Biochemical reaction during seed aging and how to mange the seed aging for long time storage, Seed aging, Characteristics of seed aging, Factors influencing seed ageing, Biochemistry of seed aging, Role of reactive oxygen species (ROS) during seed ageing, Lipid peroxidation, DNA methylation, Protein carbonylation, Programmed cell death, Mitochondrial Dysfunction , Symptoms of seed aging, Causes of seed aging, Most prone site for seed aging, Behavior of seed

1. Biochemical reaction during seed
aging and how to mange for long
time storage
BIC 501 – Plant Biochemistry (2+1)
P. Jayasankaran
2020518012
1st M.Sc.(SST)

2. CONTENT
2
 Introduction
 Seed aging
 Characteristics of seed aging
 Factors influencing seed ageing
 Biochemistry of seed aging
 Different biochemical process during seed aging
 Symptoms of seed aging
 Causes of seed aging
 How to manage for long time storage
 Case study
 Summary and conclusion
 Reference
CONTENT

3. 3
 All living organisms must eventually deteriorate and die.
 Seed viability is an important trait in agriculture which directly influences
seedlings emergence and crop yield.
 Seed needs to be stored from the day of harvest up to the time of next sowing.
 Seed in storage are the main recurring assets for growers.
 seed viability decreases over long-term storage due to seed aging.
 Our primary aims were to describe factors influencing seed aging, determine
the biochemical changes that occur during the process of seed ageing, and
explore the mechanisms involved in seed aging.
Introduction

4. Seed aging
4
“Deteriorative changes occurring within seed with time that increase its
vulnerability to external challenges and decrease the ability to survive.”
(McDonald, 1999; Ebone et al., 2019)
Deteriorative changes
enhanced
Increased exposure of
seed to external challenges
Decreases ability of the
seed to survive

5. Characteristics of seed aging
5
Inexorable process:
All living thing eventually deteriorate and die; can be retarded
Irreversible process:
Once seed deterioration has occurred, this can not be reversed
Varies among seed population:
It is now well established that certain varieties exhibit less deterioration than others.
Even within a variety, the storage potential of individual lots varies, and even within a seed lot
individual seeds have differing storage potential.
(McDonald, 1999)

6. 6
Factors influencing seed ageing
 Internal factors of seed - Genetic factors
 Maturational and harvest conditions
 Relative humidity and temperature
 Morphological, physiological, and biochemical changes

7. 7
 Seed quality is obviously dependent on genetic factors. Under the same storage conditions, seeds of
different genera, species, cultivars, or individual plants often show differences in their storability.
 Some seeds, such as Allium cepa and Lactuca sativa, inherently have short lives in storage
 Seeds of Nelumbo nucifera have been shown to survive for 1300 years (Copeland and McDonald, 2012).
Genetic factors

8. 8
 Mature seeds tolerate storage much better than immature seeds in the same seed lot .
 However, delaying harvest may also induce the ageing process under field conditions, especially
in the presence of a high seed moisture content, which decreases the later storage viability
(Bewley et al., 2013).
 The most suitable time for storage is after seeds have attained physiological maturity, or have
reached their maximum dry weight.
Maturational and harvest conditions

9. 9
 Relative humidity (RH) and temperature represent the two environmental factors that influence
seed deterioration most directly (Rajjout al., 2008).
 The RH directly affects seed moisture content during storage because seed moisture reaches an
equilibrium with the level of water vapor surrounding the seeds. Generally, the actual seed
moisture content during dry storage is frequently 5–10% (Copeland and McDonald, 2012; Baskin
and Baskin, 2020).
 An increase in relative humidity results in increased seed moisture content which in turn results
in increased seed ageing
Relative humidity and temperature

10. 10
Biochemistry of seed aging
 Free radical and ROS damage
 Lipid peroxidation
 Damage to DNA sequences
 Loss of DNA integrity
 Loss of Enzyme activity
 Macromolecular damage
 Damage to mitochondria
 Loss of membrane integrity
 Programmed cell death

11. 11
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An atom or molecule that possesses an unpaired electron is a free radical.
Formed by :-
• ionizing radiation
• splitting of oxygen by enzymes or transition metals
• normal metabolic processes
What is a free radical ?
O, O3, O2, O2
+, O2ˉ, OH ˉ are free radicals

12. 12
Role of reactive oxygen species (ROS) during seed ageing
 Reactive oxygen species are one of the byproducts of aerobic respiration.
 Low concentrations of ROS within seeds serve as important signal molecules for dormancy
alleviation, seed germination, and protection against pathogens
 However, levels of ROS can increase cumulatively if seeds are stored under unsuitable conditions
or for a long time.
 An increase in the amount of ROS has usually been identified as the most important factor that
affects seed ageing.
 ROS causes oxidative damage to lipids, proteins, and DNA, ultimately leading to a loss of viability
(Bailly, 2004; Apel and Hirt, 2004; Waszczak et al., 2018).

13. 13 Keliang Zhang et al., 2020

14. 14

15. 15
• When seeds deteriorate, one important event is the
breakdown of the plasma membrane, and this is
primarily caused by lipid peroxidation
• As seeds age, increased concentrations of ROS will
attack polyunsaturated fatty acids in membrane
phospholipids, leading the long-chain fatty acids to
break down into smaller compounds, thereby
modifying membrane permeability and leading to
membrane destruction
Lipid peroxidation

16. 16
The short lived radicals produced during autoxidation can be damaging to proteins,
enzymes and other biological compounds in their proximity.
J.F. Harrington, 1973 - The sequence of events during lipid autoxidation in seeds,
“Biochemical basis of seed longevity,"
1. Unsaturated Fats metal ions Free radicals
2. Free radicals light and other irradiation Hydroperoxides
3. Hydroperoxides Carbonyls
4. Carbonyls + Protein Inactivation of enzymes, membrane injury
5. Carbonyls + nucleic acids Chromosomal mutation

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 Progressive fragmentation of embryonic nuclear DNA occurs during seed ageing.
 Increased levels of ROS during seed ageing can also lead to genetic damage.
 Chromosomal damage, such as breaks in DNA strands, DNA methylation, and abnormal gene
expression, may be accumulated during the process of seed ageing,
 causing the frequency of affected cell damage to surpass a threshold, after which seeds lose their ability
to germinate
 For example, when the seeds of Oryza sativa and Pisum sativum were stored at 45 ◦C, all variable
seeds exhibited a decrease in the mitotic index and an increase in the frequency of chromosomal
damage during ageing; in addition, an increase in storage time resulted in increased DNA damage
(Dantas et al., 2019).
Genetic damage

18. 18
DNA methylation

19. 19 (Yin et al., 2017).
Protein carbonylation

20. 20
Programmed cell death

21. Mitochondrial Dysfunction
21 (Ewelina Ratajczak et al., 2019).

22. 22
Int. J.Mol. Sci.2019,20,1568 2of12
Table 1. The role of reactive oxygen species in dysfunction mitochondrion seeds.
Organism(Species) Processes References
Pea (Pisum sativum L.) Germination after Aspergillus ruber infection, aging [35]
Rye (Secalecereal L.) Embryogenesis, germination [36]
Soybean [Glycine max (L.) Merr] Aging [37]
Pea(Pisumsativum) Germination [25]
Soybean [Glycine max (L.) Merr] Germination,imbibition [38]
Pea (Pisum sativum cv. Jizhuang) Germination [39]
Soybean [G.max (L.) Merr] Aging [40]
Elm (Ulmus pumila L.) Aging [41]
Oat (Avena sativa L.) Aging [42]
Rice (Oryza sativa L.) Aging [7]
Rice (Oryza sativa L.) Aging [43]
Elm (Ulmus pumila L.) Aging [44]
Oat (Avena sativa L.) Aging [26]
(Ewelina Ratajczak et al., 2019).

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24. 24
 Zacheo et al., (1998) reported that the content of lipid decreased during accelerated ageing in
almond seeds. The aged seeds contained high levels of malondialdehyde, a product of the
peroxidation of unsaturated fats and increased Lipoxygenase activity, which hydrolises
unsaturated fatty acids.
 Shaban (2013) working in soyabean and sunflower and Radha et al., (2014) working in maize,
inferred that autooxidation of lipids and increase in the content of free fatty acids during storage
period are the main reasons for rapid deterioration of seed. Aged seeds show decreased vigour
and produce weak seedlings that are unable to survive once reintroduced into a habitat. Ageing
induce damage to cellular membranes, decrease in mitochondrial dehydrogenases activities,
chromosomal aberration and DNA degradation increases.
Case Study

25. 25
 Tubić et al., (2005) determined the degree of biochemical changes during accelerated and
natural aging on 5 cultivars of sunflower seed. Lipid peroxidation and decrease in superoxide
dismutase and peroxidase activities (especially pronounced in accelerated aging variant) were
caused by both types of aging.
Tubić et al., (2005)

26. 26
Morphological changes:
 Darkening of the seed coat
 Ultrastructure Changes
 Loss in integrity
 Loss of Enzyme Activity
 Reduced Respiration
 Increase In Seed Leachate
 Increase In Free Fatty Acid Content
Performance symptoms:
Symptoms of seed aging
 Reduced vigour and viability
 Loss of field emergence potential
 Decreased resistance to environmental stresses
 Morphologically abnormal seedlings may be produced

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 Seed ageing adversely affect three important physiological drivers:
Germination potential
Vigour
Viability
 Delayed seedling emergence
 Decreased environmental stress resistance in the process of germination and during the early
growth of seedlings.
 weakens the defense mechanism of the plant against various biotic and non biotic stressors.
(Radha et al., 2013 reported protein deterioration in maize seeds ageing resulting in loss in vigour
and viability.)
Causes of seed aging

28. 28
 Does not occur uniformly in seed
More damage of mitochondria will occur in the embryonic axis than in the cotyledons of seeds
during seed aging.
Most prone site for seed aging
(Ewelina Ratajczak et al., 2019., Mitochondria Are Important Determinants of the Aging of Seeds)

29. 29
seed ageing is not equal for all kinds of seeds; even in the same lot of seeds,
different individuals will show diverse levels of storage potential.
Typically, orthodox seeds have a longer lifespan, can tolerate freezing, and maybe
dried to 5% moisture content with no damage.
recalcitrant seeds have a much shorter life, cannot tolerate freezing, and cannot be
dried lower than 20–30% moisture content without injury
(Roberts.,1973, Walters et al., 2013; Ebone et al., 2019)
Behavior of seed

30. 30
Seed Ageing can’t be ignored but can be delayed
(a) Physiological maturity and Harvest Maturity
e.g., Red fruit colour of Capsicum
1. Harvesting

31. 31
(a) Threshing should be done when moisture content of seed declines to 13-18%, thereby imparting
mechanical strength.
(b) Processing unit should offer minimum damage to the seeds
Processing

32. 32
Storage:
Storage place
The climate of the place where the seed storage is located affects the life of the seed. A much
better and, more expensive seed storage is needed in tropical region like TN, Coimbatore then compared to
temperate regions like HP, Jammu & Kashmir.
Adopt good packaging material
In India, certified seeds of cereals, pulses and oilseeds are normally packed either in jute bags
or cloth bags whereas paper bags, cardboard boxes, aluminium foil pouches, polythene bags are used for
packaging flowers and vegetable seeds. Agro climatic condition should be taken into account

33. 33
• Short term: 6-9 months, storage of seed from harvest to next planting. The majority of seed
we are dealing will fall in this category (75 - 80%)
• Medium term: 16 - 36 months, storage of carryover seeds. Seeds that may not be produced
every year (20 -25%)
• Long terms: 5 -20 years, storage of germplasm and breeder seed (5%)
Seed storage
Harrington thumb rule on seed storage
 For every decrease of 1% seed moisture content, the life of the seed doubles. This rule is
applicable when moisture content between 5 and 14%.
 For every decrease of 5°C (10°F) in storage temperature the life of the seed doubles. This rule
applies between 0°C to 50°C.
Drying seed to safe moisture content
In general, cereals are dried to 10-13% and vegetables are dried to 6-8%.

34. 34
Halogenation
Antioxidant treatment (i.e. tocopherol)
Seed sanitation
Seed fumigation
Pre storage treatments

35. 35
Varieties Germination (%) Storage in months
4 8 12 16 20
Byadagi kaddi (V1) 87.43 83.05 74.90 69.95 61.06
Dyavanur local (V2) 84.24 79.23 72.66 68.85 61.48
Treatments (T)
T1 Calcium oxychloride 89.67 83.33 83.0 78.83 70.0
T2 Potasium iodide 90.83 85.5 86.0 81.33 71.66
T3 Bavistin 87.83 81.0 73.16 70.16 64.0
T4 Neem leaf 84.33 80.0 68.33 65.33 56.5
T5 Pongamia leaf 83.5 80.5 72.83 69.0 62.5
T6 Sweet flage 82.67 77.67 66.0 60.33 62.1
T7 Control 81.5 74.5 67.17 60.33 51.83
Ravi Hunje et al., 2007

36. 36
 Hydration – Dehydration
 Soaking and Drying
 Dipping and Drying
 Spraying and Drying
 Moisture equilibration and Drying
 Moist sand conditioning
Mid storage treatment

37. 37
Treatments to boost the potential expression of the seed lot
 Seed fortification
 Seed infusion/ Dry permeation
 Seed priming
Germination and vigour improvement
Before Sowing

38. 38 Ponnuswamy and Vijayalakshmi, 2011, Coimbatore

39. 39
Seed Priming Germination (%) Seedling Length (cm) Seedling Dry Weight (mg)
V1 V2
Mean (P) V1 V2
Mean (P) V1 V2
Mean (P)
PEG
( -1 Mpa )
85.50
(67.70)
74.00
(59.32)
79.75
(63.51)
17.47 16.05 16.76 134.18 123.73 128.95
KNO3 @
1%
84.00
(66.45)
71.50
(57.71)
77.75
(62.08)
16.94 15.56 16.25 131.21 123.45 127.33
KH2PO4 @
1%
83.50
(66.05)
71.25
(57.56)
77.38
(61.80)
16.92 15.47 16.19 130.00 122.18 126.09
CuSo4 @
100 ppm
75.50
(60.32)
69.00
(56.16)
72.25
(58.24)
15.15 15.39 15.27 123.83 116.35 120.09
Cocopeat 90.00
(71.62)
75.00
(59.98)
82.50
(65.80)
18.48 16.93 17.70 137.61 127.53 132.57
Perlite 91.25
(72.87)
76.00
(60.65)
83.63
(66.76)
19.58 19.58 18.49 138.20 129.50 133.85
Vermiculite 87.50
(69.31)
73.50
(58.99)
80.50
(64.15)
18.14 16.65 17.39 136.40 126.10 131.25
Soaking in
water
82.00
(64.98)
70.25
(56.93)
76.13
(60.95)
16.88 15.44 16.16 129.15 120.40 124.78
Control 74.00
(59.33)
61.00
(51.34)
67.50
(55.33)
14.21 11.61 12.91 125.38 106.53 115.95
Mean (V) 83.69
(66.51)
71.28
(57.63)
17.08 15.61 131.77 121.75
Effect of seed priming on germination, seedling length and seedling dry weight among different vigour
levels of bitter gourd seeds. (Paper Towel Method)
Mehta and Kanwar, 2013

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 Understanding the mechanisms of seed aging will lead to new methods for seed conservation
and longevity.
 It has been estimated that ca. 25% of seeds lose their viability annually, which can give rise to
billions of dollars of economic losses (McDonald and Nelson, 1986).
 which adversely affects agricultural productivity and food security for the rising population.
 The primary cause of such losses is poor storage due to high seed moisture content at harvest
and damp storage conditions.
 Moisture content of seed, storage oxygen, relative humidity and temperature are the most
important factors in storage which greatly influence the longevity and storability of seed.
Summary and conclusion

41. 41
 Seed aging has become a global issue, with consequences that are highly unfavorable for the
economy, as the loss of seed viability translates into a lack of material for reproduction.
 Understanding factors influencing of seed ageing provides effective way to delay the deleterious effect
of seed ageing and thereby improve the storage potential of the seed lot.
 Scientific and intelligent post harvest handling approach would not only delay the ageing effect but
would also reduce the deteriorative impact of ageing on seed.
 Seed longevity is a major challenge for the conservation of plant biodiversity and for crop success.
Seeds possess a wide range of systems (protection, detoxification, repair) allowing them to survive in
the dry state and to preserve a high germination ability. Therefore, the seed system provides an
appropriate model to study longevity and aging

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46. Thank You !!!
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