adaptability and yield potential of quinoa on salt affected...
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Adaptability and Yield Potential of Quinoa on Salt Affected Soils
By Shahid Iqbal
M.Sc. (Hons.) Agronomy 2006-ag-1903
A thesis submitted in partial fulfillment of the requirement for the degree of
Doctor of Philosophy in
Crop Physiology
DEPARTMENT OF AGRONOMY, FACULTY OF AGRICULTURE,
UNIVERSITY OF AGRICULTURE, FAISALABAD (PAKISTAN)
2017
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Declaration
I hereby declare that the contents of the thesis, “Adaptability and Yield potential of
Quinoa on Salt Affected Soils’’ are product of my own research and no part has been
copied from any published source (except the references, standard mathematical and
genetic models/ equations/ formulae/ protocols etc.). I further declare that this work has not
been submitted for award of any other diploma/degree. The University may take action if
information provided is found inaccurate at any stage. (In case of any default the scholar
will be proceeded against per HEC plagiarism policy).
Shahid Iqbal
2006-ag-1903
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Oh, Allah Almighty open our eyes, To see what is beautiful, Our minds to know what is true, Our heart to love what is good.
Dedicated to MY
PARENTS and my great Uncle Ch. Muhammad Akram Sahu (late)
Who provides me resources, opportunities, and a way to get success in all spheres of life
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ACKNOWLDGEMENTS
All worships and praises are only due to the Lord of creation, the most beneficent, merciful and compassionate, Whose blessings and exaltation flourished my thoughts and thrived my ambitions to have the cherish fruit of my modest effort in the form of this manuscript.
I offer my humblest thanks and countless salutations to the Holy Prophet Muhammad (PBUH), who is forever, a torch of guidance for the entire humanity.
I owe my deepest gratitude to my great supervisor Dr. Shahzad M.A Basra, Professor, Department of Agronomy, University of Agriculture Faisalabad, who in spite of his busiest tiring routine work provided his dexterous and valuable suggestions throughout research efforts.
Thanks are extended to the members of my supervisory committee Dr. Irfan Afzal, Associate Professor Department of Agronomy and Dr. Abdul Wahid, Professor, Department of Botany, University of Agriculture Faisalabad for their sincere cooperation and invigorating encouragement during the course of present investigation. I am also very thankful to my dear lab mates Muhamad Sohail Saddiq, Muhammad Bilal Hafeeez, Nauman Ali, Muhammad Amir Bhaktawer, Muhammad Idrees Faisal Muhammad Kamran, Shahbaz Khan, Muhammad Aamir Saleem and Jaam Muhammad Junaid, Department of Agronomy, University of Agriculture Faisalabad for their valuable suggestions and guidance during my research activities and thesis write up.
I can’t forget prayers of my beloved father, mother, and great supportive brothers and sisters for the strenuous efforts done by them in enabling me to join the higher ideals of life and also their financial and moral support, patience and prayers they had made for my success.
Shahid Iqbal
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Contents
Chapter Title Page No 1
Introduction
1
2
Review of literature
4
3
Material and method
29
4
Results and Discussion
45
5
Summary
93
Literature Cited
95
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Table of contents
Sr# Contents Page # Abstract xvi
Chapter 1Introdction 1 Chapter 2 Review of Literature 4
2.1 Global extent of salt-affected soils 4 2.2 Salinity Extent in Pakistan 4 2.3 Reason of increasing salinity or sodicity 5 2.4 Salinity effects on plants 5 2.5 Approaches to make salt-affected soils productive 6 2.6 Quinoa plant description 7 2.7 Quinoa Adaptation out of Latin America 8 2.8 Quinoa performance under salt regimes 8 2.8.1 Seedling establishment: 8 2.8.2 Growth and yield performance: 9 2.9 Salt-tolerance mechanisms 11 2.9.1 Morphological attributes 11 2.9.1.1 Seed Characteristics 11 2.9.1.2 Leaf salt bladders 12 2.9.1.3 Stomata 13 2.10 Na+ uptake and transport 13 2.11 Ion homeostasis and compartmentalization 14 2.12 Gas exchange relations 16 2.13 Compatible solutes, osmoprotectants and antioxidants 17 2.14 Molecular identity of Na+ transporters in quinoa 20 2.15 Salinity induced changes in nutritional profile: 23 2.16 Quinoa world trade 26 Conclusion 28 Chapter 3 Material and methods 29 Experiment 1: Identification of salt tolerant and sensitive quinoa lines. 29 3.1.1 Plant material 29 3.1.2 Raising of quinoa seedlings 29 3.1.3 Shifting of seedlings in soilless culture 32 3.1.4 Salinity Imposition 32 3.1.5 Determination of Na+ and K+ concentrations in leaves and roots 32 3.1.6 Recording of morphological and growth traits. 32 3.1.7 Leaf senescence’s number 33 3.1.8 Statistical analysis 33 Experiment: 2 Physiological performance of quinoa lines under wire house saline
conditions 33
3.2.1 Plant material 33 3.2.2 Imposition of salinity in pots 33 3.2.3 Determination of Na+ and K+ concentrations in leaves and roots 35 3.2.4 Estimation of leaf water-potential 35
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3.2.5 Estimation of leaf osmotic-potential 35 3.2.6 Estimation of leaf turgor-potential 36 3.2.7 Measurement of leaf gas-exchange relations 36 3.2.8 Leaf biochemical analysis 36 3.2.9 Leaf Chlorophyll-contents 36 3.2.10 Leaf total-phenols (mg g-1) 37 3.2.11 Free-proline determinations in leaf 37 3.2.12 Growth and yield determination 38 3.2.13 Statistical analysis 39 Experiment 3: Physiological and Agronomic performance of Quinoa under field
salinity 39
3.3.1 Experimental locations 39 3.3.2 Crop husbandry 39 3.3.3 Estimation of stand establishments 41 3.3.4 Determination of Na+ and K+ contents in leaves and roots 41 3.3.5 Assessment of Leaf water-relations 41 3.3.6 Determination of leaf Chlorophyll-contents 42 3.3.7 Determination of leaf total-phenols (mg g-1) 42 3.3.8 Free-proline determination in leaf 42 3.3.9 Determination of antioxidants 42 3.3.10 Estimation of crop-growth rate 43 3.3.11 Biomass and seed yield related traits determination 43 3.3.12 Seed mineral estimation 44 3.3.13 Seed protein estimation 44 3.3.14 Seed crude-fat estimation 44 3.3.15 Seed crude-fiber estimation 44 3.3.16 Quinoa seed saponin estimation 45 3.3.17 Statistical analysis 45 Chapter 4 Results and Discussion 47 Experiment 1: Identification of salt-tolerant and sensitive quinoa lines 47 4.1.1 Shoot growth 47 4.1.2 Root growth 47 4.1.3 Root Na+, K+ content and K+/Na+ ratio 47 4.1.4 Leaf Na+, K+ content and K+/Na+ ratio 48 4.1.5 Leaf senescence 48 Discussion 54 Experiment 2: Physiological performance of quinoa lines under wire-house saline
conditions 56
4.2.1 Plant growth and yield responses 56 4.2.2 Leaf gas relations 56 4.2.3 Leaf chlorophyll contents 57 4.2.4 Leaf water relations: 57 4.2.5 Root Na+, K+ contents and K+/Na+ ratios 57 4.2.6 Leaf Na+, K+ contents and K+/Na+ ratios 57 4.2.7 Leaf proline and phenolics 57
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Discussion 66 Experiment 3 Physiological and agronomic performance of quinoa under field
salinity 69
4.3.1 Crop stand establishment 69 4.3.2 Crop growth and yield 69 4.3.3 Root and leaf Na+ and K+ contents 69 4.3.4 Leaf water-relations 70 4.3.5 Leaf chlorophyll contents 70 4.3.6 Leaf antioxidants 70 4.3.7 Seed mineral contents 70 4.3.8 Seed proximate analysis and saponin concentration 71 Discussion 86 General Discussion 89 Summary 93 Literature cited 95
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List of Tables
Sr. # Title Page #
2.1 Global extent of salt-affected soils 4
2.2 Total geographical, cultivated and salt-affected area of Pakistan (Mha) 5
2.3 Variation in salinity tolerance of quinoa genotypes as depicted by percent yield reduction (PYR)
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2.4 Up regulated genes and their functions during salinity 22
2.5 Quinoa nutritional properties i.e. comparison with common cereals and quinoa nutritional properties influenced by salinity
26
3.1.1 Details of quinoa lines 29
3.1.2 Weather data of Faisalabad during November 2012. 30
3.1.3 Weather data of Faisalabad during December 2012. 31
3.2.1 Weather data during the experimental period 34
3.2.2 Physical and chemical characteristic of soil used in experiment 2 35
3.3.1 Details of quinoa lines 40
3.3.2 Characteristics of soil and water of two experimental locations 41
4.3.1 Score of crop density of quinoa lines affected by soil type 71
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List of Figures
Sr.# Title Page # 2.1 Relative maximum salt tolerance of common cereals and quinoa 7 2.2 Microscopic view of salt-bladders on adaxial and abaxial surface of
young quinoa leaf. 13
2.3 Summary of Na+ transport in quinoa plant 23 2.4 Quinoa word production (a) and prices (b) 27 2.5 Quinoa import demand 27 3.3.1 Weather details of experimental locations during 2013-14 40 4.1.1 Shoot length (a), shoot fresh weight (b) and shoot dry weight (c) of
quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.1.2 Root length (a), root fresh weight (b) and root dry weight (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.1.3 Root Na+ concentration (a), root K+ concentration (b) and root K+/Na+ ratio (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.1.4 Leaf Na+ concentration (a), leaf K+ concentration (b) and leaf K+/Na+ ratio (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.1.5 Leaf senescence of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.2.1 Plant height (a), main panicle length (b) and stem diameter (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.2.2 Shoot biomass per plant (a), seed yield per plant (b) and thousand seed weight (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.2.3 Leaf photosynthetic rate (An) (a), leaf stomatal conductance (gs) (b) and leaf transpiration rate (E) (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.2.4 Leaf chlorophyll a contents (a), leaf chlorophyll b contents (b) and leaf total chlorophyll contents (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.2.5 Leaf water potential (a), leaf osmotic potential (b) and leaf turgor potential (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.2.6 Root Na+ concentration (a), root K+ concentration (b) and root K+/Na+ ratio (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.2.7 Leaf Na+ concentration (a), leaf K+ concentration (b) and leaf K+/Na+ ratio (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.2.8 Leaf proline concentration (a) and leaf phenolic contents (b) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.3.1 Crop growth rate (CGR) of quinoa lines (Q1 and Q2) affected by soils type of Faisalabad (a) and Pindi bhatian (b). Q2 and Q7 indicates quinoa lines
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4.3.2 Plant height (a), Stem diameter (b) main panicle length (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.3.3 Seed yield (a), biological yield (b) and 1000-seed weight (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.3.4 Leaf Na+ (a), leaf K+ (b) and K+/Na+ (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.3.5 Root Na+ (a), root K+ (b) and root K+/Na+ (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.3.6 Leaf water potential (a), leaf osmotic potential (b) and leaf turgor potential (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.3.7 Leaf Chlorophyll a contents (a), leaf chlorophyll b contents (b) and leaf total chlorophyll contents (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.3.8 Leaf ascorbate (a), leaf total phenolics (b) and proline contents (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.3.9 Leaf Superoxide dismutase (a), leaf peroxidase (b) and leaf catalase activities (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.3.10 Seed Cu (a), seed Ca (b) and seed Fe contents (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.3.11 Seed Mg (a), seed Mn (b) and seed Zn contents (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.3.12 Seed Na+ (a) and seed K+ contents of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.2.13 Seed protein (a) and seed fat contents of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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4.3.14 Seed Crude fiber(a) and seed saponin contents of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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Abbreviation Full % Percent cm centimeter (s) CAT Catalase CGR Crop growth rate d day (s) DAS days after sowing g gram (s) g m-2 gram per square meter ha-1 per hectare K Potassium kg Kilogram kg ha-1 kilogram per hectare m Meter m-2 per square meter ml Millilitre mm Millimetre mM milli Molar MPa Mega Pascal M ha Million Hectare MINFAL, Pakistan Ministry of Food, Agriculture and Livestock, Pakistan Na Sodium POD Peroxidase ROS Reactive oxygen species SOD Superoxide dismutase SSRI Soil Salinity Research Institute UAF University of Agriculture Faisalabad
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Abstract Arable soils are decreasing over the globe including Pakistan due to escalating salinity in soils. Viable imperative option is to explore potential of halophytic plants in semi-arid and arid agro-ecologies where crop production is severely affected. Among different halophytes, quinoa is a potential option to be utilized to get benefits from salt-affected soils. Therefore, quinoa salt tolerance was explored in agroecological condition of Faisalabad and Pindi Bhatiaan, Punjab, Pakistan through series of experiments. In first phase, salt tolerance variations among four quinoa lines (Q1, Q2, Q7 and Q9) were observed in hydroponic culture amended with varying salt concentrations (0, 100, 200, 300 and 400 mM NaCl) up to one month at wire house Department of Crop Physiology, University of Agriculture, Faisalabad during 2012-13. In second phase, same lines were explored for salt tolerance up to maturity in normal and salinized soils (10, 20, 30 and 40 dS m-1) in pots in same wire house. Data regarding different salt tolerance indices indicated that tolerance was linked to high leaf turgor potential, increased leaf K+ versus N+ contents and de novo synthesis of osmoprotectants (proline and total phenolics contents) in leaves. All quinoa lines survived even at 40 dS m-1 NaCl stress and produced seeds. Quinoa line Q2 and Q7 were more salt tolerant and high yielder at all salt regimes. In third Phase; open field trials were conducted to evaluate growth and development at two locations of different salinity levels S0 (UAF Farm, Normal; ECe: 2.11 dS m-1, SAR: 5.2 (mmol L-1)1/2), S1 (Paroka Farm UAF, saline sodic; ECe: 9.8 dS m-1, SAR: 25 (mmol L-1)1/2), S2 (SSRI Farm, normal; ECe: 3.21 dS m-1, SAR: 11.9 (mmol L-1)1/2) and S3 (SSRI Farm, saline sodic; ECe: 13.9 dS m-1, SAR: 42 (mmol L-1)1/2) during 2013-2014. Two salt tolerant lines (Q-2 and Q-7) were grown in lines and were allowed to grow till maturity under RCBD split plot arrangement. Maximum seed yield (3062 kg ha-1) was achieved by Q7 at normal field (S0) soil which was statistically similar with yield of same line obtained from salt affected field S1 (2870 kg ha-1). Furthermore, low yield was seen from both lines from both S2 and S3 as compared to S0 and S1. Q7 was best under all four conditions. Minimum yield was recorded from Q-2 (1587 kg ha-
1) at S3. Q7 had higher SOD, proline, phenolic and K+ contents, and lower Na+ content in leaves as compared to Q-2. High levels of antioxidants and K+/Na+ ratio and good stand establishment of Q-7 helped to withstand salt stress and might be the cause of higher yields both under normal and salt affected soils. Seed quality (minerals and crude protein) was not affected under salt affected soils even improved in some cases, (K, Mg and Mn in Q7) and found in range of international published reports, however genotype Q-7 accumulated more Na+ in seeds than Q-2. Hence Q-2 may be considered as nutritionally superior line under problem soils. Overall quinoa produced less yield on salt affected soils of Punjab but it was still higher than world average quinoa yield (1000 kg ha-1) Abbreviations: UAF: University of Agriculture, Faisalabad; SSRI: Soil Salinity Research Institute, Pindi Bhatiaan
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Chapter 1 Introduction Increasing soil-salinity is a major cause of soil degradation over the world, 7% of
total land area is salt-affected while sodic-soils are even more prominent (Flowers et al.,
1997). Weathering of rocks (primary salinization) and anthropogenic activities (secondary
salinization) causes increased concentration of soluble salts (Wicke et al., 2011).
Secondary-salinization is aggravated by frequent use of underground brackish water and
contaminated sewerage water for irrigation purpose and clearance of forest lands for crop
production and pastures (Barret-Lennard, 2002). Globally only 15% land out of total
cultivated is irrigated but contribution in world’s food is one third (Munns, 2005).
Combined natural and human induced salt contaminated soils are estimated almost 960
Mha (Wicke et al., 2011). Approximately, 100 Mha productive land have been have turned
to saline by use of saline underground water for irrigation purpose, equating about 11% of
the worlds irrigated land (FAO, 2012). Worryingly the extent of salt-affected land and its
spread is highest and continuous in the most economically challenged and populated
countries i.e. Bangladesh (1 Mha; Hossain, 2010), Pakistan (6.18 Mha; MINFAL, 2002)
and India (7 Mha; Vashev et al., 2010) posing severe threats to sustainability of agriculture.
Increased level of salts in soil exerts three major physiological constrains on plants
those diminish its growth and yield performance: osmotic, ionic, and oxidative stresses
(Munns and Tester, 2008). The osmotic effect leads to reduced water uptake due to negative
osmotic potential caused by increased accumulation of salts in the soil. Water deficit in root
zone trigger ABA signaling which is known to induce stomatal closure by lowering turgor
of guard cells, which ultimately limits photosynthetic rate. Sodium ions cause nutrient
deficiency by decreasing the availability and absorption of other ions in soil and also
interfere with cell metabolism as a result of replacing K+ in key enzymatic reactions which
occurs in cytosol and organelles (Anschütz et al., 2014; Benito et al., 2014; Shabala and
Pottosin, 2014). Furthermore, excess accumulation of sodium in soil profiles also degrade
soil, due to dispersion of colloids because Na+ has 2700 times less flocculation capacity
than Mg+2 and Ca+2 (McKenzie, 2003). Together, the osmotic stress and Na+ toxicity lead
to generation of reactive-oxygen species (ROS) in all cell compartments causing
deleterious effects on DNA, proteins, pigments and membranes (Munns and Tester, 2008;
Bose et al., 2014).
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There are two major strategies which are being followed to improve salt tolerance;
one is introgression of tolerance traits either by breeding or genetic engineering (Apse and
Blumwald, 2002; Chinnusamy et al., 2005; Ruan et al.,2010). The alternative choice, yet
not widely exploited, is utilizing halophytes directly in agriculture (Flowers, 2004; Panta
et al., 2014).
Halophytes can grow even at salt regimes of 100-200 mM NaCl (dicots), or 50 mM
(monocots) (Flowers and Colmer, 2008). For arid and semi-arid agro ecologies, this
advocates wider irrigations with brackish water. Halophyte plant species could be
considered as “spotlight” because they not only tolerate salinity, but their growth even
improves at moderate salinities (100-200 mM). Any conventional crop species is salt
sensitive to such salt concentrations, considering, halophytes “ideal” for saline-agriculture.
Exploitation of halophytes is not a new concept but success stories are limited.
Most of the halophytes introduced already in Pakistan, have limited food value for humans,
as most of them are forages and grown for limited grazing purpose (Atriplex species; Kallar
and grass salt bush etc) (Ashraf et al., 2012). One of the promising facultative halophytes
is Chenopodium quinoa having high latent ability as a human food source and it is highly
tolerant to abiotic stresses (drought, frost and more importantly, salinity) and has
extraordinary nutritional quality that’s why Chenopodium quinoa has recently received a
considerable attention as an alternative crop in the arid and semiarid agriculture worldwide
(Jacobsen et al., 2003; Adolf et al., 2013) and the plant has been recently been studied
successfully in the Mediterranean territory (Jacobsen, 2014; Lavini et al., 2014; Benlhabib
et al., 2014). On the behalf of all these factors the Food and Agriculture Organization
(FAO), celebrated the year 2013 as “International Year of Quinoa” to focus consideration
on this crop in all over the world (Bazile et al., 2015). Quinoa is more salt tolerant than
common cereals. It has gained popularity over the world due to above mentioned features
and its cultivation is continuously increasing (Jacobsen, 2011; Bazile et al., 2015).
Quinoa is a plant species of family “Amaranthaceae” and known as pseudo-cereal
which produces grains of enormous nutritional qualities, it has origin from Latin America,
where it was grown seven-thousand years, ago (Jacobsen et al., 2003). Quinoa grows well
at moderate salt-regimes (100-200 mM NaCl), that’s why known as “true halophyte”.
Furthermore, some accessions can stand and grow even at salt-concentrations of 400 mM,
which is equal to salt-concentration of sea water (Jacobsen et al., 2003). Quinoa seed is
free of gluten, rich in all essential amino-acids, minerals (K, Fe, Ca, Mn), vitamins (A, E,
B2), carbohydrates and health supportive fatty-acids (omega 3) (Stikic et al., 2012).
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Quinoa has also been recently introduced successfully in Pakistan on normal soils
(Basra et al., 2014). But its adaptability and yield potential on local salt affected soils has
yet to be tested. Furthermore, a lot of work has been reported related on quinoa salt
tolerance but very few field studies are available especially in saline-sodic and marginally
degraded soils.
In the light of above, Quinoa was tested through series of experiments to explore the salt
tolerance of quinoa on natural salt affected fields under agroecological conditions of
Faisalabad and Pindi-Bhatiaan Pakistan with following objectives
• To explore potential of quinoa as a high salt-tolerant crop
• To investigate physiological mechanisms in quinoa
• To introduce quinoa as quality food grain crop
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Chapter 2 Review of Literature Increasing salinity is a severe threat to sustainable agriculture over the world, to live with
salinity, viable option is utilization of halophytes especially quinoa. Extensive review about
above mentioned sentence has been collected from different sources and presented in
following paragraphs.
2.1 Global extent of salt-affected soils Major factor which is contributing to soil degradation is increasing soil-salinity, 1.1
G ha of earth’s land is salt affected (Wicke et al., 2011). According to review of Wicke et
al., 2011 extent of salt-affected land over the World is given in table 2.1. Worryingly the
extent of salt-affected land and its spread is highest and continuous in the most
economically challenged and populated countries i.e. Bangladesh (1 Mha; Hossain, 2010),
Pakistan (6.8 Mha; MINFAL, 2002) and India (7 Mha; Vashev et al., 2010) posing severe
threats to sustainability of agriculture. Table 2.1 Global extent of salt-affected soils
Region Salt-affected Land (M ha) USA 77 Central America 5 South America 84 Canada 7 North Africa 161 West Africa 83 East Africa 56 South Africa 22 West Europe 1 East Europe 2 Former USSR 126 Middle East 176 South Asia 52 East Asia 98 South East Asia 6
2.2 Salinity extent in Pakistan
Pakistan is a South-Asian country, located south of Himalayan mountains, between
30.37° N, 69.34°E. Increasing salinity is a major threat to irrigated agriculture of Pakistan
and estimate of reduction in yields of major crops is 25 % (Kahlown and Azam, 2002).
According to estimate of World bank, 1.4 Mha of agricultural soils of Pakistan are salt-
affected. The salt-affected soils prevail in arid, semi-arid, sub-humid and semi-humid
5
regions of all provinces (Punjab, Sindh, Baluchistan and KPK). Moreover, deltaic and
coastal area of Sindh and Baluchistan are also salt-affected.
Pakistan’s agriculture is purely relying on Indus-Basin irrigation system (IBIS). The
contribution of IBIS in country’s GDP is 90%. Meanwhile, IBIS is creating waterlogging
and salinity due to improper drainage system. Furthermore, shortage of canal water also
compels farmers for the use of tubewell brackish water enhancing secondary salinity or
sodicity (accumulations of sodium salts in productive soils). Most of salt-affected soils of
Pakistan are saline-sodic (Murtaza et al., 2009; Sumia and Shahid, 2009). Extent of salt-
affected land of Pakistan is given in table 2.2.
2.3 Reasons of increasing salinity or sodicity
Weathering of rocks (primary salinization) and anthropogenic activities (secondary
salinization) causes increased concentration of soluble salts Wicke et al., 2011. Secondary-
salinization is aggravated by frequent use of underground brackish water and contaminated
sewerage water for irrigation purpose and clearance of forest lands for crop production and
pastures (Barret-Lennard, 2002). Globally only 15% land out of total cultivated is irrigated
but contribution in world’s food is one third (Munns, 2005). Combined natural and human
induced salt contaminated soils are estimated almost 960 Mha (Wicke et al., 2011).
Approximately, 100 Mha productive land have been have turned to saline by use of saline
underground water for irrigation purpose, equating about 11% of the worlds irrigated land
(FAO, 2012).
2.4 Salinity effects on plants
Increased level of salts in soil exerts three major physiological constrains on plants
those diminish its growth and yield performance: osmotic, ionic, and oxidative stresses
(Munns and Tester, 2008). The osmotic effect leads to reduced water uptake due to negative
osmotic potential caused by increased accumulation of salts in the soil. Water deficit in root
Table 2.2 Total geographical, cultivated and salt-affected area of Pakistan (M ha) Provinces
Geographical
Cultivated
Salt-affected Cultivated Uncultivated Total
Punjab 20.63 12.35 1.51 1.16 2.67 Sindh 14.09 5.88 1.15 0.96 2.11 Baluchistan 10.17 1.86 0.11 1.24 1.35 KPK&FATA 34.72 2.07 0.03 0.02 0.05 Total 79.61 22.16 2.80 3.38 6.18 Source: MINFAL, (2002)
6
zone trigger ABA signaling which is known to induce stomatal closure by lowering turgor
of guard cells, which ultimately limits photosynthetic rate. Sodium ions cause nutrient
deficiency by decreasing the availability and absorption of other ions in soil and also
interfere with cell metabolism as a result of replacing K+ in key enzymatic reactions which
occurs in cytosol and organelles (Anschütz et al., 2014; Benito et al., 2014; Shabala and
Pottosin, 2014). Furthermore, excess accumulation of sodium in soil profiles also degrade
soil, due to dispersion of colloids because Na+ has 2700 times less flocculation capacity
than Mg+2 and Ca+2 (McKenzie, 2003). Together, the osmotic stress and Na+ toxicity lead
to generation of reactive-oxygen species (ROS) in all cell compartments causing
deleterious effects on DNA, proteins, pigments and membranes (Munns and Tester, 2008;
Bose et al., 2014).
2.5 Approaches to make salt-affected soils productive
In present eras, research has been increasing about salinity in relation to plant. Usually,
three methodologies are practicable for enhancing the crop production from soils affected
by salinity: 1) soil reclamation, 2) introducing genotypes tolerant to salinity (Yilmaz et al.,
2004; Blumwald et al., 2004), 3) utilization of halophytes as a new crop but not exploited
widely (Flowers and Yeo, 1995; Flowers, 2004; Panta et al., 2014). Due to soil
permeability, lack of good quality water, higher cost for reclamation and widely distributed
salt affected area, practically, soil reclamation is not possible (Akhtar et al., 2010). Salt
tolerant genotypes can be helpful but it needs time and its outcome is limited. Halophytes
can be used as an alternate choice mainly in agriculture and they are not well exploited till
now (Flowers and Yeo, 1995; Glenn et al., 1999; Flowers, 2004; Panta et al., 2014). They
can grow well even at higher salt contents i.e. 50 and 100-200 mM for monocots and dicots,
respectively (Flowers and Colmer, 2008). These species are very important because they
not only survive but get benefit from saline irrigation. For saline agriculture, halophytes
are ideal because traditional crops cannot tolerate such higher amount of salt contents.
Mostly, introduced halophytes are forages and cultivated for grazing purpose, restricted
food value for people. Chenopodium quinoa is a promising C3 halophyte, highly tolerant
to abiotic stresses (frost, salinity and drought), better nutritional quality and having higher
potential as a food source for humans. Due to these properties, quinoa has gained
importance in arid and semi-arid regions of the world as an alternative crop (Jacobsen et
al., 2003; Adolf et al., 2013). In the Mediterranean region, it has been studied effectively
(Benlhabib et al., 2014; Jacobsen, 2014; Lavini et al., 2014). It can also be considered as a
7
model crop for exploiting the mechanism of tolerance to salinity in halophytes. By
considering all these factors, Food and Agriculture Organization (FAO) declared the year
2013 as an “International Year for Quinoa” to emphasis on this plant in all regions of the
world (Bazile et al., 2015). Quinoa is more salt-tolerant than common cereals (Razzaghi et
al., 2015) fig 2.1
Fig 2.1 Relative maximum salt tolerance of common cereals and quinoa
Source: (Adapted from Razzaghi et al., 2015; FAO a, 2017)
2.6 Quinoa plant description
Quinoa has been grown for about more than seven thousand years ago in Latin
America, it edible parts are grains and leaves. Chenopodium quinoa is member of
Chenopodiaceae family, now known as Amaranthaceae, which comprise many halophytic
species. Quinoa is a C3, dicot plant that’s why it is known as pseudo-cereal while most of
other common cereals species are monocot. Unique seed nutritional features and
exceptional balance of oil/fat and protein, it is also known as pseudo-oil seed (Vega-Galvez
et al., 2010). Quinoa plant attain height of 1-3 m, which is dependent on sowing-density.
Germination of quinoa seed is fast, within few hours after imbibition. Its roots can penetrate
up to depth of 30 cm in soil, Quinoa exhibits cylindrical stem with diameter of 2-3.5 cm.
Stem can be straight or branched, and of different colour i.e. red, light brown, white or
yellow depending upon genotype. Leaves shape is like goose foot. Flowers are not
complete, petal absent (Valencia-Chamorro, 2003). Seeds are flat or round, ranging in
diameter of 1.5-4 mm. Seeds also differ in colour (Mujica, 1994), i.e. black, white, grey,
red, orange and yellow. Quinoa seeds contain enormous nutritional profile (Vega-Galvez
0
10
20
30
40
50
Rice Wheat Barley Quinoa
ECe
(dS
m-1
)
8
et al., 2010), for example, proteins (De Druin, 1964), essential amino-acid (Dini et al.,
2005), minerals (Ca, Fe, Cu, Zn and Mg), and vitamins (B2, E, A) (Repo-Carrasco et al.,
2003).
2.7 Quinoa adaptation out of Latin America
Over 7000 years ago, first domestication of quinoa happened in Andean countries after
the Spanish conquests, quinoa was vetoed as “Indian food”. After, neglection for centuries,
during second half of 20th century its potential was rediscovered. Since then, increase in
number of quinoa importing countries occurred, with new quinoa producer’s country appearing
on map of globe and now it is being cultivated outside of Latin America (Bazile et al., 2016).
Due to its high adaptability and nutrition with low input requirement, quinoa has been selected
for food security in 21st century (Bazile et al., 2015).
Initial results of quinoa cultivation, in Kenya were promising due to good seed quality
and obtained yield was comparable to yield of traditional Andean region (Mujica et al., 2001).
Quinoa introduction takes place in UK in 1970s, after that experimentation also started in
Denmark. In 1993, European Union approved a project titled “Quinoa -A multipurpose crop
for EC’s agricultural diversification”. Its fields and lab trials were conducted in Netherlands,
Denmark, Italy, Scotland, France and England. Later on, other countries i.e. Sweden, Austria,
Czech Republic, Poland and Greece. also showed interest in growing of this crop (Iliadis et al.,
2001). Quinoa trials were successfully conducted in agro-ecological conditions of South-
Eastern Europe with 1.721 t ha-1 seed yield of good quality. Interest to cultivate quinoa is
growing in US, Europe and Asia. Demand for organic-quinoa has been increased (Koyro et
al., 2008). Quinoa trials has also been conducted in diverse climatic conditions of India, USA
and Canada (Bhargava et al., 2007; Pulvento et al., 2010).
In 2009, quinoa experimentation was initiated by Department of Crop Physiology,
University of Agriculture, Faisalabad. Tested quinoa accessions displayed huge variations
regarding adaptability in local conditions (Basra et al., 2014), Furthermore, locally produced
grains had nutritional composition similar to produce of native region (Nasir et al., 2015).
2.8 Quinoa performance under salt-regimes.
Quinoa performance from germination to yield is discussed in following paragraphs.
2.8.1 Seedling establishment
Stand establishment means proper formation of seedling and it is a very critical
initial growth phase of plant’s life cycle especially if plant grows under stress conditions.
Halophytes are relatively tolerant to excess salts but high concentrations of salts effect
germination in glycophytes as well as in halophytes (Debez et al., 2004). For the meantime,
9
many experiments showed that some halophytes including quinoa are also affected by salts
during seed germination and seeding emergence stages (Malcolm et al., 2003). The critical
time in quinoa phenology is stand establishment (Jacobsen et al., 1999). Gomez-pando et
al., (2010) explored variations in germination-percentage of quinoa germplasm (one
hundred and eighty-two quinoa accessions) under saline regimes (250 mM saline water)
depending upon their genetic variability and resistance to salt stress. Jacobsen et al., (2003)
reported that the Peruvian quinoa cultivar Kancolla seeds had 75% germination ability at
57 dS m-1 salt stress level, observed up to seven days. The four Chilean accession under
salt stress also showed reduction in germination percentage (Hariadi et al., 2011).
Higher concentrations of salts badly affected seedlings establishment, biomass and
growth but there was no significant effect in lower concentrations of salts (Ruiz-Carrasco
et al., 2011). High salt-tolerance of quinoa at germination stage resulted due to excessive
replacement of toxic ions (Na+ and Cl-) with compatible ions i.e. K+, Ca+, Mg+2, SO4+2 and
PO4+3. It is also concluded from many experiments that under stress condition that quinoa
tolerates high salts due to more protective seed interior (Koyro and Eisa, 2008). Seeds
viability is also dependent on plants capacity of Na+ exclusion (Hariadi et al., 2011).
In general, morphological and physiological defensive characteristics of seeds
contributes to high salt-tolerance in quinoa which is required to be studied in greater
aspects. It is concluded that under salt stress the competency of germination and seedling
establishment is also dependent on genetic material of cultivar and medium in which the
seeds germination occurs.
2.8.2 Growth and yield performance
Salinity promotes growth of quinoa but beyond certain limits growth and biomass
starts to be decreased. Quinoa grows on salt flats of the altiplano located in Bolivia and
saline coastal soils of Chile. Most experiments confirmed the halophytic behavior of quinoa
(Hariadi et al., 2011), Quinoa cv Titicaca was grown on six salinity levels up to 70 days
and significant negative impact on seed germination was observed at 80% sea level salinity,
while maximum growths were recorded between 20 and 40% sea level salinity. In another
study quinoa grain yield observed was maximum at intermediate saline regimes (10-20 dS
m-1) as reported by Jacobsen et al. (2003). At salt concentration of 25 dS m-1, yield reduced
to 50% representing that even under such high salt stress, quinoa has the ability to grow
and yield good produce while no yield was found in maximum salt stress at 51.5 dS m-1
(Razzaghi et al., 2014).
10
Pulvento et al., 2012 tested quinoa in field conditions during 2009-10 in southern
Italy in order to explore the influence salt stress by measuring quantitative and qualitative
traits of quinoa produce. Quinoa field plots were irrigated with normal and saline water,
saline water created salt stress of 22 dS m-1 in soil. Salt stress did not reduced yield
significantly in this two-year study but also increased than mean seed weight, fiber as well
as total saponin contents in seeds. The seed yield obtained was also similar as reported from
Andean areas.
In the UAE, ICBA (International Center for Bio Saline Agriculture) has been
putting efforts to investigate quinoa adaptability since 2006 as a substitute crop to be grown
on salt-affected areas. Salt-tolerant genotypes having appropriate yield potential were
further explored in other central Asian countries including Kyrgyzstan, Uzbekistan and
Tajikistan under auspices of a project sponsored by the Islamic Development bank and also
in the MENA countries, Yemen, Egypt and Jordan through collaborative scheme of IFAD
(International fund for Agricultural Development) and OFID (OPEC fund for international
development) (Choukr-Allah, 2016). In 2012-13, at Ghayathi (a location of Mediterranean
region) three quinoa lines produced average seed yield of 750 g m-2 even by use saline
water (ECw; 14-15 dS m-1) and this value of yield was similar as attained from normal
native quinoa cultivation regions (Choukr-Allah, 2016). Furthermore, mean fresh biomass
yield of these lines was 4.3 kg m-2, demonstrating quinoa’s capabilities as an alternate crop
for fodder purpose on salt-affected lands (Rao et al., 2013). Seed yields of above mentioned
quinoa lines were also obtained from trials of saline water use for irrigation purpose in
northern regions of Emirates. Levels of salinity in irrigation (dS m-1) water (ECw) were
different for three regions studied (i.e. in Dibba; 6.1; Hamraniah; 4.5 and Al-Dhaid 2.3).
Thus, average seed weight from 1 m-2 of all three quinoa lines was maximum in Al-Dhaid
(541 g), followed by 398 and 190 g in Hamraniah and Dibba respectively (Rao, 2016).
Despite higher salinity in irrigation water of western region, significant more seed yields
were obtained as compared to Northern Emirates. Furthermore, average grain yield of 4
quinoa lines was 1050 g m-2, much more as compared to seed yields of previous year noted
from same locality (750 g m-2). The yield trial results showed high variations in
performance of tested quinoa lines across different locations. Three lines of quinoa
(QS0938, QM1113, D0708) were irrigated with treated waste water cultivated in open field
exposing to different levels of salt amendments with objective to access yield response (El-
Youssfi et al., 2012). Noteworthy variations regarding yield responses of quinoa lines were
recorded at 6 dS m-1 salt stress, QM1113 was high yielder with 6.9 t ha-1 while line D0708
11
was at position second (5.65 t ha-1) when harvested from EC level of 3 dS m-1. Quinoa lines
differ in salinity tolerance (table 2.3) under new environments meanwhile their reported
yield was still comparable with the yield value of native regions (Pulvento et al., 2012).
Yield reduction is less than 50% (table 2.3) in some lines at higher salinity levels: salt levels
where most of common cereal failed to produce yield, quinoa produce seeds at 80 % sea
water salinity (table 2.3).
2.9 Salt-tolerance mechanisms
Glycophytes (salt sensitive) and halophtyes (salt tolerant) have similar type of
anatomy and physiology, but salt-tolerant plants make efficient use of mechanistic
processes to become salt-tolerant (Shabala and Mackay, 2011). However, it can also be
said, that halophytes exhibit unique salt-tolerance mechanism than glycophtyes. Does
quinoa display exceptional strategies to adapt and completion of its life span under high
salt stress? Summary of these mechanisms is compiled here from collection of extensive
work which has been done by different researchers in last few years.
2.9.1 Morphological attributes
2.9.1.1 Seed Characteristics
Results of many experiments depicts that halophytes may be salt-sensitive during
seed germination and emergence of seedlings against salt-stress (Debez et al., 2004). The
physiological mechanisms of resistance or susceptibility of seed like where sodium ions
are sequestered e.g. vacuole etc., mainly depends on ion homeostasis, distribution and
levels of various other ions in seeds and other tissues (Hasegawa et al., 2000). These
mechanisms not much affect seed and seed viability. Even though seed weight was not
affected, organic constituents (mainly (CH2O)x and other carbon comprising compounds)
Table 2.3 Variation in salinity tolerance of quinoa genotypes as depicted by percent yield reduction (PYR) Genotype Origin EC (dS m-1) % PYR Reference No. 407 Chile 6.5* 18 Karyotis et al., 2003 Colorado 407D (PI 596293) Chile 32 65
Peterson and Murphy, 2015
UDEC-1 (PI 634923) Chile 32 43 Baer (PI 634918) Chile 32 49 QQ065 (PI614880) Chile 32 73 TITICACA Denmark 20 3.7 Yazar et al., 2015 TITICACA Denmark 22 17 Pulvento et al., 2012 TITICACA Denmark 40 85 Yang et al., 2016 * EC of natural saline-sodic field EC developed in open field using salt mixture EC developed in pots, placed in green house using NaCl
12
of quinoa decreased at high NaCl concentrations and remunerated by a rise in ash content.
Moreover, the salt induced upsurge in the ash contents was observed not only by enhanced
Na+ level in plants but also by a rise in plant potassium, calcium and magnesium ion
concentrations. Even though, sodium ions were significantly increased however, the
K+/Na+ ratio was never observed lower than 1. Hence, buildup of K+ ions and other vital
nutrients, such as phosphorus and sulphur, even at higher salinity was stable. Route of toxic
cytoplasmic Na+ and Cl- to the inner parts of seeds is prohibited by seed coat as 90% Na+
and Cl- exist in it. An inquiry confirmed that seeds of quinoa grown under salt stress
provides a vital tolerance strategy related to compacted seed testa with perisperm, providing
defensive barriers ensuring the elimination of toxic Na+ and Cl-, thus, regulating
appropriate ratio of K+/Na+ inside the seed. Seeds viability is also dependent on plants
capacity of Na+ exclusion (Hariadi et al., 2011).
2.9.1.2 Leaf salt-bladders
Leaf of halophytes have distinctive characteristic of having specialized structures
called, salt bladders/glands or trichomes as presented in Fig 2.2 Accumulation and
sequestration of toxic salts in to these special anatomical structures seems to be an efficient
stratagem subsidizing to salt-tolerance in some quinoa genotypes (Agarie et al., 2007).
These structures are mainly involved in compartmentalization of incompatible ions
accumulated the ions at considerably lethal levels and thus these ions were excluded from
primary photosynthetic active mesophyll and other parts of plant. The ions may prove
themselves to be beneficial in osmoprotection and also act as a second epidermis thereby
protecting photosynthetic machinery from UV induced injury.
In a germplasm of quinoa (Chilean BO78), negligible changes in EBC numbers
were recorded in salinity control and treated plants. Also, in this case restricting ion
accumulation may not be controlled by EBCs (Orsini et al., 2011). In the salt-tolerant plant,
Mesembryanthemum crystallinum, the bladder cells (EBCs) were able to amass not only
water but also other metabolites like malate, cysteine, inositol, pinitol, flavonoids etc. (Jou
et al., 2007). Therefore, accumulation of these compounds having chaperone ability in
EBCs (epidermal bladder cells) displays a protective role from ROS attack. The
accumulation of calcium oxalate crystals in leaves of quinoa had been linked to elevated
Ca2+ under salt stress (Riccardi et al., 2014).
13
2.9.1.3 Stomata
Transpiration rate is decreased under excessive amount of salts. The decline in
stomatal conductance of gases in leaves of halophytes is assumed to be a vital parameter
for improved water use efficiency under stress condition. These adaptive responses may
occur through morphological changes, e.g. decreases in size and densities of somata or
through physiological regulation of stomatal aperture in response to ABA. Under saline
condition, drop of up to 50% in stomatal density followed by decreased size of stomata has
been described in the salt-sensitive Chilean accessions, (BO78) (Orsini et al., 2011). In
another study, 14 different quinoa lines were examined for salt-tolerance, noticeably less
stomatal densities were observed in all lines under saline regimes and these morphological
modulations were different in various lines (Shabala et al., 2013).
Fig 2.2 Microscopic view of salt-bladders on adaxial and abaxial surface of young quinoa leaf.
Source Adolf et al., (2013).
2.10 Na+ uptake and transport
Salinity tolerance has been widely linked with reduced Na+ uptake. In an
experimentation on quinoa conducted by Wilson et al., (2002) using mixed-salt solutions,
increase in sodium was observed only 3-4 folds in upper parts of plants, in another case,
even tolerant wheat genotype, sodium concentration increased to more than 6-fold. Shabala
et al. (2013) described that variations in germplasm regarding salinity-tolerance were
directly linked to Na+ uptake as highly tolerant genotypes exhibited low level of Na+ in
xylem. The 14 tested genotypes, could be categorized in to two separate groups i.e. i) Na+
excluder; ii) Na+ includers, with most salt-tolerant ones were falling in to former part. It
14
seems that, that although, quick uptake and buildup of Na+ in the leaves is mandatory for
osmotic adjustment, ions toxicity is evaded in the most tolerant germplasm of quinoa by
controlling, to some degree, Na+ charging into the xylem sap. Generally, Na+ exclusion is
thought of as a beneficial attribute in non-halophytes (Munns and Tester, 2008).
In Arabidopsis, this Na+ exclusion is mediated by Na+/H+ exchanger operative at
the plasma-membrane of epidermal root-cells (Blumwald et al., 2000) encoded by the Salt-
overly Sensitive (SOS1) gene (Qiu et al., 2002). In quinoa, SOS1 gene expression of salt-
stressed plants had been explored by several research groups (Maughan et al., 2009; Ruiz-
Carrasco et al., 2011; for details see section on “molecular identity of Na+ transpoters”).
Bonales-Alatorre et al. (2013) examined the activities of vacuolar and plasma membrane
channels and observed the genotypic variations regarding several mechanistic processes
associated with salt-tolerance. Salt-sensitive cv. Q52206 and salt-tolerant cv. Q16 of quinoa
were compared, and it significantly enhanced Na+ exclusion rate was observed in
mesophyll cells of leaf, keeping low Na+ concentrations in cytosol and decreased activity
of vacuolar channels, improved K+ maintenance in the mesophyll cells of leaf, and
enhanced activity of H+ pumps permitting mesophyll cells mesophyll cells for quick
reestablishment of membrane potential. These mechanisms are highly linked and have role
in outstanding salinity-tolerance of quinoa. Especially, the researchers linked the tonoplast
potential with sodium movement out of vacuole, faster channels of vacuole (FV) were
found inactive in older leaves of stressed plants, whereas this FV-conductance in juvenile
leaves, increased in similar saline regimes and was no less than two-fold higher,
representing the total Na+ amassed in the mesophyll cells of leaves. Additionally, under
saline-regimes, seven-fold increase in number of slow vacuolar pathways was observed in
juvenile leaves (having less Na+), shows that Chenopodium quinoa Plants have the
capabilities to adjust the activities of FV and SV-channels as confirms that considerable
amount of Na+ was compartmentalized in the vacuoles of aged leaves, thus, shielding new
emerging leaves from extreme Na+ build ups. This unique mechanism of Na+
compartmentation has been observed and elaborated in many halophytes and is a central
component of salt-tolerance (Munns et al., 2006).
2.11 Ion homeostasis and compartmentation
Ion uptake and accumulation in vacuole for the maintenance of ion homeostasis, is
vital for plant growth both during normal and stress conditions. Both halophytes and
glycophytes cannot stand to high salt concentration in cytosol. Hence, surplus salt is either
15
dumped in vacuole or transported to older leaves which ultimately senescence of these older
leaves occurs, thus protecting plant from salt-stress. Extremely saline conditions can cause
ion-toxicity because of Na+ and Cl- buildup (Munns and Tester, 2008). Whereas, some salt-
tolerant glycophtyes, limit movement of incompatible ion towards shoot, by restricting
influx of ion at root, thus avoids ion-toxicity while halophytes take up ions, transfer and
accumulate in plant’s above ground parts (Flower and Colmer, 2008).
Water uptake and transport may be facilitated by osmotic adjustment resulted by
the accumulation of inorganic ions like sodium, potassium and chloride, and thus, lowers
metabolic-energy cost essential for the organic osmolyte biosynthesis. Similarly, in quinoa
plants, of salt treated cv. Titicaca with varying NaCl levels ranging from 0 to as high as
500 mM, Hariadi et al. (2011) proved that up to 95% of cell turgor in older leaves and 80-
100% of turgor in cells of juvenile leaves was due to K+ and Na+ accumulations. High
buildup of Na+ was also recorded in cv. BO78; a cultivar from chile, when grown in NaCl
levels ranging 150 to 750 mM, as described by Orsini et al., 2011. In non-tolerant plants,
salt-stress usually triggers, K+ efflux, reduced uptake of K+ and consequential drop of K+
level in the cell which may be very harmful (Demidchik et al., 2010).
Therefore, K+ homestasis is a crucial aspect of salt-tolerance, and the capability to
maintain an optimum K+/Na+ ratio is thought to be vital for adaption or tolerance to salt-
stress (Munns and Tester, 2008). Suhayda et al., (1992) explored a strong association
between leaf tissue K+/Na+ ratio and salt-tolerance in barley, and recommended that this
attribute could be used as screening criterion for the identification of salt-tolerant
genotypes.
Wilson et al. (2002) evaluated salinity-tolerance and ion build up in wheat and
quinoa against mixed salt-stress (combinations of NaCl, MgSO4, CaCl2 and Na2SO4) using
Andean hybrid quinoa cv. and wheat cv. Yecora Rojo. No significant drop in leaf area,
plant height, and plant fresh/dry weights against salt concentrations of 11 dS m-2, (which is
equal to 110 mM) in quinoa. While wheat growth attributes were negatively affected at 11
dS m-2. Even though, increasing salt-stress caused marked reduction in K+/Na+ ratios of
quinoa leaves and stem, this ratio decline was much more in wheat. As for cv. BO78, the
increased buildups of Na+ at 150 mM NaCl, was the cause of decline in ratio of K+ versus
Na+; nevertheless, capability of K+ retention in this salt-sensitive germplasm was supported
due to facts that, when NaCl levels were raised to 600 mM, plants displayed a 3-fold
increased concentration of K+ as compared to control plants (Orsini et al., 2011). Turgor
adjustment is an important mechanism of inorganic ions and has been recorded in various
16
other halophytes (Inan et al., 2004). The documented improved values of K+ concentration
in leaf sap of juvenile leaves of quinoa (e.g. 400 to 650 mM) are consistent with the study
of Hariadi et al. (2011). In another detailed study, salt-stress modulated more uptake of Na+
and K+ up to 100 and 60 kg ha-1, respectively (Razzaghi et al., 2014). So, it is concluded
that quinoa’s keeps effective homeostatic mechanisms related to K+ retention and osmotic
adjustment, make it astonishing salt-tolerant plant.
2.12 Gas exchange relations
Abscisic acid regulates the stomatal pore opening and closing by the outward and
inward flux of inorganic ions in guard cells. Early increments in the ABA, lessened foliage
and soil Ψw (water potential) represent the water stress instigated by high salinity. Salinity
has been known to result declined gas exchange as reported by Saleem et al. (2017).
Razzaghi et al. (2011) reported that with every increase in NaCl level, significant drop in
soil Ψw which ultimately decreased Ψw and gas exchange in leaves of quinoa even if they
were irrigated thoroughly. He also reported improvement in ABA levels in plant parts thus
causing closed stomata. Likewise, the reports of Orsini et al. (2011), indicate 50 to 60%
reductions in gas exchange relations in a salt-susceptible cultivar (BO78) under medium
(150 mM) to high (300 mM) NaCl stress. Reduced gas exchange by stomata limits water
uptake by limiting transpiration, however, at the same time, it lowers CO2 uptake (Iyengar
and Reddy, 1997). Two distinct cultivars (Titicaca and Utusaya) of quinoa were analyzed
for stomatal conductance of gases and photosynthetic carbon dioxide assimilation under
salt stress. Utusaya which was originated from the Bolivian salares territory, was least
altered, with just 1/4th decrease in total CO2 fixation as compared to almost 3/4th decline in
Titicaca cultivar as recorded by Adolf et al. (2013). Gaseous exchange by stomata was
found to be declined in the former Utusaya cultivar even under control conditions and it
was conceived as a plausible tradeoff between tolerance under stress and the yield.
Regardless of the impacts of high salt stress on carbon dioxide entry from stomatal opening
and assimilation, numerous records entail the unaffected maximum photochemical efficacy
of photosystem-II (Adolf et al., 2013). However, more research is required to be done on
the fact that Photosystem-II is not a chief target of salt stress (Adolf et al., 2012)
17
2.13 Compatible solutes, osmoprtectants and antioxidants
Different researchers have reported an increase in the organic and inorganic
compatible solutes mainly glycine betaine, proline, K+ etc. (Flowers, 2004; Shabala and
Mackay, 2011). The buildup of these osmolytes or solutes is mandatory for sustaining
turgor pressure of cell which enables cell expansion in surroundings of improved
osmolality, however the role of organic vs inorganic solutes contribution to stress tolerance
is a debated topic as observed by Hasegawa et al., (2000). The accumulation of organic
solutes occurs even when there is high buildup of inorganic solutes.
Four main groups of these organic-osmolytes are identified i.e. sugars, polyols,
quaternary amines, and amino acids. All of them has been reported in tissues of quinoa
(Aguilar et al., 2003; Ruffino et al., 2010; Orsini et al., 2011; Ruiz Cararasco et al., 2011).
Morales et al., (2011) observed minor quantities of proline, sorbitol and Pinitol and huge
quantities of betaine, trehalose and particular trigonelline in cvs. Ollaque and Chipaya.
The response of young plants to high salinity w.r.t. carbohydrate metabolism indicates a
major characteristic allowing plants of quinoa to adjust osmotically to a salty environment
in its juvenile stages of development. Prado et al. (2000) found modulations in leaf glucose,
sucrose and fructose contents in normal and salt-stressed seedlings of quinoa cv.Sajama at
low temperature regimes. They stated enhanced activities of soluble acid-invertase and
sucrose-phospate synthase in leaves of salt-treated plants, indicating that more quantities
of soluble sugars are to keep osmotic balance in saline regimes. A low matric-potential
inside the seed may also limit water loss under external hypertonic conditions (high external
osmolality). Koyro and Eisa (2008) proposed that enhanced protein contents in seed taken
from plant grown under salt-stress might be contributed in lowering matric-potential.
They also stated that the high speed of germination in these seeds might be also
consequence of increased water uptake due to more accumulation of Na+ and Cl- in the seed
testa and also due to high contents of organic-solutes inside the seed. Dehydrin
accumulations were first reported in cotton seeds during later stages of embryo
development (Rorat, 2006). Furthermore, dehydrins has also been found in all vegetative
tissues of plants exposed to salt-stress (Rorat, 2006; Battaglia et al., 2008). The ectopic
expression of dehydrin in wheat had been revealed to improve salt-tolerance and
dehydration in plants of Arabidopsis thaliana, providing proof for the dehydrin’s role in
plant stress-tolerance (Saavedra et al., 2006).
18
Tissues of embryo of two quinoa accessions, Baer La Union and Sajama were
analyzed with Western blot analysis and these cultivars adapted to different environments
(one from high altitudes and one from sea level), when grown in invitro revealed the
existence of multiple bands of dehydrins in both. While the quantity of bands i.e. 32 and
30 k Da varied (Carjuzaa et al., 2008). The researchers concluded that many of dehydrins
found were of constitutive, and some of those may be linked with adaptions for multiple
environments. In another analysis, Burrieza et al., (2012) examined the influence of salts
on configuration of dehydrins in mature embryos of cultivar Hualhaus: adaptable of salty
and arid condition of altiplano, at least 4 dehydrins types were detected (using western blot
method) in seed harvested from normal and salt grown plants, in salt-treated plants, no
supplementary bands were recorded and only one band of (30 k Da) amplified under 300
and 500 mM NaCl concentration. Proposal for the modulation of dehydrins roles or
localization or both, has been described by the authors who also defined alterations in
localization in various cell compartments as well as phosphorylation of various dehydrins.
According to these scientists, these roles and localizations might depend upon the
phosphorylation of isoforms of salts. Against NaCl stress, high contents of trigonelline
(0.87 m mol g-1 dry weight) aggregated in leaves as well as roots. This concentration far
surpasses those that were recorded in other plants like, corn, soybean and tomato).
Total proline (an amino acid) accumulation under various salt regimes has been
examined comprehensively, and the functions such as compatible osmolyte, and
osmoprotectant in shielding subcellular structures and macromolecules, as signaling has
been well recognized (Szabados and Savoure, 2010). Elevated proline levels were recorded
in shoots of salt stressed quinoa cv. At sea level salinity, the increase was 10 times (up to
0.1 mmol g-1 dry weight) as compared to non-saline control (Orsini et al., 2011). Lowered
concentration range of total leaf proline (0.4-0.9 mg g-1 fresh weight) were also recorded
by Aguilar et al., (2003) in quinoa accessions from Peru from different environmental
conditions. Ruiz- Carrasco et al. (2011) observed elevated proline concentration in fifteen
days old seedlings of 4 Chilean quinoa genotypes at 300 mM NaCl stress.
These genotypes could be grouped that displayed modest (two-fold) rise, and those
who gathered 3-5-fold more total proline than non-saline conditions with the latter being
related with germplasm from more stress prone environments. However, it has been
observed that contribution of glycine betaine and total proline to cell osmotic adjustment,
accumulated in plants of quinoa exposed to salinity is negligible due to far too low
concentrations (Hariadi et al., 2011). It might be due to their osmoprotective function on
19
cellular and subcellular organelles. Also, physiologically related concentrations of free
amino acids as well as GB (Cuin and Shabala, 2005; 2007), regulate K+ movement across
the cell plasma membrane due to which, they adjust cell Ψw by indirectly regulating the
concentration of this ion. Pottosin et al. (2014) observed that a precursor of GB (choline)
clogs SV pathways in leaf and root cell vacuoles, playing a major role in Na+ sequestration
(so as to prevent Na+ flux out of the cell vacuole), so that even under apoplastic low Ψw in
salinity, plant adjusts cell turgidity which in turns, maintains growth.
Both osmotic stress and Na+ toxicity lead to overproduction of reactive oxygen
species (ROS) various cell compartments causing lethal effects on proteins, DNA,
membranes and pigments (Munns and Tester 2008; Bose et al., 2014) whereas antioxidants
detoxify reactive oxygen species (ROS) thus enabling plants to be adaptable to abiotic
stress.
Plant’s capability to reduce excessive ROS accretion under stressful environments is highly
linked to oxidative burst and its tolerance. This defense against ROS is activated in the
presence of the organic osmolytes which not only play role in osmotic adjustment but also
act as antioxidants (Szabados and Savoure, 2010). Some these organic molecules may
directly act as ROS scavengers, while some act as osmo -protectants or molecular
chaperons shielding PSII from oxidative stress (Shabala et al., 2012). To validate this
hypothesis, foliar applied Glycine betaine, was found considerably, to mitigate adverse
influence of oxidative stress induced by UV light affecting photosynthesis in quinoa
(Shabala et al., 2012)
Polyamines (PAs), i.e. spermine (Spm), Spermidine (Spd) and putrescine (Put) are
aliphatic polycations and has been recognized as plant growth regulators especially in
higher plants and according to Alcazar et al., (2010) also play role in stress responses in
crop plants. There are many reports proving that these PAs play a osmoprotective and
antioxidative role during stress conditions (Tang and Newton, 2005; Hussain et al., 2011).
Kusano et al. (2008) also observed the role of Pas in regulating ionic channels and
transporters
An opposite relationship was observed by Janicka-Russak et al. (2010) between
tissues Na+ concentration and PA representing the mechanism of these polycations in the
maintainence of ionic proportions under salt-stress, while reports indicate their protective
functions i.e. on photosynthetic apparatus as well as on membranes, done by Spm and Spd
for mitigating drought or salt-stress (Yamaguchi et al., 2007; Duan et al., 2008).
20
In another report, Ruiz-Carrasco et al. (2011) compared four Chilean quinoa
accessions against salinity levels of 150 and 300 mM NaCl. It was noticed that
(Spd/Spm)/Put ratio in BO78 accession was significantly lower as compared to other
accessions, thus indicating the salt-sensitivity of this accession. On the other hand, peak
proline accumulation categorized the salt-tolerant accession while PA response illustrated
highly salt-susceptibe one as reported by Ruiz Carrasco et al. (2011).
Plant antioxidant system is composed of enzymes (e.g. SOD, POD, CAT etc) and
non-enzymes i.e. vitamin and phenolic compounds. Amjad et al. 2015 revealed enhanced
activity of some antioxidant enzymes in leaves of cv Titicaca when exposed to 300mM
NaCl ionic as well as non-ionic (600mM mannitol) salt-stress. Together non-ionic and ionic
salt-stress caused improved activities of enzyme antioxidants in leaf tissues, superoxide
dismutase, peroxidase and catalase up to 2.33, 5.5 and 3.98 folds, respectively
In another recent study Ismail et al. 2016 explored both enzymatic and non-
enzymatic antioxidant profiles in leaves of two contrasting quinoa genotypes related to salt
tolerance. The clear trend of enhanced activity was only found for enzymatic antioxidant,
superoxide dismutase (SOD) under salinity (400m M NaCl) in the salt tolerant Utusaya ,
cultivar (1.5-2 fold increase in intermediate and old leaves, respectively than of salt
sensitive Titicaca cv). Furthermore, total antioxidant capacity (TAC) was age dependent of
leaves, young leaves displayed higher (1.5-fold) TAC than older ones meanwhile salt
tolerant cultivar Utusaya also showed more TAC. While considering non-enzymatic
component of antioxidant system, it was associated with a similar total phenol contents
(TPC) specially in cv Titicaca, For a deeper insight, comprehensive profiling of phenolic
compounds in salt stressed leaves of both cultivar rutin was the main phenol which
accumulated approximately more 27.5-times in young leaves of both cultivars. Thus,
mitigated salt stress in quinoa plants. Overall it is confirmed in halophyte quinoa that both
enzymatic and non-enzymatic components of antioxidant system operate to detoxify ROS
2.14 Molecular identity of Na+ transporters in quinoa
As mentioned before, in quinoa the capability for translocation and ion uptake
during saline regimes had been explored by leaf sap K+, Na+ along with other ionic
measurements and by monitoring the activities of transporters responsible for ionic
movements. Through using studies of molecular techniques, genes related to Na+ exclusion
had been cloned in many plant species to identify the level of salt-tolerance (Shi et al.,
2002). A gene NHX1 of Arabidopsis thaliana encodes tonoplast restricted Na+/H+
21
antiporter which is thought to be involved in Na+ locking in vacuole a critical process
known as compartmentation to avoid Na+ toxicity in cytosol. This ion also works as
additional osmoticum in cytosol for turgor maintenance and uptake of water (Flower and
Colmer, 2008).
The ionic balance in the cytosol under saline regimes is done by plasma membrane
based SOS1 a Na+/H+ exchanger (Batelli et al., 2007). Two SOS1 genes have been
characterized and cloned homologs in quinoa, found similar sequences to SOS1 homologs
of other species Maughan et al. (2009). A Bolivian accession from salares regions,
exhibited strong expression of CqSOS1B and CqSOS1A in roots as compared to leaves in
non-saline conditions. However, both genes were also observed up-regulated in leaves
under saline regimes which indicate that Na+ exclusion was not induced by salt-treatment
at root level (Maughan et al., 2009). Ruiz-Carrasco et al., (2011), also reported different
expression level of these sodium exchanger genes in root and shoot cells in 4 Chilean
genotypes having different potential of salt-tolerance. Furthermore, CqSOS1 was observed
up-regulated in root cells of salt-tolerant line suggesting sodium-tolerance may be linked
with enhanced efflux or reduced translocation of this toxic ion, also revealed in roots of
Arabidopsis plant (Jha et al., 2010). While gene expression of CqNHX1 was boosted at
300 mM sodium chloride salt- treatment, in 15-days old quinoa plants of salt-tolerant PRJ,
UDEC9 and PRP quinoa cultivars but no enhanced activity of this Na+ exchanger was
recorded in salt sensitive cultivar like BO78. In roots, different transcript levels of CqNHX1
were observed for up-regulation in highly salt-tolerant genotypes (PRJ, cultivar of salty
soils) while not in other tested salt-tolerant genotype (PRP), which suggest that salt-
tolerance may also depends upon other mechanistic processes of ion uptake, exclusion,
translocation and compartmentation. The high salinity level triggered an up-regulation of
sodium exchanger CqNHX1 in shoot tissue of salt-tolerant genotypes as compared to salt-
sensitive BO78, and this induction was also recorded in roots of highly salt-tolerant cultivar
PRJ. CqNHX1 expression in different plant parts were evaluated (i.e. leaves and roots)
sixty days after germination. Plants were exposed to 60% sea water salinity. Improved
expressions of CqNHX1 was noticed in roots of most salt-tolerant cultivar (R-49) of
northern Chile than sensitive cultivar BO78 of south in which no expression was observed
neither in shoots nor in roots. Transcriptional variations in CqNHX1 and CqSOS1 were
also evaluated in two salares ecotypes and two valley ecotypes by Morales et al. (2011)
under 450 mM NaCl treatment and during recovery when placed in normal regime. The
stable gene expression in leaves of salt-treated plants (than non-treated controls), proposed
22
that the mechanisms linked with genes remained active. In roots of different ecotypes,
noticeable difference were observed regarding SOS,1 which was highly up-regulated in
treated roots with salts in the salares-ecotypes, which suggests that cytosolic sodium was
excluding out of roots; while gene expression of NHX did not up-regulated by salt-
treatment. Furthermore, under salt-stress, up-regulation of BADH (betain-aldehyde
dehydrogenase) genes, which encodes an enzyme to be involved in gycine betaine
biosynthesis, were also observed in both ectotypes. Yet, few genes had been cloned in
quinoa against salinity. In quinoa, CqNHX and CqSOS could be taken as candidate markers
of salt-tolerance which will also aid in germplasm selection for future breeding purpose.
Article on transcriptome analysis of RNA seq was published by Maughan and coworkers
in NCBI. They analyzed gene expression in altered tissues (flowers, roots and shoots) in
response to different salt-treatments (0, 150, 300 and 450 mM NaCl). This work may prove
a great deal of future investigation of quinoa genes tangled in the responses to salt stress.
Summary of up regulated genes are presented in table 2.4 and depicted in fig 2.3
determining fate of Na+ ion.
Table 2.4 Up regulated genes and their functions during salinity
Genes Function Reference CqSOS1A Na+ exclusion in leaves Maughan et al., 2009 CqSOS1B Na+ exclusion in leaves Ruiz-Carrasco et al., 2011
CqSOS1 Na+ exclusion in roots CqNHX1 Na+ dumping in root/shoot vacuole Morales et al., 2011
BADH Encodes an enzyme, involved in glycine betaine biosynthesis
23
Fig 2.3 Summary of Na+ transport in quinoa plant
2.15 Salinity induced changes in nutritional profile of quinoa grains
Quinoa seeds are gluten free and remarkable source of food for complete nutrition,
owing to their superior protein contents, comprised of all essential amino acids, having high
mineral contents such as Mg, Fe, Zn and Ca and bioactive compounds. Whereas excess
salinity alters composition of seeds of halophytic quinoa (Aloisi et al., 2016) thus affecting
functional properties. Nutrition scientist has started examining the quality of quinoa seeds
harvested from salt regimes because knowledge of nutritional characterization under
marginal conditions will give strategic information for the sack of introduction and its
promotion in new regions and also will be helpful in identification of nutritionally superior
and stable genotypes.
24
‘Titicaca’ a genotype bred in Denmark (Hariadi et al., 2011) and a cultivar of Peru (Koyro
and Eisa, 2008), could give biomass with seeds even at salt stress of 500 mM NaCl.
However, size number of seeds, yield along with C/N ratio were found lower in seeds
harvested from above 300 mM salinity as compared to non-saline conditions. This lowered
C/N ratio was found linked with more protein content of seeds than carbohydrates. In
another proximate analysis of quinoa seeds (Choukr-Allah, 2016) harvested from three
locations Al-Dahid, Ghayathi, and Madinat-Zayed of Mediterranean region irrigated with
saline water of 2.3, 16.3 and 18.9 dS m-1 respectively, it was revealed that saline irrigation
had minimal impact (Rao, 2016) but significant alterations in mineral content were found
particularly, seeds obtained from Madina -Zayed and Ghayathi had more Na+ contents
while less Fe+2 and Ca+2 as compared to Al-Dhaid harvested seeds (Rao, 2016). These
results have serious implications for successful introduction of quinoa on salinized areas so
detailed confirmatory studies are needed.
Seed quality i.e. protein, lipid, fiber and carbohydrates contents, was determined in
a saline irrigated field experiment in southern Italy and found similar values with seeds
harvested from normal irrigated fields except fiber contents which were higher under saline
regimes possibly due to different comparative amount of hull versus rest of seed (Pulvento
et al., 2012). Wu et al. 2016 recorded more protein contents in seeds of quinoa obtained
from plant which were grown at 32 dS m-1 Na2SO4 as compared to non-saline control
plants.
Seeds of ten quinoa accessions (nine from highlands of Bolivia and one Argentinean
accession) were grown under two droughts prone agroecological locations with different
values of soil EC (2 and 7 in Encalilla and Patacamaya respectively). The outcomes showed
that seed proteins percentage was variable mainly due to different genotypes but also
affected by environment and their interactions while significant modulations were recorded
in profile of essential amino acids as compared to total protein contents and grain yield
(Gonzalez et al., 2011). Karyotis et al. 2003, evaluated seeds harvested from plants grown
in central Greece under normal (L1) and saline sodic (L2) soil conditions. Eight quinoa
accessions of different origin i.e. Denmark, Brazil, Chile, Netherlands and the United
Kingdom were under test for protein and mineral composition. Protein concentration was
statistically similar among accessions at L2 and different at L1 furthermore 20 percent
higher at L2 as compared to L1 showing negative relation among seed protein contents and
seed yield. Seed iron and phosphorous contents was also found under at soil conditions,
whereas several other minerals (Ca, Mg, K, Mn and Zn) found significantly low at saline
25
sodic conditions indicating restricted ion accumulation due to marginal soil characteristics.
Meanwhile, accessions from South America were found superior in terms of sufficient
mineral accumulations in seed under both normal and saline sodic regimes.
Recently another pot study was conducted by Aloisi et al., 2016 using three Chilian
ecotypes, to examine alterations in total phenol (TPC), Flavonoid contents (TFC), total
antioxidant activity (AA) and profile of amino acids in protein extract of quinoa seeds
harvested from plants exposed to two salt levels (100 and 300mM NaCl). Chilean landraces
under study were an ecotype belonging salares (R49) and two coastal-lowlands ecotypes,
Villarrica (VR) and VI-1. All derived amino acids from protein hydrolysis except Met and
Ala in R49 were reduced in seeds from salt affected plants particularly in ecotype VI1
while, numerous free amino acids remained stable or even increased in R49 by salinity as
compared to VI1 and VR, indicating greater salt-tolerance in ecotype from salares.
Furthermore, VR showed the maximum AA as well as TPC under non-saline conditions.
Enhanced TPC were observed in all ecotypes tested showing maximum value in R49, and
increased radical scavenging capabilities were recorded in VR and R49.
Phenolics and vitamins keeps antioxidant and harm full radicle scavenging
properties thus reduces lipid peroxidation and also contribute in the nutraceutical and
nutritional profile of chenopode quinoa. Gómez-Caravaca et al. 2012 evaluated the
influence of saline irrigation on phenolic contents in seeds of cv Titicaca. Minute changes
were observed in the level of these bioactive molecules both due to limited irrigation
without or with salt addition while in another study carried out by Gomez-Caravaca et al.
2012 reported that saline irrigation causes decrease in the free phenolic compounds.
Considering another vital class of antioxidant compounds, initial results depicts that the
modulations occurred in tocopherol (vitamin E) content of leaves and seeds of four Chilean
accessions raised on saline (300mM NaCl) regime showing increasing trend depending on
genotype.
Other interesting feature of quinoa seed is that its pericarp contains almost 5 %
saponin contents having broad spectrum properties (antifungal, antimicrobial, insecticidal
etc.) and also has industrial value and can be used as surfactants and detergents. It adds
bitter taste a deleterious feature for initial human perception. On the other side, increased
saponin production may be regarded as an asset in quinoa as a renewable and an alternative
source of saponins (Woldemichael and Wink, 2001; Carlson et al., 2012). 30 % higher
saponin content has been reported under salinity as compared to non-saline regimes
(Gómez-Caravaca et al., 2012). After two-year experimentation in field conditions using
26
cv ‘Tititicaa’ of day neutral in nature, Pulvento et al. 2012 documented linear rise in seed
saponin contents with increase in salinity. Contractedly and also interestingly Gomez-
Caravaca, et al. 2012 seed analysis showed 50% decrease in saponin content with increased
salinity.
Above mentioned reports show that salt-stress may regulate the synthesis of
bioactive compounds, thus influencing quinoa’s nutritional profile and industrial values.
Therefore, growing quinoa on salt-affected soil could be an exciting sustainable practice,
to diminish saponin contents in seeds of quinoa. In addition, this influence can be utilized
as a good tactic to avoid removal of seed outer layers where minerals and vitamins are
localized (Konishi et al., 2004). These two-opposite finding suggests further studies to full
explore the mechanistic bases linking salt-stress with saponin biosynthesis.
Overall quinoa nutritional profile did not significantly disturb by salinity even
improved in some case and found still more superior than common cereals as presented in
table 2.5
Table 2.5 Quinoa nutritional properties i.e. comparison with common cereals and quinoa nutritional properties influenced by salinity % dry weight % Ppm Crop Protein Fat Fibre Ca* P* Mg* K* Na* Fe* Cu Mn* Zn* Quinoa 17 5.0 7 0.19 0.47 0.26 0.87 115 205 67 128 50 Barley 14.7 1.1 2.0 0.08 0.42 0.12 0.56 200 50 8 16 15 Corn 8.7 3.9 1.7 0.7 0.36 0.14 0.39 900 21 - - - Wheat 12.0 1.6 2.7 0.05 0.36 0.16 0.52 900 50 7 - 14 Source: (Vega‐Gálvez et al., 2010; FAO b, 2017) Attribute of quinoa remain stable or even improved under salinity (Karyotis et al., 2003; Wu et al., 2016) *Minerals (Ca, P, Mg, K, Fe, Mn) which were found less in seeds of some quinoa genotypes harvested from saline-sodic fields but still were more than minerals contents of other common cereals (Karyotis et al., 2003 and Rao, 2016)
2.16 Quinoa world trade
The trade of quinoa grain across the globe has been increased exponentially. The
overview of quinoa import demand, world production and prices is high lightened in fig
2.4 and 2.5.
27
Fig 2.4 Quinoa word production (a) and prices (b)
Fig 2.5 Quinoa import demand
0
5
10
15
20
25
30
35
40
45
50
2001 2003 2005 2007 2009 2011
Milli
ons
of U
SD
EU-27 Canada
United States All other countries
43.7
2.4
28
Conclusion
In the light of above quinoa could be grown as an ideal halophyte crop on salinized
soils of this globe. It is evident by the inferences drawn after vast array of experiment (both
green house and open field trials) carried out under moderate to high salt regimes (10 to
750 mM NaCl). The facts and information gathered to date and summarized here shows
that the wide genetic diversity for salt-tolerance is linked to multiple tolerance mechanisms
express differentially under different agroecological (Cold, drought and salinity/sodicity
etc.). Some quinoa accessions are more salt tolerant than other, worth findings are that most
of the accessions tolerate high salinity as compared to any other conventional crop species.
This genetic diversity seems precious source for selection and breeding cultivars adaptable
of most adverse climate and soil conditions. Already information available to date about
morphological, physiological, and molecular basis of salt tolerance can assist breeders in
selection based on traits of interest. Furthermore, genetic diversity for nutritional properties
has also been reported under salinity, nutritional profile did not disturb or even improved
in some genotypes under moderate salt regimes which, offering another important criterion
for selection of genotypes for harsh growing conditions (salinity and drought). Due to its
stress tolerance and enormous nutritional benefits its worldwide demand has been increased
tremendously. All above enormous collection of phrases validates that quinoa is a crop
which could be helpful in improving the livelihoods (generate income), along with food.
29
Chapter 3 Materials and Methods
The study was conducted in following three phases under agroecological conditions
of Faisalabad (32.41° N, 73.07° E) and Pindi-Bhatiaan (31.89° N, 73.27°E) in order to
explore quinoa salt tolerance and adaptability on salt-affected soils.
Experiment 1: Identification of salt-tolerant and sensitive quinoa lines
This experiment was carried out during Nov-Dec, 2012, at wire house (open natural
environment), Department of Crop Physiology, using solution culture as growth medium,
with following details.
Experimental details
3.1.1 Plant material
Four quinoa lines were used in this study. Details of lines are presented in table
3.1.1. Lines are cited hereafter according to their respective local codes.
3.1.2 Raising of quinoa seedlings
Quinoa seedlings were raised in polythene bags (8×6 cm) containing sand, silt and
highly decomposed leaf-compost in equal ratio. Seeds of each quinoa line was sown in bags
@50 seeds per bag on 1st November 2012. After seed sowing, bags were watered with
sprinkler and placed in wire-house providing natural environment. Details of
environmental variables during course of experiment are presented in table 3.1.2 and 3.1.3.
Seedlings emerged, three days after sowing.
Table 3.1.1 Details of quinoa lines used in experiment 1
Code* G. Line** Origin PI 596293 Q1 Colorado, USA Ames 13730 Q2 New Mexico, USA Ames 13737 Q7 New Mexico, USA PI 634919 Q9 Chile (* as per the germplasm database **coding of lines made for local identification) These genotypes are well adapted and gave promising yields Source: Main source was USDA since 2008, Seeds of lines were collected from Department of Crop Physiology, University of Agriculture Faisalabad, Pakistan
30
Table 3.1.2 Weather data of Faisalabad during November 2012.
DATE TEMPERATURE R.H.
RAIN FALL
PAN EVAPORATION
Sun Shine RADIATION
ETO WIND SPEED MAX MIN. AVG.
°C °C °C % mm mm hours mm Km/h
1 28 13 20.5 69 0 3 8.75 2.6 2.6
2 28 13 20.5 63 0 2 8.50 1.7 3.1
3 28 13 20.5 69 0 3 8.50 2.6 4.9
4 28 13.5 20.8 67 0 3 8.50 2.6 3.1
5 28 13.5 20.8 70 0 2.5 8.50 2.1 4.2
6 27 13 20 70 0 3 8.50 2.6 2.6
7 27 14 20.5 72 0 2 8.50 1.7 2.9
8 26 13 19.5 74 0 2 8 1.7 2.6
9 26 13 19.5 74 0 2 8 1.7 4.5
10 26 13 19.5 78 0 2 8 1.7 3
11 25 12 18.5 80 0 2 0 1.7 1.9
12 25 11.5 18.3 80 0 2 6.50 1.7 1.5
13 25 11.5 18.3 81 0 2 6.50 1.7 1.2
14 24 11 17.5 74 0 2 7 1.7 3.5
15 24 10 17 78 0 2 7.50 1.7 3.8
16 24 10 17 74 0 3 7.50 2.6 2
17 24 11 17.5 71 0 1.5 7.50 1.3 1.9
18 24 10 17 74 0 2 7 1.7 2.5
19 24.5 10 17.3 71 0 2 7.50 1.7 4.4
20 24 11 17.5 68 0 2.5 7 2.1 5.3
21 24 12 18 63 0 3 8 2.6 2.6
22 23 9 16 65 0 2 8 1.7 3.4
23 23 9 16 72 0 3 8.50 2.6 3.1
24 23 8 15.5 83 0 2 8.50 1.7 3
25 23 7 15 73 0 3 9 1.7 4.7
26 23 6.5 14.8 73 0 3 8 2.6 2.3
27 22 7 14.5 72 0 2 8 2.6 4.5
28 22 9 15.5 72 0 2 7.75 1.7 3.4
29 22 8 15 84 0 2 7 1.7 4
30 23 7 15 76 0 2 8.50 1.7 5
AVERAGE: 24.8 10.8 17.8 73.0 0 2.3 7.6 2 3.2
31
Table 3.1.3 Weather data of Faisalabad during December 2012.
DATE TEMPERATURE
R.H. RAIN FALL
PAN EVAPORATION
Sun Shine RADIATION
ETO WIND SPEED MAX MIN AVG
°C °C °C % mm mm hours mm Km/h
1 21 5 20.5 75 0 3 8.50 2.1 5.4
2 21 4.5 20.5 75 0 2 8.50 1.7 1.8
3 21 4 20.5 75 0 2 8.50 1.7 1.8
4 21 4.5 20.8 73 0 1.5 8.50 1.3 4.4
5 22 4.5 20.8 77 0 2 8.50 1.7 5
6 23 4.5 20 80 0 2 8.25 1.7 3.7
7 23 8 20.5 73 0 3 8.25 2.6 3.8
8 24 7 19.5 74 0 3 8.25 2.6 2.7
9 25 5 19.5 71 0 2 8 1.7 2.2
10 23 5 19.5 70 0 3 8 2.6 2.9
11 23 5 18.5 76 0 2 8 1.7 2.6
12 18 9 18.3 89 0 3 0 2.6 3.6
13 18 13 18.3 89 0 1 0 .9 4.3
14 18 9 17.5 94 16 .5 2 .4 1
15 14 9 17 81 0 1 8 .9 2.5
16 17 7 17 76 0 2 8 1.7 2.5
17 18 6.5 17.5 78 0 2 8 1.7 4.7
18 17.5 9 17 77 0 2 8.25 1.7 3.4
19 18 7 17.3 76 0 2 8.50 1.7 6.8
20 19 6.5 17.5 70 0 3 8 2.6 4.1
21 19 6 12.5 76 0 1.5 8.25 1.3 4.1
22 19 5 12 73 0 1.5 8 1.3 3.2
23 19 5 12 73 0 1.5 8 1.3 2.7
24 20 5 12.5 88 0 1 8 .9 2.5
25 20 5 12.5 89 0 1 4 .9 3.3
26 20 5 12.5 89 0 1 0 .9 3
27 14.5 6 10.3 97 0 1 0 .9 3
28 11 8 9.5 100 1.2 1 0 .9 2.7
29 11 3.5 7.3 100 0 .5 0 .4 2
30 11.5 3 7.3 98 0 .5 0 .4 1.9
31 11.5 3.5 7.5 94 0 .5 0 .4 2.5
AVERAGE 18.8 6.1 12.4 81.5 17.2 1.7 5.8 1.4 3.2
32
3.1.3 Shifting of seedlings in soilless culture
At true leaf stage (7-8 days after emergence), seedlings of each line were
transplanted in plastic tubs containing 20 L half-strength Hoagland-solution culture
(Hoagland and Arnon, 1950). Transplants were rolled with foam and plugged on floating
thermophore-sheets. Continuous aeration was provided in solution-culture using electric
aquarium pump. Tubs were placed in open wire-house conditions. Twenty plants of each
quinoa line were shifted in each tub.
3.1.4 Salinity imposition
Two days after shifting of seedlings in solution culture, Salinity was imposed in
increments of 50 mM, each increment was made after 12 h, in order to avoid abrupt osmotic
stress using commercially available salt of NaCl. Total five levels of salt-stress were
developed i.e. 0 (control), 100, 200, 300 and 400 mM NaCl in separate tubs. Seedlings of
different quinoa lines were allowed to grow in treatments culture up to one month.
Dependent Variables recorded
3.1.5 Determination of Na+ and K+ contents in leaves and roots
Fully-expanded young leaves; sixth leaf from the top, three leaves, from three plants
and their respective roots of each quinoa line were collected from each salt-treated and non-
treated tub and recorded their fresh weights, leaf and root dry weights were also recorded
after oven drying. Dried leaf and roots were taken in separate glass tubes, 25 ml of 1%
HN03 was added in tubes as digestion solution. Tubes containing plant material plus
digestion solution were kept on hot plate at 80°C for 5 h. After digestion, 1 ml solution
from digestion tube was taken in another measuring cylinder and diluted by adding double
distilled water up to volume of 20 ml. Diluted solution was used to take absorbance of Na+
and K+ using flame photometer (Sherwood, UK, Model 360) (Munns and James, 2003;
Shavrukov et al., 2009). Later on, leaf Na+ and K+ concentrations were calculated by
putting absorbance of flame photometer in standard-curve equation generated from Na+
and K+ standards absorbance readings.
3.1.6 Recording of morphological and growth traits.
Twenty-five days after seedling transplantation in solution culture, five plants of
each quinoa line from each salt treated tub were un plugged to record morphological and
33
growth traits. Root and shoot lengths were measured on centimetre scale, root length was
taken from point of junction of root and shoot to the tip of most lengthy root while shoot
length was recorded from root-shoot junction to upper most leaf. Root and shoot fresh
weights were recorded by putting on digital balance. Using digital balance root and shoot
dry weights were recorded after oven drying at 60° up to persistent dry weight.
3.1.7 Leaf senescence’s number
Number of senescence leaves were counted manually from five plants of each line
from each treated and non-treated tube and then average number was calculated.
3.1.8 Statistical analysis
Each treatment was replicated four times, and data were statistically analyzed by a
two-way ANOVA analysis under a completely randomized design (CRD) comprising
factorial arrangement using statistical software “statistix” (ver8.1, Tallahasse, FL, USA).
Salinity levels and quinoa lines were taken as factor. Graphs of mean values are presented
with standard error bars provided with P values.
Experiment: 2 Physiological performance of quinoa lines under wire house saline
conditions
This study was conducted in pots placed in wire house (open natural environment),
Department of Crop Physiology, University of Agriculture during November 2012 to April,
2013 with following details
Experimental details
3.2.1 Plant material
Four quinoa lines were used in this study, same which were used in experiment 1.
3.2.2 Imposition of salinity in pots
Four quinoa lines were evaluated for their salt tolerance in a pot trial. The soil used
in this study was analysed according to standard procedures and its chemical and physical
characteristics are presented in table 3.2.2. Before filling soil, the pots were lined internally
using polythene sheet and hole at bottom was also blocked with cork in order to stop
leaching. Five salinity regimes (dS m-1). 1.51 (ambient), 10, 20, 30 and 40 were created by
adding desired amount of NaCl salt in each pot and mixing was carried out with help of
34
mechanical mixer one month prior to sowing according to protocol described in US staff
handbook-60. Twenty-five seeds of each quinoa line were sown on 25 November 2012, in
every pot comprising 13 kg soil pot-1. Every treatment was tetra replicated. Five (1.51, 10,
20, 30 and 40 dS m-1) reference pots were also maintained to notice the effect of irrigation
water on developed salinity. ECe was recorded regularly in the reference pots and from
pots containing plants at the end. No significant changes in levels of salinity was observed
at end. Pots were placed in wire-house under natural light and temperature according to
completely randomized design. Details of environmental variable are given in table 3.2.1
Each pot was supplement with P and K @ 60 kg ha-1 as basal dose using DAP and SOP
fertilizers while N dose was applied @ 75 kg ha-1 using urea half dose at sowing and half
at flowering. Pots were provided with good quality water @ 150 ml/day/pot, average of
water applied during whole experimental period. After emergence, extra plants were up
rooted to maintain equal plant population of five plants/pot.
Table 3.2.1 Weather data during the experimental period Month Rainfall
(mm) Relative Humidity (%)
Temperature (°C)
Monthly Maximum
Monthly Minimum
Daily Mean
Sunshine (h)
November-2012 0 73 24.8 10.8 17.8 7.6 December-2012 17.2 81.5 18.8 6.1 12.4 5.8 January-2013 1.5 77.6 16.8 3.5 10.2 5.1 February-2013 55 81 19.2 8.3 13.8 5.2 March-2013 1.3 61.2 27 13 20 8.6 April-2013 21.6 36.7 33.5 19.7 26.6 8.9 May-2013 4.6 24.5 39.7 24.4 32 10.4 *All the values of mean temperature, relative humidity and sunshine shown in table are the monthly averages, while rainfall values are the total rainfall received during that month; Monthly maximum and monthly minimum are the highest and lowest temperature observed during that month at any day
35
Table 3.2.2 Physical and chemical characteristic of soil used in experiment 2 Characteristics Units Values Sand % 52.1 Silt % 27.1 Clay % 19.2 Textural class - Sandy clay loam Saturation parentage % 31 pHe - 7.6 ECe dS m-1 1.41 Available phosphorus (oslon) mg kg-1 3.31 Extractable potassium (NH4OAC) mg kg-1 82 Organic matter % 0.71 Total Nitrogen % 0.06
Dependent-variables recorded
Data regarding leaf Na+ and K+ concentrations, water-relations, gas-relations, leaf
biochemical analysis and yield parameters were collected at different stages as mentioned
below.
3.2.3 Determination of Na+ and K+ contents in leaves and roots
Fully-expanded young leaves; sixth leaf from the top, three leaves, from three plants
and their respective roots of each quinoa line were collected from each treated and non-
treated pot and recorded their fresh weights, leaf and root dry weights were also recorded
after oven drying. Na+ and K+ content in leaves and roots were measured according method
followed by Shavrukov et al. 2009 also explained earlier in section 3.1.5.
3.2.4 Estimation of leaf water-potential
Forty-five days after sowing, in morning (5-6 a.m.), leaf water-potential (ѱw) was
assessed from detached, fully-expanded young leaf using Scholander-type water potential
equipment (Arimad-2-Japan). Five leaves/line/treatment were assessed to record leaf
water-potential (ѱw).
3.2.5 Estimation of leaf osmotic-potential
Same leaf used for leaf water-potential (ѱs) assessment was placed in freezer at -
30°C for 1 week. After that, frozen leaf was pressed in eppendorf tube with glass rod to
36
extract sap. Leaf osmotic-potential was determined from sap using an osmometer (Vapro,
Model 5520, USA).
3.2.6 Estimation of leaf turgor-potential
Following formula was used to calculated leaf turgor-potential (ѱp).
ѱ𝑝𝑝 = ѱw − ѱ𝑠𝑠
3.2.7 Measurement of leaf gas-exchange relations
Fifty days after emergence, C0 2 assimilation-rate (A), Stomatal-conductance (gs)
and transpiration-rate (E) were determined by placing young fully-developed leaf (usually
3rd from the top) in square leaf-chamber of an infrared gas-analyser (Analytical
development company, Hoddeson, UK). Three leaves/line/treatment were used to take
readings. Readings were taken between 100:00 am to 1:00 pm. Specifications displayed
on LED of equipment were i.e. range of leaf-chamber temperature (25-28°C), ambient C02
(370 µmol/mol), molar gas-flow rate at leaf-chamber (398 µmol/s) leaf-chamber gas-flow
rate (297ml/min), PAR at leaf-surface up to 769 µmol/m-2/s
3.2.8 Leaf biochemical analysis
Sixty-days after emergence, fully expanded-young leaves were detached from three
tagged plants of each pot. Leaf-sampling was done in the morning before 5:00 am, collected
leaves were wrapped in aluminium foil and packed in plastic zipper-bags, then put in ice
box and then placed in freezer until analysis. Following attributes regarding leaf bio-
chemical analysis were measured within two days.
3.2.9 Leaf Chlorophyll-contents
To extract chlorophyll-contents, leaf material (0.5g) was grounded in 10 ml acetone
(80%) in pestle and mortar. Grounded material was shifted in falcon tubes, falcon tubes
containing grounded material were placed in centrifuge for 10 min at 15000 rpm. Resulted
supernatant was taken in quartz-cuvette. Cuvette was placed in spectrophotometer
(UV4000) to record absorbance of supernatant at 663 and 645 nm wavelengths against 80%
acetone as blank. Calculations of leaf chlorophyll-contents were according to following
equations.
37
3.2.10 Leaf total-phenols (mg g-1)
Total phenols in leaves were estimated by method of Waterhouse, (2001). To draw
calibration-curve, standard solutions of gallic-acid (i.e. 500, 250, 150 and 100 mg L-1 gallic-
acid) were prepared. To extract phenolics, leaf material (0.5g) was grounded in 10 ml
acetone (80%) in pestle and mortar. Grounded material was shifted in falcon tubes, falcon
tubes containing grounded material were placed in centrifuge for 10 min at 15000 rpm.
Resulted supernatant (20 µL) or standards were taken in test-tube separately, (two sets were
prepared one for gallic acid standard and second for leaf extract). To start reaction 1.58 ml
DW, 100 µL Folin-Ciocalteu reagent and 300 µL sodium-carbonate (20%) was added in
each test tube. Reaction blend was kept in water-bath at 40°C for 30 min. After that
reaction-mixture was shifted to quartz-cuvette. Cuvette was placed in spectrophotometer
(UV4000) to record absorbance of reaction mixture at 760 nm wavelength against 80%
acetone as blank. Calibration-curve of standards and absorbances was plotted and total-
phenols in leaf-samples were calculated by this generated calibration-curve and reported as
gallic-acid equivalent.
3.2.11 Free-proline determination in leaf
Free-proline in leaf was measured according to method of Bates et al. 1973.
Chemical used
i. 6 M Phosphoric acid
ii. 3% Sulphosalicylic acid
iii. Acid-ninhydrin
Preparation of solutions:
To prepare 6 M phosphoric acid, 407 ml of phosphoric acid was taken in measuring
cylinder and volume was increased by adding distilled water up to mark of 1000 ml. Three
g of sulphosalicylic acid was taken in measuring in another cylinder and distilled water was
added up to mark of 100 ml to make 3%. 1.25 g of acid-ninhydrin was taken in flask ,30
ml glacial acetic-acid and 20 ml of 6 M phosphoric acid was added in flask. Flask was
placed on shaker until dissolved. The mixture was kept at 4°C and used within a day.
Chlorophyll "a" (mg g− 1) = [(0.0127×A663– 0.00269×A645)×100]/0.5
Chlorophyll "b" (mg g− 1) = [(0.0229×𝐴𝐴645− 0.00468×𝐴𝐴663)×100]/0.5
38
Extraction of leaf-proline
To extract proline, leaf material (0.5g) was grounded in 10 ml sulphosalicylic acid
(30%). Grounded material was filtered over filter-paper. 2 ml of each filtrate, acid-
ninhydrin and glacial acetic-acid was taken in test-tube. Test-tubes containing reaction-
mixture was covered with aluminium foil and shifted to water-bath for heating at 100°C
for 1 h. Immediately, after 1 h, test-tubes were placed in ice-bath for termination of reaction.
After that in each test-tube, addition of 4 ml toluene was made and vigorous shaking was
done for 10-15 seconds. The test-tubes were allowed to stay at room-temperature for few
min and pink coloured ring developed. This ring was taken in quartz’s cuvette with help of
pipette and cuvette was placed in spectrophotometer (UV4000) to take absorbance at 520
nm wavelength. Three % sulphosalicylic acid was used as blank, 2 ml sulphosalicylic acid
(3%) was taken instead of filtrate and all procedure was repeated for blank.
Preparation of proline-standard.
Proline (0.1g) was taken in measuring cylinder and volume was increased up to
mark of 1000 ml by adding double distilled water and got final concentration of stock
solution (200ug/2ml proline). Working solutions of 10-50ug/2ml of proline were prepared
by further dilutions. The absorbance of working solutions was taken at 520 nm by
spectrophotometer (UV 4000).
Plotting of Standard curve
Standard curve was plotted by taking concentration of working solutions on x-axis
and respective absorbances at Y-axis and got following equation to calculate proline.
𝑦𝑦 = 35.9𝑥𝑥 + 0.49
3.2.12 Growth and yield determination
Plant height and main panicle lengths were measured at maturity with the help of
centimetre scale, while stem diameter was measured with the help of Vernier calliper at
three points of stem i.e. upper middle and bottom than averages was calculated.
Harvesting was done from each pot on 10 April 2013 when plants were matured as
according to suggestions cited in Jacobsen and Stølen (1993) article. Inflorescences and
other plant parts were dried on filter paper at 25-30 °C. After ten days’ seeds were threshed
manually. Dry matter was determined by drying remaining plant part at 65 °C for one week,
39
and added to seed weight. Later on, thousand seeds weights were also recorded using digital
balance.
3.2.13 Statistical analysis
Each treatment was replicated four times, and data were statistically analyzed by a
two-way ANOVA analysis under a completely randomized design (CRD) comprising
factorial arrangement using statistical software “statistix” (ver8.1, Tallahasse, FL, USA).
Salinity levels and quinoa lines were taken as factor. Graphs are presented with standard
error bars provided with P values.
Experiment 3: Physiological and agronomic performance of quinoa under field
salinity
Experimental details
3.3.1 Experimental locations
Open field experiments were conducted (during 2013-14) in two locations of the
University of Agriculture, Faisalabad (elevation 184 m above sea level 31.4187° N,
73.0791° E) and Soil Salinity Research Institute Pindi-bhatian (elevation 190 m above sea
level 31.8950° N, 73.2706° E), Central Punjab, Pakistan. Two fields from Faisalabad
(normal (S0) and salt affected (S1), and two from Pindi-bhatian (normal (S2) and salt
affected (S3) were selected. Salinity and sodicity levels are given in Table 3.3.2. The
climate of the region under study is semi-arid with annual average rainfall of 325 mm and
evaporation of 1600 mm. The annual average temperature is 32 °C. Weather details of both
experimental locations are presented in fig 3.3.1.
3.3.2 Crop husbandry
Relative daylength neutral salt-tolerant quinoa lines (selected from experiment 1
and 2) were used (Table 3.3.1). Genotypes are cited hereafter according to their respective
local codes. The experimental design was randomized complete block design with split-
plot arrangements. Soil type (normal or salt affected) was used as main plot factor while
lines were assigned in sub plots (15m-2). Soil (0–30 cm depth) and tube-well water samples
were collected before sowing and analyzed according to methods cited in US staff
handbook-60 (1962) and presented in table 3.2.2. Salt affected soils were of saline-sodic
nature. Soils were prepared by two ploughings (depth 12cm) followed by planking to
40
conserve moisture suitable for germination. Seeds were sown manually in 22 cm between
rows at 2-3 cm depth at seeding rate 10 kg ha-1 on 20 November 2013, at Faisalabad and
on 22 November 2013 at Pindi Bhatian. Soil nutrient supplementation was done @
75:50:50 N: P: K kg ha-1 using Urea and DAP and SOP as fertilizer source. Full dose of P,
K and half dose of N were applied as basal dose during soil preparation while half dose of
N was applied during flowering stage. No major climatic hazard happened during crop
growth duration. Three irrigations were done equating 10 deltas of water. No insect pest
attack was found on crop at all locations. Weeds were removed manually 30 days after
sowing from normal fields of both locations, while no weeds were found in salt affected
fields.
Fig 3.3.1 Weather details of experimental locations during 2013-14
Table 3.3.1 Details of quinoa lines used in experiment 3
Code* G. Line** Origin Ames 13730 Q2 New Mexico, USA Ames 13737 Q7 New Mexico, USA (* as per the germplasm database **coding of lines made for local identification) These genotypes are well adapted and gave promising yields Source: Main source was USDA since 2008, Seeds of lines were collected from Department of Crop Physiology, University of Agriculture Faisalabad, Pakistan
41
Table 3.3.2 Characteristics of soil and water of two experimental locations
Soils S0 S1 S2 S3 EC dS m-1 2.11 9.8 3.21 13.9 SAR (mmol L-1)1/2 5.2 25 11.9 42 pH 7.4 8.1 7.9 8.8 Textural Class Sandy loam Clay loam Clay loam Clay loam Water S0 S1 S2 S3 EC dS m-1 1.32 2.53 1.52 1.52 SAR 7.41 11.4 7.62 7.62 EC, electrical conductivity. SAR, sodium adsorption ratio S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively
Dependent variables recorded
Data regarding following attributes were recoded using standard procedures
3.3.3 Estimation of stand establishments
Fourteen days after sowing, seedling-emergence count was made from 2 m-2 area
of each experimental unit to record score of crop-density according to scale described in
Table 4.3.1.
3.3.4 Determination of Na+ and K+ contents in leaves and roots
Twenty-five days after emergence, fully-expanded young leaves; sixth leaf from the
top, three leaves, from three plants and their respective roots of each quinoa line were
collected from each plot and recorded their fresh weights, leaf dry weights were also
recorded after oven drying. Na+ and K+ contents in leaves were recorded as explained
earlier in section 3.1.5
3.3.5 Assessment of Leaf water-relations
Forty-five days after sowing, in morning (5-6 a.m), leaf water-potential (ѱw), leaf
osmotic potential and leaf turgor potential were assessed from detached, fully-expanded
young leaf as explained earlier in sections 3.2.5, 3.2.6 and 3.2.7.
42
Leaf biochemical analysis
Sixty-days after emergence, fully expanded-young leaves were detached from three
tagged plants of each plot. Leaf-sampling was done in the morning before 5:00 am,
collected leaves were wrapped in aluminium foil and packed in plastic zipper-bags, then
put in ice box and then placed in freezer until analysis. Following attributes regarding leaf
bio-chemical were measured within two days.
3.3.6 Determination of leaf Chlorophyll-contents
Leaf chlorophyll was recoded according to protocol given in section 3.2.9
3.3.7 Determination of leaf total-phenols (mg g-1)
Leaf total-phenols were measured according method of Waterhouse, (2001) as
explained in details in section 3.2.10
3.3.8 Free-proline determination in leaf
Free-proline in leaf was measured according to method of Bates et al. 1973 as
explained earlier in section 3.2.11
3.3.9 Determination of antioxidants
Sixty-eight days after emergence, fully expanded-young leaves were detached from
three tagged plants of each plot. Leaf-sampling was done in the morning before 5:00 am,
collected leaves were wrapped in aluminium foil and packed in plastic zipper-bags, then
put in ice box and then placed in freezer until analysis. Antioxidants were measured within
two days. Leaf material (0.1g) was grounded in 1 ml phosphate buffer (50 mM; pH 7.8) in
pre-chilled pestle and mortar. Grounded material was shifted in pre-chilled Eppendorf
tubes, Eppendorf tubes containing grounded material were placed immediately in
centrifuge for 20 min at 15000 rpm at -4°C and the supernatant was collected to use in
assays to determine the activities of antioxidant enzymes, SOD (Giannopolitis and Ries,
1977), POD and CAT (Chance and Maehly, 1955) by recording absorbance at 560, 470 and
240, respectively. Leaf ascorbate content were measured at 525 nm following the protocol
described by Yin et al. (2008) using ascorbic acid standards as reference.
43
3.3.10 Estimation of Crop-growth rate
For estimation of crop-growth rate, quinoa plants from 0.5 m-2 area were cut
manually from base using sickle from each experimental-unit. Fresh weights of plants were
recorded using digital balance. After that leaves were separated from stem, weight of leaves
and stem was taken separately. For total dry-matter (TDM) estimation, samples of stem
(50 g) and leaves (50 g) were taken and placed in oven at 60°C until constant weight. For
estimation of crop-growth rate, first harvest was taken 20 days after sowing, and subsequent
four harvests were made after fortnight interval from each experimental unit, crop-growth
rate (CGR) was calculated in g m-2 day-1 units using following equation derived by Hunt,
(1978).
Where
W1=TDM at first-harvest
W2=TDM at second-harvest
t1= Date of first-harvest
t2= Date of second-harvest
3.2.11 Biomass and seed yield related traits determination:
Plant height and main panicle lengths were measured at maturity with the help of
centimeter scale, while stem diameter was measured with the help of Vernier caliper at
three points of stem i.e. upper middle and bottom than averages was calculated. Number of
sub panicles were counted manually
Harvesting was done from three central rows from each plot on 15 April 2014 at S0
and S2 and on 28 April 2014 at S1 and S3, when plants were maturing according to
suggestions reported by Jacobsen and Stølen (1993). Inflorescences were dried on filter
paper at 25-30 °C. After ten days seeds were threshed manually. Dry matter was determined
by drying at 65 °C for one week, and added to seed weight. Thousand grains were counted
CGR =W2−W1
t2 − t1
44
manually, from each three samples of each experimental unit and recorded their weight on
digital balance than averages were calculated.
3.3.12 Seed mineral estimation:
Seed mineral elements (K, Ca, Na, Mg, Mn, Cu, Zn and Fe) were determined using
an atomic absorption spectrophotometer (AAS; Shimadzu instruments, Inc., Spectra AA-
220, Kyoto, Japan) after digesting in a di-acid mixture (3:1) of HNO3:HCLO4 at hot plate
for two h following the rules of AOAC (1990) method No. 3.014-016.
3.3.13 Seed protein estimation
Flour of seed samples was made by grinding in coffee grinder. Nitrogen content in
flour samples was estimated by Kjeldahl’s method. Nitrogen percentage was calculated by
using following Equation
Seed protein percentage was calculated by multiplying % nitrogen with 5.7.
3.3.14 Seed crude-fat estimation
Crude fat from each sample was determined using Soxhlet apparatus as described
in respective method stated in AACC (2000). Crude fat was extracted using petroleum-
ether from 5 g quinoa grain floor at condensation-rate of 2-3 drops/sec. for 16 h. After
distillation of extra ether, the extraction-flask residue was dried at 100°C for 30 min. up to
constant-weight. Crude-fat was determined using following equation.
3.3.15 Seed crude-fiber estimation
The quinoa grain flour samples were tested to determined crude-fiber contents after
crude-fat extraction as described in respective method stated in AACC (2000). Two g fat-
free sample was digested using 200 ml boiling H2SO4 (1.25%), then filtration and washing
of samples were done. Again, digestion of samples was done for 30 min. with 200 ml
Nitrogen % =Titer of 0.1 N H2SO4 used×0.0014×250
weight of sample×vol. of aliquot×100
%Crude Fat =Wt. of ether
Wt. of flour sample X100
45
(1.25%) of boiling NaOH, followed by filtration and three time washing. The resulted
residue drying was carried out for 2 h. at 130°C and weighed. Dried-residue was burnt at
600°±15°C, after that resulted material was cooled and reweighing was done. The crude-
fiber was determined using following equation.
3.3.16 Quinoa seed saponin estimation
Saponins were estimated in quinoa grains according to standard-afrosimetric
method used by Kozial et al. (1991), 0.5-g. quinoa grains were weighed in a test-tube of
dimensions 160×16 mm. After that 5-ml. distilled-water was added in tubes, then tubes
were caped.
1-Tubes were shaken vigorously manually in up/down directions (4 shakes) for 30 s.
2-Tubes were placed in wracks in rest position for 30 min.
3-Tubes were again shaken according to no 1 and 2 points
4- After period of second rest, tubes were again shaken for 30s. by giving last shake-down
like one would shake a thermometer.
5- Tube were again placed for rest period for 5 min.
6- Measured the foam-height with centimetre scale
7- Saponin estimation was done using following equation
𝑀𝑀𝑀𝑀. 𝑆𝑆𝑆𝑆𝑝𝑝𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝑝𝑝𝑝𝑝𝑝𝑝 𝑀𝑀.𝑓𝑓𝑝𝑝𝑝𝑝𝑠𝑠ℎ 𝑤𝑤𝑤𝑤 =0.646×𝑓𝑓𝑆𝑆𝑆𝑆𝑓𝑓 ℎ𝑝𝑝𝑆𝑆𝑀𝑀ℎ𝑤𝑤(𝑐𝑐𝑓𝑓) − 0.14
𝑠𝑠𝑆𝑆𝑓𝑓𝑝𝑝𝑠𝑠𝑝𝑝 𝑤𝑤𝑤𝑤.𝑀𝑀 (𝑀𝑀)
3.3.17 Statistical analysis
Each treatment was replicated four times, and data were statistically analysed by a
two-way ANOVA analysis under a randomized complete block design (RCD) comprising
split plot arrangements. Soil type was main plot factor (A) and genotype was sub plot (B).
% Crude fiber = Loss in weight on ignition −blankWeight of sample
×100%
46
Software was “statistix” (ver8.1, Tallahasse, FL, USA). Linear regression was calculated
to find the relation between crop stand and seed yield. The two locations were analysed
separately. Graphs of mean values are presented with standard error bars provided with P
values.
47
Chapter 4 Results and Discussion The results pertaining to the experiments conducted regarding adaptability and yield
potential of quinoa on salt affected soils during year 2012-2014 are presented and discussed
below
Experiment 1: Identification of salt-tolerant and sensitive quinoa lines
Results
4.1.1 Shoot growth
Data presented in Fig 4.1.1(a, b and c) indicate that shoot length, shoot fresh weight
and shoot dry weight of quinoa lines were significantly (P ≤ 0.001) affected by salt stress.
Increased shoot lengths and shoot dry weights of all quinoa lines were found at 100 mM
NaCl saline soilless culture than onwards decreased with further increase in salt stress level
(Fig 4.1.1a, c). However, lines Q-7 and Q-2 had higher shoot lengths and shoot dry weights
than Q-1, and Q-9 at all salt regimes (Fig 4.1.1a, c). Furthermore Q-2 and Q-7 maintained
reasonable growth even at 300 mM salt stress while drastic growth reductions of Q-1 and
Q-9 were found at 300 and 400 mM NaCl stress (Fig 4.1.1a-c).
4.1.2 Root growth
Significant (P ≤ 0.001) increases in root dry weights of quinoa lines (Q1, Q2, Q7
and Q9) were observed at 100 mM NaCl salt stress when compared with 0 mM (non-saline)
(Fig 4.1.2 c). After that significant decreases were found at 300 and 400 mM. Furthermore,
lines Q-2 and Q-2 produced higher root dry weight than Q1 and Q9 at 200 and 300 mM
salt stress (Fig 4.1.2 c). Root fresh weight of all quinoa lines decreased significantly at 300
and 400 mM stress while Q2 and Q-7 had more fresh weights than Q1 and Q-9 at this level
of salt stress (Fig 4.1.2 b). Root lengths of all quinoa lines were statistically at par at all salt
levels (Fig 4.1.2 a).
4.1.3 Root Na+, K+ and Na+/K+ ratio
The influence of NaCl salt stress on root Na+ and K+ contents of quinoa lines are
shown in (Fig 4.1.3 a, b). Root Na+ contents increased with salt increments in the growth
medium in all lines (Fig 4.1.3 a). Furthermore, lines Q-2 and Q-7 had more Na+ contents
in roots at 400 mM NaCl salt stress. While root K+ contents gradually decreased with
increase in NaCl salt in the soilless growth medium (Fig 4.1.3 b). Root K+/Na+ ratio also
decreased drastically when plants were exposed to 400 mM NaCl stress (Fig 4.1.3 c).
48
4.1.4 Leaf Na+, K+ and Na+/K+ ratio
The influence of salt stress on leaf Na+ and K+ contents of quinoa lines are presented
in (Fig 4.1.4 a, b). Both Na+ and K+ contents were significantly (P ≤ 0.001) higher in leaves
of all quinoa lines with increasing salinity (Fig 4.1.4 a, b). Lines Q1 and Q2 had more Na+
in leaves than Q7 and Q9 at all incremented NaCl levels (Fig 4.1.4 a) while maximum leaf
K+ contents were found in Q2 and Q7 at 400 mM NaCl salt regime (Fig 4.1.4 b).
4.1.5 Leaf senescence
Leaf senescence number per plant was also counted and presented in Fig 4.1.5. Less leaf
senescence was observed at salinity levels above 200 mM NaCl stress in all quinoa lines.
Leaf senescence was higher in genotype Q1 and Q7 at 0 mM NaCl stress.
49
Fig 4.1.1 Shoot length (a), shoot fresh weight (b) and shoot dry weight (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
50
Fig 4.1.2 Root length (a), root fresh weight (b) and root dry weight (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
51
Fig 4.1.3 Root Na+ concentration (a), root K+ concentration (b) and root K+/Na+ ratio (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
52
Fig 4.1.4 Leaf Na+ concentration (a), leaf K+ concentration (b) and leaf K+/Na+ ratio (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
53
Fig 4.1.5 Leaf senescence of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
54
Discussion
Conduct of experiments in hydroponic culture is reliable in the way that treatments
can be maintained accurately and precisely as well as reproducible results about plant
responses can be measured. Genetic differences for abiotic stress-tolerance within species,
can be assessed with confidence. Moreover, roots in soilless-culture are easily accessible,
for investigating morphophysiological changes. Screening on salt affected soil is
confronted by heterogenous soil physiochemical properties, also due to variable rainfall
(Munns and James, 2003). Hence, majority of experimentation has been carried out in
controlled conditions for identification of salt tolerant lines in many crops (Dasgupta et al.,
2008) except quinoa. Therefore, this hydroponic study was conducted with objective to
identify salt-tolerant quinoa lines by assessing morphological modulations and ion
accumulation in leaf and root at varying salt regimes in local climatic conditions
Although quinoa behaves like facultative halophyte still huge genetic diversity
exists in quinoa lines for salt-tolerance under different salt and climatic conditions (Adolf
et al., 2012). There was increased leaf Na+ in all quinoa lines under soilless culture, while
genotype Q2 and Q7 had relatively lower Na+ in leaves than Q1 and Q9 (Fig 4.1.4 a) which
indicates salt tolerance behavior. High concentration of Na+ is damaging for both
glycophytes as well as halophytes, therefore, should be avoided (Adolf et al., 2012). It
involves an efficient Na+ dumping in leaf vacuole or Na+ translocation to older leaves. Na+
concentration was measured in young leaves in this study and usually young leaves had
lower degree of accumulation (Adolf et al., 2012). It seems that lines Q2 and Q7 responded
at whole plant level by translocating Na+ in older leaves that leads to low Na+ loads in
young leaves or it also seems restitrctions of Na+ at root paranchyma as root Na+ contents
of Q2 and Q7 were higher while growing in saline solutions especially at 400 mM NaCl
(Fig 4.1.3 a). This strategy of low accumulation of Na+ might also be linked with
preferential K+ uptake at root parenchyma and translocation to leaf, as leaf K+ concentration
was also recorded higher in Q7 and Q2 (Fig 4.1.4 a). Quinoa plants accumulate more K+ in
leaves under salt stress (Adolf et al., 2012), which was also confirmed in this study. Thus,
not only concentration of Na+, but K+/Na+ ratio is taken as very vital criterion for selection
of genotypes and had been used frequently as salinity-tolerance index (Houshmand et al.,
2005). Also in this hydroponic study, leaf K+/Na+ ratios of Q2 and Q7 were higher than Q1
and Q9. Ruiz-Carrasco et al. (2011) reported some Na+ exchangers Cq-SOS1 (location
plasma membrane: role exclusion) and Cq-NHX1 (location tonoplast: role dumping off in
55
vacuole) in both leaf and root cells of salt tolerant quinoa genotypes having direct role for
this ion homeostasis.
Low salt concentration up to 100 mM NaCl had significantly improved shoot (Fig
4.1.1 b, c) and root growth of quinoa lines (Fig 4.1.2 b, c). Therefore, this level could be
considered optimum for growth. In quinoa and several other plant species salts induced
stimulation in growth has been found at moderate salinity levels (Hariadi et al., 2011). This
might be due to increase in tissue water contents (Khan et al., 2005) which could also be
linked with relatively more ion accumulation. Noticeable less plant growth was found due
to salt increments higher than 100 mM NaCl, adversely affected at sea level salinity. Still
this plant was able to thrive at sea level concentrations. Less biomass production in
halophytes due to high salinity in growth medium is rather common (Koyro et al., 2006;
Geissler et al., 2009). These results designate that quinoa is facultative halophyte especially
lines Q2 and Q9 at moderate saline regimes potentially in relation to biomass production
(Adolf et al., 2012).
Root lengths of different quinoa lines did not effect by salt stress in this study which
might be due to less hindrances to root growth in this soil less medium as compared to soil.
Moreover, leaf senescence was more prominent in control plants which is contradictory
with previous reports of some glycophytes and thus warrants further studies to know
reasoning of this phenomena.
Conclusion
All quinoa lines survived even at very high salinity (400 mM NaCl Stress). The
growth of all quinoa lines increased at 100 mM NaCl stress than control (0 Mm NaCl),
however, reduced linearly with further increase in salinity level. Among tested lines, Q2
and Q7 performed better than Q1 and Q9 under salt stress which was linked with low leaf
Na+ contents as well as better leaf K+/Na+ ratio.
56
Experiment 2: Physiological performance of quinoa lines under wire-house saline
conditions
Results
4.2.1 Plant growth and Yield responses
Data in Fig 4.2.1(a, b) reveal that plant heights and main panicle lengths of all
quinoa lines (Q1, Q2, Q7 and Q9) were significantly (P ≤ 0.001) influenced by NaCl stress
applied in pot soil. Salt-stress of 10 dSm-1 had a substantial positive impact on increasing
plant-height and main panicle length of all lines, however further increase negative
influence. Furthermore, Q2 and Q7 had larger main panicles and higher plant heights at
maturity while growing at all salt regimes. Minimum values for these parameters were
recorded for Q1 at 40 dS m-1 salt stress while stem diameters were found statistically similar
(Fig 4.2.1c).
Fig 4.2.2 (a, b and c) indicates salt stress had significant effect on shoot biomass,
seed yield and 1000-seed weights of quinoa lines. There were significant (P ≤ 0.001)
reduction in shoot biomass and seed yield of Q1 and Q2 at 30 dS m-1 while these reductions
starts at 20 dS m-1 for Q1 and Q9 (Fig 4.2.2 a, b). Furthermore, Q2 and Q7 produced more
biomass and seed yield than Q1 and Q9 at normal and all salt regimes. Maximum shoot
biomass and seed yield was produced by Q7 at 10 dS m-1 NaCl level while minimum was
found in Q1 at 40 dS m-1 (Fig 4.2.2 a, b). Thousand-seed weight of all lines reduced
significantly at 40 dS m-1 while lines Q2 and Q7 had more seed weights as compared to Q1
and Q9 at this extreme level of salt stress (Fig 4.2.2 c)
4.2.2 Leaf gas-relations
There was significant (P ≤ 0.001) influence of increasing salt-stress on leaf
photosynthetic-rate (An), stomatal-conductance (gs) and transpiration-rate (E). NaCl stress
in growth medium significantly decreased all these parameters (Fig 4.2.3 (a, b and c)), An
was found significantly higher at 10 dS m-1 in Q1, Q2 and Q7 than decreased with further
increased salt stress. However, lines Q2 and Q7 had more An than of Q1 and Q9 at every
increased salt level (Fig 4.2.3 a). All lines showed decreasing trend for gs and E with
increase in salinity levels except Q1 which had more E at 10 dS m-1 while line Q2 and Q7
had more gs and E than other two lines (Q1 and Q9) at all salt regimes (Fig 4.2.3 b, c)
57
4.2.3 Leaf chlorophyll contents
Salt-stress had significant (P ≤ 0.001) effect on leaf-chlorophyll contents of all
quinoa lines (Fig 4.2.4). Maximum leaf Chl a contents were observed in Q9 at 40 dS m-1
while minimum were recorded in Q1 at 0 dS m-1 NaCl stress while both leaf Chl b contents
and total Chl contents increased significantly at higher salt regimes, maximum leaf Chl b
as well as total Chl were found in Q7 at 20 dS m-1. (Fig 4.2.4 b, c)
4.2.4 Leaf water relations
Fig 4.2.5 depicts that all quinoa lines had significantly linear lower (more negative)
leaf water-potential (Ψl), leaf osmotic-potential (Ψπ) and leaf turgor-potential (Ψp) when
grown at every incremented NaCl stress. However, lines Q2 and Q7 had significant better
Ψl and Ψπ compared with Q1 and Q9 at all salt regimes (Fig 4.2.5 a, c).
4.2.5 Root Na+, K+ contents and K+/Na+ ratios
The effect of NaCl salt stress on root Na+ and K+ contents of quinoa lines are shown
in Fig 4.2.6 (a, b). Higher root Na+ contents were found in all lines at every increased salt
level in pot soil (Fig 4.2.6 a). Furthermore, lines Q-1 and Q-9 had maximum Na+ contents
in roots at 400 mM NaCl salt stress. While root K+ contents gradually decreased with
increased in NaCl salt (Fig 4.2.6 b). Root K+/Na+ ratio also decreased drastically when
plants were grown in all saline medium (Fig 4.2.6 c).
4.2.6 Leaf Na+, K+ contents and K+/Na+ ratios
The influence of salt stress on leaf Na+, K+ contents and K+/Na+ ratio of quinoa lines
under non-saline and saline regimes is shown in Fig 4.2.7 (a, b and c). Significant linear
increase was found for both leaf Na+ and K+ contents at every increased salt level in all
lines (Fig 4.2.7a, b). While significant less Na+ and higher K+ contents were found in leaves
of Q2 and Q7 as compared to Q1 and Q9 at all salt regimes (Fig 4.2.7a, b). Furthermore,
leaf K+/Na+ decreased significantly in all lines at every increased NaCl level. Again, Q2
and Q7 had better leaf K+/Na+ ratios at all NaCl incremented growth regimes (Fig 4.2.7 c).
4.2.7 Leaf proline and phenolics
Fig 4.2.8 (a, b) indicates significant modulations in leaf proline and total leaf
phenolic contents of quinoa lines exposed to varying salt stress level. Total phenolic and
proline content in leaves were found significant higher at 10 dS m-1 NaCl stress and onward
stress in all quinoa lines with increasing trends compared to control. Furthermore, lines Q7
and Q2 showed maximum proline content at 80% sea level salinity (Fig 4.2.8 a)
58
Fig 4.2.1 Plant height (a), main panicle length (b) and stem diameter (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
59
Fig 4.2.2 Shoot biomass per plant (a), seed yield per plant (b) and thousand seed weight (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
60
Fig 4.2.3 Leaf photosynthetic rate (An) (a), leaf stomatal conductance (gs) (b) and leaf transpiration rate (E) (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
61
Fig 4.2.4 Leaf chlorophyll a contents (a), leaf chlorophyll b contents (b) and leaf total chlorophyll contents (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
62
Fig 4.2.5 Leaf water potential (a), leaf osmotic potential (b) and leaf turgor potential (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
63
Fig 4.2.6 Root Na+ concentration (a), root K+ concentration (b) and root K+/Na+ ratio (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
64
Fig 4.2.7 Leaf Na+ concentration (a), leaf K+ concentration (b) and leaf K+/Na+ ratio (c) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
65
Fig 4.2.8 Leaf proline concentration (a) and leaf phenolic contents (b) of quinoa lines affected by NaCl salt stress. Q1, Q2, Q7 and Q9 indicate the quinoa lines. S and G indicate salinity treatments and quinoa lines respectively, and SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
66
Discussion
Quinoa lines studied in hydroponic experiment were further evaluated in normal
and saline pots under natural wire house conditions of Faisalabad in order to explore more
physiological, growth and yield responses and to validate results of hydroponic study.
Growing season was November 2012 to April 2013 same as growing season of quinoa in
Pakistan (Basra et al., 2014).
The growth and yield of (Fig 4.2.1 and Fig 4.2.2) of quinoa lines did not affected
by 10 dS m-1 NaCl salt stress but slightly increased. This level could be considered optimum
therefore for growth in local conditions. This salt induced stimulation in growth response
in quinoa and several other plant species has been found at moderate salinity levels (Hariadi
et al., 2011). This is might be due to increase in tissue water contents (Khan et al., 2005).
However significant shoot biomass and seed yield reductions of Q1 and Q9 were found at
20 dS m-1 while these reductions for Q2 and Q7 were recorded at 30 dS m-1 (Fig 4.2.2 a, b).
While drastic reductions were found at 80% sea level salinity in Q1 and Q9 lines. Thus, it
shows wide variation among quinoa lines exists for salt tolerance when tested under
environmental conditions of Faisalabad which confirms Adolf et al., 2012 phrase that
quinoa is facultative halophyte. Regardless of this variation, all lines completed life cycle
and produced seeds even at 80 % sea level salinity. Harriadi et al. 2011 also reported this
trend in the past, it suggests that these lines could also be tested in other climatic conditions
of Pakistan and other countries as well. Thousand grain weight of all lines found less at
higher salinity levels (30 and 40 dS m-1) might be due to more seed protein contents than
carbohydrates as Koyro and Eisa (2008) found this. How quinoa lines survived at increased
salt stress and what could be the reasons of these variations among quinoa lines…? it will
be explained in following paragraphs with help of our results and previous findings of
different researchers.
Cell turgor is very vital for cell functioning which depends upon suitable water-
relations and this phenomenon of maintaining water status is crucial for plant-growth under
salt regimes in plants of quinoa (Bosque-Sanchez et al., 2003). Maintenance of water-
balance direct links with gas, ion relations and leaf morphological features (Adolf et al.,
2012). Stomatal-resistance also reduces water loss. In present study, opposite relations were
observed for stomatal-conductance and transpiration (Fig 4.2.3 b, c) due to elevated NaCl
level in growing media, which resulted less growth and low yield (Fig 4.2.1 and Fig 4.2.2).
It seems that quinoa lines regulated water-relations by increasing uptake accumulation of
inorganic osmotica (K+ and Na+) (Fig 4.2.7 a, b) in leaf, it is termed as “hypertonic”
67
condition. Moreover, less transpiration was observed in all tested quinoa lines (Fig 4.2.3 c)
at high salt regimes, which could also be supportive in regulation of water-relations, which
might be possible due to quinoa morphological adaptations i.e. fewer and smaller stomata
as also explained earlier by Orsini et al. 2011 that salt tolerant genotype BO78 responded
in this way at whole plant level. These morphological adaptions have been reported as
efficient strategy to cut down transpiration water loss but also responsible for reduction in
overall stomatal conductance of leaf (Adolf et al., 2012).
Salt stress of 20 dS m-1 and onward negatively impacted photosynthesis (Fig 4.2.3
a) in quinoa lines especially in Q1 and Q9, which was linked to stomatal conductance (Fig
4.2.3 b). Though, stomatal-closure due to salt or drought stress is indictor of plant defense
for prevention of water loss, In the meantime it leads to considerable reduction of CO2
availability to carboxylation reactions (Iyengar and Reddy, 1997). Other variables, might
also effect photosynthesis, one most important is modulations in cytosolic-K+/Na+ ratio. K+
is well known as activator of over fifty enzymes including Rubisco and enzymes which
catalyzes biosynthesis of chlorophyll (Shabala, 2003). K+ and Na+ exhibits similar physio-
chemical features (i.e. ion-hydration energy and ionic-radius), thus Na+ competes for
binding on vital sites of enzymes (Marschner, 1995). Therefore, low leaf-K+/Na+ ratio in
quinoa lines (Fig 4.2.7c) especially Q1 and Q9, might reduce the photosynthetic capability.
In this context, plant’s salt-tolerance is highly linked with prevention of shoot K+
deficiency and appropriate leaf cytosolic-K+/Na+ ratio (Shabala and Cuin, 2008). In current
study, lines Q2 and Q7 had better leaf K+/Na+ ratios at all salt regimes (Fig 4.2.7c), thus
representing salt tolerance. How this ratio maintained….? Might be it involves an efficient
Na+ dumping in leaf vacuole or Na+ translocation to older leaves. Na+ concentration was
measured in young leaves in this study and usually young leaves had lower degree of
accumulation (Adolf et al., 2013). It seems that lines Q2 and Q7 responded at whole plant
level by translocating Na+ in older leaves that leads to low Na+ loads in young leaves or it
also seems restrictions of Na+ at root paranchyma as root Na+ contents of Q2 and Q7 were
higher while growing in saline solutions especially at 400 mM NaCl (Fig 4.2.6 a). This
strategy of low accumulation of Na+ might be also linked with preferential K+ up take at
root parenchyma and translocation to leaf, as leaf K+ concentration was also found higher
in Q7 and Q2 (Fig 4.2.7 b). Quinoa plants accumulate more K+ in leaves under salt stress
(Adolf et al., 2012), which was also confirmed in this study.
Other possible elucidation might be alteration in Na+ loading in xylem. It had been
described that, nutrient-uptake at root parenchyma, radial ion transport, and loading to
68
shoot are mostly uncoupled (Plett and Moller, 2010; Wegner et al., 2011). In a recent
review on halophytes, it was claimed that, thermodynamically Na+ loading in xylem is
surely an active process, facilitated by SOS1-like Na+/H+ exchanger (Shabala and Mackay,
2011). Meanwhile, xylem-K+ loading is a passive process happens through selective
outward rectifying cation-conductance (NORC) or K+-selective (SKOR) depolarization
activated channels located at xylem parenchyma (Shabala et al., 2010). It also seems in
quinoa that xylem K+ and Na+ loadings are uncoupled and thus tolerant lines may exhibit
better activities of Na+/H+ exchangers located at the xylem parenchyma border. Validation
of this hypothesis may be included in future research programs.
Exposure to salt stress, proline concentration and leaf phenolic contents increased
exponentially at every increased stress level in all quinoa lines (Fig 4.2.8 a-b). But, the
levels of increase for both proline and total phenoles were still not enough to contribute
significantly to cell osmotic adjustment as also described by Ruffino et al. 2010. Though,
Cuin and Shabala (2005; 2007), proposed that phenols may have role in osmotic-adjustment
by proper regulation of K+ movement across plasma-membrane, therefore, preventing K+
efflux which occurs due to excess Na+. In another recent study Ismail et al., 2016 explored,
that rutin; a phenol which accumulated approximately more than 27.5-times in young
leaves of two quinoa cultivars under salt stress. Exogenous applications of rutin were also
done by same group of scientist and found rutin can mitigate salt stress in quinoa plants.
Furthermore, after detailed electrophysiological experiments after exogenous application
of rutin on quinoa leaves, beneficial impacts revealed due to K+ retention and increased
Na+ efflux out of cell. Other important role of these compounds in quinoa which has been
widely accepted are very help full to detoxify dangerous reactive oxygen species which
produce as result of salt stress (Adolf et al., 2012). As these compounds are stress indicators
and their values were not increased significantly at 10 dS m-1 in all quinoa lines. (Fig 4.2.8
a-b). It confirms that this salinity level cannot be said as stress level for quinoa.
Contrary to glycophytes, leaf chlorophyll contents increase in halophytes including
quinoa under salt stress (Yang et al., 2016). This notion was also confirmed by results of
current study especially leaf chlorophyll a and total chlorophyll contents were found higher
in leaves of all quinoa lines at every increased salt stress level (Fig 4.2.4 a, c).
Conclusion
All the quinoa lines not only survived but also produced seeds even at 40 dS m-1
salt stress. Lines Q2 and Q7 performed better and produced 25-28% more seed yield as
compared to Q1 and Q9 under control and saline conditions. With increase in salinity level
69
non-enzymatic antioxidants (proline and phenolics) increased in all quinoa lines. Salt
tolerance was linked with low Na+ and high K+ accumulation in leaves.
Experiment 3: Physiological and agronomic performance of quinoa under field
salinity
In this preliminary study of quinoa on salt-affected soils of Punjab, Pakistan, the
performance of two quinoa lines was evaluated in terms of stand establishment, growth,
crop physiology, seed yield and seed quality, after growing on normal and salt affected
soils of different locations.
Results
4.3.1 Crop stand establishment
According to data of score of crop density (table 4.3.1) crop establishment of quinoa
lines was observed different in normal and salt affected soils of both locations, less
emergence was recorded in both lines at fields (S1, S2 and S3) furthermore, most poor crop
establishment was observed for line Q-2 at S4.
4.3.2 Crop growth and yield
Growth and yield of both lines reduced at salt affected soils (Fig 4.3.1, Fig 4.3.2
and Fig 4.3.3). Statistically (P ≤ 0.001) less biological and seed yield was harvested from
salt affected soils of both locations (Fig 4.3.3 a, b). Line Q7 produced more seeds as
compared to Q2 at all fields of both locations. Relatively less yield was noted when both
genotypes were harvested from Pindi-Pindi-bhatain location. Overall line Q7 was high
yielder. Furthermore, 1000 seed weight of both lines reduced at salt affected field (Fig 4.3.3
c). Overall growth and yield performance of decreased drastically at salt affected field of
Pindi-bhatian.
4.3.3 Root and leaf Na+, K+ contents
Na+ concentration was observed significantly (P ≤ 0.001) higher in leaves and roots
of both lines when sampled from salt affected fields of both locations meanwhile line Q-2
was emerged as hyper accumulator of Na+ at S2 and S3 (Fig 4.3.4 and Fig 4.3.5). Increased
concentration of K+ was also observed in leaves of both genotypes when harvested from
salt affected field of Faisalabad (S1) while root Na+ concentration was higher in Q7 (Fig
4.3.5 a) at S1 and S3, furthermore, higher K+ accumulation was found in leaves of genotype
Q-7 grown on S1. Root and leaf K+/Na+ ratios drastically reduced in plants of both lines
grown on salt affected soils (S1 and S3).
70
4.3.4 Leaf water-relations
Leaf water-potential, leaf osmotic-potential and leaf turgor-potential of quinoa lines
were significantly (P ≤ 0.001) affected by soil type (Fig 4.3.6). Leaf water-potential and
leaf osmotic-potential were recorded more negative in both lines, at S1 and S3 while leaf
turgor potential was also observed low at S1 and S3
4.3.5 Leaf chlorophyll contents
Leaf chlorophyll contents, leaf chlorophyll b content and leaf total chlorophyll
contents were also influenced by soil type in quinoa lines. All type of leaf chlorophyll
contents increased at S1 and S2 and S3 in both quinoa lines while maximum leaf
chlorophyll contents were recorded at S3 in both lines (Fig 4.3.7).
4.3.6 Leaf antioxidants
Data presented in Fig 4.3.9 are depicting the activities of antioxidants enzymes,
superoxide dismutase (SOD), catalase (CAT), and peroxidases (POD) in leaves of quinoa
genotypes when grown on normal and salt affected soils. Activities of POD and CAT were
recorded statistically similar in both lines (Q2 and Q7) when grown on normal as well as
salt affected soils, however, significantly (P ≤ 0.001) increased activity of SOD was noted
in leaves of both lines when grown on salt affected soils (S1 and S4).
Non-enzymatic antioxidants (leaf ascorbate, leaf total phenolic and leaf proline)
levels in leaves of quinoa lines were also recorded and presented in Fig 4.3.8. Significantly
(P ≤ 0.001) increased level of ascorbate was recorded in leaves of both lines when grown
on salt affected soils while leaf total phenol contents were found higher in line Q7 at S1
and also higher in both lines at S4. Leaf proline concertation also increased in both lines in
salt affected soils while maximum value of this parameter was found in leaves of line Q7
at S3.
4.3.7 Seed mineral contents
Mineral concentrations (Ca, Cu Mg, Mn, Fe, Zn, K and Na) were also recorded in
seeds of both lines harvested from normal and salt affected soils of both locations and
presented in Fig 4.3.10, Fig 4.3.11 and Fig 4.3.12. Seed K+ concentration affected by soil
type in both location. Maximum seed K+ concentration was found in line Q7 at S0 which
was statistically similar with values of seed K+ in all other line/soil type treatment
combinations except minimum value of seed K+ found in line Q7 at S0 while in case of
Pindi-bhatian location maximum K+ concentration was found in seeds of line Q-2 at S3
followed by at S2 while minimum was found in line Q7 at S2. Furthermore, seed Na+
71
concentration was found higher in line Q7 when harvested from S1, S2 and S3. Mg+2
concentration was found higher in seeds of line Q7 when harvested from all normal and
salt affected fields of both locations. In case of Zn concentration line Q7 accumulated more
zinc in its seeds when harvested from S0 and S1. Seed manganese concentration was found
higher in line Q7 at S2 while for Pindi-bhatian location maximum seed Mn concentration
was found in line Q2 at S3. Concentration of other minerals (Ca, Cu and Fe) found
statistically similar in seeds of both lines harvested from both locations.
4.3.8 Seed proximate analysis and saponin concentration
Data in Fig 4.3.13 (a, b) reveals that seed protein contents did not affected by soils
type while seed fat % decreased in both lines at S1, S2 and S3 while seed crude fiber and
seed saponin contents was also found similar at all was also observed similar at all soil
conditions (Fig 4.3.14 a, b).
Table 4.3.1 Score of crop density of quinoa lines affected by soil type
S0 S1 S2 S3
Q1 1.2 3.1 2.1 4
Q2 1 2.2 2.2 3.1
2Crop density (m-2),
1 = 90 % Emergence or more (very good),
2 = 80–89 % (good),
3=70–79 % (acceptable),
4=60-69 % (poor) S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1
and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian
respectively, Q2 and Q7 indicate the quinoa lines.
72
Fig 4.3.1 Crop growth rate (CGR) of quinoa lines (Q1 and Q2) affected by soils type of Faisalabad (a) and Pindi bhatian (b). Q2 and Q7 indicates quinoa lines
73
Fig 4.3.2 Plant height (a), Stem diameter (b) main panicle length (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
74
Fig 4.3.3 Seed yield (a), biological yield (b) and 1000-seed weight (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
75
Fig 4.3.4 Leaf Na+ (a), leaf K+ (b) and K+/Na+ (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
76
Fig 4.3.5 Root Na+ (a), root K+ (b) and root K+/Na+ (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
77
Fig 4.3.6 Leaf water potential (a), leaf osmotic potential (b) and leaf turgor potential (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
78
Fig 4.3.7 Leaf Chlorophyll a contents (a), leaf chlorophyll b contents (b) and leaf total chlorophyll contents (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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Fig 4.3.8 Leaf ascorbate (a), leaf total phenolics (b) and proline contents (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
80
Fig 4.3.9 Leaf Superoxide dismutase (a), leaf peroxidase (b) and leaf catalase activities (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
81
Fig 4.3.10 Seed Cu (a), seed Ca (b) and seed Fe contents (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
82
Fig 4.3.11 Seed Mg (a), seed Mn (b) and seed Zn contents (c) of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
83
Fig 4.3.12 Seed Na+ (a) and seed K+ contents of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
84
Fig 4.2.13 Seed protein (a) and seed fat contents of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
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Fig 4.3.14 Seed Crude fiber(a) and seed saponin contents of quinoa lines affected by soil type. S0 and S2 indicate normal soil of Faisalabad and Pindi-Bhatian while S1 and S3 indicate salt affected soil of Faisalabad and Pindi-Bhatian respectively, Q2 and Q7 indicate the quinoa lines. S and G indicate soil type and quinoa lines respectively, SxG indicates interactive effect. Bars are customized with standard error, s.e (n=4)
86
Discussion
Seedling establishment is the most critical stage of quinoa (Jacobsen et al., 1994,
1999)., affected by abiotic stresses (Bohnert et al., 1995). Poor crop stand establishment of
quinoa line Q2 was observed at salt affected soils of both locations (Faisalabad and Pindi
Bhatian) which might be quinoa line Q2 is sensitive to saline-sodic soils, which have
features of compactness, low exchangeable Mg++ and K+ and high Na+ (U.S. Salinity,
Laboratory-Staff, 1954). Excess Na+ accumulation in soil-profiles degrades soil,
generating compact sticky structure, due to colloids dispersion since Na+ has 2700 times
less flocculating capability than Ca+2 and Mg+2 which create hindrance to emerging
seedling. Furthermore, quinoa seed is small dicotyledonous (Bazile et al., 2015) and has
epigeal type emergence which may be disturbed due to crust formation on surface of saline-
sodic soils as also reported for cotton. Overall germination and crop stand establishment
under saline-sodic conditions depend on genotype. One interesting thing is that no weeding
was needed on salt affected soils as no weeds were found. In these adverse conditions
quinoa plants were still growing.
Quinoa lines performed differently under salt-affected field conditions. Growth (Fig
4.3.1) as well as yield performance (Fig 4.4.3) declined. 10-40 % yield reduction was
observed while growing on salt-affected soils, might be due poor crop stand (table 4.3.1).
Thinkable reasons of poor crop stand are discussed in above paragraph. Crop density-score
and seed yield correlated negatively, indicating less score was associated to high yield. At
salt- affected soil of Faisalabad, correlation was negative but weak and poor yield was
harvested from salt-affected soil of Pindi-Bhatian might be also linked with reason that
quinoa lines were exposed first time to this new set of environment while lines were
adaptable of Faisalabad. Furthermore Na+ toxicity might contribute to yield reduction.
Overall Q7 had higher yield than Q-2.
Leaf Na+ concentration increased in both lines, but more in Q-2 than Q7 (Fig 4.3.4
a) which indicates tolerance. High concentration of Na+ is damaging for both glycophytes
as well as halophytes, and therefore should be avoided (Adolf et al., 2013). It involves an
efficient Na+ dumping in leaf cell vacuoles and Na+ translocation to older leaves. In this
study, Na+ concentration was measured in young leaves, which usually have a lower degree
of sodium accumulation (Adolf et al., 2013). Q7 might responded to salt by translocating
Na+ in older leaves. This strategy of low accumulation of Na+ might be also linked with
87
preferential K+ up take at root parenchyma and translocation to leaf, as leaf K+
concentration was also higher in Q7 (Fig 4.3.4 b). Quinoa plants accumulate more K+ in
leaves under salt stress (Adolf et al., 2013) which was confirmed in this study. K+
availability decrease in saline-sodic soils, but both quinoa lines accumulated substantial
amount of K+ in their leaves while growing on these marginal soils of high pH. These
finding warrants further study how quinoa manages this K+ uptake and accumulation.
Stomatal-closure due to osmotic effect of salinity and excessive Na+ accumulation
in cytosol under saline regimes decrease capability of plant to make use of light which is
absorbed by photosynthetic-pigments, and leads to excessive generation of reactive-oxygen
species (ROS) (Tavakkoli et al., 2011; Shabala et al., 2013). These excessive ROS must be
detoxified or scavenged to become safe from detrimental effects i.e. lipid peroxidation,
protein denaturation, DNA damage, pigment break down, and impairment in activities of
various enzymes (Amjad et al., 2015). Two major antioxidant systems operate in plant cells
to limit the excess accumulation of ROS in cell, i) enzymatic and ii) non-enzymatic (Adolf
et al., 2012). Both these systems were found operative in leaves of both tested quinoa
genotypes (Fig 4.3.8, Fig 4.3.9). Similar level of activities enzymatic antioxidants i.e. POD
and CAT were found in leaves of lines both under normal and salt regimes. except SOD
whose increased activity was found in leaves of both lines under salt regimes (Fig 4.3.9).
Other non-enzyme component were ascorbate, phenolic acid and proline, which were
higher in leaves of both genotypes grown on salt affected soils (Fig 4.3.8).
Cell turgor is very vital for cell functioning which depends upon suitable water-
relations and this phenomenon of maintaining water status is crucial for plant-growth under
salt regimes in quinoa (Bosque-Sanchez et al., 2003). Modulations were observed in plant
water relations in both quinoa lines (Fig 4.3.6) which indicates abilities to extract water
from hypertonic external soil conditions.
Relatively more leaf chlorophyll contents were observed on salt affected soils (Fig
4.3.7) which confirms previous finding of Yang et al. 2016 that under high sodium chloride
stress chlorophyll contents increases.
Seed quality (mineral and protein concentration) was not affected under saline
conditions, and even improved in some cases, mainly K, Mg and Mn, also reported in Vega-
Gálvez et al. (2010). Q7 accumulated more Na+ in seeds than Q2.
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Conclusion
Overall salt tolerance of quinoa grown on salt affected soils of Pakistan was linked
with better crop stand establishment, low Na+ accumulation in leaves as well as improved
activities of non -enzymatic and enzymatic antioxidants. Quinoa lines produced lower yield
on salt affected soils, but with 1 t ha-1 it must be regarded as acceptable. Q7 had higher
yield but higher sodium content.
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General Discussion
By the commencement of 21st century, human population, is facing shortage of
water, environmental-pollution and escalated water and soil salinization. Escalating
human-population and reduction in cultivated area are two major threats for sustainable
agriculture (Shahbaz and Ashraf, 2013). Various abiotic stresses i.e., elevated temperatures,
soil salinity/sodocity, floods followed by long droughts have impacted cultivation and
production of crops, out of these, salinity and drought are major devastating abiotic-stress,
which is main reason of reduction in cultivable soils, crop-production and produce quality
(Yamaguchi and Blumwald, 2005). Generally, a saline soil is characterized by having ECe
more than 4 dS m-1 and SAR less than 15% while sodic soil has ECe less than 4 and SAR
more than 15 and has further characterized by disturbed soils structure. Furthermore saline-
sodic soils are combination of both saline and sodic soils, these are the most devastated
soils. Considerable yield reductions have been observed in many crops when ECe and SAR
of soil exceeds more than 4 and 15 respectively (Munns, 2005; Jamil et al., 2011). Globally,
it is estimated that 20% of total-cultivated area and 33% irrigated soils are salt-affected
(Shrivasta and Kumar, 2015). Furthermore, salinized land is increasing @ 10% per annum
due to various reasons, i.e. more surface evaporation as compared to precipitation, rock
weathering, irrigation with brackish water, and poor cultivation practices and it is expected
that 50% of cultivable land will be salinized up to year 2050 (Jamil et al., 2011).
Worryingly the extent of salt-affected land and its spread is highest and continuous in the
most economically challenged and populated countries i.e. Bangladesh (1 Mha; Hossain,
2010), Pakistan (6.18 Mha; MINFAL, (2002). and India (7 Mha; Vashev et al., 2010)
posing severe threats to sustainability of agriculture. Most of salt affected soils of Pakistan
are saline-sodic.
Salt-stress creates water-stress due more negative water-potential in growing media,
thus plants become unable to uptake water which leads to nutrient deficiency or toxicity.
Excess salt concentration also induces excess ROS production which damages
macromolecules lipid, proteins and DNA, Na+ replaces Ca+2 located between lipids of
membranes (Pattanagul and Mayasaya, 2008). Al these effects collectively leads to yield
reduction. Plant tolerates salt stress by osmotic adjustments due to accumulation of
inorganic, osmotic (K+, Ca+2 and Na+) or de novo synthesis by organic osmolytes (amino
acids, polyols, sugars, quaternary ammonium compounds (Adolf et al., 2012) in cytosols.
90
Further more tolerant plants, tolerates salt induced oxidative stress by strong antioxidant
system of both enzymes and non-Enzymes (Adolf et al., 2012).
In present eras, research has been increasing about salinity in relation to plant.
Usually, three methodologies are responsible for enhancing the crop production from soils
affected by salinity: 1) soil reclamation 2) introducing genotypes tolerant to salinity
(Yilmaz et al., 2004; Blumwald et al., 2004) 3) utilization of halophytes as a new crop but
not exploited widely (Flowers and Yeo, 1995; Flowers, 2004; Panta et al., 2014). Due to
soil permeability, lack of good quality water, higher cost for reclamation and widely
distributed salt affected area, practically, soil reclamation is not possible (Akhtar et al.,
2010). Salt tolerant genotypes can be helpful but it needs time and its outcome is limited.
Reasons of limited salt-tolerant genotypes are variation in environmental conditions, lack
of genetic knowledge, and complex polygenic morpho-physiological traits. Salinity-
tolerance will only be achieved when all key characters are collectively considered in a
complementary way (Shahbaz and Ashraf, 2013). Therefore, it should be realized that there
is no existence of such thing which can be considered as a silver bullet that can resolve
problems of salinity; focusing only one character (gene) will not result any substantial
improvements. Halophytes can be used as an alternate choice mainly in agriculture and
they are not well exploited till now (Flowers and Yeo, 1995; Glenn et al., 1999; Flowers,
2004; Panta et al., 2014).
Quinoa is a plant species of family “Amaranthaceae” and known as pseudo-cereal
which produces grains of enormous nutritional qualities, it has origin from Latin America,
where it was grown seven-thousand years, ago (Jacobsen et al., 2003). Quinoa grows well
at moderate salt-regimes (100-200 mM NaCl), that’s why known as “true halophyte”.
Furthermore, some accessions can stand and grow even at salt-concentrations of 40 dS m-1
which is equal to salt-concentration of sea water (Jacobsen et al., 2003; Koyro and Eisa,
2008). The interest about this crop is increasing over the world, both due to grain superior
nutritional qualities and its abiotic stress-tolerance (Repo-Carrasco et al., 2003; Stikic et
al., 2012). Quinoa seed is free of gluten, rich in all essential amino-acids, minerals (K, Fe,
Ca, Mn), vitamins (A, E, B2), carbohydrates and health supportive fatty-acids (omega 3)
(Repo-Carrasco et al., 2003; Stikic et al., 2012).
Quinoa has been also recently introduced successfully in Pakistan on normal soils
(Basra et al., 2014). But its adaptability and yield potential on local salt affected soils has
yet to be tested. Furthermore, a lot of work has been reported related on quinoa salt
tolerance but very few field studies are available especially in saline-sodic and marginally
91
degraded soils. Therefore, the preliminary studies were carried out to examine the salt
tolerance variations of four quinoa genotypes with the aim to identify lines suitable for
cultivation on salt affected soils.
Identification of salt-tolerant and sensitive lines is very vital for further cultivation
on salt-affected soils and future breeding program. Therefore, in first experiment four
quinoa lines (Q1, Q2, Q7 and Q9) were initially tested in hydroponic culture (wire house
conditions) to examine tolerance level and variation among lines against various levels of
salt stress (0,100, 200, 300, 400 mM NaCl). Results showed that quinoa growth increased
at 100 mM NaCl stress confirming its halophytic behaviour, while linear reduction in
growth was observed at higher levels, meanwhile this reduction was more prominent in Q1
and Q9. Even at 200 mM NaCl stress, Q2 and Q9 showed reasonable growth and this
tolerance was highly linked with low Na+ accumulation in leaves which indicated that
tolerant line had Na+ exclusion capabilities. (Adolf et al., 2012).
In second experiment, same lines which were used in first experiment were further
tested in pots under same above-mentioned wire house conditions. Varying level of salt
stress (normal EC of soil used, 10, 20, 10, 40 dS m-1) was created before sowing using NaCl
salt. Outcomes obtained were encouraging in the way that, all lines not only survived but
produced seeds even at 40 dS m-1. Furthermore, lines Q1 and Q9 emerged as hyper
accumulators of Na+ but low yielders, all the lines produced substantial amount of
phenolics and proline in leaves at levels above than 20 dS m-1. All quinoa lines showed
improvement in growth up to 10 dS m-1 which indicates halophytic behaviour. Q2 and Q7
were proved as high yielder because of better water and gas exchange relations and low
Na+ in leaves.
In third study, trials were conducted at normal and salt affected fields at two
locations, Faisalabad and Pindi bhatian. Normal soil was at main campus farm of University
of Agriculture, Faisalabad (UAF) farm and salt affected fields selected were at proka farm
(25 km away from main campus), while one normal and one salt affected plot was selected
at Soil Salinity Research Institute (SSRI) Pindi-bhatian research farm (75 km away from
main campus). Overall all salt affected soils were of saline sodic nature and also
underground was unfit for irrigation. Salt tolerant lines (Q2 and Q9) of experiment 1 and 2
were used in this study, Results of these open field trials revealed that adverse conditions
of saline sodic soils affected quinoa germination which ultimately caused yield reductions
in both lines at salt affected soils, furthermore low stand establishment was observed in Q2
than Q7 and Q7 was higher yielders at all normal and salt affected soils owing to improved
92
leaf K+ and antioxidants activities specially SOD. Overall all 10-40% less yields were
obtained from salt affected soils but were still higher than world average yield (1000 kg ha-
1) (Bazile et al., 2015). Minimum yield was obtained (1587 kg ha-1) by Q2 at salt affected
field of Pindi Bhatian. Seed quality (mineral and protein concentration) did not affected
under salt affected soils even improved in some case, (K, Mg and Mn) and found in range
of international published reports (Vega-Gálvez et al., 2010), however line Q7 accumulated
more Na+ in seeds than Q-2 hence Q-2 may be said as nutritionally superior quinoa line.
It can be concluded that quinoa can be grown on salt affected soils, moderate
salinity even improves growth and yield (10 dS m-1). Salinity levels above 20 dS m-1. High
sodicity, causes low stand establishment which ultimately reduces yield up to 40% but still
better than reported world average yield (Bazile et al., 2015).
93
Summary Salinity is leading cause of low productivity and threat to burgeoning world
population. Cultivation of halophytes is gaining popularity to live with salinity. Quinoa, a
high value facultative halophyte is recent introduction in Pakistan and has been grown
successfully in Pakistan on normal soils. But its adaptability and yield potential on local
salt affected soils has yet to be tested. Therefore, quinoa was tested as salt tolerant crop
through following wire house and field trials under agroecological conditions of Faisalabad
and Pindi bhatian
In first experiment four quinoa lines (Q1, Q2, Q7 and Q9) were initially tested in
hydroponic culture (wire house conditions) to examine tolerance level and variation among
lines against various levels of salt stress (0,100, 200, 300, 400 mM NaCl). Results showed
that quinoa growth increased at 100 mM NaCl stress confirming its halophytic behaviour,
while linear reduction in growth was observed at higher levels, meanwhile this reduction
was more prominent in Q1 and Q9. Even at 200 mM NaCl stress, Q2 and Q9 showed
reasonable growth and this tolerance was highly linked with low Na + accumulation in
leaves which indicated that tolerance line had Na+ exclusion capabilities.
In second experiment, same lines which were used in first experiment were further
tested in pots under same above-mentioned wire house conditions. Varying level of salt
stress (normal EC of soil used, 10, 20, 10, 40 dS m-1) was created before sowing using NaCl
salt. Outcomes obtained were encouraging in the way that, all lines not only survived but
produced seeds even at 40 dS m-1. Furthermore, lines Q1 and Q9 emerged as hyper
accumulator of Na+ but low yielders, all the lines produced substantial amount of phenolics
and proline in leaves at levels above than 20 dS m-1. All quinoa lines showed improvement
in growth up to 10 dS m-1 which indicates halophytic behaviour. Q2 and Q7 were proved
as high yielder because of better water and gas exchange relations and low Na+ in leaves.
In third study, trials were conducted at normal and salt affected soils of two
locations, Faisalabad and Pindi bhatian. Normal soil plots were at University of Agriculture
Faisalabad (UAF) main campus farm and salt affected plots were at proka farm, while one
normal and one salt affected plot was selected at Soil Salinity Research Institute (SSRI)
Pindi-bhatian research farm. Overall all salt affected soils were of saline sodic nature and
also underground was unfit for irrigation. Salt tolerant lines (Q2 and Q9) of experiment 1
and 2 were used in in this study, Results of these open field trials revealed that adverse
conditions of saline sodic soils affected quinoa germination which ultimately caused yield
94
reductions in both lines at salt affected soils, furthermore low stand establishment was
observed in Q2 than Q7 and Q7 was higher yielders at all normal and salt affected soils
owing to improved leaf K+ and antioxidants activities specially SOD. Overall all 10-40 %
less yields were obtained from salt affected soils but were still higher than world average
yield (1000 kg ha-1). Minimum yield was obtained (1587 kg ha-1) by Q2 at salt affected
field of Pindi bhatian. Seed quality (mineral and protein concentration) did not affected
under salt affected soils even improved in some case, (K, Mg and Mn) and found in range
of international published reports, however line Q7 accumulated more Na+ in seeds than
Q-2 hence Q-2 may be said as nutritionally superior quinoa line.
Quinoa can be grown on salt affected soils, low salinity even improves growth and
yield. salinity levels above 20 dS m-1. High sodicity, causes low stand establishment which
ultimately reduces yield up to 40% but still better than reported world average yield.
Future prospects
• Under sodic soil conditions, poor stand establishment of quinoa was found. Seed enhancement techniques may be tested to improve it (seed priming, seed coating and seed inoculation using halotolerant bacteria).
• Salt tolerant quinoa lines should be tested in other agroecological zones of country.
• Local breeding for salt-tolerance should be started using huge genetic diversity of quinoa.
• Saponin washing is a major issue in processing for marketing. Saponin free quinoa may be screened out.
95
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