BOTANY project work REPORT

Submitted by: vineela devarakonda

b.sc.microbiology
 PHYSICAL METHODS OF SEED
INVIGORATION ADVANTAGES AND
CHALLENGES
 PHYSICAL METHODS OF
SEED INVIGORATION
ADVANTAGES AND
CHALLENGES
SUBMITTED BY
D.Vineela
3RD.B.Sc.Mirobiology
20033442054

D.K.GOVT COLLEGE OF WOMEN(a)

NELLORE-524002
december-2022

Project report certificate

 This is to certify that the project report
entitled”PHYSICAL METHODS OF SEED
INVIGORATION ADVANTAGES AND
CHALLENGES” Submitted by DEVARAKONDA
VINEELA 3RD.B.Sc.Microbiology to the D.K.GOVT
COLLEGE OF WOMEN(A)NELLORE Affiliated to
VIKRAM SIMHAPURI UNIVERSITY,NELLORE For
the partial fulfillment for the award of the degree of
bachelor of science is a bonafide record of project work
carried out by her under my mam Dr.B.Rajaeshwari
mam lecturer in Botany.

D.K.govt college of women(A)

Nellore,524001

Date:
PROJECT GUIDANCE
DR.B.Rajeshwari mam

Declaration
 I hereby declare that this project entitled
physical methods of seed Invigoration
advantages and challenges is original and
bonafide work done and submitted by me
during the year 2022-23 under the guidance of
DR.B.Rajeswari mam Lecturer in the
Department of Botany D. K. Govt. College for
Women (A), Nellore.

Acknowledgement
I would like to express my sincere thanks to Dr.D.GIRI,
Principal,for providing me necessary facilities and beautiful
college atmosphere to carry out my project work.
First and foremost I would like to thank my Head of
Department Dr.P.VICTORIA RANI Lecturer in Botany for
her valuable guidance and constant help which enabled the
successful completion of my project work.
I would like to specially thank to Smt. DR.B.Raja Rajeswari
mam for her constant suggestions during my project work.
I would like to thank Department of Botany for providing this
project and K.V.Ramana mam, botany work they constant
help during my work.
I take immense pleasure to express my thanks to each and
every faculty member for giving their valuable suggestions. I
thank all my colleagues and friends for giving their moral
support.
I would like to express my humble gratitude to my beloved
parents for their blessings throughout my academic career.
I gratefully thank D.K.GOVT DEGREE COLLEGE(A) for
the beautiful environment.

VINEELA
DEVARAKONDa
TABLE OF CONTENTS
 S..NO ACTIVITY PAGE
NO
1

abstract
In the context of seed technology, the use of physical methods for
increasing plant production offers advantages over conventional
treatments based on chemical substances. The effects of physical
invigoration treatments in seeds can be now addressed at multiple
levels, ranging from morpho-structural aspects to changes in gene
expression and protein or metabolite accumulation. Among the
physical methods available, “magneto-priming” and irradiation with
microwaves (MWs) or ionizing radiations (IRs) are the most
promising pre-sowing seed treatments. “Magneto-priming” is based
on the application of magnetic fields and described as an eco-friendly,
cheap, non-invasive technique with proved beneficial effects on seed
germination, vigor and crop yield. IRs, as γ-rays and X-rays, have
been widely regarded as a powerful tool in agricultural sciences and
food technology. Gamma-rays delivered at low dose have showed to
enhance germination percentage and seedling establishment, acting as
an actual ‘priming’ treatment. Different biological effects have been
observed in seeds subjected to MWs and X-rays but knowledge about
their impact as seed invigoration agent or stimulatory effects on
germination need to be further extended. Ultraviolet (UV) radiations,
namely UV-A and UV-C have shown to stimulate positive impacts on
seed health, germination, and seedling vigor. For all mentioned
physical treatments, extensive fundamental and applied research is
still needed to define the optimal dose, exposition time, genotype- and
environment-dependent irradiation conditions. Electron paramagnetic
resonance has an enormous potential in seed technology not fully
explored to monitor seed invigoration treatments and/or identifying
the best suitable irradiation dose or time-point to stop the treatment.
The present manuscript describes the use of physical methods for seed
invigoration, while providing a critical discussion on the constraints
and advantages. The future perspectives related to the use of these
approaches to address the need of seed technologists, producers and
trade markers will be also highlighted.

INTRODUCTION
One of the biggest challenges that humanity is now facing is
improving the sustainability of agriculture while reducing its
environmental impact, to meet the food demands of the
growing global population (Edmondson et al., 2014). The idea of
agricultural sustainability relies on the need to develop technologies
and practices with no adverse effects on environmental goods and
services but still leading to improvements in food productivity
(Pretty et al., 2006). High vigor seeds are proxy of crop
establishment and sustainable productivity. In 2012, the value of the
EU seed market reached around €7 billion corresponding to 20% of
the global market ranking third after USA and China (Ragonnaud,
2013). In this economically competitive context, innovative
biotech/molecular tools, treatments, or products are crucial to speed-
up the consolidation process of the seed industry.

Despite contemporary agriculture largely uses chemical

compounds, the use of physical factors might represent a good
alternative to raise the yield of agricultural production while
improving plant protection and storage Physical methods for seed
invigoration offer several advantages over conventional treatments
based on chemical substances. First, they reduce the use of
fertilizers, thus decreasing pollution of on-farm produced raw
materials. Another advantage is that physical methods may be also
used for seed disinfection before sowing and during the storage
(Aladjadjiyan, 2012). Possible approaches include the treatment with
electromagnetic waves (EWs), magnetic fields (MFs), the
ultrasounds (US), and ionizing radiations (IR). Food treatment with
IR, which enhances its microbiological safety and storability, has
been one of the most studied technologies of the 20th century as a
result of the general concern about the potential role of food
irradiation in the safety of food supply (Farkas and Mohácsi-Farkas,
2011). Also, some of recent advances on the impact of IR on living
organisms have been driven by the Space research. Among space
factors, the ionizing radiation (including both gamma and X
radiation) is one of the main growth constraints of organisms (Arena
et al., 2014; Wolff et al., 2014).

In this context, the cultivation of higher plants in space involves

the development of new agro- technologies for the design of
ecologically space greenhouses and a deep understanding of the
effects of space factors on biological systems (De Micco et al.,
2014). Importantly, the long term effects of the extraterrestrial
environment on plant growth and development still needs to be fully
addressed (Wolff et al., 2014).

The impacts of physical treatments in plants are being addressed at

multiple levels, from morpho-structural aspects to changes in gene
expression. Plant systems are excellent models for the
characterization of biological responses to several environmental
factors, which includes the genotoxic effects of physical and
chemical reagents (Zaka et al., 2002; Balestrazzi et al., 2011;
Confalonieri et al., 2014; Faè et al., 2014; Macovei et al., 2014).
Physical treatments impacts in plants depend on several factors:
radiation or MF (e.g., type, total dose, and dose rate) and plant
features, such as species, cultivar, age, ploidy, and complexity of
the target organ or tissue (De Micco et al.,

Low-vigor seeds can be improved using a wide range of

treatments, some of them well characterized such as hydropriming
and osmopriming. In the context of seed technology, physical
methods have showed several advantages over conventional
osmopriming protocols (Bilalis et al., 2012). One of the most
investigated physical pre- sowing seed treatments in agriculture is
based on the use of MFs (Aguilar et al., 2009).

MFs have been described as eco- friendly, cheap, and non-

invasive technique (Bilalis et al., 2012; Efthimiadou et al., 2014).
Additionally, the impact of other types of physical treatments, such
as, gamma (γ), and X ionizing, UV, and microwave (MW)
radiation will be addressed in this review.

Despite thermopriming being another important physical

treatment used for improving seed vigor, it will not be covered
here since it has been recently addressed by Paparella et al. (2015).
The present manuscript attempts for reviewing the use
of physical methods for seed invigoration, while providing a critical
discussion on the constraints and advantages. The future
perspectives related to the use of these approaches to address the
need of seed producers, trade markers and consumers will be also
highlighted.

“MAGNETO-PRIMING”: A
SUITABLE INVIGORATION
PROTOCOL
The effects of MFs in living organisms have received
considerable attention. Indeed, the Earth’s MF (50 μT) is a
natural component of the environment (Belyavskaya, 2004). One
Tesla (T) unit of magnetic flux density corresponds to 1 kg s-2A-
1, where A stands for Ampere, the unit of electrical current or the
current that flows with an electric charge of one Coulomb per
second. Krylov and Tarakanova (1960) reported the effects of
MFs in plants for the first time. In their work, a definition for
magnetotropism came out when referring to the auxin-like effect
exerted by MFs on germinating seeds the case of the Earth’s
magnetic field or by industrial processes, EMFs are generated by
electrically charged objects being extended indefinitely
throughout space (Mitchell and Cambrosio, 1997)

In the seed research context, the impacts of MFs on seeds have

been studied with the overall goal to understand their suitability
to derive new seed treatments. The beneficial effect of pre-
sowing magnetic treatments for improving germination
parameters and biomass accumulation has been described for a
wide range of plants (Table 1) and recently reviewed by Teixeira
da Silva and Dobránszki (2015). In these studies, different MF
strengths have been tested ranging from 0 to 300 mT. Magneto-
primed seeds showed improved germination rates, vigor and
seedling biomass or root development. Another interesting
feature of MFs-treatments is that they appeared to enhance
tolerance to biotic (De Souza et al., 2006) or abiotic stresses
(Javed et al., 2011; Anand et al., 2012) as result of the antioxidant
response activation.

Increased antioxidant enzyme activities of superoxide dismutase

(SOD), catalase (CAT), and glutathione reductase (GR) were
described in magneto-primed cucumber (Cucumis sativus L.)
seeds (Bhardwaj et al., 2012). In agreement with these findings,
Baby et al. (2011) highlighted a reduced production of
superoxide radicals (O2-) in magneto-primed soybean (Glycine
max (L) Merr. var Js-335) seeds. Consequently, MFs-treatments
have the additional potentiality to be used for minimizing the
drought- or disease-induced adverse effects on crop productivity.
Summary of magnetic fields (MFs) effects on seed and seedling
performance.
Based on the previous assumption, some interest has been
devoted to understand the physiological, biochemical, and
molecular mechanisms underlying the improved performance of
plants grown from MFs-treated seeds (Vashisth and Nagarajan,
2010; Baby et al., 2011; Javed et al., 2011; Anand et al., 2012).
Vashisth and Nagarajan (2010) showed that, in sunflower
(Helianthus annuus L.) seeds, exposure to magnetic fields
(SMFs) appear to act like priming, with similar enhancement
effects on seed performance. Indeed, these authors detected
enhanced alpha-amylase, dehydrogenase and protease activities
in magneto-primed seeds during imbibition when compared to
non-treated ones. The higher activities of hydrolyzing enzymes
were then linked to the enhanced seed germination, seedling
vigor, and rooting traits of SMFs-treated seeds.

Environmental factors, also known as interdependent factors,

such as light, humidity and temperature are implicated in
modulating horticultural and crop seed performance but the
impact of their combined application with MFs remains unclear.
This resulting knowledge could be relevant to develop new
locally adapted seed treatments. Poinapen et al. (2013) studied
how the combined impact of MFs and interdependent factors
affected seed viability and performance in tomato (Solanum
lycopersicum L. var. MST/32) under laboratory conditions.
Relative humidity showed to be key factor modulating seed
performance in magneto-primed seeds, especially during early
stages of seed germination/imbibition. These findings are
relevant for determining the most suitable combination of
physical parameters to develop improved magneto-priming
protocols.

Ionizing Radiation Treatments

Gamma Radiation Is a Promising
Invigoration Approach
Ionizing radiation is a powerful tool in the field of agricultural
sciences and food technology, being frequently utilized to
address food microbiological safety and storability issues
(Jayawardena and Peiris, 1988). Gamma (γ) radiation is a high-
energy type of the IR able to penetrate and interact with living
tissues. It is usually delivered by means of Cobalt-60 (60C)
sources (Moussa, 2006). The absorbed dose of IRs is expressed in
units of Gray (Gy), in which 1 Gy dose corresponds to 1 Joule
radiation energy adsorbed per kilogram. When interacting with
biological material (organism, organ, tissue), the absorbed dose
of IRs could be also expressed in Sievert units (Sv), in which 1
Sv dose corresponds to 1 Joule radiation energy adsorbed per
kilogram of organ or tissue weight. Another key parameter for
establishing IR treatments is the dose rate (rate of energy
deposition, expressed as Gy h-1). Currently, γ-rays are
predominantly used in situation in which a high sterilization level
is required.
Gamma-irradiation treatments are widely used to abolish
microbial contamination or control insect pests and pathogens,
thus acting in disease prevention. Besides safety issues, γ-
irradiation is also used to delay fruit ripening and vegetable
sprouting by hampering the activation of key enzyme activities,
contributing to extended crop shelf-life (Mokobia and
Anomohanran, 2005; Moussa, 2006). In another agricultural
context, γ-rays represent an effective mutagenic tool for plant
breeders who want to add new traits into commercially valuable
crops and develop new varieties (Irfaq and Nawab, 2001).

Among the different radiobiology aspects, the characterization of

the γ-rays effects on seeds is a topic that is recently receiving
evident attention. Studies undertaken have been particularly
focused on the impacts of low dose rate and/or low total dose γ-
irradiation on germination yield and seedling performance.
Gamma-rays directly interact with the cell components at
multiple levels, reaching membranes, proteins, and nucleic acids
(Kovács and Keresztes, 2002).

However, an indirect action is also well reported through the

generation of reactive oxygen species (ROS) from water
radiolysis (Borzouei et al., 2010; Esnault et al., 2010). ROS
diffuse and damage cellular macromolecules and organelles.
Nevertheless, the biological effects γ-rays have been
demonstrated to be strongly dependent on the intensity, dose-rate
and exposure time. Concerning seed treatments, γ-rays delivered
at low dose enhance germination percentage and seedling
establishment, acting like an actual ‘priming’ treatment.
. The transcriptomic data obtained showed that a differential
response between acute or chronic treatments is observed. In
response to acute radiation, plant metabolism is shifted to
immediate repair of the damage, activation of pro-survival
mechanisms and possibly the inhibition of cell division/cell
differentiation. In contrast, chronic irradiation triggered a totally
different response, in which the expression of genes belonging to
general stress and nucleic acid metabolism categories is
activated. Moreover, the outcomes of this study also showed that
chromatin modifications and changes in methylation patterns are
associated to an adaptation to chronic irradiation, both having a
trans-generational nature.

All above mentioned studies provided cumulative evidences that

gamma-irradiation is a valuable tool in seed technology, since
low-dose treatment can act efficiently as a seed invigoration
treatment. However, more studies are needed to understand the
molecular basis underlying the improved growth/development
response observed in irradiated seeds. Despite the advantageous
aspects mentioned, gamma-irradiation treatments in a seed
technology context depend on the proper establishment of γ-
irradiation facilities able to work at an industrial scale. The
application of this seed invigoration method still requires an
extensive research in order to define the optimal treatment
conditions (total dose, dose rate) which depend on crop species,
genotype and environmental context.

Effects of X-Rays in Seed Germination

and Seedling Development
The effects of X-rays on organisms are still not fully understood.
Among IR sources, X-rays have a wavelength in the range of
0.01 to 10 nm of the electromagnetic spectrum, corresponding to
frequencies in the range 30 to 30000 PHz (1 Petahertz
corresponds to 1015 Hertz) and energies in the range 120 eV to
120 keV (Kotwaliwale et al., 2014). Soft X-rays with energies of
about 0.12 to 12 keV are the most suitable X-radiation to be used
on agricultural products, due to low penetration power and ability
to reveal the internal density changes (Kotwaliwale et al., 2014).
Food treatment with X-rays has been performed to improve food
microbiological safety and storability (Farkas and Mohácsi-
Farkas, 2011). While the impact of X-rays is well studied in
humans due to its general use in medical practice, research on the
impact of X-radiation on plants had only a boost during the
1930–1960 timeframe (Benedict and Kersten, 1934; Bless, 1938;
Smith, 1950; Caldecott et al., 1952; Yagyu and Morris, 1957;
Beard et al., 1958).

To the best of our knowledge, very few works focused on the

impacts of X-ray irradiation on seed performance were published
after the 60′s decade (Al-Enezi et al., 2012; De Micco et al.,
2014; Einset and Collins, 2015; Pérez-Torres et al., 2015).
Increasing X-ray irradiation doses were seen to reduce seed
germination percentage and root growth of date plam (Phoenix
dactylifera L.) seeds (Al-Enezi et al., 2012). However, these
authors noticed a stimulatory effect on leaf growth when seeds
were irradiated with a 0.65 Gy dose. Another interesting study
was recently conducted in S. lycopersicum L. cv. Microtom (De
Micco et al., 2014). In this study, anatomical and
ecophysiological features (e.g., growth traits, leaf anatomy) of
tomato plants grown from seeds irradiated with increasing X-rays
doses were investigated. The overall results showed that
germination and development of functional leaves was not
significantly hampered by increasing irradiation dose, which
suggested some resistance of this cultivar to irradiation. Further,
the radio-resistance of the Microtom cultivar was supported by
the slight structural perturbations observed in leaves with minor
impairment of the photosynthetic efficiency, namely when seeds
were irradiated with high doses of X-rays. The identification of
radio-resistant candidate species or seed lots can be an important
achievement for the design of space-oriented agriculture (Arena
et al., 2014).

The current knowledge about the effects of X-rays in plants is

still scarce, focused on few aspects of the plant physiology and
needs to be extended. New clues about molecular and
physiological mechanisms underlying the resistance of plant
tissues to X-rays could be unveil by using global profiling
techniques (e.g., Omics).

“Priming” Seeds with Ultraviolet

Radiation: A Difficult Task
The amount of solar UV radiation reaching the Earth’s surface is
increasing as consequence of the depletion of the stratosphere
ozone layer. According to the wavelength range, solar UV
radiation is classified into UV-C (200–280 nm), UV-B (280–320
nm), and UV-A (320–400 nm). A large number of studies have
been focused on the global impact of UV exposure on plants at
multiple levels, from the ecosystem to the whole plant (Kovács
and Keresztes, 2002; Heisler et al., 2003). On the other hand,
very few studies have been dedicated to investigate UV impact in
seed biology and germination

Although UV-C radiation is extremely harmful to organisms, it is

not relevant under natural conditions of solar irradiation
(Hollósy, 2002). UV-C radiation is non-ionizing and it penetrates
superficially into the plant tissues, which supports its potential as
a germicidal agent. Seed treatments with low doses of UV-C (3.6
kJ m-2) were used to elicit host resistance to black rot in cabbage
(Brassica oleracea L.) (Brown et al., 2001).

This UV-C seed treatment also improved the quality and growth
response of cabbages under greenhouse conditions. In another
study, Ouhibi et al. (2014) investigated the impact of UV-C pre-
sowing treatments in lettuce (Lactuca sativa L. ‘Romaine’).
Lettuce seeds were UV-C- treated by exposure to 0.82 and 3.42
kJ m-2 doses and resulting seedlings were challenged with salt
stress. The results showed that UV-C treated seedlings were able
to mitigate the impact of excessive salinity, possibly as result of
the enhanced free radical scavenging activity detected in their
leaf tissues (Ouhibi et al., 2014). Additionally, the authors also
showed that a dose-dependent response occurs: seedlings derived
from seeds treated with the lowest UV-C dose showed higher
tolerance to salinity conditions.

The impacts of increasing solar UV radiation on terrestrial

ecosystems have been extensively reviewed under the present
Climate Changes context. Similarly to what is described for MFs,
the impact of UV irradiation can significantly differ, depending on
the target organism and radiation component used. Positive effects
of UV- A and UV-C radiation have been highlighted on seed
germination/seedling vigor, as well as, seed health. However, a
deeper insight is still requested to elucidate about the molecular
mechanisms underlying the improvements occurring in the UV-
treated seeds.

Microwaves Potentialities in Seed

Technology
Microwaves (MWs) are components of the electromagnetic
spectrum. MWs include radiation ranging in frequency from 300
MHz (300 million cycles per second) to 300 GHz (300 billion
cycles per second), which correspond to a wavelength range from
1 m down to 1mm (Banik et al., 2003). Despite the initial
controversy, it is now generally accepted that the absorbed non-
ionizing electromagnetic radiation as MWs induces thermal and
non-thermal effects in biological systems (Banik et al., 2003).
Mounting evidences show that MWs cause different biological
effects depending on field strength, frequencies, wave forms,
modulation and duration of exposures (Vian et al., 2006). While
the effects of MWs on humans and animals were widely
investigated, a very limited number of published studies have
addressed the MWs-mediated effects on plants (Jayasanka and
Asaeda, 2014). Interestingly, most of the studies currently
available describe the impact of the 2.45 GHz radiation, which is
absorbed by water molecules in living cells (Iuliana et al., 2013).
When the MW radiation is absorbed by living tissues, it causes
ionic movement, dipole rotation and distorsion of the electron
orbit which ultimately results into fast and selective heating.

In a seed technology context, non-lethal MWs treatments have

been extensively used for seed disinfection before sowing or
storage (Reddy et al., 1995, 1998; Scialabba and Tamburello,
2002; Aladjadjiyan, 2010; Knox et al., 2013). Interestingly,
deleterious MWs treatments have been used for inhibiting
germination of weed seeds buried in the soil (Velázquez-Martí et
al., 2006; Sahin, 2014). MWs treatments applied caused soil
heating (up to 80°C) and weed germination was totally inhibited.
Consequently, MWs emerged as a valid non-chemical alternative
for weed control in greenhouses from horticultural/ornamental
plant nurseries.

Physical Methods to Study the Impact of

Physical Seed Invigoration: Electron
Paramagnetic Resonance as a Case
Study
Electron paramagnetic resonance (EPR), also known as electron
spin resonance (ESR), is technique for the qualitative and
quantitative analysis of short-lived (10-9–10-1 s) free radical
species, such as ROS. This powerful tool, currently regarded as
one of the most specific and sensitive for this purpose, rise from
the original studies in quantum mechanics carried out by Zeeman
(1897). Subsequently, these studies were interpreted by Uhlenbeck
and Goudsmit (1926), who introduced the concept of ‘spin’
(quantitized angular moment) as intrinsic feature of the electron.
Although the first EPR spectrum was reported by Zavoisky
(1945), EPR was only made available to the research community
in the 1980s when the equipment became cheaper and
commercially available. This was essential to support research on
biological systems containing organic-based radicals and
transition metals

Paramagnetic species profiles are emerging as promising

biomarkers for screening stress tolerance in cereal grains.
Labanowska et al. (2012) used EPR to analyze the long lived
stable radicals stabilized by starch and other carbohydrates present
in grains of five wheat genotypes with different drought stress
tolerance. Increased amounts of carbohydrate and semiquinone
radicals were found in seeds from drought sensitive genotypes,
which also correlated with higher amounts of starch determined by
biochemical analysis.
These authors also proposed that those EPR radical profiles can
be used as indicators of stress tolerance. Kurdziel et al. (2015) also
used EPR to investigate free radical profiles in wheat whole grains
but also in grain specific components (embryo, seed coat, and
endosperm) from four wheat genotypes with different degree of
drought tolerance.
Again, the level of carbohydrate radicals was significantly higher
in the drought-sensitive genotypes, corroborating their use as a
reliable marker of the plant ability to withstand water deficit.
More recently, EPR was used by Labanowska et al. (2016) to
investigate the influence of short-term ozone application on wheat,
oat and barley grains in two contrasting genotypes in terms of
oxidative stress tolerance. EPR revealed that the character and the
number of paramagnetic species [transition metal ions: Fe(III),
Cu(II), Mn(II), and stable organic radicals] changed upon ozone
exposure, depending on the cereal species, stress tolerance of a
particular genotype and the part of grain studied.

Moreover, the patterns of stable organic radicals (semiquinone,

phenoxyl, and carbohydrate) significantly changed in response to
ozone treatment, showing a stronger enhancement of these
paramagnetic species in the embryo of the tolerant cultivars.
Interestingly, oxidized iron species (Fe2+) in the embryo were
stabilized by the organic matrix while in seed coat and endosperm
increased free radical levels correlated with the amount of
transition metal ions (Fe2+, Cu2+, Mn2+). The results obtained
from this study also corroborated the suitability of EPR to screen
oxidative stress tolerant cultivars of cereals.
The knowledge so far acquired through EPR-based studies in food
processing or seeds, as mentioned previously, support the
application of this technique to investigate the effects of physical
invigoration methods on seeds. ESR was used to investigate the
biochemical changes induced in maize seeds upon irradiation with
increasing doses of gamma-radiation (Marcu et al., 2013). In this
work, a correlation between the relative concentrations of
paramagnetic species as a function of the absorbed irradiation
dose was established. This reflects the increase of ROS generated
through water radiolysis. The results suggest that EPR
spectrometry is a relatively fast and simple technique to measure
free radicals generated upon irradiation. This has an enormous
potential on a seed technology context, since EPR can be used to
monitor seed invigoration treatments.

Conclusion
High vigor seeds are proxy of crop establishment and sustainable
productivity. Physical seed invigoration methods are an
alternative approach – to current chemical based – to develop
new biotech-based solutions for the growing world seed market.
The use of physical methods for increasing seed germination and
seedling vigor offers eco-friendly advantages and the possibility
to be used in a high throughput scale. Promising approaches
include the treatment with MFs, MWs, and IRs.

Besides the existence of adequate facilities to perform the

physical treatments, the current gap of knowledge on the pre-
germinative metabolism is hampering its successful application
of priming treatments, as seen for chemical treatments. So far, the
research conducted to understand the molecular mechanisms
governing physical seed invigoration lacks deepness. Most of the
studies were restricted to the assessment of the impact of a range
of radiation treatments applied, with very little information on
biochemical or gene expression changes occurred. The lack of
biomarkers associated with the best suitable irradiation dose or
time-point to stop the treatment is hampering the implementation
of physical seed invigoration protocols at the industry level.

Nevertheless, researchers from Academia and Industry are

currently focused on overcoming these issues. The recent
advances on molecular high-throughput techniques (e.g., Omics)
combined with the recent release of new genomic resources on
target species or crops are expected to lead, in a short-term,
research on this topic. More studies are needed to indentify the
molecular players triggered during the seed response to physical
invigoration treatments, especially in radio-tolerant species or
cultivars. On the other hand, by expanding the number of
species/genotypes tested with each different approach, it will be
possible to identify those targets best suitable for a specific
physical treatment, preventing deleterious conditions. The study
of the impact of the environmental conditions in modulating the
response of seed to radiation treatments could not be neglected,
constituting it self a promising research avenue. This knowledge
is crucial to develop new strategies to design new biotech-based
treatments to modulate and improve seed germination and
invigoration.

An integrated and multi-disciplinary approach is needed to speed

up basic and translational research in seed technology, finally
producing guidelines for the seed operators.

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