Impact of Hydro and Osmo-Priming of Soybean on seed germination, seedling

emergence and biochemical traits

By

Muhammad Shumail Shahid

(FA19C1BA232)

A report submitted in partial fulfillment of the requirement for the degree of

BACHELOR OF SCIENCE (HONS.)

IN

AGRICULTURE (AGRONOMY)

Department of Agronomy

Faculty of Agriculture and Environment

The Islamia University of Bahawalpur, Bahawalpur
2022
DECLARATION
I hereby declare that the research work entitled “Impact of Hydro-priming
& Osmo-priming of Soybean on seed germination, seedling emergence and
biochemical traits” Is the product of original work done by me and no part has
been copied from any published source (Except the References, mathematically
standard or genetic models/equations/formulas/protocols, etc.)I further declare
that this work has not been submitted for the granting of any diploma/degree. If
the information submitted is determined to be inaccurate at any time, the
institution may take action. In case of any fault, the scholar shall be prosecuted by
HEC’s plagiarism policy.

Muhammad Shumail Shahid

ACKNOWLEDGEMENTS

All the praises and thanks to Almighty ALLAH, the compassionate, the merciful
and the only creator of the Universe, Who blessed me with health, talented
teachers, affectionate parents and sympathetic friends and bestowed me with the
potential and ability to contribute a little to the existing ocean of scientific
knowledge. I offer my humblest thanks to the Holy Prophet HAZARAT
MUHAMMAD (P.B.U.H.) who is forever a torch of guidance and knowledge for me
to perceive the higher ideas of life.

I would like to express my sincere appreciation and profound gratitude to

my supervisor Dr. Muhammad Latif Malik and for his continuous, prolonged
reconsideration and critical assessments, inspiring guidance and helpful
suggestions in the conduct of this work and particularly in reviewing the
manuscript. I also thank lab assistant for his affectionate attitude, valuable
suggestions and the best co-operation throughout the research.

I wish to record my sense of admiration for all my colleagues and friends

who cooperated with me in any way during my B.Sc. studies. I am profoundly
indebted for the prayers and sacrifices of my affectionate father, loving mother,
sincere and caring brothers and sisters. May Allah bless them with all the
pleasures of this world and beyond (Amin).

Muhammad Shumail Shahid

CERTIFICATE

I certify that the contents and form of this project, submitted by Muhammad
Shumail Shahid in the report titled “Impact of Hydro and Osmo-Priming on
Soybean Seed germination and emergence” is based on the result of the research
study conducted by the candidate under my supervision. No portion of this work
has been formally being offered for higher in this university on any other institute
of learning and to the best of the author’s knowledge, and no material has been
used in this research report which is not their work, except where due
acknowledgment has been made. He has fulfilled all the requirements and is
qualified to submit this report in partial fulfillment of the degree of Bachelor of
Science (Hons.) in agriculture agronomy in the Faculty of Agriculture “at the
Islamia University of Bahawalpur”.

________________________

Dr. Muhammad Latif Malik

Professor /Supervisor
Department of Agronomy
Faculty of Agriculture and Environment,
The Islamia University of Bahawalpur
Introduction

Soybeans(Glycine max (L.) are a widely consumed agricultural commodity around

the world in many forms, such as the whole soybean, soy oil and soy meal
markets (Fearnside, 2001). However, only 6% of the world’s total soy production
is used in the form of whole beans, while the remaining 94% is crushed (Oliveira
and Schneider, 2016). Whole soybeans may be processed for industrial usage
(e.g., biofuel and edible oils) or crushed to produce food products (e.g., soy milk,
animal feed, and soy sauce) (WWF, 2014). In addition, compared with the other
crops, soybean has the potential for greater resource efficiency. Taking land-use
efficiency, for instance, a soy yield of 3000 kg per hectare can be used to produce
343 kg of cattle meat, 600 kg of pork or 1200 kg of poultry (Garrett and Rausch,
2016).Otherwise, without soy meal, cattle production can only produce one head
and 250 kg of cattle meat on average on 1 ha of land (Walker et al., 2013).

Soybean is a globally important Crop providing fixed N and high-quality

protein and oil (Holmberg 1973; Kamp et al. 2010; Kusano et al.
2015;Lewandowska and Michalak 2019). Crop rotation with Soybean increased
yield of many crops (Lauer et al.1997; Kelley et al. 2003), improved the energy of
crop Production systems (Rathke et al. 2007), and reducedWeed occurrence
(Jedruszczak et al. 2005). According to Lewandowska (2016), for many farmers the
production of This plant is a “godsend” of defective rotation to which Belongs
mainly cereals, maize, and rapeseed. The inclusion of soybean cultivation in crop
sequences is an Important and valuable element (Michalak et al. 2018).
 Soybean is the richest, cheapest and easiest source of best quality protein
and fat. It is also called “Gold of soil”. It is major source of edible oil and protein.
Hence, the crop is called as vegetarian meat and wonder crop.

Soybean is one of the most important contributors to protein and world’s

edible vegetable oils. Soybean is the world’s most important seed legume, which
contributes to 25 % of the global edible oil, about two-thirds of the world’s
protein concentrate for livestock feeding. Soybean meal is a valuable ingredient in
formulated feeds for poultry and fish. The cultivation and use of soybean could be
traced back to the beginning of China’s agricultural age. Chinese medical
compilations, dating back 6,000 years, mention its utilization for human
consumption [12]. To the populace of China, Japan, Korea, Manchuria,
Philippines, and Indonesia, for centuries, soybean has meant to be meat, milk,
cheese, bread, and oil. This could well be the reason, why in these countries, it
has earned epithets like ‘‘Cow of the field’’ or ‘‘Gold from soil’’ [8]. Owing to its
amino acids composition, the protein of soybean is called a complete protein. Its
nutrition value in heart disease and diabetes is well known. It is significant that
Chinese infants using soybean milk in place of cow’s milk are practically free from
rickets. Today, USA, Brazil, and Argentina are the ‘‘Big-3’’ producers of the world
(Table 1). Versatility of soybean was recognized in the West quite recently.
Around 1921, China produced about 80 % of the world’s soybean [4]. In India,
Soybean was introduced from China in tenth century AD through the Himalayan
routes, and also brought in via Burma (now Myanmar) by traders from Indonesia.
As a result, soybean has been traditionally grown on a small scale in Himachal
Pradesh, the Kumaon Hills of Uttar Pradesh (now Uttaranchal), eastern Bengal,
the Khasi Hills, Manipur, the Naga Hills, and parts of central India covering
Madhya Pradesh. It has also been reported that the Indian continent is the
secondary center for domestication of the crop after China [9, 11, 18]. At present,
India ranks fifth in the area and production in the world after USA, Brazil,
Argentina, and China (Table 1).

Pakistan depends a lot upon imports of soybeans; the country imports

about 2-2.5 million mt of soybeans every year for poultry and livestock . Brazil
and the USA are the main suppliers, where Brazil takes the biggest share of the
import. There is huge potential to tap the local demand of soybean by
commercializing soybean crop in Pakistan. Moreover, the crop has been
neglected for one reason or the other resulting in decline of its area under
cultivation. Soybean Crop and two other non-conventional oilseed crops, viz.
Sunflower and Safflower were introduced in mid-sixties and commercial
cultivation began in 1970-71. Coordinated Research work was started at Pakistan
Agricultural Research Council (PARC) and Provincial Research Institutes in 1977-78
and eight soybean varieties were developed. Efforts were being made to
commercialize soybean crop among farmers and the area under soybean
cultivation reached to 5980 hectares during 1986-87. Still the cropping area
increased with very slow pace and frequent variations during the period of 1970-
71 to 1997-98 and later declined to few hectares after 2005-06 (Figure 2). In the
absence of a coherent production policy like other major crops, soybean could
not be commercialized and area under its cultivation reduced gradually.

Mainly issues like lack of marketing of produce and its by-products,

absence of adequate skills, knowledge and production technology and low
economic return hindered soybean commercialization [4]. Most importantly, the
unavailability of high yielding, climate ready and pest resistant soybean varieties
is the major bottleneck in crop adoption. The traditional varieties (William-82,
Ajmeri, NARC-I, NARC-II and Rawal-I) introduced or developed in 1980s and 1990s
have lower yield potential.

Moreover, the yield per unit area of the crop is still low in Pakistan. Poor
germination and low seed viability are among the serious problems in the
production of soybean. The use of high quality seed with appropriate seed rate is
essential to establish a suitable plant population in a soybean field for better
returns. Vigorous seeds germinate rapidly, uniformly and are able to withstand
environmental adversity after sowing (Ajouri et al., 2004; Marwat & Nafziger,
1990).

Seed quality may be improved by production techniques or by seed

pretreatments with water (hydro-priming), osmotic solutions (osmo-priming) and
matric materials (matri-priming) (Hur, 1991; Harris et al., 1999; Kaya et al., 2008;
Pill et al., 1991).Pre-sowing seed treatment including priming is known to improve
seed performance in the field (Pill et al. 1991; Parera and Cantiliffe1994; Pill and
Necker 2001; Nawaz et al. 2013; Paparella Et al. 2015).Seeds can be primed in
different media such as water(hydropriming), aerated low water potential
solutions such as polyethylene glycol, or salt solution (KNO3, KCl,K3PO4, KH2PO4,
MgSO4, CaCl2and NaCl)(osmopriming),solid matrix(matripriming) and priming
with plant growth regulators and polyamines (Farooq etal. 2008).Seeds are then
removed, rinsed 2–3 times and re-dried nearer to original weight to permit
routine handling and storage if required (Farooq et al. 2007).
 Seed priming is a treatment commonly applied In agriculture, horticulture,
and forestry to improve the Germination of the seeds. Seed priming has been
successfully demonstrated to improve germination and emergence in seeds of
many crops, particularly seeds of vegetables and small seeded grasses (Heydecker
& Coolbaer, 1977; Bradford, 1986). Seed priming is a suitable method to enhance
seed and seedling vigor, leading to better stand establishment and yield (Bruggink
et al., 1999; Khalil et al., 2010).

The beneficial effects of seed priming have been demonstrated for many
field crops such as wheat (Parera and Cantliffe, 1994), sweet corn (Chiu et al.,
2002), mung-bean (Khan et al., 2005), barley (Abdulrahmani et al.,2007), lentil
(Ghassemi-Golezani et al., 2008), cucumber (Ghassemi-Golezani and Esmaeilpour,
2008) and winter rapeseed (Ghassemi-Golezani et al., 2010). However, seed
priming decreased germination percentage in barley and corn (Sharif et al., 2006)
and reduced rate of seedling emergence and plants per unit area in sunflower
(Hussain et al., 2006). However, few detailed studies have been reported on the
performance of osmotically treated seed under field condition. Park et al., (1997)
reported that priming the aged seed of soybean resulted in good germination and
stand establishment. Harris et al., (2001a) reported that the direct benefits of
seed priming in all Crops included faster emergence, better, more and uniform
stands, less need to re-sow, more vigorous plants, better drought tolerance,
earlier flowering, earlier harvest and higher grain yield. Moreover, the
effectiveness of the priming process depends mainly on the selection of
appropriate conditions for seeds of a given species or even a genotype. Factors
affecting the success of conditioning include the following: light, temperature,
time, and drying method of seeds after application (Cantliffe et al. 1981; Chiu et
al. 2005; Di Girolamo and Barbanti 2012; Rajjou et al. 2012). It is necessary to add
that the amount of time it takes to prime seeds is also dependent on the plant
species, because the priming process is customized to each relevant crop (Kubala
et al. 2013). According to McDonald (2000), drying affects the efficiency of seed
conditioning more than any other factor. Storage of seeds after priming precedes
their drying process to the initial level of moisture content. It should be
emphasized that both the time and temperature of drying have a decisive
influence on the subsequent development of seeds, because benefits of
conditioning may be lost during the dying process (Schwember and Bradford
2005).

There are more reports about seed priming effects on different plants. It was
reported that soybean seed-priming made better seedling emergence and yield
improvement (Arif et al., 2008).In this regard, hydro priming and osmo priming
with different chemicals is carried out for 12 h. This research was carried out to
investigate the effects of hydro and osmotic priming on Soybean variety…………. to
check seed germination, seedling emergence and biochemical traits at different
treatments.
Review of Literature

The need for increased seed quality has become a priority necessary to face the
current demand for high standards in the agricultural market. Achieving rapid and
uniform seedling emergence is a key point for crop performance since slow
germination rates frequently expose plantlets to adverse environmental
conditions and soil-borne diseases (Osburn and Schroth 1989). ‘Priming’ is a well-
established treatment for enhancing seed quality. Seeds subjected to priming
show increased germination rates which result in high levels of biotic/abiotic
stress resistance and crop yields. All these features that improve product
competitiveness directly correlate to seed vigour, a complex agronomic trait
controlled by multiple genetic and environmental factors (Rajjou et al. 2012; Jisha
et al. 2013).

Priming is a water-based technique that allows controlled seed rehydration

to trigger the metabolic processes normally activated during the early phase of
germination (‘pre-germinative metabolism’), but preventing the seed transition
towards full germination. Thus, priming treatment must be stopped before loss of
desiccation tolerance occurs. The benefits of seed priming have been widely
reported. Along with synchronous and fast emergence, primed seeds show
reduced photo- and thermo-dormancy, a wider range of germination
temperatures and better capacity to compete with weeds and pathogens (Ellis et
al. 1988; Hill et al. 2008). Moreover, the use of priming permits growers to
efficiently manage water usage and harvest scheduling (Hill et al. 2008). The
success of seed priming is strongly correlated to plant species/genotype and
physiology, seed lot and vigour, as well as to the priming method applied (Parera
and Cantliffe 1994). Priming is routinely used to treat vegetables seeds,
particularly carrot (Daucus carota L.), leek and onion (Genus Allium) (Dearman et
al. 1987), celery (Apium graveolens L.), lettuce (Lactuca sativa L.), endive
(Cichorium endivia L.), pepper (Genus Capsicum) and tomato (Solanum lycoper-
sicum L.) (Parera and Cantliffe 1994; Di Girolamo and Barbanti 2012). Priming
contributes to enhance product quality in the flower seed industry where it is
frequently used on elite varieties of Petunia hybrida L. (Di Girolamo and Barbanti
2012, Momin 2013). Herbs, e.g. Rosmarinus officinalis L. and Salvia splendens L.,
also benefit from seed priming (Di Girolamo and Barbanti 2012). Large scale
routine priming of commercial cereal seeds is more difficult to achieve, although
advantages are well known (Murungu et al. 2004). Furthermore, priming provides
optimal conditions to accelerate seed germination when requested by the cereal
grain and brewing industry (Yaldagard et al. 2008). In most cases, the priming
procedures commercially used are proprietary and managed by specialised agri-
seed companies.

Theophrastus (371–287 B.C.) investigated the seed response during

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

Effects of seed priming on Soybean

In the case of Soybean, seed priming has been shown to have a positive effect on
various physiological and biochemical processes like antioxidants enzymes
production in the seed and seedling. Some of the most significant effects of seed
priming on soybean include:

• Enhanced water uptake and imbibition: Seed priming helps to increase the
water uptake capacity of the seed, which is essential for germination. It also
leads to better water imbibition and mobilization during germination,
which results in higher seedling vigor and faster emergence.
• Improved enzymatic activity: Seed priming can trigger the activation of
various enzymes involved in the process of seed germination, such as α-
amylase, protease, and lipase. This helps to break down stored seed
reserves into simpler forms that are more readily available for seedling
growth.
 • Increased antioxidant capacity: Seed priming has been shown to enhance
the antioxidant capacity of Soybean seeds, which helps to protect them
from oxidative damage during germination and early seedling growth.
• Higher nutrient uptake: Seed priming can enhance the uptake of essential
nutrients, such as phosphorus and nitrogen, from the soil by improving the
root system’s development and activity.

Overall, these effects of seed priming on Soybean result in better seedling growth,
increased crop establishment and higher yields.

Hydropriming

In hydro-priming, the seeds are simply soaked in water prior to sowing for a
defined period of time depending upon the radical protrusion time of each plant
species. Aeration may or may not be provided to the seeds. It is a very simple,
low-cost technology as simple water is used in this priming technique.
Hydropriming is followed by surface drying or redrying of seeds to their original
weight. Many studies have reported that hydropriming may improve the stand
establishment, seedling vigor, and productivity of field crops under optimal and
suboptimal conditions. For example, Roy and Srivastava (1999) found that
hydropriming of wheat seeds improved the seed germination in a saline soil. In
another study, seed emergence and plant growth at vegetative and reproductive
stages were improved in maize due to hydropriming (Nagar et al. 1998). Many
studies have reported that hydropriming of seeds improved the seed germination
and seedling emergence of wheat (Harris et al. 2001; Afzal et al. 2007; Nawaz et
al. 2017). Hydro-priming is a simple and economical technique in which seeds are
soaked in water for a specific period and dried to a certain moisture level before
sowing (Singh et al. 2015a, b). This technology is useful in areas with adverse
environmental conditions including high heat and drought stress. Hydro-priming
improves the water uptake efficiency and seed hydration under such conditions
(McDonald 2000). However, it is critical to maintaining optimum humidity and
temperature to avoid radical projection, as hydro-priming may result in
uncontrolled water uptake (Taylor et al. 1998). The key technique of hydro-
priming is “drum priming” invented by Rowse (1991). Drum priming consists of a
drum containing seed lot connected to a boiler producing vapours. The vapours
upon entering the drum condense into water droplets. During treatment, the
increase in relative seed mass is measured along with strict control of the volume
of the water and time required for seed hydration (Warren and Bennett 1997).
Various research studies have explained that during drought stress conditions,
hydro-priming increases the germination and seedling growth by 3–4 times as
compared to non priming (Kaur et al. 2002). Likewise, Sung and Chiu (1995)
reported that watermelon seeds when subjected to hydro-priming result in fast
germination and seedling growth. In addition to several food crops such as Allium
porrum, coriander, pyrethrum, and wheat, hydro-priming resulted in a uniform
early germination in many desert plants like desert cacti (Dubrovsky 1996)In
conclusion, hydro-priming improves seed germination, seedling emergence, and
productivity of field crops.

Hydro-priming is a technique which uses water to soak seeds, drying it for

dehydrating and then sowing the next day. This leads to increase in the process of
germination, accelerates seedling growth and strength [10]. [5] carried out hydro-
priming of aged chickpea seeds and found increased rate of germination and dry
weight of seedling. Priming increased the quantity of grains/plant/unit area from
low vigour seeds in comparison with high vigour seeds and also restored the
quality of aged and damaged seeds enhancing the production in the field. It also
increased germination percentage and seedling emergence which cause
productive use of resources (soil, light) to increase the amount of grains. Decrease
in seed vigour is an indication of electrolyte leaching due to an increase in
membrane permeability. Electrolytes such as inorganic ions, amino acids and
sugars are leached, which have high impact on metabolism and different activities
involved in synthesis causing decrease in germination and overall growth of
seedling. Priming seeds might have repaired seeds by reducing electrolyte
leakage, repair of biomolecules such as DNA, RNA, proteins, enzymes and
membranes. Similarly, [11] have evaluated the effect of different priming
methods such as osmo-priming, hydro-priming (Zinc sulphate with 100 mM Zn
and Monopotassium phosphate with 50 mM P) and halo-priming (1% potassium
nitrate solution) on corn. Good results in terms of metabolic processes, higher
germination percentage and rate, seed yield and yield components were seen in
primed seeds. Hydro primed seeds showed maximum results suggesting that ion
accumulation in the seed has no toxic effect on the embryo. This treatment thus
can be used to increase tolerance in drought conditions and enhance growth
leading to environment-friendly techniques for improving crop yields.

Osmopriming

Osmopriming (‘osmotic priming’, ‘osmotic conditioning’)is a wide-spread pre-

sowing procedure that involves treatment with osmotic solutions at low water
potential facilitating the control of water uptake. During imbibition, water entry
into the seed associates with progressive ROS accumulation and oxidative damage
of cellular components (lipid membranes, proteins, nucleic acids). The main goal
of osmopriming is to limit the ROS-mediated oxidative injury by delaying water
entry. Thus, the water potential of the osmotic agent is a crucial parameter
(Heydecker and Coolbear 1977; Taylor et al. 1998).

Seed priming has been successfully demonstrated to improve germination

and emergence in seeds of many crops, particularly seeds of vegetables and small
seeded Grasses (Heydecker & Coolbaer, 1977; Bradford, 1986). The beneficial
effects of priming have also been demonstrated for many field crops such as
wheat, sugar beet, maize, Soybean and sunflower (Parera & Cantliffe 1994; Singh,
1995;Sadeghian & Yavari, 2004). However, few detailed studies have been
reported on the performance of osmotically treated seed under field condition.
Park et al., (1997) reported that priming the aged seed of soybean resulted in
good germination and stand establishment.

Priming treatment has beneficial effects on the vigor and viability of seeds
which is manifested by improved germination performance (increased
germination rate, reduced time to achieve 50% germination – T50, increased total
germination percentage, and greater uniformity of germination) and seedling
growth especially under adverse environmental conditions. A wider discussion on
the impact of priming on seed germination, seedling growth, and development
was addressed by Kubala et al. (2013)

Osmopriming is a pre-sowing seed treatment technique that involves soaking seeds

in an osmotic solution to enhance seed germination and vigor. This technique is
commonly used in soybean cultivation to improve crop establishment and yield.
During osmopriming, the osmotic agent creates an osmotic gradient between the
seed and the surrounding solution, causing the seed to absorb water and the
osmotic agent into its cells. This process triggers the activation of various enzymes
and metabolic pathways, which promote seed germination and early seedling
growth.

The process of osmopriming involves the following steps:

1. Seed selection: Choose healthy, mature seeds with no physical damage or

disease.
2. Osmotic treatment: Soak the seeds in an osmotic solution containing a
specific concentration of solutes such as sodium chloride (NaCl). This
treatment is carried out for a specific period, typically ranging from 12 to 24
hours.
3. Drying: Remove excess water from the seeds by placing them on a dry
surface or using a paper towel.
4. Storage: Store the primed seeds in a cool, dry place until sowing.

Compounds are currently used for osmopriming includes, NaCl, Ascorbic Acid,
CaCl2, H2O2, FeSO4.7H2O, Copper Sulphate, Urea & KNO3. Priming with these
compounds can either be effective as the PEG-based treatment or it can
trigger considerable different responses, depending on seed morphology. The
presence of a semi-permeable outer layer found in certain seeds is the main
structural feature influencing priming effectiveness (Pill 1995).This outer layer,
consisting of amorphous tissue placed between pericarp and seed coat, can
limit or avoid ion/solute exchange, therefore controlling seed permeability to
water and priming Agents (Zhou et al. 2013). Treatment is preferentially
carried out in a sealed container that permits air circulation, avoiding excessive
evaporation.The procedure avoids all the technical problems associated with
 aeration and allows the simultaneous treatment of large amount of seeds at
limited costs.

Overall, osmopriming is a simple and effective technique that can be used to

improve the performance of soybean seeds, leading to higher crop yields and
better crop establishment.

Chapter 3

Material and method

The current experiment was conducted to evaluate the effect of seed priming of
Soybean on crop germination and anti-oxidant enzymes production.

3.1. Experimental Area:

The experiment was conducted at the Agronomy Research Laboratory, Faculty of

Agriculture and Environmental Science. The Islamia University of Bahawalpur,
Pakistan

3.2. Growing condition:

The germination test was conducted in wirehouse at Faculty of Agriculture and
Environmental Science. The Islamia University of Bahawalpur, Pakistan having an
average temperature range of 25 OC to 30 OC and relative humidity ranging from
59% to 70%. Experiment was laid out in pots. Seeds were sown in pots filled with
Sandy soil and Clay-Loam soil separately as growth medium.

3.3. Chemicals used for seed priming:

5. Distilled water
6. NaCl
7. Ascorbic acid
8. CaCl2
9. H2O2
10.FeSO4.7H2O
11.CuSO4
12.Urea Solution
13.KNO3
14.Potassium phosphate

3.4. Treatments:

T0= Control

T1= 1L Distilled Water

T2= 5g Nacl

T3= 5g Ascorbic acid

T4= 10g CaCl2

T5= 10ml H2O2

T6= 10g FeSO4.7H2O

T7= 10g CuSO4

T8= 5g/L Urea Solution

T9= 10g KNO3

T10= 10g Potassium phosphate

In control only dry seeds are used, while in other treatments seed priming was
performed with distilled water and chemicals with 1:5 volume.

3.5. Soybean variety:

3.6 Soil texture:

S1= Sandy soil

S2=Sandy-Loam soil

3.7. Experimental design:

This experiment was designed according to Complete Randomized Design (RCBD)

having 66 treatments which was replicated 6 times(3 times for each soil).

3.8. Date of sowing:

 21 Feb 2023
3.9. Data Analysis:
ANOVA was used to analyze the data. Computer software program Microsoft
Excel 2010 was used to represent the comparison of means among treatments.

3.10. Planting material:

Clean and healthy seeds of soyabean were taken and seeds divided into 11
groups. The first group is considered as control and only dry seeds without any
priming used. In second group distilled water is used as priming agent. In third
group NaCl is used as priming agent. In forth group CaCl 2 is used as priming agent.
In fifth group Ascorbic is used as priming agent. In sixth group H 2O2 is used as
priming agent. In seventh group Iron Sulphate is used as priming agent. In eight
group Urea is used as priming agent. In nine group Copper Sulphate is used as
priming agent. In tenth group KNO3 is used as priming agent. In eleventh group
Potassium phosphate is used as priming agent.

3.11. Preparation of priming solutions

3.11.1. Distilled water:

Take 1L volume of distilled water in a volumetric flask.

3.11.2. NaCl Solution:

Take 5g of NaCl in a volumetric flask. Add water in flask and make the solution
having total volume upto 1000ml.

3.11.3. Ascorbic acid Solution:

Take 10g of Ascorbic acid in a volumetric flask and add water in flask and make
the solution having total volume of 1000ml.

3.11.4. CaCl2 Solution:

Take 10g of CaCl2 in a volumetric flask and add water in flask and make solution
having total volume of 1000ml.

3.11.5 H2O2 solution:

Take 10ml of H2O2 in a volumetric flask and add water in flask and make the
solution having total volume of 1000ml.

3.11.6. Ferrous Sulphate:

Take 10g of Ferrous sulphate in a volumetric flask and add water in flask and
make the solution having total volume of 1000ml.

3.11.7. Copper Sulphate solution

Take 10g of Ascorbic acid in a volumetric flask and add water in flask and make
the solution having total volume of 1000ml.

3.11.8. Urea Solution

Take 10g of Ascorbic acid in a volumetric flask and add water in flask and make
the solution having total volume of 1000ml.

3.11.9. KNO3 Solution

Take 10g of KNO3 in a volumetric flask and add water in flask and make the
solution having total volume of 1000ml.
3.11.10. Potassium phosphate Solution

Take 10g of Potassium phosphate in a volumetric flask and add water in flask and
make the solution having total volume of 1000ml.

Priming Procedure

Above mentioned solutions are taken separately in a well aerated box and 200
grams well clean and healthy seeds of soybean are soaked in each solution for 12
hours and after the 12 hours seeds are taken out from the solutions and placed
for drying.

3.12. Observations:
1. Germination Percentage
2. Mean Germination Time
3. Germination Index
4. Coefficient of Velocity of germination time
5. Mean germination rate
6. Relativized percentage

Procedure and Formulation for recording observation

3.12.1.1. Germination Percentage:

Germination percentage is an estimate of the viability of a population of seeds.

The equation to calculate germination percentage is:
GP = seeds germinated/total seeds x 100
The germination rate provides a measure of the time course of seed
germination.

3.12.1.2. Mean Germination Time

Mean time to germination (MGT) is a measure of the rate and time-spread of
germination.

Mean germination time (MGT) was calculated by using the equation:

MGT = ∑ (n × d) / N

Where n = number of seeds germinated on each day

d = number of days from the beginning of the test

N = total number of seeds germinated at the termination of the experiment

(Ellis and Roberts,
1981)

3.12.1.3. Germination Index

The germination index (GI), which is a measure of the percentage and speed of
germination.

GI = ΣG/T

Where G is the percentage of seed germinated per day, and T is the

germination period

3.12.1.4. Coefficient of Velocity of germination time CVG

The coefficient of velocity of germination (CVG) (Jones and Sanders, 1987) gives
an indication of the rapidity of germination. Its value increases when the
number of germinated seeds increases and the time required for germination
decrease

CVG = N1 + N2 + · · · + Nx/100 × N1T1 + · · · + NxTx

N = No. of seeds germinated each day, T=No. of days from seeding
corresponding to N

3.12.1.5. Mean germination rate

It is the reciprocal of the mean germination time
v¯=1/ t¯
Where; t¯= Mean germination time

Mean germination rate (MGR) = CV/100 = 1/T

Where T is mean germination time and CV: coefficient of velocity

3.12.1.6. Relativized percentage

The germination percentage can be relativized by the following equation (Fitch et
al., 2007):

R (%) =AP/HP×100

AP= actual percentage

HP= highest percentage amongst group of data.
CAT activity (g-1FW

Sandy soil Clay Loam soil

6

0
V1T0

V1T1

V1T2

V1T4

V1T5

V1T6

V1T7

V1T10
V1T3

V1T8

V1T9

Control Hydro Priming Priming Priming Priming Priming Priming Priming Priming Potas-
priming with NaCl with with with with with with Urea with sium
Ascorbic CaCl2 H2O2 FeSO4. Copper KNO3 Phos-
Acid 7H2O Sulphate phate
priming
CAT g/FW
POD activity (mg/protein/min

Sandy soil Clay Loam soil

100
90
80
70
60
50
40
30
20
10
0
V1T0

V1T4

V1T5

V1T6

V1T7

V1T8
V1T1

V1T2

V1T3

V1T9

V1T10

Control Hydro Priming Priming Priming Priming Priming Priming Priming Priming Potas-
priming with NaCl with with with with with with Urea with sium
Ascorbic CaCl2 H2O2 FeSO4. Copper KNO3 Phos-
Acid 7H2O Sulphate phate
priming
POD (mg/protein/min)
APX(g-1FW

Sandy soil Clay Loam soil

2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
V1T2

V1T3

V1T4

V1T5

V1T6
V1T0

V1T1

V1T7

V1T8

V1T9

V1T10

Control Hydro Priming Priming Priming Priming Priming Priming Priming Priming Potas-
priming with NaCl with with with with with with Urea with sium
Ascorbic CaCl2 H2O2 FeSO4. Copper KNO3 Phos-
Acid 7H2O Sulphate phate
priming
APX (g/FW)
 SOD activity g-1 FW

Sandy soil Clay Loam soil

1.6

1.4

1.2

0.8

0.6

0.4

0.2

0
V1T2

V1T3

V1T4

V1T5

V1T6
V1T0

V1T1

V1T7

V1T8

V1T9

V1T10
Control Hydro Priming Priming Priming Priming Priming Priming Priming Priming Potas-
priming with NaCl with with with with with with Urea with sium
Ascorbic CaCl2 H2O2 FeSO4. Copper KNO3 Phos-
Acid 7H2O Sulphate phate
priming
SOD (g/FW)
 Sandy soil Clay Loam soil
7

0
V1T0

V1T1

V1T2

V1T6

V1T7

V1T8
V1T3

V1T4

V1T5

V1T9

V1T10
Control Hydro Priming Priming Priming Priming Priming Priming Priming Priming Potassium
priming with NaCl with with CaCl2 with H2O2 with with with Urea with KNO3 Phosphate
Ascorbic FeSO4. Copper priming
Acid 7H2O Sulphate
Carotenoids (ug/ml)