effect of hydropriming on physiological and biochemical activities of high and low vigour seed lots of cereals

2. EFFECT OF HYDROPRIMING ON
PHYSIOLOGICAL AND BIOCHEMICAL
ACTIVITIES OF HIGH AND LOW
VIGOUR SEED LOTS OF CEREALS
PRESENTED BY
KIRAN DASANAL
PALB 7319

3. 1.High vigour : Produces
normal, vigorous seedling in a
wide range of conditions.
(Ahirwar, 2012)
3.Low vigour : Produce
weak,abnormal seedling even
under favourable conditions.
2.Medium vigour :
Produces normal seedling
under favourable conditions.
Non-viable seed
The vigour scale

4. SEEDQUALITYENHANCEMENT
Definition:
Post harvest treatment that
improve germination or seedling
growth or facilitate the delivery of
seeds and other materials required
at the time of sowing
(Tayloret al.,1998)

6. WHERE& WHENIT ISNEEDED?
• Problematic seed
• High value of seed
• Biotic stresses
• Direct seeding
• Adverse climatic conditions

7. SEED PRIMING PROCESS
Priming allows some of the metabolic processes necessary for
germination to occur without germination take place.
This prevents the seeds from absorbing in enough water for
radicle protrusion, thus suspending the seeds in the lag phase.
(Taylor et al.,1998)
This hydration is sufficient to permit pre-germinative metabolic
events but insufficient to allow radicle protrusion through the seed
coat. (Heydecker et al.,1975)
Controlled
Imbibition
Dehydration
Enzymes
Activation
Imbibition
in field
Radicle
emergence

8. Physiological basis of seed priming.
(Agarwal, 2002)

9. HYDRO PRIMING
 Soaking of seeds in pure water and re-drying to original
moisture content prior to sowing.
(Stanley et al., 2016)

10. Time duration for hydro-priming
Crop seeds Duration (hour) Reference
Maize 6 (Satish et al., 2012)
Sorghum 12-24 (Moradi,
Younesi, 2009)
Wheat 12 (Kalpana et al., 2013)
Paddy 48 (Farooq et al., 2006)

11. WHY ?
• Simplest method of seed priming
• Low-cost and environmentally friendly
• No use of additional chemical substances as
a priming agent
• Useful for resource-poor farmers in
marginal tropical environment
(Stanley et al., 2016)

12. There are two types of hydro-priming
1) Drum priming
2) On-farm priming
Methods of hydro-priming

13. 1. DRUM PRIMING
• In this technique, seeds are gently rotating in drum and
gradually hydrated by addition of water in vapor form.
Drum priming allows seed imbibition in a controlled
manner and could be an attractive alternative to
conventional hydro priming.
(Stanley et al., 2016)

14. 2. ON-FARM PRIMING
• Soaking of seeds in water followed by surface
drying and subsequent sowing.
• The duration of treatment obligatorily cannot be
longer than “safe limit”(Around 6 -12 hours).
• On-farm priming is especially useful for resource-
poor farmers in marginal tropical environment .
(Harris et al., 2002)

15. ADVANTAGES
• Hastening and synchronization of germination.
• A faster seedling establishment resulting from
priming may contribute to a yield from 10-45%.
• It increases the GA / ABA ratio.
• Hydro-priming improves the imbibition in field
condition.
• It helps in synchronization in endosperm
weakening, embryo cell elongation and reserve
mobilization.
(Stanley et al., 2016)

16. Cont….
• Higher the ethylene production during
priming may promote endo-β-mannase.
• It initiates the repair and reactivation of
mitochondria and to initiates biogenesis of
new ones.
• Higher the vigour.
• Salinity and drought resistance.
(Stanley et al., 2016)

17. DISADVANTAGES
• Uncontrolled water uptake by seeds.
• Unequal degree of seeds hydration thus
leading to lack of simultaneous
metabolic activation within seeds
followed by unsynchronized
emergence.
(Stanley et al., 2016)

18. PRIMING AND WATER CONTENT
• Water transport across the cell membrane is essential
for to initiates the metabolic process.
• The aquaporins are a play a key role in transcellular
and intracellular plant water transport.
https://en.wikipedia.org/wiki/Aquaporin
(Stanley et al., 2016)

19. AQUAPORINS
• These are intrinsic trans-membrane proteins that
facilitates rapid and passive water transport across
the cell membrane.
• The major intrinsic proteins are
1. Plasma Membrane Intrinsic Proteins(PIP’S)
2. Tonoplast Intrinsic Proteins(TIP’S)
https://en.wikipedia.org/wiki/Aquaporin
(Stanley et al., 2016)

20. PRIMING AND CELL CYCLE
https://www.albert.io/blog/g1-g2-phases-cell-cycle/

21. CONT….
• Synchronization of embryonic cells in G2
phase of cell cycle.
• DNA synthesis and repair mainly through
Base and nucleotide excision repair.
• Advancement of cell cycle from G1 to G2
phase
• Activation of cyclin dependent protein kinase
and proliferating cell number antigens.
• Higher expression of genes encoding
aquaporins (PIP/TIP)
(BENAMAR et al., 2003)

22. DURING DEYDRATION
• LEA’s are stabilize the cell structure and
macromolecules upon cell dehydration by preventing
the inactivation and aggregation of proteins and the
loss of membrane intigrity (Stanley et al., 2016).
• LEA proteins participate in protecting cellular
components from dehydration (Reyes et al., 2005).
https://www.naro.affrc.go.jp/archive/nias/anhydrobiosis/Sleeping%20Chironimid/e-taisei.html

23. PRIMING AND SEED ULTRA STUCTURE
• Abundant α and β tubulin.
• Accumulation of β tubulin.
• Cellular cyto-skeleton reorganization.
• Weakening of tissues surrounding elongating radicle
by cell seperation.
• Hydrolysis of seed endosperm due to increasing in
the activity of Endo-β-Mannase.
• Improved integrity of outer membrane of
mitochondria.
(Stanley et al., 2016)

24. GENERAL DIAGRAM OF CEAREAL
(Zhenguo ma et al., 2017)
https://ars.els-cdn.com/content/image/1-s2.0-S2214514117300909-gr2_lrg.jpg

25. • Activation of respiration and rapid ATP production
is primary metabolic event occurs during priming
• Abundance of low molecular weight heat shock
proteins
• Synthesis of L-isoaspartyl methyl transferase.
• Increased activity of endo-beta-mannase (weakening
of endosperm)
• Enhanced synthesis and accumulation of enzymatic
and non-enzymatic antioxidant.
BIOCHEMICAL CHANGES
(BENAMAR et al., 2003)

26. CONT….
• Increased levels of soluble sugars.
• Synthesis and Activation of enzymes (alpha
amyalase, isocitrate lyase etc.) for mobilization of
stored food
• Increased solubilisation of Beta subunit of 11S-
GLOBULIN.
• Rapid ATP production and increased ATP/ADP ratio
and energy charge.
• Accumulation of dehydrins and LEA proteins.

27. Management of oxidative status
• The biochemical and cellular events triggered by water
uptake and subsequent loss are accompanied by a
generation of reactive oxygen species (ROS).
• During seed imbibition and early stages of germination,
ROS production occurs mainly through respiratory
activities of mitochondria, activities of β-oxidation
pathways and enzymes such as NADPH oxidases,
extracellular peroxidases, and oxalate oxidases.
• ROS accumulation and associated oxidative damage can
be regarded as a source of stress that may affect the
successful completion of germination.
(Stanley et al., 2016)

28. WHATAREFREERADICALS?
 Any molecule containing one or
more unpaired electrons.
 Highly reactive
 Very short half-life
 Generate new radicals by chain
reaction
 Cause damage to biomolecules,
cells and tissues.
Types of free radical

29. DURING REDRYING
• Controlled hydration of the seeds and drying
back to the initial moisture content. Seed pre-
hydration followed by re-drying during
priming treatment, exerts changes in moisture
content, which leads to ROS production and
activation of the antioxidant system.
(Stanley et al., 2016)

30. ACTIVATION OF ANTIOXYDANT SYSTEM
• H2O2 (A ROS) can act as signaling molecules, seeds
must be endowed with a ROS removing system that
tightly regulates their concentration. Scavenging of
ROS is carried out by antioxidant system.
• It’s a multifunctional machine, which includes
enzymes (i.e., catalase (CAT), superoxide dismutase
(SOD), and ascorbate peroxidase (APX)) as well as
non-enzymatic compounds (i.e., ascorbic acid (AsA)
or reduced glutathione (GSH).
(Stanley et al., 2016)

31. CASE STUDIES

32. Effect of hydropriming on physiological and biochemical
activities of high and low vigour seed lots of a maize hybrid
and its parental lines
• Study was conducted to evaluate the
influence of hydropriming on high and low
vigour seed lots of maize hybrid (VQPM 9)
and its parental lines VQL 1 (female) and
VQL 2 (male).
• Seeds were subjected to hydropriming (30
hour) followed by re drying to original
moisture content.
(Heena and Shiv, 2013)
Case study 1

33. Effect of hydropriming on germination (%), seedling length, seedling dry
weight, Seedling vigour indies's (I & II) of high (I) and low (II) lots of
maize hybrid VQPM 9 and its parental lines.
Increase in the germination percentage of VQL 1 (I) and VQL 1 (II) BY
12.6% and 3.8%; that of VQL 2 (I) and VQL 2 (II) 16.1% and 3.3% fair as
in VQPM 9 (I) and (II) by 4.2% and 4.8% respectively.
Increase in vigour indices paralleled that of seedling length and dry
weight increase in vigour index I ranged from 5-11.3% and 14.4-54.8% in
low and high vigour seed lots respectively.
Similarly the increase in vigour index II ranged from 3.7 - 9.5% and
10.3 - 42.7% in low and high vigour seed lots respectively.
It was concluded that priming of VQPM9 maize hybrid and its parental
line seeds with water for 30 H was found to improve the physiological seed
quality parameters
(Heena and Shiv, 2013)

34. Tab 1. Effect of hydro-priming on antioxidant enzymes of high (I) and low
(II) vigour seed lots of maize hybrid VQPM 9 and its parental lines.
Genotypes Treatments SOD activity
(unit / g seed /
min)
POX activity
(i moles/cm/min/g
seed)
CAT activity
(i mole/min/g
seed)
VQL 1 (I)
Control 5.720 12.827 14.210
Hydropriming 6.100 15.160 29.473
VQL 1 (II)
Control 2.983 9.813 9.997
Hydropriming 3.470 11.280 21.053
VQL 2 (I)
Control 5.563 25.270 14.947
Hydropriming 6.910 38.983 26.840
VQL 2 (II)
Control 3.137 13.767 8.943
Hydropriming 4.487 18.890 17.370
VQPM 9 (I)
Control 5.583 12.523 20.000
Hydropriming 7.743 16.743 31.580
VQPM 9 (II)
Control 3.387 9.397 14.210
Hydropriming 5.927 14.067 24.207
(Heena and Shiv, 2013)

35. Geno-type Treatment EC ( iS /cm /g
of seed )
Amylase activity (ig
starch hydrolysed / g
seed / min)
Dehydrogenase
activity (OD units /
20 seeds)
VQL 1(I) Control 0.037 9.860 0.457
Hydropriming 0.026 10.273 0.487
VQL 1(II) Control 0.044 9.620 0.445
Hydropriming 0.027 9.867 0.459
VQL 2(I) Control 0.041 28.367 0.844
Hydropriming 0.031 30.953 0.915
VQL 2(II) Control 0.037 10.967 0.485
Hydropriming 0.031 11.087 0.492
VQPM 9(I) Control 0.033 43.620 0.899
Hydropriming 0.016 48.740 0.956
VQPM 9(II) Control 0.038 31.427 0.885
Hydropriming 0.019 40.273 0.936
Tab 2. Effect of hydro-priming on EC and hydrolytic enzyme activity of
high(I) and low (II) vigour seed lots of maize hybrid VQPM 9 and its parental
lines.
(Heena and Shiv, 2013)

36. • Objective of this study to assess the influence of seed
priming on biochemical traits of maize (Zea mays L.).
• Nine seed priming treatments in maize cv. African tall viz.,
water, ZnSO4 @ 0.5%, KNO3 @ 0.5% &KH2PO4 @ 0.5%
for 6 and 12 hrs and control which were assessed for
biochemical studies.
Effect of Seed Priming on Biochemical traits of
Maize (Zea mays L.)
( Kotambari et al., 2018)
Case study 2

37. Tab 1. Effect of seed priming in biochemical constituents in
maize
Treat
ments
Seed priming treatments
Protein
(%)
Total
Crude
Fiber
(%)
Carbohydrates
(%)
Fat
(%)
Ash
(%)
T1 H2O for 6 hours 9.49 6.07 68.37 3.34 1.50
T2 H2O for 12 hours 10.51 5.80 73.46 3.33 1.77
T3 ZnSO4 @ 0.5 % for 6 hours 10.51 6.07 63.45 3.34 1.50
T4 ZnSO4 @ 0.5 % for 12 hours 8.67 4.90 62.42 3.27 1.63
T5 KNO3 @ 0.5 % for 6 hours 8.01 5.30 71.18 3.05 1.47
T6 KNO3 @ 0.5 % for 12 hours 8.38 4.80 71.05 2.99 1.61
T7 KH2PO4 @ 0.5 % for 6 hours 7.98 5.70 65.73 3.12 1.43
T8 KH2PO4 @ 0.5 % for 12 hrs 6.32 4.30 67.56 3.01 1.27
T9 Control 7.21 5.10 60.74 3.03 1.52
( Kotambari et al., 2018)

38. Effect of different seed priming treatments on germination, growth,
biochemical changes and yield of wheat varieties under sodic soil
 Experiments were conducted during rabi season
of 2012 and 2013 to study the effect of seed
priming on germination, growth, biochemical
changes and yield of tolerant KRL 210 and
susceptible HD 2733 varieties under sodic soil.
 Seed priming was done by soaking the seeds for
12 hours in distilled water, KNO3 (3%), KCl (1%),
GA3 (150 ppm) and cycocel 500 ppm.
(Kalpana et al., 2013)
Case study 3

39. Treatments Germination
(%)
Starch content
(mg/g)
Control 68.75 93.63
Hydro-priming 73.08 102.06
Priming with KNO3 80.92 113.83
Priming with KCl 77.75 108.71
Priming with GA3 76.25 105.62
Priming with Cycocel 74.50 103.09
(Kalpana et al., 2013)
Table 1. Effect of seed priming with chemicals and PGRs on
germination and biochemical changes of wheat varieties under
sodic soil

40. Treatments Plant height
(cm)
Number of tillers
per plant
Dry biomass
plant (g)
No. of grains
per ear
Control 82.89 6.45 27.05 33.97
Hydro priming 85.38 6.74 28.95 37.28
Priming with KNO3 93.70 7.92 29.59 44.78
Priming with KCl 90.65 7.33 30.43 41.50
Priming with GA3 89.48 7.33 29.38 39.36
Priming with cycocel 81.17 7.12 29.29 38.17
Tab 2. Effect of seed priming with chemicals and PGRs on yield
and yield contributing traits of wheat varieties under sodic soil
(Kalpana et al., 2013)

41. Effects of Osmo- and Hydro-priming on seed parameters
of grain sorghum (Sorghum bicolor L.)
• The influence of priming treatments and subsequent
accelerated aging on seed parameters of grain
sorghum was investigated.
• Seeds of sorghum were treated by osmo-priming
(polyethylene glycol 6000 solution with osmotic
potential -1.5 MPa) and hydro-priming for different
time (12, 24 and 36 hours).
(Moradi and Younesi, 2009)
Case study 4

42. Seed treatment Time of treatment
(h)
Non-aged (days) Aged (days)
Control 0 87.5 78.7
Hydro-priming 12 88.7 75.0
Hydro-priming 24 91.5 68.5
Hydro-priming 36 78.4 55.3
Osmo-priming 12 88.0 77.0
Osmo-priming 24 95.9 76.1
Osmo-priming 36 84.0 68.8
Tab 1. Emergence percentage of sorghum seeds as affected by priming
treatment for different time and subsequent accelerated aging (%)
(Moradi and Younesi, 2009)

43. Seed treatment Time of treatment(h) Non-aged (days) Aged (days)
Control 0 10.7 12.2
Hydro-priming 12 9.8 12.5
Hydro-priming 24 8.6 11.6
Hydro-priming 36 11.1 14.2
Osmo-priming 12 9.5 11.6
Osmo-priming 24 7.6 10.0
Osmo-priming 36 9.9 12.3
Tab 2. Mean emergence time (MET) of sorghum seeds as affected by
priming treatment for different time and subsequent accelerated aging
(Moradi and Younesi, 2009)
The seeds primed for 36 hours failed to improve emergence
percentage and mean emergence time (MET).

44. Inference
• Both osmo- and hydro-priming improved the percentage and
mean emergence time (MET) of seeds at sub- optimal
temperature of 15 C.
• Seed treatment for 12 and 24 hours had a positive statistically
significant effect on percentage and speed emergence.
• After receiving accelerated aging, osmo- and hydro - primed
seeds showed a lower emergence percentage and longer mean
emergence time (MET) than their non – aged counterparts.
(A. Moradi,O.Younesi.,2009)

45. INFLUENCE OF PRIMING TECHNIQUES ON EMERGENCE AND
SEEDLING GROWTH OF FORAGE SORGHUM (Sorghum bicolor L.)
• This study was designed to evaluate the effects of different
seed priming techniques, un-soaked seed (control), Hydro-
priming (soaked with distill water for 10 hour), Halo-priming
with KNO3 and CaCl2 (1% solution), on seed emergence and
seedling growth of three sorghum genotypes (Hegari, JS-263
and JS-2002).
• Experiment was conducted in wire house under natural
climatic conditions during 2008.
( Shehzad et al., 2012)
Case study 5

46. Treatments FEP (%) MET in days (VI I)
Varieties Priming
Hegari Dry 50.97 3.65 2580.21
Hydro priming 75.50 3.63 3545.48
CaCl2 77.73 3.12 4174.10
KNO3 59.87 3.12 2879.75
JS-263 Dry 62.20 3.51 2610.53
Hydro priming 62.17 3.55 2718.69
CaCl2 77.63 3.01 3838.03
KNO3 68.63 3.03 2987.46
JS-2002 Dry 44.40 3.51 1870.57
Hydro priming 59.97 3.62 3038.08
CaCl2 82.20 3.11 4074.65
KNO3 53.30 3.01 2747.08
Tab 1. Effect of seed priming on seedling emergence in sorghum
( Shehzad et al., 2012)

47. Tab 1. Effect of hydro-priming duration on
germination and seedling vigour of rice (oryza sativa)
Hydro-priming
treatments
Germination (%)
8 hrs 73.00
16 hrs 74.50
24 hrs 77.25
32 hrs 81.75
40 hrs 86.75
48 hrs 87.75
56 hrs 69.00
64 hrs 55.00
72 hrs 49.25
control 68.75
(Prasad et al., 2012)
Case study 6

48. Tab 1. Biochemical changes due to seed priming in
maize hybrid COH(M) 5
Case study 7
Priming
treatment
Germination
(%)
Shoot
length (cm)
Root length
(cm)
Vigour
index
Control 92 18.23 20.55 3569
Water for 6 hours 94 20.32 23.04 4123
1% KH2PO4 for
6 hours
98 23.45 26.09 4839
(Satish et al., 2012)
The objective of the present study was to ascertain the changes in
seed profile due to priming and the changes in DNA,phytate and
minerals.

49. CHANGES IN THE PROTEIN PROFILE
M:protein marker
T1: Control
T2: Seeds primed with water for 6 hours
T3: Seeds primed with 1% KH2PO4 for 6 hours
(Satish et al., 2012)

50. CHANGE IN THE DNA CONTENT
Priming treatment At 6 hours (μg) At 12 hours (μg) At 48 hours (μg)
control 53.9 65.1 249
Water for 6 hours 66.3 124.2 259.1
1% KH2PO4 for 6 hours 66.8 125.3 259.5
(Satish et al.,2012)

51. CHANGE IN THE PHYTATE CONTENT
Priming treatment At 6 hours (g) At 24 hours (g) At 48 hours (g)
control 1.12 0.55 0.27
Water for 6 hours 1.02 0.28 0.11
1% KH2PO4 for 6 hours 1.02 0.26 0.10
(Satish et al.,2012)

52. EFFECT OF HYDROPRIMING METHOD ON MAIZE (Zea mays)
SEEDLING EMERGENCE
• The hydro-priming methods were-
T1 : Non-priming
T2 : 14 hours soaking + drying + storing (2 months)
T3 : 18 hours soaking +drying + storing (2 months)
T4 : 22 hours soaking + drying + storing (2 months)
T5 : 14 hours soaking+ surface drying (2 hour)
T6 : 18 hours soaking + surface drying (2 hour)
T7 : 22 hours soaking + surface drying (2 hour)
• Effect of different hydropriming methods on seedling emergence
performance of maize was evaluated at two moisture levels viz.,
30% and 60% moisture of saturated sand in the experiment.
Ahammad et al., 2014
Case study 8

53. Effect of hydropriming methods on germination
percentage and mean germination time of maize seed.
Treat
ment
Germination (%)
30% moisture 60% moisture
T1 40 76
T2 43 81
T3 54 87
T4 49 84
T5 78 90
T6 84 95
T7 81 93
Treat
ment
Mean Germination time (day)
30% moisture 60% moisture
T1 5.19 4.75
T2 4.99 4.39
T3 4.76 4.21
T4 4.85 4.26
T5 4.13 3.42
T6 3.56 3.22
T7 3.67 3.32
Ahammad et al.,2014
Table 1 Table 2
T6 : 18 hours soaking + surface drying

54. Tab 1. Optimization of hydropriming techniques for
rice seed invigoration
Hydro-priming MGT
(days)
Germination
(%)
MET
(days)
Field Emergence
(%)
control 2.70 66.33 5.41 41.41
Hydropriming 12
h
2.66 74.00 5.23 62.03
Hydropriming 24
h
2.40 83.00 3.67 71.97
Hydropriming 36
h
2.03 86.33 3.05 64.19
Hydropriming 48
h
1.91 100.00 2.44 74.19
Hydropriming 60
h
2.91 68.56 5.31 39.75
(Farooq et al., 2006)
Case study 9