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PROCESS AND SYNTHESIZER
FOR
MOLECULAR ENGINEERING OF MATERIALS
USED IN THE
MODIFICATION OF SEED GERMINATION PERFORMANCE
(U.K.Patent No.GB2397782,INDIA Patent No.200286)
(Rights Recorded in U.S.A, CANADA, JAPAN, CHINA,PHILIPPINES & PCT Countries)
Seed germination characteristics have been modified in agricultural species by coating the surface of the seeds with macromolecules from the cold plasma process of the patented process and synthesizer. The source gas entering the wave guide during the reaction process determined the type of coating, and coatings were typically much less than 5.0 µm in thickness. To delay germination different hydrophobic source gases, carbon tetrafluoride (CF4) or octadecafluorodecalin (ODFD) are used. Seeds of radish and two pea cultivars treated with CF4, resulted in a significant delay in germination compared with untreated controls.
Plasma treatment significantly delays germination in soybean , corn and bean seeds. The degree of delay is dependent on the amount of coating applied, with an increased thickness of coating resulting in a greater delay in germination. To enhance germination seeds are treated with cyclohexane, or with a gas such as aniline or hydrazine. Seeds treated with cyclohexane will result in a significant acceleration in germination percentage for soybean but not corn seeds, while hydrazine-treated corn seed will show a small acceleration over control seed. However, both aniline-treated soybean and corn seed will have a significant acceleration in germination percentage. Tests of water uptake determined that the major mode of action of the plasma coatings was largely on the rate of imbibition. The low temperature cold plasma in the synthesizer is a potentially important technique to modify seed germination characteristics in agricultural plant species.
Plasma Gases
The following materials and conditions are recommended:
1.Fluorocarbon—CF4.
2.Nitrogen-containing—aniline.
3. Hydrazine.
4.Carbon—cyclohexane.
Base pressure—30-40 mT.
Pressure in the absence of plasma—250 mT.
Drum
speed—30 rpm.
Treatment of Seeds with Fluorocarbon Plasmas
A study on the PLASMA TREATMENT of the seeds was carried out by:
John C. Volina, Ferencz S. Denesb, Raymond A. Youngc and Scott M.T. Parkd
a Div. of Science, Florida Atlantic Univ., 2912 College Avenue, Davie, FL 33314 USA
The BRIEF SUMMARY of the RESULTS published by them is as under.
TABLE -1.
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Results |
Treatment of Seeds with Fluorocarbon Plasmas
Fluorocarbon plasmas deposited chemically and
thermally inert (Teflon-like) macromolecular layers on the seed
substrates. It was observed that the fluorine:carbon (F:C)
ratios of specific fluorocarbon derivatives (rather than the
experimental parameters) control the macromolecular-structure forming
processes; high values of this parameter shift the mechanisms towards
ablation (etching) reactions, while low values of these ratios lead
to fluorocarbon-layer depositions. Numerous reaction mechanisms have
been suggested for the formation of fluorocarbon-type structures
(Denes et al., 1997,
1999).
Plasma treatment with CF4
results in no significant (P < 0.05) differences in seed
germination from the control seed for all species tested. For the two
pea cultivars, viability was reduced 25% compared with control seed,
resulting in a final germination percentage of 75% (P = 0.08
for both pea cultivars) (Fig.
2) . For radish, viability was not negatively affected at all in
the plasma treatment, resulting in a 87% compared with 88% final
germination for the untreated-control seed (P = 0.71) (Fig.
2). However, after the first day from sowing, plasma treatment
with the hydrophobic CF4 resulted in a highly significant
(P = 0.009) reduction in germination for treated compared with
control radish seeds. For instance, by Day 1, 75% of control seeds
had germinated compared with only 20% of CF4-treated seeds
(Fig. 2).
By Day 2, there was no significant difference between treated and
control seeds. Control radish seeds achieved their greatest percent
germination 2 d after sowing, while treated seeds did not achieve a
comparable maximum percent germination until Day 6. Both cultivars of
peas also had a tendency for a delayed germination as a result of
plasma treatment with CF4, although this was only
significantly different for Little Marvel control seeds on Day 3 (P
= 0.03). Maximum germination (100%) in control seeds occurred by Day
3 for both pea cultivars, while treated seeds did not achieve their
maximum germination until Day 5 and Day 6 for pea cultivars Alaska
and Little Marvel, respectively (Fig.
2). Both T50 and T10–90, were similar to
germination percentage, in that T50 and T10–90 were
increased by CF4 in radish seeds, while the response was not
as pronounced in the pea seeds (Table
2) .
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Table 2 Mean time to germination (T50) and germination span (T10–90), of several agricultural species subjected to different plasma-treatments versus untreated controls
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Corn and soybean seeds were also treated with hydrophobic ODFD. For
both species, germination was significantly delayed in seeds treated
with ODFD compared with untreated control seeds (Fig.
3 , Table
2). Moreover, both species showed a significant decrease in
germination percentage between treated and non-treated seeds by the
end of the study (P < 0.0001 and P = 0.046 for corn and
soybean, respectively). Corn control seeds achieved their maximum
germination by Day 3 (100%), while ODFD-treated seeds achieved
maximum germination (47%) by Day 7 (Fig.
3). Likewise, ODFD-treated soybean seeds reached maximum
germination (80%) by Day 7, while control seeds reached maximum
germination (100%) 3 d earlier.
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Corn and bean seeds were also treated with ODFD for 0-, 2-, 5-, or
20-min at a lower pressure than in the previous study. ODFD-treated
corn seeds showed a significant delay in germination (P <
0.0001 for all treatment comparisons) for all three plasma treatments
compared with the non-treated seeds for the first 2 d after sowing (Fig.
4 , Table
2). By Day 3 there were no significant differences between the
control seeds and those that had been treated with ODFD for 2 min (P
= 0.15), while there continued to be significant differences in
germination between the control and the 5- and 20-min ODFD-treated
seeds through Day 4. However, by Day 5 there were no significant
differences in final germination percentages between control and
5-min ODFD-treated seeds (P = 0.06), although there remained a
significant difference in germination for control compared with
20-min ODFD-treated corn seeds throughout the study (P <
0.0001). In fact, with increasing treatment time, there was a
progressive decrease in seed germination, resulting in final
germination percentages of 99, 85, 81 and 27% for the 0-, 2-, 5-, and
20-min treated corn seeds, respectively (Fig.
4). Bean seeds showed a similar pattern in that an increase in
treatment time showed a linear increase in the delay in germination.
Unlike corn seeds there were no differences in final germination
percentages among bean seeds treated under differing lengths of
plasma exposure (final germination was 87% for all three treatment
times) (Fig.
4). T50 and T10–90 were significantly
greater in the 5- and 20-min ODFD-treated seeds compared with the
control and 2-min ODFD-treated seeds (Table
2).
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Water uptake studies conducted on the corn seeds showed a slower rate
of water uptake for ODFD-treated compared with control seeds, where
hydration rate was K = 0.096 versus 0.102, 24 h after sowing.
This corresponded to an average (±SE) 99 ± 5.5% versus 114 ± 6.8%
increase in moisture content for the ODFD-treated compared with the
control seeds.
Survey ESCA data collected from
plasma-modified corn (Fig.
5A) and bean (Fig.
5B) substrates indicated the presence of fairly high relative
fluorine (corn: 41.8%; bean: 54.7%) and carbon (corn: 45.1%; bean:
39.7%) atomic concentrations and the existence of a lower oxygen
content (corn: 13.1%; bean: 5.5%). It should also be noted that the
relative surface oxygen atomic concentrations at the surface of corn
seeds is significantly higher in comparison to the bean substrate,
which might be related to a ab initio different atomic composition at
the surface of the two substrates.
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High resolution ESCA analysis of non-equivalent C1s functionalities of
plasma modified corn and bean surfaces (Fig.
6A and B) clearly indicate that fluorinated macromolecular layers
were deposited under ODFD-RF-plasma environments on both substrate
surfaces. CF3 (294.1 eV), CF2 (291.7 eV),
C*HF-CH2 (288.0 eV), and C*-CF2 (286.6 eV)
functionalities can be identified both in the corn and bean ESCA
diagrams, in addition to the substrate origin C-C (285.0 eV) and C-O
(286.6 eV) linkages. Although the survey ESCA data indicate
comparable relative surface carbon atomic concentrations for the two
seed species, there is a significant difference between the CF2/C-C
relative peak area ratios which is not of plasma-deposited
macromolecular-layer-thickness origin. This, indicates that the
composition of the layers from the recombination of plasma-generated
ODFD-molecular fragments is dependent on the nature of the
substrates.
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SEM images were also taken for the ODFD plasma treated seeds. SEM
allows detailed analysis of the morphology of the seed coat surfaces.
Although some debris-like contamination was noted on the plasma
seeds, the comparative SEM images of both plasma-treated corn and
bean, show significantly smoother surfaces for the treated seeds,
which indicates the presence of a deposited macromolecular structure (Fig.
7A–D) .
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Treatment of Seeds with Nitrogen-Containing Plasmas
Incorporation of nitrogen onto the surfaces of seeds by plasma processing
was carried out to evaluate the potential effect on germination.
Although nitrogen is usually not limiting, it is an important
macronutrient for all plant material and therefore worthy of further
evaluation. We evaluated two nitrogen-containing organic compounds,
aniline and hydrazine, for plasma mediated incorporation of
nitrogen-containing films onto the seed surfaces.
Soybean seeds plasma treated
with aniline showed a highly significant (P < 0.0001)
acceleration in germination compared with control seeds after the
first day from sowing, resulting in 83% germination in treated seeds
compared with 12% in control seeds (Fig.
8) . Similarly, corn treated with aniline showed a highly
significant (P < 0.0001) increase in germination by Day 1
with 53% treated seed germination compared with 8% control seed
germination. However, for both species there were no significant
differences in treated and control seed germination by Day 2. T50
was significantly greater for the aniline-treated soybean seeds,
although not in the aniline-treated corn. Moreover, there was no
significant differences between treatments in germination span (T10–90)
for either the aniline-treated soybean or corn seeds (Table
2).
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Similar to the T50 results, 24 h after sowing, water uptake in
soybean was slightly greater in plasma-treated compared with control
seeds, although not in corn. Hydration rate was K = 0.115
versus 0.112 and K = 0.098 versus 0.101 for soybean and corn
aniline-treated versus control seeds, respectively. By 48 h after
sowing, moisture content had increased 412 ± 17.2% in plasma-treated
soybean seeds, compared with 380 ± 15.3% in control seeds, while in
corn moisture content was 217 ± 11.0% versus 212 ± 3.6% for
plasma-treated versus untreated seeds, respectively.
Corn seeds treated with
hydrazine showed a slight acceleration in germination by Day 1, which
was significantly different (P = 0.03) compared with control
seeds; however, the germination percentage of treated seeds was only
5% after the first day from sowing compared with 0% for control seed.
By Day 2 there were no significant differences in the percent
germination between treatments. Final germination was 98% and 100%
for hydrazine-treated and control corn seeds, respectively, and there
was no difference in either T50 or T10–90 (Fig.
9 , Table
2). Consistent with the germination percentage after 24 h
moisture content was 124 ± 1.7% versus 115 ± 6.8% for
hydrazine-treated versus untreated control seeds. By 48 h, hydration
rate was similar for both treatments at K = 0.065 for treated
versus K = 0.064 for untreated seeds.
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Treatment of Seeds with Cyclohexane Plasma
Cyclohexane is a hydrophobic organic compound composed only of carbon
moieties. In a plasma, cyclohexane will fragment and reform a
crosslinked polyethylene type film at the seed surface, which was
anticipated to delay germination. However, etching can also occur in
any plasma-mediated process, which would be expected to increase
water uptake as a result of increased seed coat permeability, thereby
increasing the rate of germination. It was uncertain which of these
two processes would predominate with the cyclohexane plasma
treatment.
Corn and soybean seeds were
plasma treated with cyclohexane at two different pressures. For corn
seeds, there were no significant differences among treatments, with
all three treatments achieving maximum germination (98–99%) by Day
3 (Fig. 10)
. Water uptake after 48 h in both treated and control corn seeds were
similar resulting in hydration rates of K = 0.063 and 0.064, respectively.
In contrast, in soybean there were significant differences in
germination percentage between cyclohexane plasma-treated versus
control seeds as well as between treated seeds exposed to cyclohexane
under different pressures. For instance, by Day 1 both plasma
treatments resulted in a significant (P < 0.0001 for both
pressures) acceleration in germination compared with the control
treatment. This significant (P < 0.0001) pattern was still
evident in Day 2. However, by Day 3 there were no significant
differences in germination among all three treatments, resulting in
maximum germination percentages by Day 4 for all treatments (Fig.
10). In addition, on Day 1 there was a significant (P =
0.004) acceleration in germination for soybean seeds treated at 150
mT pressure compared with those treated at 70 mT, although there was
no significant difference by Day 2 between the two plasma treatments
(P = 0.34) (Fig.
10). This same trend, although not significant, was also found
for T50 (Table
2). Among treatments, water uptake was not significantly
different, although by 48 h after sowing, moisture content in
cyclohexane-treated soybean seeds was 381 ± 7.2% compared with 369
± 5.0% for control seeds.
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Conclusion |
Our first objective was to test if seeds from several important agricultural
species would survive treatment in a rotating cold plasma reactor.
This objective was met in all six studies for five different crop
species under various plasma gas treatments and conditions. The only
exception was corn seeds plasma treated with ODFD under pressures of
300 mT or with a long plasma exposure time of 20 min. Therefore, in
most cases overall seed viability was not significantly impacted in a
cold plasma treatment and suitable viability could be obtained by
proper selection of the plasma parameters. Cold plasmas are
nonthermal or equilibrium plasmas that have high kinetic energy
electrons but the kinetic energy of the atomic and molecular species
is low. In contrast, the kinetic energy of all species is high in a
hot plasma, which would damage or destroy organic materials (Denes,
1997).
The rotating plasma reactor
allowed for the plasma-reacted deposition of a selected material on
the seeds, as was found in the ESCA and SEM images. The rotating drum
of the plasma reactor allowed for the seeds to gently tumble during
the plasma reaction process so that all surfaces of the seeds
received a uniform exposure to the plasma species. Depending on the
nature of plasma gases/vapors and the selected plasma parameters,
etching, surface-functionalization, and deposition processes can be
developed. Plasma species usually only penetrate surface layers to a
depth of around 10 nm, and even in the cases of deposition processes,
due to the relatively short treatment time periods, the
plasma-created layers are fairly thin (0.5–2 µm). The main
advantage of plasma-mediated surface modification processes is that,
in addition to the generation of charged and neutral gas phase
reactive species, the surfaces contained in the discharge [e.g.
substrates (seeds)] are also activated under the action of plasma
particles. These processes result, in most of the cases, in a
covalent attachment of plasma-origin molecular fragments, and
consequently in good adhesion of the deposited layers. In contrast,
some conventional seed coating processes can result in poor adherence
of the coating to the seed or in nonuniform application, or they may
produce a significant amount of dust, which is ultimately hazardous
to those handling the seeds (Robani,
1994).
Unlike alternative field
applications, seed treatment technologies provide for an economical
and less polluting delivery system because the amount of material
applied is relatively small and it is in immediate contact with the
target site (Taylor
and Harman, 1990). These advantages are particularly pronounced
with the plasma treatment of seeds. In addition, plasma-treated seeds
are exposed to a dry gas during the plasma process. Therefore, no
additional moisture is introduced into the seeds, limiting the need
for drying after pelleting or other wet coating applications.
After successfully reaching our
first objective, we introduced several different surface
modifications to seeds via cold plasma-mediated deposition of
materials. Although a myriad of different seed modifications could
have been investigated, we focused on delayed and accelerated
germination. In the case of delayed germination, the seed coat
characteristics were modified via plasma-deposition of hydrophobic
materials that would decrease water absorption. In contrast, to
accelerate germination, either hydrophilic materials were deposited,
or predominant etching processes were initiated (e.g., cyclohexane),
that promoted uptake of water. We also applied a macronutrient such
as nitrogen that could improve early nutrient uptake and therefore
accelerate the germination process and/or enhance early plant growth,
although nitrogen is generally not deficient in the early stages of
seedling development for these species.
Treatment of Seeds with
Fluorocarbon Plasmas
Hydrophobic coatings have been studied to delay imbibition, especially
for seeds sown in cold wet soil, where seeds with low moisture
content are particularly susceptible to imbibitional chilling injury (Herner,
1986). Priestly
and Leopold (1986) found that coating soybean and cotton seeds
with lanolin in acetone reduced water uptake and resulted in greater
emergence than non-treated controls, although there was no
significant advantage for treated corn seeds. Similarly, Taylor
(1987) coated snap beans with an aqueous preparation of a
hydrophobic polymer that showed a slight improvement in germination.
This may have been improved with an even coating of the polymer,
since scanning electron microscopy showed a non-uniform application
of the coating thereby resulting in an uneven uptake of water. In our
studies, pea, radish, corn, soybean and bean treated with either CF4
or ODFD resulted in a hydrophobic layer on the seed which reduced
water uptake, thereby delaying germination in all species. In fact, a
longer plasma exposure resulted in a greater delay in germination.
There are several advantages to delayed germination such as in
planting under high moisture conditions and potential use in space
flights.
Carbon tetrafluoride is a
non-depositing gas under common cold-plasma conditions. However,
plasma implanted CFx functionalities can create
hydrophobic surface characteristics. Specifically, CF4 plasmas
do not deposit fluorinated macromolecular layers under common RF cold
plasma conditions due to the intense etching effects related to the
high plasma-generated fluorine atomic concentrations. However, the
presence in the gas mixture of fluorine atom scavengers (e.g.,
hydrogen) allows the deposition of fluorinated macromolecular layers (Denes
et al., 1997). Thus, under appropriate conditions, source gases
such as CF4 can be utilized to deposit material on
surfaces rather than etch them.
Unlike, CF4, ODFD
plasmas do not primarily etch, but rather deposit a polymer film on
the seed surface. Since seeds were kept under vacuum for 24 h prior
to plasma treatment, this may have allowed for some absorption of the
gases into the seeds during the plasma reaction. This would explain
why some species, especially seeds with thin seed coats or embryos
near the seed surface had reduced viability. Moreover, this would
explain why these species showed an increased reduction in
germination percentage and increased T50 with increased
plasma treatment duration. Germination may not be as affected by
plasma reaction if seeds are not put under vacuum conditions until
the reaction takes place. In this situation, the vacuum would cause a
slight loss of water from the seed during the plasma reaction, which
would likely prevent the plasma gas from entering the seed, thereby
limiting the deposition to the seed coat. However, this hypothesis
needs to be tested in future studies.
Treatment of Seeds with
Nitrogen-Containing Plasmas
Seeds treated with either aniline or hydrazine, which are nitrogen-based compounds,
showed a small acceleration in seed germination. Previous work in our
laboratory has demonstrated that aniline, in a plasma gas, is much
more efficient for implantation of amine groups on substrate surfaces
compared with hydrazine. This may explain the superior germination of
seeds treated with aniline plasmas, although we do not have direct
evidence that amine groups accelerate germination. Acceleration in
germination may improve early plant growth, as has been found for
sulfur phosphate and molybdenum-coated legume seed (Scott
and Archie, 1978) and for phosphate-treated grass seed (Silcock
and Smith, 1982). For both aniline- and hydrazine-treated seeds
there are limitations to the amount of nitrogen that could be applied
via plasma treatment, and its usefulness would likely primarily pertain
to a very early augmentation in plant nutrition. Since micronutrients
are required in substantially lower concentrations compared with
macronutrients, such as nitrogen, it may be advantageous in future
studies to incorporate micronutrients in the plasma coating.
Treatment of Seeds with
Cyclohexane Plasma
In our studies we had mixed results when treating seeds with cyclohexane.
Cyclohexane-treated seeds did have a shorter germination time for
soybean but not for corn. In addition, soybean seeds treated at
higher pressure resulted in a more pronounced acceleration in
germination than those treated at lower pressure. An increase in
water uptake, as a result of etching of the seed coat would help
explain why soybean treated seed had accelerated germination over
control seed. However, it is unclear why soybean seed treated with
150mT pressure had greater acceleration in germination compared with
those treated at 70mT. An increase in pressure may promote etching
versus film deposition, although this will need to be investigated in
further studies. Corn seeds treated with cyclohexane did not result
in any difference in germination percentage or T50
compared with the untreated control seeds, which was likely due to
their equal imbibition rates. Etching processes may have countered
the hydrophobic nature of the cyclohexane, thereby resulting in
little response. However, ESCA and SEM will have to be conducted in
future studies of cyclohexane-treated seeds to determine the nature
of the material that is applied to each species of seed, as well as
to provide a detailed analysis of their seed surface morphology.
0.05.
