Cold plasma poration and corrugation of pumpkin seed coats

Abstract

The treatment of seeds and plants by electrically generated cold atmospheric pressure plasma can accelerate seed germination and radicle growing rates. The plasma generated reactive oxygen and nitrogen species, UV photons, and high frequency electromagnetic fields can penetrate into seed coats and modify their surface properties. Atomic force microscope data shows that cold helium or argon plasma induces strong corrugation of pumpkin seed coats, produces pores and surface defects. These structural deformations and poration enhance water uptake by seeds during the imbibing process, accelerate seeds germination, and increase seed growth. The cold atmospheric pressure plasmas treatment of pumpkin seeds also decreases the apparent contact angle between a water drop and the seed surface, thereby improving the wetting properties of seeds surfaces. Magnetic resonance imaging studies show acceleration of water uptake in pumpkin seeds exposed to a cold plasma jet. Reactive nitrogen and oxygen species, high frequency electromagnetic fields and photons emitted by the plasma jets accelerate germination of pumpkin seeds both independently and synergistically. These results show that cold plasma can be used in agriculture for acceleration of seed germination, increasing growth of plants seedlings, poration and corrugation of the bio-tissue surfaces.

Introduction

Cold atmospheric-pressure plasma (CAPP) is a partly ionized gas operated at atmospheric pressures, consisting of charged particles (electrons, ions) and neutral particles (atoms, molecules) as well as photons. CAPPs typically have low gas temperatures around 300 K, but high electron temperatures or energies of several electron-volts (1 electron-volt = 11,600 K). CAPPs produce various atomic or molecular species if interacting with molecular gases such as air. For example, CAPP in air can produce reactive oxygen and nitrogen species (RONS) including NO_x, OH, O, and O₃. These CAPP products lead to the activation of surface modifying processes [[1], [2], [3], [4]]. There has been an increased number of applications of CAPP in agriculture and plant biology in the last two decades, so much that the application of cold plasma in agriculture for treatments of seeds, plants and fruits is now called plasma agriculture [[5], [6], [7], [8], [9], [10], [11], [12], [13], [14]].

It is known that reactive oxygen species (ROS) participate in plant developmental processes by acting as signaling molecules for cell proliferation and differentiation, programmed cell death, seed germination, gravitropism, root hair growth, pollen tube development, and senescence [[15], [16], [17]]. However, the specific mechanisms responsible for the regulatory action of ROS at various plant developmental stages remain largely unknown. RONS produced by CAPP in atmospheric air appear to be the primary cause of activation of phytosensors and phytoactuators in plants and seeds [[18], [19], [20]]. Some of these RONS are known to be signaling molecules, which control plants' developmental processes [[21], [22], [23]] as well as plant cell death [24].

For seeds, which are the focus of this study, CAPP have been shown to disinfect and sterilize the surfaces of seeds by RONS and UV radiation, which can increase the percentage of germinated seeds [3,7,25]. The plasma can also increase the seed germination rate and speed-up plant growth. Typical seed germination is triggered by absorption of water which leads to activation of metabolism, ion channels, and other processes [[26], [27], [28]]. It is also known CAPP can modify the wettability of plant seeds such as lentils, beans, and wheat [29]. As seed germination is dependent on water absorption, the effect of plasma on the surface characteristics and structure, thus wettability, of the seeds are important and the focus of this paper.

In this work, we studied the effect of CAPP on the surface characteristics of pumpkin seeds. The objective was to determine how plasma modifies the seed surface to encourage water absorption and thus germination and growth. We selected pumpkin seeds because of their large surface area and economic importance in agriculture and medicine [30]. The pumpkin plant has been used for its medicinal and nutritious benefits for centuries. Pumpkin seeds have beneficial effects on prostate glands, blood glucose level, cholesterol, learning disabilities, immunity, liver, bladder, parasite inhibition, depression and cholesterol [30,31]. Electrophysiology and electrical signaling in pumpkin plants were analyzed by Vodeneev et al. [32]. Work has been done to study the enzymes and voltage gated ion channels in germinating pumpkin seeds [[26], [27], [28],33]. In Cucurbita pepo L., cv. Cinderella imbibed seeds, Volkov et al. [28] found the presence of resistors with memory known as memristors. The analysis was based on cyclic voltammetry where a memristor should manifest itself as a nonlinear two-terminal electrical element, which exhibits a pinched hysteresis loop on a current-voltage plane for any bipolar cyclic voltage input signal. Dormant pumpkin seeds have very high electrical resistance without memristive properties [28]. The dormant state helps pumpkin seeds to conserve energy. Seed dormancy is caused by endogenous characteristics of the embryo. The electrostimulation by bipolar sinusoidal or triangular periodic waves induces electrical responses in imbibed pumpkin seeds with fingerprints of memristors. Tetraethylammonium chloride, an inhibitor of voltage gated K⁺ channels, transforms a memristor to a resistor in imbibed pumpkin seeds. There is an electrophysiological difference between imbibed and dormant pumpkin seeds: absorption of water by pumpkin seeds activates voltage gated K⁺-channels and decreases electrical resistance inside seeds. The discovery of memristors in pumpkin seeds created a new direction in the understanding of electrophysiological phenomena in germinated seeds.

CAPP has also been used in medicine for transdermal drug delivery due to poration of the human skin [[34], [35], [36], [37]]. Poration is the formation of pores in a surface, or a pattern of such pores. The cause may be that the magnitude of the electric fields generated by plasma could exceed the threshold for electroporation. When applied to seed surfaces, CAPP may also induce poration and/or corrugation of the dormant pumpkin seed to improve germination and water imbibition due to intracellular penetration of electric fields and RONS.