Plasma Chem Plasma Process (2017) 37:207–221

DOI 10.1007/s11090-016-9763-9

ORIGINAL PAPER

Seed Germination and Early Growth Responses to Seed

Pre-treatment by Non-thermal Plasma in Hemp
Cultivars (Cannabis sativa L.)

B. Sera1,2 • M. Sery3 • B. Gavril4 • I. Gajdova5

Received: 2 October 2016 / Accepted: 14 November 2016 / Published online: 22 November 2016
Ó Springer Science+Business Media New York 2016

Abstract The two key questions addressed in this paper were whether different cultivars
of hemp (Cannabis sativa L.) have the same reactions to non-thermal plasma seed pre-
treatments and whether different plasma sources have different effects on the seeds. Seed
germination and early growth of hemp in design of hierarchical analysis of variance was
conducted. Differences in response among seeds of three hemp cultivars (‘Finola’,
‘Bialobrzeskie’, ‘Carmagnola’) to the non-thermal plasma pre-treatment generated by two
apparatuses (gliding arc and downstream microwave devices) in four time expositions (0,
180, 300, 600 s) were found. The high importance was found in type of apparatus and time
exposition. A positive/neutral effect was observed in all measured characteristics after
gliding arc plasma pre-treatment. Gliding arc pre-treatment increased the length of seed-
lings, seedling accretion and weight of seedling in both cv. ‘Finola’ and cv. ‘Bialobrzeskie’
hemp. On the other hand, the downstream microwave apparatus had an inhibiting effect on
all tested hemp cultivars. It was the first time when significant differences in response to
non-thermal pre-treatment were found in taxonomically close plants. The results obtained
in this study describes different effect of various plasma treatment on germination and
early growth of hemp seeds. The direct pre-treatment of non-thermal plasma discharge in
condition of atmospheric pressure was better. Results of our experiment show that the use
of non-thermal plasma pre-treatment may increase survival of some hemp cultivars during

& B. Sera
sera@fns.uniba.sk
1
Department of Landscape Ecology, Faculty of Natural Sciences, Comenius University, Ilkovicova
6, Bratislava, Slovakia
2
Department of Biology, Faculty of Education, University of South Bohemia, Jeronymova 10,
Ceske Budejovice, Czech Republic
3
Department of Physics, Faculty of Education, University of South Bohemia, Jeronymova 10,
Ceske Budejovice, Czech Republic
4
Department of Electrical Engineering and Electrical Machines, Gheorghe Asachi Technical
University of Iaşi, Judetul Iaşi, Romania
5
Obránců mı́ru 817, Bechyně, Czech Republic

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208 Plasma Chem Plasma Process (2017) 37:207–221

seedlings establishment in a drier period and may be used in new agro-technical measures
in unconventional agriculture.

Keywords Hemp cultivars  Nested ANOVA  Non-thermal plasma  Plasma device  Seed
germination

Introduction

Hemp (Cannabis sativa L., Cannabaceae family) is one of the oldest sources of textile
fiber. It is a multi-purpose plant that has been domesticated for bast (phloem) fiber in the
stem, fixed oil in fruits, and intoxicating resin secreted by epidermal glands. It has been
used for fibre (textile and paper industry, engineering), for fruit and oils (food, engineer-
ing), for medicinal purposes, and as a soft drug [1–4].
Some non-frequent cultural plants are used in unconventional agriculture, such as
‘‘industrial hemp’’. A renewed interest in hemp as a source of cellulose fibre and fruit oil in
western European countries, Australia, the US and Canada share a need for prof-
itable arable non-food crops. All EU countries, except France, have never had a hemp
industry, or have interrupted it for decades. A substantial hemp industry has survived only
in eastern Europe, the former Soviet-Union and China [5]. Recently, there has been a
growing interest of farmers in unconventional plants and alternative procedures [6]. Hemp
seems to be a re-discovered plant well used in energetic and textile industry [3, 7–10].
In conventional agriculture, synthetic chemicals (plant pests, herbicides, insecticides,
pesticides and fertilizers) are used to increase the growth of cultivation and crop yield. In
Europe, current tendencies are moving away from mass use of synthetic products. The non-
conventional (organic, alternative) farming is more environmental and it differs from the
conventional farming in a number of characteristics. New technology, including nano or
biotechnology, may help to increase healthy crop yield rather than using chemicals. Usage
of non-thermal plasma pre-treatment may change the seed preparation and sowing. The
number of applications of plasma technology in many fields including polymer engi-
neering, biotechnological applications, metallurgy, microelectronics, inconvenient agron-
omy, and medical engineering is growing rapidly. The plasma contains active particles
(electrons, ions, excited and metastable particles, radicals and photons) in non-equilibrium
states; thus they may interact with various material surfaces. It was found that plasma
treatment modify seed wettability [11–17]. Seed surfaces affected by plasma treatment are
often documented by SED; deeply cracks and various erosion of seed surface treated by
plasma are obvious in comparison with the control [11, 13, 14, 18, 19]. Treated seeds got
wet faster, had better germination (higher number of germinated seeds), and started to
germinate in a shorter time than untreated seeds [11–17, 19]. Non-thermal plasma pre-
treatment may positively affect the vitality of growing seeds [11–13, 20, 21], probably in
cases of short-term drought during the initial seed growth.
Hemp does best on a loose, well-aerated loam soil with high fertility and abundant
organic matter. Good soil moisture is necessary for seed germination, and plenty of rainfall
is needed for good growth, especially during the first 6 weeks [3]. Non-thermal plasma pre-
treatment can be probably used in seeds for increasing the seed wettability [11, 14, 15],
seed germination [11, 14, 15, 22, 23] and seedling root growth [16, 17, 21, 24, 25].

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Plasma Chem Plasma Process (2017) 37:207–221 209

However, no information is available on the evaluation of non-thermal plasma pre-treat-

ment in various cultivars of hemp seed.
The aim of this experiment was to test three morphologically variable cultivars of hemp
species and to find the way how to pre-treat hemp seeds by plasma before sowing. The
objectives were (a) investigate non-thermal plasma pre-treatment on seed germination and
characteristics of early growth of hemp seeds and (b) to explore how a type of plasma
apparatus, time duration of pre-treatment and type of hemp cultivars will change the seed
germination and growth characteristics in hemp seed. Germinating and growth parameters
were measured.

Materials and Methods

Plant Material

Three cultivars of industrial hemp seeds were obtained from the civil association Konopa.
There term ‘‘seed’’ is used for fruit (achene) in this paper. Here are short characteristics of
the investigated varieties (varieties signed according to OECD [26]):
‘Finola’ (FI, 453) began in Finland and was registered in 1995. It is early blooming,
short (less than 2 m) and morphologically different from all EU varieties. It is primarily
grown for grain and it also produces fiber/biomass (http://www.finola.com).
‘Bialobrzeskie’ (PL, 1368) was registered in 1968 in Poland. It is a result of a multiple
crossing of dioecious and monoecious strains ((‘LKCSD’ 9 ‘Kompolti’) 9 ‘Bredemann
18’) 9 ‘Fibrimon 24’ [27]. This cultivar was bred for fibre content [26].
‘Carmagnola’ (IT, 1345) is an old dioecious variety from the northern Italian land race.
It is a quite high plant (1.5–4 m) that blooms in the end of summer. The EU recognizes
‘Carmagnola’ as a low THC technical cultivar grown for its fiber (http://www.
centennialseeds.com).

Apparatus Description

For plasma pre-treatment we used two plasma devices. The first one was the commercial
apparatus Plasonic AR-550-M, which is a low-pressure and low-temperature microwave
apparatus generating plasma using a stationary-wave resonator (Fig. 1). In this paper this
apparatus is marked DMP according to the downstream microwave plasma processing. The
second apparatus constructed in the laboratory was the gliding arc device consisting of two
electrodes (marked GA, Fig. 2). Both devices are described in more detail in [28–30].

Plasma Pre-treatment

The parameters used in DMP were: pressure 140 Pa, frequency 2.45 MHz, magnetron
power 500 W, oxygen gas-flow rate 50 ml/min, argon gas-flow rate 50 ml/min, tempera-
ture about 25 °C. A glass Petri dish (20 cm in diameter) with tested seeds was placed on
the bottom of the vacuum chamber, ca. 10 cm under the plasma output. The influence of
low-pressure in DMP was not tested separately, because a small effect of low-pressure on
seed germination was confirmed [11].
The working gas was humid air in the GA apparatus. The distance between electrodes
and the treated seeds laying on the glass Petri dish (9 cm diameter) was 250 mm. Other

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Fig. 1 The diagram of downstream microwave plasma apparatus (DMP) and position of the seed during
plasma exposition

Fig. 2 The diagram of gliding arc device (GA) and position of the seed during plasma exposition

used parameters: power frequency 50 Hz, gas-flow rate 10 l/min, temperature not more
than 50 °C. Positions of seeds during plasma pre-treatment are obvious in the Figs. 1 and
2.
In both apparatuses, three different time expositions were used: 180, 300, 600 s. A
reference (control) sample represents a result of seeds without plasma pre-treatment (0 s).

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Germination and Early Growth Tests

There were used 30 seeds of one pre-treated cultivar subsample, KA 0/80 filter paper, 6 ml
of distilled water for one plastic Petri dish of 9 cm in diameter. Each subsample pre-
treatment was repeated five times, so 150 seeds were used for one plasma time exposure
and one plant cultivar. The germination and early-growth tests were run in growing box
(temperature 22 °C, night condition).
The data about the number of germinating seeds (germination was considered if 1 mm
radicle occurred) and the length of the seedlings (including the embryonic leaves) were
collected on the 3rd, 4th and 5th day of cultivation. The length of seedlings was measured
without cotyledon. The seedling mass was collected on the 5th day of cultivation and
weighed on laboratory scale (balance sensitivity 0.001 g). The length of the seedling
accretions was calculated on the 4th and 5th day of cultivation.
List of investigated characteristics:
i Index of petri dish
k Day of cultivation
j Index of germinated seed
NPi (j) Number of germinated seeds per i-petri dish in j day
lk (j) Length of seedling k-germinated seed in j day
wk Weight of seedling k-germinated seed
Mean of seed germination in j day
P5
i¼1 NPi ð jÞ
5
Mean length of seedling k-germinated seed in j day
!
P5 Pi ð jÞ
NP
lk ð jÞ
i¼1 NPi ð jÞ
k¼1

5
Mean accretion of seedling k-germinated seed in j day
P5 PNPi ð jÞ
i¼1 k¼1 ½lk ð jÞ  lk ðj  1Þ
5
Mean weight of seedling k-germinated seed
P P 5 NPi ð5Þ wk

i¼1 k¼1 NPi ð5Þ

Statistical Analysis

All data were analyzed using the statistical package STATISTICA at the significance level
of 0.05. Logarithmic transformation (y = log(x)) of the basic data was used for normal-
ization before statistical analyses.
Nested ANOVA (fixed factors) was used to evaluate the influence of the type apparatus
(factor Plasma), hemp cultivars (factor Cannabis) and time of plasma pre-treatment (factor

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Time). The hierarchical structure: four possibilities of time of plasma pre-treatment (0,
180, 300, 600 s) were nested into hemp cultivars (‘Carmagnola’, ‘Bialobrzeskie’, ‘Fi-
nola’); the highest level was a type of apparatus with two characters: DMP and GA. The
dependent variances were the data obtained from the early growth testing: seed germi-
nation, length of seedlings, seedling accretion, and weight of seedlings.
The detailed testing of experimental variances from each other was done using the one-
way ANOVA test followed by the Tukey HSD test for multiple comparisons.

Results

Nested ANOVA Analyses

Overall nested analyses of variance results are reported in Table 1, which shows the
proportions of variance of each factor in the hierarchical structure. All the factors and their
combinations (excluding the combination of Plasma 9 Cannabis 9 Time) were important,
because all of them observed many significant differences in the measured characteristics
(Table 1). The importance of the plasma apparatus character is obvious in Fig. 3, where
high difference in seed responses to GA and DMP apparatuses are shown. The highest
variability was found in weight of dried biomass, where all factors including all possible
combinations observed significant differences (Table 1; Fig. 3).
All recorded characteristics are shown in percentage according to control samples
(100%) in Fig. 3. Significant differences (Tukey test, p \ 0.05) among measured char-
acteristics according to the lowest level of hierarchy are summarized in the Table 2.

Seed Germination

Samples of cv. ‘Finola’ and cv. ‘Carmagnola’ seeds pre-sowed by GA germinate better
than untreated samples (Fig. 3a), but not on a significant level (Table 2). Seeds of cv.
‘Finola’ and cv. ‘Bialobrzeskie’ germinated more unified than seeds of cv. ‘Carmagnola’
(Fig. 3a). The highest number of germinated seeds had cv. ‘Carmagnola’ after 180 and
300 s time expositions by GA plasma on the 3rd day of cultivation (122 and 118%
respectively, Fig. 3a; Table 2).
Seed germination is one of the measured characteristics, where seeds pre-treated by
DMP did not have a destructive reaction (Fig. 3), but all pre-sowed samples had a small
number of germinated seeds than control samples (Fig. 3b; Table 2). Seeds of cv. ‘Car-
magnola’ after DMP pre-treatment (all times of exposures) had significantly smaller seed
germination on 4th and 5th days than control samples (Tukey test, p \ 0.05). Just as it was
at seeds of cv. ‘Bialobrzeskie’ after DMP pre-treatments on 3rd day of cultivation
(Table 2).

Length of Seedling

The length of seedlings of all cultivars pre-sowed by GA were usually bigger than control
samples (Fig. 3c), but without a significant difference (Table 2). The highest length of all
tested hemp had cv. ‘Finola’ seedlings after seed pre-treatment with 300 s on the 5th day of
cultivation (153% in comparison with a control sample, Fig. 3c). Seeds of cv. ‘Finola’

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 Table 1 Results of factorial analyses: relationship among the type apparatus (factor Plasma), hemp cultivars (factor Cannabis) and time of plasma pre-treatment on seed
reaction (factor Time) in relation to seed germination and characteristics of early growth
Factor DF Seed germination 3rd day Seed germination 4th day Seed germination 5th day

SS F p SS F p SS F p

Plasma 1 612.01 98.84 0.000 425.63 76.92 0.000 323.41 78.72 0.000
Cannabis 2 2958.07 238.87 0.000 412.55 37.28 0.000 402.52 48.99 0.000
Time 3 135.49 7.29 0.000 218.03 13.13 0.000 177.63 14.41 0.000
Plasma 9 cannabis 2 41.27 3.33 0.040 60.52 5.47 0.006 49.82 6.06 0.003
Plasma 9 time 3 218.49 11.76 0.000 146.70 8.84 0.000 115.56 9.38 0.000
Cannabis 9 time 6 95.93 2.58 0.023 128.32 3.86 0.002 145.95 5.92 0.000
Plasma 9 cannabis 9 time 6 61.13 1.65 0.143 38.35 1.16 0.337 35.72 1.45 0.204

Factor DF Length of seedling 3rd day Length of seedling 4th day Length of seedling 5th day
Plasma Chem Plasma Process (2017) 37:207–221

SS F p SS F p SS F p

Plasma 1 18.79 10.30 0.002 606.60 111.71 0.000 7691.20 441.40 0.000
Cannabis 2 90.86 24.89 0.000 246.44 22.69 0.000 908.53 26.07 0.000
Time 3 11.73 2.14 0.100 88.45 5.43 0.002 1241.47 23.75 0.000
Plasma 9 cannabis 2 15.39 4.21 0.018 33.97 3.13 0.048 45.58 1.31 0.275
Plasma 9 time 3 11.30 2.06 0.110 223.66 13.73 0.000 2688.66 51.43 0.000
Cannabis 9 time 6 16.77 1.53 0.176 65.79 2.02 0.070 487.90 4.67 0.000
Plasma 9 cannabis 9 time 6 12.58 1.15 0.341 14.64 0.45 0.844 42.84 0.41 0.871

Factor DF Seedling accretion 4th day Seedling accretion 5th day Weight of seedling 5th day

SS F p SS F p SS F p

Plasma 1 440.14 483.18 0.000 3973.02 563.14 0.000 310,177.89 526.12 0.000
Cannabis 2 38.23 20.98 0.000 253.31 17.95 0.000 26,182.52 22.21 0.000
Time 3 43.41 15.89 0.000 706.59 33.38 0.000 106,695.62 60.32 0.000
213

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 Table 1 continued
214

Factor DF Seedling accretion 4th day Seedling accretion 5th day Weight of seedling 5th day

SS F p SS F p SS F p

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Plasma 9 cannabis 2 8.44 4.63 0.012 11.22 0.80 0.454 16,524.99 14.01 0.000
Plasma 9 time 3 152.60 55.84 0.000 1369.51 64.71 0.000 105,334.30 59.56 0.000
Cannabis 9 time 6 11.22 2.05 0.066 335.25 7.92 0.000 44,332.84 12.53 0.000
Plasma 9 cannabis 9 time 6 9.57 1.75 0.117 44.84 1.06 0.393 8512.20 2.41 0.033

Significant differences are in bold

Plasma Chem Plasma Process (2017) 37:207–221
Plasma Chem Plasma Process (2017) 37:207–221 215

Fig. 3 Percentage of measured characteristics in hemp cultivars after plasma pre-sowing. Hemp cultivars:
Fin—cv. ‘Finola’, Bia—cv. ‘Bialobrzeskie’, Car—cv. ‘Carmagnola’. Time of measurement: light shade 3rd
day, dark shade 4th day, medium shade 5th day of cultivation

seedlings were cv. ‘Bialobrzeskie’ (141%) after 600 s pre-treatment and cv. ‘Carmagnola’
(126%) after 300 s pre-treatment on the 4th day of the measurement (Fig. 3c).
Almost all obtained characteristics after DMP plasma pre-treatment were significantly
smaller than control samples (100%, Table 2). Seeds of cv. ‘Finola’ pre-treated by DMP
plasma grow in the range of 24–46% and seeds of cv. ‘Carmagnola’ in the range 6–59%
(Fig. 3d). All measured characteristics of cv. ‘Bialobrzeskie’ looked like as cv. ‘Finola’
and cv. Carmagnola, but one big peak after 600 s plasma pre-treatment on the 3rd day of
cultivation was obtained (Fig. 3d).

Seedling Accretion

Positive effects after GA plasma pre-sowing were recorded (Fig. 3e; Table 2). The high
rates of growing were found in cv. ‘Finola’ (164%) after 300 s pre-treatment on the 5th day
and in cv. ‘Carmagnola’ (163%) after 600 s on the 4th day (Fig. 3e). Both results were on a
significant level (Table 2).
On the other hand, DMP plasma pre-sowing had only an inhibitive effect, above all in
cv. ‘Carmagnola’ and cv. ‘Bialobrzeskie’ seedlings (Fig. 3f). All obtained results were on a
significant level (Table 2).

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Table 2 Seed germination and characteristics of early growth on the 5th day of plant cultivation
Plasma Cannabis Time (s) Seed germination (1) Length of seedling (mm) Seedling accretion (mm) Weight of
seedling (mg)

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3rd day 4th day 5th day 3rd day 4th day 5th day 4th day 5th day 5th day
Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD

GA ‘Finola’ 0 22.6 ± 1.9 a 24.0 ± 2.6 a 24.4 ± 2.2 a 3.4 ± 0.4 a 8.5 ± 1.6 a 22.8 ± 3.1 b 5.1 ± 1.2 a 14.4 ± 1.8 b 119.8 ± 7.9 a
180 24.2 ± 2.2 a 25.2 ± 1.6 a 26.0 ± 0.7 a 3.3 ± 0.6 a 10.1 ± 2.7 a 31.1 ± 7.3 b 6.8 ± 2.1 a 21.0 ± 4.8 a 143.1 ± 12.3 a
300 24.6 ± 1.5 a 25.4 ± 1.7 a 25.4 ± 1.7 a 3.8 ± 0.5 a 11.4 ± 0.7 a 34.9 ± 2.5 a 7.6 ± 0.4 a 23.6 ± 1.8 a 151.8 ± 5.5 a
600 24.6 ± 3.6 a 25.6 ± 2.6 a 26.2 ± 2.3 a 3.2 ± 0.6 a 9.1 ± 1.7 a 27.7 ± 4.2 b 5.9 ± 1.2 a 18.6 ± 2.5 b 137.6 ± 11.1 a
‘Bialobrzeskie’ 0 25.4 ± 1.3 a 26.8 ± 2.3 a 27.0 ± 2.1 a 2.4 ± 0.3 a 6.7 ± 1.1 a 22.5 ± 5.3 a 4.4 ± 1.0 b 15.7 ± 4.3 a 135.0 ± 9.3 a
180 24.2 ± 2.2 a 26.4 ± 1.1 a 27.0 ± 1.2 a 1.9 ± 0.3 a 4.1 ± 1.1 a 23.4 ± 3.6 a 5.2 ± 0.9 b 16.3 ± 2.6 a 144.4 ± 6.9 a
300 24.2 ± 1.8 a 26.6 ± 1.1 a 27.0 ± 0.7 a 2.1 ± 0.4 a 8.3 ± 1.6 a 25.9 ± 5.7 a 6.2 ± 1.2 b 17.6 ± 4.3 a 150.5 ± 13.6 a
600 24.8 ± 1.3 a 26.4 ± 1.1 a 26.4 ± 1.1 a 2.2 ± 0.4 a 9.3 ± 2.5 a 28.2 ± 6.1 a 7.1 ± 2.2 a 18.8 ± 3.6 a 156.6 ± 11.6 a
‘Carmagnola’ 0 12.0 ± 2.5 a 25.0 ± 1.4 a 25.6 ± 1.3 a 0.6 ± 0.1 a 4.8 ± 0.6 a 22.4 ± 2.7 a 4.3 ± 0.5 b 17.5 ± 2.2 a 222.8 ± 21.2 a
180 14.6 ± 3.0 a 22.2 ± 1.6 a 22.6 ± 1.1 a 0.6 ± 0.2 a 4.6 ± 0.9 a 20.1 ± 2.9 a 3.9 ± 0.8 b 15.5 ± 2.3 a 177.8 ± 13.6 a
300 14.2 ± 4.1 a 25.2 ± 2.9 a 25.4 ± 2.5 a 0.6 ± 0.2 a 6.1 ± 1.1 a 24.5 ± 4.9 a 5.6 ± 1.0 a 18.4 ± 3.9 a 207.0 ± 18.3 a
600 11.2 ± 2.6 a 21.6 ± 1.3 b 21.6 ± 1.3 b 0.4 ± 0.1 a 4.5 ± 0.7 a 17.4 ± 2.0 b 4.0 ± 0.7 b 12.9 ± 1.5 b 157.7 ± 15.4 a
DMP ‘Finola’ 0 22.6 ± 1.9 a 24.0 ± 2.6 a 24.4 ± 2.2 a 3.4 ± 0.4 a 8.5 ± 1.6 a 22.8 ± 3.1 a 5.1 ± 1.2 a 14.4 ± 1.8 a 119.8 ± 7.9 a
180 18.6 ± 2.3 a 21.2 ± 3.3 a 22.0 ± 2.8 a 1.2 ± 0.3 b 2.7 ± 0.9 b 10.5 ± 4.4 b 1.5 ± 0.6 b 7.8 ± 3.5 a 60.3 ± 10.0 b
300 20.4 ± 3.0 a 23.4 ± 2.9 a 14.0 ± 2.6 a 1.1 ± 0.2 b 2.3 ± 0.6 b 7.9 ± 3.1 b 1.3 ± 0.5 b 5.6 ± 2.6 b 46.3 ± 5.4 b
600 18.6 ± 3.7 a 21.6 ± 2.4 a 22.6 ± 1.5 a 0.9 ± 0.2 b 2.1 ± 0.3 b 7.3 ± 1.8 b 1.2 ± 0.6 b 5.2 ± 1.5 b 44.4 ± 2.4 b
‘Bialobrzeskie’ 0 25.4 ± 1.3 a 26.8 ± 2.3 a 27.0 ± 2.1 a 2.4 ± 0.3 a 6.7 ± 1.1 a 22.5 ± 5.3 a 4.4 ± 1.0 a 15.7 ± 4.3 a 135.0 ± 9.3 a
180 16.4 ± 3.0 b 21.6 ± 3.4 b 23.0 ± 2.4 b 0.6 ± 0.1 b 0.9 ± 0.2 b 1.8 ± 0.4 b 0.4 ± 0.1 b 0.9 ± 0.3 b 6.5 ± 1.0 b
300 16.8 ± 1.3 b 23.2 ± 1.9 a 24.2 ± 1.9 a 0.6 ± 0.1 b 1.0 ± 0.1 b 2.1 ± 0.6 b 0.4 ± 0.1 b 1.1 ± 0.5 b 7.0 ± 2.4 b
600 15.4 ± 0.5 b 22.4 ± 1.5 a 23.2 ± 1.1 b 3.4 ± 6.5 a 5.4 ± 9.9 a 6.5 ± 9.8 b 04 ± 0.1 b 1.2 ± 0.6 b 6.8 ± 2.7 b
‘Carmagnola’ 0 12.0 ± 2.5 a 25.0 ± 1.4 a 25.6 ± 1.3 a 0.6 ± 0.1 a 4.8 ± 0.6 a 22.4 ± 2.7 a 4.3 ± 0.5 a 17.5 ± 2.2 a 222.8 ± 21.2 a
180 6.6 ± 1.9 b 14.0 ± 4.4 b 15.0 ± 3.2 b 0.2 ± 0.1 b 0.7 ± 0.3 a 1.3 ± 1.0 b 0.4 ± 0.3 b 0.7 ± 0.8 b 6.6 ± 3.8 b
300 10.0 ± 2.0 a 16.2 ± 3.0 b 17.4 ± 3.0 b 0.3 ± 0.1 a 0.6 ± 0.2 a 1.5 ± 1.0 b 0.3 ± 0.2 b 0.8 ± 0.8 b 10.2 ± 3.1 b
Plasma Chem Plasma Process (2017) 37:207–221
 Table 2 continued

Plasma Cannabis Time (s) Seed germination (1) Length of seedling (mm) Seedling accretion (mm) Weight of
seedling (mg)
3rd day 4th day 5th day 3rd day 4th day 5th day 4th day 5th day 5th day
Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD

600 9.6 ± 3.9 a 15.8 ± 2.3 b 16.8 ± 2.8 b 0.3 ± 0.1 a 0.8 ± 0.2 a 2.0 ± 1.1 b 0.5 ± 0.2 b 1.2 ± 0.9 b 18.2 ± 5.4 b

Tukey HSD test was used. Significant differences at p \ 0.05 are indicated by different letters. DMP downstream microwave plasma apparatus, GA gliding arc device, more
detail in Materials and Methods
Plasma Chem Plasma Process (2017) 37:207–221
217

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218 Plasma Chem Plasma Process (2017) 37:207–221

Weight of Seedling

Seeds pre-treated by GA plasma had very a positive effect on biomass creation (Fig. 3g).
The weight of seedlings in cv. ‘Finola’ ranged between 115 and 127% and cv. ‘Bialo-
brzeskie’ between 107 and 116% in comparison with control samples (100%, Table 2).
The variability in response to GA plasma pre-treatment was high. No significant differ-
ences were found (Tukey test, p \ 0.05, Table 2). A negative effect was found in cv.
‘Carmagnola’ (Fig. 3g; Table 2).
DMP plasma pre-sowing had high inhibitive and destructive effects in the dry weight of
seedlings, above all in both cv. ‘Carmagnola’ (3–8%) and cv. ‘Bialobrzeskie’ (5%)
seedlings (Table 2; Fig. 3h).

Discussion

Effect of Devices

An obvious difference in seed reaction after plasma treatment generated in different

apparatuses was observed (Fig. 3). Hunnekens et al. [31] studied the effects of three
different discharges on surface properties of wood-polymer composites. The results
obtained confirmed the relation between the plasma source used and surface wettability.
The GA plasma pre-treatment had a neutral or positive effect on both seed germination
and seedling growth. A positive effect was found in seedling accretion and dried weight of
seedlings, above all in cv. ‘Finola’ and cv. ‘Bialobrzeskie’. The best time exposure was
300 s (Fig. 3). In accordance with this, short time of plasma pre-treatment was documented
in previous experiments [11, 12, 19]. The seeds treated by GA apparatus were activated by
direct exposure to the discharge at atmospheric pressure (in comparison to the DMP
apparatus, see Figs. 2, 3). Atmospheric pressure processes are more attractive for seed pre-
treatment applications, because of their lower cost and ability to operate without vacuum
systems. Plasmas sustained at atmospheric pressure are commonly used to improve the
wetting and adhesion properties of polymer materials [32, 33], and have been successfully
tested also for the seed surface treatment [11–17]. We attribute the better seed germination
after GA plasma treatment to the increased water absorption.
On the other hand, the DMP plasma pre-treatment had an inhibiting effect after all times
of durations in all tested hemp cultivars. Above all, a strong negative effect was found in
cv. ‘Carmagnola’ in seed germination and in both cv. ‘Bialobrzeskie’ and cv. ‘Carmag-
nola’ in the length and weight of seedlings (Table 2). Generally, seeds after DMP plasma
germinated less than 100%, so the number of seedlings available for other measured
characteristics was smaller too. This fact caused a high variability in data obtained from
seeds pre-sowed by DMP. So a type of plasma apparatus is important factor for seed pre-
treatment. This result corresponds to the paper describing four plasma devices used for pre-
sowing in the experiment with buckwheat seeds [34].

Reaction of Hemp Cultivars

It is interesting that large differences in response to plasma treatment were found among
three cultivars of hemp, within one plant taxon (species hemp, Cannabis sativa). It was the
first time when differences among various seed cultivars were tested in relation to non-
plasma pre-treatment. Previous comparative experiments with non-thermal plasma pre-

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treatment were conducted among species that were taxonomically different

[11, 14, 18, 20, 28].
The differences in sensitivity to plasma pre-sowing were obvious in all tested hemp
cultivars (Fig. 3). The cultivars can be sorted in ascending order of sensitivity as follows: cv.
Carmagnola, cv. Bialobzeskie, and cv. Finola. Seeds of both cv. ‘Finola’ and cv. ‘Bialo-
brzeskie’ hemp cultivars seem suitable for plasma pre-treatment by a gliding arc apparatus.
The best taxon of the tested hemp cultivars appeared to be cv. Finola, because it
germinated and grew after GlidArc pre-treatment better than the other cultivars and the
control sample. On the 5th day, the length and weight of cv. ‘Finola’ seedlings were 153
and 127% respectively (after 300 s plasma exposition). After DMP plasma pre-treatment,
the measured values for cv. ‘Finola’ were the least destructive in comparison with the other
tested cultivars.

Measured Variables

The length of seedlings may be caused by reaction to stress, therefore this plant parameter
may be badly evaluated [35]. Seedling accretions were calculated on the 4th and 5th days
as a difference between seedling lengths on the 3rd and 4th days and on the 4th and 5th
days respectively (Table 2). Therefore seedling accretion reflects seedling growth in time
better than the length only. The most important characteristics of early growing seeds were
a number of germinating seeds, accretion of seedlings and weight of biomass.
Plasma pre-sowing in cv. ‘Finola’ and cv. ‘Bialobrzeskie’ caused increasing the dry
weight of biomass in the range between 15–20 and 7–16% respectively (Table 2). Bigger
seedlings can be more successful in a short-term water deficit condition during the initial
stage of crop establishment on high water-holding capacity soils. Early growth and bigger
biomass of hemp seedlings connected with correct cultivation [6] may help to improve
seedlings development in temporarily dry conditions.
Seeds of industrial hemp are used to make hemp oil from fruit (and seed). Elderly fat
seeds germinate worse, because the fat content affects seed germination in older seeds.
Hemp seeds fall into the same category as fatty poppy seeds. In comparison to poppy seeds
[29], hemp seeds had comparable germination and early growth parameters after non
thermal plasma pre-treatment.

Benefits

Many scientists, companies and farmers show an increasing interest in investment, agri-
culture, processing and manufacturing of hemp [3, 6, 8, 10, http://news.bio-based.eu/
worldwide-growth-industrial-hemp-fibres-shivs-seed-oil-pharmaceuticals]. In Europe all
three investigated cultivars are discussed as important seed source of industrial hemp
[7, 9, 27]. The result showed that non-thermal plasma pre-sowing should be studied as a
new technology of seed pre-treatment in agricultural crops (Fig. 3). Plasma pre-treatment
may be used not only for seed stimulation, but probably against some diseases [36] and
contaminations [37, 38]. Using plasma pre-treatment is not connected with a chemical way,
so it may be used in non-conventional agriculture [29] and in fresh food [38, 39]. Due to
the potential use of non-thermal plasma pre-sowing, unconventional farming is very
interesting for the production of hemp. But according to experimental data (Tables 1, 2),
not all apparatuses and hemp cultivars are suitable for non-thermal pre-treatment. The fact,
that the effectiveness of different types of plasma apparatus for seed pre-treatment are
different, is very significant finding.

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Conclusion

This work confirms that the non-thermal plasma pre-sowing can be used as clear tech-
nology for seed stimulation. The results have bring two new findings. (1) Different
apparatus generated non-thermal plasma have big differences on hemp seed vitality
(germination and early growth). It is reasonable to attribute the positive effect of the GA
apparatus to the direct exposure to the discharge at atmospheric pressure. (2) Various
cultivars of hemp seeds have significant sensitivity to the non-thermal plasma pre-
treatment.

Acknowledgements We are thankful to prof. Petr Spatenka and prof. Eugen Hnatiuc for their lending the
both plasma apparatus. The preparation of the manuscript was funded by the both Faculty of Natural Science
Comenius University and Faculty of Education University of South Bohemia.

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