**3. Priming methods and priming agents**

Several methods of seed priming have been developed in order to invigorate seeds and alleviate the environmental stresses. A common feature of water-based priming techniques, which distinguishes them from other pre-sowing treatments, is partial seed pre-hydration and the activation of early germination events in seed. Priming efficiency is affected by many factors and strongly depends on treated plant species and chosen priming technique. Physical and chemical factors such as osmotica and water potential, priming agent, duration, temper‐ ature, presence or absence of light, aeration, and seed condition also influence priming success and determine germination rate and time, seedling vigor, and further plant development [15, 16].

#### **3.1. Hydropriming**

Hydropriming is the simplest method of seed priming, which relies on seed soaking in pure water and re-drying to original moisture content prior to sowing. No use of additional chemical substances as a priming agent makes this method a low-cost and environmentally friendly. The main disadvantage of hydropriming is uncontrolled water uptake by seeds. This is a consequence of free water availability to seeds during hydropriming, so that the rate of water uptake depends only on seed tissue affinity to water [17]. Moreover, this technique may result in unequal degree of seeds hydration thus leading to lack of simultaneous metabolic activation within seeds followed by unsynchronized emergence [18]. Considering these limiting factors, it is highly important to define accurate treatment duration, temperature, and water volume used in hydropriming to ensure desired level of seed hydration and to prevent radicle protrusion. Despite the aforementioned limitations, many reports indicated beneficial effect of hydropriming on seed germination and seedling growth under both optimal and stress conditions, in various crop plants such as chickpea, maize [19], wheat [20], Indian mustard [21], canola [22], sunflower [23], rice [24], mung bean [25], capsicum [26], and durum wheat [27].

One of the commercially used types of hydropriming is the system named "drum priming", patented in the early 1990s [28, 29]. In this technique, seeds are gently rotating in drum and gradually hydrated by addition of water in vapor form. Drum priming allows seed imbibition in a controlled manner and could be an attractive alternative to conventional hydropriming. Specially designed apparatus enables monitoring of the seed weight, precise regulation of time, and water amount during hydration process, what ultimately results in an appropriate and uniform moisture level of the seeds [30]. Drum priming with 24-epibrassinolide shows positive effect on germination time and seedling growth of bell pepper concomitant with improved superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX) activities [31]. Another variant of hydropriming, so-called "on-farm priming", consist of seed soaking in water followed by surface drying and subsequent sowing. The duration of treatment obligatorily cannot be longer than "safe limit" (maximum time of priming without risk of seed or seedling damage by premature germination) [32]. The positive impact of this method on crop emer‐ gence and yield was confirmed by Harris et al. [33]. On-farm priming is especially useful for resource-poor farmers in marginal tropical environment [34].

#### **3.2. Osmopriming**

compounds (including tyrosine and asparagine) before and during germination. The discov‐ ery of plant hormones in the 1920s underlined the crucial role of these compounds in seed desiccation tolerance, reserve mobilization, as well as cell division and cell elongation occur‐ ring during germination. The possibility to influence final germination as a consequence of pre-sowing treatment has led to a wide range of empirical methods for numerous cultivated

4 New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology

Several methods of seed priming have been developed in order to invigorate seeds and alleviate the environmental stresses. A common feature of water-based priming techniques, which distinguishes them from other pre-sowing treatments, is partial seed pre-hydration and the activation of early germination events in seed. Priming efficiency is affected by many factors and strongly depends on treated plant species and chosen priming technique. Physical and chemical factors such as osmotica and water potential, priming agent, duration, temper‐ ature, presence or absence of light, aeration, and seed condition also influence priming success and determine germination rate and time, seedling vigor, and further plant development [15,

Hydropriming is the simplest method of seed priming, which relies on seed soaking in pure water and re-drying to original moisture content prior to sowing. No use of additional chemical substances as a priming agent makes this method a low-cost and environmentally friendly. The main disadvantage of hydropriming is uncontrolled water uptake by seeds. This is a consequence of free water availability to seeds during hydropriming, so that the rate of water uptake depends only on seed tissue affinity to water [17]. Moreover, this technique may result in unequal degree of seeds hydration thus leading to lack of simultaneous metabolic activation within seeds followed by unsynchronized emergence [18]. Considering these limiting factors, it is highly important to define accurate treatment duration, temperature, and water volume used in hydropriming to ensure desired level of seed hydration and to prevent radicle protrusion. Despite the aforementioned limitations, many reports indicated beneficial effect of hydropriming on seed germination and seedling growth under both optimal and stress conditions, in various crop plants such as chickpea, maize [19], wheat [20], Indian mustard [21], canola [22], sunflower [23], rice [24], mung bean [25], capsicum [26], and durum wheat

One of the commercially used types of hydropriming is the system named "drum priming", patented in the early 1990s [28, 29]. In this technique, seeds are gently rotating in drum and gradually hydrated by addition of water in vapor form. Drum priming allows seed imbibition in a controlled manner and could be an attractive alternative to conventional hydropriming. Specially designed apparatus enables monitoring of the seed weight, precise regulation of time, and water amount during hydration process, what ultimately results in an appropriate and

plant species during the year 1970s [14].

16].

[27].

**3.1. Hydropriming**

**3. Priming methods and priming agents**

Osmopriming involves soaking seeds in osmotic solution with low water potential instead of pure water. Due to low water potential of osmotic solutions, water enters seed slowly which allows gradual seed imbibition and activation of early phases of germination but prevents radicle protrusion [35]. Usually water potential of priming agent varies from −1.0 down to −2.0 MPa [36]. However, values of water potential together with duration of the priming treatment should be always adjusted to species, cultivar, and sometimes seed lot. Different compounds are used in osmopriming procedure including polyethylene glycol (PEG), mannitol, sorbitol, glycerol, and inorganic salts such as NaCl, KCl, KNO3, K3PO4, KH2PO4, MgSO4, and CaCl2 [37]. Priming with salt solutions is often referred as "halopriming". Most common chemical employed in osmopriming treatment is PEG, mainly owing to its specific characteristic. Large molecular size of PEG prevents its penetration into the seed thus avoiding induction of potential cytotoxic effect and reduction of osmotic potential within seed [35]. Nevertheless, PEG exhibits some undesirable features including high viscosity, which restrict diffusion of oxygen in the solution so in PEG priming aeration system is preferred [2]. Seed priming with PEG has been shown as an effective method to improve seed germination, seedling emergence, and stress tolerance of several crop plants under unfavorable conditions such as salt, water, chilling, and nano-ZnO stresses [1, 3, 36–38].

#### **3.3. Solid matrix priming**

Solid matrix priming (SMP, matriconditioning), in which water uptake by seeds is controlled, has been developed as an alternative method to osmopriming because of high cost of osmotic agents and technical problems with aeration [2]. During solid matrix priming, seeds are mixed and incubated with wet solid water carrier for a certain period. Afterward, seeds are separated from matrix, rinsed, and back-dried. The use of solid medium allows seeds to hydrate slowly and simulates natural imbibition process occurring in the soil [18]. To successfully accomplish SMP, materials utilized as matrices should possess specific physical and chemical features such as low matrix potential, minimal water solubility, high water holding capacity and surface area, no toxicity to seeds, and ability to adhere to seed surface. In fact, vermiculite, peat moss, charcoal, sand, clay, and some commercially offered substrate such as Celie or Micro Cell are exemplary solid carries applied in solid matrix priming [2, 35]. In order to obtain the best priming performance, time of treatment and optimal water content must be determined separately for each matrix [39].

Positive effects of SMP on crop seeds have been noted in many reports. Solid matrix priming enhanced field performance of carrot [40] as well as improved germination and seed vigor of soybean [41]. Study on onion showed that matriconditioning improved seed germination rate, seedling emergence, and growth under optimal and low temperature conditions [42]. Sand priming increased the activities of antioxidant enzymes such as catalase (CAT), peroxidase (POX), and soluble sugar content in waxy maize concomitant with improved rate of germi‐ nation and seedling growth under high-salt stress conditions [43].

It is well established that integration of SMP with biological and chemical factors may greatly enhance seed performance [18]. Adoreoli and de Adnrade [44] indicated that inclusion of gibberellins/fungicide/*Bacillus subtilis* to matriconditioning leads to improved stand establish‐ ment and productivity of some vegetable crops under tropical conditions. Similarly, matri‐ conditioning with GA3 enhanced the quality of hot pepper seeds [45]. More recently published data demonstrated that solid matrix priming with *Trichoderma viride* improved seedling emergence and yield of okra under low temperatures [46].

#### **3.4. Hormopriming**

During hormopriming, seeds imbibition occurs in the presence of plant growth regulators, which can have direct impact on seed metabolism. The following regulators are commonly used for hormopriming: abscisic acid, auxins, gibberellins, kinetin, ethylene, polyamines, and salicylic acid (SA). Gibberellic acid (GA3) and PEG priming improved photosynthetic proper‐ ties, antioxidant system, seedling emergence, and growth of white clover on heavy metal polluted soil [47]. Priming spring wheat seeds with GA3 increased grain yield and salt tolerance by modulating hormone homeostasis together with alterations of ion uptake and accumulation between shoots and roots [48]. Enhanced salt tolerance, growth, and grain yield of wheat were also observed after kinetin-priming [49]. Among the different techniques of seed priming (hydro-, osmo-, and halopriming), spermidine pretreatment appeared to be the most effective method for induction drought tolerance in rice [50]. High efficiency of polyamines-priming on the improvement of rice tolerance to drought has been demonstrated also by Farooq et al. [51]. Critical role of phytohormones exogenously supplied into seeds for plant response to salinity stress was stated in wheat seeds primed with ascorbic acid and salicylic acid, as this pretreat‐ ment method increases the ability of wheat to grow successfully under salt stress, whereas hormonal priming with ABA was not effective in this case [52].

#### **3.5. Biopriming**

Biopriming involves seed imbibition together with bacterial inoculation of seed [53]. As other priming method, this treatment increases rate and uniformity of germination, but additionally protects seeds against the soil and seed-borne pathogens. Hydration of seeds infected with pathogens during priming can result in a stronger microbial growth and consequently impairment of plant health. However, applying antagonistic microorganisms during priming is an ecological approach to overcome this problem [54]. Moreover, some bacteria used as biocontrol agents are able to colonize rhizosphere and support plant in both direct and indirect way after germination stage [55]. It was found that biopriming is a much more effective approach to disease management than other techniques such as pelleting and film coating [56]. Nowadays, the use of biopriming with plant growth-promoting bacteria (PGPB) as an integral component of agricultural practice shows great promise [57, 58]. In pearl millet, biopriming with *Pseudomonas fluorescens* isolates enhanced plant growth and resistance against downy mildew disease [59]. Biopriming with rhizobacteria improved germination parameters of radish seeds under saline conditions [60].

#### **3.6. Others**

priming performance, time of treatment and optimal water content must be determined

Positive effects of SMP on crop seeds have been noted in many reports. Solid matrix priming enhanced field performance of carrot [40] as well as improved germination and seed vigor of soybean [41]. Study on onion showed that matriconditioning improved seed germination rate, seedling emergence, and growth under optimal and low temperature conditions [42]. Sand priming increased the activities of antioxidant enzymes such as catalase (CAT), peroxidase (POX), and soluble sugar content in waxy maize concomitant with improved rate of germi‐

It is well established that integration of SMP with biological and chemical factors may greatly enhance seed performance [18]. Adoreoli and de Adnrade [44] indicated that inclusion of gibberellins/fungicide/*Bacillus subtilis* to matriconditioning leads to improved stand establish‐ ment and productivity of some vegetable crops under tropical conditions. Similarly, matri‐ conditioning with GA3 enhanced the quality of hot pepper seeds [45]. More recently published data demonstrated that solid matrix priming with *Trichoderma viride* improved seedling

During hormopriming, seeds imbibition occurs in the presence of plant growth regulators, which can have direct impact on seed metabolism. The following regulators are commonly used for hormopriming: abscisic acid, auxins, gibberellins, kinetin, ethylene, polyamines, and salicylic acid (SA). Gibberellic acid (GA3) and PEG priming improved photosynthetic proper‐ ties, antioxidant system, seedling emergence, and growth of white clover on heavy metal polluted soil [47]. Priming spring wheat seeds with GA3 increased grain yield and salt tolerance by modulating hormone homeostasis together with alterations of ion uptake and accumulation between shoots and roots [48]. Enhanced salt tolerance, growth, and grain yield of wheat were also observed after kinetin-priming [49]. Among the different techniques of seed priming (hydro-, osmo-, and halopriming), spermidine pretreatment appeared to be the most effective method for induction drought tolerance in rice [50]. High efficiency of polyamines-priming on the improvement of rice tolerance to drought has been demonstrated also by Farooq et al. [51]. Critical role of phytohormones exogenously supplied into seeds for plant response to salinity stress was stated in wheat seeds primed with ascorbic acid and salicylic acid, as this pretreat‐ ment method increases the ability of wheat to grow successfully under salt stress, whereas

Biopriming involves seed imbibition together with bacterial inoculation of seed [53]. As other priming method, this treatment increases rate and uniformity of germination, but additionally protects seeds against the soil and seed-borne pathogens. Hydration of seeds infected with pathogens during priming can result in a stronger microbial growth and consequently impairment of plant health. However, applying antagonistic microorganisms during priming

nation and seedling growth under high-salt stress conditions [43].

6 New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology

emergence and yield of okra under low temperatures [46].

hormonal priming with ABA was not effective in this case [52].

separately for each matrix [39].

**3.4. Hormopriming**

**3.5. Biopriming**

Chemical priming refers to seed treatment with different chemical solutions used as priming agents. This approach includes priming with wide range of both natural and synthetic compounds such as antioxidants (ascorbic acid, glutathione, tocopherol, melatonin, and proline), hydrogen peroxide, sodium nitroprusside, urea, thiourea, mannose, selenium, chitosan, fungicide etc. Positive impact of chemical priming with various priming agents in a wide range of environmental conditions was indicated by numerous studies [26, 61–64]. Seed priming with β-amino butyric acid increased drought and salt tolerance of green gram [65]. Application of ascorbic acid as a seed priming agent induced drought and salt resistance of wheat [66, 67]. Analysis conducted by Fercha et al. [67] revealed that priming with ascorbate counteracts the negative effects of salinity stress by changes in abundance of proteins involved in metabolism, protein destination, and storage.

Nutripriming is a technique in which seeds are soaked with solutions containing the limiting nutrient instead of pure water. The idea of this method is to obtain nutritional effect together with biochemical advantages of priming in order to improve seed quality, germination parameters, and seedling establishment [68]. Seed priming with Zn improved productivity of chickpea and wheat [69], germination and early seedling growth of rice [70], development and root growth of maize seedling exposed to low root zone temperatures [71], while K-priming brought favorable effect on growth and nutrient status of cotton seedling under saline conditions [72]. Some nutripriming techniques are commonly used by seed companies in the process of seed production and preparation for growers. One of this methods, broad spectrum nutrient seed priming (BSN), is based on imbibing seeds in mixture of minerals, such as zinc, copper, manganese, molybdenum, and phosphorus, which has been proved to fertilize the seed and provides the nutrients for early growth, which positively affects germination, seedling vigor, and root system development (http://seedprimer.com/).
