WO2021094755A1

WO2021094755A1 - Gas plasma activated water seed treatment

Info

Publication number: WO2021094755A1
Application number: PCT/GB2020/052876
Authority: WO
Inventors: Giles GRAINGE; Tina Steinbrecher; Kazumi NAKABAYASHI; Gerhard LEUBNER
Original assignee: Royal Holloway And Bedford New College
Priority date: 2019-11-14
Filing date: 2020-11-12
Publication date: 2021-05-20
Also published as: GB201916576D0

Abstract

The disclosure relates to a seed priming technology which retains seed storage resilience without the need for an additional post-priming treatment. This allows the primed seeds to be stored for longer, whilst still retaining good seed vigour. There is provided a method for priming seeds, the method comprising the steps of imbibing a seed in gas plasma activated water and drying the seed. Seed priming is a process of controlled hydration of a seed which permits pre-germinative metabolic activity to proceed but prevents actual emergence of the radicle.

Description

Gas Plasma Activated Water Seed Treatment

Field of the Invention

The present invention relates to seed priming treatment with gas plasma activated water (GPAW) to enhance germination performance combined with retaining seed storability resilience.

Background to the Invention

The challenge to sustainably intensify global food production to keep pace with the world’s expanding population is aggravated by climate change. This includes the higher risk of erratic and extreme weather events which threaten the sustainable intensification of crop production. It is preferable to broaden the climates we can cultivate in and to increase the harvest yield from the current arable land through strategic and technological advances. The vast majority of crops produced in world agriculture begin with the sowing of a seed to establish a new seedling in the field. This is the first critical step for crop production as the plant is at its most vulnerable to abiotic stresses such as drought, heat or flooding. The successful completion of germination and seedling establishment is an ultimate requirement for achieving maximum yield potential. High crop seed quality is, therefore, a critically important agronomic trait and is achieved, at least in part, by seed treatment and enhancement technologies such as seed priming.

A key issue within the seed industry, which heavily impacts yield efficiency, is irregular germination timing and the vulnerability of germination at the early seedling stages. High-quality commercial crop seed refined by priming provides enhanced seedling performance, with rapid and uniform emergence even upon abiotic stress. The intense competition in the global seed market, which is expected to reach USD 78.8 billion by 2022, is also driven by advanced and innovative seed treatment technologies to increase crop seed quality. The seed treatment market is projected to reach a value of USD 9.8 billion by 2021 at a compound annual growth rate (CAGR) of 10.8%. Seed priming and other seed technologies are applied to commercial seeds including vegetables, sugar beet, oilseed rape, flowers, and other agricultural and horticultural crop species. Seed priming involves activating the seed metabolism by allowing imbibition, but by limiting the water uptake, germination completion is prevented. Classical seed priming is by far the most common method utilised across the seed technology industry to enhance the vigour of commercial seeds. The International Seed Testing Association (ISTA 2015) defines seed vigour as 'the sum of those properties that determine the activity and performance of seed lots of acceptable germination in a wide range of environments'. The seed technology industry currently focuses on hydro-priming, which is typically conducted in priming drums. This is the process of elevating a seeds’ moisture content with a limited amount of water for a defined period, before drying the seeds off and placing them in storage prior to being sold to farmers.

The priming technology provides the benefit of enhanced seed vigour, by widening the temperature window for germination, and in some cases alleviating both the light requirement for germination and physiological dormancy. Lettuce is a well-studied vegetable crop seed for which priming is conducted commercially to improve the vigour, remove photo-dependent and physiological dormancy, and increase thermo tolerance. However, the longevity of stored seeds in most cases, including lettuce, is depleted due to accelerated aging (deterioration) during storage, and storability is therefore heavily compromised by classical seed priming leading to reduced vigour. To reintroduce seed storability into primed seed, some companies apply an additional post-priming treatment such as heatshock which requires adding another step and additional equipment into the seed processing pipeline. Due to the additional cost, time and effort for the post-priming treatment to re-introduce storage resilience, this is only conducted by some companies to achieve more than single season storability and to provide superior seed quality to their product. The major problem of the classical seed priming technology is therefore that in many cases it compromises seed storability due to the increased aging sensitivity of the primed seeds. This effect causes the primed seeds vigour to deteriorate rapidly during storage in the dry state, limiting the time window the treated seed remains an acceptable quality for customers. Seed companies, therefore, can’t store primed seeds for more than one season and extending this storage resilience to at least two seasons is a priority. Gas plasma is considered to be the fourth state of matter. Current applications for gas plasma involve cleaning and treating manufactured surfaces for a variety of industrial and medical processes. Gas plasmas are associated with the formation of a myriad of free radicals such as highly reactive oxygen species (ROS) and reactive nitrogen species (RNS) which can react with macromolecules in their proximity. With non-thermal (cold) atmospheric-pressure gas plasma (NTAGP) technologies, this is achieved without heat generation and at atmospheric pressure allowing for the treatment of sensitive biological tissues. This property has been proven useful in developing new medical applications for dentistry and oncology, and additionally provide an excellent sterilization tool for food and food packaging. New trends of sustainable agricultural applications of NTAGP, including plant, seed, and food treatments, are currently emerging as a rapidly developing innovative field called "plasma agriculture". This includes the inactivation/sterilisation of seed-borne microorganisms, as well as the stimulation of seed germination and seedling growth by NTAGP treatment. In these methods, seeds are soaked and treated with NTAGP. Soaking of the seeds consequently leads to germination. However, NTAGP treatment has never been used in seed priming, or to improve the tolerance and storage resilience of a seed.

Summary of the Invention

The present invention relates to a seed priming technology which retains seed storage resilience without the need for an additional post-priming treatment. This allows the primed seeds to be stored for longer, whilst still retaining good seed vigour.

The present invention provides a method for priming seeds, the method comprising the steps of imbibing a seed in gas plasma activated water and drying the seed.

Seed priming is a process of controlled hydration of a seed which permits pre-germinative metabolic activity to proceed but prevents actual emergence of the radicle by drying the seed. This is in contrast to seed soaking where the seed proceeds to germination and radical emergence. The seed may be any suitable seed for which it is desired to enhance the seed vigour, whilst also retaining seed storage resilience. The seed may be any seed from a seed producing plant, such as the gymnosperm and angiosperm plants. The seed may be from a fleshy fruit or a dry fruit. The fruit may be dry at maturity and constitute a dispersal and harvest unit, such as sugar beet, parsnip, lettuce and cereal crop species. The seed may be a seed from a monocotyledonous or dicotyledonous plant, including the basal angiosperms and Caryophyllidae. The seed may be produced on a small scale or on a large scale. The seed may be selected from nuclear seed, breeder seed, foundation seed, registered seed and certified seed. The seed may be selected from food crop species and non-food crop species including horticulture, floriculture, agriculture and industrial crop species. The seed may be a cereal crop species, such as wheat, maize, rice, sorghum, barley, rye, oats, amaranth, quinoa or millet. The seed may be a vegetable crop species, including: leafy or stem vegetables such as cabbages, broccoli, cauliflower, brassicas, lettuce, asparagus, Pak Choi, cress, chard and spinach; fruit-bearing vegetables such as cucumbers, courgettes, squashes, melons, tomatoes, peppers, aubergines and pumpkins; root, bulb or tuberous vegetables such as carrots, parsnips, turnips, beetroot, kohlrabi, radishes, celery, celeriac, fennel, swede, bulb onions, shallots, salad onions, garlics, and leeks. The seed may be a fruit and nut crop species, including: citrus fruits including oranges, limes, lemons and grapefruit; grapes including grapevine; berries including gooseberry, strawberry, raspberry, loganberry, blackberry, blueberry, bilberry, goji berry, cranberry, acai berry, mulberry and dewberry; pome fruits and stone fruits, such as apples, pears, peaches, nectarines, cherries, apricots and plums; and nuts such as peanuts, walnuts, almonds, cashews and macadamia. The seed may be an oilseed crop species, such as soya beans, groundnuts, castor bean, linseed, rapeseed, sesame and sunflower. The seed may be a root, tuber or bulb crop species, such as potatoes, sweet potatoes, tuberous begonia, cyclamen, cassava and yams. The seed may be a beverage and spice crop species, such as coffee, tea, mate, cocoa, chilies and peppers, nutmeg, cinnamon, ginger and vanilla. The seed may be from herbs such as basil, coriander, chives, dill, parsley, salad rocket, sweet fennel, cress, thyme, lemon balm, mint, oregano, rosemary, sage, French tarragon, marjoram, anise, mustard and sorrel. The seed may be a leguminous crops species, such as beans, broad beans, chick peas, French beans, runner beans, cow peas, lentils, lupins, peas and pigeon peas. The seed may be a sugar crop species, such as sugar beet, sugar cane and sweet sorghum. The seed may be another crop species, such as grasses and other fodder crops, fibre crops such as cotton, catch and cover crops, green manure, medicinal, aromatic, pesticidal or similar crops, rubber, tobacco or cannabis. The seed may be an ornamental species including garden flowers and grasses grown from seed, such as Achillea, Alchemilla, Allium, Ageratum, Antirrhinum, Begonia, Beilis, Brassica, Calceolaria, Calendula, Canna, Celosia, Cleome, Cosmos, Dahlia, Dianthus, Geranium, Helianthus, Heliotrophium, Impatiens, Lavendula, Lobelia, Nemesia, Nicotiana, Osteospermum, Pelargonium, Petunia, Penstemon, Phlox, Primula, Ranunculus, Rudbeckia, Salvia, Scabiosa, Senecio, Tagetes, Trollius, Verbena, Viola, cornflower (Centaurea cyanus), poppies ( Papaver spp., Eschscholzia spp.), sweat pea (Lathyrus spp.) species. The seed may be ornamental, lawn, sports turf or meadow grass seed, such as Stipa, Pennisetum, Molinia, Miscanthus, Hakonechloa, Cortaderia, Anemathele, Agrostis, Briza, Festuca, Poa, Lolium and Zoysia species.

In particular embodiments, the seed may be a crop seed selected from:

• cereals, including wheat ( Triticum aestivum ), rice ( Oryza sativa ), maize (Zea mays), oat ( Avena sativa), barley ( Hordeum vulgare), rye ( Secale cereale), sorghum ( Sorghum bicolor), millet ( Panicum spp., Pennisetum spp., Setaria spp., Digitaria spp,, Echinochloa spp., Eleusine spp.);

• cruciferous plants, including oilseed rape (. Brassica napus), thale cress (. Arabidopsis thaliana), Lepidium spp., rocket ( Eruca vesicaria), Brassica oleracea (cabbage, broccoli, cauliflower, kale, Brussels sprouts, collard greens, savoy, kohlrabi), radish ( Raphanus sativus), turnip ( Brassica rapa) and swede ( Brassica napus),

• amaranth/goo sefoot species, including Beta vulgaris (sugar beet, red beet, fodder beet, chard), spinach ( Spinacia oleracea), quinoa ( Chenopodium quinoa), and Amaranthus spp.;

• curcubitous and solanaceaous vegetables and fruits, including tomato ( Solanum pimpinellifolium, Lycopersicon esculentum), peppers and chillies ( Capsicum annum and other Capsicum spp.), eggplant ( Solanum melongena), Cucurbita spp. (squash, pumpkin, zucchini, some gourds), Citrullus spp. (watermelon), Cucumis spp. (cucumber, melons); • umbelliferous and solanaceaous root/tuber vegetables, including carrot ( Daucus carota ), parsnip ( Pastinaca sativa), celery {Apium graveolens ) and potato (Solanum tuberosum),

• herbs and spices, including parsley {Petroselinum crispum), coriander {Coriandrum sativum), dill {Anethum graveolens), fennel {Foeniculum vulgare), cumin {Cuminum cyminum), caraway {Carum carvi), and anise {Pimpinella anisum),

• legumes, including soybean {Glycine max), peanut {Arachis hypogaea), bean {Phaseolus spp.), chickpea {Cicer arietinum), cowpea {Vigna unguiculata), pea {Pisum sativum) and lentil {Lens culinaris or Lens esculenta),

• ornamentals, including Begonia spp., Viola spp., Allium spp., Geranium spp., Nicotiana spp., Helianthus spp., Pelargonium spp., Petunia spp., Primula spp., Penstemon spp., Trollius spp., Verbena spp.; and

• grasses, including Festuca spp., Poa spp., Lolium spp., Stipa spp., Zoysia spp., Pennisetum spp., Miscanthus spp., and the species lettuce {Lactuca sativa), sunflower {Helianthus annuus), cotton {Gossypium spp.), tobacco {Nicotiana tabacum), flax {Linum usitatissimum), grapevine (Vilis vinifera), and Allium spp. (bulb onions, shallots, salad onions, garlics, leeks, ornamentals). In some embodiments, the seed may be selected from Brassica napus (oilseed rape), Brassica oleracea (cabbage, broccoli, cauliflower, kale, Brussels sprouts, collard greens, savoy, kohlrabi), Brassica rapa (turnip), Raphanus sativus (radish), Triticum aestivum (wheat), Oryza sativa (rice), Zea mays (maize), Hordeum vulgare (barley), Beta vulgaris (sugar beet, red beet, fodder beet, beetroot, chard), Spinacia oleracea (spinach), Solarium pimpinellifolium and Lycopersicon esculentum (tomato), Capsicum annum and other Capsicum spp. (peppers and chillies), Cucurbita spp. (squash, pumpkin, zucchini, some gourds), Citrullus spp. (watermelon), Cucumis spp. (cucumber, melons), Daucus carota (carrot), Pastinaca sativa (parsnip), Apium graveolens (celery), Petroselinum crispum (parsley), Coriandrum sativum (coriander), Begonia spp., Viola spp., Allium spp. (bulb onions, shallots, salad onions, garlics, leeks, ornamentals), Geranium spp., Nicotiana spp., Helianthus spp., Pelargonium spp., Petunia spp., Lactuca sativa (lettuce), Helianthus annuus (sunflower), Nicotiana tabacum (tobacco), Gossypium spp. (cotton), and the grasses Festuca spp., Poa spp. and Lolium spp.

Imbibition is the absorption of water by a seed. The seed is imbibed in the gas plasma activated water, wherein the seed absorbs a volume of the gas plasma activated water.

The gas plasma activated water may be generated using any method known to one skilled in the art. For example, suitable methods are described in Kong, M. and Shama, G, 2014 (Cold Atmospheric Gas Plasmas. 10.1016/B978-0- 12-384730-0.00366-9). Such methods include contact of gas plasma with water, for example by discharging gas plasma directly on water or hydrated surfaces/biological material, and also discharge of gas plasma and bubbling the plasma afterglow (resulting ionised gas) through water. These methods may employ radio-frequency discharges, microwave discharges, gliding discharges or surface discharges.

The gas plasma used to generate the gas plasma activated water may be thermal plasma or non-thermal plasma. Alternatively, the gas plasma may be a combination of thermal plasma and non-thermal plasma. The gas plasma may be generated using any plasma generation apparatuses known to one skilled in the art. Such apparatuses include plasma jet systems, vacuum reactors and dielectric barrier discharge (DBD) plasma devices.

The gas plasma used to generate the gas plasma activated water may be generated using any feed gas. The feed gas may be composed of a single gas species or multiple gas species. The feed gas may comprise atmospheric gases, non- atmospheric gases or a combination of both atmospheric gases and non- atmospheric gases. The feed gas may comprise any noble gases such as argon, helium, neon, krypton, xenon, radon and oganesson, atmospheric gases including oxygen, carbon dioxide and nitrogen, and also other gases such as cyclohexamine, carbon tetrafluoride, octadecafluorodecalin, aniline and hydrazine. The feed gas may be any mixture of these gases in combination. In some embodiments, the feed gas is selected from helium, neon, argon, oxygen, carbon dioxide and nitrogen, or combinations thereof. The feed gas may be air. The gas plasma activated water may be air-gas plasma activated water. Air-gas plasma activated water may be generated with gas that is at least 95% air, preferably at least 96% air, more preferably at least 97% air, more preferably at least 98% air, more preferably at least 99% air, more preferably 99.5% air, more preferably at least 99.9% air, and even more preferably 100% air. The air used in the plasma feed may be synthetic air or natural air.

The gas plasma activated water may be He/C -gas plasma activated water, preferably wherein the gas is 90-99.5% helium, more preferably wherein the gas is 95-99% helium, more preferably wherein the gas is 97-98% helium. Preferably the gas is 0.5-10% oxygen, more preferably the gas is 1-5% oxygen, more preferably the gas is 2-3% oxygen. In a preferred embodiment, the He/0₂ gas is 98% helium and 2% oxygen.

Gas plasma activated water differs considerably to the gaseous form of non-thermal (cold) atmospheric-pressure gas plasma (NTAGP), at least in terms of reactive species and oxidative species. The plasma gas phase chemistry and the liquid phase (GPAW) chemistry differ considerably in their primary and secondary production of distinct sets of reactive species including reactive oxygen and nitrogen species. A method employing the gaseous form of NTAGP would not result in the benefits associated with the claimed method.

Before priming, the seed typically has a moisture content of 5-10%. The seed may be imbibed in the gas plasma activated water until the moisture content of the seed is 10- 90%. In some embodiments, the seed may be imbibed in the gas plasma activated water until the moisture content of the seed is 20-80%, 30-80%, 40-80%, 50-80%, 50-70%, or about 60%.

The seed may be imbibed in the gas plasma activated water until the moisture content of the seed is 2-20% lower than the moisture content required for germination of the specific seed lot of a species being treated. Preferably, the seed is imbibed in the gas plasma activated water until the moisture content of the seed is 3-15% lower than the moisture content required for germination of the specific seed lot of a species being treated. More preferably, the seed is imbibed in the gas plasma activated water until the moisture content of the seed is 4-10% lower than the moisture content required for germination of the specific seed lot of a species being treated. Even more preferably, the seed is imbibed in the gas plasma activated water until the moisture content of the seed is 4-5% lower than the moisture content required for the completion of germination of the specific seed lot of a species being treated.

The moisture content required for germination is specific and is determined for a specific seed lot of a species to allow a controlled water uptake by the seed up to the end of water uptake phase II, just before the radicle protrudes from the seed or fruit coat. This point is determined empirically for each seed lot of a species and often internal knowledge within a seed company of those skilled in the art of seed priming. The principle of this required moisture content usually 4-5% lower than the moisture content required for the completion of germination by radicle emergence is for example well described by the International Seed Testing Association (ISTA) in the article by Corbineau & Come "Priming: a Technique for improving Seed Quality" in Seed Testing International 132:38- 40 (2006). The mechanisms underpinning this moisture content at the end of water uptake phase II, is that seeds are desiccation tolerant up to this stage, the germination process can therefore be arrested by drying. The moisture content for the optimal seed priming is therefore specific for a specific seed lot of a species.

The moisture content of the seed may be tested by any suitable method. For example, the change in moisture content over time can be determined by weighing. Alternatively, a seed moisture analyser can be used. Suitable moisture analysers are well known to those skilled in the art, such as those manufactured by Mettler Toledo, for example, the model HB43-S. These methods for the moisture analysis are, for example, described by the ISTA Manual with the International Rules for Seed Testing (2015, ISTA, Bassersdorf, Switzerland).

The seed may be imbibed in the gas plasma activated water for 20 min - 12 days. The seed may be imbibed in the gas plasma activated water for 1 h - 11 days. The seed may be imbibed in the gas plasma activated water for 6 h - 10 days. The seed may be imbibed in the gas plasma activated water for 12 h - 9 days, 1 day - 8 days, 2 days - 7 days, 3 days - 6 days, 4 days - 5 days. Preferably, the seed is imbibed in the gas plasma activated water for the length of time, ± 3 days, which causes the maximum increase in vigour of the specific seed lot of a species being treated. More preferably, the seed is imbibed in the gas plasma activated water for the length of time, ± 2 days, which causes the maximum increase in vigour of the specific seed lot of a species being treated. Even more preferably, the seed is imbibed in the gas plasma activated water for the length of time, ± 1 day, which causes the maximum increase in vigour of the specific seed lot of a species being treated. Most preferably, the seed is imbibed in the gas plasma activated water for the length of time which causes the maximum increase in vigour of the specific seed lot of a species being treated. The seed imbibition is conducted in a way that it does not allow the completion of seed germination. This may be achieved by restricting the volume of gas plasma activated water provided to the seed to only allow a maximum seed moisture content that is lower than the moisture content required for the completion germination by radicle emergence. Alternatively, this may be achieved by restricting the duration of the imbibition of the seed by restricting the length of time to before the onset of radicle protrusion within the seed population. Optimal increase in seed vigour is obtained by either of these methods and the increase in seed vigour is quantified with the gas plasma activated water primed and dried seeds using germination assays as described below.

The length of time which causes the maximum increase in vigour of the specific seed lot of a species being treated can be measured by conducting an experiment testing a range of gas plasma activated water priming times for the specific seed lot of a species, for example between 12 h to 20 days. Following priming, the seeds are dried (as described later) and a germination assay is conducted on each treatment group and an untreated control. The number of germinated seeds of the population are counted at regular time intervals, starting at the time point when the seeds are first imbibed and continuing until the seeds reach the maximum germination percentage. The completion of seed germination is defined by when the embryonic root (radicle) protrudes from the seed covering layers (seed coats and/or fruit coats depending on the species). The germination percentage of the population is calculated at each time point and a hill curve is fitted to the data points of each treatment group. The time required for each treatment group to complete 50% or another percentage of germination is calculated from the hill curve. The length of time which causes the maximum increase in seed vigour can then be calculated by selecting the treatment group which displayed the shortest time to reach 50% or the defined other percentage of seed germination.

The temperature of the water used for imbibing the seed during the priming treatment with gas plasma activated water is preferably between 10 °C and 40 °C. In some embodiments, the temperature of the water used for imbibing the seed is between 10 °C and 30 °C.

The seed may be dried by any technique known to one skilled in the art. The drying technique may be natural, such as air-drying. The drying technique may be artificial, such as drying with drying beads, rice or silica gel, using a dehumidifier or fan, or drying at higher temperatures, for example using an oven or a heater. The drying technique may use systems such as deep-layer dryers, shallow-layer dryers and in-sack dryers.

The drying step may comprise drying the seed at 10-20% relative humidity, preferably 15% relative humidity. The drying step may comprise drying the seed for 1 hour - 3 days at 10-35°C, preferably 2 hours at 29°C. The drying step may further comprise drying the seed for an additional 1-3 days above silica gel at ambient room temperature.

The drying step preferably lowers the moisture content of the seed to less than 40%, preferably 1-30%, more preferably 2-20%, more preferably still 3-15%, and even more preferably about 3-10%.

The seeds may be additionally be dried prior to the imbibing step. The seed may be dried by any technique known to one skilled in the art. The drying technique may be natural, such as air-drying. The drying technique may be artificial, such as drying with drying beads, rice or silica gel, using a dehumidifier or fan, or drying at higher temperatures, for example using an oven or a heater. The drying technique may use systems such as deep- layer dryers, shallow-layer dryers and in-sack dryers. The seed may be dried at 10-20% relative humidity, preferably 15% humidity. The drying step prior to imbibing preferably lowers the moisture content of the seed to less than 40%, preferably 1-30%, more preferably 2-20%, more preferably still about 3- 15%, and even more preferably about 3-10%.

The method may further comprise a step of storing the seed. The seed may be stored at ambient conditions or in conditioned storage where the temperature and/or relative humidity may be controlled. Preferably, the seed is stored in conditioned storage. More preferably, the seed is stored at low relative humidity (e.g. less than 20% relative humidity) and at 10-20°C. Even more preferably, the seed is stored at 10% relative humidity and at 12-15°C. Alternatively, the seed is stored at between -15°C and -80°C, preferably at -20°C in a dry state and container which prevents moisture uptake.

During storage, the seed may become aged by natural or artificial means, preferably by natural means. Natural aging of seed includes seed storage for a period of time at ambient conditions or in dry storage conditions (for example, 15°C, low relative humidity). Artificial aging of seed includes seed storage at high relative humidity and/or high temperature relative to ambient conditions. The seed may be stored for any length of time. Preferably, the seed may be stored for at least 1 month. More preferably, the seed may be stored for at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 8 months, a least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, at least 25 years, at least 50 years.

Following storage, the method may further comprise a step of germinating the seed. The seed may be germinated by any technique known to one skilled in the art. The germination step may comprise imbibing the seed with water. The germination may be achieved with or without using soil. The water may be selected from distilled water, double-distilled water or gas plasma activated water. The gas plasma activated water may be air-gas plasma activated water. Air-gas plasma activated water may be generated with gas that is at least 95% air, preferably at least 96% air, more preferably at least 97% air, more preferably at least 98% air, more preferably at least 99% air, more preferably 99.5% air, more preferably at least 99.9% air, and even more preferably 100% air. The gas plasma activated water may be He/Ch-gas plasma activated water, preferably wherein the gas is 90-99.5% helium, more preferably wherein the gas is 95-99% helium, more preferably wherein the gas is 97-98% helium. In the preferred embodiment, the He/0₂ gas is 98% helium and 2% oxygen.

Without being restricted to any particular theory, it is thought by the inventors that the gas plasma activated water promotes embryo growth, endosperm weakening and reducing the resistance of any existing seed coat (testa) or fruit coat (pericarp). The gas plasma activated water may physically reduce the resistance of the endosperm, testa and pericarp to the threshold typically required for the completion of germination.

The present invention also provides a seed produced by the above method. The seed produced by the above method may be more tolerant to aging and retain seed vigour.

Methods of the prior art directed to the priming of seeds compromise the storability of the seed and as a result, seed vigour is reduced following storage. Post-priming treatments are often used to reintroduce storage resilience into the seed. An advantage provided by the method described herein is that seed vigour is improved while retaining seed storage resilience, or storability. Seeds treated by the method can be stored for longer periods of time without compromising germination. As such, the seeds treated by the method are tolerant to aging and storage. Furthermore, additional post-priming treatments to reintroduce seed storage resilience are not required, therefore reducing the cost, time and effort of the seed processing pipeline.

Brief Description of the Drawings

The invention will now be described in detail by way of example only with reference to the figures in which: Figure 1 shows a schematic presentation of classical seed priming and the problem of rapid seed vigour loss by aging during dry storage of primed seed. The slow and non- uniform germination of unprimed seeds (A). During hydro-priming, the seeds metabolism is activated but the completion of germination and transition to seedling growth is blocked by returning the seed to its storage moisture content (B). If the dried primed seed is sown immediately it provides fast and uniform germination (C). However, if the classical primed dry seed is stored longer (D) it ages rapidly and loses its vigour resulting in poor seed quality with slow and unreliable germination.

Figure 2 shows a schematic demonstrating the advantages of extending and maintaining storability of the primed seed by EPOWER. (A) The black line demonstrates the relationship between seed vigour and priming intensity. The dashed grey line demonstrates the relationship between storability and priming intensity; the solid black line represents a conventional (with “normal” water) hydro-primed seed batch, the dashed black line represents an EPOWER primed seed batch. These data highlight the significantly reduced loss of storability following EPOWER priming. (B) The grey bars shows seed vigour levels whilst the black bar shows storability levels following hydro- and EPOWER priming. These data highlight the advantage of EPOWER priming when maximum vigour is achieved.

Figure 3 shows a diagram of the bubble reactor used to produce gas plasma activated water (GPAW). GPAW is required for EPOWER priming seed technology. The bubble reactor includes 12 high voltage AC electrodes in a dielectric material fixed below a gas permeable stainless-steel membrane. Above the membrane is a tank containing 100 ml of double-distilled water (ddlEO). Carrier gas flows past the electrode, and then through the membrane and ddfEO. For activation, plasma is formed between the electrodes and the membrane within the carrier gas and then flows through the membrane bubbling up through the water to produce the GPAW.

Figure 4 displays the results for the chemical characterisation of both Air-GPAW and He/0₂-GPAW produced from double-distilled water. (A, B) The concentration of NO2 , NO3 and H2O2 versus the NTAGP discharge time (30, 45 or 60 minutes) of He/0₂-GPAW (98% helium, 2% oxygen as gas) and Air-GPAW (natural air as gas) production, respectively. (C) OH· production of Air-GPAW and He/0₂-GPAW versus NTAGP discharge time. (D) OH· secondary chemical synthesis post-discharge during incubation of Air-GPAW and He/0₂-GPAW in a vessel.

Figure 5 shows the comparison of Lactuca sativa cv. Grand Rapids (lettuce) seed aging sensitivity with classical and EPOWER priming. The germination of unprimed control seed is compared to classical priming (with dd¾0) and to EPOWER priming with either Air-GPAW or He/0₂-GPAW. (A) The germination kinetics of unaged lettuce seed demonstrates that classical and EPOWER priming both equally increase the germination performance. (B) The germination kinetics of 3 days aged lettuce seed demonstrates that the aging tolerance of EPOWER primed seed is significantly higher compared to classically primed seed. (C) The Germination Rates (50%) demonstrate that EPOWER and classical priming both increase the germination speed. (D) The Germination Rates (50%) demonstrate that compared to classically primed seeds, the EPOWER primed seeds are significantly more tolerant to aging. EPOWER priming provides positive priming effects without negative effects on the aging sensitivity. (E) Relative seed storability (aging resilience) of EPOWER primed and classically primed seed as compared to the unaged, unprimed control. In contrast to classical priming, our EPOWER priming retains the seed storability and therefore delivers increased shelf life.

Detailed Description of the Invention

The inventors have developed a seed priming method referred to as EPOWER priming (Enhanced Priming Outcome With Enhanced Retainment of shelf life (seed storability)). This priming technology retains seed storage resilience without the need of an additional post-priming treatment.

EPOWER priming has the advantage that it improves seed vigour (rapid and uniform germination and robust seedling establishment under varying environmental conditions including abiotic stress), removes the light requirement for germination, and alleviates thermoinhibition and residual dormancies, whilst critically not compromising the storability of the seeds. Further to this, it is applicable to conventional and organic seeds as it does not involve any addition of chemicals. EPOWER-priming technology therefore combines all the positive effects of classical seed priming without any of the known negative consequences on the seed storability.

The EPOWER priming seed technology is an invention which offers a practical, cost- effective and high through-put method of applying gas plasma activated water (GPAW) to commercial crop seeds, resulting in improved seed vigour whilst maintaining aging tolerance and thereby seed storability (Figure 2). Water is treated in a chamber by a plasma reactor, creating a chemistry of integral ions and radicals of the resultant GPAW. The GPAW is then distributed evenly in a large rolling drum (‘normal’ hydro-priming equipment) which contains the crop seeds of interest. As in any priming treatment a limited amount of water, in our case GPAW, is used; it is enough to activate the seeds but will not be enough to allow the completion of germination. The seeds after a defined (seed variety depending) activation period with the GPAW are then dried back to a moisture content suitable for seed storage. Within the details of this invention, the underlying molecular mechanism of how GPAW alleviates seed dormancy and enhances the vigour has in part been proved and understood with our research. We also have produced novel physiological data which demonstrates the maintenance of storability and removed light requirement for germination of EPOWER primed seed as described in detail in the examples.

Loss of seed storability post-classical-priming causes several logistical problems for seed companies due to the significant loss of shelf-life. EPOWER priming’ s ability to maintain storability facilitates companies to distribute seeds further afield or store for longer, beyond one season, whilst maintaining higher levels of viability and vigour (Figure 2). In addition, many companies have introduced further additional post-priming treatments, which means additional costs, time and efforts. EPOWER seed priming can remove this requirement for companies and provide a superior primed seed product. The EPOWER priming is conducted as follows: Water is treated in a chamber by a bubble reactor used to produce gas plasma (Figure 3), creating a chemistry of integral ions and radicals of the resultant GPAW. Typical composition and concentration of GPAW integral ions and radicals are described in detail in the examples. For conducting the EPOWER priming, the GPAW is then distributed evenly in a large rolling drum (classical hydro-priming equipment) which contains the crop seeds of interest. As in any priming treatment a limited amount of water, in our case GPAW, is used; sufficient to activate the seeds but not sufficient to allow the completion of germination. The seeds after a defined (seed variety depending) activation period with the GPAW are then dried back to a moisture content suitable for seed storage. This GPAW seed priming treatment increases the seed vigour, alleviates the seeds dormancy (light and non-light dependent dormancy) and - most importantly - does this while maintaining the seed storability which is lost in conventional priming methods.

The productivity of existing seed priming facilities can also be increased by using EPOWER. The schedule for the treatment and sale of crop seeds is dependent on environmental conditions, therefore priming drums are only in operation during the sowing seasons. Extending the storability of primed seeds broadens the time window priming drums can be in operation, increasing productivity, whilst also removing some of the logistical difficulties associated with inconsistent treatment schedules. Logistical issues are also alleviated due to a reduction in priming time required to maximise seed vigour. This is achieved due to EPOWER priming’s germination stimulating effect which results in less priming time to maximise seed vigour.

In tandem with the improvement of seed storability, we have shown EPOWER has a clearly defined germination stimulating effect, offering an advantage over other treatment processes. Significantly higher viability and prolonged retainment of seed vigour was observed, combined with improved seed stress tolerance. Furthermore, the most common use of gas plasma technology in industry is to sterilise, demonstrated by its use to sterilise food packaging, medical equipment, wounded skin and the extensive research being conducted for water treatment facilities with the aim of removing harmful pathogens. EPOWER priming will also help reduce microorganisms on the seed’s outer layers and possibly internally through the treatment process and hence help prevent cross contamination through the drums. This process does not involve any chemicals and leaves no residual chemicals on the seed deeming the treatment suitable for the organic seed industry. Seed technology is of prime importance in fruit, vegetable and crop seed production (agriculture) as well as wild species (restoration) and ornamental plants (horticulture). EPOWER seed priming can make significant vigour-related improvements to the uniformity, stress tolerance and speed of germination, removing the light requirement, whilst maintaining seed storability as a major advantage (Figure 2). Because priming with EPOWER can stimulate germination, it has the potential to also provide high seed quality for wild species and for cultivated species which have not been fully domesticated by breeding. Most importantly, EPOWER priming of seeds comes with maintaining the seed storability allowing companies to store their primed seed for longer, beyond one season, which is a breakthrough and high priority for seed companies.

Within the details of this invention, the underlying molecular mechanism of how GPAW stimulates germination and enhances the vigour, has in part been proved and understood with our research. We also have produced novel physiological data which demonstrates the maintenance of shelf life and retainment of aging resilience during dry storage of EPOWER primed seed. Seed germination is dependent on two opposing forces, the expansion of the radicle (embryonic root) and the structural integrity of the seed's covering layers, with the covering tissues being the micropylar endosperm, testa (seed coat) and in some cases the pericarp (fruit coat). Stimulation of embryo growth as well as weakening of the covering layers are regulated and achieved by a shift in the balance of phytohormones (contents and signalling sensitivities) leading to the localised production of cell-wall remodelling proteins (CWRP) and ROS. These molecular processes initiate germination and are imperative to synchronizing seedling establishment. We have demonstrated GPAW treatment and the EPOWER seed technology have a significant effect, on hormonal and cell wall loosening mechanisms as well as on endosperm weakening as described in detail in the examples.

Examples Here the inventors demonstrate that EPOWER priming retains the aging tolerance (storability or "shelf life") of treated seeds in contrast to the prior art, classical seed priming. The experiment was carried out with freshly harvested mature seeds of Lactuca sativa (lettuce) cultivar ‘Grand Rapids’ (Figure 5) and with a large number of diverse seeds (Table 1). Lettuce is commonly primed to remove its light requirement for germination as it produces photo-dependent, physiologically dormant seeds. Lettuce is a prime example for which classical seed priming is reducing seed storability due to increased aging tolerance. We show here that EPOWER priming improves the germination combined with retaining the storability and aging tolerance of lettuce and other crop seeds.

MATERIAL AND METHODS Seed material of different crop species

Lactuca sativa (lettuce) cultivar ‘Grand Rapids’ was grown at 20/18°C in a 16/8 h day/night cycle. Lettuce seeds were harvested at maturity, dried down at 15% relative humidity (RH) and stored above silica gel or at -20°C in air-tight containers. The moisture content of these freshly harvested mature dry seeds was determined by weighing before and after heating at 120°C (Mettler Toledo HB43-S).

Production of gas plasma activated water (GPAW)

The bubble reactor engineered to produce GPAW (Figure 3) consists of 12 high voltage AC electrodes covered in a dielectric material fixed below a gas permeable stainless- steel membrane. Above the membrane is a tank containing 100 ml of double-distilled water (dd!LO). Carrier gas flows past the electrode at 1 standard litre per minute (1SLPM), and then through the membrane and dd!LO. For activation, plasma is formed between the electrodes and the membrane within the carrier gas. The NTAGP after-glow then flows through the membrane bubbling up through the water facilitating radical and ion diffusion into the water, producing the GPAW. Two GPAW regimes were used distinguished by the carrier gas used. The first with air flowing at 1SLPM with the plasma discharge power at 15.8 kV (Air-GPAW), the second with helium (98%) and oxygen (2%) (He/0₂-GPAW) mixture flowing at 1SLPM with the plasma discharge powered at 8.5 kV. Compressed air, helium (BOC, N4.6), oxygen (BOC, N5.0) and nitrogen (BOC, N5.5) gases were used for all treatments and both their mixture and flow rate were controlled through Alicat MC- series mass flow controllers. Voltage and frequency measurements for all devices were recorded using a Tektronix P6015A probe and TBS 1102B Digital Oscilloscope. GPAW Chemical quantification

H2O2 was quantified colourimetrically using the titanium sulphate method (Eisenberg 1943). Peroxotitanium (IV) complex is formed by the reaction of H2O2 with titanyl ions under acidic conditions; absorbance was measured at 407 nm. A standard curve constructed using pure H2O2 standards and then was used to calculate a molar extinction coefficient. NO2 and NO3 were quantified simultaneously using Griess and vanadium (III) chloride (VCI3) reagents in an assay described in detail by Garcia-Robledo et al. (2014) OH^* radicals were quantified through the hydroxylation of the chemical probe terephthalic acid, the resultant 2-hydroxy terephathalic (HTA) acid product is fluorescent (excitation 315 nm, and emission at 425 nm), standards of HTA were used for quantification (Sahni & Locke 2006). OH^* radical synthetic rate what quantified both during plasma discharge and post-discharge. EPOWER priming and classical priming of seeds

The change of moisture content compared to the dry seed was quantified over time during imbibition by weighing. Lettuce seeds were bucket primed via imbibition with 3 ml of priming solution within a 6 cm plastic Petri-dish at room temperature for 4 h, resulting in 60% moisture content before being dried for 2 days at 29°C and then 2 days on silica gel at room temperature (15% RH). Classical priming was conducted by using ddH₂0, whereas EPOWER priming was by using GPAW as the priming solution. The two different GPAW regimes delivered either Air-GPAW (with air used as carrier gas) or He/0₂-GPAW (with the He/0₂ mixture used as carrier gas). A general scheme for the classical seed priming and drying process and subsequent seed dry storage is presented in Figure 1.

Artificial aging treatments

Artificial aging treatments were conducted to compare the aging sensitivities of non- primed with classically primed and GPAW primed lettuce seeds. To age seeds, they were subjected to elevated humidity and temperature in contrast to ambient conditions (room temperature and "normal" relative air humidity) and seed dry storage in company warehouses (ca. 15 °C and low relative air humidity). Lithium chloride in solution was used to create an elevated and controlled relative humidity of 70% above the solution at 50°C (5.9 mM LiCl, 500 ml in an airtight plastic container). Seeds were placed on a platform above the solution within the container to subject them to the aging environment for 3 days. Germination assays were subsequently conducted to quantify the aging sensitivity, as compared to the unaged states.

Germination assays

For each germination assay, 30 seeds were used for each replicate and were imbibed in 6 cm diameter Petri dishes containing two filter papers and 2 ml of ddH20. The Petri dishes were subsequently sealed with parafilm and incubated in constant light. Germination was assayed at either non-optimal (29°C) or at optimal (24°C) constant temperature. To score germination, triplicate replicates were used for each treatment group, and the completion of germination by visible radicle protrusion through all covering layers (pericarp, testa and endosperm for lettuce) was scored visually through stereomicroscopy; the visible protrusion of the radicle (embryonic root) was scored as the visible completion of the germination process.

Statistical analysis

Germination curves were graphed and analysed using Prism 7.01 software (GraphPad Software, Inc., USA). For each treatment group, the mean seed population percentage between replicate Petri dishes were plotted with SE. Curves were fitted via Hill functions. To quantify differences in germination performance ('speed') their germination rates at 50% germination were compared. Germination Rate (GR50%) is defined as the inverse of time taken to reach 50% germination in a population (t50^_1), this was plotted with SE and compared through ANOVA and Tukey's analysis; t50^_1 values were obtained through the fitted Hill functions of individual replicates.

RESULTS

The bubble reactor GPAW was characterised for NO2 , NO3 and H2O2 under two treatment regimens (Air-GPAW and He/0₂-GPAW) (Figure 4). NO2 in Air-GPAW revealed concentrations of 56.5 mM, 47.3 pM and 35.4 pM for 30 min, 45 min and 60 min treatment times respectively (Figure 4B). NO3 concentration in Air-GPAW was 3.4 mM, 5.0 mM and 6.2 mM for 30 min, 45 min and 60 min respectively. H2O2 concentration was 47.8 mM, 33.3 pM and 30.2 pM for 30 min, 45 min and 60 min respectively (Figure 4B).

He/C -GPAW showed low NO3 concentrations of 22.7 pM 18.1 pM and 15.7 pM (30 min, 45 min and 60 min respectively) and NO2 was not detectable. H2O2 was recorded in concentrations of 180.4 pM, 294.9 pM and 387.7 pM for 30 min, 45 min and 60 min respectively (Figure 4A). During plasma discharge, hydroxyl radicals were generated at a rate of 1.8 pm/min and 1.3 pm/min for He/0₂ and Air-GPAW respectively (Figure 4C/D). Post-discharge, He/0₂ showed a clear production of OH^* through secondary reactions at a rate of 0.1363 pM/h. Air-GPAW displayed a negligible production of OH^* post-plasma discharge of 0.0113 pM/h (Figure 4D).

For the classical seed priming (with dd¾0) of freshly harvested mature lettuce seeds, Figures 5A and 5C confirm what is known from the literature namely that the germination performance is significantly (p<0.05) improved and the aging tolerance significantly (p=0.0002) decreased (Figure 5B) leading to poor storability (Figure 5E). The 3 day aging treatment was chosen because it affects the germination speed of lettuce without any negative effects on the maximum germination and viability of the seeds in any of the treatments (Figure 5B). Our EPOWER priming of lettuce seed with either Air-GPAW or He/0₂-GPAW resulted in an increase in germination performance, akin to classical priming (Figures 5A and 5C). However, in contrast to classical priming, the EPOWER priming did not lead to a massive reduction in germination performance upon aging (Figure 5B). While classical (with ddH₂0) priming followed by 3 days of aging led to a 42% reduction in GR50% (p=0.0002), the GR50% of Air-GPAW and He/0₂-GPAW primed seed followed by 3 days of aging was only 25% (p=0.0059) and 20% (p=0.0215) reduced, respectively (Figure 5D). The classical priming caused a high aging sensitivity resulting in only ca. 65% relative storability compared to the unaged and unprimed control (Figure 5E). Compared to this, the Air-GPAW and He/0₂-GPAW primed seeds demonstrated a significantly improved retainment of the aging tolerance at an 85-90% level (Figure 5E). EPOWER priming, therefore, combines the positive effects of priming with retaining the aging tolerance and therefore without a significant reduction in the seed storability (shelf life).

The above finding with lettuce seeds was verified with a larger list of seeds covering different species including horticultural (vegetables, flowers) and agricultural crops (Tables 1 and 2). Air-GPAW was used in these experiments for the EPOWER priming treatment and the relative priming effect of EPOWER priming compared to classical hydropriming was calculated from the subsequent germination assays (Table 1). The GP AW -priming/Hydro-priming values in Table 2 are between 0.93 and 1.09 demonstrating that EPOWER priming provides at least an equally strong priming enhancement to all the seeds investigated. To compare the effects on the seed storability, aging assays were conducted with the air-GPAW and classically primed seeds. Because species differ in their sensitivities the precise conditions of the aging assays (temperature, relative humidity, duration) were optimised for each species (Table 1). The seed storability of EPOWER primed seed was calculated by dividing the germination percentages at a defined time during imbibition of the aged GPAW-primed seed to the germination percentages of the control seed (unaged and unprimed). The seed storability of classically primed seed was calculated in the same way at the same time of imbibition. The obtained values (Table 2) show that compared to the control seeds EPOWER retains most of the storability or even provides better storability compared to the control seeds. Table 1 also shows that the seed storability of the GPAW-primed seeds is always much better compared to the hydro-primed seeds. Calculation of the ratios "GPAW-primed / hydro-primed" indicates that the EPOWER priming provides 1.4- to 3.0-fold better relative seed storabilities (Table 2). EPOWER priming, therefore, combines the positive effects of priming with retaining the aging tolerance and therefore without a significant reduction in the seed storability (shelf life) as demonstrated with a large number of species. Table 1. Comparative germination results of control seeds either "Not primed", "GP AW -primed" and "Hydro-primed" as compared to the seeds being subjected to artificial aging by incubation at the conditions (temperature, relative humidity, time) to quantify the ageing sensitivity and storability.

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Table 2. Comparative seed priming effect and seed storability (aging resilience) of seeds either "GP AW -primed" or "Hydro-primed". Note that the column "Seed priming" provides a value for the priming effect of the GPAW-priming as compared to the Hydro-priming demonstrating that the GPAW-priming effect is either similar (values around 1) or better (Swede: value 2 means twice as good). Note further that the column "Seed storability GPAW/Hydro" provides a value for the seed storability of the priming seed; values above 1 indicate better storability of the GPAW- primed seeds.

DISCUSSION

During seed development, seeds on the mother plant firstly and progressively gain the ability to germinate, and then during late-maturation they progressively develop seed vigour ("germination performance") and seed longevity ("storability or shelf-life") until reaching physiological maturity (PM), which is the point of maximum seed quality. The acquired seed longevity is the lifespan of the seed lot, as determined by a complex expression of physiological traits including cellular mobility, storage compound composition, endogenous protective compounds such as antioxidants, and the ability to resist and repair damage. Interestingly, seed vigour can initially continue to increase during post-harvest after-ripening dry storage, but eventually, it decreases during storage due to seed aging and deterioration processes. The speed of the seed aging during storage depends on the intrinsic seed lot properties combined with the ambient storage conditions with RH (generating defined seed moisture), temperature and oxygen, these being the three most decisive factors. In general, higher RH, higher temperature, and higher oxygen concentration accelerate seed aging during storage. Biochemical deterioration processes are underpinning this and lead to a decrease and eventually a loss of seed vigour. Vigour loss occurs first and the seed aging is subsequently followed by a decrease in the number of normal (usable) seedlings, and finally by seed viability loss. Biochemical mechanisms which cause the decrease in seed vigour and eventual viability include oxidative damage to DNA, mRNA, proteins, lipids and cellular structures by Reactive Oxygen Species

(ROS) if they are produced in excess. Their major forms include singlet oxygen ('qi), H2O2, the superoxide anion (O2^* ) and the highly reactive hydroxyl radical (OH^*). The ability of repair of the accumulated damage, for example during seed imbibition, is crucial.

During seed germination cell-wall remodelling protein (CWRP) are induced endogenously within the micropylar endosperm by gibberellins (GA) and it is now clear that apoplastic (cell wall) ROS are key effectors in this mechanism (Liszkay et al. 2004; Miiller et al. 2009). In L. sativum and A. thaliana it was shown that treatment with O2^* synthesis inhibitor diphenyleneiodonium chloride (DPI) delayed radicle emergence, and through puncture force analysis it was confirmed that the ROS scavenging of DPI reduced and delayed endosperm weakening in contrast to both untreated samples (Miiller et al. 2009). ROS appears to act in similar fashion in roots as O2^* , H2O2 and OH^* production have been located via histochemical assays and electron paramagnetic resonance spectroscopy in growing zones (Liszkay et al. 2004). Miiller at al. (2009) provided direct in vivo evidence for hydroxyl radical (OH^*)-mediated cell wall loosening during plant seed germination and seedling growth. This work showed that OH^* is generated in the cell wall during radicle elongation and endosperm weakening of Lepidium sativum precedes for radicle emergence, as demonstrated by direct biomechanical measurements. Distinct and tissue-specific target sites of OH^* attack on polysaccharides were evident for the radicle and the endosperm. Plant cell wall loosening by OH^* is a controlled action of this type of reactive oxygen species (ROS). This work, therefore, showed that direct action of ROS on the endosperm tissue causes its weakening evident from the reduction in puncture force. These ROS species are also produced in Air-GPAW which is a source of OH^* amongst many other reactive compounds (Figure 4). Both Air-GPAW and He/0₂- GPAW therefore act by physically reducing the resistance of the seed's covering layers to the threshold typically associated the completion of germination.

The major problem with classical priming is that while it provides the benefits of increased seed vigour, it has been shown in many species to decrease the seed storability, by causing an increased aging sensitivity. It has been shown that classically primed lettuce seeds aged 2-5 fold faster compared to the unprimed control seeds. Other results showing compromised storability were obtained for many vegetable and flower seeds; this highlights the clear limitation of classical seed priming. That classical priming decreases seed storability by increasing the aging sensitivity is also apparent for the seeds of cereals and of sugar beet.

EPOWER priming does not appreciably increase the aging sensitivity (Figure 5). The desired result of any priming (Figure 1) is to advance metabolic processes such as respiratory pathways initiating DNA repair mechanisms and alter concentrations of specific phytohormones to unify the germination of the seed batch and ultimately enhance seed vigour. Our results with the classical and the EPOWER priming technology are aligned with this understanding and the observed benefits for the field performance of seeds and seedlings. However, in contrast to non-primed seeds, classically primed seed showed a significant loss of germination speed and uniformity due to their increased aging sensitivity during storage; highlighting the frustration experienced in the industry. This is very different for EPOWER primed seeds in which storage resilience was retained: Against the industry standard (classical primed seeds), Air-GPAW and He/Ch-GPAW showed increased lettuce seed aging tolerance (Figure 5B). In summary, EPOWER priming, therefore, combines the benefits of classical priming on germination performance and seedling establishment with the benefit of retaining a high level of aging resilience and therefore provide an increased seed storability.

Seeds have several mechanisms to cope with stressful (oxidative) environments. Salt stress, for example, causes ionic imbalances which result in the accumulation of ROS due to interference with the electron transport chain. This over accumulation results in damage similar to that experienced by aged seeds, namely; oxidative damage of the membrane lipids, proteins and nucleic acids. Halophytes have mechanisms to counteract this, they detoxify plant cells by accumulating a range of enzymatic and non-enzymatic antioxidants such as glutathione reductase and phenolics. Upregulation of these antioxidant systems have shown to correlate with ROS and NO accumulation, both products are chemically synthesised in the GPAW. Enhanced antioxidant systems, including tocochromanols, glutathione, ascorbic acid, and the corresponding enzymes are hallmarks of stress resilient seeds. ROS is also involved in hormonal signalling and interacts with the hormonal network which affects germination.

This indicates possible explanations on how GPAW improves aging tolerance during EPOWER priming. In addition to these antioxidant systems, the increased production of heatshock and LEA proteins in oxidative environments during the EPOWER priming could be another mechanism for the increased storage resilience of EPOWER primed seeds. Heatshock proteins (sHSPs) are believed to have a role in aging tolerance. They accumulate during late seed maturation, a process which in part is triggered by ABA, and have been shown in transgenic tobacco seeds which over express sHSPs, specifically HaHSFA4a and HaHSFA9, to decrease sensitivity to aging. LEA proteins are also synthesised in late embryogenesis and provide a level of desiccation tolerance by influencing the stability of membranes during freezing or drying; therefore, they significantly influence the longevity of stored seed. Both sHSPS and LEA protein are good targets for our investigation into how EPOWER priming enhances storability against the industry standard. It has been identified there is an irregular delay between transcription and protein synthesis of LEA proteins suggesting the mechanism is influenced by post-translational modification; of the oxidative species synthesised in GPAW, NO is a known post-translational modifier, causing nitrification and nitrosylation of amino acids, and could, therefore, influence this regulation. EPOWER could therefore also be active by interfering with post-translational modification. The concept of enhancing antioxidant mechanisms though EPOWER priming alongside enhancing seed vigour during metabolism activation offers an enticing prospect for the seed industry, as it limits the loss of ‘shelf-life’ implicit with classical seed priming.

Claims

1. A method for priming seeds, comprising the steps of:

(1) imbibing a seed in gas plasma activated water; (2) drying the seed.

2. A method according to claim 1, wherein the gas plasma activated water is air-gas plasma activated water.

3. A method according to claim 1, wherein the gas plasma activated water is He/C - gas plasma activated water.

4. A method according to any preceding claim, wherein the seed is imbibed in the gas plasma activated water until the moisture content of the seed is 30-70 %.

5. A method according to any preceding claim, wherein the seed is imbibed in the gas plasma activated water until the moisture content of the seed is about 2-20 % below the required moisture content for germination of the specific seed lot of a species.

6. A method according to any preceding claim, wherein the seed is imbibed in the gas plasma activated water for 12 hours to 8 days.

7. A method according to any preceding claim, wherein the seed is imbibed in the gas plasma activated water for the length of time ± 3 days which causes the maximum increase in vigour of the specific seed lot of a species being treated.

8. A method according to any preceding claim, wherein imbibition of the seed does not stimulate germination.

9. A method according to any preceding claim, wherein the drying step comprises drying the seed until the moisture content of the seed is 3-20 %.

10. A method according to any preceding claim, further comprising an additional step of drying the seed prior to step (1).

11. A method according to any preceding claim, further comprising the step of: (3) storing the seed.

12. A method according to claim 11, wherein the seed is stored for at least 3 months.

13. A method according to claims 11-12, further comprising the step of: (4) germinating the seed.

14. A seed produced by the method of any preceding claim.