Use of Low-dose Gamma Radiation to Promote the Germination and Early Development in Seeds

Abstract

The study of the effect of low doses of ionizing radiation on the germination and initial growth of different seeds is a recent area of research, with gamma rays and X-rays receiving the most attention. The use of this type of energy can generate an increase in germination percentages, an increase in germination speed, and changes in the length and area of roots and shoots, which will depend both on intrinsic factors of the nature of the energy (dose, dose rate, energy, etc.) as well as aspects of the irradiated seeds (water content, sensitivity, etc.). In addition to morphological effects, radio-stimulation due to low doses of ionizing radiation (a phenomenon also described as radio-hormesis) generates changes at physiological, biochemical, metabolic, and molecular levels. Despite the evidence that has been accumulating, it is still necessary to deepen the knowledge about these phenomena in order to establish the use of ionizing radiation with the aim of using radio-stimulation as a real impact tool in the agroforestry sector.

1. Introduction

The mutagenic effect of ionizing energy has been widely studied, being the most commonly used physical agent to irradiate seeds (and other plant materials) in order to generate heritable mutations in plant breeding programs [1]. For this purpose, it is necessary to define the highest possible dose to induce high frequency mutations and, at the same time, the least negative effects dose, a concept named Lethal Dose 50 (LD50) [2]. However, in recent years, the non-mutagenic effect related to low doses (below LD50) of ionizing radiation (IR) is an area that has attracted special attention, a phenomenon known as radio-stimulation or radio-hormesis (from the Greek “hormaein”, which means to stimulate) [3]. The vast majority of plant foods are produced by crops that are propagated by seeds, but the germination process is highly vulnerable to external conditions, which can cause delayed and/or uneven germination or even weak seedlings, which will inevitably end up affecting the yield and production quality [4]. In search of alternatives to reduce the difficulties associated with seed quality, low doses of IR have begun to be applied to speed up the germination processes, increase seedling quality, and to improve tolerance to biotic and abiotic stressors [5].

Within the different types of IR (X-rays; alpha and beta particles; neutrons), the majority of research has been done using gamma radiation due to the safety and ease of operation of equipment such as Gammacell irradiators.

Gamma rays can engage with cell components like atoms or molecules. This interaction affects the structure and biochemical processes and, in this way, modifies the overall plant behavior. Seed germination, seedling growth, secondary metabolite synthesis, and biotic/abiotic stress responses are some of the main processes that are modified in response to gamma ray exposure [5, 6].

Considering the large amount of information accumulated to date on this topic, the purpose of this chapter is to summarize the most recent and more relevant results on the radio-stimulation of seeds.

2. Physical nature of IR and its interaction with plant tissues

To understand the stimulating effect of IR, it is necessary to recall basic aspects of atomic structure. All atoms are made up of protons and neutrons (forming the atomic nucleus) and electrons that “orbit” around the nucleus. When the nuclear forces that keep the protons and neutrons together are strong enough to overcome the electric repulsion between particles of the same charge (protons), the atom will remain stable. Conversely, an atom will be unstable when the number of neutrons exceeds a limit (z = 82), causing the excess energy to be released in the form of radioactivity to maintain the integrity of the nucleus [7]. The radioactivity emitted from the nucleus can be in the form of alpha particles, beta particles, gamma radiation, or a combination of the three. Alpha particles are composed of 2 protons and 2 neutrons (structurally equivalent to a positively charged Helium nucleus), while beta particles are electrons that come from the nucleus (the product of the transformation of a neutron into a proton and an electron). The formed electron is released from the nucleus as a result of the decay [8]. Due to the release of alpha and beta particles, a rearrangement occurs inside the nucleus, which in turn results in the release of electromagnetic energy or gamma radiation [9].

2.1 Interaction of IR with matter

Both alpha and beta particles, as well as gamma radiation, have the ability to interact with matter and deposit enough energy to “knock out” an electron and thereby ionize the matter [10]. This ability to alter the atomic structure (ionization) is what differentiates the effect of alpha particles, beta particles, and gamma radiation from the effect of UV light and other electromagnetic radiations (visible light, infrared, microwaves, etc.), since the latter are capable of modifying the behavior of matter but do not have enough energy to alter its structure [7]. To understand the differences between alpha particles, beta particles, and gamma radiation, it is necessary to define two main components that largely determine the ability of each of these to interact with matter and, consequently, modify its behavior: Penetrability and Linear Energy Transfer (LET). Penetrability refers to the ability of radiation to pass through matter, which is directly related to the mass and electric charge of the radiation. Alpha particles have a mass (2 protons +2 neutrons) and an electric charge of +2, which means that they are highly reactive particles and therefore have very low penetrability (they are blocked by a paper sheet) [8]. Beta particles, with a fraction of the mass of a proton and an electric charge of −1, penetrate material to a greater depth than alpha particles [9] and can be blocked by aluminum foil. Gamma radiation, due to its electromagnetic nature, does not have mass, and therefore it can penetrate deeply into matter. To block gamma radiation, dense materials such as concrete or lead are required [11]. Linear Energy Transfer (LET) refers to the energy dissipated per unit length along the tracks of the ionizing particles [12]. Gamma rays have low linear energy transfer (LET), while alpha particles have high linear energy transfer (LET) [13]. The radiation source, along with penetrability and linear energy transfer (LET), determines the amount of energy absorbed by matter or absorbed dose, which is expressed in Gy (gray), with 1 Gy equivalent to 1 Joule of energy absorbed per kilogram [14].

At the level of plant tissues or cells, the interaction of IR can occur in two ways. When the energy of the radiation is deposited directly onto macromolecules (DNA, membranes, etc.) [15], it generates “damage” that, depending on the dose, can become lethal [16, 17]. However, the main effect of IR on plant cells occurs indirectly and it is mediated by the ionization of the water molecule (radiolysis), which results in the production of Reactive Oxygen Species (ROS) [18, 19]. Most of the responses described below, related to the stimulating effects of low doses of IR applied to seeds, are ultimately mediated by the accumulation of ROS.

For more detailed information on the physical, physicochemical, and chemical processes triggered by the interaction of IR with the water molecule, please refer to [20, 21].

3. The effects of low doses of IR on germination, plant growth, and development

Before presenting reports on the effect of treating seeds with low doses of IR, it is important to emphasize that the range of what is called low doses of IR is species-specific and depends not only on the particular radio-sensitivity of each species but also on the type of radiation (alpha particles, beta particles, gamma rays, X-rays, etc.), the rate of dose (acute or chronic irradiation), the pre-treatment of the material to be irradiated (moisture content), and the ontogenic state of the irradiated material [22]. In this way, the stimulating dose of IR found in the literature can vary from less than 1 Gy to doses of more than 1 KGy [23, 24], which highlights the difficulty of establishing cross-sectional ranges of stimulating thresholds as these must be defined on a case-by-case basis. In view of the above, the data and results presented below aim only to compile the growing and recent information on radiation stimulation by IR in seeds and visualize the differential effect (stimulation or inhibition) that different IR treatments have on different plant species. A simplified scheme summarizing the overall seed radio-stimulation response to low doses of IR is presented in Figure 1.

Figure 1.

Overall radio-stimulation response of seed treated with low doses of ionizing radiation. Created with BioRender.com.

3.1 IR and effect on germination

The effects of low doses of IR on plants have been studied for several decades. These studies have been conducted on different species and varieties, applying IR to different structures (seeds or vegetative structures), under different systems (in vivo or in vitro), on different ploidy levels and/or ages of the organ or tissues and using different dose and dose/rate combinations [25]. All of these parameters make it difficult to define a standard protocol to apply to a given species or situation.

Despite the above, in literature, some authors have made efforts to define low doses like the one that falls between 5 and 20 Gy for seeds and 1 to 5 Gy for vegetative material [26]. These authors mention only stimulatory effects of low doses, on seeds of several crops, like Capsicum annuum, Arabidopsis thaliana, Phaseolus vulgaris, Cajanus cajan, Triticum sp. and in vegetative stages of Arabidopsis thaliana. Another study on tomato seeds [27] defines 150 Gy as a low dose that stimulates parameters like germination, fruit number, and total production up to 86%.

Nevertheless, it has been observed that doses ranging from 5 to 800 Gy of gamma radiation have had stimulatory effects on growth of dry seeds [28]. This wide range of possibly stimulating doses is related to seed radio-sensitivity, which in terms depends on genetic characteristics and on seed moisture content. Table 1 shows the latest works on the stimulation effect caused by IR.

Plant species	Stimulatory dose (Gy)	Reference
Sugarcane	4.7–5.7	[29]
Datura innoxia	5	[30]
Eucalyptus nitens	10	[31]
Hordeum vulgare	4–20	[32, 33, 34]
Cucumis sativus	50	[35]
Abelmoschus esculentus (seeds, seedlings)	50	[35]
Chenopodium quinoa	50	[36]
Vicia faba	<100	[37]
Lathyrus chrysanthus	50–150	[38]
Vigna unguiculata	100	[39]
Physalis peruviana	125–200	[40]
Abelmoschus esculentus	400	[41]
Sophora davidii	800	[42]

Table 1.

Low-dose IR on some seeds.

It has been reported repeatedly that low dose rate and/or low total dose gamma irradiation impact germination yield and seedling performance, acting like an actual priming treatment [14]. Due to this well-documented effect, efforts have been made to investigate the molecular mechanism activated in seeds as a response to this physical treatment. Doses lower than 100 Gy of gamma rays positively stimulated the germination index, seedling growth, primary root length, and fresh weight on A. thaliana seeds. In that work, 50 Gy was the dose that showed the maximal positive effect on all growth parameters [43].

Seed germination, vigor, and seedling growth in wild oat (Avena fatua L.) [44], garden cress (Lepidium sativum L.) [45], deadly nightshade (Atropa belladonna L.) [46], okra (Abelmoschus esculentus L. Monch.) [47], and rocket (Eruca vesicaria L. subsp. sativa) [48] have been stimulated by low-dose gamma rays. All these works provided cumulative evidence that small doses of γ-rays result in beneficial action in physically treated seeds. These works point to several mechanisms as responsible for the effect of low-dose gamma rays on seeds and early growth. Among the triggering factors of the response are the ROS produced, which act as signaling molecules to respond under stress conditions; increased enzymatic activity; nucleic acids and protein synthesis in treated seeds. These changes in metabolism could explain the boost in germination, break of dormancy, and plant development. Conversely, exposing seed to high doses of gamma rays has been demonstrated to alter protein synthesis, hormonal equilibrium, enzyme activity, and leaf gas, and water exchange [49].

3.2 IR and its effect on growth

There are reports showing that low gamma irradiation doses led to positive effects on growth and plant yield in tomato hybrids [50]. Nevertheless, El-Sayed et al. [51] reported that 12 krad (equivalent to 120 Gy) gamma rays increased plant height, yield, chlorophyll a and b and carotenoids in tomato hybrids. Studies on Jerusalem artichoke (Helianthus tuberosus) show a stimulatory effect at 5 Gy dose on plant height, number of branches, fresh and dry shoot weight [52].

The effect of gamma rays on plant growth and development is explained by cytological, genetical, biochemical, physiological, and morphogenetic changes in cells and tissues [53]. These changes are commonly reported as more vigorous vegetative growth [54], early maturity [55], and higher yield [56].

While a definitive explanation regarding positive impacts of low-dose gamma radiation remains elusive, researchers suggest a theory that these irradiation levels stimulate growth by altering the hormonal signaling network within plant cells. Besides, it is proposed that increased cells’ antioxidative capacity could allow better performance over stressful fluctuations in light intensity and temperature conditions [57]. Conversely, the growth suppression due to high doses of irradiation has been linked to cell cycle alterations in the G2/M phase, as well as several impairments across the entire genome [58].

Recent studies have shown that not only the dose is important but also the dose rate. Even when the final dose was the same, long-term exposure to gamma rays produced more free radicals than shorter exposure. When exposed for short periods, wheat shoot and root lengths showed minor decreases compared to control samples, however, longer periods resulted in substantial growth reduction. The expression of genes associated with antioxidants and DNA repair showed a reduction in response to long-term gamma ray exposure [59].

3.3 IR and seed priming

Seed priming corresponds to the induction of a particular physiological state through the application of treatments (physical, chemical, thermal, etc.) prior to germination, which allows the plant to better respond to the subsequent presence of abiotic and biotic stresses [60]. In recent years, priming (particularly at the seed level) has emerged as a strategy for stress management without significantly affecting plant development [61]. Many of the advances in understanding and elucidating the stimulatory effect of low doses of ionizing radiation have their origin in the study of priming through physical stimuli [14]. A study on the effect of low doses of gamma radiation and the induction of tolerance to stress caused by Cadmium and Lead in Arabidopsis seeds [43], reported that doses up to 100 Gy induced better germination rates and initial growth. It also demonstrated that doses of 50 Gy induced a better response of tolerance to stress caused by these metals, such as a decrease in the presence of H₂O₂, higher activity of antioxidant enzymes, and greater accumulation of proline compared to non-irradiated seeds. Similar results were observed when irradiating Hordeum vulgare seeds with doses up to 300 Gy, where those seedlings derived from seeds irradiated with 50 Gy improved their tolerance to the presence of heavy metals (lower contents of H₂O₂ and improvement in the ultrastructure of chloroplasts) [62]. Doses of 50 Gy applied to Arabidopsis seeds stimulated the tolerance of seedlings to thermal stress (improved growth rates, reduced ROS levels, higher antioxidant enzyme activity, etc.) [63], while exposure to 100 Gy applied to Vicia sativa L. seedlings (alone or in combination with salt and drought stress) generated significant increases in dry matter accumulation, higher antioxidant enzyme activity (CAT, SOD and APX), higher proline contents, and decreases in relative water content [6]. Doses between 500 and 1000 Gy decreased the incidence of fungal diseases in Pennisetum glaucum grains, and despite the high doses applied, no effects on the germination percentage of these grains were observed [64].

3.4 IR and metabolic effects

Due to its penetrability and linear energy transfer, as gamma radiation passes through different plant structures and tissues, it generates a broad spectrum of modifications affecting biochemical, physiological, and molecular processes. As the effect of IR is mainly mediated by the increase in ROS, a significant part of the literature focuses on describing the processes that are directly modified by these molecules, such as processes associated with photosynthesis or processes associated with the plant antioxidant system [26].

3.4.1 Effect on photosynthesis

IR affects various components of the photosynthetic apparatus, such as the content of pigments responsible for the absorption of visible radiation; enzymes responsible for CO2 reduction; thylakoids structure, etc. [26]. Regarding the chlorophyll content, various studies show contradictory trends or dynamics. Studies in Arabidopsis thaliana show that chlorophyll concentration remains stable up to doses of approximately 60 Gy [65], while similar doses (50 Gy) generated a significant increase in the total chlorophyll content in cowpea [66].

On the other hand, a recent study on soybean seeds and seedlings reported a positive relationship between the content of chlorophyll a, chlorophyll b, and carotenoids at a dose of 12 Gy when compared to non-irradiated seeds [67]. Another study suggests that, in general, the effect of IR on photosynthetic pigments follows a pattern of mild stimulation at low IR doses, while as the doses increase, the concentrations may initially increase but then drop in the long term [26]. The activity of the Rubisco enzyme, a central component of the CO2 fixation process, shows stimulation at low doses (5 and 25 Gy) in wheat seedlings [68], while higher doses decrease the specific activity of this enzyme in Arachis hypogaea L [69].

The radio-sensitivity studies of this enzyme are particularly complex since the different subunits that form this enzyme are encoded by both nuclear and plastidial genetic material [70]. However, despite the stimulatory effect of low doses of radiation on Rubisco activity, the effect of radiation on the rate of CO2 assimilation appears to be negative even at doses as low as 0.12 Gy [71] and 1.2 Gy [72], while higher doses have resulted in prolonged inhibition of the CO2 assimilation rate [68].

3.4.2 Antioxidant metabolism

The increase of ROS after exposure to IR has been thoroughly discussed. Seeds and plants subjected to IR show high levels of ROS that remain elevated for times ranging from several hours to several days post-irradiation. Since ROS are short-lived compounds, the increase of these components observed in plant tissues days or hours post-irradiation would not be the product of the direct process of radiolysis of water but rather the result of an imbalance between the processes of generation and use of these compounds [26]. In this way, it is expected that IR affects the content and/or activity of various enzymes involved in these processes. For example, irradiation with doses between 25 and 200 Gy stimulates the activity of ascorbate peroxidase and glutathione reductase enzymes in rice seeds [73], while peroxidase activity increased in orange seeds (doses of 10–50 Gy) [74] and in bean seeds (150 and 200 Gy) [75]. Irradiation of red pepper seeds with doses of 2 to 16 Gy resulted in an increase in superoxide dismutase activity but, at the same time, led to a decrease in glutathione reductase activity [57].

Several studies have established that ROS production mediated by IR is dose-dependent and follows a trend close to linearity [73, 76, 77, 78]. However, the existence of multiple pathways for ROS production and utilization (in response to biotic and abiotic stimuli) makes it very complex to identify or describe a specific pathway of response to IR mediated by ROS in plants [18]. Besides, these pathways are being influenced or affected differentially depending on the radiation dose [79].

3.4.3 Effect on secondary metabolism

One of the most widely studied processes is the stimulating effect of IR on secondary metabolism, and more specifically on metabolites with antioxidant capacity. It is postulated that the increase in antioxidant compounds would be triggered by the high concentration of ROS resulting from the radiolysis of water through two different pathways [80]. On one hand, the increase in ROS would stimulate the expression of genes that encode for key enzymes in the biosynthetic pathways of these compounds, as demonstrated by the results in Rosmarinus officinalis [81] and Arabidopsis thaliana [82]. On the other hand, studies on different medicinal plants show that the presence of ROS would directly stimulate the activity of these enzymes and, even more interestingly, demonstrate that the increase in activity of enzymes such as phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) is dose-dependent [83]. Solanum melongena seeds treated with 50 Gy gamma rays showed increased growth that led to increased levels of flavonoid and tannin contents in pulp, peel, and whole fruits [84].

3.4.4 Molecular response to low doses of IR

As detailed in the previous sections, the radio-stimulative effect of gamma radiation on seeds encompasses a wide range of processes and responses, ranging from promoting germination to modifications in plant fruiting. This diversity of effects has led to the absence, to date, of a single or consensus mechanism that molecularly modulates these responses [28]. Apart from the damage (repairable or unrepairable) that IR causes at the DNA level (associated with higher doses, which finally led to mutations), IR also causes modifications in the regulation of the genome in processes related to oxidative stress, signal transduction, transcription factors, hormone response, metabolism transport, energy, development and morphogenesis, and cell cycle [26, 82, 85]. Due to the extensive nature of a section that would describe the background information available in the literature regarding this topic (including plant species, radiation dose, acute vs. chronic exposure, etc.), Table 2 presents some relevant reports of genes that modify their expression in response to low doses of IR in seeds. For further information, the reader is encouraged to review [21, 26, 85, 90].

Gen name	Plant sp.	Dose	Function	Reference
Apetala 1 (AP1)	Arabidospis thaliana	3 cGy and 17 cGy (chronic exposure); 15 Gy (acute exposure)	Promote floral meristem identity	[86]
Ascobate peroxide (APX), Catalase (CAT), and Glutathione Reductase (GR)	Oryza sativa	25, 50, 100, and 200  Gy	Antioxidant defense	[73]
Heat Shock Protein 90 (HSP90)	Arabidopsis thaliana	100, 250, 500, and 750 Gy	Signal transduction, cell cycle, DNA repair, and stress response	[87]
Lipid Transfer Protein (EARLI 1)	Hordeum vulgare	15, 20, and 25 Gy	Germinability and early seedling development	[34]
Proliferating Cell Nuclear Antigen (PCNA)	Hordeum vulgare	1.6, 2.6, and 4.2 μSv/h	Cell cycle regulation	[77]
Superoxide Dismutase (SOD) and Guaiacol Peroxidase (GPOX)	Hordeum vulgare	2, 4, 6, 8, 10, 13, 16, 20, 25, and 50 Gy	Antioxidant defense	[33]
Suppressor of Gamma Response 1 (SOG1)	Arabidopsis thaliana	1 to 540 mGy/h	Master regulator of DNA damage response	[88]
Terpene Synthase 30 (TPS30)	Oryza sativa	100, 200, 300, 400, and 500 Gy	Secondary metabolism	[89]
X-Ray Repair Cross-Complementing Protein (XRCC)	Oryza sativa	100, 300, and 500 Gy	DNA damage response	[59]

Table 2.

Seed gene expression modified by IR.

4. Effect on germination and growth of other physical agents

Much of the research on the effects of physical agents on seed growth and development is carried out in self-shielded equipment with 60Co as a gamma radiation emitting source (i.e., Gammacell 220R). However, there are other types of physical agents not necessarily derived from nuclear reactions that have also been used to study their possible radio-stimulating effect.

4.1 X-rays

X-rays correspond to a non-nuclear IR of electromagnetic nature (similar to gamma rays) that has also been used to modify the behavior and development of various plant species. Regarding their use as a radio-stimulant agent, shorter germination times have been reported in Hibiscus [91], increased vigor of coffee seedlings [92], increased leaf area in Phaseolus plants [93], and increased leaf and plant size in Tomato [25] in response to doses ranging from 0.1 Gy to doses greater than 100 Gy.

4.2 Protons

It corresponds to corpuscular radiation (hydrogen nuclei) typical of extraterrestrial environments (part of solar particles). The study of the effect of protons on the growth and development of plant species is crucial to evaluating potential species to be used in space missions or settlements [94]. In this regard, there are reports of higher germination rates and increases in chlorophyll and ascorbate peroxidase (APX) contents in soybean plants irradiated with protons [95], as well as stimulation of seedling growth and greater plant height [96] and root elongation [97] in rice seeds irradiated with low doses of this type of ionizing energy.

4.3 Electron beam

Low-energy electron beam (LEEB) beta radiation consists of a beam of accelerated electrons. This form of ionizing radiation operates within a range spanning from a few up to around 300 kGy. The accelerated electrons display enough energy to remove electrons from atoms or molecules producing ions [98]. LEEB application to lentil seeds accelerated seed germination, defined by the percentage of hypocotyl and leaf emergence at 3 days [99]. There are also reports that low doses of this type of radiation, applied to barley seeds, induced higher germination rates [100] while it improved the height and weight in wheat plants [101].

4.4 Non-thermal plasma (NTP)

By increasing the internal energy of a material, it will go from solid to liquid to gas and finally to an ionized gas state (where electrons separate from the elements), giving rise to the fourth state of matter or “plasma”, which has unique properties [102]. Depending on the conditions (working pressure, type of energy, amount of energy) required for plasma formation, it will have different properties [103], with non-thermal plasma (NTP) being the most studied for inducing changes at the seed or seedling level because the low temperatures generated do not alter the behavior of the material subjected to such plasma. Since the effect depends not only on the dose or energy imparted or absorbed by the irradiated material but also on the conditions under which the NTP state is induced or reached, it is complex to summarize the results obtained (and beyond the scope of this article). As an example, a report from 2005 studied the effect on the growth and development of tomato plants obtained from seeds irradiated with NTP [104], reporting higher seedling emergence, greater antioxidant enzyme activity, and a higher number of fruits and fruit biomass per plant. Stimulating effects of different doses and configurations of NTP have also been reported in cereals [105], legumes [106, 107], oilseeds [108], vegetable crops [109, 110], among others.

An alternative to the direct exposure of plant material (usually seeds) to NTP is the exposure of water to this type of energy, resulting in what is known as plasma-activated water (PAW) or plasma-treated water (PTW), which induces several chemical and physical modifications resulting in different biological effects on plant material [111]. The response to PAW involves change in redox potential, conductivity, pH and ROS and reactive nitrogen species content [112]. By using this technology, Vigna radiata seeds exposed to different treatments with PAW (different time exposition of water to NTP) resulted in the maximization of parameters such as germination rate and growth parameters, as well as an increased content of flavonoids and total phenols in seedlings [113]. Wheat seeds treated with PAW resulted in better germination, faster seedling growth, higher photosynthetic pigments in leaves, and soluble protein content in roots [114].

5. Conclusions

According to statistics, 90% of edible crops are cultivated from seeds [5]. Low germination and poor seedling growth often result in huge crop losses and therefore, developing strategies aimed at improving processes related to seed germination and crop establishment is a primary way to ensure food security [4, 115]. The necessity to develop strategies applied to seeds that aim to improve these processes while being environmentally friendly has driven studies on the use of low doses of IR (mainly gamma radiation) as a stimulating agent, a phenomenon called radio-stimulation or radio-hormesis. In the last two decades, increasing evidence has accumulated of the radio-stimulatory effect of gamma radiation (a safe, non-polluting, and sustainable form of ionizing radiation) on seed germination and associated processes.

The range or dose limit in which the stimulatory effect of gamma radiation is observed depends on intrinsic factors of the applied energy (LET, dose, dose rate) as well as the irradiated material (species; tissue; state of development, etc.). This makes it difficult to use transversal concepts regarding dose limits, that can be called “low doses” and rather, the stimulatory effect must be studied and defined on a case-by-case basis.

In the same way, the physiological processes and metabolic responses that are modified as a result of the application of stimulating doses of IR are also diverse and include changes in hormonal balance, activation of antioxidant protection systems, modifications of parameters associated with photosynthesis, stimulation of secondary metabolites, etc. However, despite decades of research, the precise mechanism of beneficial plant response to IR remains elusive [28], although there seems to be a consensus that all responses are associated (directly or indirectly) with changes in the content of ROS species.

Recently, the use of new techniques such as ion beam and especially the use of NTP have emerged as cost-effective and non-polluting alternatives capable of stimulating germination and modifying the development of some plant species [4].

Despite the advances and studies on the stimulating effect of gamma radiation on seed germination and eventually on the growth and development of plants, the use of this technique is still mostly associated with scientific studies and little progress has been made on its operational use. Therefore, it is necessary to continue researching to elucidate the response and molecular pathways that modulate the interaction between the ionizing radiation and plant development with the aim of making use of radio-stimulation as a real impact tool in the agroforestry sector.

Acknowledgments

This work has been supported by the Grant FONDECYT-PAI 7818I20007 granted by Research & Development National Agency (ANID).