IBRAHIM OLAJIDE ORUNSOLU 

 

 

 

 

 

 

SEED PRIMING IMPROVES SEED GERMINATION AND 

SEEDLING GROWTH OF SUNFLOWER UNDER SALINE 

CONDITIONS 

 

 

 

 

 

 

 

 

 

 

LAVRAS – MG  

2023



 
 

IBRAHIM OLAJIDE ORUNSOLU 

 
 

 

SEED PRIMING IMPROVES SEED GERMINATION AND SEEDLING GROWTH 

OF SUNFLOWER UNDER SALINE CONDITIONS 

 

 

 
Dissertation for master’s qualification 

presented to the Federal University of 

Lavras as part of the requirements of the 

Graduate Program in Agronomy/Plant 

Physiology, to obtain the Master's 

degree. 

 

 

 

Prof.ª Dr.ª Elisa Monteze Bicalho 

Advisor 

 

Dr.ª Aline Aparecida Silva Pereira 

Co-Advisor 

 

 

 

 

LAVRAS - MG 

2023



 

 
 

Ficha catalográfica elaborada pelo Sistema de Geração de Ficha Catalográfica da Biblioteca 

Universitária da UFLA, com dados informados pelo (a) próprio (a) autor (a). 

 

 
Orunsolu, Ibrahim Olajide. 

Seed priming improves seed germination and seedling growth of sunflower 

under saline conditions/ Ibrahim Olajide Orunsolu. - 2023. 
78.p.: il. 

 

Orientador (a): Elisa Monteze Bicalho 

Coorientador (a): Aline Aparecida Silva Pereira 

Dissertação (mestrado) - Universidade Federal de Lavras, 2023. 
 Bibliografia. 

 

1. Abiotic stresses 2. Short-term storage. 3. Salinity. 4. 

Halopriming 5. Enzymatic antioxidant system 

I. Bicalho, Elisa Monteze. II Pereira, Aline Aparecida Silva. 

IV. Título 

 

 

 

 

 

 

 

 



 
 

IBRAHIM OLAJIDE ORUNSOLU 

 

 

SEED PRIMING IMPROVES SEED GERMINATION AND SEEDLING GROWTH 

OF SUNFLOWER UNDER SALINE CONDITIONS 

 

PRIMING DE SEMENTES MELHORA A GERMINAÇÃO DE SEMENTES E O 

CRESCIMENTO DE MUDAS DE GIRASSOL EM CONDIÇÕES SALINAS 

 

Dissertation for master’s qualification 

presented to the Federal University of 

Lavras as part of the requirements of the 

Graduate Program in Agronomy/Plant 

Physiology, to obtain the Master's 

degree. 

 

 

 

 

APPROVED ON 11 OF JULY 2023.  

 

Dr.ª Elisa Monteze Bicalho - UFLA 

Dr.ª Shakira Shaik - UKZN 

Dr.ª Mariana Silva Artur - WUR 

 

 

 

 

Prof.ª Dr.ª Elisa Monteze Bicalho 

Advisor 

 

Dr.ª Aline Aparecida Silva Pereira 

Co-Advisor 

 

 

LAVRAS-MG 

2023 



 
 

DEDICATION 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I dedicate this research work to Almighty 

ALLAH, who gave me the opportunity to 

achieve remarkable academic success.



 

 
 

ACKNOWLEDGMENT 

 

 

 

 

 I express my sincere gratitude to Almighty ALLAH, who guaranteed me the opportunity to 

witness another success in my academic journey. My sincere appreciation goes to my advisor 

Prof. (Dr.) Elisa Monteze Bicalho for her patience in guiding me through the course of my 

research, and providing me with all the necessary instructions on how to achieve the 

objective. Also, I appreciate my co-advisor, Dr. Aline Aparecida Silva Pereira. I 

acknowledge the efforts of my dear colleagues in person of Mateus Moreira Bernardes, 

Melina Karla Arantes, Tais Teixera das Neves and Surajo Isah Aliyu for their kind assistance 

during the course. Thank you to my parents, Alhaji and Alhaja Orunsolu, my siblings, and 

friends, who stood by me throughout this course. I appreciate the efforts of my landlady and 

her family for welcoming me with open arms and providing me with shelter when I arrived 

Brazil, and all Nigerian Scholars in UFLA, may GOD reward you abundantly.I appreciate the 

Forum for Agricultural Research in Africa (FARA) and Tertiary Education Trust Fund 

(TETfund) who through the Agricultural Research and Innovation for Africa (ARIFA) 

funded this programme. Finally, I thank Joyce and the staff of the International Relation 

Office (DRI) of the UFLA. 

 

 

 

 

 

 

 

 

 



 

 
 

ABSTRACT 

 

Sunflower (Helianthus annuus L.) is an important oil seed crop that is produced 

mainly due to its high oil content that is consumed by humans in different ways. However, 

because of the high lipid content in sunflower, the seeds are more liable to reduce their 

quality during short-term storage as a result of oxidation due to temperature changes or 

moisture content. Additionally, salinity can hinder sunflower productivity. In this study, we 

evaluated the effect of seed halopriming on the germination of sunflowers after storage 

(manuscript one) and seedling establishment during the early growth stage under salinity 

conditions (manuscript two). The results from the first manuscript indicated that seed 

halopriming improved the germination of primed seeds compared to control seeds, and also 

increased antioxidant enzymatic activity in primed seeds compared to the control. The results 

of the second manuscript illustrate that seedlings from primed seeds were more tolerant to 

higher salinity concentrations by increasing their enzymatic activity. Altogether, it can be 

concluded that seed halopriming is a cost-effective, simple, and efficient technique for 

improving the germination and seedling growth of sunflowers under stressful conditions.  

 

 

Keywords: Abiotic stresses. Short-term storage. Salinity halopriming. Enzymatic antioxidant 

system. 

 

 

 

 

 

 

 

 

 



 

 
 

RESUMO 

 

O girassol (Helianthus annuus L.) é uma importante oleaginosa produzida 

principalmente devido ao seu alto teor de óleo que é consumido pelo homem de diversas 

formas. No entanto, devido ao alto teor de lipídios no girassol, as sementes são mais 

propensas a reduzir sua qualidade durante o armazenamento a curto prazo como resultado da 

oxidação devido a mudanças de temperatura ou teor de umidade. Além disso, a salinidade 

pode prejudicar a produtividade do girassol. Neste estudo, avaliamos o efeito do 

halocondicionamento de sementes na germinação de girassóis após armazenamento 

(manuscrito um) e estabelecimento de plântulas durante o estágio inicial de crescimento em 

condições de salinidade (manuscrito dois). Os resultados do primeiro manuscrito indicaram 

que o halocondicionamento das sementes melhorou a germinação das sementes 

condicionadas em comparação com as sementes controle e também aumentou a atividade 

enzimática antioxidante nas sementes condicionadas em comparação com o controle. Os 

resultados do segundo manuscrito ilustram que mudas de sementes condicionadas foram mais 

tolerantes a concentrações de salinidade mais altas, aumentando sua atividade enzimática. Ao 

todo, pode-se concluir que o halopriming de sementes é uma técnica econômica, simples e 

eficiente para melhorar a germinação e o crescimento de mudas de girassóis sob condições 

estressantes. 

 

 

Palavras-chave: Estresse abiótico. Armazenamento de curto prazo. Salinidade. Halopriming. 

Sistema antioxidante enzimático. 

 

 

 

 

 

 

 

 

 

 



 

 
 

LIST OF FIGURES 

ARTICLE ONE: 

Figure 1: Cumulative germination of control seeds (A) and (B) in primed seeds. Bars are 

means ± standard error (n = 4) ………………………………………………………………37 

Figure 2: Germination percentage (A); germination speed index (B); and time taken for fifty 

percent germination (C) of seeds of Helianthus annuus (control and primed). Bars are means 

± standard error (n = 4). Capital letters compare differences among storage time and lower 

letters compare differences between treatments (control and primed). The same letters or ns 

(not significant) do not show any significance difference by Tukey’s test at 5% significance. 

…………………………………………………………………………………………………………………38 

Figure 3: Viability of seeds of Helianthus annuus (control and primed). Bars are means ± 

standard error (n = 4). Capital letters compare differences among storage time and lower 

letters compare differences between treatments (control and primed). The same letters or ns 

(not significant) do not show any significance difference by Tukey’s test at 5% 

significance…………………………………………………………………………………...39 

Figure 4: Estimated levels of lipid peroxidation (A); and hydrogen peroxide (B) of 

Helianthus annuus seeds. Bars are means ± standard error (n = 4). Capital letters compare 

differences among storage time and lower letters compare differences between treatments 

(control and primed). The same letters or ns (not significant) do not show any significance 

difference by Tukey’s test at 5% significance ……………………………………………….40 

Figure 5: Specific superoxide dismutase activities, SOD (A); catalase, CAT (B) and ascorbate 

peroxidase, APX (C) in control and primed seeds of Helianthus annuus. Bars are means ± 

standard error (n = 4). Capital letters compare differences among storage time and lower 

letters compare differences between treatments (control and primed). The same letters or ns 

(not significant) do not show any significance difference by Tukey’s test at 5% 

significance…………………………………………………………………………………………………...41 

 



 

 
 

LIST OF FIGURES 

ARTICLE TWO: 

Figure 1: Length of shoot (A); length of root (B); dry mass of shoot (C); dry mass of root 

(D); and leaf area (E) of Helianthus annuus seedlings. Bars are means ± standard error (n = 

4). Capital letters compare differences among salinity concentrations and lower letters 

compare differences between treatments (control and primed). The same letters or ns (not 

significant) do not show any significance difference by Tukey’s test at 5% significance. 

…………….............................................................................................................................64 

Figure 2: Estimated levels of chlorophyll a (A); chlorophyll b (B); carotenoids (C) of 

Helianthus annuus seedlings. Bars are means ± standard error (n = 4). Capital letters compare 

differences among salinity concentrations and lower letters compare differences between 

treatments (control and primed). The same letters or ns (not significant) do not show any 

significance difference by Tukey’s test at 5% significance………………………………... 65 

Figure 3: Estimated levels of reducing sugars (A) and total soluble sugars (B) of Helianthus 

annuus seedlings. Bars are means ± standard error (n = 4). Capital letters compare differences 

among salinity concentrations and lower letters compare differences between treatments 

(control and primed). The same letters or ns (not significant) do not show any significance 

difference by Tukey’s test at 5% significance……………………………………………….65 

Figure 4: Estimated levels of proline (A) and proteins (B) of Helianthus annuus seedlings. 

Bars are means ± standard error (n = 4). Capital letters compare differences among salinity 

concentrations and lower letters compare differences between treatments (control and 

primed). The same letters or ns (not significant) do not show any significance difference by 

Tukey’s test at 5% significance……………………………………………………………...66 

Figure 5: Estimated levels of lipid peroxidation – MDA (A); hydrogen peroxide – H2O2 (B); 

ascorbate peroxidase – APX (C) and superoxide dismutase – SOD (D) of Helianthus annuus 

seedlings. Bars are means ± standard error (n = 4). Capital letters compare differences among 

salinity concentrations and lower letters compare differences between treatments (control and 

primed). The same letters or ns (not significant) do not show any significance difference by 

Tukey’s test at 5% significance………………………………………………………………67



 
 

 
 

SUMMARY 

 

1. INTRODUCTION……………………………….……………..……………….. …12 

2. LITERATURE REVIEW …………………………………..……………………...14 

REFERENCES…………………………………………………………….………..20 

ARTICLE 1: SEED HALOPRIMING BEFORE STORAGE HELP MAINTAIN 

SEED QUALITY AND IMPROVE GERMINATION OF 

SUNFLOWER………………………………………………………...…………….29 

1. INTRODUCTION……...……………………………………………..…………….30 

2. MATERIALS AND METHODS……………………………..…………………….32 

2.1 Plant material………………………………………………...……………………….33 

2.2 Priming procedure……………………………………………………...…………….33 

2.3 Storage conditions………………………………………………………………...….33  

2.4 Germination parameters…………………………………………………...…………33 

2.5 Sampling…………………………………………………….…………..……………34 

2.6 Biochemical analysis………………………………………………………...……….34 

2.6.1 Hydrogen Peroxide (H2O2)……………………………………..………………….34 

      2.6.2 Lipidic Peroxidation ……………………………………………………...………..34 

      2.6.3 Antioxidant Enzymatic System Analysis…………………….……...……..………34 

2.7 Statistical analysis……………………………………………………………...…….35 

3. RESULTS AND DISCUSSION…………………………………………………….36 

4. CONCLUSIONS…………………………………………………………………….40 

REFERENCES………………………………………………………………...……41 

ARTICLE 2: SEED HALOPRIMING IMPROVES SEEDLING GROWTH OF    

SUNFLOWER UNDER SALINE CONDITIONS………………………..………53 

1. INTRODUCTION ...………………………………………………………….…….55 

2. MATERIALS AND METHODS…………………………………………………...55  

2.1 Planting material and priming procedure…………………………………………….55 

2.2 Preparation of salt concentrations and soil incubation……………………….………55 

2.3 Experimental conditions……………………………………………………………...55 

2.4 Sampling……………………………………………………………………………...56 

2.5 Growth parameters………………………………………………...…………………56 

2.6 Biochemical analysis………………………………………………...……………….56 

2.6.1 Hydrogen Peroxide (H2O2) …………………………………………...……………56 

2.6.2 Lipidic Peroxidation (MDA Content)…………………………………...…………56 

2.6.3Antioxidant Enzymes…………………………………………………………...…. 57 

2.6.4 Proline quantification…………………………………………….………..……….57 

2.6.5 Determination of pigment content………………………………….……..…..…....57 

2.6.6 Determination of reducing sugars content…………………………..……………..58 

2.6.7 Determination of total soluble sugars content……………………...………………58 

2.6.8 Quantification of proteins…………………..………………………………………58 

2.7 Statistical analysis………………………………………………………...………….58 

3. RESULTS……………………………………………………………………...…….59



 
 

 
 

4. DISCUSSIONS ………………………...………………………………..………….65 

REFERENCES……………………………………………………………………...68 

FINAL CONSIDERATIONS………………………………………...…………….77 

 

 



12 
 

 
 

1. INTRODUCTION  

Sunflower (Helianthus annuus L) is an oilseed crop that belongs to the family 

Asteraceae and is indigenous to North America. It is grown all over the world (YEGOROV et 

al., 2019) because it can acclimate to various climatic and soil conditions. This has improved 

its cultivation as an oilseed plant worldwide (FORLEO et al., 2018). As a source of high-

quality oil and dietary fiber that considerably benefits human health, sunflower is one of the 

key oilseed crops cultivated worldwide (KHAN et al., 2015). 

The demand for edible sunflower seeds, oil, and byproducts has increased in response 

to the ongoing rise in the human population, and to meet this demand, efforts must be 

intensified to boost sunflower production (TAHER et al., 2017). However, there is an 

extended duration of storage during which seed quality deteriorates before the seeds are 

utilized for germination or oil extraction. Therefore, it is essential to conduct comprehensive 

examinations of all changes that occur during storage (ARC et al., 2011).  

Seed storage typically causes seed aging, which manifests as permanent cellular and 

metabolic alterations such the oxidation of proteins, nucleic acids, and lipids. Enzyme 

inactivation, membrane disruption, and impairment of RNA and its production are further 

effects of seed aging (EL-MAAROUF-BOUTEAU et al., 2011; HU et al., 2012; KIBINZA et 

al., 2006). Given that oilseeds are more prone to seed deterioration than cereal grains, storage 

of oilseeds poses a major challenge. 

Also, there are several abiotic stressors, such as salinity, cold, drought, flood, and 

high temperature, that have a significant impact on global agricultural productivity 

(SHRIVASTAVA; KUMAR, 2015). Soil salinity is one of these abiotic stresses that has the 

worst environmental effects, reducing the amount of arable area, crop productivity, and crop 

quality. Commonly, due to the accumulation of soluble salts in the soil, salinization of 

agricultural land occurs (BHARTI et al., 2016), especially the sodium (Na+) and chloride (Cl-

) ions. As a result of higher Na+ ion assimilation and a decrease in the Na+/K+ ratio as a result 

of decreased osmotic potential in plant roots, salinity stress results in osmotic stress and ion 

toxicity (ARIF et al., 2020). 

Furthermore, these ionic imbalances impair critical plant processes and activities by 

interfering with the uptake and transport of other significant necessary ions in target cells 

(ARIF et al., 2020). Salinity lowers the viability of the seeds, hinders the establishment of the 



13 
 

 
 

seedlings, causes stunted plant growth, and poor reproductive development, and eventually 

lowers crop output (TURAN et al., 2009).  

In order to minimize the harm caused by storage and salinity while preserving 

important resources for growth and reproduction, seeds, seedlings and mature plants have 

evolved unique mechanisms that enable them to sense precise environmental changes and 

respond to complicated stress circumstances. A range of plant responses is brought on by the 

perception of salt stress, including the activation of enzymatic and non-enzymatic antioxidant 

mechanisms (SUBRAMANYAM et al., 2012) to reduce ROS build-up and oxidative stress. 

Therefore, increasing plants’ antioxidant capacity is a possible strategy to offset the negative 

effects of ROS-induced oxidative damage (KASOTE et al., 2015).  

To reduce the negative effect storage and salinity on crop productivity, methods like 

seed priming has been adopted. Seed priming is the process of carefully hydrating seeds such 

that pre-germinative metabolic activity can continue but that real radicle emergence is halted. 

Seed priming allows pre-germinative physiological and biochemical changes to take place 

(SADDIQ et al., 2019). Seed priming enhances germination processes including defense, 

which give seeds the ability to withstand environmental challenges that arise during 

germination (FAROOQ et al., 2006, 2009).  

To stimulate the seeds and reduce environmental stress, numerous kinds of seed 

priming techniques have been created and are currently being used successfully, these include 

hydropriming, halopriming, osmopriming, biopriming, hormonal priming, solid matrix 

priming, etc. with our main focus on halopriming technique (VENKATASUBRAMANIAN; 

UMARANI, 2007). Halopriming is imbibing seeds in salt solutions that are not organic, like 

NaCl or KCl. Halopriming encourages uniform germination and higher crop production even 

in unfavorable environmental conditions including temperature extremes and oxygen 

deprivation.  

Halopriming boosts seed metabolism by generating osmolytes and/or signaling 

molecules, promoting germination and enhancing growth and yield capabilities 

(VENKATASUBRAMANIAN; UMARANI, 2007). Osmolytes are compatible solutes that 

build up inside plant cells and act as osmoticum to store water by reducing the osmotic 

potential, shielding macromolecules and cell membranes from oxidative stress and 

dehydration (FAHAD et al., 2017; TABASSUM et al., 2018; VERSLUES; JUENGER, 



14 
 

 
 

2011). 

Sunflower is an important oilseed crop with a lot of health and economic benefits. 

Therefore, it is important to improve the quality of seeds and also minimize the negative 

effects of abiotic stresses. There is still a wide gap to be filled regarding the use of 

halopriming technique to mitigate the effect of abiotic stresses as a result of storage on the 

germination of sunflower seeds. The main objective of this study was to investigate the effect 

of seed halopriming on germination and seedling establishment of sunflowers after storage 

(manuscript one) or during early growth stage at salinity conditions (manuscript two). We 

hypothesize that halopriming procedure will improve the seed germination of sunflowers 

after storage, and we also hypothesize that seed halopriming technique will improve seed 

germination and seedling growth of sunflowers under saline conditions by enhancing the 

activities of enzymes and non-enzymatic antioxidants.  

2. LITERATURE REVIEW  

Sunflower is the third-most significant oilseed crop in the world (53.48 million metric 

tons), behind soybeans (337.48 million metric tons) and rapeseed (68.02 million metric tons), 

which are the most lucrative and economical (SHABANDEH, 2020). It is mainly produced 

for the creation of high-oil varieties by plant scientists under optimum conditions for 

maximum yield and productivity, which calls for fertile soil, sufficient rainfall, and ideal 

environmental conditions. Climate, edaphic, and management factors, along with nutrient-

limiting environmental circumstances, can reduce the production of sunflower products such 

as seeds, oil content, and other goods. Sunflower output and quality may be affected by the 

addition of synthetic fertilizer and organic manure (ENEBE; BABALOLA, 2018). The 

availability of crucial micronutrients like potassium boosts crop output and increases a crop's 

resistance to salinity and other environmental stresses.  

Sunflower crop yields fluctuate every year based on the climate and market prices. 

Sunflower plants thrive in a variety of soil types, making them a competitive alternative to 

other cereal grains like maize, sorghum, or cowpea. Sunflowers grow most vigorously in hot, 

dry climates compared to other cereal grains. The plant's long, deep taproot system conserves 

moisture, allowing it to recover from moisture loss quickly and to survive in stressful 

situations such as salinity and drought (HUSSAIN, Mubshar et al., 2018). Sunflower is a 

profitable and promising agricultural crop with numerous advantages for enhancing valuable 



15 
 

 
 

market goods, serving as a source of revenue, and reducing poverty (ERYILMAZ et al., 

2016). 

Sunflower oil is generally classified as a non-volatile obtained during the processing 

of sunflower seeds. It is frequently used as the main component in the preparation of food 

such as frying, and creation of cosmetics (emollient) (LAI et al., 2017). Sunflower oil mostly 

consists of linoleic acid (polyunsaturated lipid) and oleic acid (mono-saturated). The 

difference in the fatty acid and composition of sunflower oil may be caused by the type of 

plant used and the processing method used to produce it (AVNI et al., 2016). 

The oil is known for its intriguing flavour and light amber colour. Vitamins and 

minerals are abundant in the oil. To meet consumer demand for high quality oil, the 

production and commercialization of sunflower oil can be improved with the use of modern 

technology (AVNI et al., 2016). The plants may thrive in colder or drier climates where other 

non-oilseed crops cannot because they are hardy and drought-resistant. Sunflower seeds are 

typically revealed in the apical section of the plant once they mature (ISLAM, R. T. et al., 

2016) 

Sunflower kernel which makes about 80% of the achene’s total weight and covers the 

seeds, is enclosed within lignin and cellulolytic material shell. Raw sunflower seeds typically 

contain 25% oil, nut plant breeding has improved that to 40%. Cold extraction and hot 

pressing are two methods for obtaining the seed oil. Sunflower seeds are consumed by birds 

as they mature in the open fields because of the protein and lipids they contain (ISLAM, R. T. 

et al., 2016).  

The need to supply the growing worldwide population, which is expanding at a pace 

of about 1.05% per year, with food is a significant problem for world agriculture (MODI, 

2019). Seed storage and abiotic stressors have a significant negative impact on plant 

development, productivity, yield, and food quality (SINGH et al., 2018; WANG, Yanhong et 

al., 2017). Drought, salinity, temperature, and excessive rainfall are some of the abiotic 

stressors. Soil salinization is the most harmful of all abiotic stresses (DALIAKOPOULOS et 

al., 2016), and it is regarded as one of the major impediments to agricultural output and food 

security. 

The two main factors that induce seed deterioration as a result of seed storage are a 

high moisture content and a high temperature during storage (KIBINZA et al., 2006). Seeds' 



16 
 

 
 

stored lipids degrade mostly as a result of (i) oxidation brought on by high temperatures and 

moisture content, (ii) hydrolysis, where fat is broken down into fatty acids, and (iii) 

contamination (ABDELLAH; ISHAG, 2012). One of the main variables contributing to seed 

degeneration is lipid peroxidation, according to numerous reports. Reactive oxygen species 

(ROS), a class of chemicals, are a significant source of these harmful alterations (ROACH et 

al., 2010). The seed deterioration is one of the challenges of food production.  

Another important challenge of crop production around the world is the salinization. 

While the main cause of salinization of agricultural land is the buildup of salts in the soil 

(BHARTI et al., 2016; WANG, Yanhong et al., 2017), especially sodium (Na+) and chloride 

(Cl-) ions. Salinity has the biggest negative impact on the productivity and quality of crops by 

modifying physiological processes as well as plant biochemical processes. High Na+ buildup 

restricts soil porosity, aeration, and water conductivity, and the microbial diversity within and 

around plant roots is significantly impacted by soil salinity stress. Additionally, the rate of 

photosynthesis, stomatal conductance, and enzyme activity in seedlings and plants are all 

impacted by excessive salt concentrations (KUMAR et al., 2017).  

Salinity stress and seed deterioration as a result of storage can both increase the 

likelihood of oxidative damage by reactive oxygen species formation (ROS), can cause harm 

to nucleic acids (DNA, RNA), proteins, lipids, and cell membranes, and may potentially 

result in programmed cell death. Due to the excessive accumulation of Na+ and Cl- ions, 

salinity also causes hypertonic stress (ZHANG et al., 2018). Salinity slows down the 

commencement of seed germination, lowers seedling development and dispersal of 

germination events, and decreases seedling metabolism, all of which harm plant growth and 

crop yield (SABAGH et al., 2019).  

During salinity, the negative effect on most plants is apparent in the early stages of 

plant development, especially during seed germination and seedling establishment, as both 

stages are the most responsive and critical stages of plant development (HUSSAIN, S. et al., 

2019). Osmotic stress, ionic imbalance, and oxidative stress are the three main causes that 

reduce crop production when there is salinity stress.  

In plants lifecycle, seed germination begins with the imbibition of water is a dynamic 

and crucial stage. Seed germination commonly implies three distinct phases; consisting of 

Phase I: seed hydration process associated with passive imbibition of dry tissues associated 



17 
 

 
 

with water movement first occurring in the apoplastic spaces; Phase II: activation phase 

associated with the re-establishment of metabolic activities and repairing processes at the cell 

level; and Phase III: initiation of growing processes associated with cell elongation and 

leading to radical protrusion. Phases I and III both include an increase in water content, 

whereas Phase II maintains hydration. Germination is usually thought to be a reversible 

process before the completion of phase II (BEWLEY; BLACK, 2013). 

Salinity reduces seed germination either by osmotic pressure or stress that hinders 

water absorption or results in ionic toxicity.  These consequences as a whole prevent cell 

division and growth, and also modify the activity of some important enzymes; thus, 

decreasing the use of the seed reserves (EL-HENDAWY et al., 2019), therefore salinity has a 

detrimental impact on the process of germination by modifying the typical germination 

pathway that diminished plant development and growth, finally led to a fall in economic 

return.  

Additionally, in whole plants, excessive salt reduces the photosynthetic efficiency, 

leaf area, and chlorophyll content of the leaves. During salinity stress, the activity of 

photosystem II (PSII), a crucial site of the electron transport chain (ETC), is inhibited 

(KALAJI et al., 2011; MEHTA et al., 2010). Salinity stress has an impact on several cellular 

enzymes, including RNAse, DNAse, proteases, and enzymes involved in nitrogen 

metabolism and amino acid synthesis (SIDDIQUI et al., 2008). The accumulation of ROS, 

such as singlet oxygen, superoxide radicals, and H2O2, is also indirectly triggered by salinity 

stress. Under salinity stress conditions, the ETC in the chloroplast and the mitochondria are 

the primary sources of ROS generation (GILL; TUTEJA, 2010). 

There are several treatments that mitigate salt effect in seed germination and early 

growth. We can consider seed priming as one of them. Seed priming is the partial hydration 

of seeds until phase II and the attainment of appropriate metabolic activity that enables the 

seeds to commence crucial pre-germination processes. Phase II displays a steady level of 

hydration. Up until phase II, germination is still reversible allowing the seeds to be dried out 

once more, stored in viable state, and re-start germination in a suitable environment 

(FAROOQ et al., 2007). The primary alterations that occur during seed priming include 

membrane repair, DNA and RNA synthesis and repair, immature embryo growth, alterations 

to the endosperm tissues that surround the embryo, dormancy breakage, and enrichment of 

pre-germination metabolism (FAROOQ et al., 2007). Primed seeds have a higher germination 



18 
 

 
 

rate, early germination, uniformity in germination, superior growth qualities, faster 

emergence, and better stand establishment.  

These enzymes are essential for breaking down macromolecules for the embryo's 

growth and development, which eventually leads to quicker and more vigorous seedling 

emergence (FAROOQ, et al., 2007). According to some research, priming makes it easier to 

repair chromosomal damage (SIVRITEPE; DOURADO, 1995), boosts RNA and de novo 

protein synthesis, enables early DNA replication and repair, and decreases metabolite leakage 

(BLACK; BEWLEY, 2000). Thus, seed priming significantly increased total seed protein, 

peroxidases, polyphenol oxidases, RNA, and de novo protein synthesis. 

It is hypothesized that germination-related processes like respiration, energy 

metabolism, and mobilization of the initial reserve also take place during priming. To cover 

the energy pool and hasten germination, greater respiratory activity is needed. There has been 

a link between pre-sowing treatments and increased respiratory activity (FORTI et al., 2020). 

Seed priming raised the standard deviation of germination responses, decreased the oxygen 

time constant, and enhanced the respiratory activity of seeds (FORTI et al., 2020). Storage 

proteins must be activated during seed germination to support seedling growth since they 

provide a source of reduced nitrogen and inorganic minerals (LUTTS et al., 2016). 

A crucial component of the physiology of primed seeds is the control of oxidative 

state. The antioxidant response is triggered by priming, and the seeds are modified for 

potential stressors like salinity (KIBINZA et al., 2011). Reactive oxygen species (ROS) are 

largely produced during the initial phases of seed ingestion and germination because of the 

respiratory activity of mitochondria, enzymes like NADPH oxidase, extracellular peroxidase, 

and oxalate oxidase, as well as activities in the β-oxidative pathway (JIN et al., 2016).  

Antioxidants are crucial for effective germination because they reduce excessive ROS 

during early endocytosis, especially in stressful situations like salinity. Seed priming in 

tomato seeds exhibited increased activity of antioxidant enzymes such as ascorbate 

peroxidase, catalase, peroxidase, glutathione reductase, and superoxide dismutase 

(COOLBEAR; SLATER; BRYANT, 1990).  

Halopriming has been shown to increase watermelon seed germination and seedling 

vigor (DEMIR; MAVI, 2004), rice (FAROOQ et al., 2006), maize (KUMARI et al., 2017), 

many other crops. Seed priming with various inorganic salts, including NaCl, KCl, and 



19 
 

 
 

CaCl2, has a favorable effect on pepper germination when exposed to salinity stress by 

accelerating imbibition and enabling the seeds to perform metabolic processes more quickly 

(ALOUI et al., 2014). When primed with KCl and NaCl, the salt-sensitive and salt-tolerant 

wheat genotypes increased germination %, stimulated physiological traits like chlorophyll 

concentration, and decreased mean germination time (SADDIQ et al., 2019). Sesame seeds 

(Sesamum indicum L.) primed with CaCl2 boosted germination by up to 88.75% and 

improved vigor and yield (TIZAZU et al., 2019).  

Sesame seeds and Vigna mungo L. were found to have an enhanced salt tolerance 

index (STI) based on growth factors such as shoot length, root length, and relative water 

content in NaCl primed seeds(BISWAS; SAHA, 2021; MOHAMMADI et al., 2013). When 

tomato (Solanum lycopersicum cv. 'Ro Grande) and fenugreek seeds (Trigonella foenum-

greacum) were primed with NaCl, the harmful effects of salt stress were greatly reduced 

(GONZÁLEZ-GRANDE; SUÁREZ; MARÍN, 2020; MOHAMMADI et al., 2013).  

After halopriming, CAT, POD, and APX antioxidant enzyme activities of the salt-

tolerant Lu26s and salt-sensitive Lasani-06 wheat (Triticum aestivum L.) cultivars 

considerably increased (ISLAM, F. et al., 2015). The antioxidant activity in chicory was 

considerably boosted by halopriming with KCl (SADEGHI; ROBATI, 2015). Halopriming 

significantly raised the SOD, CAT, APX, and GPOX (guaiacol peroxidase) activities in 

primed seeds compared to non-primed seeds of various Oryza sativa cultivars (SEN; 

PUTHUR, 2021). Therefore, the use of priming as seed treatment technique, can improve 

seed germination and all physiological related metabolism even in seeds or in seedlings. 

  



20 
 

 
 

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Article 1: Seed halopriming before storage help maintain seed quality and improve 

germination of sunflower 

 

Manuscript in the journal's rules Journal of Seed Science (Impact factor: 1,4) 

  



29 
 

 
 

SEED HALOPRIMING BEFORE STORAGE HELP MAINTAIN SEED QUALITY 

AND IMPROVE GERMINATION OF SUNFLOWER 

ABSTRACT: Sunflower (Helianthus annuus L.) is an important oil seed crop mainly due to 

its high oil content, which is consumed by humans in different ways. However, because of 

the high lipid content in sunflower, the seeds are more liable to reduce their quality during 

short-term storage because of oxidation due to temperature changes or moisture content. We 

aimed to evaluate the effect of seed halopriming on the germination, reactive oxygen species, 

and antioxidant enzymes of sunflower hybrid seeds during storage (0, 7, 14, and 30 days). 

Priming improved germination by enhancing germination percentage (G%), germination 

speed index (GSI), and time taken for 50 percent germination (T50%), even in stored seeds. 

In non-primed seeds, storage time decreased all these parameters. Primed seeds had a lower 

hydrogen peroxide content than non-primed seeds at all storage durations. However, 

increased levels of the lipid peroxidation marker (MDA) were observed in primed seeds at 14 

and 30 days of storage, even though there was no signal of oxidative stress. The superoxide 

dismutase enzyme activity was higher in control seeds than in primed seeds at 0 and 7 days of 

storage, while at 14 and 30 days, the activity was the same between control and primed seeds. 

Catalase and ascorbate peroxidase enzyme activities were higher in the control seeds than in 

the primed seeds across all storage durations. Therefore, it can be concluded that: (I) 

sunflower seeds can be sensitive to short-term storage, and halopriming can increase seed 

germination parameters even in stored seeds; (II) halopriming maintained controlled levels of 

reactive oxygen species, and anticipated lipid storage reserve mobilization with respect to 

MDA levels. 

Keywords: Seed germination, oxidative damage, seed storage, oilseed, antioxidant enzymes  

  



30 
 

 
 

1. INTRODUCTION  

 Sunflower (Helianthus annuus L) is an oilseed crop that belongs to the family 

Asteraceae and is indigenous to North America. It is grown all over the world (YEGOROV et 

al., 2019) because it can acclimate to various climatic and soil conditions. This has improved 

its cultivation as an oilseed plant worldwide (FORLEO et al., 2018). The cultivation of these 

species is considered to have a short life cycle with extensive acclimatization to variations in 

altitude and photoperiod, in addition to being resistant to cold and high temperatures 

(CARVALHO et al., 2016). However, there is an extended duration of storage during which 

seed quality deteriorates before the seeds are utilized for germination or oil extraction. 

Therefore, it is essential to conduct comprehensive examinations of all changes that occur 

during storage (ARC et al., 2011). 

Seed storage typically causes seed aging, which manifests as permanent cellular and 

metabolic alterations such as the oxidation of proteins, nucleic acids, and lipids. Enzyme 

inactivation, membrane disruption, and impairment of RNA and its production are further 

effects of seed aging (EL-MAAROUF-BOUTEAU et al., 2011; HU et al., 2012; KIBINZA et 

al., 2006). Given that oilseeds are more prone to seed deterioration than cereal grains, storage 

of oilseeds poses a major challenge. Seeds' stored lipids degrade mostly as a result of (i) 

oxidation brought on by high temperatures and moisture content, (ii) hydrolysis, where fat is 

broken down into fatty acids, and (iii) contamination (ABDELLAH; ISHAG, 2012). One of 

the main variables contributing to seed degeneration is lipid peroxidation, according to 

numerous reports. Reactive oxygen species (ROS), a class of byproducts, are a significant 

source of these harmful alterations (ROACH et al., 2010). 

Antioxidants found in plants act as a line of defense against dangerous chemicals.  

Numerous enzymes in the enzymatic antioxidant system regulate the amount of ROS and 

antioxidants in the body, removing any excess ROS. Superoxide dismutase (SOD), catalase 

(CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) are a few of the key 

antioxidant enzymes in plants that scavenge free radicals that are harmful to seed viability, 

according to research by (BAILLY, 2004). These antioxidant enzymes become compromised 

by prolonged seed storage or synthetic aging. To minimize the negative effect of abiotic 

stresses caused by storage on seeds, methods like seed priming have been adopted.  

Seed priming enhances germination processes including defense, which give seeds the 

ability to withstand environmental challenges that arise during germination (FAROOQ et al., 



31 
 

 
 

2006, 2009). In primed seed, pre-germination metabolic processes include structural 

(membrane protection during imbibition) and genetic repair, RNA and protein synthesis, and 

antioxidant mechanisms (SADDIQ et al., 2019). These processes guarantee appropriate seed 

germination and seedling development (SADDIQ et al., 2019). To stimulate the seeds and 

reduce environmental stress that can be caused due to seed storage, numerous kinds of seed 

priming techniques have been created and are currently being used successfully, these include 

hydropriming, halopriming, osmopriming, biopriming, hormonal priming, solid matrix 

priming, etc. with our main focus on halopriming technique (VENKATASUBRAMANIAN; 

UMARANI, 2007).  

Halopriming is imbibing seeds in salt solutions that are not organic, like NaCl or KCI. 

Halopriming encourages uniform germination and higher crop production even in 

unfavorable environmental conditions including temperature extremes and oxygen 

deprivation. Halopriming is an easy and affordable priming method that ensures better 

emergence and crop stand synchronization under a variety of environmental 

situations(KUMARI et al., 2017).  

There is still a wide gap to be filled regarding the use of halopriming technique to 

mitigate the effect of abiotic stresses as a result of storage on the germination of sunflower 

seeds. The main objective of this study is to investigate the effect of seed halopriming on 

seed germination of sunflowers seeds after storage. For this, we hypothesis that halopriming 

procedure will improve the seed germination of sunflowers after storage conditions by 

enhancing the activities of enzymatic and non-enzymatic antioxidants. 

  



32 
 

 
 

2. MATERIALS AND METHODS 

2.1 Plant Material 

 The experiment and assays were performed at the Plant Growth and Development Lab 

and the Seed Central Lab at the Federal University of Lavras (UFLA), located in the state of 

Minas Gerais. Seeds were obtained commercially for the experiment. The sunflower seeds 

used were hybrid species produced in 2021 by a company named Sakata, Brazil.  

2.2 Priming Procedure 

 For the imbibition curve, 20 seeds per replicate (n = 4) were used. The seeds were 

placed in germination boxes with paper moistened with water. The seed mass was weighed 

every hour to determine phases I and II of imbibition.  

For the dry curve, the seeds were washed with distilled water and placed in an oven at 27 °C 

to dry back to their initial mass, which was determined prior to priming. The seeds took 

approximately 4 hours to dry back to their initial masses. Seeds were primed with 50 mM 

NaCl (BISWAS; SAHA, 2021).  

2.3 Storage Conditions 

 After priming, both non-primed and primed seeds were divided into four groups and 

stored for different periods (0, 7, 14, and 30 days). Seeds were placed into paper bags, sealed 

with a stapler, and labelled according to each storage period. Each paper bag was placed in 

polyethylene bags, and the bags were kept in a refrigerator (5-8oC). Non-primed seeds were 

used for the germination experiment immediately after the priming procedure. 

2.4 Germination Parameters  

 Seeds were placed in a gearbox containing germination paper that had been moistened 

with sterile water. Afterwards, seeds were kept in a germination chamber at 25 °C with a 12 

hours photoperiod. Seeds were considered germinated at 2mm radicle protrusion and 

germination was checked daily for 7 days to reach a constant count and result was expressed 

as percentage (germination %) based on seed analysis rules (BRASIL, 2009) 

 The germination speed index (GSI) was determined according to (SCOTT; JONES; 

WILLIAMS, 1984): GSI = ∑TiNi/S. The time for 50% germination (T50%) was also 



33 
 

 
 

calculated according to ((SCOTT; JONES; WILLIAMS, 1984): T50% is given by the 

following formula; 

T50 = ti + [(N / 2) − − ni] (tj – ti)/ (nj – ni) 

2.5 Sampling  

 For the biochemical analysis, the seeds were collected after 7 hours of which marked 

the beginning of phase II of germination. After collection, seeds were macerated using liquid 

nitrogen and prepared for biochemical analysis.  

2.6 Biochemical Analysis: 

2.6.1 Hydrogen Peroxide (H2O2) 

The levels of H2O2 were measured according to (VELIKOVA; YORDANOV; 

EDREVA, 2000), in which 100mg of the seeds sample was grinded in liquid nitrogen and 

then homogenized with 1 mL of 0.1% Trichloroacetic Acid (TCA). After centrifuging the 

samples, the reactions were carried out with 10 mM potassium phosphate buffer pH 7 and 1M 

potassium iodide (KI). The samples were read using a spectrophotometer at 390 nm, and a 

standard curve were used to calculate the quantities of H2O2 in the samples. 

2.6.2 Lipid Peroxidation 

 Lipid peroxidation estimates was performed by quantifying Malondialdehyde (MDA) 

as described by (DU; BRAMLAGE, 1992), 100mg of the seed sample was grinded in liquid 

nitrogen and then homogenized with 1 mL of 80% ethanol ( three times). The reaction was 

carried out using 1 mL of the extraction and 1 mL of % TCA, 0.65 % thiobarbituric acid 

(TBA), and 0.1 percent beta-hydroxytoluene (BHT). At wavelengths of 600, 440, and 532 

nm, a spectrophotometer was used to measure the estimation of lipid peroxidation. 

2.6.3 Antioxidant Enzymatic System Analysis 

 The enzymatic antioxidant system activity (SOD, CAT, and APX) were quantified. 

The extracts performed according to BIEMELT; KEETMAN; ALBRECHT, (1998) in which 

100mg of seed sample was grinded in liquid nitrogen with PVPP (Polyvinylpyrrolidone) 

added to 1.5 mL of extraction buffer composed of 400 mM potassium phosphate (pH 7.8), 10 



34 
 

 
 

mM EDTA, and 200 mM ascorbic acid. The solution was centrifuged at 13.000g for 10 

minutes at 4°C and the supernatant collected to quantify enzyme activities. 

 SOD activity was based on the enzyme capacity of inhibiting the photoreduction of 

nitro blue tetrazolium (NBT) (GIANNOPOLITIS; RIES, 1977). Absorbance readings was 

made at 560 nm. A unity of SOD corresponds to the number of enzymes able to inhibit 50% 

of NBT photoreduction in the assay conditions.  

 HAVIR; MCHALE, (1987) findings were used to determine the CAT activity. Based 

on the hydrogen peroxide consumption and the decrease in absorbance at 240nm, every 15 

seconds for 3 minutes, this enzyme activity was estimated. We assumed a molar extinction 

coefficient of 36 mM-1 cm-1 (AZEVEDO et al., 1998). According to NAKANO; ASADA, 

(1981), APX activity was examined using ascorbate oxidation at 290 nm and a molar 

extinction coefficient of 2.8 mM-1 cm-1. 

2.7 Statistical Analysis  

The statistical analysis were performed with the software R-Bio (BHERING, 2017). 

Data was subjected to two-way ANOVA and, in case of normal distribution, Tukey means 

test was selected at 5% significance. Data without a normal distribution was evaluated by a 

GLM analysis, assuming normality by observation of the qq- plot graphs and then applying 

the Tukey test at 5% of significance. 

  



35 
 

 
 

3. RESULTS AND DISUSSIONS 

3.1 Effect of Priming on Germination of Sunflower seeds  

 For the germination results, non-primed seeds at zero and 7 days of storage were 

more synchronized, uniform, and had better germination percentage, while there were delays 

and reductions in the number of germinated seeds at 14 and 30 days of storage (Fig. 1A). 

Primed seeds from 14 and 30 days of storage presented a delay in germination in the first 24 -

72 hours, but they reached the same germination percentage as primed seeds d from zero and 

7 days of storage.  

    Fig. 1. Cumulative germination of control seeds (A) and (B) in primed seeds. Bars are means ± standard error 

(n = 4).  

 

  



36 
 

 
 

In general, priming increased seed germination, speed index, and time to reach 50% 

germination, even in stored seeds compared to non-primed seeds (Fig. 2). The results showed 

that the overall seed germination percentage (G%) in primed seeds did not differ with 

increasing storage time when compared to control seeds, which had a reduction in overall 

germination percentage with increasing storage time. Primed seeds had a better overall 

germination percentage than the control (non-primed) seeds at each storage time (Fig. 2A).  

The germination speed index (GSI) was significantly different between treatments and 

storage times. Seeds with no storage had the highest germination speed index (GSI). GSI 

decreased with increasing storage time for both primed and non-primed seeds.  

 The time taken for 50 percent of germination to occur (T50%) differed between 

primed and non-primed seeds. There was no difference in the T50% of primed seeds with 

increasing storage time. At zero and 7 days of storage, non-primed seeds showed a decrease 

in T50% compared to 14 and 30 days of storage. Primed seeds had a lower T50% than non-

primed seeds during each storage period. The results G%, GSI and T50% illustrate 

physiological effect of priming was more evident in GSI as priming reduces the time for 

imbibition, hasten germination and enhance respiratory activity of seeds (FORTI et al., 2020) 

Fig. 2 Germination percentage (A); germination speed index (B); and time taken for fifty percent germination 

(C) of seeds of Helianthus annuus (control and primed). Bars are means ± standard error (n = 4). Capital letters 

compare differences among storage time and lower letters compare differences between treatments (control and 

primed). The same letters or ns (not significant) do not show any significance difference by Tukey’s test at 5% 

significance.



37 
 

 
 

 3.2 Effect of Priming on Viability of Sunflower Seeds 

 The results showed that the viability of sunflower seeds was maintained in both 

control and primed seeds at zero and seven days of storage (Fig. 3). At 14 and 30 days, the 

viability of sunflower seeds was reduced by 12% and 22% in control seeds and by 6% and 

11% in primed seeds, respectively. The reduction in seed viability observed at 14 and 30 days 

of storage was due to the storage effect and not priming. Primed seeds also had almost 100% 

germination after 30 days of storage, which shows that priming increased the germination 

capacity of sunflower seeds (ZULFIQAR, 2021). 

Fig. 3 Viability of seeds of Helianthus annuus (control and primed). Bars are means ± standard error (n = 4). 

Capital letters compare differences among storage time and lower letters compare differences between 

treatments (control and primed). The same letters or ns (not significant) do not show any significance difference 

by Tukey’s test at 5% significance. 

  



38 
 

 
 

3.4 Effect of Priming on Oxidative stress of Sunflower Seeds 

Lipid peroxidation (MDA) and hydrogen peroxide levels showed no evidence of 

oxidative stress as a result of seed storage (Fig. 4). The results indicated that the 

concentration of MDA in primed seeds was lower than that in non-primed seeds at zero and 

seven days of storage (Fig. 4A). At 14 and 30 days of storage, primed seeds had a higher 

concentration of MDA than  non-primed seeds. This MDA increase is probably due to the 

process acceleration of lipid storage mobilization that was meant to occur during phase III of 

germination and was brought forward to phase II. This possibility has been previously 

discussed in an oil seed species in which increasing levels of MDA followed imbibed and 

early germinated seeds (BICALHO et al., 2015).  

Fig. 4 Estimated levels of lipid peroxidation (A); and hydrogen peroxide (B) of Helianthus annuus seeds. Bars 

are means ± standard error (n = 4). Capital letters compare differences among storage time and lower letters 

compare differences between treatments (control and primed). The same letters or ns (not significant) do not 

show any significance difference by Tukey’s test at 5% significance.  

 We may conclude there was no evidence of oxidative stress in the seeds, as the level 

of H2O2 was relatively controlled by both primed and non-primed seeds. The result for 

hydrogen peroxide (H2O2) differed from that of MDA. Primed seeds had a lower H2O2 

concentration than non-primed seeds at all four storage times. The results show the 

importance of priming, as priming of seeds helps to control the formation of reactive oxygen 



39 
 

 
 

species (RAHIMI, 2013). The low level of H2O2 in primed seeds also explains why primed 

seeds had better G%, GSI, and T50 values than non-primed seeds (Fig. 4B). 

3.5 Effect of Priming on the Enzyme activity of the Antioxidant System of Sunflower 

Seeds 

 Among the specific activities of the enzymes of the antioxidant system, it was 

observed that superoxide-dismutase (SOD) had higher enzymatic activity in non-primed 

seeds than in primed seeds at zero and 7 days of storage. The results showed a different 

dimension at 14 days of storage, where SOD activity was higher in primed seeds than in non-

primed seeds (Fig. 5A) For the enzymatic activity of catalase (CAT) and ascorbate 

peroxidase (APX), the results illustrated that non-primed seeds had higher CAT and APX 

activity than primed seeds at all four storage times (Fig. 5B&C).  

 The difference in SOD enzymatic activity between primed and non-primed seeds at 

zero and 7days storage could be a result of an increase in superoxide anion (O2
.-) oxidation in 

primed seeds. The difference at 14 days may be due to the higher catalysis of superoxide 

anions (O2
.-) by SOD (WANG Ying et al., 2018). The increase in CAT and APX in non-

primed seeds shows there was no evidence of oxidative stress and may also indicate non-

primed seeds regulated the level of H2O2 and this process involves the conversion of H2O2 

by CAT and APX into water (WANG, Ying et al., 2018) 



40 
 

 
 

Fig. 5 Specific superoxide dismutase activities, SOD (A); catalase, CAT (B) and ascorbate peroxidase, APX (C) 

in control and primed seeds of Helianthus annuus. Bars are means ± standard error (n = 4). Capital letters 

compare differences among storage time and lower letters compare differences between treatments (control and 

primed). The same letters or ns (not significant) do not show any significance difference by Tukey’s test at 5% 

significance.  

 

4. CONCLUSIONS 

 This study demonstrated that haloprimed sunflower seeds had a better germination 

performance when compared to non-primed seeds. Furthermore, it was also observed that the 

side effect of storage was more evident in control seeds with increasing storage time than in 

primed seeds.  

Short-term storage also reduced the viability of sunflower seeds at 14 and 30 days in 

both treatments, but halopriming helped improve germination capacity. 

The results indicated that physiological sunflower seeds were able to tolerate the 

negative effects of short-term storage. We may conclude that there was no evidence of 

oxidative damage in either the control or primed seeds.  

Enzymatic activities were also observed to be high in the control seeds, which also 

proved that the level of oxidative stress was relatively controlled by the seeds, especially in 

primed seeds.  

With more studies, halopriming technique can be used to improve the germination 

rate of sunflower seeds after short-term storage, as it was evident in this study.  

Farmers and consumers can also adopt halopriming of sunflower seeds as it is a 

simple, cost-effective, and efficient technique.  

  



41 
 

 
 

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Article 2: Seed halopriming improves seedling growth of sunflower under saline 

conditions 

Manuscript in the journal's rules Brazilian Journal of Botany (Impact factor: 1,36) 

  



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SEED HALOPRIMING IMPROVES SEEDLING GROWTH OF SUNFLOWER 

UNDER SALINE CONDITIONS 

ABSTRACT: The maximum production of sunflowers (Helianthus annuus L.) can be 

limited by salinity stress by reducing germination, early seedling growth, and overall quality. 

In this study, we evaluated the effects of seed halopriming on seedling growth, pigments, 

antioxidant enzymes, oxidative stress, osmolytes, proteins, and sugars in sunflower hybrids 

under saline conditions (0mM, 60mM, 120mM and 240mM). Seedlings from primed seeds 

had higher enzymatic activity of superoxide dismutase (SOD) and ascorbate peroxidase 

(APX) and higher protein and chlorophyll a content than the control at higher salinity 

concentrations. Priming also improved root length and root dry matter at higher salinity 

concentrations than in the control. The reducing sugar concentration was also improved by 

priming at higher salinity concentrations than the control.  It can be concluded that priming 

technique is useful for increasing salinity tolerance in sunflower seedlings by the recruitment 

or permanency of some osmolytes, such as soluble sugars, and is capable of increasing 

antioxidant capacity by enhancing SOD and APX activity under high salinity.  

Keyword: Helianthus annuus, salinity, halopriming, enzymatic antioxidant system, osmolyte 

  



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1. INTRODUCTION   

Sunflower (Helianthus annus) is one of the most important oilseed crops grown 

worldwide because it provides high-quality oil and dietary fiber that significantly improves 

human health (KHAN et al., 2015). Several abiotic stressors, such as salinity, cold, drought, 

flood, and high temperature, have a significant impact on global agricultural productivity 

(SHRIVASTAVA; KUMAR, 2015). Commonly, due to the accumulation of soluble salts in 

the soil, salinization of agricultural land occurs (BHARTI et al., 2016), especially the sodium 

(Na+) and chloride (Cl-) ions. As a result of higher Na+ ion assimilation and a decrease in the 

Na+/K+ ratio as a result of decreased osmotic potential in plant roots, salinity stress results in 

osmotic stress and ion toxicity (ARIF et al., 2020).  

During the early stages of salinity effects, an osmotic imbalance causes roots' capacity 

to absorb water to drop and their transpiration rate to increase, which results in hyperosmotic 

stress (MUNNS, 2005; YANG; GUO, 2018). Additionally, these ionic imbalances interfere 

with the uptake and transport of other important and essential ions in target cells, which 

negatively impacts crucial plant processes and activities (ARIF et al., 2020). Salinity also 

damages the photosynthetic system, the cell's ultrastructure, and the membrane structure 

(HASANUZZAMAN et al., 2014) by increasing the production of reactive oxygen species 

(ROS), which oxidative stress enables to be harmful to cell membranes, proteins, lipids, and 

nucleic acids (DNA, RNA). 

Several techniques have been used to decrease the detrimental impact of salt and other 

abiotic stressors on crop productivity. Seed priming is the process of hydrating seeds until 

phase II, drying before radicle emergence for sowing them. Seed priming allows pre-

germinative physiological and biochemical changes to take place and improve germinative 

parameters (HUSSAIN, M; FAROOQ; LEE, 2017). Among seed priming, the halopriming is 

imbibing seeds in salt solutions that are not organic, like NaCl or KCI. Halopriming promotes 

uniform germination and higher crop production even in unfavorable environmental 

conditions including temperature extremes and oxygen deprivation. Halopriming is an easy 

and affordable priming method that ensures better emergence and crop stand synchronization 

under a variety of environmental situations (KUMARI et al., 2017).  

There is still a wide gap to be filled regarding the use of halopriming technique to 

mitigate the negative effect of salinity on seed germination and seedling growth of 

sunflowers. The objective of this study is to investigate the effect of seed halopriming on 



54 
 

 
 

seedling growth of sunflowers under saline conditions. For this, we hypothesize that 

halopriming procedure will improve the seedling growth of sunflower under saline conditions 

by enhancing the activities of enzymatic and non-enzymatic antioxidant.  

 

2. MATERIALS AND METHODS  

2.1 Plant material and Priming Procedure 

 The experiment and assays were performed at the Plant Growth and Development Lab 

at the Federal University of Lavras (UFLA) located in the state of Minas Gerais. The seeds of 

sunflower (Helianthus annus L.), obtained commercially (Sakata, Brazil), were used for the 

experiment. The seeds were imbibed for 7 hours (phase II of the imbibition curve) in 50mM 

of NaCl according to (BISWAS; SAHA, 2021) and dried in an oven at 27°C until they 

returned to their initial mass, which was determined prior to priming. 20 seeds in four 

replicates were used for this procedure. After the priming procedure, the seeds were sowed in 

7 L pots containing oxisoil for greenhouse experiments.   

2.2 Preparation of Salt Concentrations and Soil Incubation 

 Thirty-two pots (7 L) were used, with 16 pots for control seeds and 16 pots for primed 

seeds. The pots were transferred to a greenhouse after they were filled with oxisoil. NaCl was 

used to prepare four concentrations - 0mM, 60mM, 120mM and 240mM - (KAIWEN et al., 

2020) of salinity that were used for the experiment. Salt solutions were added to pots 

containing soil, and each concentration of salinity had four replicates. The soil was left for a 

period of twenty-one days to allow for proper incubation of the salts into the soil and to avoid 

damage to the seeds during sowing (YAN et al., 2015) 

2.3 Experimental Conditions 

 After twenty-one days of soil incubation, five primed seeds or control seeds were 

sown in each pot. The experiment consisted of a factorial 2x4, of two treatments (control and 

primed) seeds and four concentrations of salinity (0mM, 60mM, 120mM and 240mM). 

Irrigation was performed regularly, and seedlings were thinned into three seedlings per pot 

after 14 days of sowing.  



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2.4 Sampling  

 The Seedlings were collected after the development of the first four expanded leaves, 

which took approximately twenty-nine days after sowing. Seedling roots were washed to 

remove the soil. One seedling from each replicate was used to determine growth parameters, 

such as shoot length, root length, leaf area, and dry mass. The other two seedlings were 

macerated using liquid nitrogen and prepared for biochemical analysis.  

2.5 Growth Parameters 

 The seedlings were separated into roots and shoots. The dry matter content of the 

shoots and roots was calculated using the formula by (SHIPLEY; VU, 2002). Image-J 

software (SCHNEIDER; RASBAND; ELICEIRI, 2012) was used to determine the leaf area 

and plant height (shoot and root).  

2.6 Biochemical Analysis: 

2.6.1 Hydrogen Peroxide (H2O2) 

The levels of H2O2 was measured according (VELIKOVA; YORDANOV; EDREVA, 

2000) approach. In brief, 100mg of the seedling sample was grinded in liquid nitrogen and 

then homogenized with 1 mL of 0.1% Trichloroacetic Acid (TCA). After centrifuging the 

samples, the reactions were carried out with 10 mM potassium phosphate buffer pH 7 and 1M 

potassium iodide (KI). The samples were read using a spectrophotometer at 390 nm, and a 

standard curve were used to calculate the quantities of H2O2 in the samples. 

2.6.2 Extension of Lipid Peroxidation (MDA content) 

 Lipid peroxidation was determined by malondialdehyde (MDA) quantification 

(HODGES et al., 1999). Samples of 100mg of seedlings were grinded in liquid nitrogen and 

homogenized