thesis-AS-v3 (Repaired)

EFFECT OF PRIMING ON SEED GERMINATION AND SEEDLING
ESTABLISHMENT OF MAIZE (Zea mays) IN OKHALDHUNGA, NEPAL
AVINASH SHRESTHA
AUGUST 2018
i
EFFECT OF PRIMING ON SEED GERMINATION AND SEEDLING
ESTABLISHMENT OF MAIZE (Zea mays) IN OKHALDHUNGA, NEPAL
AVINASH SHRESTHA
RESEARCH REPORT
SUBMITTED TO THE
FACULTY OF AGRICULTURE
AGRICULTURE AND FORESTRY UNIVERSITY
RAMPUR, CHITWAN
NEPAL
BACHELOR OF SCIENCE IN AGRICULTURE
AUGUST 2018
This research report entitled “EFFECT OF PRIMING ON SEED GERMINATION
AND SEEDLING ESTABLISHMENT OF MAIZE IN OKHALDHUNGA, NEPAL”
prepared and submitted by Mr. AVINASH SHRESTHA, under Learning Entrepreneurial
Experience Program as an integral part of Bachelor of Science in Agriculture.
_____________________ _______________
Asst. Prof. Pankaj Raj Dhital Mr. Dal Prasad Pudasainy
Major Supervisor Member Supervisor
Date: Date: 27 August, 2018
Forwarded:
____________________________
Prof. Kalyani Mishra Tripathi, PhD
Assistant Dean (Academic)
Faculty of Agriculture
Agriculture and Forestry University
Date:
Accepted:
___________________
Prof. Jay Prakash Dutta
Dean
Faculty of Agriculture
Agriculture and Forestry University
Date:
Dedication
I would like to dedicate this to my beloved family, friends and teachers who helped me
accomplish this. This is how I would like to express my gratefulness for your love and support
for my success in life.
Acknowledgement
First of all I would like to express my gratitude to my supervisor Mr. Pankaj Raj Dhital
for helping me and providing me assistance in each and every steps of this research program. I
would like to thank my brother Mr. Asis Shrestha for his diligent support in analysis and other
technical fields of my research.
Prime Minister Agriculture Modernization Project (PM-AMP) has provided me with this
opportunity to conduct an action research in farmer’s field, helping me to understand the real
problem and a strategy to cope with it. Hence I would like to thank PM-AMP and my university
AFU for this opportunity under Learning and Entrepreneurial Experience (LEE).
I Acknowledge the support from faculty members of department of Agronomy, AFU

specially Asst. Prof. Mr. Suman Dhakal for their immense support in this priming research.
I would like to thank Dr. Dhruba Bahadur Thapa of National Agriculture Research
Council (NARC) for providing me with the idea of priming and for supporting me at different
stages of research. I would also like to express my sincere gratitude to Dal Prasad Pudasainy,
Senior Agri-Economist of Ministry of Agricultural Development, Nepal for his support and
guidance.
I will always remember the help and support provided my members of District
Agriculture Development Office (DADO) and my fellow colleague Ms. Shreena Pradhan, Jeny
Shrestha, Animesh Lamichhane, Diwash KC, Anamkika Kandel and Dharti Poudel.
Finally I must express my appreciation to my family for unyielding support and love
throughout my course of studies.
Achieving this feat would not have been possible without these amazing people I
mentioned. Thank you all.
v
TABLE OF CONTENTS
LIST OF TABLES ix
LIST OF FIGURES: x
LIST OF APPENDICES: xii
ABSTRACT xiii
1. INTRODUCTION 1
1.1 Objectives of the research 3
1.2 Research questions and hypothesis 3
1.3 Scope of the research 4
1.4 Limitations of the research 4
2. LITERATURE REVIEW 5
2.1 General Introduction to maize 5
2.2 Status of maize in Nepal 6
2.3 On farm seed priming 7
2.3.1 Osmopriming 8
2.3.2 Hydropriming 9
2.3.3 Priming with plant extract and urine 9
3. METHODOLOGY 11
3.1 Site selection 11
3.2 Meteorological data collection and soil testing 11
3.3 Reviewing of literature 12
3.4 Procuring maize seeds and priming reagents 12
3.5 Calculation of amount priming reagents 12
3.6 Constructing an experimental setup 13

3.7 Field Preparation 15
3.8 Seed selection and Priming 15
3.9 Sowing of maize and tagging 16
3.10 Data collection 16
3.11 Data Analysis 18
3.11.1 Mean performance 18
3.11.2 Analysis of Variance (ANOVA) 19
3.13.3 Germination Parameter 19
4. RESULTS 20
4.1 Germination 20
4.2 Shoot length 22
4.3 Leaf Number 23
4.4 Leaf length 25
4.5 Leaf Width 26
4.6 Root Length 27
4.7 Shoot Dry Weight 29
4.8 Root Dry Weight 31
4.9 Correlation analysis 32
5. DISCUSSION 36
6. SUMMARY AND CONCLUSION 39
7. REFERENCES 41
APPENDICES 48
LIST OF TABLES
Table 1: Trend of maize area, production and productivity during 2007 to 2014, Nepal 7
Table 2: Treatment Combination for Research 13
Table 3: Analysis of Variance table for RCBD 18
Table 4: Table representing germination percentage, germination index and mean
germination time for variety A (Manakamana-3) and variety B (Nutan-IL60) 19
Table 5: Pairwise Pearson’s correlations coefficients between variables measured in variety
Nutan-IL60 at 21 DAS on phenotypic traits for different priming reagents. 29
Manakamana-3 at 21 DAS on phenotypic traits for different priming reagents. 31
Manakamana-3 at 28 DAS on phenotypic traits for different priming reagents 31
Table 10: Pearson’s correlations coefficients between variables measured in variety
Manakamana-3 at 35 DAS on phenotypic traits for different priming reagents 32
LIST OF FIGURES
Figure 1: Trend of increase in Maize yield 2
Figure 2: Growth stages of maize in on basis of leaf number in relation with number
of days from emergence 6
Figure 3: Conceptual Framework 10
Figure 4: A conceptual model showing the relationships between current problems and
outcome through priming intervention 10
Figure 5: Rainfall and temperature status of research site (Source: http://mfd.gov.np/) 11
Figure 6: Diagrammatic representation of research field 14
Figure 7: Priming of seeds in different reagents 15
Figure 8: Counting, weighing and bagging of seeds 15
Figure 9: Maize in research field at 23 DAS 15
Figure 10: Line sowing of maize 15
Figure 11: Measurement of shoot length root length and keeping in shade for drying 17
Figure 12: Effect of seed priming methods on Germination Percentage of maize
Nutan-IL60 and Manakamana-3. 20
Figure 13: Effect of seed priming methods on shoot length of maize Nutan-IL60
andManakamana-3 at different growth stages. 21
Figure 14: Effect of seed priming methods on Leaf number of maize Nutan-IL60 and
Manakamana-3 at different growth stages. 22
Figure 15: Effect of seed priming methods on Leaf length of maize Nutan-IL60 and
Figure 16: Effect of seed priming methods on Leaf width of maize Nutan-IL60 and
Figure 17: Effect of seed priming methods on Root length of maizeNutan-IL60 and
Figure 18: Effect of seed priming methods on Shoot dry weight of maize Nutan-IL60 and
Figure 19: Effect of seed priming methods on Root dry weight of maize Nutan-IL60 and
ix
LIST OF APPENDICES
Appendix 1: Mean Table of shoot length for different DAS with assigned DMRT values 46
Appendix 2: Mean Table of leaf length for different DAS with assigned DMRT values 47
Appendix 3: Mean Table of leaf number for different DAS with assigned DMRT values 48
Appendix 4: Mean Table of leaf width for different DAS with assigned DMRT values 49
Appendix 5: Mean Table of root length for different DAS with assigned DMRT values 50
Appendix 6: Mean Table of shoot dry weight for different DAS with assigned DMRT values 52
Appendix 7: Mean Table of dry root weight for different DAS with assigned DMRT values 53
Appendix 8: ANOVA of shoot length for different DAS 55
Appendix 9: ANOVA of leaf number for different DAS 55
Appendix 10: ANOVA of leaf length for different DAS 56
Appendix 11: ANOVA of leaf width for different DAS 57
Appendix 12: ANOVA of root length for different DAS 58
Appendix 13: ANOVA of shoot dry weight for different DAS 58
Appendix 14: ANOVA of root dry weight for different DAS 59
List of abbreviations
% Percentage
ANOVA Analysis of Variance
Cm Centimeter
CV Coefficient of Variance
DADO District Agriculture Development Office
DAS Days after Sowing
DMRT Duncan Mean Range Test
DRW Dry Root Weight
DSW Dry Shoot Weight
FGP Final Germination Percentage
FYM Farm Yard Manure
GDP Gross Domestic Product
GI Germination Index
H Hour
HSD Honest Significant Difference
K Potassium
K3PO4 Potassium Phosphate
Kg Kilogram
KH2PO4 Potassium Di-hydrogen Phosphate
KNO3 Potassium Nitrate
LEE Learning and Entrepreneurial Experience

LL Leaf Length
LSD Least Significant Difference
LW Leaf Width
M2 Square Meter
MGT Mean Germination Time
MMT Million Metric Tons
MOP Murate of Potash
N Nitrogen
NaCl Sodium Chloride
NARC Nepal Agriculture Research Council
NMRP National Maize Research Council
NOL Number of Leaves
NUE Nitrogen Use Efficiency
OPV Open Pollinated Variety
P Phosphorous
PM-AMP Prime Minister Agriculture Modernization Project
RCBD Randomized Complete Block Design
RL Root Length
SL Shoot Length
T50 Time for 50% Germination
Ton/ha Ton per Hectare
TOR Terms of Reference
Zn Zinc
ABSTRACT
Priming enhances early emergence and stand establishment which enable the crop to capture
more soil moisture, nutrient and solar radiation. Rapid and uniform field emergence is an
essential pre-requisite to reach the yield potential and quality of maize. An experiment was
conducted in a farmer’s field in Okhaldhunga, Nepal from February to May, 2018 to study the
effect of different priming treatments on seedlings of two maize varieties. Different
morphological and germination parameters were tested to see if priming reagents had any effect.
Two varieties were experimented in Randomized Complete Block Design with three replications.
Observations were taken for quantitative traits i.e. emergence, germination, shoot length, root
length, leaf number, leaf length, leaf width, dry shoot weight and dry root weight. The mean
performances were evaluated and correlation analysis was performed among the studied traits.
Significant differences were observed among the varieties and priming reagents for most of the
traits. The only trait unaffected by priming reagents was found to be number of leaves. Priming
showed better results in Nutan-IL60 compared to Manakamana-3 based on individual assessment
of traits for all the treatments. Also, nitrogen treatment (Urea and Urine) for both varieties was
found most favorable for attaining a higher value of all the traits studied. Based on all the
treatments observed and varieties tested, nitrogen based on-farm priming treatment had the most
desirable change in the studied traits. The trait most relatable to stress tolerance; root length and
dry root weight was found higher in nitrogen based priming in both the varieties. Also, Nutan-
IL60 seemed more responsive to priming treatments as compared to Manakamana-3.
xiii
1. INTRODUCTION
Maize is the leading crop in the world, with production of 825 million metric tons (MMT)
however maize is mostly used as animal feed, with only 15% of grain used for food [ CITATION
Awi11 \l 1033 ]. It is considered that maize was one of the first plants cultivated by farmers
between 7000 and 10,000 years ago which was originated in Mexico supported by the discovery
of fossil pollen and cave corncobs in archaeological areas support the position that maize while
other theories describe maize as originating in the region of the Himalayas in Asia [ CITATION
Ran14 \l 1033 ]. Nepal which lies in belt of Himalayas have maize production of 2,231,517
metric ton and productivity of 2.5 ton/ha after rice with production 4,299,078 metric ton and
productivity of 3.14 ton/ha [ CITATION Min17 \l 1033 ]. The trend of maize consumption varies
from one geographical location to another as per capita consumption of maize is 15 kg in
America while east and south African countries have per capita consumption of 90-180 kg
[ CITATION Awi11 \l 1033 ] and also in hills of Nepal maize is staple crop though rice dominates
production and consumption in national basis [CITATION Kar15 \t \l 1033 ]. The world
population is increasing exponentially and is predicted to reach 9 billion by 2050 [ CITATION
God10 \l 1033 ]. Increasing population leads to decrease in cultivable area but need of increased
food materials can only be met only with increased productivity of crops.
Nepal is a land locked country that lies in between two nations: China in North and India in
remaining three faces. 65.6% population in country is directly involved in agriculture which
contributes 32.6% of gross domestic product (GDP) [CITATION Gov16 \l 1033 ]. The growth rate
of agricultural sector in year 2007/008 was 2.13% while in the same year population growth rate
was 2.25% showing agriculture growth trend is falling behind to population growth rate
[ CITATION Pau101 \l 1033 ]. Though production of maize is increasing in Nepal, it still lacks
behind overall maize productivity of the world i.e. 4.8 ton/ha [ CITATION Agr15 \l 1033 ] with
production of 2067522 kg in 906253 ha land with productivity 2281 kg/ha [CITATION Agr68 \l
1033 ]
Nepal is one of the least developed countries in the world; a common feature that under
developed country face is more population involved in agriculture but with less productivity.
1
Also within Nepal, plains produces more than enough for itself while Hills lack behind as Plains
contribute 22% of total maize production while Hills contribute 70% [ CITATION Pat01 \l 1033 ].
Low productivity has made Nepal dependent on neighbor India for supply of foods such as
maize as there is about 40-45% of maize import from India every year [CITATION Bha11 \l 1033 ]
and low production of maize in Nepal can be accounted for it.
Figure 1: Trend of increase in Maize yield
Source: [CITATION MoA091 \l 1033 ]
Nepal is divided into three agro ecological belts: plains, hills and mountains of which
hills occupy 68% land area followed by 17 % of plains and contribution of plains in maize
production is 22% while that of hills is 70% as it is staple crop in the area [CITATION KCG151 \t \l
1033 ] . The increase in maize productivity during the past decade (2005 to 2014) was only 0.439
ton/ha but still the present yield level is quite low to fulfill the country’s demand.
Eastern hills of Nepal faces three major yield reducing factor: poor germination, drought
stress and Turcicum leaf blight [ CITATION Tiw01 \l 1033 ] . Seasonal drought and poor seed quality
are further aiding factor in low productivity of maize. There are several ways to cope with the
mentioned factors and priming is one of those. Priming treatment consists of soaking seeds in
water for certain amount of time followed by surface drying which helps to gain rapid
germination, better establishment and increasing plant yield in unfavorable environmental
2
conditions [ CITATION Mou12 \l 1033 ] . The problem of poor germination can be combated by seed
priming which in turn gives proper crop stand and increased yield [CITATION Kha92 \l 1033 ].
While priming seeds which are damaged or infested with pests float up and can be discarded so
we can select the best seeds thus helping reduce the problem of poor quality seed. With seed
priming the rate of hydration increased which helps to combat the drought stress during
germination and other stages of growth [ CITATION Tia14 \l 1033 ] . Priming enhances early
emergence and stand establishment which enable the crop to capture more soil moisture, nutrient
and solar radiation. Rapid and uniform field emergence is an essential pre-requisite to reach the
yield potential and quality of annual crops [ CITATION Par94 \l 1033 ]. Priming has shown
promising result in different vegetable and maize. Priming treatment significantly affected the
emergence and growth of maize varieties [CITATION Aji13 \l 1033 ]. Maize being the most
important crop in hills of Nepal still faces low production compared to developed nations and
this study focuses on improving maize productivity using priming method to combat current
problem of poor plant stand or low germination percentage, seasonal drought, and poor seed
quality.
1.1 Objectives of the research

General objective:
The general objective of the research is to study the effect of different types of priming on the
emergence and vigor of Maize Seedling.
Specific objectives:
The specific objectives of the research were to:

 To study priming as a tool to improve productivity of maize
 To identify if priming treatment help in a hardier crop stand
 To select the best priming solution for maize seed
1.2 Research questions and hypothesis

The study will aim to answer the following questions:
 Which of the varieties of maize grown in Okhaldhunga is more responsive to priming
treatments in establishment phase?
 Which of the priming treatments have the most desirable effects in the studied traits?
3
 Can morphological characters be used to study stress tolerance?
The null and alternate hypotheses to guide the study are:
Null Hypotheses:
1. Different priming treatments will perform similarly for all the studied traits.
2. There will be no change in response of plant when primed and not primed.
Alternate Hypotheses:
1. There will be significant difference among performances of different priming treatments
leading to identification of most desirable reagent.
2. Change in morphological traits will be seen when primed and not primed.
1.3 Scope of the research
This research will be useful for students, government and non-government agencies and
educational institutions planning to go deeper into the effect of priming treatments in stress
tolerance and yield determination of maize. It will be a documentation of physical performance
of maize varieties as influenced by different priming treatments in Okhaldhunga, hence leading
the way for further research. Also this provides information to cope the problem of low
germination in Eastern hills of Nepal.
1.4 Limitations of the research
This research was carried out to find out what traits were affected by different priming
treatments and how significantly these effects were seen in the two maize varieties studied. Due
to time and budget limitations, performance up to 45 Days after sowing was recorded. This study
was carried out in hills of Okhaldhunga and could be helpful for researches conducted under
similar conditions. This study may not actually prove the existence of water stress tolerance
because of priming treatments. This study may not be useful in predicting the grain yield based
on priming treatments but will serve as documentation for further research.
4
2. LITERATURE REVIEW
2.1 General Introduction to maize
Maize belongs to family Poaceae (grass) under the genus Zea. It is a cross pollinating
crop with 10 pair of chromosomes [ CITATION Kyn01 \l 1033 ]. Although the ancestral pedigree of
maize has not been fully resolved yet [ CITATION Hal10 \l 1033 ]there are two main hypothesis
regarding the origin of maize “The Teosinte Hypothesis” and “Tripsacum – Z. diploperennis
Hypothesis” with final verdict falling in favor of the teosinte [ CITATION Mot06 \l 1033 ]. Other
theories suggest that teosinte (Zea mexicana) is the wild progenitor of corn; the other is that a
wild pod corn, now extinct, was the ancestor of domesticated corn [ CITATION Bro85 \l 1033 ] . With
domestication there is significant increase in number of races (300) with wider availability
ranging from sea level to 1200 feet altitude and maturity ranging from 6 week to 13 months
[ CITATION Bro85 \l 1033 ]. Corn variation may be artificially defined according to kernel type as
follows: dent, flint, flour, sweet, pop and pod corn of which dent has yellow endosperm in most
cases and white in few [ CITATION Bro85 \l 1033 ]. Maize is known to be one of the few major
cultivated species indigenous to the Western Hemisphere about 7,000–10,000 years ago
[ CITATION Wil04 \l 1033 ]. Most cultivated maize in Nepal are monecious with life span ranging
from 90 to 175 days [CITATION Agr18 \l 1033 ].
F
igure 2: Growth stages of maize in on basis of leaf number in relation with number of days
from emergence
5
Source:[ CITATION Pri17 \l
1033 ]
2.2 Status of maize in Nepal

Maize originated in Mexico. Also some sources of pollen suggest some varieties of maize
originated in Himalayas [ CITATION Ran14 \l 1033 ].Maize (Zea mays L.) is main crop after Rice in
Nepal in term of area and production [ CITATION MOA14 \l 1033 ]. Maize (Zea mays L.) is the most
important cereal crop in the hills of Nepal, where the grain is used for human consumption and
the stover for animal fodder [ CITATION Ran03 \l 1033 ]. Although in global basis maize is widely
used as feed only about 15% used as food source [ CITATION Awi11 \l 1033 ], but in context of
Nepal in hills more than 86% maize production has been used for human consumption and 80%
maize production in the Plains is used for poultry and animal feed [CITATION Gur11 \l 1033 ]
justifying it is food source for people of hills. The proportion of maize area to total cereals was
30% in the high hills, 40% in the mid hills and about 11% in the plains while maize production
as a proportion of total cereal production was 33% for the high hills, 39% for the mid hills and
9% for the plains [ CITATION Pau01 \l 1033 ]. It is reported that the demand for maize has been
growing by 5% over the last decade [ CITATION Sap10 \l 1033 ].
Recheck the Font size and settings of the table 1.
Table 1: Trend of maize area, production and productivity during 2007 to 2014, Nepal
Particular
s 2007 2008 2009 2010 2011 2012 2013 2014
870401 90625
Area (ha) 870166 875428 875660 871387 849635 982761
1 3
Productio 181992 187864 193066 185518 20675 217941 199901
2283222
n (mt) 5 8 9 4 2 4 0
Yield
2091 2159 2205 2119 2281 2501.1 2353 2458
(kg/ha)
Source: [CITATION KCG151 \t \l 1033 ]
Okhaldhunga has maize yield varying from 1.9 ton/ha in rain fed and 2.3 ton/ha in
irrigated condition both of which is below national average of 2.5 ton/ha [ CITATION DAD17 \l 1033
]. Like other parts of the country, the farming system here comprises of subsistence type and the
6
poor yield of maize is probably due to the same three factors that affect the other hilly regions of
Eastern Nepal [ CITATION Tiw01 \l 1033 ]. Eastern hills of Nepal faces three major yield reducing
factors poor germination, drought stress and Turcicum leaf blight [ CITATION Tiw01 \l 1033 ].
2.3 On farm seed priming
Seed priming is often considered as method to increase production in certain crops

[ CITATION Pir11 \l 1033 ], [ CITATION Bas03 \l 1033 ] including hydro-priming, hardening, osmo-
conditioning, osmo-hardening, hormo-priming, matri-priming, and others [ CITATION Dre97 \l
1033 ]. Seed priming is defined as a pre-sowing treatment in osmotic solution, which allows
seeds to imbibe water to proceed to the first stage of germination but prevents radicle protrusion
through the seed coat [ CITATION Hey73 \l 1033 ]. The result of some research have been shown
that priming could increase germination traits, accelerate germination, improve seedling
establishment and enhance plant growth [ CITATION Nag98 \l 1033 ] which can solve the problem
of poor germination and poor seedling establishment in hills of Nepal. According to research
conducted in western hills of Nepal, plant stand after germination in primed treatment was found
better as reported by 80% of the respondents. Seventy eight percent of the respondents claimed
earlier maturity in Manakamana-5 whereas more than 60% respondents claimed the same for rest
of the varieties [ CITATION Koi17 \l 1033 ] which could be effective in eastern hills of Nepal as
well. Many evidences have shown seed priming could improve germination and early seedling
growth under stress conditions compared to plants grown from untreated seed [ CITATION Tia14 \l
1033 ]. Poor crop stand is one of the major biotic reasons for low yield of crop and this problem
can be solved by help of proper seed priming methods [ CITATION Har96 \t \l 1033 ]. Although the
germination is not completed, metabolic activities that prepare seeds for radicle protrusion may
be initiated during priming [ CITATION Soe05 \l 1033 ]. The increase grain yield may be due to the
fact that priming advances the metabolism of the seed and the seed protein is synthesized which
has a direct affect in increasing seed performance and hence yield [ CITATION Var10 \l 1033 ].
Rapid and uniform field emergence is an essential prerequisite to reach the yield potential,
quality, and ultimately profit in annual crops [ CITATION Parera \l 1033 ] . With seed priming the
rate of hydration increased which helps to combat the drought stress during germination and
other stages of growth [ CITATION Tia14 \l 1033 ] which is another problem that can occur in rain
fed area.
7
2.3.1 Osmo-priming
Osmo-priming is a type of priming in which seeds are primed in osmotic solution of seed
priming where reagents like PEG, KNO3, K3PO4, KH2PO4, NaCl etc. are used at low osmotic
potentials [ CITATION Ghi08 \l 1033 ] . Priming treatment with PEG reagent significantly increased
shoot biomass compared to the control group; the shoot biomass was also significantly higher
when presoaking with NaCl reagent compared to control [ CITATION Tia14 \l 1033 ] . By priming
seeds with nutrients deficit in soil we can improve the yield of seeds, for example the crop maize
responded well to seed priming with P and Zn solutions as experimental soil was deficient in
both the nutrients [ CITATION Kum14 \l 1033 ] . Seed priming with KNO3 and NaCl decreased time
to 50% germination and increased final germination of sorghum [ CITATION Tir09 \l 1033 ].
Treatment with 0.5% KNO3 was very effective and showed more rapid germination compared to
other treatments at low temperature in case of maize [ CITATION Pet16 \l 1033 ]. Maximum
coleoptile length was note in seeds osmo-primed in urea solution for 96 h which followed by
seeds hydro-primed for 36 h [ CITATION Dez08 \l 1033 ]. Urea can be used as source priming
reagent which showed highest germination percentage while priming for abiotic stress in maize
[ CITATION Ano11 \l 1033 ]. Urine is locally available material in farming system which as several
use as fertilizer, insecticide etc. Both human and animal urine can be collected and used as a
source of priming material which in turn has proved effective. Study on NaCl priming
demonstrated that germination recorded from primed seeds was non-significantly different from
non-primed treatments of both cultivars when exposed to different salinity levels [ CITATION
Bak11 \l 1033 ].
2.3.2 Hydro-priming
The duration for seed to be soaked in water may vary with crop and variety of crop as
earlier germination was recorded for hydro-primed seeds as indicated by lower value of T 50 (time
by which 50% germination occurred) and mean germination time (MGT) or by higher
germination index (GI). Seeds hydro-primed for 36 h had lowest values (T50 and MGT),
followed by 24 h and 48 h seed treatments, whereas the lowest germination rate that was
indicated by higher T50 and MGT was observed in osmo-primed seeds which were subjected to
PEG-6000 solution [ CITATION Dez08 \l 1033 ]. Harris, Joshi, Khan, Gothkar , & Sodhi (1999)
8
revealed that on-farm seed priming (soaking seeds overnight in water) markedly improved the
establishment and early vigor of maize resulting in faster development, earlier flowering,
maturity and higher yields. Hydro-priming had more positive effect than potassium nitrate
(KNO3) while priming for stress condition tolerance in maize [ CITATION Pir11 \l 1033 ].
2.3.3 Priming with plant extract and urine
Seed priming with P had highest increase in maize yield (relative yield 1.71 t/ha)
followed by human urine (1.45 t/ha), cow urine (1.28 t/ha) and Zn (1.24, P = 0.08) treatments,
whereas hydro-priming proved ineffective in a research conducted at Meghalaya, Northeast India
[ CITATION Kum14 \l 1033 ]. Yield improvement due to seed treatment with urine can be ascribed
to the presence of a range of plant nutrients therein, including N, P, K and micronutrients
[ CITATION Sch02 \l 1033 ] , and hence proposed as an alternative fertilizer in crop production
[ CITATION Ric02 \l 1033 ];[ CITATION Sri11 \l 1033 ].
Damaged seeds/ poor quality seeds

Drought Lack of proper seed technology Pests and diseases
Low germination and seedling vigor
Possible remedy
Reduced yield
Seed priming
Selection
Increased root growth and of undamaged seeds
Proliferation Metabolic activities for Increased initiated
speed and
radicle protrusion synchrony of germination
Seed vigor enhancement
Improved germination and seedling vigor
9 Increased yield
Figure 3: Conceptual Framework

3. METHODOLOGY
3.1 Site selection
Okhaldhunga district was selected as the research site. The research was conducted in
Siddhicharan Municipality. The field area is located at 28 o 65’ N latitude and 82o 16’ E longitude
in Eastern Nepal at an altitude of 1700 meters above sea level with sub-tropical climate. Soil
type is sandy loam and acidic with greyish white color.
Figure 4: Map depicting action research site
3.2 Meteorological data collection and soil testing

The research location shows characteristics of climate ranging from sub-tropical to
temperate. The climatic data of the research period (March to July, 2018) is presented in figure 5.
10
Figure 5: Rainfall and temperature status of research site (Source: http://mfd.gov.np/)
3.3 Reviewing of literature
A thorough review was conducted to select appropriate priming reagent suitable for
existing agro-climate. Based on various review common salts, MOP, urea, urine and normal
water were selected as priming reagents. Time to prime seeds was taken as 12 hour and 24 hour
based on study by [ CITATION Dez08 \l 1033 ]. Concentration of osmotic solution for priming was
common salt (50 millimoles), urea solution (200 millimoles), MOP solution (1 percent) and urine
(2.5 times dilution).
3.4 Procuring maize seeds and priming reagents

Test was performed on two varieties with 11 different treatments. Nutan-IL60 and Arun-6
were selected at first but due to failure of germination ofArun-6 overall research was conducted
again using the variety Manakamana-3. Seeds of Nutan-IL60 were brought from Agro-vet, Arun-
6 was procured from National Maize Research Program (NMRP) and that of Manakamana-3 was
provided by Agriculture Section Siddicharan Municipality, Okhaldhunga.
Since priming reagents should be easily accessible to farmers, locally available materials
were selected.
 Urea and Mop from Kathmandu

 Salt from local household
 Urine of cow from local farmer
3.5 Calculation of amount priming reagents

A priming solution of 200 mL was made for common salt solution (50 millimoles), urea
solution (200 millimoles), MOP solution (1 percent) and urine (2.5 times dilution). Chemical
11
equations were used to calculate mentioned concentrations of solution which resulted in 0.6 gram
salt, 1.2 gram urea, 2 gram MOP and 80 ml urine.
3.6 Constructing an experimental setup

Design: Randomized complete block design (RCBD)
Treatments: Total 11 treatment.
Plant material: Manakamana-3 and Nutan-IL60
Replication: Three
Plot size: 2.25m × 2.75 m
Spacing: 75 cm×25 cm between row to row and plant to plant respectively.
Each plot accommodated 2 rows. Each row proceeds up to 22.75 m vertically.
Spacing between plots = 50 cm
Spacing between replications = 1m
Total area of field = 221.81 m2 for one variety
Table 2: Treatment Combination for Research
Variety Treatments Treatment Combinations
Nutan-IL60 (V1) Control (C1) V1/V2*C1

Manakamana-3 (V2) Urea, 12 hours (C2) V1/V2*C2
Urea, 24 hours (C3) V1/V2*C3

Salt, 12 hours (C4) V1/V2*C4
Salt, 24 hours (C5) V1/V2*C5
Urine, 12 hours (C6) V1/V2*C6
Urine, 24 hours (C7) V1/V2*C7

MOP, 12 hours (C8) V1/V2*C8

MOP, 24 hours (C9) V1/V2*C9

Water, 12 hours (C10) V1/V2*C10

Water, 24 hours (C11) V1/V2*C11
12
REP 1 REP 2 REP 3 REP 1 REP 2 REP 3
V1T1 V1T11 V1T2 V2T1 V2T11 V2T2

2.25 m
V1T2 V1T10 V1T4 V2T2 V2T10 V2T4
V1T3 V1T9 V1T6 V2T3 V2T9 V2T6
V1T4 V1T8 V1T10 V2T4 V2T8 V2T10

75 cm
V1T5 V1T7 V1T11 V2T5 V2T7 V2T11
V1T10 V1T2 V1T5 V2T10 V2T2 V2T5
V1T11 V1T1 V1T3 V1T11 V2T1 V2T3
2.75 m 50 cm
NUTAN MANAKAMANA-3
Figure 6: Diagrammatic representation of research field

Where V = Variety T1 to T11= Treatments
3.7 Field Preparation

Field was ploughed once and left for solar sterilization for 3 days. Furadan (Insecticide)
was applied in the field at the time of ploughing as the area was highly susceptible for white
grub. FYM 450 kg, urea 2 kg, DAP (Di-Ammonium phosphate) 2kg and MOP 1 kg were
applied a day earlier to sowing. A total of 225 m2 square meter of land was separated for each
variety with row to row spacing of 75 cm and plant to plant spacing 25 cm. The area was divided
into three blocks and each plot was measured and separated with help of bamboo pegs and nylon
wire.
13
3.8 Seed selection and Priming
There were 10 treatments and one control making 11 plots per replication. Each plot
contained 2 rows and 10 hills per row and two seeds were sown per hill. Same procedure was
followed for both Manakamana-3 and Nutan-IL60.
Priming was conducted after rainfall as sowing date was based on rainfall as research was
based on rain-fed system. Solutions were made as such first reagent was kept in beakers and
water was poured to volume to 200ml. Each reagent had two duration of priming: 12 and 24
hours.
Figure 8: Counting, weighing and Figure 7: Priming of seeds in different

bagging of seeds reagents
3.9 Sowing of
maize and tagging
Sowing was done on last of February. There were three rows per plot and 10 plants per row.
Figure 10: Line sowing of maize Figure 9: Maize in research field at 23

DAS
14
3.10 Data collection
Seeds emerged 8 days after showing in urea, urine, hydro-primed seeds for 12 hours at
most. Number of seeds emerged, shoot length, root length, leaf number, leaf length, leaf width,
fresh mass of shoot and root and dry mass of root and shoot were the data taken. Sheets were
prepared in excel for each data to be taken and data was taken on printed sheets.
3.10.1 Emergence
Data for emergence was taken after first emergence and was taken on daily basis until
constant number of plants was recorded for same plot. After similar result data was taken on day
interval. Data on emergence was taken up to 23 days after sowing (DAS).
3.10.2 Shoot length
First data on shoot length was taken 21 DAS and three more data were taken on 28 DAS,
35 DAS and 45 DAS. Data for shoot length were taken till knee high stage (KHS). Data for shoot
length was taken by help of measuring tape taken length from base to tallest point of the leaf.
Five plants per plot were selected.
3.10.3 Root length
Data for root length were taken for three times at 21 DAS, 28 DAS and 35 DAS. Data for
root length was taken by digging deep around the plant about 20 cm X 50 cm to avoid damage.
Four plants per plot were selected.
3.10.4 Data on leaf length leaf width
Data on leaf length and leaf width were taken on the same day as shoot length. Length of
two leaves per plant was taken and its average was recorded. Leaf width was measured on three
places of mid region of a leaf and its average was taken. Five plants per plot were taken for data.
15
3.10.5 Number of leaves
Number of leaves was counted for same day as shoot length. Those leaves were only
counted whose collar region was visible. Data was taken till knee till knee high stage. Five plants
per plot were taken for data.
3.10.6 Data on dry weight
Data for dry weight was taken for plants of 21 DAS, 28 DAS and 35 DAS. Separate
weight of roots and shoots were taken. Dry weight was taken by air drying due to lack of
availability of oven. For taking dry weight plant was placed on shade and weight was taken on
regular basis at interval of 12 hours until there was constant mass for 3 consecutive intervals.
Four plants per plot were taken for data.
Figure 11: Measurement of shoot length root length and keeping in shade for drying
3.11 Data Analysis
Data entry and processing was carried out using Microsoft Excel 2013 and Microsoft
Word 2013 software. Germination indices were calculated in Microsoft Excel. Mean and
Standard deviations, Analysis of variance (ANOVA), mean performance, Pearson’s correlation
co-efficient, Duncan Multiple Range Test (DMRT) and growth profile analysis was calculated
by using R-Studio version 3.1.1 with the help of “Agricolae” package. Multiple comparisons
were performed using post hoc Tukey test.
16
3.11.1 Mean performance
On the basis of individual plant observations, the mean performance for each character
was computed as follow:
n
x́=∑ xi
i=0
Where, 𝑥̅ = mean performance, 𝑥𝑖 = individual value, n = number of observations
3.11.2 Analysis of Variance (ANOVA)

The analysis of variance for different characters was performed on mean data separately
in order to partition the variability due to different sources. The method given by [ CITATION
Hun07 \l 1033 ]was followed. The structure of ANOVA is presented in Table.
Table 3: Analysis of Variance table for RCBD
Source of Degree of Sum of Mean sum of Square Computed Tabulated F

1% 5%
variation freedom Square F
Replication (r-1) RSS RMS=RSS/(r-1) RMS/EMS
Treatment (t-1) TSS TMS=TSS/(t-1) TMS/EMS
Error (r-1)(t-1) ESS EMS=ESS/(r-1)(t-1)
Total (rt-1)
3.13.3 Germination Parameter
Seed germination was recorded from day 3 after sowing till about 80 % seeds emerged. Mean
germination time, germination percentage and germination index was calculated [ CITATION
Mor12 \l 1033 ].The germination index (GI) will be calculated as described in the [ CITATION
Ass83 \l 1033 ]by following formula
No . of germinated seed No .of germinated seed

 Germination Index (GI) = +…+
Days of first count Days of last count
17
 Mean Germination Time (MGT =
∑f .x where, f = number of seeds germinated of day
∑f
x
total number of seeds germinated
 Final Germination Percentage = ×100 %
number of seeds sown
18
4. RESULTS
The analysis of variance, mean performance, stress indices, correlation coefficients of

quantitative traits and the description on qualitative traits are presented below.
Overall germination performance and nine phenotypes viz. plant height, leaf length, leaf
number, leaf width, root length, fresh root and shoot biomass and dry shoot and dry biomass
were evaluated.
4.1 Germination Parameters

Table 4: Table representing germination percentage, germination index and mean germination
time for variety A (Manakamana-3) and variety B (Nutan-IL60)
Treatment Germination Percentage Germination Index Mean Germination Time
Man-3 Nutan-IL60 Man-3 Nutan- Man-3 Nutan-IL60
IL60
Control 75.0 72.9 121.33 90.00 11.53 14.63
MOP-12 75.0 70.8 134.67 98.33 10.11 14.49
MOP-24 75.0 69.8 152.33 117.33 9.75 12.99
Salt-12 76.0 63.5 155.33 87.00 9.77 14.61
Salt-24 77.1 64.6 151.33 81.67 10.19 15.19
Urea-12 88.5 85.4 178.00 155.33 10.29 12.32
Urea-24 83.3 87.5 168.67 169.00 10.01 11.82
Urine-12 80.2 86.5 157.00 159.00 10.16 12.22
Urine-24 84.4 84.4 161.67 170.67 10.53 11.20
Water-12 83.3 81.3 169.67 97.00 9.76 14.55
Water-24 82.3 72.9 170.33 115.00 9.71 13.53
Beneficial effect of treatment method on seed germination is indicated by three different

indices; final germination percentage (FGP), mean germination time (MGT) and germination
index (GI) adopted from [ CITATION Kad051 \l 1033 ] . FGP indicated the percentage of germinated
seeds and does not consider speed and uniformity of germination. FGP was highest for urea
treated samples for both Manakamana-3 and Nutan-IL60 followed by urine treated plots. It was
highly suppressed in salt and MOP treated plots (Table 4 and Fig 12). Another index MGT is a
measure of time taken for lot to germinate, but it fails to consider uniformity and germination
19
percentage. Lower MGT indicates better effect. MGT was lowest lots soaked in urea and urine
for 24 hours while, it was highest in lots soaked in salt solution for 24 hours in Nutan-IL60.
However, MGT in Manakamana-3 was lower in water soaked seed lot. Third parameter, GI is the
most comprehensive measurement which considers germination percentage including speed and
uniformity of germination. Higher GI indicated beneficial effect. In Manakamana-3, seed lot
soaked in water and nitrogen source (urea and urine) produced better performance while salt and
MOP treated lot were comparable to control lot. However, Nutan-IL60 showed better response to
treatments with nitrogen source (urea and urine). Other treatments were comparable to control.
Figure 12: Effect of seed priming methods on Germination Percentage of maize varieties (A)
Nutan-IL60 and (B) Manakamana-3.
Mean values and standard errors (n=3) are plotted. Indexed letter above the bars indicate
significant difference (p<0.05) between treatments not sharing the same letter by Tukey’s HSD
test. Abbreviation: days after sowing (DAS).
Germination percentage (GP) was calculated for each plot there was significant varietal,
treatment and interaction effect on germination percentage. GP was higher in urea and urine
primed plots while it was suppressed in salt priming treatment for both Nutan-IL60 and
Manakamana-3. Interestingly, the level of GP was much lower in Nutan-IL60 compared to
Manakamana-3 which showed some signs of salinity tolerance in Manakamana-3. However, for
other treatments, GP between Nutan-IL60 and Manakamana-3 was comparable.
20
4.2Shoot length
There was significant genotype, treatment and genotype treatment interaction effect on
shoot length. However, hours of soaking for a given treatment did not influence the shoot length
(Appendix 8).
A: Nutan 21 28 35 45
120 110.31 105.6 107.64 103.74

100 90.19 86.49 91
84.86
Shoot Length (cm)
79.66 78.43 79.34

80 65.54
62.11 65.08 64.98
60 45.6 47.2 48.71 47.73
43.95 42.59 44.73
40 25.88 34.7 34.45 35.41 33.29
29.01 24.81 26.07 24.47 24.43
22.84 22.07 24.35 22.91 23.18
20 15.9 13.03 11.84 13.97 12.84 14.6 14.44
0
Control MOP-12 MOP-24 Salt-12 Salt-24 Urea-12 Urea-24 Urine-12 Urine-24 Water-12 Water-24
Treatment
B: Manakamana-3 21 28 35 45
140
120 107.4 112.73 117.2 105.4 115.8 113.5
103.73
Shoot Length (cm)
95.3 100.13 93.87 94.8

100
80 61.57 67.03 60.73 64 57 60.43
60 51.17 52.2 52.67 45.97 48.7 46.93 49.43
38.57 38.2 38.93 37.63 36.67 42.3 42.77 38.27 40
40 26.4 31.93 27.6 25.83 24.07
16.4 22.13 17.93 22.57 18.6 21.8
20
0
Treatment
Figure 13: Effect of seed priming methods on shoot length of maize varieties (A) Nutan-IL60
and (B) Manakamana-3 at different growth stages.
Significant treatment effect was observed in shoot length when data was recorded at
different time points. Overall, higher shoot length was observed in urea and urine treated plots
for both varieties. Likewise, the salt and MOP treated samples were grouped together to control
plots. Water soaked seeds showed intermediate response (Figure 13). At 45 DAS, plant height
21
was did not differ significantly between the treatments for Manakamana-3, although, average
shoot length was still higher in urea and urine soaked samples (Figure 13).
4.3 Leaf Number
A: Nutan 21 28 35 45
7 6.44 6.22 6.44 6.11 6.22 6.11
6 6 5.89 5.78 5.89
6
4.89 4.89 4.89 4.56
5
Leaf Number
4.11 4
4
3.89 3.67 3.66 3.67 3.78 3.89
3.44 3.67 3.334
2.89 3.22 3.22 3
2.44 2.56 2.45 2.78
3 2.11 2.22 2.33 2.11 2.11
1.89 1.66 1.78 1.78
2
1
0
Treatment
7 6.22 6.11 6.11 6.22 6.22
5.89 5.89 6 5.89 6 5.78
6
4.89 5 4.89 4.78 4.67
5 4.44 4.33 4.33 4.33 4.44 4.56
Leaf Number
3.78 4 4 3.89 3.89 3.78

4 3.44 3.44 3.56 3.56
3.11 2.78
3 2.22 2.33 2.33 2.33 2.67 2.67 2.44 2.56 2.56 2.44
2
1
0
Treatment
Figure 14: Effect of seed priming methods on Leaf number of maize varieties (A) Nutan-IL60
There was significant varietal, treatment and interaction effect on leaf number (Appendix
9). Significant result were seen for 28, 35 and 45 DAS for Nutan-IL60 with better performance
22
from urea and urine treatments for 12 hours while other had similar response to priming
treatment (Figure 14). However, in Manakamana-3 there was no treatment effect for leaf number
except five weeks after sowing where urea and urine treated plots showed better effects (Figure
14).
4.4 Leaf length
A: Nutan 21 28 35 45
80 71.37
70 66.53 66.17 64.67 64.23
61.83 61.67
55.87
Leaf Length (cm)
60 50.87 53.73 50.27

47.73 49.37 48.17
50 41.37
40 33.57 35.43
31.33 28.47 26.93 30.03
30 23.03 26.07 25.47 23.3
18.83 18.3 19.2 17.63 21.17
20 16.93 16.63 14.83 14.77 14.5 15.63 15.77
7.27 8.5 6.17 7.63 6.87 8.57 8.1
10
0
Treatment
100
86.73 82.23
77.93 80.27 81.63 81.87 81.63
72.77 76.63 76.9
80 70.2
Leaf Length (cm)
60 49.83 51.83 48.87

42.43 42.33 46.03 45.83
40.1 39.23 36.6
38.63 38.33 39.07 40.5
40 27.33 28.2 29.17 32.2 31.1 28.33 32.07
26
17.47 19.23 20.57
20 14.53 14.37 13.67 14.8 15.57 15.67 13.5
10.6
0
Treatment
Figure 15: Effect of seed priming methods on Leaf length of maize varieties (A) Nutan-IL60 and
(B) Manakamana-3 at different growth stages.
23
There was significant varietal, treatment and interaction effect on leaf length (Appendix
10). Urine treatments showed best response for leaf length in all DAS while water treatments and
control had highly suppressed leaf length for overall period. Salt and MOP treatments showed
intermediate response at later stages though being suppressed at earlier stages in Nutan-IL60
(Figure 15).
In contrast to Nutan-IL60, Mankamana-3 had similar response for all treatments at later
stages (35 and 45 DAS). MOP, Control and Salt had poor response in earlier stages with Urea
showing best followed by Urine treatment while remaining showed intermediate results (Figure
15).
24
4.5 Leaf Width
A: Nutan
21 28 35 45
10 8.93 9.03 8.93
8.57 8.5 8.2 8.27
7.83
8 7.07 6.83
Leaf Width(cm)
6.23
6 4.87
4.2 4.5
4 3.63 3.23 3.37
2.33 2.73 2.53
2.3 2.1 2.13 1.93 2.17 2.2
1.5
1.37 1.67 1.63
1.27 1.3 1.37 1.3 1.6 1.63
1.5 1.6
2 0.93 1.13 0.9 0.87 1.07 1.2
0
Treatment
10 9.4
8.43 8.27 7.97 7.97
7.43 7.67 7.43
8 7.03 6.83 6.97
Leaf Width (cm)
5.47 5.6 5.73

6 5.1 5.17 4.87 5 5.2
4.6 4.33 4.37
4 3.47 3.57 3.1
2.53 2.77 2.7 2.67 3 2.8 2.97
2.07
1.57 1.6 1.57
2 1.2 1.23 1.27 1.3 1.4 1.43 1.2 1.3
0
Treatment
Figure 16: Effect of seed priming methods on Leaf width of maize varieties (A) Nutan-IL60 and
There was significant varietal, treatment and interaction effect on shoot dry weight
(Appendix 11). Leaf width was consistently suppressed in salt primed treatments across all DAS
in Nutan-IL60. Best response was observed in urea treatment. Interestingly, MOP priming
showed slightly better response in leaf width compared to other observed traits. Water and urine
priming also showed positive response compared to control plots (Figure 16).
25
Also, in Manakamana-3 urea-24 showed the best response for leaf width especially at 45
DAS. Salt and MOP priming were comparable to control condition while other treatment showed
intermediate response for leaf width in Manakamana-3 (Figure 16)
4.6 Root Length
A: Nutan 21 28 35
40
30.6
Root Length (cm)
30 24.47 26.2
19.1 18.97 20 19.87 18.87 17.53
20 14.47 15.43 15.37 13.6 15.6 14 14.4
10.57 10.13 10.13 11.97 12.53 11.33 10.27
8.57 9.13 9.97
10 6.77 5.93 6.2 6.1 5.43 6.23 6.2
0
Treatment
B: Manakamana-3
30 27.5 27.1 27.4 28.23 21 28 28 3527.27
26.6 26.43 25.4 25.4 24.3 24.77
25 22.23 22.2 22.17 22.33 22.67 22.2 21.9 21.93 23.23
21.27 21.4
Root Length (cm)
19.3 20.37 19.1

20 17.83 17.37 18.33 18.57 18.33 17.8 18.07
15
10
5
0
Treatment
Figure 17: Effect of seed priming methods on Root length of maize varieties (A) Nutan-IL60 and
There was significant varietal, treatment and interaction effect on shoot dry weight
(Appendix 12). Surprisingly, root length was highly suppressed in water primed plots for all
DAS in Nutan-IL60 compared to other treatments. It was highest in urine-24 followed by urine-
26
12 and salt-24. Other treatments showed intermediate response towards root length compared to
control in Nutan-IL60 (Figure 17).
In contrast to Nutan-IL60, Manakamana-3 did not show great variation for root length in
response to different priming methods. Root length was significantly higher in urea-24 treatment
at 21 DAS in Manakaman-3. For other DAS, the root length phenotype did not vary significantly
from control treatment (Figure 17).
4.7 Shoot Dry Weight
A: Nutan
21 28 35
6
5.03
Shoot Dry Weight (g)
5 4.11 4.18 4.38

4.01 3.78
4 3.55 3.43 3.58
3.05
3 2.7
2.17 2.23 2.32 2.06
2 1.63 1.37 1.45 1.55 1.39
1.3 0.98 1.27
0.91 0.61 0.78 0.94 0.75 0.68
1 0.38 0.54 0.5 0.47
0
Treatment
B: Mankamana-3
6 21 28 35
4.86
Shoot Dry Weight (g)
5 4.21 4.38
4 3.57 3.75 3.67 3.56
3.33 3.32
2.81 2.89
3
2.09
2 1.44 1.61 1.64 1.39 1.55
1.21 1.04 1.04 1.25
1.14
0.71 0.9 0.66 0.71
1 0.49 0.59 0.61 0.57 0.55
0.44 0.49
0
Treatment
Figure 18: Effect of seed priming methods on Shoot dry weight of maize varieties (A) Nutan-
IL60 and (B) Manakamana-3 at different growth stages.
27
Also, nitrogen primed samples showed better performance in terms of shoot biomass at
all days of data collection in Nutan-IL60. The most prominent effect was observed in urine
treated samples. Water primed samples did not show any beneficial effect compared to control
plots. In contrast, MOP and salt primed plots showed intermediate response to shoot dry weight
in Nutan-IL60 (Figure 18)
Urea primed plots showed best performance for shoot weight in Manakaman-3 and the
effect was strongest at 35 DAS. Like Nutan-IL60, water primed plots did not perform good
especially at 35 DAS, however, 24 hour water soaking was better in early stages in particular.
Also, salinity and MOP priming showed intermediate phenotype in Manakamana-3 (Figure 18).
28
4.8 Root Dry Weight
A: Nutan
21 28 35
2.5 2.31 2.24
1.83 1.86
Root Dry Weight (g)
2 1.7 1.71
1.63 1.54 1.55
1.42 1.51 1.48
1.5 1.37 1.37
1.1
0.98
1 0.75 0.84 0.73
0.67 0.66 0.67 0.7
0.63 0.67 0.72
0.59 0.53
0.46 0.38 0.4 0.41
0.5 0.33
0
Treatment
B: Manakamana-3
2.5 2.25 2.25 21 28 35
Root Dry Weight (g)
2 1.73
1.57 1.54 1.51 1.61
1.34 1.45
1.5 1.25 1.33
0.92 0.85 0.84 0.87
1 0.79 0.78 0.76 0.72 0.79 0.78 0.73
0.63 0.67
0.58 0.64
0.47 0.49 0.54 0.43 0.43 0.45
0.5 0.39
0
Manakamana-3
Figure 19: Effect of seed priming methods on Root dry weight of maize varieties (A) Nutan-IL60
There was significant varietal, treatment and interaction effect on root dry weight
(Appendix 14). Root dry weight was significantly high for plots sown with urea and urine treated
seeds with urine being the best for Nutan-IL60. However, other treatments were comparable to
control treatment for all days of observation (Figure 19).
Similarly, Mankamana-3 also showed better response to urea and the response being
distinctly visible especially at 35 DAS. Other treatments were not significantly different from
control condition for root dry biomass (Figure 19).
29
4.9 Correlation analysis:
Pairwise Pearson’s correlation was performed for 21, 28 and 35 DAS for both varieties.
Mean values for each treatment genotype combination were correlated. In each correlation table
upper triangle indicates statistical significance (ns not significant, * P<0.05, **P<0.01,
***P<0.001), while lower triangle indicates correlation coefficients. Traits correlated include SL
(shoot length), NOL (number of leaves), LL (leaf length), LW (leaf width), RL (root length),
DSW (dry shoot weight) and DRW (dry root weight).

Nutan-IL60 at 21 DAS on phenotypic traits for different priming reagents.
SL NOL LL LW RL DSW DRW

SL 1 *** *** *** *** *** ***
NOL 0.6 1 *** ** ** ** *
LL 0.98 0.61 1 *** *** *** ***
LW 0.78 0.52 0.79 1 *** *** ***
RL 0.92 0.54 0.91 0.76 1 *** ***
DSW 0.67 0.48 0.7 0.83 0.72 1 ***
DRW 0.63 0.42 0.66 0.67 0.68 0.9 1
Leaf number of Nutan-IL60 is significantly correlated at (* P<0.05) with dry root mass
and at **P<0.01 with Dry Shoot Weight, Root Length, Root Length and Leaf Width at 21 DAS.
All other traits are correlated with each other highly significantly at ***P<0.0001. (Table 5)


SL 1 *** *** *** *** *** ***
NOL 0.65 1 *** *** ** ** **
LL 0.93 0.64 1 *** *** *** ***
LW 0.8 0.7 0.78 1 *** ** **
RL 0.83 0.51 0.88 0.7 1 ** ***
DSW 0.64 0.41 0.58 0.47 0.54 1 ***
DRW 0.65 0.47 0.64 0.5 0.62 0.91 1
At 28 DAS leaf number have positive and significant relation with root length, dry shoot
weight and dry root weight at P<0.01 also leaf width with dry shoot weight and dry root weight,
30
root length with dry shoot weight exhibit similar correlation. All remaining traits show positive
and highly significant correlation with each other at P<0.001.


SL 1 *** *** ** ** ** **
NOL 0.7 1 *** ** NS ** **
LL 0.72 0.68 1 *** *** *** ***
LW 0.46 0.52 0.71 1 NS *** **
RL 0.52 0.29 0.73 0.3 1 *** ***
DSW 0.48 0.49 0.77 0.56 0.75 1 ***
DRW 0.52 0.52 0.82 0.47 0.7 0.75 1
Root length of Nutan-IL60 showed non-significant correlation with leaf number and leaf
width at 35 DAS. Shoot length showed positive correlation with leaf width, root length and dry
masses at P<0.001, similar correlation can be seen in leaf number with leaf width and dry
masses. Dry shoot weight is positively correlated with shoot length and leaf number at P<0.001
also dry root weight exhibit similar relation with leaf width as addition to previous two. All
remaining traits have highly significant positive correlation at P<0.0001. (Table 7)

Manakamana-3 at 21 DAS on phenotypic traits for different priming reagents.

SL 1 * * * ** ns ns
NOL 0.41 1 Ns ns * * *
LL 0.41 0.24 1 ns ** ** **
LW 0.39 0.07 0.22 1 * * ns
RL 0.52 0.39 0.47 0.4 1 ** **
DSW 0.26 0.42 0.45 0.33 0.42 1 ***
DRW 0.28 0.44 0.45 0.23 0.46 0.9 1
At 21 DAS shoot length of Mankamana-3 had positive significant correlation with leaf
number, leaf length and leaf width at P<0.05 and with root length at P<0.001. Leaf number had
positive significant relation with root length and dry masses at P<0.05. Leaf length had positive
significant relation with root length and dry masses at P<0.001. Leaf width had significant
31
positive correlation with root length and dry shoot mass at P<0.05. Root length had positive and
highly significant relation with dry masses at P<0.001 while dry masses were correlated
P<0.0001. (Table 8)

Manakamana-3 at 28 DAS on phenotypic traits for different priming reagents

SL 1 *** *** * ns ns *
NOL 0.58 1 Ns ns * * *
LL 0.76 0.26 1 *** ns ns *
LW 0.4 0.13 0.59 1 ns ns *
RL 0.2 0.31 0.05 0.13 1 ns Ns
DSW 0.1 0.3 0.23 0.28 0.04 1 ***
DRW 0.35 0.41 0.45 0.42 0 0.81 1
In Manakamana-3 at 28 DAS leaf number and leaf length were positively correlated with
shoot length at high significance at P<0.0001 while leaf width and dry root mass was correlated
at P<0.05. Leaf number was positively correlated with root length and dry masses at P<0.05.
Leaf length and leaf with were positively correlated with high significance at P<0.001 dry
masses exhibited similar correlation. (Table 9)
Table 10: Pearson’s correlations coefficients between variables measured in variety

Manakamana-3 at 35 DAS on phenotypic traits for different priming reagents

SL 1 *** *** * ns ns *
NOL 0.58 1 ns ns * * *
LL 0.76 0.26 1 *** ns ns **
LW 0.4 0.13 0.59 1 ns ns *
RL 0.2 0.31 0.05 0.13 1 ns Ns
DSW 0.1 0.3 0.23 0.28 0.04 1 ***
DRW 0.35 0.41 0.45 0.42 0 0.81 1
At 35 DAS highly significant positive correlation at P<0.001 was expressed by shoot length
with leaf number and leaf length, leaf width with leaf length and dry root weight with dry shoot
weight. Positive significant correlation at P<0.05 was exhibited by shoot length with leaf width
and dry root weight, leaf number with root length and dry masses, and dry root weight with leaf
width. (Table 10)
32
33
5. DISCUSSION
The research was conducted primarily to evaluate the effect of different priming reagents on
germination and phenotypic characters of two maize varieties Nutan-IL60 (hybrid) and
Manakamana-3 (locally cultivated). Priming reagents were chosen as chemical (urea and MOP)
and locally available (water, salt and urine). The interaction between reagents and duration of
time was found statistically insignificant. The agro-morphological attributes studied were plant
height, leaf length, leaf number, root length, dry shoot weight and dry root weight. Germination
is one of the major attributing factors to yield as increasing the rate of crop emergence could
increase crop stand establishment, efficient use of resources such as water and light to produce
good yields [ CITATION Chi93 \l 1033 ].
Priming has been shown to enhance seedling establishment under stressful conditions
([ CITATION Ash05 \l 1033 ] ; [ CITATION Afz05 \l 1033 ] ). Both varieties performed well with
increased germination percentage and germination index for Nitrogen treatments (Urea). Urine
which is highly rich in nitrogen content and comprise other nutrients like phosphorus and
potassium [ CITATION Kum14 \l 1033 ]also showed positive effect on germination index.
Statistically significant differences were found in germination percentage and germination index
in Nutan-IL60 between treatments while statically insignificant variation was seen between
treatments in Manakamana-3. This implies that in Nutan-IL60 seed priming improved seed
germination by speeding up imbibition, which possibly contributed to facilitate emergence phage
after raining in the semiarid area. Nutan-IL60 responded well to Nitrogen treatment as hybrids
can be more responsive to Nitrogen treatment; high nitrogen use efficiency (NUE) is recorded
for hybrid varieties [ CITATION Mas18 \l 1033 ]. Also hydro-priming improved the germination
percentage and have greater germination index compared to control in both the varieties and
similar results can also be seen in seedling emergence of lentil [ CITATION Sag10 \l 1033 ] . Also
hydro-primed seeds of maize for 36 hours had highest T50 value and mean germination
[ CITATION Dez08 \l 1033 ]. This was in coherence to this study as both varieties when hydro
primed had better mean germination time than in control. There is increased GP, GI and reduced
MGT in Manakamana-3 for NaCl treatments as priming with NaCl has proved to increase final
germination percentage and reduce T50 value [ CITATION Tir09 \l 1033 ] . However, NaCl had
negative response on emergence parameters on Nutan-IL60 suggesting Manakamana-3 is more
34
saline stress tolerant compared to Nutan-IL60. Priming with cow urine increased the seed
germination percentage in sorghum [ CITATION Ama15 \l 1033 ] as it did for both Manakamana-3
and Nutan-IL60. MOP priming reduced MGT and increased GI value (Table 3) in case of
Manakamana-3 while it wasn’t very significant for Nutan-IL60. This can be attributed to the
study conducted by [ CITATION Sey17 \l 1033 ] which stated that priming with based fertilizers
solution significantly increased germination percentage and speed.
Priming reagents (especially for Urea and Urine priming) had the beneficial effects on root
length and shoot length at every time point. This was mainly due to the accelerated metabolism
occurring in primed seeds, which increases the imbibition speed as compared to unprimed seeds
(control)[ CITATION Pri10 \l 1033 ] . Similar results were also reported by[ CITATION Gua09 \l 1033 ] .
However this was not the case with Manakamana-3 in this study as root and shoot length
performed similarly with other treatments. Salt and MOP priming had comparably lower root
and shoot length which in case of Manakamana-3 was statistically similar with that of control
while in case of Nutan-IL60 was statistically lower than that of control suggesting prevalence of
moisture stress.
Morphological traits except leaf number and dry masses were similar for both varieties while
leaf length of Nutan-IL60 was higher when compared to Manakamana-3. Dry biomass is a trait
taken significantly correlated to grain yield[ CITATION Rey94 \l 1033 ] . The dry biomass was
always higher in case of Urea and Urine priming for both the varieties while it was lower in case
of Salt and unprimed seeds. This suggests that Urea and Urine priming has more pronounced
effect on increasing the dry biomass of maize seedling (whether hybrid or OPV) and hence
possibly the yield.
In a bigger picture, Nutan-IL60 seems to have more variation in traits between different
priming reagents suggesting Urea and Urine priming to be more promising in performance and
possibly yield. Manakamana-3 on the other hand performed more stably for most of the traits
regardless of the different priming treatments. However, a slight statistical advantage was seen
among the traits such as dry biomass and root length when it came Nitrogen-based priming.
Since root length is a trait associated with water stress hardiness [ CITATION Bru66 \l 1033 ] and dry
biomass is a trait associated with grain yield, Urea and Urine priming has a comparative
advantage to other priming reagents in Manakamana-3 as well.
35
Manakamana-3 is the recommended open pollinated variety (OPV) for the mid-hills by
government of Nepal [ CITATION NMR13 \l 1033 ]due to its higher adaptability to the agro climatic
area. This study provided an overview of the effect of different priming reagents in different
morphological traits during different growth stages of two maize varieties. It found that Nitrogen
based priming had a comparable advantage to other priming methods. With further
documentation of similar effects on later stages of maize development, prediction of yield and
yield contributing factors as affected by priming reagents can be identified. It could help in
identifying a more profitable practice for specific geographical areas and might play a significant
role in possibly increasing the grain yield and maintaining a hardier crop stand.
36
Appendices
6. SUMMARY AND CONCLUSION
Maize is the second most important staple food crop both in terms of area and production
after rice in Nepal. There is a big yield gap of maize in Nepal as affected by various
technological and socio-economic factors like the expansion of maize cultivation into less
suitable terrain, declining soil fertility, prolonged drought, loss of soil nutrients, and reluctant
adoption of improved management practices. The main production constraint in eastern hills
after labor shortage is the lack of enough selection of varieties for various stresses such as
drought. This study was conducted with the aim to characterize different priming reagents
according to their performance in two maize varieties (OPV and hybrid) and study the
morphological changes that the plant goes through.
Field experiment was carried out on 27th of February 2018. Two varieties of maize viz. OPV
and hybrid were sown in Randomized Complete Block Design with three replications each.
Priming of both maize varieties were done with 5 different reagents and two different timings.
The primed seeds were air-dried for 12 hours before sowing. Observations were taken for
quantitative traits such as days to germination, shoot length, root length, leaf number, leaf length,
leaf width, fresh shoot weight, fresh root weight, dry shoot weight and dry root weight starting
from germination to 45 Days after Sowing. The mean performance was obtained, DMRT was
done and correlation analysis was performed between dry biomass and other traits. Duration of
priming did not have significant response in morphological traits and hence it was overlooked.
Since priming foremost improves the germination index and percentage, it was seen that
Nitrogen based priming had the best result in case of Nutan-IL60 while Nitrogen based priming
and hydro-priming performed similarly in case of Manakamana-3. Based on quantitative traits
DMRT result especially of shoot length, root length and dry biomass, Nitrogen primed maize
seedlings were found to be better performing. These traits can be used to identify possible
drought tolerance of a variety. These traits were in general higher and better performing in
Nitrogen-primed seeds of both varieties and hence priming using this as priming reagents would
help in survival chance of maize seedlings in adverse water stress conditions.
37
This study discussed about the effects different priming reagents on different morphological
traits of seedlings of two maize varieties in Okhalhdunga, Nepal. It has shown the comparative
advantage of different priming reagents in two maize varieties in order to observe germination
behavior change and morphological trait response. It is seen that Nitrogen based priming not
only improves the germination index but also those morphological traits that have the most direct
relation with water stress tolerance (longer root length)and possibly higher grain yield (higher
dry biomass). Since both Nitrogen based priming treatments performed statistically similar,
Urine based priming is seen more feasible in eastern hills considering its wide availability.
With further study, it can be identified whether these traits as affected by the priming
reagents have a role in altering the grain yield and hardy crop stand establishment. With further
study it can be studied if priming treatments only have effect on early growth stages or during
harvest as well.
38
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45
APPENDICES
Appendix 1: Mean Table of shoot length for different DAS with assigned DMRT values
Shoot length
21 DAS 28 DAS 35 DAS 45 DAS
Treatment
Control 16.150abc 32.225b 48.383ab 87.482a
MOP-12 17.583bcd 30.522ab 48.073ab 95.162ab
MOP-24 14.888a 30.500ab 47.628ab 96.132ab
Salt-12 18.267cd 30.992ab 45.348a 86.150a
Salt-24 15.722ab 29.788a 47.950ab 87.072a
Urea-12 27.705f 40.817de 61.840c 111.523c
Urea-24 28.372f 41.942e 66.288c 111.400c
Urine-12 26.833f 38.857cd 62.905c 106.522bc
Urine-24 24.505e 38.030c 64.488c 109.772c
Water-12 19.335d 31.368ab 52.857b 99.993bc
Water-24 18.118cd 32.213b 54.083b 97.368ab
LSD 2.201 2.238 6.843 11.826
Pr(>F) 0.000*** 0.000*** 0.000*** 0.000***
Variety
Man-3 23.206b 40.882b 56.497b 105.442b
Nutan-IL60 18.154a 27.710a 52.566a 92.480a
LSD 0.938 0.954 2.918 5.042
Pr(>F) 0.000*** 0.000*** 0.009** 0.000***
Interaction
Man-3 x Control 16.400abcd 38.567bcde 51.167abcde 95.300abcd
Man-3 X MOP 12 22.133defg 38.200bcde 52.200abcde 100.133abcd
Man-3 x MOP 24 17.933bcde 38.933bcde 52.667abcde 107.400abcd
Man-3 X Salt 12 22.567efg 37.633bcde 45.967abc 93.867abcd
Man-3 X Salt 24 18.600bcdef 36.667bcd 48.700abcde 94.800abcd
Man-3 X Urea 12 26.400ghi 46.933fg 61.567bcde 112.733cd
Man-3 X Urea 24 31.933i 49.433g 67.033e 117.200d
Man-3 X Urine12 27.600ghi 42.300def 60.733abcde 105.400abcd
Man-3 X Urine 24 25.833gh 42.767ef 64.000cde 115.800cd
Man-3 X Water 12 24.067fgh 38.267bcde 57.000abcde 113.500cd
Man-3 X Water 24 21.800cdefg 40.000cde 60.433abcde 103.733abcd
Nutan-IL60 X Control 15.900abc 25.883a 45.600ab 79.663ab
46
Nutan-IL60 X MOP 12 13.033ab 22.843a 43.947ab 90.190abcd
Nutan-IL60 X MOP 24 11.843a 22.067a 42.590a 84.863abc
Nutan-IL60 X Salt 12 13.967ab 24.350a 44.730ab 78.433a
Nutan-IL60 X Salt 24 12.843ab 22.910a 47.200abcd 79.343ab
Nutan-IL60 X Urea 12 29.010hi 34.700bc 62.113bcde 110.313bcd
Nutan-IL60 X Urea 24 24.810gh 34.450bc 65.543de 105.600abcd
Nutan-IL60 X Urine 12 26.067ghi 35.413bc 65.077de 107.643abcd
Nutan-IL60 X Urine 24 23.177efgh 33.293b 64.977de 103.743abcd
Nutan-IL60 X Water 12 14.603ab 24.470a 48.713abcde 86.487abcd
Nutan-IL60 X Water 24 14.437ab 24.427a 47.733abcd 91.003abcd

Pr(>F) 0.000*** 0.002** 0.291 0.487
Grand mean 20.680 34.296 54.531 98.961
CV 9.133 5.601 10.770 10.256
Appendix 2: Mean Table of leaf length for different DAS with assigned DMRT values
Leaf Length
Treatment
Control 10.900ab 22.133ab 29.467a 61.817a
MOP-12 11.433b 22.417ab 38.000cd 72.233bc
MOP-24 11.817b 22.000ab 38.883a 71.050bc
Salt-12 9.117a 20.383a 35.283bcd 71.400bc
Salt-24 10.267ab 25.550c 33.550cde 61.967a
Urea-12 21.133d 32.633e 45.600e 71.650bc
Urea-24 19.433d 32.567e 49.783ef 75.700c
Urine-12 17.000c 31.117e 50.950f 74.133c
Urine-24 16.600c 28.283d 49.567ef 73.050bc
Water-12 12.117b 21.983ab 33.500bcd 65.500ab
Water-24 10.800ab 23.917bc 31.900ab 65.200ab
LSD 1.874 2.098 5.301 7.995
Pr(>F) 0.000*** 0.000*** 0.000*** 0.005**
Variety
Man-3 15.452b 31.673b 44.148b 78.982b
Nutan-IL60 11.933a 19.779a 35.212a 59.873a
LSD 0.799 0.894 2.260 3.409
Pr(>F) 0.07742 0.000*** 0.000** 0.000***
Interaction
Man-3 x Control 14.533cde 27.333bc 40.100defg 72.767def
Man-3 X MOP 12 14.367cde 28.200bc 42.433defgh 77.933ef
47
Man-3 x MOP 24 17.467def 29.167bc 42.333defgh 80.267ef
Man-3 X Salt 12 10.600abc 26.000b 39.233defg 76.633ef
Man-3 X Salt 24 13.667cd 36.600de 38.633defg 70.200bcdef
Man-3 X Urea 12 19.233efg 38.333e 49.833gh 81.633ef
Man-3 X Urea 24 20.567fg 39.067e 51.833gh 86.733f
Man-3 X Urine12 14.800cde 32.200cd 46.033efgh 76.900ef
Man-3 X Urine 24 15.567cdef 31.100bcd 48.867fgh 81.867ef
Man-3 X Water 12 15.667def 28.333bc 45.833efgh 81.633ef
Man-3 X Water 24 13.500bcd 32.067cd 40.500defg 82.233ef
Nutan-IL60 X Control 7.267a 16.933a 18.833a 50.867abc
Nutan-IL60 X MOP 12 8.500ab 16.633a 33.567bcde 66.533abcdef
Nutan-IL60 X MOP 24 6.167a 14.833a 35.433cdef 61.833abcde
Nutan-IL60 X Salt 12 7.633a 14.767a 31.333abcd 66.167abcdef
Nutan-IL60 X Salt 24 6.867a 14.500a 28.467abcd 53.733abcd
Nutan-IL60 X Urea 12 23.033g 26.933bc 41.367defg 61.667abcde
Nutan-IL60 X Urea 24 18.300defg 26.067b 47.733efgh 64.667abcde
Nutan-IL60 X Urine 12 19.200efg 30.033bc 55.867h 71.367cdef
Nutan-IL60 X Urine 24 17.633def 25.467b 50.267gh 64.233abcde
Nutan-IL60 X Water 12 8.567ab 15.633a 21.167ab 49.367ab
Nutan-IL60 X Water 24 8.100a 15.767a 23.300abc 48.167a

Pr(>F) 0.000*** 0.000* 0.000*** 0.025*
Grand mean 13.692 25.726 39.680 69.427
CV 11.748 6.999 11.465 9.883
Appendix 3: Mean Table of leaf number for different DAS with assigned DMRT values
Leaf Number
Treatment
Control 2.053a 3.167ab 4.277ab 5.945ab
MOP-12 1.998a 3.333abc 4.110ab 5.945ab
MOP-24 2.055a 3.502ab 4.000a 5.945ab
Salt-12 2.222ab 3.055a 3.998a 5.835a
Salt-24 2.222ab 3.000a 4.057ab 6.055bc
Urea-12 2.612c 4.000e 4.890c 6.277de
Urea-24 2.613c 3.890de 4.945c 6.165cde
Urine-12 2.333abc 3.890de 4.890c 6.332e
Urine-24 2.445bc 3.667cde 4.668c 6.165cde
Water-12 2.333abc 3.167ab 4.113ab 6.110bcd
Water-24 2.277abc 3.555cd 4.335b 5.945ab
LSD 0.379 0.336 0.285 0.200
48
Pr(>F) 0.0186* 0.000*** 0.000*** 0.000***
Variety
Man-3 2.485b 3.677b 4.606b 6.101a
Nutan-IL60 2.090a 3.273a 4.173a 6.030a
LSD 0.162 0.143 0.122 0.085
Pr(>F) 0.000* 0.000*** 0.000** 0.101
Interaction
Man-3 x Control 2.220ab 3.443bcde 4.443bcde 5.890a
Man-3 X MOP 12 2.333ab 3.443bcde 4.330abcde 5.890a
Man-3 x MOP 24 2.333ab 3.780cde 4.330abcde 6.000ab
Man-3 X Salt 12 2.333ab 3.110abcde 4.333abcde 5.890a
Man-3 X Salt 24 2.667ab 3.557bcde 4.443bcde 6.220ab
Man-3 X Urea 12 2.667ab 4.000e 4.890e 6.110ab
Man-3 X Urea 24 2.780b 4.000e 5.000e 6.110ab
Man-3 X Urine12 2.443ab 3.890de 4.890e 6.220ab
Man-3 X Urine 24 2.557ab 3.890de 4.780de 6.220ab
Man-3 X Water 12 2.557ab 3.557bcde 4.557bcde 6.000ab
Man-3 X Water 24 2.443ab 3.777bcde 4.670cde 5.780a
Nutan-IL60 X Control 1.887ab 2.890abc 4.110abcd 6.000ab
Nutan-IL60 X MOP 12 1.663a 3.223abcde 3.890ab 6.000ab
Nutan-IL60 X MOP 24 1.777ab 3.223abcde 3.670a 5.890a
Nutan-IL60 X Salt 12 2.110ab 3.000abcd 3.663a 5.780a
Nutan-IL60 X Salt 24 1.777ab 2.443a 3.670a 5.890a
Nutan-IL60 X Urea 12 2.557ab 4.000e 4.890e 6.443b
Nutan-IL60 X Urea 24 2.447ab 3.780cde 4.890e 6.220ab
Nutan-IL60 X Urine 12 2.223ab 3.890de 4.890e 6.443b
Nutan-IL60 X Urine 24 2.333ab 3.443bcde 4.557bcde 6.110ab
Nutan-IL60 X Water 12 2.110ab 2.777ab 3.670a 6.22ab
Nutan-IL60 X Water 24 2.110ab 3.333abcde 4.000abc 6.110ab

Pr(>F) 0.6715 0.044* 0.016* 0.031*
Grand mean 2.288 3.475 4.389 6.065
CV 14.226 8.298 5.573 2.823
Appendix 4: Mean Table of leaf width for different DAS with assigned DMRT values
Leaf Width
Treatment
Control 1.067a 1.950b 3.050a 7.250bc
MOP-12 1.183ab 2.217d 4.367d 8.500fg
MOP-24 1.267bc 2.167cd 4.267d 7.767cde
Salt-12 1.233bc 1.683a 3.733bc 6.533a
49
Salt-24 1.083a 2.017bc 3.350ab 6.900ab
Urea-12 1.850e 3.100g 4.983ef 8.600gh
Urea-24 1.850e 3.050g 5.233f 9.217h
Urine-12 1.350c 2.467ef 4.050cd 7.750cd
Urine-24 1.517d 2.633f 4.550de 8.083defg
Water-12 1.133ab 2.150cd 3.317ab 7.850efg
Water-24 1.250bc 2.283de 3.700bc 8.450
LSD 0.135 0.185 0.513 0.688
Pr(>F) 0.000*** 0.000*** 0.000*** 0.000***
Variety
Man-3 1.370a 2.876b 5.039b 8.036a
Nutan-IL60 1.318a 1.800a 3.070a 7.764a
LSD 0.057 0.079 0.219 0.293
Pr(>F) 0.07742 0.000*** 0.000** 0.000***
Interaction
Man-3 x Control 1.200abcde 2.533ef 4.600defgh 7.433abc
Man-3 X MOP 12 1.233bcdef 2.767fg 5.100fgh 8.433bcd
Man-3 x MOP 24 1.267cdefg 2.700fg 4.333defg 7.033ab
Man-3 X Salt 12 1.567fg 2.067cde 5.167fgh 6.833ab
Man-3 X Salt 24 1.300defg 2.667fg 4.367defgh 6.967ab
Man-3 X Urea 12 1.600g 3.467hi 5.467fgh 8.267bcd
Man-3 X Urea 24 1.567fg 3.567i 5.600gh 9.400d
Man-3 X Urine12 1.400defg 3.000fgh 4.867efgh 7.667abcd
Man-3 X Urine 24 1.433efg 3.100ghi 5.733h 7.967abcd
Man-3 X Water 12 1.200abcde 2.800fg 5.000efgh 7.433abc
Man-3 X Water 24 1.300defg 2.967fg 5.200fgh 7.967abcd
Nutan-IL60 X Control 0.933abc 1.367a 1.500a 7.067ab
Nutan-IL60 X MOP 12 1.133abcde 1.667abc 3.633cde 8.567bcd
Nutan-IL60 X MOP 24 1.267cdefg 1.633abc 4.200def 8.500bcd
Nutan-IL60 X Salt 12 0.900ab 1.300a 2.300abc 6.233a
Nutan-IL60 X Salt 24 0.867a 1.367a 2.333abc 6.833ab
Nutan-IL60 X Urea 12 2.100h 2.733fg 4.500defgh 8.933cd
Nutan-IL60 X Urea 24 2.133h 2.533ef 4.867efgh 9.033cd
Nutan-IL60 X Urine 12 1.300defg 1.933bcd 3.233bcd 7.833abcd
Nutan-IL60 X Urine 24 1.600g 2.167de 3.367bcd 8.200bcd
Nutan-IL60 X Water 12 1.067abcd 1.500abc 1.633a 8.267bcd
Nutan-IL60 X Water 24 1.200abcde 1.600ab 2.200ab 8.933cd
Pr(>F) 0.000*** 0.0168* 0.000*** 0.093

Grand mean 1.344 2.338 4.055 7.900
CV 8.601 6.800 10.857 10.857
50
Appendix 5: Mean Table of root length for different DAS with assigned DMRT values
Root length
21 DAS 28 DAS 35 DAS
Treatment
Control 12.300a 15.918a 20.533a
MOP-12 11.650a 16.183a 22.767ab
MOP-24 12.750a 15.383a 22.183ab
Salt-12 12.217a 15.250a 22.700ab
Salt-24 12.000a 15.650a 24.383bc
Urea-12 15.533b 18.883bc 23.683b
Urea-24 17.600c 20.067c 22.983ab
Urine-12 17.500c 19.867c 26.800dc
Urine-24 14.833b 18.750bc 29.417d
Water-12 12.017a 15.950a 21.000a
Water-24 12.133a 16.750ab 20.833a
LSD 1.233 2.342 2.645
Pr(>F) 0.000*** 0.000*** 0.000***
Variety
Man-3 18.979b 22.236b 26.694b
Nutan-IL60 8.391a 12.064a 20.085a
LSD 0.526 0.999 1.128
Pr(>F) 0.000*** 0.000*** 0.000***
Interaction
Man-3 x Control 17.833c 21.267def 26.600def
Man-3 X MOP 12 17.367c 22.233ef 26.433def
Man-3 x MOP 24 19.300cd 22.200ef 25.400bcdef
Man-3 X Salt 12 18.333cd 20.367def 25.400bcdef
Man-3 X Salt 24 18.567cd 22.167ef 24.300bcdef
Man-3 X Urea 12 19.100cd 22.333ef 27.500f
Man-3 X Urea 24 22.667e 24.767f 27.100ef
Man-3 X Urine12 21.400de 22.200ef 27.400f
Man-3 X Urine 24 18.333cd 21.900ef 28.233f
Man-3 X Water 12 17.800c 21.933ef 28.000f
Man-3 X Water 24 18.067c 23.233ef 27.267f
Nutan-IL60 X Control 6.767a 10.570abc 14.467a
Nutan-IL60 X MOP 12 5.933a 10.133abc 19.100abc
Nutan-IL60 X MOP 24 6.200a 8.567a 18.967ab
Nutan-IL60 X Salt 12 6.100a 10.133abc 20.000abcde
Nutan-IL60 X Salt 24 5.433a 9.133ab 24.467bcdef
Nutan-IL60 X Urea 12 11.967b 15.433cd 19.867abcd
Nutan-IL60 X Urea 24 12.533b 15.367bcd 18.867ab
Nutan-IL60 X Urine 12 13.600b 17.533de 26.200cdef
51
Nutan-IL60 X Urine 24 11.333b 15.600cd 30.600f
Nutan-IL60 X Water 12 6.233a 9.967abc 14.000a
Nutan-IL60 X Water 24 6.200a 10.267abc 14.400a

Pr(>F) 0.000*** 0.002** 0.000***
Grand mean 13.685 17.150 23.389
CV 7.736 11.720 9.707
Appendix 6: Mean Table of shoot dry weight for different DAS with assigned DMRT values
Shoot Dry Weight

Treatment
Control 0.438a 1.368abcd 3.558c
MOP-12 0.567b 1.258a 3.883e
MOP-24 0.660c 1.632c 3.720cde
Salt-12 0.555b 1.290ab 3.822de
Salt-24 0.908d 1.422bcd 3.627cd
Urea-12 0.922d 1.298abc 4.282f
Urea-24 1.207e 1.708e 4.320f
Urine-12 0.660c 1.445d 3.852e
Urine-24 0.710c 1.433cd 4.297f
Water-12 0.480a 1.468d 2.755a
Water-24 0.692c 2.075f 2.972b
LSD 0.069 0.136 0.205
Pr(>F) 0.000*** 0.000*** 0.000***
Variety
Man-3 0.699a 1.311a 3.670a
Nutan-IL60 0.718a 1.670b 3.800b
LSD 0.029 0.058 0.088
Pr(>F) 0.197 0.000*** 0.005**
Interaction
Man-3 x Control 0.493abc 1.440def 3.567defg
Man-3 X MOP 12 0.593bcdef 1.607ef 3.753efgh
Man-3 x MOP 24 0.707def 1.637f 3.333bcde
Man-3 X Salt 12 0.610bcdef 1.210bcd 4.213hi
Man-3 X Salt 24 1.040ij 1.390cdef 3.670efgh
Man-3 X Urea 12 0.903ghi 1.043bc 4.383ij
Man-3 X Urea 24 1.140jk 1.247bcde 4.860jk
Man-3 X Urine12 0.570bcd 0.657a 3.323bcde
Man-3 X Urine 24 0.440ab 0.550a 3.563defg
Man-3 X Water 12 0.490ab 1.550def 2.810ab
52
Man-3 X Water 24 0.707def 2.093g 2.893abc
Nutan-IL60 X Control 0.383a 1.297cdef 3.550def
Nutan-IL60 X MOP 12 0.540abcd 0.910ab 4.013fghi
Nutan-IL60 X MOP 24 0.613bcdef 1.627f 4.107ghi
Nutan-IL60 X Salt 12 0.500abc 1.370cdef 3.430cde
Nutan-IL60 X Salt 24 0.777fgh 1.453cdef 3.583defg
Nutan-IL60 X Urea 12 0.940hi 1.553def 4.180hi
Nutan-IL60 X Urea 24 1.273k 2.170g 3.780efgh
Nutan-IL60 X Urine 12 0.750efg 2.233g 4.380j
Nutan-IL60 X Urine 24 0.980ji 2.317g 5.030k
Nutan-IL60 X Water 12 0.470ab 1.387cdef 2.700a
Nutan-IL60 X Water 24 0.677cdef 2.057g 3.050abcd
Pr(>F) 0.000*** 0.000*** 0.000***

Grand mean 0.709 1.491 3.735
CV 8.337 7.815 4.722
Appendix 7: Mean Table of dry root weight for different DAS with assigned DMRT values
Dry Root Weight

Treatment
Control 0.398a 0.732b 1.378b
MOP-12 0.475c 0.720b 1.602cd
MOP-24 0.460bc 0.835c 1.685d
Salt-12 0.417ab 0.648a 1.637cd
Salt-24 0.695g 0.775bc 1.528c
Urea-12 0.653fg 0.838c 1.883e
Urea-24 0.887h 1.118d 2.057f
Urine-12 0.622ef 1.077d 1.878e
Urine-24 0.552d 1.065d 1.927ef
Water-12 0.430abc 0.757b 1.175a
Water-24 0.585de 1.052a 1.520c
LSD 0.048 0.068 0.133
Pr(>F) 0.000*** 0.000*** 0.000***
Variety
Man-3 0.568a 0.754a 1.622a
Nutan-IL60 0.554a 0.995b 1.700b
LSD 0.020 0.029 0.057
Pr(>F) 0.177 0.000*** 0.008**
Interaction
Man-3 x Control 0.467bcde 0.793bcdef 1.340abc
53
Man-3 X MOP 12 0.490bcde 0.780bcdef 1.570bcde
Man-3 x MOP 24 0.537cdef 0.917f 1.540bcde
Man-3 X Salt 12 0.430abc 0.627b 1.733de
Man-3 X Salt 24 0.763ij 0.853def 1.507bcde
Man-3 X Urea 12 0.720hij 0.840cdef 2.253f
Man-3 X Urea 24 0.793j 0.867ef 2.250f
Man-3 X Urine12 0.577defg 0.673bcd 1.450abcd
Man-3 X Urine 24 0.387ab 0.430a 1.613cde
Man-3 X Water 12 0.450abcd 0.780bcdef 1.253ab
Man-3 X Water 24 0.637fghi 0.730bcde 1.333abc
Nutan-IL60 X Control 0.330a 0.670bc 1.417abcd
Nutan-IL60 X MOP 12 0.460bcde 0.660bc 1.633cde
Nutan-IL60 X MOP 24 0.383ab 0.753bcdef 1.830e
Nutan-IL60 X Salt 12 0.403ab 0.670bc 1.540bcde
Nutan-IL60 X Salt 24 0.627fgh 0.697bcde 1.550bcde
Nutan-IL60 X Urea 12 0.587efg 0.837cdef 1.513bcde
Nutan-IL60 X Urea 24 0.980k 1.370g 1.863e
Nutan-IL60 X Urine 12 0.667ghij 1.480g 2.307f
Nutan-IL60 X Urine 24 0.717hij 1.700h 2.240f
Nutan-IL60 X Water 12 0.410abc 0.733bcde 1.097a
Nutan-IL60 X Water 24 0.533cdef 1.373g 1.707de

Pr(>F) 0.000*** 0.000*** 0.000***
Grand mean 0.561 0.874 1.661
CV 7.349 6.663 6.878
54
Appendix 8: ANOVA of shoot length for different DAS
Shoot Length
21 DAS
Mean
Factors
Df Sum Sq Sq F value Pr(>F)
Variety 1 421.2 421.2 118.081 8.86E-14 ***
Treatment 5 1517.39 303.48 85.0791 < 2.2e-16 ***
Replication 2 8.52 4.26 1.1947 0.312868
Variety:Treatment 5 147.61 29.52 8.2767 1.65E-05 ***
Residuals 42 149.81 3.57
28 Das
Mean
Factors
Variety 1 2862.83 2862.83 775.804 < 2.2e-16 ***
Treatment 5 1264.52 252.9 68.5348 < 2.2e-16 ***
Replication 2 11.64 5.82 1.5768 0.218616
Variety:Treatment 5 108.85 21.77 5.8992 0.000324 ***
Residuals 42 154.99 3.69
35 Das
Mean
Factors
Df Sum sq Sq F value Pr(>F)
Variety 1 255 255 7.3932 0.009478 **
Treatment 5 3619.9 723.99 20.9906 1.85E-10 ***
Replication 2 80.3 40.13 1.1635 0.322253
Variety:Treatment 5 401.3 80.26 2.3271 0.059341
Residuals 42 1448.6 34.49
45 Das
Mean
Factors
Variety 1 2772.3 2772.27 26.9117 5.78E-06 ***
Treatment 5 5641.4 1128.29 10.9528 8.65E-07 ***
Replication 2 202.2 101.09 0.9813 0.3832
Variety:Treatment 5 505.6 101.13 0.9817 0.4402
Residuals 42 4326.6 103.01
Appendix 9: ANOVA of leaf number for different DAS
Leaf Number
21 DAS
Factor Df Sum Sq Mean Sq F value Pr(>F)
55
Variety 1 2.5685 2.56849 24.2538 1.36E-05 ***
Treatment 5 2.5926 0.51853 4.8963 0.00128 **
Replication 2 0.0303 0.01516 0.1431 0.86707
Variety:Treatmen
5 0.4075 0.08149 0.7695 0.57706
t
Residuals 42 4.4478 0.1059
28 Das
Variety 1 2.6923 2.69226 32.3815 1.11E-06 ***
Treatment 5 6.9245 1.3849 16.6571 4.77E-09 ***
Replication 2 1.5602 0.78009 9.3826 0.00043 ***
Variety:Treatmen
5
t 0.6502 0.13003 1.564 0.1912
Residuals 42 3.492 0.08314
35das
Variety 1 3.0983 3.09833 51.7686 7.66E-09 ***
Treatment 5 8.4871 1.69742 28.3614 1.88E-12 ***
Replication 2 0.1643 0.08216 1.3727 0.26456
Variety:Treatmen
5
t 1.4021 0.28043 4.6855 0.00173 **
Residuals 42 2.5137 0.05985
45das
Variety 1 0.08226 0.08226 2.805 0.1014
Treatment 5 1.14361 0.22872 7.7997 2.90E-05 ***
Replication 2 0.3019 0.15095 5.1476 0.01001
Variety:Treatmen
5
t 0.46501 0.093 3.1715 0.01616 *
Residuals 42 1.23163 0.02933
Appendix 10: ANOVA of leaf length for different DAS
Leaf Length
21 DAS
Variety 1 204.23 204.23 78.9289 3.39E-11 ***
Treatment 5 987.21 197.442 76.3052 < 2.2e-16 ***
Replication 2 0.9 0.452 0.1747 0.84034
Variety:Treatmen
t 5 317.85 63.57 24.568 1.76E-11 ***
Residuals 42 108.68 2.588
56
28 Das
Variety 1 2334.19 2334.19 720.067 < 2.2e-16 ***
Treatment 5 1166.31 233.26 71.9582 < 2.2e-16 ***
Replication 2 10.62 5.31 1.6378 0.20657
Variety:Treatmen
t 5 287.4 57.48 17.7316 2.03E-09 ***
Residuals 42 136.15 3.24
35 Das
Variety 1 1317.7 1317.67 63.6677 5.98E-10 ***
Treatment 5 3674.4 734.88 35.5084 4.81E-14 ***
Replication 2 42.3 21.15 1.0218 0.3687
Variety:Treatmen
t 5 1319.7 263.94 12.753 1.46E-07 ***
Residuals 42 869.2 20.7
45 Das
Variety 1 6025.1 6025.1 127.974 2.49E-14 ***
Treatment 5 1120.8 224.2 4.7613 0.00155 **
Replication 2 143.2 71.6 1.5208 0.23033
Variety:Treatmen
t 5 933.6 186.7 3.9659 0.0049 **
Residuals 42 1977.4 47.1
Appendix 11: ANOVA of leaf width for different DAS
Leaf Width
21 DAS
Variety 1 0.0438 0.04379 3.277 0.07742 .
Treatment 5 4.4917 0.89835 67.2306 < 2.2e-16 ***
Replication 2 0.2121 0.10606 7.9374 0.00119
Variety:Treatmen
5 1.8754 0.37508 28.0699 2.21E-12 ***
t
Residuals 42 0.5612 0.01336
28 DAS
Variety 1 19.0947 19.0947 755.502 < 2e-16 ***
Treatment 5 11.252 2.2504 89.0393 < 2e-16 ***
Replication 2 0.0585 0.0292 1.157 0.32424
57
Variety:Treatmen
5 0.3453 0.0691 2.7325 0.0317 *
t
Residuals 42 1.0615 0.0253
35 DAS
Variety 1 64.015 64.015 330.336 < 2.2e-16 ***
Treatment 5 27.664 5.533 28.5513 1.69E-12 ***
Replication 2 0.121 0.06 0.312 0.73369
Variety:Treatmen
5 14.896 2.979 15.3732 1.39E-08 ***
t
Residuals 42 8.139 0.194
45 DAS
Variety 1 1.227 1.2273 3.5225 0.0675
Treatment 5 32.946 6.5892 18.9124 8.27E-10 ***
Replication 2 0.274 0.1368 0.3927 0.67769
Variety:Treatmen
5 3.915 0.783 2.2475 0.06712
t
Residuals 42 14.633 0.3484
Appendix 12: ANOVA of root length for different DAS
Root Length
21 DAS
Variety 1 1849.7 1849.7 1650.55 < 2.2e-16 ***
Treatment 5 272.46 54.49 48.625 < 2.2e-16 ***
Replication 2 0.65 0.32 0.2881 0.75114
Variety:Treatmen
5 67.74 13.55 12.0898 2.76E-07 ***
t
Residuals 42 47.07 1.12
28 DAS
Variety 1 1707.39 1707.39 422.602 < 2.2e-16 ***
Treatment 5 194.64 38.93 9.6354 3.51E-06 ***
Replication 2 8.23 4.11 1.0181 0.37001
Variety:Treatmen
5 122.62 24.52 6.0698 0.00026 ***
t
Residuals 42 169.69 4.04
35 DAS
Variety 1 720.72 720.72 139.808 6.00E-15 ***
58
Treatment 5 399.89 79.98 15.5142 1.23E-08 ***
Replication 2 29.24 14.62 2.8361 0.06993
Variety:Treatmen
5 393.98 78.8 15.2853 1.49E-08 ***
t
Residuals 42 216.51 5.16
Appendix 13: ANOVA of shoot dry weight for different DAS
Shoot Dry Weight

21 DAS
Variety 1 0.00601 0.00601 1.7215 0.1966
Treatment 5 2.25823 0.45165 129.289 < 2.2e-16 ***
Replication 2 0.00575 0.00287 0.8228 0.4462
Variety:Treatmen
5 0.54515 0.10903 31.2114 4.03E-13 ***
t
Residuals 42 0.14672 0.00349
28 DAS
Variety 1 2.1276 2.12761 156.761 9.12E-16 ***
Treatment 5 1.3143 0.26285 19.3667 5.91E-10 ***
Replication 2 0.1173 0.05865 4.3211 0.01966
Variety:Treatmen
5 8.2694 1.65388 121.856 < 2.2e-16 ***
t
Residuals 42 0.57 0.01357
35 DAS
Variety 1 0.2802 0.28015 9.0075 0.00451 **
Treatment 5 14.5821 2.91641 93.7692 < 2.2e-16 ***
Replication 2 1.0172 0.50862 16.3534 5.59E-06
Variety:Treatmen
5 7.101 1.4202 45.6628 6.48E-16 ***
t
Residuals 42 1.3063 0.0311
Appendix 14: ANOVA of root dry weight for different DAS
Root Dry Weight

21 DAS
Variety 1 0.00321 0.00321 1.8846 0.17709
Treatment 5 0.83041 0.16608 97.6292 < 2.2e-16 ***
59
Replication 2 0.00488 0.00244 1.4357 0.24938
Variety:Treatmen
5 0.21987 0.04397 25.8494 8.05E-12 ***
t
Residuals 42 0.07145 0.0017
28 DAS
Variety 1 0.96002 0.96002 282.93 < 2.2e-16 ***
Treatment 5 1.15599 0.2312 68.1366 < 2.2e-16 ***
Replication 2 0.01642 0.00821 2.4198 0.1012
Variety:Treatmen
5 2.82155 0.56431 166.309 < 2.2e-16 ***
t
Residuals 42 0.14251 0.00339
35 DAS
Variety 1 0.0993 0.0993 7.6093 0.00856 **
Treatment 5 3.5821 0.71642 54.9008 < 2.2e-16 ***
Replication 2 0.0352 0.0176 1.3484 0.27067
Variety:Treatmen
5 2.6575 0.5315 40.7294 4.71E-15 ***
t
Residuals 42 0.5481 0.01305
60

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EFFECT OF PRIMING ON SEED GERMINATION AND SEEDLING

ESTABLISHMENT OF MAIZE (Zea mays) IN OKHALDHUNGA, NEPAL

BACHELOR OF SCIENCE IN AGRICULTURE

I Acknowledge the support from faculty members of department of Agronomy, AFU

LIST OF APPENDICES: xii

1.1 Objectives of the research 3

1.2 Research questions and hypothesis 3

1.3 Scope of the research 4

1.4 Limitations of the research 4

2.1 General Introduction to maize 5

2.2 Status of maize in Nepal 6

2.3 On farm seed priming 7

2.3.3 Priming with plant extract and urine 9

3.1 Site selection 11

3.2 Meteorological data collection and soil testing 11

3.3 Reviewing of literature 12

3.4 Procuring maize seeds and priming reagents 12

3.5 Calculation of amount priming reagents 12

3.6 Constructing an experimental setup 13

3.8 Seed selection and Priming 15

3.9 Sowing of maize and tagging 16

3.10 Data collection 16

3.11 Data Analysis 18

3.11.1 Mean performance 18

3.11.2 Analysis of Variance (ANOVA) 19

3.13.3 Germination Parameter 19

4.2 Shoot length 22

4.3 Leaf Number 23

4.4 Leaf length 25

4.5 Leaf Width 26

4.6 Root Length 27

4.7 Shoot Dry Weight 29

4.8 Root Dry Weight 31

4.9 Correlation analysis 32

6. SUMMARY AND CONCLUSION 39

LEE Learning and Entrepreneurial Experience

Figure 1: Trend of increase in Maize yield

Source: [CITATION MoA091 \l 1033 ]

1.1 Objectives of the research

The specific objectives of the research were to:

1.2 Research questions and hypothesis

1.3 Scope of the research

1.4 Limitations of the research

2.1 General Introduction to maize

2.2 Status of maize in Nepal

Source: [CITATION KCG151 \t \l 1033 ]

2.3 On farm seed priming

Seed priming is often considered as method to increase production in certain crops

2.3.3 Priming with plant extract and urine

Damaged seeds/ poor quality seeds

Low germination and seedling vigor

Improved germination and seedling vigor

Figure 3: Conceptual Framework

3.1 Site selection

Figure 4: Map depicting action research site

3.2 Meteorological data collection and soil testing

3.4 Procuring maize seeds and priming reagents

 Urea and Mop from Kathmandu

3.5 Calculation of amount priming reagents

3.6 Constructing an experimental setup

Table 2: Treatment Combination for Research

Variety Treatments Treatment Combinations

Pr(>F) 0.000* 0.000* 0.000***