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SEED PHYSIOLOGY, PRODUCTION & TECHNOLOGY

Modification of Seed Germination Performance through Cold Plasma Chemistry Technology

^a Div. of Science, Florida Atlantic Univ., 2912 College Avenue, Davie, FL 33314 USA
^b Center for Plasma-Aided Manufacturing, Univ. of Wisconsin-Madison, 1410 Engineering Res. Center, Madison, WI 53706 USA
^c Dep. of Forest Ecology and Management, 1630 Linden Drive, Madison, WI 53706 USA
^d Div. of Science, Florida Atlantic Univ., 2912 College Ave., Davie, FL USA

jvolin{at}fau.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION Materials and methods Results Discussion Conclusions REFERENCES

 
This study was conducted to determine if an alternate seed treatment approach based on plasma chemistry would offer a more viable alternative over traditional seed coating technologies. Seed germination characteristics were modified in five agricultural species by coating the surface of the seeds with macromolecules from a cold plasma process using a rotating plasma reactor. The source gas entering the plasma chamber during the reaction process determined the type of coating, and coatings were typically much less than 5.0 µm in thickness. To delay germination we utilized two different hydrophobic source gases, carbon tetrafluoride (CF₄) or octadecafluorodecalin (ODFD). Seeds of radish (Raphanus sativus) and two pea cultivars (Pisum sativum cv. Little Marvel, P. sativum cv. Alaska) treated with CF₄, resulted in a significant delay in germination compared with untreated controls. Similarly, plasma treatment with ODFD significantly delayed germination in soybean [Glycine max (L.) Merr.], corn (Zea mays L.), and bean (Phaseolus vulgaris L.) seeds. The degree of delay was dependent on the amount of coating applied, with an increased thickness of coating resulting in a greater delay in germination. To enhance germination we treated seeds with cyclohexane, or with a gas such as aniline or hydrazine. Seeds treated with cyclohexane resulted in a significant acceleration in germination percentage for soybean but not corn seeds, while hydrazine-treated corn seed showed a small acceleration over control seed. However, both aniline-treated soybean and corn seed had 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. These results demonstrate a potentially important technique to modify seed germination characteristics in agricultural plant species.

Abbreviations: carbon tetrafluoride, CF₄ • octadecafluorodecalin, ODFD • radio frequency, RF • electron spectroscopy for chemical analysis, ESCA

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Materials and methods Results Discussion Conclusions REFERENCES

 
A PRODIGIOUS AMOUNT of research has been conducted on alteration of seed germination characteristics. Typically, the underlying objectives of seed treatments are focused on alleviating stress associated with the soil environment and/or on directly increasing plant growth (Taylor and Harman, 1990). The most common seed treatments alleviate biotic stress by reducing the damage caused by seed or soil borne pests and pathogens (e.g., insects and fungi) on seeds and seedlings (Silininger et al., 1996; Cliquet and Scheffer, 1997; Shah-Smith and Burns, 1997). Alleviation of abiotic stress is another objective in seed treatment research, and has been achieved by modifying the soil water-oxygen relations around a germinating seed (Leaver and Roberts, 1984; Huang and Hsiao, 1987).

To increase seedling growth directly, a seed treatment may involve direct application of nutrients or plant growth regulators to seeds (Silcock and Smith, 1982; Taylor and Harman, 1990; Zarnstorff et al., 1994). One such treatment is seed coating, the direct application of material to a seed surface. Seeds can be treated by mixing them with a slurry of chemicals and water or by applying a water-based mist. Depending on application technology, this treatment may result in an uneven application to the seed. Typically, seed coating refers to the application of useful material(s) to a seed without changing its general shape or size, whereas pelleted seed refers to seed to which inert fillers have been added to increase the apparent size and weight of the seed. Coated seed may, in some circumstances, be produced by a dry powder process, which can have several disadvantages, such as poor adherence, non-uniform application, generation of significant amounts of dust, etc. (Jeffs and Tuppen, 1986; Kaufman, 1991).

Pelleting is a net process resulting in a substantial increase in seed moisture content. Pelleted seeds must be dried to avoid reduced shelf life. In fact, one cause of accelerated aging of seeds is high levels of seed moisture (Carpenter et al., 1995). An alternative method to reduce this effect is film coating of seeds. In film coating, the active materials are dispersed or dissolved in a liquid adhesive, which is applied to the seeds either by a fluidized bed treatment or by a pharmaceutical coating drum. There are typically three major components in film coating: a polymer, a plasticizer, and a pigment. Film coatings can be applied in multiple layers and typically increase seed weight by 1 to 10%. Generally, film coatings are less than 0.1 mm in thickness (Kaufman, 1991; Robani, 1994; Taylor et al., 1994, 1998).

Seeds produced by commercial seed companies are commonly treated with insecticides and fungicides in attempt to enhance the survivability of the planted seed. Because film coating provides a polymer that encapsulates a seed, it reduces the potential health hazards during transportation, storage, and planting compared with more conventional seed treatment coatings.

Seed treatments have also been used to enhance germination. For instance, seeds have been coated with peroxide compounds that provide oxygen to seeds planted under anoxic or near-anoxic soil conditions (Leaver and Roberts, 1984), as may occur in a flooded environment. In other instances, delayed germination has been investigated through retardation of imbibition to prevent inhibitional chilling damage of seed sown in a cold, wet soil (Priestly and Leopold, 1986; Taylor et al., 1992). Other seed technologies have focused on the application of macro- or micronutrients or beneficial bacteria to improve early plant growth (Scott and Archie, 1978; Glick, 1995; Bashan, 1998). To enhance nutrient uptake, some investigators have treated seeds with beneficial microorganisms that can proliferate on the seed and improve nitrogen fixation (Tonkin, 1984; Kanvinde and Sastry, 1990). In this study, we describe the use of a novel method for the treatment of seeds which has several advantages over current technology as described below. We used plasma chemistry for seed surface modification of several important agricultural species. Cold plasmas are being utilized for an ever-increasing number of applications. This is related to the many advantages associated with the use of plasmas for modification of materials. These include environmental benefits because the technique involves dry chemistry and thus does not produce contaminated aqueous waste water, the active species only penetrate about 10 nm deep so they do not alter the base structure, and even the most inert surfaces can be functionalized with plasma techniques (Denes et al., 1999).

The energy levels of the active species in plasmas are sufficiently high to dissociate all chemical bonds of organic compounds, and consequently the resulting multifunctional species can lead, through recombination processes, to the formation of macromolecular structures. Plasma species can also become attached to polymer substrate surfaces creating new functional groups and characteristics. The possibility of modifying even the most inert inorganic- and organic-material surfaces places macromolecular plasma chemistry in the sphere of advanced technology (Denes et al., 1999; Denes, 1997).

The most commonly used plasma reactors for the synthesis of macromolecular structures and surface modification of various substrates are radio frequency (RF) installations. The discharges can be excited and sustained even by insulated electrodes located inside or outside the reaction chamber (Denes, 1997). A schematic diagram of a capacitively coupled, rotating RF-plasma reactor is illustrated in Fig. 1 . A unique feature of the work presented in this paper is the use of this rotating reactor to treat seeds uniformly with plasma gases.

View larger version (28K): [in this window] [in a new window]   Fig. 1 Diagram of a 13.56-MHz-RF-plasma rotating reactor: 1—reservoirs; 2—valves; 3—flow-meters; 4,10—ferrofluidic feed-through; 5,9—connecting rubber and stainless steel rings; 6—semicylindrical copper electrodes; 7—Pyrex glass chamber; 8—13.56-MHz RF power supply and matching network assembly; 11—gate valve; 12—electric engine; 13—transmission system

	Materials and methods

TOP ABSTRACT INTRODUCTION Materials and methods Results Discussion Conclusions REFERENCES

 
Plasma Reactor
The rotating plasma reactor can be used to create coatings on the surface of seeds, although if the plasma gas is not polymerizable then only functionalization of the surface takes place. To plasma-process the seeds, an original 13.56-MHz RF-plasma reactor was utilized (Fig. 1) (Denes et al., 1999). The reactor is composed of a Pyrex glass chamber [7] provided with connecting rubber and stainless steel rings [5, 9] on both ends. The vacuum-tight connection of the reactor to the monomer- and gas-supply system and to the vacuum line is assured with the aid of two ferrofluidic feed-throughs [4, 10]. The hollow shaft of the stainless steel chambers is made of special magnetic material and they are a part of the ferrofluidic sealing system. The RF power is transferred to the reactor through two semi-cylindrical copper electrodes [6] located outside from a 1000 W, 13.56-MHz RF power supply and matching network assembly [8]. A large cross-section gate valve [11] separates the reactor from the vacuum line and allows the control of the out-flow of the plasma gases. The vapor and gas flow into the reactor is controlled through individual flow controllers [2, 3]. The rotation of the reactor at various angular velocities is assured by a digitally speed-controlled electric engine-transmission system [12, 13].

In a typical experiment, the seeds are introduced into the reactor, the system is closed, and the base pressure is created, where seeds remain under vacuum conditions 24 h before plasma gas is introduced. Control samples were run for all seeds under the same 24-h vacuum conditions to be certain that this treatment was not affecting germination. There were no significant differences in seed moisture between control and plasma treated seeds, averaging across treatments 8.4 ± 0.4% and 8.5 ± 0.6% seed moisture for control and plasma-treated seeds, respectively. We did not detect any changes in the seed germination as a result of the vacuum treatment. Upon introduction of plasma gas, the rotation of the reactor was started at the selected speed and the selected gas-flow and pressure conditions were established, the plasma was ignited and sustained for the desired treatment time. At the end of the reaction the RF-power supply was disconnected, the system was evacuated, and air was introduced into the reactor until atmospheric pressure is reached. The plasma-treated seeds were then removed from the reactor and stored in sealed plastic bags until analytical and germination tests were initiated.

Plasma Gases
The following materials and conditions were used: fluorocarbon—CF₄, ODFD; nitrogen-containing—aniline, hydrazine; carbon—cyclohexane; base pressure—30-40 mT; pressure in the absence of plasma—250 mT; angular speed—30 rpm.

Treatment of Seeds with Fluorocarbon Plasmas
The first phase of the investigation was designed to test the effect of plasma treatment on seed survival. In addition, the ability to delay germination was tested by using a hydrophobic gas in the plasma treatment that would react with the surface of the seed. Seeds of radish (Raphanus sativus) (n = 31) and two cultivars of pea (Pisum sativum cv. Little Marvel, and Pisum sativum cv. Alaska) (n = 16 for each cultivars) were treated with CF₄, under a RF cold plasma condition within a rotating plasma reactor (see above). RF power applied to the plasma was 150 W, with a pulsed application of 500 µs pulse period and 30% duty cycle. The pressure in the reactor during the discharge was 200 mT with a plasma flow rate of 6 standard cubic centimeters (sccm). Treatment time was 5 min (Table 1) . These conditions provided a plasma-mediated deposit on the surfaces of the seeds.

Seeds of corn (n = 400) and bean (Phaseolus vulgaris) (n = 400) were again treated with ODFD, but for various lengths of time: 0-, 2-, 5- or 20-min. Pressure was maintained at 200 mT. High resolution electron spectroscopy for chemical analysis (ESCA) and scanning electron microscopy (SEM) imaging were conducted on the both the corn and bean seeds to compare the surface deposition of treated seeds with the vacuum control seeds. Both survey and high resolution (HR) ESCA multiplex spectra were taken with a PHI 5400 Spectrometer—Perkin Elmer Corporation, Norwalk, CT (Mg source; 15 kV and 300 W; angle: 45°). Carbon (C1s), oxygen (O1s), and fluorine (F1s) atomic compositions were evaluated and the nonequivalent positions of carbon linkages were analyzed. In order to correct surface-charge-origin binding energy, shift calibrations were performed on the basis of the well-known C1s peak.

SEM images of gold sputtered, ODFD-plasma treated virgin corn and bean substrates were produced using a JSM-840 SEM instrument (JEOL U.S.A., Inc., Peabody, MA) and a, Perk-1 sputter coater (Denton Vacuum, Inc., Moorestown, NJ).

Treatment of Seeds with Nitrogen-Containing Plasmas
Seeds of soybean (n = 200) and corn (n = 200) were treated with aniline gas under the same plasma treatment protocol described in the initial plasma treatments (Table 1). Aniline is a nitrogen-based compound that was tested for its potential ability to shorten the time of germination. Likewise, another nitrogen-containing compound, hydrazine, was used for plasma treatment of corn seeds (n = 200) under 200 mT for 20 min.

Treatment of Seeds with Cyclohexane Plasma
For the final part of the work, seeds of corn (n = 300) and soybean (n = 300) were also surface deposited by plasma treatment with cyclohexane. In this case, seeds were treated for 10 min under two different pressures: 70 and 150 mT. All other plasma reaction conditions were consistent with the first part of the study (Table 1).

Seed Germination
For all studies, plasma treated and untreated control seeds were placed in transparent acrylic seed germination trays (11 by 11 by 3.5 cm, Hoffman Manufacturing, Inc., Albany, OR). Control samples consisted of seeds that had been kept under vacuum condition only. By means of sterile techniques and a laminar flow hood, treated and untreated seeds were transferred from polyethylene bags into germination trays (5 seeds/tray). Each tray contained moist seed germination paper folded over to completely cover the seeds. Germination paper was maintained in a moist condition by misting with distilled water as required. Germination trays were placed in a germination growth chamber (Hoffman Manufacturing, Inc.) with consistent environmental conditions consisting of a temperature of 28/25°C, and a photoperiod of 16/8 day/night, respectively. Average photosynthetic photon flux was 100 µmol m^-2 s^-1 at the seed surface. The seeds were examined every 24 h under a laminar flow hood for signs of germination; when radicle emergence was 0.5 cm, seeds were considered germinated. Seeds were continuously counted until the germination percentage was consistent over a 72-h period. Seeds that did not germinate were tested for viability; across treatments it was determined that seeds not germinating, although fully hydrated, were in all cases dead.

The mean time to germination, expressed as hours to 50% of final germination (T₅₀), and germination span, in number of hours elapsed between 10 and 90% germination (T_10–90), were calculated and analyzed as described by Furutani et al. (1985).

Water uptake was tested in a subset of plasma-treated and control seeds. For instance, aniline- and cyclohexane-treated corn and soybean seeds, and ODFD- and hydrazine-treated corn seeds were examined. Seeds were monitored for water uptake every 24 h for 6 d following sowing. Hydration rate was expressed as K (lnMC h^-1) where K = [(lnMC - lnMCo)t^-1], and K is the hydration rate, MC is the moisture content at Time t, MCo is the initial moisture content, and t is time in hours (Taylor et al., 1992).

Over the course of the study, seed germination of treated and control seeds was determined by the frequency of seeds that showed radicle emergence. A chi-square analysis was performed on the counts determined in each study. Control seeds were used to determine expected frequencies, while plasma treated seeds generated the observed frequencies. Chi-square analysis of treatment main effects was conducted for each study by species and by day.

	Results

TOP ABSTRACT INTRODUCTION Materials and methods Results Discussion Conclusions REFERENCES

 
Treatment of Seeds with Fluorocarbon Plasmas
Fluorocarbon plasmas were investigated to deposit chemically and thermally inert (Teflon-like) macromolecular layers on the seed substrates. It has been demonstrated 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 resulted 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 T_10–90, were similar to germination percentage, in that T₅₀ and T_10–90 were increased by CF₄ in radish seeds, while the response was not as pronounced in the pea seeds (Table 2) .

View larger version (16K): [in this window] [in a new window]   Fig. 2 Percent germination of CF4-treated and untreated control seeds of radish (Raphanus sativus) and two cultivars of pea (Pisum sativum cv. Little Marvel, P. sativum cv. Alaska). Control seeds were under vacuum pressure for 24 h. Treatment main effects were significant (P = 0.009) for radish on Day 1 and for pea cv. Little Marvel (P = 0.03) on Day 3. All other treatment effects were not significant at P < 0.05

View larger version (18K): [in this window] [in a new window]   Fig. 3 Percent germination of ODFD-treated and untreated control seeds of corn (Zea mays) and soybean (Glycine max). Treatment main effects were significant (P < 0.05) for both species after Day 1

View larger version (23K): [in this window] [in a new window]   Fig. 4 Percent germination of ODFD-treated and untreated control seeds of corn (Zea mays) and bean (Phaseolus vulgaris). Plasma treated seeds were conducted at three different exposure time duration's: 2-, 5-, and 20-min. For corn, ODFD-treated seeds were significantly different (P < 0.0001) from control seeds for the first 2 d after sowing. Twenty-min ODFD-treated corn seeds maintained a significant difference from control seed throughout the study. For bean, ODFD 5-min treated seeds were significantly different (P = 0.004) from control seeds from Day 2 through 4, while 20-min treated seeds were significantly different (P = 0.008) from Day 2 through 6. All other treatment effects were not significant at P < 0.05

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.

View larger version (15K): [in this window] [in a new window]   Fig. 5 Survey electron spectroscopy for chemical analysis (ESCA) for ODFD-plasma-treated (A) corn, and (B) bean seeds

View larger version (23K): [in this window] [in a new window]   Fig. 6 High-resolution electron spectroscopy for chemical analysis (ESCA) analysis of non-equivalent C1s functionalities of ODFD-plasma-treated (A) corn, and (B) bean seeds

View larger version (147K): [in this window] [in a new window]   Fig. 7 Scanning electron microscopy images of ODFD-plasma-treated (A) and untreated (B) corn seed and ODFD-plasma-treated (C) and untreated (D) bean seed

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 (T_10–90) for either the aniline-treated soybean or corn seeds (Table 2).

View larger version (16K): [in this window] [in a new window]   Fig. 8 Percent germination of aniline-treated and untreated control seeds of soybean (Glycine max) and corn (Zea mays). Treatment main effects were significant at P < 0.0001 for both species on Day 1 after sowing. Treatment main effects were not significant (P < 0.05) after Day 1

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 T_10–90 (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.

View larger version (18K): [in this window] [in a new window]   Fig. 9 Percent germination of hydrazine-treated and untreated-control seeds of corn (Zea mays). Treatment main effects were significant at P = 0.03 on Day 1 after sowing. Treatment main effects were not significant (P < 0.05) after Day 1

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.

View larger version (20K): [in this window] [in a new window]   Fig. 10 Percent germination of cyclohexane-treated and untreated-control seeds of corn (Zea mays) and soybean (Glycine max). Plasma treated seeds were conducted at two different pressures: 70 and 150mT. Treatment main effects were not significant (P < 0.05) for corn throughout the study, while for the first 2 d after sowing there were significant differences (P < 0.0001) in germination among the soybean seed treatments, except between the plasma treatments at different pressures on Day 2 (P = 0.34). After Day 2, there were no significant differences (P < 0.05) among treatments for soybean seeds

	Discussion

TOP ABSTRACT INTRODUCTION Materials and methods Results Discussion Conclusions REFERENCES

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 CF_x 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.

	Conclusions

TOP ABSTRACT INTRODUCTION Materials and methods Results Discussion Conclusions REFERENCES

 
We have modified seed germination percentage and rate by applying different materials to seeds via a rotating cold plasma reactor system. In this series of studies, we found that we could achieve either delayed or accelerated germination in several different agricultural species without significantly affecting their percent germination. For instance, by proper choice of gas and cold plasma conditions germination was delayed in two cultivars of pea, radish, soybean, corn and bean seed, while germination was accelerated in soybean and corn seed.

Plasma treatment of seeds for delayed germination could be intermixed with regular germination seeds to provide phased germination of seeds in field plantings for successive cropping. On the other hand, accelerating germination could increase the earliness in crop emergence, especially advantageous for slow-to-germinate seeds, and increase seedling development, which would alleviate a problem in some species (Zarnstorff et al., 1994).

The advantage of the plasma reaction process is that the technique is not deleterious to the seed, practical for crop species, and it is environmentally safe. Although an economic analysis has not been performed, it is probable that the plasma approach would be equivalent or less costly than current technology based on current plasma technology. These criteria are considered a necessity for future development and commercial use of seed-treatment technologies (Taylor and Harman, 1990).

	ACKNOWLEDGMENTS

 
Appreciation is expressed to Angie Beechey of Kaltenberg Seed Farms, Waunakee, WI, for the generous gift of seeds utilized in this investigation. Moreover, the authors wish to thank Sorin Manolache for his contribution to both the ESCA and SEM analyses.

Received for publication October 13, 1999.

	REFERENCES

TOP ABSTRACT INTRODUCTION Materials and methods Results Discussion Conclusions REFERENCES

 

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