Plant Cell Rep DOI 10.1007/s00299-015-1784-y

REVIEW

Seed priming: state of the art and new perspectives S. Paparella1 • S. S. Arau´jo2,3 • G. Rossi4 • M. Wijayasinghe4 D. Carbonera1 • Alma Balestrazzi1

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Received: 6 February 2015 / Revised: 15 March 2015 / Accepted: 16 March 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Priming applied to commercial seed lots is widely used by seed technologists to enhance seed vigour in terms of germination potential and increased stress tolerance. Priming can be also valuable to seed bank operators who need improved protocols of ex situ conservation of germplasm collections (crop and native species). Depending on plant species, seed morphology and physiology, different priming treatments can be applied, all of them triggering the so-called ‘pre-germinative metabolism’. This physiological process takes place during early seed imbibition and includes the seed repair response (activation of DNA repair pathways and antioxidant mechanisms), essential to preserve genome integrity, ensuring proper germination and seedling development. The review provides an overview of priming technology, describing the range of physical–chemical and biological treatments currently available. Optimised priming protocols can be designed using the ‘hydrotime concept’ analysis which provides the theoretical bases for assessing the relationship between water potential and germination rate. Despite the Communicated by N. Stewart. & Alma Balestrazzi [email protected] 1

Department of Biology and Biotechnology ‘L. Spallanzani’, via Ferrata 1, 27100 Pavia, Italy

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Plant Cell Biotechnology Laboratory, Instituto de Technologia Quı´mica e Biolo´gica Anto´nio Xavier, Universidade Nova de Lisboa (ITQB-UNL), Avenida da Repu´blica, EAN, 2780-157 Oeiras, Portugal

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Present Address: Department of Biology and Biotechnology ‘L. Spallanzani’, via Ferrata 1, 27100 Pavia, Italy

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Department of Earth and Environmental Sciences, via S. Epifanio 14, 27100 Pavia, Italy

efforts so far reported to further improve seed priming, novel ideas and cutting-edge investigations need to be brought into this technological sector of agri-seed industry. Multidisciplinary translational research combining digital, bioinformatic and molecular tools will significantly contribute to expand the range of priming applications to other relevant commercial sectors, e.g. the native seed market. Keywords DNA repair  Pre-germinative metabolism  Priming  Reactive oxygen species  Seed vigour

Introduction The need for increased seed quality has become a priority necessary to face the current demand for high standards in the agricultural market. Achieving rapid and uniform seedling emergence is a key point for crop performance since slow germination rates frequently expose plantlets to adverse environmental conditions and soil-borne diseases (Osburn and Schroth 1989). ‘Priming’ is a well-established treatment for enhancing seed quality. Seeds subjected to priming show increased germination rates which result in high levels of biotic/abiotic stress resistance and crop yields. All these features that improve product competitiveness directly correlate to seed vigour, a complex agronomic trait controlled by multiple genetic and environmental factors (Rajjou et al. 2012; Jisha et al. 2013). Priming is a water-based technique that allows controlled seed rehydration to trigger the metabolic processes normally activated during the early phase of germination (‘pre-germinative metabolism’), but preventing the seed transition towards full germination. Thus, priming treatment must be stopped before loss of desiccation tolerance occurs. The benefits of seed priming have been widely

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reported. Along with synchronous and fast emergence, primed seeds show reduced photo- and thermo-dormancy, a wider range of germination temperatures and better capacity to compete with weeds and pathogens (Ellis et al. 1988; Hill et al. 2008). Moreover, the use of priming permits growers to efficiently manage water usage and harvest scheduling (Hill et al. 2008). The success of seed priming is strongly correlated to plant species/genotype and physiology, seed lot and vigour, as well as to the priming method applied (Parera and Cantliffe 1994). Priming is routinely used to treat vegetables seeds, particularly carrot (Daucus carota L.), leek and onion (Genus Allium) (Dearman et al. 1987), celery (Apium graveolens L.), lettuce (Lactuca sativa L.), endive (Cichorium endivia L.), pepper (Genus Capsicum) and tomato (Solanum lycopersicum L.) (Parera and Cantliffe 1994; Di Girolamo and Barbanti 2012). Priming contributes to enhance product quality in the flower seed industry where it is frequently used on elite varieties of Petunia hybrida L. (Di Girolamo and Barbanti 2012, Momin 2013). Herbs, e.g. Rosmarinus officinalis L. and Salvia splendens L., also benefit from seed priming (Di Girolamo and Barbanti 2012). Largescale routine priming of commercial cereal seeds is more difficult to achieve, although advantages are well known (Murungu et al. 2004). Furthermore, priming provides optimal conditions to accelerate seed germination when requested by the cereal grain and brewing industry (Yaldagard et al. 2008). In most cases, the priming procedures commercially used are proprietary and managed by specialised agri-seed companies. A representative list of patented seed priming treatments, currently available in the market, is shown in Table 1. Some of the reported priming treatments (e.g. EasyPrime, EmergisÒ, PROMOTORTM) target horticultural species, ensuring improved seed germination, faster and more uniform seedling emergence, and

reduced frequency of abnormal plantlets. Other technological products (e.g. EasyDormex, ThermocureTM, SPLITKOTEÒSPECIAL) specifically address the issues of seed thermo- and photo-dormancy, while in some cases (e.g. AdvantageÒ and XbeetÒ) the technology has been developed to improve seed germination/seedling emergence of elite vegetables under stress conditions (Table 1).

A brief history of seed priming Attempts to improve seed germination date back to ancient Greeks as reviewed by Everari (1984) in his historical description. Theophrastus (371–287 B.C.) investigated the seed response during germination, observing that cucumber (Cucumis sativus L.) seeds soaked in water prior to sowing resulted into faster germination (Theophrastus, Enquiry into Plants, Book VII, I.6). The Roman naturalist Gaius Plinius Secundus (A.D. 23–79), also named Pliny the Elder, collected most of the knowledge on seed physiology in his Encyclopaedia, the Naturalis Historia. In his work, he refers to the relevance of presoaking cucumber seeds in water and honey to improve germination (Gaius 1949– 1954). Subsequently, the French agronomist and botanist Oliver de Serres (1539–1619) described the effectiveness of the treatment used by farmers on grains (Triticum, Secale and Ordeum spp.), in which seeds were soaked for 2 days in manure water and then dried in the shade before sowing. Charles Darwin (1809–1882) focused on seed germination as a part of his interest on plant development (Darwin 1855a, b). He tested osmopriming conditions by submerging cress (Lepidium sativum L.) and lettuce seeds in salty sea water and demonstrated that the treatment was able to enhance germination (Darwin 1855c, d). Later on, these issues were afforded by Ells (1963) who highlighted

Table 1 List of some patented seed priming treatments commercially available Patented protocol (trading name)

Company

Description and target plants

EasyPrime

ATLAS s.r.l. (Italy)

Priming method to improve seed germination (faster, uniform, reduced abnormal seedlings). Targets: tomato, pepper, eggplant, melon, leek, Brassica

Germains Seed Technology (United Kingdom)

Priming technology for safer crop emergence, earlier plant establishment, improved root shape/size and increased stress tolerance/yield. Target: sugar beet

EasyDormex

Priming method to remove seed thermo- and photo-dormancy. Targets: lettuce, endive AdvantageÒ XbeetÒ

Improved speed germination, promotes uniform emergence and stronger plant establishment. Targets: all vegetables, flowers, herbs

EmergisÒ ThermocureTM SPLITKOTEÒSPECIAL PROMOTORTM IMPROVERTM

INCOTEC Europe BV (The Netherlands)

Priming method to remove seed thermo-dormancy. Targets: lettuce Priming method to alleviate seed photo-dormancy in photo-sensitive varieties, increase temperature tolerance, improve germination to obtain uniform seedling establishment. Target: lettuce, endive, escarole, radicchio Priming method to improve germination efficiency/uniformity under stress conditions. Targets: onion, carrot, tomato, Brassica Primed seeds are selected based on the X-Ray image of seed interior. Targets: tomato

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some key points in seed treatment, paving the way to the modern concept of seed priming. He noticed that tomato seeds treated with a nutrient solution showed increased germination rate, while May et al. (1962) observed that seeds dried at different time points following imbibition resulted in quicker germination. Measurement of the germination process is extremely complex as several mathematical models have been used until now, often with contrasting results and interpretations. However, Ranal and Garcia de Santana (2006) have provided a comprehensive review which describes germination rate (expressed using the coefficient of velocity or the reciprocal of the average germination time) as the fundamental parameter. According to International Seed Testing Association (ISTA), seed vigour is defined as ‘the sum total of those properties of the seed which determine the level of activity and performance of the seed or seed lot during germination and seedling emergence’ (ISTA 1995). At that time, an increasing number of reports indicated that ‘imbibing and drying’ pretreatments, in some cases named ‘hardening’ or ‘advancing’, exerted beneficial effects on seed germination in a wide range of horticultural and cereal species, particularly under stress conditions (Austin et al. 1969; Hegarty 1970; Berrie and Drennan 1971). In the attempt to improve germination efficiency, the controlled imbibition carried out with defined amounts of water was replaced by treatments with salt solutions, a condition able to promote RNA accumulation according to Koehler (1967). Subsequently, Heydecker et al. (1973) used the high molecular weight polyethylene glycol (PEG), H–(O–CH2–CH2)n–OH, for priming to avoid some constrains associated with ‘hardening’ and treatments with salts. These osmotic treatments, in which different combinations of osmotic potential and temperature were tested, produced an increase in germination percentage of onion (Allium cepa L.) seeds and also the uniformity of germination was positively affected. Since then, seed priming has been adopted by the agri-seed companies as a routine tool to improve seed vigour.

Pre-germinative metabolism and seed priming At the physiological level, the positive effects of priming are due to specific metabolic changes induced in the seed when water up-take starts (Osborne 1983; Dell’Aquila and Taranto 1986; Dell’Aquila and Bewlwy 1989; Bray et al. 1989; Bray 1995). As a consequence of rehydration, main cellular processes are triggered, such as the de-novo synthesis of nucleic acids and proteins, ATP production, accumulation of sterols and phospholipids, activation of DNA repair and antioxidant mechanisms (‘pre-germinative metabolism’). Up-to-date knowledge on the metabolic dynamics that accompany the transition from the quiescent

state of dry seeds to the active proliferating state of germinating seeds/seedlings has been provided at different levels using ‘omics’ tools, e.g. proteomics (Tan et al. 2013; Galland et al. 2014). Within this context, the seed repair response plays a key role in preserving seed vigour (Rajjou and Debeaujon 2008; Oge` et al. 2008). Seeds are exposed to environmental stresses not only during maturation on the mother plant and post-dispersal storage but also during the early phase of germination, and these conditions lead to extensive oxidative damage of lipids, proteins, and nucleic acids (Kranner et al. 2010; Ventura et al. 2012). DNA repair functions must be kept at proper levels in the embryo to preserve seed vigour and increase the chances of successful germination. When DNA damage is properly repaired, embryo cells resume cell cycle progression and undergo DNA replication but when repair mechanisms are defective, oxidative injury leads to cell death (Waterworth et al. 2010, 2011; Kranner et al. 2010; Balestrazzi et al. 2011a; Ventura et al. 2012). DNA repair is a key component of the ‘pre-germinative metabolism’ activated when water up-take starts and throughout imbibition, accompanied by uncontrolled ROS accumulation. All the main DNA repair pathways (e.g. Base- and Nucleotide-Excision Repair, BER and NER) are activated during the early phase of seed imbibition to maintain genome integrity (Macovei et al. 2010, 2011a, b; Balestrazzi et al. 2011a, 2012; Chen et al. 2012; Co´rdobaCan˜ero et al. 2014). Transcription Coupled-NucleotideExcision Repair (TC-NER), a subpathway which specifically recognises and removes lesions from the transcribed strands of transcriptionally active genes (Shuck et al. 2008), has been investigated during seed germination in rice (Oryza sativa L.) by Macovei et al. (2014) who highlighted the requirement for DNA helicases to facilitate proper unwinding of the DNA duplex during transcription. Furthermore, microRNAs targeting helicases in the context of the DNA damage response and repair in rice seeds/ seedlings have been also investigated under gamma rayinduced oxidative stress conditions by Macovei and Tuteja (2013), expanding the current knowledge on the seed response to treatments mediated by ionising radiation or ‘physical vigorization’ (Grover and Khan 2014). Proper DNA repair relies on the efficient ligase-mediated rejoining of broken strands and DNA ligase VI has been identified as the major determinant of seed longevity in Arabidopsis (Waterworth et al. 2010). Reactive oxygen species (ROS) play key roles in the regulation of seed germination but the molecular events underlying their function as signalling molecules still need to be fully elucidated (Diaz-Vivancos et al. 2013). It has been suggested that short-lived radicals would act at the site of their production, while long-lived ROS would be translocated to distant sites (Moller et al. 2007). Several components of the

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ROS-mediated signalling pathways, e.g. MAP kinases and calcium-binding proteins (Foyer and Noctor 2005), have been so far characterised in plants but their role in seeds still deserves investigation. On the other hand, ROS react with biomolecules resulting into severe oxidative damage. DNA lesions can be removed by specific repair functions but RNA is highly sensitive to ROS-mediated oxidative injury, due to the absence of a dedicated repair mechanism while damage at the protein level can be both reversible (e.g. cysteine and methionine oxidation) and irreversible (e.g. in case of carbonylation) (El-Maarouf-Bouteau et al. 2013). The increased activity of antioxidant enzymes allows to control ROS accumulation during water uptake (Bailly et al. 2000; McDonald 2000; Hsu et al. 2003). The seed activity in terms of ROS scavenging is expressed as the seed antioxidant potential, a critical requirement to withstand stress and improve germination (Liu et al. 2007). The seed antioxidant response can be evaluated by monitoring the expression profiles of genes encoding antioxidant enzymes such as Superoxide Dismutase (SOD, EC 1.15.1.1) involved in the early scavenging of superoxide radicals (Yao et al. 2012). Similarly, the antioxidant functions Ascorbate Peroxidase (APX, EC 1.11.1.11), Catalase (CAT, EC 1.11.1.6) and Glutathione Reductase (GR, EC 1.8.1.7) were enhanced at transcript and protein/enzyme activity level, indicating the activation of the antioxidant defence (Macovei et al. 2014; Chen et al. 2014). Other useful indicators of the antioxidant response in germinating seeds are metallothionein (MT) genes encoding different isoforms of the ROS scavenger protein metallothionein (Balestrazzi et al. 2011b; Leszczyszyn et al. 2013). The MT2 gene, encoding a type 2 MT typically induced by oxidative stress, was up-regulated in Silene spp. during seed rehydration (Dona` et al. 2013), while Zhou et al. (2012) demonstrated that transgenic Arabidopsis seeds overexpressing the NnMT2a and NnMT3 genes from sacred lotus (Nelumbo nucifera Gaertn.) showed improved resistance to accelerated ageing. Improved germination rates under salt stress were reported in Arabidopsis seeds overexpressing the rice rgMT gene (Jin et al. 2014). All these protective functions are activated during priming treatments which allow seeds to undergo through the main physiologic and metabolic changes typical of the pre-germinative phase up to the first cell division, paving the way to increased germination and enhanced seedling growth rate once the seeds will be sowed.

An overview of priming techniques Hydropriming During hydropriming, seeds are soaked in water under optimal temperature conditions (usually in a range from 5

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to 20 °C). Seeds are submerged in water with or without aeration, an additional parameter that in some cases improves germination. This process is especially useful in those agricultural areas where crop cultivation is impaired by adverse climate conditions, e.g. drought, and it is required to improve water uptake efficiency minimising exposure to agri-chemicals (McDonald 2000). Distilled water is used to achieve partial seed hydration corresponding to 10–20 % of full imbibition (Pill 1995). Hydropriming results into non-controlled water up-take, since the process depends on seed affinity to water and the main critical point is to find and maintain optimal temperature and humidity conditions to avoid radicle protrusion (Taylor et al. 1998). Another limiting factor of hydropriming is the lack of homogeneous seed hydration which can lead to uneven germination (McDonald 2000). The main variant of hydropriming is ‘drum priming’, patented by Rowse (1991) in which a drum that contains seeds is connected with a boiler generating vapour. Once inside the drum, the vapour condenses into liquid water. The machine is able to measure the increase in seed relative mass during the treatment. The time and water volume required to complete seed rehydration are strictly controlled to reach gradual and even seed imbibition (Warren et al. 1997). Hydropriming is the most ancient type of priming, since the benefits of this presowing treatment have been known for a long time, however it is now applied less frequently in comparison with other methods. Osmopriming Osmopriming (‘osmotic priming’, ‘osmotic conditioning’) is a wide-spread pre-sowing procedure that involves treatment with osmotic solutions at low water potential facilitating the control of water uptake. During imbibition, water entry into the seed associates with progressive ROS accumulation and oxidative damage of cellular components (lipid membranes, proteins, nucleic acids). The main goal of osmopriming is to limit the ROS-mediated oxidative injury by delaying water entry. Thus, the water potential of the osmotic agent is a crucial parameter (Heydecker and Coolbear 1977; Taylor et al. 1998). The osmotic agent mainly used is PEG, a polyether compound with several industrial applications, commercially available with a wide range of molecular weights. The chemical features of PEG are recognised as extremely useful in pre-sowing treatments since the inert nature of the molecule avoids cytotoxic effects and the large molecular size (6000–8000 Daltons) prevents absorption of the molecule into the seed (Michel and Kaufman 1973; Heydecker and Coolbear 1977). Despite these favourable features, PEG shows some disadvantage when used in bulk, due to high costs and extremely high viscosity which limits oxygen transfer

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within the solution. To date, all these technical constraints can be overcome using effective aeration systems (Bujalski and Nienow 1991). Other compounds are currently used for osmopriming, e.g. inorganic salts of sodium, potassium and magnesium (most commonly NaCl, NaNO3, MnSO4, MgCl2, K3PO4 and KNO3) (‘halopriming’), and organic molecules (e.g. glycerol and mannitol) that are less expensive, easier to aerate and remove compared to PEG. Priming with these compounds can either be effective as the PEG-based treatment or it can trigger considerable different responses, depending on seed morphology. The presence of a semi-permeable outer layer found in certain seeds is the main structural feature influencing priming effectiveness (Pill 1995). This outer layer, consisting of amorphous tissue placed between pericarp and seed coat, can limit or avoid ion/solute exchange, therefore controlling seed permeability to water and priming agents (Zhou et al. 2013). If the osmotic agent is not properly chosen in relation to permeability of the treated seeds, ions released from salts in the priming solution can easily penetrate the seed, disrupting the endogenous osmotic equilibrium. Uncontrolled ion accumulation within the seed results into cytotoxic effects and nutritional imbalance (Bradford 1995). Reports on the use of sodium propionate, Na(C2H5COO), a food preservative, or synthetic seawater (a mixture of mineral salts which mimics seawater), as an alternative to PEG or salts in osmopriming are available (Pill 1995). Solid matrix priming Solid matrix priming (‘matriconditioning’) has been developed as an alternative to overcome the high costs of osmopriming, which needs large volumes of osmotic solution and expensive systems for the control of aeration and temperature. Furthermore, there are concerns related to the environment-friendly removal and waste disposal of the osmotic agent after treatment. Osmopriming is performed in liquid solutions (‘liquid priming’), while during solid matrix priming seeds are mixed with solid (organic or inorganic) material (‘solid priming’) able to properly adjust the moisture content, allowing to control water uptake (Harman and Taylor 1988). The substrate used as matrix must be chosen carefully, since it can significantly influence priming performance. Specific features are needed for the matrix which has to be inert and non-toxic, with high surface:volume ratio, high adhesiveness to seeds, low matric potential (wm) (Whalley et al. 2013), high capacity to retain water, negligible water solubility while it should be also easy to remove from seeds after treatment (Khan 1991). Many natural substances have been used as matrices for solid priming, e.g. coal, sawdust, vermiculite, calcined kaolin, charcoal and commercial substrates such as AgroLigÒ (a humic acid-based product used to improve soil

features). Treatment is preferentially carried out in a sealed container that permits air circulation, avoiding excessive evaporation. Solid matrix priming simulates the natural imbibition process occurring in soil, leading to the same results obtained with liquid priming, but at reduced costs. Furthermore, solid matrix priming can be integrated by adding chemical or biological agents known to improve seed performance (McDonald 2000). The procedure avoids all the technical problems associated with aeration and allows the simultaneous treatment of large amount of seeds at limited costs. To improve the control of imbibition, the matrix can be also moistened with an osmotic solution instead of pure water (Khan 1991). Biopriming The priming mixture is integrated with beneficial microorganisms or bioactive molecules. Is it known that the association of plants with specific fungi or bacteria results into extremely convenient outcomes, since these microorganisms are able to establish endophytic relationships with the plant, leading to plant growth enhancement and phytohormones production, improvement of biotic/abiotic stress resistance (Waller et al. 2005). The strains mainly used for biopriming belong to Trichoderma spp., Enterobacter spp., Pseudomonas spp. and Bacillus spp. (Niranjan et al. 2004). Effective biopriming treatments were achieved in vegetable seeds using the Trichoderma harzianum strain, followed by Trichoderma pseudokoningii, Bacillus spp., Gliocladium spp. and Pseudomonas fluorescens (Ilyas 2006). Biopriming is also performed by adding secondary metabolites to the priming mixture and in most cases, phytohormones such as salicylic acid (SA), abscisic acid (ABA) or gibberellic acid (GA) have been used (Hamayun et al. 2010). These phytohormones control key biochemical processes during seed maturation/germination and throughout plant development, while exogenous application improves the antioxidant response (Radhakrishnan et al. 2013). Treatments with biological agents or metabolites can be applied to both liquid priming or solid matrix priming (‘bio-matri-conditioning’). Chemopriming Chemopriming is achieved by adding conventional disinfectants, such as sodium hypochlorite (NaOCl) or hydrochloric acid (HCl), natural substances, and agrichemicals (e.g. fungicides, pesticides) to the priming solution to prevent microbial contaminations (Parera and Cantliffe 1990). Although treatments with NaOCl and HCl solutions reduce losses in germination caused by pathogens, several parameters (disinfectant concentration, treatment duration, solution temperature, seed age) need to

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be carefully evaluated (Khah 1992). Natural compounds with broad-spectrum antimicrobial activity, including organic acids, essential oils, crude plant extracts and dairy products are used for seed disinfection mainly in organic agriculture (Van der Wolf et al. 2008). Fungicide-based products for seed treatments have been tested, as in the case of Thiram (dimethylcarbamothiosulfanyl-N,Ndimethylcarbamodithioate) mixed with PEG polymers at different molecular weights to identify the best suitable combinations able to limit seed deterioration (Kaushik et al. 2013). Similarly, the triazole systemic fungicide Tebuconazole [(RS)-1-(4-chlorophenyl)-4,4-dimethyl-3(1H, 1, 2, 4-triazol-1-ylmethyl)pentan-3-ol] tested on maize (Zea mays L.) seeds in the form of ethyl cellulosebased microcapsules resulted in enhanced emergence rates, shoot/root fresh weight, and carotenoid/chlorophyll content (Yang et al. 2014). The current chemopriming technology relies on the use of innovative materials for seed treatment, e.g. those based on pesticide–collagen hydrolysate mixtures with bioactive properties which allow to reduce pesticide dispersal in the environment. More ecological friendly tools are envisaged, such as those made of microencapsulated plant extracts with insecticidal and fungicidal properties (Gaidau et al. 2014).

bases for a widely used application in seed testing (Bradford and Still 2004). ‘Hydrotime’ is measured in MPa h-1 or MPa d-1 and it is defined as the time required to see radicle emergence (Gummerson 1986). According to this model, w represents the water potential of seed/seed environment, while wb is the minimum w value that allows germination to be completed (physiological threshold for radicle emergence). The wb value varies among individual seeds, and this lack of uniformity can be quantified as the wb standard deviation or rwb. The hydrotime constant hH indicates the speed of germination within a seed lot. By combining these parameters, hydrotime analysis allows prediction of the pattern of seed germination in terms of speed, stress tolerance and uniformity. According to the following equation: hH ¼ ½w  wb ðgÞ tg

Seed treatment at different temperatures carried out before sowing is also known as ‘thermopriming’. This technique has been widely demonstrated to improve germination efficiency under adverse environmental conditions, reducing thermo-inhibition of seed germination (Huang et al. 2002). Thermopriming is achieved by pre-sowing seeds at different temperatures. Low temperatures generally provide the best results. Although not so widely applied, thermopriming at high temperatures has been used in some species, resulting in advantages in germination especially for plants adapted to warm climates (Khalil et al. 1983). Thermopriming on lettuce seeds has produced slight improvements in seed development and germination rates in saline soil (Ashraf and Foolad 2005). Thermopriming combined with other treatments resulted in beneficial effects on germination parameters of white spruce (Picea glauca L.), enough to improve nursery practices for commercial seedling production (Liu et al. 2013).

The hydrotime constant hH is represented as a function of w, wb(g) (the threshold water potential expressed in MPa defined for the specific fraction g of the total seed population) and tg, the time expressed in hours, required for radicle emergence in the same fraction g of the total seed population. Experimental analyses have shown that wb(g) values change within a seed population according to normal or Gaussian distribution (Bradford 2002). Based on these calculations, the hH value is expected to increase with the time required for radicle emergence. Indeed, the hH value for carrot seeds which germinate in seven to ten days is higher than the hH value for lettuce seeds whose germination normally require 1–2 days. Thus, an increase in hH value is considered an indicator of seed ageing while successful seed priming accelerates germination, reducing hH. The different parameters of the hydrotime model can change depending on cultivar, seed lot, and treatment. On the other hand, hydrotime analysis is useful to evaluate the physiological status of a seed lot since all the different parameters can disclose any possible abnormal response in terms of seed germination/seedling establishment under stressful conditions (Bradford and Still 2004). This technique is helpful in supporting the breeding programmes by providing the opportunity to select those plant lines with improved seed performance and stress tolerance. Hydrotime analysis is however time consuming, due to the need for repeated observations throughout the germination time period. These constrains might be overcome with the help of innovative technical tools as automated seed imaging for scoring germination.

‘Hydrotime concept’ and analysis for seed priming assessment

Seed priming and crop productivity under adverse environmental conditions

The ‘hydrotime concept’ provides a unifying model for the description of the relationship between water potential (w) and seed germination rate/percentage and the theoretical

Due to global climate changes, worldwide crop production systems are currently exposed to intermittent or terminal drought conditions with effects ranging from

Thermopriming

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impairment of plant growth to premature plant death. The molecular processes underlying the plant response to drought in terms of stress perception, signalling pathways, modulation of gene expression and consequent physiological responses are still poorly understood (Bhargava and Sawant 2013). It has been reported that Selenium (Se) enhances plant growth, playing a key role in the adjustment of plant water status under drought stress, improving seed germination and radicle elongation. Nawaz et al. (2013) investigated the effects of Se-mediated priming in wheat (Triticum spp.) seeds, demonstrating that these treatments enhance germination efficiency and improve drought tolerance. An increase in the amount of soluble sugars was observed with increasing rates of Se-mediated priming (Nawatz et al. 2013). The positive role played by Se is possibly due to modulation of the sugar flux that might act as a molecular signal for the activation of the plant response under drought stress (Hartikainen 2005; Kishor et al. 2005). Primed seeds result into improved field performance of seedlings, optimised stand establishment of plants and more efficient/prolonged use of soil-derived nutrients and light. Enhanced chlorophyll content was measured in chickpea (Cicer arietinum L.) plants obtained from seeds treated with hydropriming (Hosseinzadeh-Mahootchi and Ghassemi-Golezani 2013). Furthermore, chickpea plants from primed seeds revealed improved stomatal conductance under irrigation treatments, another distinctive feature of stress tolerance (Ghassemi-Golezani et al. 2012). In a different study, Dias et al. (2009) evaluated the effects of osmopriming on carrot seed germination under water stress and extreme temperature conditions, showing improved field seedling emergence. Li and Zhang (2012) demonstrated that rice seeds subjected to hydropriming, PEG-mediated osmopriming and SA-mediated priming were able to provide enhanced stress tolerance to seedlings. When these seedlings were challenged with PEG-mediated osmotic stress and, the root length of seedlings derived from PEG- and SA-primed seeds was significantly increased, compared to hydroprimed seeds (Li and Zhang 2012). Similarly, net photosynthetic rate and water use efficiency were higher in seedlings obtained from PEG- and SA-primed seeds. The SA treatment was more effective in improving seedlings performance under stress conditions, resulting in enhanced content of photosynthetic pigments and Chla/ Chlb ratio, triggering SOD and APX enzyme activities. Proline content was also enhanced in plants derived from SA-primed seeds (Li and Zhang 2012). This finding is in agreement with the role of SA in promoting photosynthesis rate by increasing chlorophyll pigment production, RuBisCO activity and water absorption (Shekari et al. 2010).

Digital Image Technology applications for assessing seed vigour Digital image technology (DIT), a non-invasive method allowing precise and automated assessment of seed morphological and physiological features, is exploited by seed technologists for different purposes, e.g. purity analysis, taxonomic screening, and vigour assessment (Dell’Aquila 2009). Using DIT, the seed response is monitored throughout imbibition, revealing the progressive seed enlargement caused by water uptake until radicle protrusion is observed. DIT allows high-resolution analysis of small seeds and accurate measurements of germination parameters (e.g. germination speed and seedling length) on a single seed basis. The observed changes in size and shape occurring and any possible differences within the same or different seed populations can be analysed by means of mathematical models (Fiorani and Schurr 2013; Lei et al. 2014). Digital images of seeds are captured using hardwares, such as a flat-bed scanner or a charged coupled device (CCD)-camera, and subsequently undergo fast processing, thus replacing human visual inspection. New algorithms are now available that enable the extraction of numerical data from the acquired images, thus several seed features (among which are size and shape, colour density and growth rate) can be easily measured and integrated with biochemical and molecular data (Fiorani and Schurr 2013; Lei et al. 2014). Data acquired from DIT are converted into curves that provide information on the spatiotemporal response of seeds during germination under physiological conditions, environmental stress as well as priming agents. The effectiveness of osmopriming mediated by PEG and potassium nitrate (KNO3), respectively, on germination and establishment of a seeded bermudagrass (Cynodon dactylon L. Pers.) was investigated using a DIT-based approach. Digital images were taken weekly for 6 weeks and subsequently batch-analysed (Siebert and Richardson 2002). Similarly, Dutt and Geneve (2007) monitored the effects of priming on Impatiens (Impatiens walleriana Hooker F.) and petunia (Petunia hybrida Hort. Vilm.) seeds using a flat-bed scanner interfaced with a personal computer. Images were captured every hour to measure radicle protrusion and seedling growth as indicators of seed vigour. DIT was also used by Mahajan et al. (2011) to speed up the analysis of the seed response to hydropriming and osmopriming in rice, allowing the calculation of several key parameters such as germination index, seedling vigour index and leaf area index. Besides its priming-specific applications, the use of DIT has proved to be a valuable tool in several other context. Imbibition in white cabbage (Brassica oleracea L.) seeds was monitored using an image analysis system able to

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determine a range of parameters (area, perimeter, width, length and eccentricity) which describe the quantitative changes in seed morphology during water uptake (Dell’Aquila et al. 2000). Digital imaging was used to analyse seed vigour by Sako et al. (2001) and Hoffmaster et al. (2003), who tested a scanner designed to contain germinating seeds and seedlings of lettuce and soybean (Glycine max L.). The acquired numerical data representing several morphological parameters were further elaborated to obtain a vigour index. Computer-assisted analysis of digital images was also used to rank seed lot vigour in the genus Impatiens spp., based on seedling length (Oakley et al. 2004). Accelerated ageing tests were performed on Impatiens seed lots with different vigour and the germination response was then captured through a flat-bed scanner. The resulting digital images were used to calculate seedling size and growth rate and data were further analysed to measure the population uniformity (Oakley et al. 2004). The same technology was tested with cotton (Gossypium hirsutum L.) seeds (Xu et al. 2007). An exhaustive update of the current imaging technology is provided by Fiorani and Schurr (2013). Imaging techniques need to be carefully designed, depending on the target species, growth location and requirements. Phenotyping platforms are now available, that can measure watershortage use by gravimetric methods to assess plant evapotranspiration and then start automatic irrigation at specific sites (Berger et al. 2010). Automated evaluation of rice panicle yield-related traits (1000 plants per day) has been reported using seed imaging and weighting (Duan et al. 2011). Future advances in seed phenotyping technology will contribute to expand the number of seed-related traits useful for assessing plant productivity. Similarly, new diagnostic ‘omics’ methods analysing genetic/epigenetic factors, gene expression, and protein/metabolite analysis by high-throughput technologies will help to establish advanced protocols for seed quality testing.

Seed priming towards the native wild plant conservation Currently, tens of thousands of wild plant species are rare or endangered and potentially face extinction worldwide in different habitats (Weinig et al. 2014). Land use change and climate change make the conservation of biodiversity even more of a challenge (Thuiller 2007; Jackson and Kennedy 2009). In this context, in situ seed conservation is considered an important way to prevent plant extinction, but pressures on the environment are so intense in many areas that it is not always possible to conserve plants in their natural habitats. In these cases, ex situ seed conservation act as a backup for species which might be

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threatened or lost in nature (Li and Pritchard 2009). While we cannot always guarantee the safety of a plant even in the best-protected nature reserve (in situ conservation), seeds can be kept safely for hundreds of years in a seed bank (Van Slageren 2003). Collecting and storing seeds under seed bank conditions is, therefore, recognised as an effective strategy to safeguard species and to provide propagation materials to re-establishment of wild plant populations. Hence, several seed banks, such as the Millennium Seed Bank (http://www.kew.org/science-con servation/millennium-seed-bank) (Van Slageren 2003), have been established in the past decades with the aims to collect and conserve seeds of wild species. However, the storage of seeds for a long time, the lack of knowledge on storage conditions and the low availability of seeds of native wild plants are major drawbacks for successful management of seed banks and restoration. There is urgent need for innovation in native seed science, to strengthen the current knowledge in seed conservation and use, to support and increase the capacity building in local seed companies for large-scale native seed production. In this regard, novel techniques will be developed to monitor and improve the life span of native plants of greatest likely interest for commercial production (e.g. for use in ski trucks restoration). Attention will be focused on tools to monitor and improve the longevity of short-lived seeds, such as those of alpine plants (Mondoni et al. 2011). Indeed, all seeds stored even under air dry low temperature conditions will have suffered a degree of deterioration, but if the damage is not too severe, repair will be possible under proper conditions (Butler et al. 2009). As for priming, most of the research has been so far conducted on crop varieties/species, however studies on wild species have revealed that priming may have a rejuvenating effects on aged seeds, as shown in Digitalis purpurea L. (Butler et al. 2009) and Ranunculus sceleratus L. (Probert et al. 1991).To date, the effects of priming on seed longevity in wild species remain poorly investigated and the possibility that its effectiveness will be highly species-dependent and might vary across plant populations, cannot be ruled out.

Main limitations of current priming techniques Several types of priming protocols have been designed so far, all of them optimised by accurate timing to stop the treatment before the occurrence of radicle emergence. Many protocols developed include at the end of the treatment, a rapid re-dry of seeds for storage purposes (Halmer 2004). However, in some cases, desiccation can alter the beneficial effects of priming, which are lost during storage (Heydecker and Gibbins 1978). Indeed, reduced seed longevity is a well-reported disadvantage of seed priming

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(Chiu et al. 2002). When Tarquis and Bradford (1992) applied different priming methods on L. sativa seeds, high susceptibility to deterioration during storage was observed, compared to non-primed seeds. However, other reports demonstrate that priming ameliorates seed longevity (Probert et al. 1991; Butler et al. 2009). The deleterious effects of priming have been ascribed to prolonged treatments that cause the loss of seed desiccation tolerance (Sliwinska and Jendrzejczak 2002). Importantly, the conditions in which seeds are kept immediately after priming (storage temperature, humidity, air composition) negatively affect seed viability (Schwember et al. 2005). To overcome this problem, several post-priming treatments have been developed. Bruggink et al. (1999) showed how to obtain the desired seed longevity by keeping primed seeds under mild water and temperature stress. In addition, Gurusinghe and Bradford (2001) noted that incubation of seeds under slow drying conditions followed by exposure to high temperature resulted in enhanced longevity.

Molecular perspectives in seed priming The complex trait of seed quality influences harvest rate and uniformity and positively correlates with the plant response to adverse field conditions. In most cases, loss of seed vigour is not recorded by producers and it will be detected only by farmers, as a sowing failure. Predictive methods for the early diagnosis of seed quality are needed and the novel technology will be then converted into innovative quality control procedures throughout the trade. DNA repair and antioxidant mechanisms are activated in seeds upon rehydration to minimise growth inhibition and deleterious mutagenesis during seedling development. The study of the effects of the priming agent on DNA repair mechanisms is essential to define optimised priming protocols (Balestrazzi et al. 2010, 2015; Macovei et al. 2011a, b). Indeed, prolonged treatment results into an unpredicted enhancement of oxidative DNA injury which irreversibly affects seed viability (Balestrazzi et al. 2010). The choice of the correct timepoint to stop the priming treatment and dehydrate the seed is a critical step still difficult to monitor. Seed operators working in seed companies and germplasm banks require reliable indicators able to predict in large advance the seed response to industrial treatments, saving time and costs. Due to the complexity of physical, chemical, and molecular factors that contribute to seed viability, improved knowledge of the regulatory network that control the DNA damage response, and prooxidant/antioxidant balance during seed germination will be gained only using translational research projects with multidisciplinary features. These highly competitive technological platforms will be able to provide information that will be

immediately translated into innovative, reliable priming protocols.

Systems Biology approaches: new avenues to understanding the complexity of seed priming Seed priming elicits complex cellular responses that have been elucidated by considerable progresses made in exploring and understanding the molecular, physiological and biochemical mechanisms underlying seed priming, as described previously. Proteome analyses of seed priming and germination have proven invaluable in identifying changes between primed and non-primed seeds in various plants (Wang et al. 2015). These studies provided a comprehensive metabolic and biochemical understanding of the increased seed vigour observed in primed seeds of Arabidopsis (Gallardo et al. 2001; Rajjou et al. 2006), alfalfa (Medicago sativa L.) (Yacoubi et al. 2013), wheat (Fercha et al. 2013, 2014). During the last decade, the ‘reductionistic’ molecular biology and functional biology approaches are being progressively replaced by the ‘holistic’ approach of Systems Biology (Duque et al. 2013). The development of integrative approaches, based on global analysis of transcriptomes, proteomes and metabolomes integrated in solid bioinformatics platforms, has noticeably changed our knowledge and expanded our way to address seed priming research. One of the earliest reports of the use of combined postgenomics platforms in understanding the molecular mechanisms underlying seed priming is described in the work of Barba-Espı´n et al. (2011). These authors investigated the role of H2O2 during pea seed germination using a combined proteomic and hormone profiling approach. Specific changes at the proteome and hormonal levels in response to H2O2 treatments resulted in an acceleration of the germination process, most probably because of invigoration of the seeds. These findings have practical implications, suggesting a key role of H2O2 as a regulator of seed germination and its potential use in seed priming technologies to invigorate low vigour seeds. The beneficial effect of PEG-mediated osmopriming on rapeseed (Brassica napus L.) germination was studied using a global proteome and transcriptome expression profiling (Kubala et al. 2015). Up to 952 genes and 75 proteins were differentially expressed during the main phases of priming procedure and final germination of primed seeds. Distinct specific pathways are triggered, since only a minority of genes and proteins are involved in all phases while a vast majority is involved in only one single phase. The differences noticed between transcriptome and proteome data set emphasised the importance of the regulation of mRNA translation and post-translational processes occurring during priming and post-priming germination.

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Summarising, System Biology-based approaches are providing new avenues to understand seed priming, as well as, bringing novelty in identifying new molecular markers of seed quality and viability. The use of combined platforms representing the multiple layers of gene expression (transcripts, protein and metabolite) is crucial to find regulatory mechanisms controlling gene expression. The modulation of such transcriptional, post-transcriptional or post-translational mechanisms of gene expression constitutes a promissory strategy to engineer seeds with desired traits and enhanced quality.

Conclusions Advanced molecular tools applied to translational research programmes in seed science will address the current expectations of seed industry. Within this context, Academia and Industry need to reinforce their involvement to build up high-impact translational research networks that will bring seed operators strong advantages in seed quality management. This will provide early hallmarks of the seed response to industrial treatments, particularly as concerns priming technology for the rationale management/production of competitive commercial seed lots. The availability of novel indicators will also facilitate investigations on innovative molecules/materials to be included in industrial formulations designed to improve seed quality. Author contribution statement Stefania Paparella, Malaka Wijayasinghe and Susana Araujo collected background information together. Alma Balestrazzi drafted the manuscript. Graziano Rossi and Daniela Carbonera helped to revise the manuscript. All authors read and approved the final manuscript. Acknowledgments This work was supported by Regione Lombardia D.G. Istruzione, Formazione e Lavoro, Struttura Asse V—Interregionalita` e Transnazionalita` POR FSE 2007-2013, Project ID 46547514 ‘Advanced Priming Technologies for the Lombardy AgroSeed Industry-PRIMTECH’ (Action 2). S.P. has been awarded by a Research Fellowship from Regione Lombardia within the same project. S.A. has been awarded by a Research Contract (Recruited Foreign Scientist) funded by CARIPLO Foundation within the same project (Action 3) Code 2013-1727. M. W. has been awarded by a PhD Fellowship within the NASSTEC (The NAtive Seed Science TEchnology and Conservation Initial Training Network) project, proposal Number 607785 (FP7-PEOPLE-2013-ITN). Conflict of interest of interest.

The authors declare that they have no conflict

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