and rate of green bean seeds was ~X~mi ~d
using water as control. The green bean was of the type
Spartan Arrow Bush Bean Lot #1987-3, produced by
Northrup King Co. Three different test solutions were
prepared for the experiments as follows:
Test solution pH
A deionized water 7.01
B betaine (0.02 g/l) 6. 34
C betaine (2 g/l) 6.80
Twenty green bean seeds were soaked for 24 hours in 330
ml of one of the afo~ ~ntioned test solutions. The
seeds were then dried on stainless steel screens and
sown into soil with two seeds placed in each container.
The cont~n~s were then placed on a window ledge with
a southern exposure to the sun, and they were watered
daily with deionized water.
The early development of the seeds was followed
by determining both the rate and frequency of
germination. The first measurements were performed ten
days after the experiment began, and the second set of
measurements was conducted 19 days after the beginning
of the experiment. The results are shown in Table 4.
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Table 4
Effect of betaine application on the early development
of green bean seeds
Betaine Germination Average shoot
5~on~ntrationfrequency (%) length
(g/ Day Day (inch) (% of
19 control)
0 (control) 0 40 2.25 100
0.02 80 100 4.82 214
2 20 100 3.88 172
The results show that betaine promotes faster
germination in green beans. Betaine also produced
changes in the look of plants, for example the colour
of leaves became dark green. The best results were
achieved with the lower betaine rate of 0.02 g/l.
Example 5
Effect of betaine application on pea yield
under stress conditions
The effect of betaine application on the yield
of peas growing under stress conditions was ~x~;ned in
the following way. Peas were sown in 5 1 plastic pots
containing a mixture of peat and vermiculite in a ratio
of 1:1. The plants were grown in greenhouses at a mean
day/night temperature of 28~C/12~C and a relative
humidity of between 42 and 45%. Supplemental
illumination was provided for 17 hours a day with
tungsten lamps (PAR 434 ~mol m~Zs~l). Twenty seeds were
sown per pot and they were later thinned to 10 plants
per pot. The total number of pots used was twenty-four,
twelve of which were exposed to drought, i.e. water
stress, and twelve to high salinity, i.e. salt stress.
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18
A completely r~o~;sed design was utilized in the
experiment, with 4 replications.
Water stress (pF3) was imposed on half of the
plants four weeks after see~l; ng emergence. The pots
were then grouped into 3 sets, each of which consisted
of 4 pots, and each set was sprayed with either 25 ml
of distilled water, O.lM betaine solution or 0.3M
betaine solution two weeks after the stress imposition.
To induce salt stress, 200 ml of 100 mM NaCl
solution was applied to half of the pots every four days
for five weeks after s~e~l ing emergence. The pots were
grouped in 3 sets of 4 pots in each set, and they were
sprayed with 25 ml of distilled water, O.lM betaine
solution or 0.3M betaine solution after the first
A~m; n; ~tration of the NaCl solution. The NaCl treatment
was repeated six more times after the betaine
application.
At harvest, the total number of nodules, number
of active nodules, number of pods, and leaf dry matter
content were determined. The results are shown in Tables
5 and 6.
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19
O ~ ~ C O O O
o
~ ~ o o
~ ~ ~e
~ O O o a~
~ O
o ~U~~ ~ 0~ ~ ._
O
~,,
O O _ O~ ~ O
~e ~O ~ ~ ~ ~
~~0 ~0
~ e ~
a~ ~~ ~ O
_~ O G 0o
e ~ D,~
.C ~~
O ~ C C ~ ~ o
C ~ o 0~ ~C,~ C
D g ~)
~? ~ql ~ ~O <~' ~
t.~ ~ ~ ~
G ~ ~ c ~--
a~ G .. o ~~ ~ q ~
C o o o C
C o
O .~ O -_
J G ~ ~ ~ G ~ ~ ~ ~
G
~ ~ g ~
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The smaller betaine rate of O.lM thus had a positive
effect both on the number of active nodules, number of
pods and the leaf dry matter content when the pea was
growing under dry conditions. The positive effect on pea
growing under salt stress was even clearer. The higher
betaine content of 0.3M had a positive effect on the
number of nodules and leaf dry matter content of peas
growing under salt stress.
Example 6
Effect of betaine application on the y o~h
rate of pea
The experiment of Example 5 was repeated by
utilizing betaine solutions of 0 (control), 0. 05M, 0. lM
and 0. 2M. Water stress was induced in the manner
described in Example 5, whereas salt stress was not
ex~m; ned in this experiment. In order to ~~X~ir~e the
recovery of plants, the stressed plants were divided on
day 28 of the experiment into two groups one of which
still remained under water stress, and the other one was
irrigated and its recovery was followed. Samples were
taken on days 21, 28, 35 and 42. Peas growing under
optimum conditions (sufficient irrigation) were used as
control. The best results were obt~;n~ with the betaine
rate of 0.05M. The results concerning the relative
growth rate of pea and the dry weight of the shoot are
shown in Figures 1 and 2, respectively.
Example 7
Effect of betaine application on faba bean
yield under stress conditions
The experiments described in Example 5 were
repeated utilizing faba bean. Ten seeds of faba bean
were sown per pot and they were later thi nne~ to 3
plants per pot. The other parameters of the experiments
corresponded to those described in Example 5.
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The positive effect on faba bean was apparent
especially for the number of pods, which increased under
water stress from a control value of 3.13 to 3.50 with
the O.lM betaine solution, and to 3.63 with the 0.3M
betaine solution. The results correspond to values 112
and 116 in percentages of the control (100). The leaf
dry matter content increased from a control value of
2.04 g to 2.21 g with the O.lM betaine solution, but
decreased to 1.67 g with the 0.3M betaine solution.
Example 8
Effect of betaine application on soybean yield
The effect of betaine application on soybean
yield under normal and dry conditions was investigated
in field conditions on a farm where the soil is very
light and sandy and can retain about 35 mm of rain for
a few days, but where water stress will occur in a few
days even after a heavy rain. There were overhead
irrigation facilities on the farm to guarantee
sufficient irrigation of the control areas. In addition
to the growth of soybean, the experiment also determined
its nitrogen fixation capacity.
The experiments were conducted with a 3-Factor
R~n~om;zed Complete Block Design with watering level
(main factor), cultivar (subplot) and betaine
concentra~ion (split) as factors. The cultivars were
Biloxi and Cook, which have a different drought
tolerance; cultivar Cook has a more drought tolerant
symbiosis system than cultivar Biloxi. The betaine
levels applied included 0 (control), 3 kg/ha, and 6
kg/ha. Betaine was applied by spraying. The season had
an early drought period followed by very heavy rains and
again a drought period. The betaine application was
repeated after the rains before the second drought
period with the same application levels. The results
co~c~rning the leaf dry weight are shown in Table 7.
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22
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The nitrogen fixation capacity of soybean was
determined by measuring the nitrogenase activity with
an acetylene reduction test wherein acetylene is reduced
to ethylene. The experiment was conducted 10 weeks after
sowing. In order to perform the measurement in the
field, metal cylinders of 10 cm in diameter and 20 cm
in depth were placed in the soil around a soybean plant.
The plant was l vad from the soil in the cylinder and
the shoot was cut off. The roots were then quickly
placed in an airtight contAine~ of 1000 ml. 150 ml of
acetylene was then injected in the cont~;ne~, and a 6.5
ml gas sample was taken by a syringe 5, 10 and 15
minutes after incubation, and the samples were then
subjected to gas chromatography. It has been established
that acetylene reduction is linear for about 20 minutes
from the acetylene introduction. The results obt~ln~A
after 15 minutes are shown in Table 8.
.
Table 8
Effect of betaine application on the nitrogen fixation
of soybean
betaineethylene concentration
concentration(~mol/2 plants)
(kg/ha)
Cv. Cook Cv. Biloxi
dry normal dry normal
stress stress
0 (control) 0.29 0.55 0.42 0.68
3 0.60 0.68 0.53 0.79
6 0.34 0.52 0.35 0.38
The application of betaine at the rate of 3 kg/ha thus
clearly increases the nitrogen fixation or plants.
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24
Example 9
Effect of betaine application on s~' -n yield
The acetylene reduction test described in
Example 8 was repeated in greenhouses utilizing the
betaine rate of O.lM and cultivar Biloxi. The experiment
was conducted by closing plant roots (2 per pot) in a
glass cont~; n~ (l l) and by sucking 150 ml of air out
of the cont~i n~, whereafter the air was replaced with
a corresponding amount of acetylene gas in the manner
described in Example 8. (Reference: Denison, R.F.,
Sinclair, T.R., Zobel, R.W., John~o~, M.N. & Drake, G.M.
1983. A non-destructive field assay for soybean nitrogen
fixation by acetylene reduction. Plant & Soil 70; 173-
182; Vessey, J.K. 1994. Measu~. - t o~ nitrogenase
activity in legume root nodules; In defense of the
acetylene reduction assay. Plant & Soil 158; 151-162).
The nitrogen fixation of soybeans of 4 weeks
was assayed in greenhouse tests 2 days after the betaine
application. The results are shown in Table 9.
Table 9
Effect of betaine application on the nitrogen fixation
of soybean
betaine ethylene concentration (ppm)
concentration
(M)
normal water stress
0 (control) 3.029 2.193
0.1 3.642 2.690
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Ex ~ ple lO
Effect of betaine application on soybean yield
The effect of betaine application on the
photosynthesis of soybean was ~XA~i ne~ in greenhouses
utilizing simulated water stress conditions.
Six (inoculated) seeds of nodulating or 15
(inoculated) seeds of non-nodulating soybean cultivar
were sown in 5 1 plastic pots cont~n;n~ a mixture of
peat, vermiculite and sand in the ratio of 1:2:1. After
10 seedling emergence the plants were th;nne~ to 3 per pot.
Half of the pots used in the experiment cont~ine~ the
nodulating cultivar and half the non-nodulating
cultivar. Water stress (pF3) was imposed on the plants
15 days after seedling emergence. The water-stressed
15 plants were divided into three groups, one of which
(control) was treated with distilled water, the second
one was sprayed with betaine at the rate of 2 kg/ha and
the third one with betaine at the rate of 6 kg/ha a day
after stress imposition. The photosynthetic activity of
20 the plants was deteL ;ne~ with the Li-cor Li-1600-Steady
State Porometer. The apparatus and its use are described
in the following references: Campbell, G.S. 1975.
Steady-state diffusion porometers. In: Measurement of
stomatal aperture and diffusive resistance. Coll. Agric.
25 Res. Center Bull. 809. p. 20. Washington State Univ.
Pullman, Wash, and B; ngh;l , G.E. ~i Coyne, P.I. 1977. A
portable, temperature-controlled steady-state porometer
for field measurements of transpiration and
photosynthesis. Photosynthetica 11(1): 148-160. The
30 results are shown in Table 10.
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26
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Betaine considerably improved the
photosynthetic activity of soybean both under normal and
water stress conditions with both application rate~.
Example 11
Effect of betaine application on the growth of
~L_an
The effect of betaine on the photosynthesis of
soybean and on the water potential situation of soybean
plant leaves was eY~;ne~ in greenhouses with the
betaine ~onc~ntrations of 0 (control), 0.05M, O.lOM and
0.15M. The soybean cultivar was Biloxi. At the time of
spraying, the plants were about six weeks old and the
measurements were performed five days after the
spraying. The photosynthesis and the water potential of
the plants, i.e. the stomatal resistance and stomatal
conductance and the temperature difference of leaves
were measured with the Li-cor Li 6200 Portable
Photosynthesis System. The xy~ l is based on a method
described by Ball et al. (A Model predicting stomatal
conductance and its contribution to the control of
photosynthesis under different condltions. Progress in
Photosynthesis Research IV. Martinus Nijhoff, Drodrect,
The Netherlands, 1987, pp. 221-224, (Ed.) J. Biggins).
Water potential parameters indicate the state of opening
or closing of the guard cells of the plant. High
resistance and low conductance mean that the plant has
a poor intake of carbon dioxide and is stressed. The
numerical results are shown in Table 11, a graphical
illustration is provided in Figure 3.
-
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Table ll
Effect of betaine on the photosynthesis and leaf water
potential of soybean
betaine photo- stomatal stomatal leaf
~O~C~n_ Syn~h~;~ ~.on~ctance resistance tempe-
tration (~molm2s-l) (ms~1)(scm~l) rature
(M) diffe-
rence
0 (con- 2.70 0.02 18.16 -2.81
trol)
0.05 4.01 0.05 11.21 -3.01
0.10 14.01 0.26 6.30 -1.55
0.15 12.83 0.12 4.46 -1.72
The results clearly show that ~; n~ betaine can reduce the
stress of plants. The photosynthetic activity of the plant
thereby increases, thus le~; ng to a higher yield.
