Proposal on

Effect Of Seed Priming by Mannitol On Germination

Characteristic Of Maize In Artificial Drought Condition

Submitted by: Submitted to:

Roll No. : 42-50 Roshan Subedi
Sixth Semester Asst. Professor
IAAS, Lamjung IAAS, Lamjung
Abstract
Drought stress during the imbibition phase of germination is the primary reason for both inhibition
or delayed seed germination and seedling establishment. Rapid germination of seed and emergence
of seedling is pre-requisite for the good maize production under adverse environmental stresses
like salinity, drought soil and high and low temperature which has been reported under seed
priming in osmoticum such as mannitol, polyethylene glycol (PEG). To enhance the pre sowing
metabolic activities priming is done that ensures the high germination percentage. An experiment
will be conducted in Lab on new variety 940 of Maize primed with Mannitol of 2%, 4%, 6% and
8% (w/v) concentrations with artificial drought condition created by using different concentration
of salt solutions viz. 1.5 bar, 4.9 bar, 10.3 bar, 17.6 bar to study the effect of priming by mannitol
on germination of maize in water stress environment performed as factorial experiment under
completely randomized design (CRD) with three replications.

Introduction
Background
Water stress due to drought is probably the most significant abiotic factor limiting plant and also
crop growth and development (Khalilli et al., 2013). Seed germination and early seedling growth
are potentially the most critical stages for water stress. Many studies on plant responses to drought
stress with regard to seed germination and seedling growth have been recently reported, including
studies of agricultural crops, Oryza sativa, Zea mays, Spinacia oleracea, Amaranthus
mangostanus and helianthus annuus (Ahmad et al., 2009). Uneven or poor germination and
subsequently uneven seedlings growth can lead to a great financial losses, by reducing crop
production and lower prices of uneven plant batches which can be addressed by seed priming
resulting increased speed and uniformity of germination.
Seed priming is a presowing strategy for influencing seedling development by modulating pre-
germination metabolic activity prior to emergence of the radicle and generally enhances
germination rate and plant performance. Seed priming is soaking of seeds in a solution of any
priming agent followed by drying of seeds that initiates germination related processes without
radical emergence (McDonald, 2000). There are reports that seed priming permits early DNA
replication, increase RNA and protein synthesis, enhances embryo growth, repairs deteriorated
seed parts and reduces leakage of metabolites. Seed priming is seen as a viable technology to
enhance rapid and uniform emergence, high vigor and better yields in some field crops (Basra et
al., 2002; Chiu et al., 2002; Harris et al., 1999; Murungu et al., 2004). Earlier works showed that
the success of seed priming is influenced by the complex interaction of factors including plant
species, water potentiality of the priming agent, duration of priming, temperature, seed vigour and
dehydration, and storage conditions of the primed seed. Germinating the seeds in solutions of
different water potentials is a convenient method to study the responses of seeds at germination
against low water potential (Naylor, 1992).
Problem Statement
Being drought sensitive crop, maize is affected at each and every stage of growth and development
by lesser moisture availability. Drought stress during the imbibition phase of germination is the
primary reason for both inhibition or delayed seed germination and seedling establishment. The
detrimental drought effects lose in yield and quality; and the death of plan. The germination and
establishment of seedlings to drought stress is very sensitive, in such a huge extent that drought
stress decreases seed germination and seedling have a non-uniform establishment (Soltani et al.,
2007). Drought stress is seriously affecting the maize crop resultantly hindering the productivity.
Insufficient seed germination and inappropriate seedling establishment has been a very
troublesome factor for the cultivation of maize in the rainfed areas. Maize grain size is greater than
other cereals so water requirement is greater for maintenance of osmotic potential and conversion
of stored food into consumable form for proper germination. Water absorption, imbibition and
metabolic enzymatic activation are hindered under limited water availability which reduces the
maize grain germination. After germination, water deficiency significantly reduced the plumule
and radicle growth which resulted in unusual seedling growth (Aslam, 2015).

Rationale of Study
Various seed enhancing technique are widely used to overcome such deleterious effect of drought
stress with the main aim of increasing the germination of seed. Priming is one of the most important
physiological method which boosts up the germination process of seed right up to the radicle
emergence stage ensuring the germination of the seed. Seed priming is a technique that allows
water uptake by the seed before planting to control the level of the activity is the initial buds
initiated germination that prevents of out roots, until the seeds of dry maintainability are able to
grow. Rapid germination of seed and emergence of seedling is pre-requisite for the good corn
production under adverse environmental stresses like salinity, drought soil and high and low
temperature. It has been reported that seed priming in osmoticum such as mannitol, polyethylene
glycol (PEG), and Sodium chloride increases the yield of chickpea, maize and wheat under
semiarid condition because of the rapid seed germination and seedling emergence. Also priming
of seed improves speeds, synchrony and percentage of seed germination. Numerous biochemical
changes have been reported in osmo-primed seed of different crops like a space is developed in
osmoprimed seed of tomato that facilitates water uptake, thereby accelerating the speed of
germination. And also, the ebryo expands considerably and compreses the endosperm, deforming
the tissues that have lost flexibility due to hydration It also has been shown to induce nuclear DNA
synthesis in the radical tip cells in tomato and several other plant species, including corn (Zea
mays)and leek (Aslam, 2015).
Objectives
Broad objective
 To evaluate the effect of seed priming by mannitol on germination characteristics of maize
in water stress environment.

Specific Objectives
 To calculate the different germination parameters
 To find out the effect of seed priming by mannitol in water stress environment

Literature Review
Seed priming is a process in which seeds are imbibed in water or osmotic solutions followed by
drying before radicle emergence (McDonald, 2000). Seed priming has been reported to improve
germination, reduce seedling germination time, and improve stand establishment, increase
emergence, earlier flowering, earlier maturing and higher grain yield (Harris et al., 1999). Hydro
priming involves allowing seeds to absorb sufficient water to initiate metabolic process but
insufficient water to allow completion of germination. When seeds are imbibed, the lag period
before radicle emergence is considerably reduced and improved the rate and uniformity of
germination.
The paper ”Comparative effect of polyethylene glycol and mannitol induced drought on growth
(in vitro) of canola (Brassica napus), cauliflower (Brassica oleracea) and tomato (Lycopersicon
esculentum) seedlings” published by Fazal Hadi and et al. has clearly stated that PEG 6000 is the
best for assessment of drought tolerance potential of plants at early growth stage. Polyethylene
glycol (PEG6000) and Mannitol were used for induction of drought stress and compared their
effects on seed germination, seedling growth and biomass and water contents. Seed germination
of all the species decreased significantly with the increasing concentration of the chemicals. The
lowest was found at the highest concentration. Similarly, shoot and root length and biomass
decreased with increasing concentration of PEG or Mannitol. The comparison of PEG and
mannitol showed that growth parameters got highly reduced in mannitol than that of PEG and the
toxic effect was also more in mannitol as compared to PEG. (Fazal, 2014).
Seed priming has been reported to enhance vigor, rapid and uniform emergence, and improved
yields of vegetable, ornamental and field crops under wide ranging field conditions. Many
physiological, biochemical and molecular changes have been found as the possible basis of this
performance. These include earlier and increased protein synthesis, aldolase, and isocitrate lyase
activity and greaterglucose-6-phosphate dehydrogenase and decreased alcohol dehydrogenase
activity. During priming, de novo synthesis of α-amylase is also documented, since increased α-
amylase activity has been correlated with improved metabolic activities and higher seed vigor.
(Dara, 2013). It has also been stated in the paper titled “Drought Stress in Plants: An Overview”
published by M. Farooq, M. Hussain, Abdul Wahid and K. H. M. Siddique that plant growth
substances such as salicylic acid, auxins, gibberellins, cytokinins, and abscisic acid modulate plant
responses toward drought. Though the exact mechanism of this is not known but it is believed that
plant hormones produce enzymes that act as antioxidants and reduce adverse effects of water
deficit. (Farooq, 2008).

In an experiment, performed at laboratory conditions of Division of Genetics and Plant Breeding

SKUAST-K FoA Wadura with thirteen corn cultivar the effect of stress levels on length of radicle
and radicle weight were significant. (Dar, 2018). It was also seen that in relation to various levels
of induced water stress, the root-shoot length, root shoot fresh weight and root dry weight of 6
different cultivars of maize responded significantly (Khan, 2009). Water stress in plants reduces
nutrient uptake and the weight gain, due to the limited transpiration and nutrient absorption as well
as inhibited root transport mechanisms (Tanguilig et. al., 1987). In the case of the Z. mays the
significant inhibition of growth was observed through changes in dry mass for both aboveground
and underground organs of plants treated with osmotic drought stress. The largest differences were
observed in plants grown from grains germinated on distilled water and watered with mannitol in
the growth phase
After the first day, there was a small percentage of germinated grains on the Petri’s dishes with
distilled water (control). In the other two cases, there were no germinated Z. mays grains. After 48
hours, the share of germinated individuals were about 64% (control), 44% (-0.5MPa) and 12% (-
1.5MPa). After another day results were as follows: 92%, 92% and 28% (the control group, -
0.5MPa and - 1.5MPa, respectively). After 96 hours, 100%, 100% and 68% of maize grains had
germinated. Statistically significant differences in the seedling length were observed in both
concentrations of mannitol (-0.5MPa and -1.5MPa) in relation to the seedlings growing on the
Petri’s dishes with distilled water (control). In the case of high concentration of mannitol, seedlings
reached about 1.5cm, which accounted for approximately 34% of the length of maize seedlings
from control. In low concentration, the length of the seedlings was about 60% of the control
seedling length.
Plants watered with mannitol solution at a concentration of -0.5MPa during growth had the shortest
roots (6.13cm) in comparison with control group (19.03cm). Significant differences in the length
of section from the root to the first leaf was found only between Z. mays plants which had grown
from grains germinating on dishes with -0.5MPa mannitol and plants watered with -1.5MPa
mannitol about concentration in growth phase. Generally, watering with increasing concentrations
of mannitol in germination phase and growth phase showed stimulatory effect on the length of Z.
mays 1 st leaf with. Additionally, statistical analysis showed their inhibitory effects on the growth
of the 2nd, 3rd and 4th Z. mays leaves. The highest concentrations of mannitol in both phases of
growth completely inhibited the development of the 5 th and 6th of the Z. mays leaves. Plants
watered with mannitol during growth had the shortest remainder of shoot in comparison with
control. The experiment showed a slight decrease in the length of the root of Z. mays in the group
treated with mannitol in the germination phase and a significant decrease in the group watered
with mannitol in the growth phase. The length of the ground organs of Z. mays was reduced in
each of the two phases. Statistically significant differences were demonstrated especially among
the plants watered with solutions of mannitol in the growth phase (Możdżeń, Katarzyna et. al,
2015)
According to the results of dry matter and water content in maize seedlings, it was indicated that
mannitol-induced osmotic stress significantly affects water uptake by germinating maize grains.
The biggest difference in the percentage content of water in comparison with control (distilled
water) was recorded for the seedlings in the highest concentration of mannitol (-1.5MPa). In the
case of the dry weight, seedlings germinated on mannitol had slightly higher mass values.
Determination of dry matter of Z. mays organs showed statistically significant inhibition of weight
gain after watering with mannitol. Analysis of water content in the organs of Z. mays revealed a
statistically significant reduction of water content in the root, the section from the root to the first
leaf and the first leaves of plants watered with mannitol in the growth phase. The remaining organs
of maize plants watered with mannitol in germination phase and in growth phase observed an
increase of water content compared to the water content in the organs of control plants. (Możdżeń,
Katarzyna et. al, 2015)

Methodology
The above entitled research "Effect Of Seed Priming by Mannitol On Germination
Characteristic Of Maize In Artificial Drought" will be conducted out in petridishes in the
laboratory of Agronomy -Institute of Agriculture and Animal Science, lamjung campus. The site
is located in the Lamjung district, Gandaki zone of the Western Development Region and borders
with Gorkha district in East, Kaski in West, Manang in North and Tanahun in South. The district
(Latitude: 28º03'19" to 28º30'38" N and Longitude: 84º11'23" to 84º38'10" E) covers an area of
1692 sq.km with an altitude range from 385 m to 8162 m.
Materials
Seeds of maize (Zea mays ) of variety NEW 940, Petri dishes, Germinator, Salt, Tray, Mannitol,
Forceps, Filter paper, Cotton, Spirit, etc. will be used.

Seed priming
Mannitol of 2%, 4%, 6% and 8% (w/v) concentrations will be used for seed priming. Three
replications of 25 seeds each will be used for each treatment. Seed priming will be done for 12 hr.
Following the priming, seeds will be dried back to their original moisture contents at room
temperature for 24 hours. The unprimed dry seeds will be used as control (CK).
Concentration w/v
2% 50 gm in 250 ml water
4% 100 gm in 250ml water
6% 150 gm in 250 ml water
8% 200 gm in 250 ml water

Measurement of germination and physiological characteristics of seedlings

After priming, seed germination tests will be carried out. Twenty five seeds each for each treatment
will be placed in Petri dishes. Artificial drought condition will be created by using different
concentration of salt solutions viz. 1.5 bar, 4.9 bar, 10.3 bar, 17.6 bar and a treatment with tap
water is taken as control.
 Drought level NaCl (g/l)
1.5 bar 1.76
4.9 bar 5.74
10.3 bar 12.07
17.6 bar 20.88

Finally we will get 75 plots (Petri dishes) [i.e., 5 treatments of priming* 5 treatments of drought
condition* 3 replications].
Then, seeds will be incubated in a germination chamber at 24°C for 7 days, with regular
supervision. Seeds will be considered germinated when there was a visible coleoptile protrusion
of more than 5mm in length through the seed coat. The germinated seeds will be counted daily for
7 days. On the seventh day 10 samples from each petridish will be taken and root length and shoot
height of the sample seeds will be measured manually with a ruler and fresh weight of the seeds
along with the shoot and root will be measured. Then the petridishes with the maize seedlings will
be filled with water and kept for 12 hours.
After 12 hours the turgid weight of the seeds along with the shoot and root will be measured by
removing the extra water of the dish with the help of tissue paper. Then the seedlings will be
wrapped in the envelope and kept in hot air oven. The dry weights of shoot and root will be
determined after drying at 105°C for 24 h.
All data will be analyzed by means of analysis of variance (ANOVA) using Statistical Analysis
System.

Timeline
Gantt Chart:
Date 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Onwards
Activities
Literature
Review
Priming
Seed Drying
Germination
Count
Root and
Shoot length
Measurement
Wetting
Wet Weight
Drying
Dry Weight
Data Analysis
Report
Writing
Budget
S.N PARTICULARS ESTIMATED AMOUNT(Rs)
1. Literature review 5,000
2. Lab Materials 15000
3. Lab Cost 5,000
4. Internet and Communication 2,000
5. Data analysis 5,000
6. Stationary/Print/Photocopy 6,000
7. Report writing/Preparation/Presentation 4,500
8. Sub total 42,500
9. Contingency(5%) 2,125
10. Overhead(10%) 4,250
11. Grand total 48, 875

Log Frame
Summary Objectively Means of Assumptions
verifiable verification
indicators (MOV)
Goals Germination test of Germination N/A
Improvement in seeds treated in count
germination in primed Mannitol
seed in drought
environment
Purpose Germination test Germination Normal and uniform
Increase in seed count germination of seeds
germination in drought No error in counting of
stress environment seeds

Expected outputs Growth of shoot Measurement of Uniform growth of

Radical and plumule and root length Root and Shoot plumule and radicle
growth in drought Length without any restriction
condition
Activities Availability of seed
-Priming of seed with materials, chemicals
concentration of Mannitol and lab equipment.
-Creating artificial Sterilized lab
drought condition in environment
laboratory Germination of seed in
-Germination count -More than 5mm -Measurement the medias.
-Shoot and Root weight emergence of radical
measurement
-Dry matter calculation
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