Nutrient seed priming improves abiotic stress tolerance in Zea mays

Institute of Crop Science, Nutritional Crop Physiology

University of Hohenheim

Prof. Dr. Günter Neumann

Nutrient seed priming improves abiotic stress tolerance in

Zea mays L. and Glycine max L.

Dissertation

Submitted in fulfilment of the requirement of the degree “ Doktor der

Agrarwissenschaften”

(Dr. sc. agr. /Ph.D. in Agricultural Sciences)

to the

Faculty of Agriculture Sciences

Presented by

Muhammad Imran

2012!

!!!!!!!!!!!!!!!!!!!!!!!!!!This thesis was accepted as a doctoral dissertation in fulfillment of the

requirements for the degree “ Doktor der Agrarwissenscheften” (Dr. sc.

Agr. / Ph.D in Agricultural Science) by the Faculty of Agricultural

Sciences of the University of Hohenheim.

Date of Examination: 14.02.2013

Examination Committee

Dean and Head of the Committee: Prof. Dr. R. Mosenthin

Supervisor and Reviewer: Prof. Dr. Günter Neumann

Co- Reviewer: Prof. Dr. Michael Kruse

Co-Examiner: Prof. Dr. Uwe Ludewig

Dedicated

to

My loving mother and father

Eidesstattliche Erklärung

Ich erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation

selbständig angefertigt, nur die angegebenen Quellen und Hilfsmittel

benutzt und inhaltlich oder wörtlich übernommene Stellen als solche

gekennzeichnet habe. Ich boch noch keinen weiteren Promotionsversuch

unternommen.

DECLARATION OF ORIGINALITY

I declare that this thesis is description of my own research and this work

has not previously been submitted for the doctoral degree at any other

institution.

Stuttgart, den Muhammad Imran

Table of Contents Summary……………….…………………………..………….………...……1 Zusammenfassung……………………….…….…………...……………….3 General introduction……………………………….…………………….…..6 Impact of nutrient seed priming on germination, seedling development, nutritional status and grain yield of maize ..............................................................................................................46 Nutrient seed priming improves seedling development and increases grain yield of maize exposed to low root zone temperatures during early growth ..............................................................................................................70 Accumulation and distribution of Zn and Mn in soybean seeds after nutrient seed priming and its contribution to plant growth under Zn and Mn deficient conditions ………………………………………………………………………..……...94 General discussion…………………….………………………………….118 References………………………….……………………………….…….141 Curriculum vitae………………..…………………………………………179 Acknowledgements...…………………………………………………….183

List of Figures

Figure Title Page No.

2.1 Schematic diagram of normal germination and seed priming process.

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2.2 Time course of major events associated with germination and subsequent post germination growth (Bewley, 1997).

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2.3 A conceptual diagram depicting relationships between plant and soil temperature for nutrient uptake. Soil temperature affects nutrient uptake directly by altering root growth, morphology and uptake kinetics. Indirect effects include altered rates of decomposition and nutrient mineralization, mineral weathering and nutrient transport processes (mass flow and diffusion).

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2.4 Schematic diagram of the pore system of the apparent space. DFS= Donan free space, WFS = water free space.

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2.5 Primary effects of a short chill in the light and the dark on photosynthesis in thermophilic plants.

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2.6 Effect of high and low seed P reserves on shoot and root biomass accumulation in wheat.

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3.1 Effect of water priming for different time periods on germination rate (a) and seedling growth (b) of maize. Data points represent means and SE of 4 replicates. Different characters indicate significant differences between treatments.

54

3.2 Shoot (a) and root (b) dry biomass of maize plants, grown in nutrient solution after nutrient seed priming with sequential harvests at 2, 3 and 4 weeks after sowing. Data represent means and SE of 4 replicates at each harvest. Different characters indicate significant differences between treatments.

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3.3 Nutrient concentrations in shoots of maize plants, grown in nutrient solution after nutrient seed priming with sequential harvests at 2, 3 and 4 weeks after sowing. Data represent means and SE of 4 replicates at each harvest. Different characters indicate significant differences between treatments.

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3.4 Nutrient concentrations in shoots of maize plants, grown in nutrient solution after nutrient seed priming with sequential harvests at 2, 3 and 4 weeks after sowing. Data represent means and SE of 4 replicates at each harvest. Different characters indicate significant differences between treatments.

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3.5 Shoot (a) and root (b) dry biomass of 4 weeks old maize plants grown in soil culture after nutrient seed priming. Data represent means and SE of 4 replicates. Different characters indicate significant differences between treatments.

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3.6 Minimum and maximum temperature at research farm (°C) recorded in April-May, 2010.

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4.1 Five weeks old maize plants grown in nutrient solution under low RZT stress.

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4.2 Roots of maize plants grown along the root windows in rhizo-boxes after 3 weeks of low RZT stress.

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4.3 Root lengths of maize plant roots grown along the root windows after 1 and 3 weeks of low RZT stress. Data represent means and SE of 4 replicates. Different characters indicate significant differences between treatments.

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4.4 Minimum temperature in Hohenheim (°C) recorded in April-May, 2010 and 2011.

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4.5 Shoot fresh and dry biomass of maize plants harvested in the field 5 weeks after sowing (year 2011). Data represent means and SE of 4 replicates. Different characters indicate significant differences between treatments.

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5.1 (a) Radical emergence of soybean seeds, water primed between layers of moist filter paper for different time duration. Data points represent means of 4 replicates. (b) Abnormal seedlings in the germination test after water priming with different methods and time durations. Bars show means and SE of 4 replicates. Different characters indicate significant differences between treatments.

103

5.2 Soybean seedlings grown for 7 days on peat culture substrate after water priming with different methods and priming durations. AC-Method = volume of priming solution according to water absorption capacity of seeds; FP-Method = priming between moist filter paper; SP-Method = submerged priming.

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5.3 (a) Shoot and (b) root dry biomass production of 5 weeks old soybean plants grown in nutrient solution. Bars show means and standard errors of four replicates. Significant differences (Duncan's Multiple Range Test, p= 0.05) are indicated by different characters. (NS = Nutrient solution; WP = water priming; NS – Zn = nutrient solution without Zn; NS – Mn = nutrient solution without Mn; NS – (Zn+Mn) = nutrient solution without Zn and Mn).

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5.4 Shoot Zn and Mn (a) concentrations and (b) contents in 5 weeks old soybean plants grown in nutrient solution. Deficiency thresholds for Zn and Mn are marked by horizontal lines..Bars show means and standard errors of four replicates.

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5.5 (a) Shoot and (b) root dry biomass of 5 weeks old soybean plants grown in soil culture. Deficiency thresholds for Zn and Mn are marked by horizontal lines..Bars show means and standard errors of four replicates. Significant differences (Duncan's Multiple Range Test., p= 0.05) are indicated by different characters.

110

5.6 Shoot Zn and Mn (a) concentrations and (b) contents in 5 weeks old soybean plants grown in soil culture. A line across the bars indicates critical deficient levels of Zn and Mn. Deficiency thresholds for Zn and Mn are marked by horizontal lines. Bars show means and standard errors of four replicates. Significant differences (Duncan's Multiple Range Test., p= 0.05) are indicated by different characters.

111

6.1 Effect of B seed priming on 2 weeks old wheat seedlings grown under salt stress (8dSm-1).

128

6.2 Effect of B seed priming on root growth of 4 weeks old wheat seedlings grown under salt stress (8dSm-1).

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6.3 Effect of B seed priming on shoot and root fresh biomass of 4 weeks old wheat plants grown under salt stress (8 dSm-1). Bars show the means and SE of 4 replicates. Different characters show the significant differences between the treatments.

129

6.4 Effect of nutrient seed priming on stomatal conductance (left) and leaf area (right) of 1st fully developed leaf of 5 weeks old maize plants grown in nutrient solution at low root zone temperature (10°C for water, Fe and Zn). Bars represent the means and SE of 4 replicates. Different characters show the significant differences between the treatments.

133

6.5 Effect of nutrient seed priming on rate of photosynthesis (left) and net photosynthesis (right) in 1st fully developed leaf of 5 weeks old maize plants grown in nutrient solution at low root zone temperature (10°C for water, Fe and Zn). Bars represent the means and SE of 4 replicates. Different characters show the significant differences between the treatments.

133

6.6 Effect of nutrient seed priming on shoot Zn concentration of 4 weeks old wheat plants grown under salt stress (8dSm-1). Bars show the means and SE of 4 replicates. Different characters show the significant differences between the treatments.

134

6.7 Effect of P and micronutrients (Zn, Mn and B) seed priming on shoot growth of 4 weeks old soybean plants.

139

List of table

Table Title Page No.

2.1 Effect of high and low seed P (HSP and LSP) reserves and P fertilization on P accumulation in shoot and whole wheat plant.

41

3.1 Concentrations and salts used for maize nutrient seed priming treatments.

51

3.2 Mineral nutrient contents of maize seeds after nutrient seed priming. Data represent means of 4 replicates. Different characters indicate significant differences between treatments.

55

3.3 Nutrient concentrations and contents in young leaves and the remaining shoot of maize plants grown in soil culture. Data represent means of 4 replicates +SE. Different characters indicate significant differences between treatments in similar shoot portion.

61

3.4 Final grain yield of maize grown (kg ha-1). Data represent means +SE of 4 replicates. Different characters indicate significant difference between treatments.

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4.1 Seed mineral nutrient concentrations and contents after nutrient seed priming. Data represent means and SE of 4 replicates. Different characters indicate significant differences between treatments.

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4.2 Seed priming treatments and root zone temperature regimes of maize seedlings grown in nutrient solution and rhizo-boxes.

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4.3 Fresh shoot and root biomass, and root/shoot ratio of maize plants grown in nutrient solution and rhizo-boxes after nutrient seed priming. Data represent means and SE of 4 replicates. Different characters indicate significant differences between treatments.

81

4.4 Total root lengths (cm plant-1) and root lengths in different diameter lasses of maize plants grown in nutrient solution and rhizo-boxes after nutrient seed priming. Data represent means and SE of 4 replicates. Different characters indicate significant differences between treatments.

84

4.5 Shoot mineral concentrations of maize plants grown in nutrient solution and rhizo-boxes under low RZT stress. Data represent means and SE of 4 replicates. Different characters indicate significant differences between treatments.

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4.6 Shoot mineral contents of maize plants grown in nutrient solution and rhizo-boxes under low RZT stress. Data represent means and SE of 4 replicates. Different characters indicate significant differences between treatments.

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Table Title Page No.

4.7 Shoot mineral concentrations and contents of maize plants grown in the field for 5 weeks. Data represent means of 4 replicates and SE Different characters indicate significant differences between treatments.

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4.8 Final grain yields of maize grown in the field (2010-2011). Data represent means and SE of 4 replicates. Different characters indicate significant differences between treatments.

89

5.1 Zinc and manganese contents in complete soybean seeds after nutrient seed priming. Data represent means and SE of 4 replicates. Different characters indicate significant differences between priming treatments.

100

5.2 Effect of seed priming with different Zn and Mn concentrations on abnormal seedling development. Data represent means and standard errors of four replicates. Significant differences (Duncan's Multiple Range Test, p= 0.05) are indicated by different characters.

103

5.3 Distribution of Zn and Mn in soybean seeds after nutrient seed priming. Data represent means and standard errors of four replicates. Column values followed by different letter are significantly different by Duncan's Multiple Range Test, p= 0.05).

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6.1 Effect of nutrient seed priming on total phenolic compounds and proline in the roots of 4 weeks old maize plants grown in soil culture at low zone temperature (10°C for water, Fe and Zn+Mn treatments). Data represents the means and SE of 4 replicates. Different chracters show the significant differences between the treatments.

136

Chapter(1((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((Summary(

( 1(

Chapter 1

Summary

Seed reserves are the primary source of mineral nutrients for early

seedling development. ‘Nutrient seed priming’ is a technique in which

seeds are soaked in nutrient solution and subsequently dried back to

initial moisture content for storage. It is an efficient approach to increase

seed nutrient contents along with priming effects to improve seed

quality, germination speed and seedling establishment. Various abiotic

stresses, such as sub-optimal temperature, drought, submergence and

soil pH extremes can seriously affect seedling establishment and

nutrient acquisition at early growth stages. This thesis focused on the

role and contribution of nutrient seed priming in plant growth and

nutritional status in maize and soybean under conditions of limited

nutrient availability and low root-zone temperature.

Protocols for nutrient seed priming with Zn, Mn, Zn+Mn, B and P were

optimised for application in maize and soybean seeds (B and P priming

in maize only). Optimum priming durations of 24 h (maize) and 12 h

(soybean) were identified for both plant species but in instead of

submerging seeds in priming solutions slow imbibition between filter

papers was essential for soybean to minimise development of abnormal

seedlings to avoid imbibition damage. Nutrient concentrations were

calculated according to water uptake to double the natural seed

reserves of the respective micronutrients and 50% increase in

phosphorus. However, final uptake of the micronutrients was generally

much higher (+500-1000%) while it was lower for P (+20%). In case of

soybean this could be attributed to a high Zn and Mn binding capacity of

the seed coat, which adsorbed up to 60% of the primed nutrients.

Particularly, Zn and Zn+Mn priming stimulated plant growth in

hydroponic culture systems and to a lower extent also on a soil with low

availability of P, Zn and Fe. This was associated with a high shoot

translocation of the primed nutrients (Zn and Mn), which was most

Chapter(1((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((Summary(

( 2(

expressed in the hydroponic culture system. Combined priming

treatments with Zn and Mn were usually less effective than Zn priming

alone, suggesting an antagonistic interaction. By contrast, mobility of

primed B was extremely low and B priming was completely ineffective.

In soil culture also P priming moderately increased shoot biomass

production by 10-20 %. However the efficiency of P priming was largely

limited by the high P demand of the plants.

Low root zone temperature (RZT) at early spring is a limiting factor for

maize production in Central and Northern Europe. Nutrient acquisition,

nutrient uptake and particularly root growth are severely affected at low

RZT and the consequences of these growth depressions are often not

completely compensated until final harvest. Model experiments in

hydroponics and soil culture revealed that maize nutrient seed priming

with Zn, Mn and Fe is a promising strategy to diminish the deficiency of

specific nutrients, such as Zn, Mn and also P and to maintain plant

growth under low RZT stress. This was mainly attributed to significantly

increased root growth and particularly fine root production in plants

grown from nutrient-primed seeds. Improved net photosynthesis of

primed plants was mainly related with increased leaf area and

preliminary results suggest a higher tolerance to oxidative damage due

to increased production of protective phenolics.

Two independent field experiments under conditions of suboptimal

temperatures during germination and early growth revealed an increase

in grain yield of 10 – 15 % for plants derived from Zn+Mn and Fe primed

seeds. This finding demonstrates long-lasting persistence of priming

effects. The molecular and physiological mechanisms behind require

further investigation.

Chapter(1((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((Summary(

( 3(

Zusammenfassung

Samenreserven sind die primäre Quelle für die Mineralstoffversorgung

während der frühen Keimlingsentwicklung. Nährstoffpriming ist ein

methodischer Ansatz, bei dem Saatgut in Mineralstofflösungen

vorgequollen und anschließend zur weiteren Lagerung auf Ausgangs-

feuchte zurückgetrocknet wird. Hierdurch ist es möglich, neben

vorquellungsbedingten Primingeffekten auch die Mineralstoffreserven

zur Verbesserung der Saatgutqualität zu erhöhen sowie die Auflauf-

Geschwindigkeit und die Keimlingsentwicklung zu verbessern, die durch

verschiedenste Stressfaktoren, wie suboptimale

Temperaturbedingungen, Trockenheit, Staunässe und pH-Extreme

beeinträchtigt werden kann.

Die vorliegende Arbeit beschäftigt sich mit der Wirkung von

Nährstoffpriming auf das Wachstum und den Ernährungsstatus von

Mais und Soja unter Bedingungen eingeschränkter

Nährstoffverfügbarkeit und niedriger Wurzelraumtemperatur.

Für die Saatgutapplikation von Zink (Zn), Mangan (Mn), Mn+Zn, Bor

(B), Eisen (Fe) und Phosphat (P) bei Mais und Soja wurden

Primingprotokolle entwickelt. Bei beiden Pflanzenarten ergab sich eine

optimale Behandlungsdauer von 24 h. Jedoch war es notwendig, das

Soja-Saatgut anstelle von submerser Inkubation in den Nährlösungen,

langsam in Nährlösungsgetränktem Filterpapier einzuquellen, um die

Entwicklung abnormaler Keimlinge aufgrund von Quellungsschäden zu

vermeiden. Anhand der Wasseraufnahme der Keinlinge wurden die

Nährlösungskonzentrationen so berechnet, das sich nach Einquellen

eine Verdopplung der natürlichen Mineralstoffreserven im Samen

ergeben sollte. Jedoch wurden für die Mikronährstoffe erheblich höhere

Aufnahmeraten gemessen (+500 - 1000%), während die Aufnahme von

P geringer war (+ 20%). Bei Soja konnte gezeigt werden, das die

erhöhten Mikronährstoffgehalte auf eine hohe Bindungskapazität der

Chapter(1((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((Summary(

( 4(

Samenschale zurückzuführen waren, die bis zu 60 % der

aufgenommenen Mineral-stoffe adsorbierte.

Das Pflanzenwachstum wurde besonders durch Zn und Zn+Mn-Priming

stimuliert, sowohl in Nährlösungskultur und in geringerem Umfang auch

auf einem Boden mit geringer P-, Zn-, und Fe-Verfügbarkeit, was mit

einer besonders ausgeprägten Translokation der zugesetzten

Nährstoffe ins Sprossgewebe, besonders in Nährlösungskultur

verbunden war. Dabei zeigten Kombinationsbehandlungen (Zn+Mn)

geringere Effekte als alleiniges Zn-Priming, was auf antagonistische

Interaktionen hinweist. Im Gegensatz dazu ergab sich für B eine

besonders geringe Mobilität innerhalb der Pflanze und entsprechend

war B-Priming wirkungslos. In Bodenkultur wurde auch durch P-Priming

ein moderater Wachstums-effekt (+10-20 % Biomasseproduktion)

erzielt. Die Effizienz des P-Priming wurde jedoch stark durch den hohen

P-Bedarf der Pflanzen eingeschränkt.

Niedrige Wurzelraumtemperaturen (NWT) im zeitigen Frühjahr limitieren

den Maisanbau in Zentral und Nordeuropa. Die Nährstoff-aneignung,

Nährstoffaufnahme und besonders das Wurzelwachstum werden durch

NWT stark beeinträchtigt und die Folgen können häufig bis zur Ernte

nicht mehr kompensiert werden. Modellexperimente in Nährlösungs-,

und Bodenkultur ergaben für Saatgutpriming mit Zn, Mn und Fe

deutliche Hinweise darauf, das bei NWT Mangelerscheinungen

bestimmter Nährstoffe, wie Zn, Mn und auch P vermindert werden

können. Dies konnte in erster Linie auf ein signifikant verbessertes

Wurzelwachstum und besonders auf vermehrte Feinwurzelbildung

zurückgeführt werden. Eine erhöhte Nettophotosyntheserate ergab sich

hauptsächlich aus einer vergrößerten Blattfläche und erste Vor-

untersuchungen weisen auch auf eine höhere Toleranz gegenüber

oxidative Schädigungen durch erhöhte Produktion von Phenolen mit

Schutzfunktion hin.

Chapter(1((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((Summary(

( 5(

Zwei unabhängige Feldversuche bei suboptimaler Temperatur während

der Keimphase und des Jugendwachstums ergaben Kornertrags-

steigerungen von 10-15 % bei Mais nach Saatgutpriming mit Zn+Mn

oder Fe. Diese Ergebnisse belegen die Langzeitwirkung der Priming-

effekte. Die molekularen und physiologischen Grundlagen erfordern

allerdings noch weitere Untersuchungen.

Chapter(2( ((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((General(Introduction(

( 6(

Chapter 2

General introduction

Early and healthy seedling development is essential for optimal growth and

maximum yield in agricultural crops. Strong and well-developed seedlings can

tolerate several biotic and abiotic stresses during various growth stages.

Healthy and vigorous seedlings are also important to overcome the adverse

environmental effects on crops grown under temperate climate conditions

(Lütke-Entrup and Oehmichen, 2000).

The germination process is the crucial step determining the vigor and success

of the life cycle of a crop plant. It includes the events that start with imbibition

by, water uptake, quiescent dry seeds and ends with the elongation of the

embryonic axis (Bewley and Black, 1994). Accelerated and homogeneous

germination is necessary for better crop establishment, efficient usage of seed

reserves and ultimately increased final yields (Harris, 1996). Different methods

and strategies are used to improve seed quality and germination e.g. use of

biotechnology, hybrid seeds, mechanical seed treatments i.e. polishing off or

removal of the seed coat (testa), sorting the seeds into different seed size

classes and various seed treatments like seed coating, pelleting or soaking in

different solutions (Kolotelo et al., 2001). All these various techniques are

applied to reduce the time between seed sowing and seedling emergence and

also to avoid biotic and abiotic stresses during germination phase. Soaking of

seeds into water, also termed as, seed priming is getting very popular and a

successful method to improve seed germination and crop establishment in

developing countries. It is an easy to use and cost effective technique for

resource poor farmers and farming systems (Harris, 1996).


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2.1 Seed priming

Seed priming is a technique in which seeds are soaked in water for a certain

period of time and subsequently dried back to storage moisture contents

before sowing. Seedling emergence from water-primed seeds is faster and

more vigorous compared to emergence from unprimed seeds (Rashid et al.,

2002; Arif et al., 2005; Miraj, 2005). Soaking of seeds into water stimulates a

variety of biochemical changes in the seed that are essential to initiate the

germination process e.g. water imbibition, breaking of dormancy, activation of

certain enzyme etc. (Ajouri et al., 2004). A number of processes stimulating

germination are activated by seed priming and persist following the re-

desiccation of the seed (Asgedom and Becker, 2001). Therefore, upon sowing,

the primed seeds can rapidly imbibe and restore the seed metabolism,

resulting in an increased germination rate, decreased physiological

germination heterogeneity and better seedling development (Rowse, 1995).

The germination process can be divided into three phases: (i) initial fast

imbibition (ii) starting of metabolic processes in the seed and (iii) subsequent

radicle emergence and resumption of growth. In the process of seed priming,

seeds are soaked in water for the estimated time period to complete the first

two phases of the germination process. Drying the seed after the initiation of

radicle emergence causes damage to seed vigor and viability. Figure 2.1

shows the diagrammatic processes of normal seed germination and seed

priming.


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Fig. 2.1: Schematic diagram of normal germination and seed priming process.

Seed priming has shown beneficial effects on seed germination and seedling

development in many plant species. Harris (1996) demonstrated increased

germination rate and better establishment in tropical crops such as rice, maize,

chickpea and sorghum by water priming. In, different rice cultivars water

priming for 12-24 hours in West Africa has shown a 50% reduction in

germination time (Harris and Jones, 1997). Besides increasing germination

rate, priming also leads to faster crop growth, early flowering and higher grain

yields than unprimed seeds (Harris et al., 1999). Similarly, water priming of

barley seeds has also resulted increased germination rate and early seedling

emergence, moreover, priming also enhanced seedling growth, nutrient uptake

and drought tolerance (Ajour et al., 2004).

Several factors could be involved or responsible for the stimulation of early

seed germination after seed priming. During initial stages of seed priming,

seed water contents are rapidly increased to a certain level during imbibition

and therafer, there is a little change in water contents until radical emergence

(Bardford, 1986). Seed priming up to this stage (before radicle emergence)

can be beneficial. Further extended priming duration may have negative


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effects on seed germination due to death of the seedling on drying (Ajouri et

al., 2004). During priming, rapid and uniform stimulation of several

physiological changes occurring in the seeds enhance germination speed (Fig.

2.2).

Fig. 2.2: Time course of major events associated with germination and subsequent post

germination growth (Bewley, 1997).

In Arabidopsis, polypeptide contents increased in the seeds during the priming

period. These peptides were identified as degradation products (Gallardo et

al., 2001) of storage proteins. Similar results were obtained during the priming

process of sugar-beet seeds (Job et al., 1997). Degradation of 12S-cruciferin-

ß-subunits may play an important role in adaptation of embryo metabolism to

the oxidative conditions encountered during germination (Job et al., 2005). In

addition, seed priming causes the hydrolysis of abscisic acid (ABA) and the

leaching of different biochemicals e.g. coumarin, and various phenolic

compounds from the seed to the priming solution (Hopkins, 1995). These

compounds are reported as germination inhibitors when present in the seeds.

Priming, on the other hand, also improves membrane integrity of the

germinating seed. Short-chain fatty acids (C6-C12) are known for their function

to inhibit seed germination. During imbibition, the position of certain fatty acids


( 10(

associated with seed dormancy is changed and does not revert upon drying

(Bewley and Black, 1982). Hence, after seed priming, all the necessary

physiological and biochemical changes occurring during the priming process

persist upon drying. Therefore, when a primed seed is sown, it avoids phase

(i) & (ii), as shown above, and a little absorption of water can initiate the radicle

emergence. Instead, an unprimed seed has to go through all phases of seed

germination after sowing, which can be a possible reason for reduced

germination rate and seedling growth compared to primed seeds.

Seed nutrient contents play an important role in germination and early seedling

development. During early establishment stages, seedlings obtain mineral

nutrients partly from seed reserves and partly from soil (Rengel and Graham,

1995). During the germination process, nutrients stored in the seeds are

degraded by enzymes and transported to the growing embryo (Fincher, 1989).

A young seedling passes through various stages in nutrient uptake during

growth, based on the internal and external nutrient supplies and on crop

nutrient requirements (Scaife and Smith, 1973). In the beginning, the plant

utilizes nutrients from the seed reserves; it is the stage when external sources

have a partial effect, and afterwards nutrient uptake by roots is responsible for

plant growth with no further contribution from seed reserves (Whalley et al.

1966). Therefore, sufficient mineral nutrient reserves in the seed are

necessary to maintain seedling growth until the root system starts supplying

the nutrients. This is particularly important for crops grown in soils with low

available nutrients for plant growth (Asher, 1987). In rice, plants from seeds

with high Zn contents were better able to grow under Zn deficient conditions

compared to those grown from low Zn seeds (Rengel and Graham, 1995;

Hacisalihoglu and Kochian, 2003; Tehrani et al., 2003). Previous reports have

shown that wheat seeds with low Mn contents produced less vigorous

seedlings and ultimately resulted in low yields at harvest (Marcar and Graham,

1986; Singh and Bharti, 1985). Delayed germination and seedling

development was also observed in barley and lupin with low Mn seed contents

(Genc et al., 2000; Longnecker et al., 1991; Crosbie et al., 1994). It has been

shown that the percentage of abnormal seedlings increased with decreasing


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level of Boron (B) in soybean seeds (Rerkasem et al., 1993) and in green and

black grams (chickpea) (Rerkasem et al., 1989; Bell et al., 1989). High seed

Phosphorous (P) contents have improved early seedling growth and dry matter

production in clover (Thomson and Bolger, 1993), barley (Zhang et al., 1990),

spring wheat (Bolland and Baker, 1988) and rice (Ros et al., 1997). Seed P

contents also affected the number and mass of the nodules in bean plants

(Teixeria et al., 1999). Zhu and Smith (2001) have shown that wheat plants

grown from high seed P contents were able to accumulate more P in the shoot

compared to plants grown from low P seeds.

Different strategies are used for the continuous supply of micronutrients to the

crops, like direct soil application, foliar sprays and/or seed treatments. Direct

soil application of fertilizers is a widely adopted method but finding a good

quality micronutrient fertilizers and uniform application in the field is a difficult

and cost in-effective method. Foliar sprays of micronutrients have been used

as an effective technique to overcome micronutrient deficiencies in crop plants

(Wilhelm et al., 1988; Savithri et al., 1999) but it is expensive, time consuming

and more laborous (Johnson et al., 2005). Seed treatment with mineral

nutrients, on the other hand, is an easy, cost-effective and less time

consuming method (Robert, 1973; Ros et al., 2000). In case of barley

production in China on calcareous soils, seed soaking in P solution stimulated

seedling growth, and plants used the added P as early as three days after

application (Zhang et al., 1998). Yilmaz et al., (1998) have reported that Zn

seed dressing significantly increased wheat yield on Zn deficient soils.

2.2 Nutrient seed priming ‘Nutrient seed priming’ is a technique in which seeds are soaked in nutrient

solutions instead of water. Nutrient priming is an approach to increase seed

nutrient contents along with priming effects to improve seed quality for better

germination and seedling establishment. Priming seeds in solutions of macro

or micronutrients has been shown to improve yield of rice (Peeran and

Natanasabapathy, 1980), wheat (Khalid and Malik, 1982; Marcar and Graham,

1986; Wilhelm et al., 1988) and forage legumes (Sherrell, 1984), but the


( 12(

potential to damage the seed and inhibit germination by priming at high

nutrient concentrations has also been reported (Roberts, 1948; Ajouri et al.,

2004). In barley, nutrient priming of seeds with P and Zn enhanced

germination, seedling growth and uptake of these elements by plants (Ajouri et

al., 2004). Priming of maize seeds with 1% ZnSO4 solution improved plant

growth, final grain yield and increased seed Zn contents in the harvested

grains on soils with limited or deficient Zn supply (Harris et al., 2007). Soybean

seed priming with micronutrients i.e. Zn, Mn and B significantly increased

germination rate and concentrations of primed nutrients in young seedlings

grown under salt stress conditions (Imran, 2006).

2.3 Soil temperature

Soil temperature is an important abiotic factor involved in plant growth, as it

constantly affects other biotic and abiotic components of the soil–plant system

(Pregitzer et al., 2000). Soil temperature has a vital role in determining the soil

moisture contents, rate constants of different chemical reactions as well as

nutrient transport and availability in the soil. Besides that, it also affects plant

physiological processes involved in nutrient uptake, root growth and the soil

microbial activities (Pregitzer and King, 2005). In fact, nearly all processes

taking place in soil, from weathering of primary minerals to release of mineral

nutrients into soil solution, accumulation of soil organic carbon, are strongly

predicted by soil temperature. A number of chemicals, physical and biological

processes involved in the acquisition of mineral nutrients by plants are all

sensitive to temperature. Thus, soil temperature has a large influence on the

plant growth (figure 1.3).

There is a great variation in soil temperature with depth and over time. Air

temperature, irradiation and radiant heat transfer are the key factors

responsible for the large fluctuations in the temperature of surface soil layers

compared to more stable temperature in the deeper soil layers (Marschner,

2011). Root growth is severely affected by soil temperatures (Kasper and

Bland, 1992), and the optimum temperature for better root growth is mainly

under genetic control (Klepper, 1987). Optimum temperature for root growth is


( 13(

usually lower than required for shoot growth and also varies among the

species (Sattelmacher et al., 1990c, Cooper, 1973). For example, roots of oats

can grow normally at a temperature as low as 5 °C, but in maize at

temperature below 15 °C root growth already starts declining (Singh, 1998) for

maximum biomass production.

Fig. 2.3: A conceptual diagram depicting relationships between plant and soil temperature

for nutrient uptake. Soil temperature affects nutrient uptake directly by altering root growth, morphology and uptake kinetics. Indirect effects include altered rates of decomposition and nutrient mineralization, mineral weathering and nutrient transport processes (mass flow and diffusion). (Pregitzer and King, 2005).

Low or suboptimal temperature is the temperature at which plant growth is

inhibited. According to Larcher and Baur (1981) low temperature stress can be

defined as “any decline in temperature that can induce reversible or

irreversible functional disturbances or fatal injuries to plant resulting in growth

inhibition”. Therefore, low root zone temperature (RZT) stress can be

associated with decrease in soil temperature responsible for inhibited root

growth, reduced availability and transport of mineral nutrient in the soil for

optimal plant growth.


( 14(

2.3.1 Effect of root zone temperature on nutrient transport in the soil

In soil, most of the chemical reactions and nutrient transport take place in soil

solution; therefore, soil temperature directly affects the nutrient uptake in

plants. Soil nutrients are transported to the root surface by diffusion and mass

flow. Viscosity of the soil solution is inversely related to soil temperature,

almost, in a linear fashion (r2 = 0.94) in a wide range of soils worldwide (0 to

~70 °C), from 1.15x10–3 to 0.41x10–3 N s m–2 (Brown and LeMay, 1981).

Increased viscosity of soil water at low temperature is responsible for the

decreased water uptake rate by roots and transport within the plants (Johnson

and Thornley, 1985; Kramer and Boyer, 1995; Wan et al., 2001). This leads to

the reduced transport of nutrients to the roots by mass flow. Similarly, low root

zone temperature reduces the transport of nutrients from sites of high to low

concentration by the process of diffusion (Pregitzer and King, 2005). Hillel

(1998) has discussed the diffusion of gases in the soil atmosphere or solutes

in the soil solution by Fick’s first law:

Jd =−Ds θdc/dx

Where Jd is the flux rate of ion d, Ds is the diffusion coefficient of that ion

accounting for the tortuosity of the particular soil pore space, θ is the fractional

water volume, and dc/dx is the concentration gradient of species d. The

magnitude of the concentration gradient is considered as the main driving

force of the flux rate in the process of diffusion, but temperature has significant

influence on Jd because Ds increases linearly with increasing absolute

temperature and vice versa (Weast, 1978). Therefore, it can be concluded that

nutrient transport by the process of diffusion will be reduced at low root zone

temperature compared to warm soil conditions.

2.3.2 Effect of low soil temperature on root growth and functions

Under low temperature conditions growth of primary and secondary roots is

severely inhibited (Pahlavanian and Silk, 1988; Gladish and Rost, 1993) but

shoot growth is usually more inhibited than root growth, resulting in a high

root/shoot dry weight ratio (Huang et al., 1991). Exposure of plant roots to low


( 15(

soil temperatures usually causes chilling-induced drought stress with following

leaf wilting due to reduced root hydraulic conductance, limiting water uptake

(Allen and Ort, 2001, Bloom et al., 2004). Particularly, impaired stomatal

function is observed under low temperature stress since the hydraulic

conductivity of the roots is decreased due to the higher viscosity of water at

low temperature and changes in the physiological activity of roots (Melkonian

et al., 2004). The root physiological activities of chill-sensitive genotypes are

characterized by stronger inhibition of water uptake and root respiration than in

chill-tolerant genotypes when exposed to low temperature (Sowinski and

Maleszewski, 1989).

The rate of root respiration decreases at low soil temperature conditions as the

enzyme activity is remarkably reduced in cold soil (Covey-Crump et al., 2002).

Sink capacity declines under low root zone temperature conditions, which

could be responsible for inhibited root growth at early spring in temperate

climate region and at high altitudes (Alvarez-Uria and Korner, 2007). At low

soil temperatures, extensibility of cell walls is reduced in the extension zone, -

a possible reason for a decreased rate of root elongation under cold stress

(Marschner, 2011). In maize, cell wall extensibility in the root elongation zone

is reduced by 25 % by lowering the temperature from 30 to 15°C (Pritchard et

al., 1990). Root anatomy is also greatly affected by low root zone

temperatures.

Shoot and leaf elongation rate is inhibited at low root zone temperature, even

without disturbing leaf water potential (Milligan and Dale, 1988), most probably

due to an increase in ABA concentration in the leaves (Smith and Dale, 1988).

Atkin et al., (1973) have reported an almost two fold increase in ABA

concentration in the xylem exudates of maize plants when soil temperature

was shifted from 28 to 8°C. Production of cytokinins in the roots and its export

is also depressed at low root zone temperature. In maize plant, there was 15%

decline of cytokinin production at 18°C compared to 28°C (Atkin et al., 1973).

Although, lipid composition differs between cellular membranes (Yoshida and

Uemura, 1986), plant tissues and plant species (Staehelin and Newcomb,

2000), extreme growth conditions also strongly influence their composition in


( 16(

different plant parts. For example, clear changes in sterol concentrations in the

leaves under low temperature stress have ben reporte by Westerman and

Roddick (1981). Low temperature causes changes in the membrane lipid

composition (Welti et al., 2002; Penfield, 2008), and phospholipids are

replaced by di-galatosyl-di-acyglycerol (DGDG) and sulphoquinovosyl-di-

acylglycerol (SQDG) in leaves of P deficient plants (Hölzl and Dörmann, 2007;

White and Hammond, 2008). Low root zone temperature also causes changes

of similar nature in the root membranes (Cakmak and Marschner, 1988c).

Usually, the changes in lipid composition are considered for the adaption of a

plant to growing conditions. Plants growing at low soil temperature have

changes in their membranes; have more phospholipids with charged head

groups and low saturated shorter fatty acid chains, and higher sterol content

compared with plants growing at higher temperatures (Staehelin and

Newcomb, 2000; Wallis and Browse, 2002; Penfield, 2008). These kinds of

changes are necessary for the alteration of the freezing point (i.e. the transition

temperature) of membranes to a lower temperature and could be important for

the maintenance of membrane functions at low root zone temperatures

(Marschner, 2011).

2.3.3 Effect of low root zone temperature on Nutrient uptake

In the plant cell walls the ‘apparent free space’ consists of (i) the ‘water free

space’ (WFS) available to individual ions, charged and uncharged molecules,

and (ii) the ‘Donnan free space’ (DFS) for cation exchange and anion repulsion

(Fig 1.4). The term ‘apparent free space’ (AFS) was introduced by Hope and

Stevens (1952).


( 17(

Fig. 2.4: Schematic diagram of the pore system of the apparent space in plant cell walls.

DFS= Donan free space, WFS = water free space. (Marschner, 2012)

Temperature has a little effect on physical processes e.g. exchange adsorption

of cations in the AFS (Q10 = 1.1–1.2, where Q10 refers to the change in a

reaction or process forced by a change in temperature by 10 °C) (Marschner,

2011). On the other hand, temperature has much greater effect on chemical

and biochemical reactions with Q10 = 2 and Q10 > 2, respectively. In the

physiological temperature range, the value of Q10 often raises beyond 2 for the

uptake of ions from low concentration solutions (Clarkson et al., 1988; Wang et

al., 1993). In maize, it has been observed that the process of ion uptake is

more severely inhibited by low temperature than respiration, especially at

temperatures below 10°C (Bravo and Uribe, 1981).

Soil temperature affects ion uptake in plants in two different ways: (i) the short-

term effects of a sudden change in root zone temperature, which take place

instantly and show the direct effects of temperature on the ion uptake system,

and (ii) the long-term effects, where plants are exposed to particular root

temperature for a longer growth period. The long term effects of low

temperature can be observed after several days or weeks of growth at low root


( 18(

zone temperature and involve adaptive responses e.g. changes in the

properties of root membranes. Low root zone temperature influences cell

division and cell elongation in the shoots (Marschner, 1995).

Effect of low root zone temperature on nutrient uptake differs between

nutrients. For example, P uptake at low root zone temperature is drastically

reduced as compared to other nutrients (Engels and Marschner, 1992a;

Engels, 1993). In cold-sensitive plants at low root zone temperature, uptake

rate of nitrate is strongly reduced compared to ammonium (Tachibana, 1987),

but in cold-tolerant species, such as barley and ryegrass, low root zone

temperature does not affect the strong preference for ammonium compared

with nitrate uptake (Macduff and Jackson, 1991; Clarkson et al., 1992).

Potassium uptake is more affected by low root zone temperature as compared

to Ca and Mg.

At low root zone temperature, Zn uptake and translocation from root to the

shoot is usually more depressed than growth, resulting in lower Zn

concentration in the shoot compared to roots (Ellis et al., 1964; Sharma et al.,

1968; Schwartz et al., 1987). Higher supply of P fertilizers at low root zone

temperature may also enhance the Zn deficiency (Martin et al., 1965).

Similarly, Mn concentration in the shoot is also decreased at low root zone

temperature (Moraghan, 1985; Moraghan et al., 1986). Furthermore, Fe

uptake and translocation rate was decreased with decreasing root zone

temperature in tomato plants (Riekels and Lingle, 1966).

The roots of soil-grown plants have to explore for many immobile soil nutrients

(Engels and Marschner, 1990; Rengel, 2001; Lynch, 2007; White and

Hammond, 2008; White and Broadley, 2009). In plants grown at low root zone

temperature, therefore, nutrient uptake is not only affected by low nutrient

availability but also by detrimental effects on root growth rate, root system

morphology and functionality.

2.3.4 Effect of low temperature on plant growth

Low temperature affects a number of metabolic (physiological and

biochemical) and structural (anatomical and morphological) changes in the


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plants (Cooper, 1973; Levitt, 1980; Pollock and Eagles, 1988). Effects of low

temperature on physiological and biochemical processes may include:

reactions in the rate of cell division, photosynthesis and assimilate transport,

rate of respiration, synthesis and translocation of hormones, lipid synthesis,

production and activities of proteins and enzymes (Berry and Raison, 1981;

Graham and Patterson, 1982; Long and Woodward, 1988; Howarth and

Ougham, 1993, Engles and Marschner, 1992). Structural changes may affect

branching of roots, elongation, and cell wall properties (Cooper, 1973; Bowen,

1991; Marschner 1995 and 2012; McMichael and Burke, 1998; Rapacz, 1998;

Equiza et al., 2001). Under the conditions of low temperature stress, the plant

nutrient demand is also altered by effects on root to shoot ratio and other

biochemical processes (Clarkson et al., 1988).

The sensitivity of different plant growth processes to low temperature is

variable. Some are more sensitive to low temperature than the others, and the

sensitivity of the same process varies between and even within plant species

(Miedema, 1982; Paul et al., 1990). For example, in sunflower, leaf expansion

is more severely inhibited than cell division by low temperature (Granier and

Tardied, 1998). There was a significant decrease in shoot dry matter

production in barley plants grown at 5°C due to reduced water uptake rather

than to lower photosynthesis (Sharratt, 1991). Low root zone temperature

causes chill-induced drought stress with subsequent leaf wilting due to

inhibited root hydraulic conductance limiting water uptake (Allen and Ort, 2001;

Bloom et al., 2004, Melkonian et al., 2004).

Photosynthetic capacity and efficiency is decreased in plants grown at

suboptimal growth temperature (Nie et al., 1992), particularly, in chilling

sensitive genotypes (Fracheboud et al., 1999). The reduced photosynthetic

activity of the leaves developed under low temperature stress does not resume

to its optimal level, even after shifting plants to normal growth temperature

(Haldimann, 1996; Nie et al., 1992), which indicates structural changes of the

photosynthetic machinery. In contrast to photosynthetic activity, chlorophyll

content of the cold-stressed leaves recovers immediately after shifting to

optimal temperature but chlorophyll pigment composition analyses have


( 20(

shown a great reduction in the chlorophyll a/b ratio at suboptimal growth

temperature, especially in cold-tolerant species (Haldimann et al., 1995;

Haldimann, 1998). Effects of chilling on photosynthesis are illustrated in fig.

2.5.

Fig. 2.5: Primary effects of a short-term chilling periods in the light and the dark on

photosynthesis in thermophilic plants. (Allen and Ort, 2011)

The ratio of LHC-II (light-harvesting (or antenna) complex) to the reaction

centre (RC, a complex of several proteins, pigments and other co-factors

assembled together to execute the primary energy conversion reactions of

photosynthesis) increases at suboptimal growth temperature (Caffarri et al.,

2005). Furthermore, the number of minor antenna (predominantly contains

chlorophyll b, xanthophylls, and carotenoids) complexes decreased in

comparison to LHCII when maize seedlings were grown at suboptimal

compared to optimal temperature but to a lesser extent than RCII (Leipner and

Stamp, 2009).

Particularly, when seedlings are grown at low temperature conditions, plastid-


( 21(

encoded proteins of the RC fail to accumulate in the mesophyll thylakoids (Nie

and Baker, 1991; Robertson et al., 1993). A typical ultra structure of the

mesophyll chloroplasts reflects the reduced accumulation of certain thylakoid

proteins. Mesophyll chloroplasts in leaves developed at chilling temperature

are a little bigger and more round in shape (Kutík et al., 2004; Robertson et al.,

1993). Low temperature causes disruption in the granal arry (mesophyll

chloroplast) due to extensive vesiculation, particularly in maize genotypes

sensitive to chilling (Pinhero et al., 1999). Furthermore, growth at low

temperature disturbs the fatty acid composition of the chloroplast membranes

by increasing the unsaturated fatty acids (Fracheboud, 1999).

Regarding the activity of enzymes from the C4 cycle and the Calvin cycle,

contents of rubisco and phosphoenolpyruvate carboxylase were reduced in

leaves that developed at suboptimal temperature, whereas the NADP malate

dehydrogenase activity increased compared to leaves grown at optimal

temperature (Kingston-Smith et al., 1999). Chilling stress significantly reduces

the amount of Rubisco in chilling-sensitive plant compared to chilling-tolerant

plants (Pietrini et al., 1999). However, an increase in the activation state of

rubisco may compensate the decline in its activity caused by chilling stress

(Kingston-Smith et al., 1999).

In the C4 photosynthetic system, assimilates are transported between the

mesophyll and the bundle sheath cells. Low temperature stress, in the leaves,

causes the development of a larger area between mesophyll cells and the

bundle sheath and increases the number of plasmodesmata between these

cell types (Sowinski et al., 2003), particularly in cold- tolerant genotypes. This

increase in plasmodesmata density plays an important role in the efficient

exchange of photosynthetic metabolites as well as export of sucrose at low

temperatures responsible for reduced diffusion rate from cell to cell (Sowinski

et al., 2003).

Older or fully developed leaves are less sensitive to chilling than newly

developing leaves (Leipner and Stamp, 2009). The shoot apex is very

sensitive to low root zone temperature; furthermore, low root zone temperature

also adversely affects growth rate and leaf expansion (Engels, 1994; Stone et


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al., 1999). Photosynthetic performance of maize seedlings is strongly inhibited

at low soil temperature, especially in chill-sensitive genotypes (Chassot, 2000;

Hund, 2003), that could be due to delay in leaf development (Sowinski et al.,

2005). Cold-induced growth inhibition of the leaves is caused by a strong

prolongation of cell cycle and by a reduction of cell production (Rymen et al.,

2007). Spatial analysis of maize leaf blades, exposed to low temperature

stress (6°C, 400 µmol m-2 s-1) for four days, showed two zones of chlorosis.

These cholrotic areas were tracked on the leaf meristem and the leaf surface

which were just exposed to light (Leipner and Stamp, 2009). Therefore, it is

concluded that (i) chilling induced damages during cell production limits the

development of fully functional cells, and (ii) the combined effect of high light

intensity and low temperature leads to the impaired development of

chloroplast.

2.4 Functions of Mineral nutrients in plants

2.4.1 Zinc (Zn) in plants

2.4.1 .1 Zinc uptake and transport in plants

Zinc is an essential micronutrient vital for plant growth and development.

Under normal conditions, zinc is taken up as a divalent cation (Zn2+) whereas

uptake in the monovalent form (ZnOH+) is preferred under high pH conditions.

During long-distance transport in xylem, Zn is either bound to organic acids or

may be translocated as free divalent cations (Marschner, 2012). Zinc

concentrations are fairly high in phloem sap, most probably due to its binding

to low-molecular-weight organic solutes (Kochian, 1991). Zinc does not take

part in redox reactions as it exists only as ZnII in plants and other biological

systems. The metabolic functions of Zn depend on its strong tendency to form

tetrahedral complexes with (nitrogen anions) N-, (oxygen anions) O-, and

particularly Sulfur S-ligands for its catalytic and structural role in enzymatic

reactions (Marschner, 1995).

The functions of Zn and its deficiency symptoms in plants have been


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comprehensively discussed and reviewed (Thorne, 1957; Cakmak and

Marschner, 1988; Welch and Norvell, 1993). Zinc has a critical role in

maintaining plasma membrane structure and its proper functioning in plant

cells (Welch and Norvell, 1993). Cakmak (2000) has discussed importance of

Zn in protecting leaf tissues from oxidative stress.

2.4.1 .2 Morphological effects of Zn deficiency in plants

In dicotyledonous plant species, Zn deficiency causes reduction in leaf size,

shortening of internodal distance leading to stunted plant growth. In oil seed

rape, total number and size of leaves was depressed by Zn deficiency (Hedley

et al., 1982a, b). In dicots generally, Zn deficiency causes intervenial chlorosis

of young leaves starting at tips and margins of leaves, followed by

development of irregular intervenial chlorotic spots (Roberth and Francis,

1983), associated with reduced plant growth and decreased dry biomass

production (Hewitt, 1984). Zinc deficiency symptoms may vary depending on

the plant species, age and growing conditions (Snowball and Robson, 1983;

Grundon et al., 1987).

In monocots, Zn deficiency causes short internodes, which results in stunted

plant growth, delayed flowering and maturity (Grundon et al., 1987). In cereals,

like in wheat, Zn deficiency causes reduced shoot growth and development of

whitish-brown necrotic patches on older leaves while young leaves becomes

yellowish green in colour but now showing necrotic lesions (Cakmak, 1996a).

Chlorosis and necrosis in older leaves of Zn-deficient plants could be

secondary effects due to P or B toxicity, or by photo-oxidation because of

impaired translocation of photosynthates (Cakmak, 2006). Generally, under Zn

deficiency, both, in mono and dicots, shoot growth is more inhibited compared

to root growth (Zahng et al., 1991a), which may be a consequence of

increased translocation of photosynthates to roots due to a shift in sink

location from developing shoot tissues to growing roots (Cumbus, 1985;

Cakmak, 1996b). Abiotic stress factors such as low soil water contents and

low soil temperature have been reported to increase in Zn deficiency


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symptoms (Lindsay, 1972). Under conditions of reduced soil moisture

contents, Zn uptake is limited due to reduced diffusion rate of Zn to the root

surface (Marschner, 1993). Low soil temperature impairs root growth, root

activity and reduces Zn translocation from root to the shoot (Moraghan and

Mascagani, 1991; Engels and Marschner, 1992). Moreover, high light intensity

also increases the severity of Zn deficiency symptoms in leaves (Cakmak et

al., 2000).

2.4.1 .3 Physiological and biochemical effects of Zn

Zn is involved in a vast range of physiological and biochemical processes in

plants. It is associated with metabolism of carbohydrates, proteins, enzyme

activities, structural and functional integrity of cell membranes (Römheld and

Marschner, 1991; Cakmak, 2000; Marschner, 2011).

i. Role of Zn in enzymatic functions

Zinc is the only metal, with functions in all six enzyme classes including oxido-

reductases, transferases, hydrolases, lyases, isomerases and ligases, in

biological systems (Sousa et al., 2009). Four different functions of Zn have

been identified in these enzymes: (i) catalytic, (ii) structural, (iii) co-catalytic

and (iv) protein synthesis in determining the biological activity of various

enzymes (Marschner, 2012). The involvement of Zn in structural and functional

activities of such a wide range of enzymes suggests that Zn deficiency may

inhibit the processes involved in the metabolism of carbohydrate, protein,

auxin and reproduction (Römheld and Marschner, 1991; Brown et al., 1993).

The role of Zn in function of some of the key enzymes is reviewed below.

Alcohal dehydrogenase

Alcohol dehydrogenase (ADH) is an exceptional Zn enzyme with 2 atoms per

molecule, one with catalytic and the other with structural functions, while

mostly Zn enzymes have only 1 Zn atom in their molecules (Colman, 1992;


( 25(

Auld and Bergman, 2008). ADH is one of the two proteins responsible for the

reduction of cytotoxic acetylaldehyde to ethenol in the ethanolic fermentation

pathway, resulting in continuous regeneration of NAD+ in the cytoplasm

(Chung and Ferl, 1999). Thus, induction of ADH can improve plant growth

under submerged conditions (Johnson et al., 1994). Proper root functions

could be impaired as a consequence of reduced ADH activity due to Zn

deficiency (Moore and Patrick, 1988).

Carbonic anhydrase

Carbonic anhydrase (CA) is a zinc-containing metallo-enzyme that catalyzes

the interconversion of CO2 and HCO3- in many organisms. In plants, both,

chloroplasts and cytoplasm contain CA, which catalyses the reversible

hydration of CO2 :

CO2 + H2O → HCO3- + H+

In C3 plants, plant Zn nutritional status does not have a direct relationship

between CA activity and photosynthetic CO2 assimilation (Atkin et al., 1972;

Brown et al., 1993). CA activity is completely inhibited under extreme Zn

deficiency, but maximum photosynthetic rate may be attained even when CA

activity is low. However, in C4 plants, it is evident that high CA activity is

required in the mesophyll chloroplasts to convert CO2 to bicarbonate (HCO3−).

Consequently, HCO3− is fixed via phosphoenolpyruvate carboxylase (PEPC)

into C4 compounds (e.g. malate) for the shuttle into the bundle sheath

chloroplasts for decarboxylation (Kanai and Edwards, 1999). Hence, Zn

deficiency may impair the photosynthetic rate in C4 plant as compared to C3

plants (Burnell et al., 1990).

Superoxide dismutase (SOD)

The most abundant superoxide dismutase enzyme found in plant cells is CuZn

superoxide dismutase (CuZnSOD); where Cu (copper) is the catalytic metal

component while Zn is part of the structural unit. Localization and functions of


( 26(

CuZnSOD have been discussed in details by Cakmak (2000). In different plant

species, under Zn deficiency, CuZnSOD activity is significantly reduced

(Marschner, 1995), and enzyme activity is immediately restored after Zn re-

supply (Cakmak and Marschner, 1993). Yu and Rengel (1999) have reported

that Zn deficiency in lupin decreased the total SOD and CuZnSOD activity, but

MnSOD activity was unaffected. Under Zn deficiency, decreased SOD activity

is responsible for the increase in the rate O2− generation and leads to the

membrane lipids peroxidation and increase membrane permeability (Cakmak

and Marschner, 1988; Cakmak and Marschner, 1993). Hence, abiotic stress

tolerance in transgenic plants can be increased by the over expression of

CuZnSOD (Cakmak, 2010; Kim et al., 2010).

ii. Role of Zn in carbohydrate metabolism

A number of Zn depending enzymes are involved in the carbohydrate

metabolism in plants at various levels (Marschner, 1995). The association of

Zn in carbohydrate metabolism is linked to its role in photosynthesis and sugar

transformation. In different plant species, net photosynthesis could be

depressed up to 70%, depending on the level of Zn deficiency (Brown et al.,

1993). Under Zn deficiency, activity of fructose-1,6-bisphospahte declines

quite rapidly, while the activities of other enzymes is effected to lesser extent

(Marschner, 2011). Sugars and starch often accumulate in the leaves of Zn

deficient plants (Sharma et al., 1982). Cakmak et al., (1989) found that re-

supply of Zn could restore the phloem loading of sucrose. Most probably, the

restoration of phloem loading of sucrose by Zn supply can be attributed to the

role of Zn in the structural integrity of bio-membranes (Welch et al., 1982).

iii. Role of Zinc in protein metabolism

Zinc is an integral component of the ribosomes and for their structural integrity.

Protein synthesis is dramatically impaired in Zn deficient plants but the

composition is almost unchanged (Obata et al., 1999). This decrease in


( 27(

protein synthesis can be associated to increase in concentration of free amino

acids under Zn deficiency, as the concentration of free amino acids rapidly

decreases after Zn resupply (Cakmak, 1989). Under Zn deficiency, level of

free RNA and ribosomes is depressed as a result of reduced transcription

(Kitagishi and Obata, 1986; Kitagishi et al., 1987). RNase activity is

significantly increased under zinc deficiency (Sharma et al., 1982). There is an

inverse relationship between Zn supply and RNase activity, and also between

RNase activity and protein content (Johnson and Simons, 1979). Zn is

essential for RNA polymerase activity and prevents ribosomal degradation by

ribonuclease ( Fachuk et al., 1978; Jendrisk and Burgess, 1975). Under Zn

deficiency activity of RNase increase, resulting in stunted plant growth and

changes in leafe anatomy (Dwivedi and Takkar, 1974).

2.4.1.4 Zinc in membrane integrity

Zinc has a critical role in the maintaining integrity and functions of the bio-

membranes (Bettger and O’Dell, 1981; Pinton et al., 1994). Zn deficiency in

plant cells causes damages in plasma membrane structure and increases the

permeability of inorganic ions (Welch et al., 1982). Role of Zn in membrane

stability is not coupled with the function of Ca, as increased external supply of

Ca could not reduce the increased membrane permeability caused by Zn

deficiency (Cakmak and Marschner, 1988c). Zinc protects membrane lipids

and proteins from oxidative damages by binding to phospholipids and

sulfhydryl (Powel, 2000) or form tetrahedral complexes with cysteine residues

of polypeptide chains (Cakmak and Marschner, 1988c). Zinc interference with

NADPH oxidation and also as a metal component of CuZnSOD controls the

generation of toxic oxygen radicals and prevents the membrane damages

against reactive oxygen species (ROS) (Cakmak and Marschner, 1988a,b;

Cakmak, 2000). Redox status of the reactive –SH (sulfhydryl) groups present

in the ion channel proteins controls the activity of the cannel (Cakmak, 2000).

In isolated vacuoles from sugar beet, a shortly induced Zn deficiency strongly

reduced the activity of Ca-activated slow vacuole –type anion channels


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(Hedrich and Kurkdjian, 1988).

2.4.1.5 Zinc localization in seeds

Zinc, in seeds and grains, is mostly localized in protein bodies in the form of

special particles, the globoid crystals (Lott and Buttrose, 1978; Welch, 1986).

Phytate (salts of phytic acid) is the main constituent of these globoids. In

wheat seeds, Zn is highly concentrated (600 µgg-1dry weight) in the scutellum

(Mazzolini et al., 1985). Mostly, Zn, Fe and proteins are co-localized in the

seed tissues (Cakmak et al., 2010b) and concentrations of Zn, Fe and proteins

are highly correlated to each other in seeds of a number of germplasms

(Peterson et al., 1986; Morgounov et al., 2007; Zhao et al., 2009; Cakmak et

al., 2010b). It could be suggested that proteins act as a sink for Zn and Fe.

Phytic acid has a high affinity to bind divalent cations such as Zn, due to the

negative charged phosphate residues, and form insoluble or unavailable Zn-

phytate complexes in the seeds (Lönnerdal, 2002; Schlemmer et al., 2009)

causing major problems by reducing the bioavailability of Zn for monogastric

animals and humans.

2.4.2 Iron (Fe) in plants

2.4.2.1 Iron uptake in plants

Iron (Fe) is one of the most abundant metal elements present on earth.

Dominant forms of soluble Fe present in soils and nutrient solutions are

chelates of Fe (III) and Fe (II) (Marschner, 2012). Under alkaline conditions,

the presence of Fe ions in the soil solution is severely reduced due to the

formation of Fe hydroxides, oxyhydroxides and oxides (Lemanceau et al.,

2007). Depending on the plant species (strategies I and II), uptake of Fe (II) is

favored as compared to Fe (III) in strategy I plants. There is a predominance

of Fe (III) complexes in the long distance transport in xylem. Iron as a

transition element, easily changes its oxidation state and also forms octahedral

complexes with various ligands. Redox potential of Fe (II or III) widely depends


( 29(

on the complexing ligands. Due to this character, Fe plays a significant role in

biological redox systems. Presence of low molecular weight Fe chelates and

free iron (Fe3+ or Fe2+) in aerobic systems produces reactive oxygen species

(ROS), such as superoxide and hydroxide radicals and other related

compounds (Halliwell and Gutteridge, 1986; Halliwell, 2009). These extremely

lethal radicals are responsible for membrane lipid and protein peroxidation.

Binding or incorporation of Fe into structures (e.g., heme and non-heme

proteins) is necessary for controlled reversible oxidation–reduction reactions,

to prevent cell damages from ROS.

2.4.2.2 Iron in plant proteins.

Cytochromes are well known heme proteins containing heme Fe-porphyrin

comlex as a prosthetic group. Cytochromes are essential compenents of the

redox systems in chloroplasts (Hil and Scarisbrick, 1951; Hil 1954), in the

electron transport in the respiratory chain of mitochondria and also involved in

the redox chain in nitrate reductase (Cava et al., 2008).

Catalases and peroxidases are other heme enzymes; the activity of these

enzymes declines under the low Fe supply (Marsh et al., 1963; Chouliaras et

al., 2005). Catalases play an important role in the detoxification of

photorespiratory Hydrogen peroxide (H2O2) to water and CO2 (Kendall et al.,

1983; Willekens et al., 1997; Corpas, Barroso and del Río, 2001;

Vandenabeele et al., 2004). Additionaly catalases also have other essential

functions in plant tissues at different developmental stages: catalases take part

in the signal transduction pathways of various stress responses (Hu et al.,

2010; Fukamatsu, Yabe and Hasunuma 2003; Verslues et al., 2007).

Peroxidases exists in the plants in various forms (Shanon, 1948). They mainly

catalyse the following reactions:

XH2 +H2O2 →X+2H2O

and

XH+XH+H2O2 → X---X+2H2O


( 30(

For example, scavenging of H2O2 in chloroplasts catalysed by ascorbate

peroxidase (Foyer and Halliwell, 1976; 1977) represents the first type of

reaction, while the polymerization of phenols to lignin is catalysed by cell-wall

bound peroxidase is related to the second type. Impaired peroxidase activity

may alter cell wall formation, as the presence of peroxidase in the cell walls of

the epidermis (Hendricks and Van Loon, 1990) and rhizodermis (Codignola et

al., 1989) is vital for the biosynthesis of lignin and suberin. Phenolic

compounds and H2O2 are required as substrates for the synthesis of both

compunds.

Peroxidase activity is severely reduced in Fe-deficient roots (Sijmons et al.,

1985; Ranieri et al., 2001). This leads to an increase in the production of H2O2

and accumulation of phenolic compounds in the root cell (Ranieri et al., 2001;

Römheld and Marschner, 1981a). It is reported that phenolics are also

released at higher rates from the roots of Fe-deficient compared with Fe-

sufficient plants (Hether et al., 1984; Marschner et al., 1986a; Jin et al., 2007).

It has been proposed that release of high amounts of phenolics contribute to

the utilization and remobilization of apoplastic Fe from the roots (Jin et al.,

2007).

Ferredoxin is an example of non-heme Fe-S proteins; these proteins are

characterized by the presence of Fe-S clusters containing sulfide-linked di-, tri-

, and tetra Fe centers in variable oxidation states. In plants, ferredoxins

function mainly in the process of photosynthesis; they transmit electrons from

photoreduced Photosystem I to ferredoxin NADP+ reductase, where NADPH is

generated for CO2 assimilation. Moreover, ferredoxins also act as electron

transmitters in various ferredoxin-dependent enzymes for the assimilation of

inorganic N, S, N2 and also regulate the CO2 assimilation cycle (Fukuyama,

2004). Iron-superoxide dismutase (Fe SOD) is another example of Fe-S

proteins. Superoxide dismutases degrade superoxide anion free radicals (O2-)

by formation of H2O2 and may contain Cu, Zn, Mn or Fe as metal components

(Fridovich, 1983; Sevilla et al., 1984). FeSOD is mainly present in the

chloroplast (Kwiatowsky et al., 1985) and could be also present in the


( 31(

mitochondria and peroxisomes in the cytoplasm (Droillard and Paulin, 1990).

In Fe-deficient plants reduced activity of FeSOD (Iturbe-Ormaetxe, 1995)

causes increase in the activity of CuZnSOD, which increases H2O2 production

(Tewari et al., 2005).

There are also a number of other enzymes where Fe takes part as a metal

component in the redox reactions or as bridging element between enzyme and

substrate (Marschner, 2012). Reduced activity of these enzymes due to Fe

deficiency in plants leads to vital changes in the metabolic processes.

In the process of ethylene biosynthesis, methionine acts as a primary

precursor. Iron(II) catalyses the two step 1 oxidation, during the biosynthetic

pathway in the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to

ethylene. In Fe-deficient cells, formation of ethylene is strongly inhibited, but

immediately restored after resupply of Fe, involving protein synthesis

(Bouzayen et al., 1991).

Lipoxygenases enzymes contain a single Fe atom per molecule (Hildebrand,

1989), catalyse the peroxidation of linolic and linolenic acid, i.e. of long chain

polyunsaturated fatty acids. Hence, high lipo-oxygenase activity is essential for

fast growing plant tissues and organs, and could be vital for membrane

stability.

2.4.2.3 Iron in chloroplast Development and Photosynthesis

Size of the chloroplast and protein contents per chloroplast are severely

affected by Fe deficiency in plants as compared to leaf growth, cell number per

unit area, or number of chloroplasts per cell (Terry, 1988). Fe status of the leaf

cells has a vital role in determining the number of ribsomes and protein

synthesis (Lin and Stocking, 1978). Iron deficiency in maize leaves, causes a

25% reduction in total protein contents, while chloroplast production was

decreased by 82% (Perur et al., 1961), as there is a high Fe requirement of

chloroplastic mRNA and rRNA (Spiller et al., 1987). Iron is required for RNA


( 32(

synthesis, in sugar beet leaves, a strong inhibition of protein synthesis is linked

to Fe deficiency in the leaves (Nishio et al., 1985). Decrease in leaf protein

contents under Fe deficiency are attributed to the Rubisco protein, which is

approximately 50% of the chloroplast soluble proteins (Ellis, 1979).

There are about 20 Fe atoms directly involved in the electron transport chain in

the thylakoid membranes. Because of higher Fe contents (12 atoms of Fe per

complex) Photosystem (PS) I is a major sink for Fe compared to PS II (3

atoms of Fe per complex) and the Cyt bf complex (5 atoms of Fe per complex)

(Raven et al., 1999). Iron is required in high amounts for the structural and

functional integrity of the thylakoid membranes. Fe deficiency in the leaves

does not decrease, equally, all the photosynthetic pigments and different

components of the electron transport chain (Pushnik and Miller, 1989). Under

Fe deficiency, the activity of PS I is more depressed than that of PS II (Pushnik

and Miller, 1989). The electron-transmitting ability of the PS I is strongly

increased upon re-supplying Fe to chlorotic leaves than that of PS II. Under

severe Fe deficiency, the activity of PS II also decreases and is more difficult

to restore (Terry, 1980; Morales et al., 1991).

Photosynthetic activity in the leaves is generally low under Fe deficiency but

light energy absorbed per chlorophyll molecule increases than the required for

photosynthesis under Fe deficiency, particularly under high radiation (Abadía

et al., 1999). This increases the risk of photoinhibitory and photooxidative

damages in Fe-deficient leaves.

Low concentrations of starch and sugars are a typical characteristic of Fe

deficient leaves (Arulanathan et al., 1990). This is usually attributed to the

reduced chlorophyll and ferredoxin concentrations, impairment of

photosynthetic electron transport and the decreased regeneration of reduced

ferredoxin (Marshner, 2012). Reduced photosynthetic activity is a typical

physiological response of plants to Fe deficiency. Re-supply of Fe to Fe-

deficient plants rapidly increases the photosynthesis (Larbi et al., 2004). Under

Fe deficiency, reduction in photosynthetic activity is attributed to reduced

photosynthetic electron transport and inhibition of carboxylation process due to


( 33(

decreased availability of ATP and NADPH for the Calvin cycle (Larbi, 2006).

Another important reason for the low photosynthesis is low concentration of

rubisco proteins (Larbi et al., 2004; Timperio et al., 2007).

2.4.2.4 Iron in seeds

Iron is mainly stored as phytoferritin (plant ferritin) in plants. The structure

resembles a hollow protein shell, where up to 5000 atoms of Fe (III) can be

stored. Phytoferritin has a proposed formula (FeO. OH)8. (FeO.OPO3H2),

which has usually a well-defined crystalline structure (Seckbach, 1982).

Ferritin is present in high abundance in seeds. In pea plants, more than 90%

of the total Fe present in the seed embryo is in the form of ferritin-bound Fe,

indicating that ferritin could be a major form of Fe storage in the seeds

(Marentes and Grusak, 1998). However, Fe concentration in seed as ferritin-

bound Fe largely varies among the plant species. For example, in legume

species, ferritin-Fe concentration in kidney beans ranges from 15% of the total

Fe up to 69% in lentils (Hoppler et al., 2009). Microanalysis of wheat seeds

has shown that Fe is present in high concentration in the aleurone and

scutellum layer of the embryo, while very low concentration in the endosperm

(Mazzolini et al., 1985). In barley sesds, approximately, 70% of the total Fe is

located in the endosperm and aleurone while 7 - 8% is found in the embryo

(Duffus and Rosie, 1976). In monocots, most of the Fe is deposited in the

protein storage vacuole (PSV) of the embryo and the aleurone layer and forms

specialized structures ‘globoids’ after complex formation with phytate (Søren et

al., 2009). The protein storage vacuoles are complex structures, consisting of

a protein matrix with globoid inclusions containing phytate, niacin,

carbohydrates and phenolics (Becraft, 2007). Phytic acid is a P storage

compound in most seeds. It has a strong chelating ability that readily binds

metal cations such as Ca, Zn, Mg, Fe, and Mn, making them insoluble and

decrease in their bioavailability in food (Lönnerdal, 2000; Urbano et al., 2000;

Jiang et al., 2001; Ficco et al., 2009).


( 34(

2.4.3 Manganese (Mn) in plants

Manganese ions have a high capacity to change the redox state. Mn2+ is the

more dominant form and easily oxidized to Mn3+ or Mn4+, and therefore, in the

plant Mn plays an important role in redox reactions. In biological systems,

Mn2+ and Mn4+ ions are fairly stable while Mn3+ is unstable (Hughes and

Williams, 1988).

2.4.3.1 Role of Mn in enzyme structure and activation

Mn2+ is required for the activation of a large number of enzymes but there are

only a small number of Mn-containing enzymes, namely the Mn-protein in PS

II, the Mn-containing superoxide dismutase (MnSOD) and oxalate oxidase

(Marschner, 2011). Oxalate oxidase is a Mn-containing multimeric

glycosylated enzyme (Kanauchi et al., 2009). Oxalate oxidase catalyses the

conversion of oxalate and dioxygen to CO2 and H2O2 (Lane, 1994).

Superoxide dismutases (SOD) are produced in all aerobic life systems and

play a vital role in protecting against reactive oxygen species (Elstner, 1982;

Fridovich, 1983). The Mn SOD is an important member of SOD isoenzymes

along with others i.e. FeSOD and CuZnSOD. MnSOD is not commonly found

in higher plants (Sandmann and Böger, 1983) and largely located in

mitochondria and peroxisomes. Production of MnSOD in the chloroplast of

numerous transgenic plants has increased abiotic stress tolerance e.g., salinity

(Tanaka et al., 1999) drought (Wang et al., 2005a) and Mn deficiency (Yu et

al., 1999a).

Polypeptide (33kDa) of the water-splitting system in PS II is an important Mn-

containing enzyme. Water splitting system contains six components of the

Mn4Ca, in which four Mn atoms arranged as a cluster, that are very important

for oxidation of 2 water molecules (Nikos et al., 1971).

There are almost 35 enzymes where Mn acts as a cofactor (Burnell, 1988).

For example enzymes which catalyse oxidation-reduction, decarboxylation and

hydrolytic reactions (Marschner, 1995). Manganese is essential for the PEP


( 35(

carboxykinase enzyme in the bundle sheath chloroplasts of C4 plants, where it

catalyses the process of decarboxylation. Manganese is required for the

activation of several enzymes of the shikimic acid pathway, and subsequent

pathways, resulting in the biosynthesis of aromatic amino acids (such as

tyrosine), different secondary products (such as lignin, flavonoids) and IAA

(Burnell, 1988; Hughes and Williams, 1988). For example, Mn by stimulating

and combining with peroxidase work as diffusible redox shuttle in lignin

biosynthesis (Önnerud et al., 2002), stabilizes the activity of phenylalanine

ammonia lyase (PAL) (Wall et al., 2008). Indole acetic acid (IAA) oxidase

activity is increased in the leaves suffering from deficient or toxic levels of Mn

(Morgan et al., 1976).

2.4.3.2 Role of Mn in Photosynthesis

In higher plants, photosynthesis is severely depressed under Mn deficiency.

Particularly, Mn deficiency causes a strong inhibition in photosynthetic O2

evolution in PS II (Shenker et al., 2004; Husted et al., 2010). In subterranean

clover, Mn deficiency has little effect on chlorophyll concentration or leaf dry

biomass, but there was more than 50% reduction in Photosynthetic O2

evolution (Nable et al., 1984). Photosynthetic O2 evolution was restored to

normal rate with one day after resupplying the Mn to deficient leaves

(Marscher, 2011). Manganese deficiency causes the changes in the

ultrastructure of thylakoid membranes, responsible for the loss of PS II

functional units in the stacked areas of thylakoid membranes (Husted et al.,

2010). Number of the PS II protein- pigment units in the thylakoid membranes

is restored upon resupplying the Mn (Simpson and Robinson, 1984; Gong et

al., 2010).

Under severe Mn deficiency, concentration and contents of chlorophyll is

significantly low in the leaves and ultrastructure of thylakoids is considerably

changed (Kroengier et al., 1993). Restoration or reversibility of these

ultrastructural changes of thylakoids is very difficult, and are most probably a

consequence of biosynthetic inhibition of lipids and carotenoids, and not of

enhanced photooxidation (lipid peroxidation) of the thylakoids and chlorophyll


( 36(

(Marschner, 2011).

2.4.3.3 Role of manganese in cell division and elongation

In Mn-deficient plants, shortage of carbohydrates and also direct requirement

of Mn for growth is responsible for limited root growth (Campbell and Nable,

1988; Sadana et al., 2002). It is observed that the rate of cell elongation is

more suppressed under Mn deficiency than the rate of cell division (Abbott,

1967). Under normal Mn deficiency, the growth rate is rapidly restored on re-

supply of Mn. Formation and development of lateral root is strongly inhibited

under Mn deficiency (Abbott, 1967). In the roots of Mn-deficient plants,

development of a large number of small non-vacuolated cells in the roots

compared to Mn sufficient plants indicates Mn deficiency hinders the cell

elongation more strongly than cell division (Neumann and Steward, 1968).

2.4.4 Boron (B) in plants

Boron (B) is found in all subcellular compartments of the plant cells e.g. the

apoplasm, cell walls, cytosol and vacuoles, whereas greater part of the cellular

B is bound in the cell walls (Dannel et al., 2002). In the cells, B is present in

water-soluble and water insoluble forms (Matoh, 1997). Water-soluble B is

present in the apoplastic space in the form of boric acid and its concentration

decreases under limited supply of B to the plants (Skok and McIlrath, 1958;

Shive and Barnett, 1973). In some plant species, another water-soluble B

fraction is phloem-mobile B present in the form of B-polyol complexes (Hu et

al., 1997; Hu and Brown, 1997b). Although, size of the mobile B pool is much

smaller relative to total B, but it has vital role in plant growth (Brown et al.,

1999).

The water insoluble B fraction is largely present as a complex with

rhamnogalacturonan-II (RG-II) in higher plants and mostly situated in cell walls

(O’Neill et al., 1996). Mostly, B in the cell walls is coupled with pectin via the B-

RG-II complex, a borate diester (Kobayashi et al., 1996). It has been shown


( 37(

that B-RG-II and homogalacturonan (HG) are covalently bound together in

pectin (Ishii and Matsunaga, 2001). Accordingly, RG-II is the sole B containing

polysaccharide found in the biological materials (O’Neill et al., 1996) and most

likely the unique B form structurally complexed within the cell walls (Matoh,

1997).

Parr and Loughman (1983) have postulated 10 important roles of B in plants,

such as involvement in (i) transport of sugar compounds, (ii) synthesis of cell

wall, (iii) lignification, (iv) cell wall structure (v) carbohydrate metabolism, (vi)

RNA metabolism, (vii) respiration, (viii) indole acetic acid (IAA) metabolism, (ix)

phenol metabolism, and (x) functions of membranes. This long list shows the

immense role of B in plants but it still needs more research to fully understand

the functions of B in these processes. Functions of B in higher plants have

been reviewed by many authors (Goldbach, 1997; Blevins and Lukaszewski,

1998; Dannel et al., 2002; Brown et al., 2002). The role of B in cell wall

structure is well described (Matoh, 1997; Matoh and Kobayashi, 1998).

Although, the B function in plasma membrane is also well explained (Cakmak

and Römheld, 1997; Mühling et al., 1998; Kobayashi et al., 1999).

2.4.4.1 Role of B in cell wall structure

Boron is an important constituent of the cell wall structure. Durst (1988) stated

that 96 to 99% of the total B in cultured carrot cells is localized in cell walls,

while only 0.7 to 4.1% is present in the protoplasts. According to the Loomis

and Durst model (1992) for the role of B in cell wall structure, borate is cross-

linked in the middle lamella, probably associated with calcium (Ca) cross-

linking. Boron and Ca are vital to maintain the integrity of cell walls through

binding to the RG-II (rhamnogalacturaon II) (Matoh and Kobayashi, 1998).

Borate and Ca2+ cross-linking in the RG-II region (B-RG-II complex contains

two molecules of boric acid, two molecules of Ca2+ and two chains of

monomeric RG-II) play important role in retaining the chelator-soluble pectic

polysaccharides in cell walls (Kobayashi et al., 1999).

There is no evidence that B is directly involved in cell wall synthesis. However,


( 38(

B may influence the assimilation of proteins, pectins and/or their precursors

into the present and extending cell wall (Brown et al., 2002). Boron deficiency

rapidly increases the cell wall pore size, which leads to cell death upon

entering into the elongation phase of growth (Fleischer et al. 1999). The ability

of cells to form a pectin network with proper pore size is impaired under B

deficiency, which may influence important physiological processes, e.g.

polymer incorporation in the cell wall and transportation of wall-modifying

proteins or enzymes to their substrates and also the transport of polymers

from the protoplast into the cell wall (Fleischer et al., 1999; Brown et al., 2002,

Marschner, 2011). Boron could be crucial for cell-to-wall adhesion and to

organise the architectural integrity of the cell (Bassil et al., 2004). 2.4.4.2 Role in Boron Cellular membranes

Boron plays an important role in membrane structure and functional integrity.

The effect of B on plasma membranes may be similar to that in cell walls

(Brown et al., 2002). Accordingly, B is a key element in maintaining proper

structure of the membranes by forming cross-links with glyco-proteins and

glyco-lipids in the membrane “rafts”, membrane sub-domains rich in these

compounds (Brown et al., 2002).

The amount of B is located in the membranes is smaller relative to the total B

pool in the cell (Pollard et al., 1977; Torchia and Hirsh, 1982; Parr and

Loughman, 1983; Tanada, 1983). The B form in membranes is probably the

same as present in the cell walls (Cakmak and Römheld, 1997; Blevins and

Lukaszewski, 1998). Restoration of membrane functions inhibited due to B

deficiency and of B-deficient tissues after B re-supply is fast, and normally

occurs within a few minutes (Goldbach et al., 1990; Schon et al., 1990; Barr et

al., 1993). These functions may include membrane potential, release of

ferricyanide-dependent H+, activity of adenosine triphosphatase (ATPase),

limited nicotinamide adenine dinucleotide (NADH) oxidase activity, transport of

ions, and membrane permeability (Cakmak and Römheld, 1997; Blevins and

Lukaszewski, 1998).


( 39(

Boron may have direct functional roles in the cellular membranes, probably by

regulating the activity of membrane-related enzymes (Pfeffer et al., 1998).

Boron deficiency reduces the uptake rate of ions across the plasma membrane

(Pollard et al., 1977). Direct or indirect effects of B on membrane structure

control effects of B on ion uptake and also the functions of different membrane

transport processes, including plasma membrane-bound H+-pumping ATPase

(Goldbach and Wimmer, 2007). In suspension-cultured tobacco cells, indole

acetic acid (IAA) is required for the effect of B on the H+-ATPase, and IAA

induced enhanced H+ excretion requires B (Goldbach et al., 1990). In several

species, typical effects of B on plasma membrane integrity and H+ pumping

activity has been observed in vitro with membrane vesicles from B-sufficient

and B-deficient roots (Goldbach and Wimmer, 2007).

2.4.4.3 Role of B in root elongation and shoot growth

Boron plays a critical role in shoot growth and root elongation. Boron

deficiency causes a fast inhibition or termination of root elongation and shoot-

meristematic growth (Marschner, 1995). Boron deficiency results in stubby or

bushy appearance of the root, while in shoots it may lead to complete

inhibition of meristematic growth or even cause meristem death in different

species (Marschner, 2012). In squash plants, interrupting the B supply for 24 h

completely ceased the root elongation and 12 h after B re-supply, root

elongation again started to increase (Bohnsack and Albert, 1977).

Furthermore, stopping the supply of B increased the activity of IAA oxidase in

the roots, which decreased quickly on B re-supply.

Shoot growth inhibition is characteristic symptom of B deficiency. This growth

inhibition may lead to tissue death in some species and re-supply of B results

in a bushy shoot development as lateral shoots emerge (Marschner, 1995). In

root tips, B deficiency causes alterations in cell division from a normal

longitudinal to a radial direction, which results in a decrease of elongation

growth (Robertson and Loughman, 1974). Enhanced cell division in a radial

direction with a proliferation of cambial cells and impaired xylem differentiation


( 40(

are also features typical of the subapical shoot tissue of B-deficient plants

(Marschner, 2012).

2.4.5 Phosphorus (P) in plants

Phosphorus is an essential mineral nutrient, which is an important constituent

of many key structural compounds and also catalyzing a number of various

biochemical reactions taking place in plants. Phosphorus is very important for

some specific growth factors such as, stimulated shoot and root growth,

flowering and seed production, uniform and early crop maturity, biological N-

fixing capacity of legumes and increased disease resistance in crop plants.

Phosphorus is a major structural unit of nucleic acids, amino acids and various

proteins. Both DNA and RNA structures are linked together by phosphate to

form macromolecules. The structural form of P is also high in phospholipids of

biomembranes, where it links different molecules like amino acids, amines,

and alcohols to diglyceride (Marschner, 2012).

In seeds, phytic acid is the main storage form of P (also known as phytate)

(Lott et al. 1995; Park et al. 2006), and its concentration varies from 0.5% to

5.0% in different cereal and legume seeds (Greiner and Egli 2003; Laboure et

al. 1993). In seeds and grains, phytate is the main storage form of different

mineral nutrients like K, Mg, Ca, Mn, Zn and Fe (Lott et al., 1985; Raboy et al.

1989; Ravindran et al. 1994). Phosphorus as a component of different cellular

membranes, DNA and RNA is also found in the seeds (Raboy et al. 2001). In

maize seeds, more than 90% phytate is accumulated in the embryo and

aleurone layers (Lott et al. 1995; Raboy et al. 2000). Phytate plays two

important roles in the seeds (i) it serves as a pool of inositol phosphate and

cations and (ii) it controls the homeostasis of inorganic P during seed

development and also during germination and seedling development (Lott et

al. 1995). Concentration of P and phytic acid in the seeds largely depends on

cultivar, plant nutrient status and climatic condition during seed developmental

stage (Horvatic and Balint 1996; Miller et al. 1980; Raboy et al. 1991). High

seed P reserves have been shown to improve early seedling development and


( 41(

increased dry matter production in various crops, such as spring wheat (De

Marco, 1990; Derrick and Ryan, 1998), barley (Zhang et al., 1990), rice (Ros

et al., 1997) and clover (Thomson and Bolger, 1993), and also increased the

number of nodules and their mass in bean plants (Teixeira et al., 1999). In

wheat plants, it has been shown that high seed P reserves increased root and

shoot biomass production and P uptake during early seedling development

(Table 1.1; Fig.1.6)

Table 2.1: Effect of high and low seed P (HSP and LSP) reserves and P fertilization on P accumulation in shoot and whole wheat plant.

Fig. 2.6: Effect of high and low seed P reserves on shoot and root biomass accumulation in wheat. (Zhu and Smith, 2001)

2.5 Maize

Maize (Zea mays L.) is a major cereal crop besides wheat and rice. Low

temperature has a critical role in geographical distribution and productivity of

maize (Allen and Ort, 2001). Maize plant requires high temperature for its

growth starting from germination till final harvesting (Miedema, 1982).

Temperature below 15°C adversely affects the early development of maize

plants (Stamp, 1984). Therefore, low temperature at early spring in Northern

latitudes is a major factor limiting early seedling development (Verheul et al.,


( 42(

1995). Low temperature adversely affects plant growth rate causing

development of weak seedlings and prolonged growth duration.

Vigorous and well-developed maize seedlings are required to tolerate the cold

and wet conditions of Central and Northern Europe and also for its integration

to no till or conservation tillage systems of temperate region where soil

warming is relatively slow (Tollner et al., 1984; Arshad and Azooz, 1996). In

zero tillage cultivation systems, low soil temperature is the most dominant

physical stress factor which limits the early development of maize seedlings

(Chassot, 2000), where the shoot apex of the maize seedlings which remains

below the soil surface upto the 6-leaf stage (Blacklow, 1972; Erbach et al.,

1986; Stone et al., 1999). Therefore, soil temperature has a direct effect on

both, the shoot and root meristems. Shoot development is impaired by a direct

effect of soil temperature on shoot meristems or by limited supply of mineral

nutrients e.g. P, Zn, Mn etc. through roots (Engels and Marschner, 1990). It

has been found that soil temperature directly controls the early leaf

development of maize plants grown under conditions climatic (Beauchamp and

Lathwell, 1966) and also in the field experiments (Giauffret et al., 1995; Stone

et al., 1999). Existing maize genotypes for cold tolerance are differentiated on

the basis of shoot development (Lee et al., 2002) and root development

(Stamp, 1984; Richner et al., 1997). In a study on roots related traits to low

temperature tolerance, there was a strong link between the length of the lateral

roots on the primary roots and the traits associated with photosynthesis and to

the dry biomass (Hund et al., 2007).

Uptake and translocation of mineral nutrients is markedly reduced in maize at

low soil temperature (Engels et al., 1992). Furthermore, maize has a low

morphological ability for adaptation to low root zone temperature due to limited

biomass allocation to the root. It was further suggested that reduced allocation

of biomass to roots could be responsible for greater susceptibility of maize to

nutrient deficiency at low root zone temperature. Engels and Marschner (1996)

found that in maize plants soil temperature inhibits the translocation of Zn and

Mn form root to shoot irrespective of the chemical nutrient availability in the

rhizosphere or by shoot meristem temperature, while net translocation of Fe is


( 43(

regulated according to the internal growth related shoot demand per unit of

roots.

2.6 Soybean

Soybean (Glycine max L.) is an important crop for protein and oil production

worldwide. In 2009, soybean was cultivated on more than 90 million hectares

of agricultural lands. It is grown all over the world in varying climatic and soil

conditions. Zinc deficiency in agricultural soils is a world wide problem,

especially in calcareous soils (Takkar and Walker, 1993; White and Zasoski,

1999) and limits the crop yields (Grewal et al., 1997; Cakmak, 2000). Global

survey conducted by FAO and other studies have shown that about 50% of the

cultivated soils were either Zn deficient (Sillanpa, 1982) or low in available Zn

for plant growth (Graham et al., 1992; Welch, 1993).

Manganese (Mn) deficiency in soils is also another serious problem for

soybean production in many areas throughout the world (Reuter et al., 1988)

and responsible for decreased crop yields. Manganese deficiency is

particularly more prominent in soils with low inherent Mn availability, relatively

high pH levels and under dry soil conditions (drought).

2.7 Aims and objectives

Previous studies have reported positive effects of nutrient seed priming in

different crop plants, but knowledge on the specific contribution of primed

nutrients in plant growth and nutritional status is limited. This thesis focused on

investigating the specific role of nutrient seed priming in maize and soybean

plants grown under different abiotic stress conditions. Main objectives of this

thesis were as follows:

Objective 1.


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To investigate the effect of nutrient seed priming on early seedling

development, plant growth, nutrient uptake and final yield of maize at low root

zone temperature.

Following study questions were addressed to achieve the objective 1:

i. What is the optimum priming duration for maize seeds?

ii. How much Zn, Mn, Fe, B and P can be added into maize seed

via nutrient seed priming?

iii. What are effects of nutrient seed priming on early seedling

development and how long primed nutrients can support plant

growth at early stages?

iv. Can nutrient seed priming improve shoot and root growth of

maize at low root zone temperature stress?

v. Does nutrient seed priming improve the nutrient status of maize

plants under various nutrient availability conditions e.g.

deficient, low availability and low root zone temperature stress

conditions?

vi. Can nutrient seed priming improve plant growth, nutrient

uptake and the grain yield of maize grown in the temperate

climate of Germany?

Objective 2.

To study the effect of nutrient seed priming on soybean seedling development

and plant nutrient status under low Zn and Mn available conditions.

Following study questions were established to achieve the objective 2.

i. What is the optimum priming duration for soybean?

ii. How soybean seed should be primed to avoid damage during the

priming process.

iii. How does Zn and Mn priming effect shoot and root growth of

soybean seedlings under Zn and Mn deficient conditions?

iv. Can Zn and Mn seed priming improve plant nutrient status under

nutrient deficient conditions?


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Chapter(3(((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((Maize(Nutrient(Seed(Priming(

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Chapter 3

Impact of nutrient seed priming on germination, seedling

development, nutritional status and grain yield of maize (Journal of Plant Nutriton: Accepted, 2012)

Authors

Imran Muhammad, Maria Kolla, Römheld Volker, Neumann Günter

Institute of Crop Science, Nutritional Crop Physiology (340h),

Universität Hohenheim, 70593 Stuttgart, Germany

Corresponding author: Imran Muhammad

Institute of Crop Science, Nutritional Crop Physiology (340h)

Universität Hohenheim, 70599, Stuttgart, Germany

Phone: 0045 71536841 Fax: 0049 711 459 23295

E-mail: [email protected]

Contribution in the manuscript:

Planning of experiments, Preparation of experimental materials,

Nutrient analysis, Root length measurements, Conducting field trial,

Statistical analysis, Manuscript writing.


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3.1. Abstract

Effects of seed priming with zinc (Zn) plus manganese (Mn), boron (B)

and phosphate (P) on growth and nutritional status of maize were

studied. Nutrient seed priming significantly increased seed contents of

primed nutrients. In nutrient solution (NS) lacking Zn and Mn, growth of

maize plants primed with Zn+Mn increased by more than 50% and

100%, respectively, as compared to control treatment. The primed

nutrients were efficiently translocated to the growing shoot and could

maintain Zn and Mn supply for at least three weeks of the culture

period.

In soil culture, plants suffered from P and Zn deficiency, which was

mitigated to some extent by P and Zn+Mn priming. Particularly,

translocation of Zn seed reserves to the shoot tissue was negatively

affected by the highly calcareous soil. In the field experiment, Zn+Mn

seed priming increased grain yield by 15 %, demonstrating the potential

for long-lasting effects of nutrient seed priming.

Keywords: Zea mays L., nutrient seed priming, Zinc, Boron,

Phosphorus, maize grain yield


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3.2. Introduction

Early seedling development and uniform crop stand establishment is an

important determinant for crop yield (Harris, 1996). Different abiotic

stresses such as drought, flooding, low temperatures salinity and other

toxicities can delay germination and increase abnormal seedling

development (Bourgne et al., 2000). Poor crop stand can promote weed

populations causing competition for water, light and limiting nutrients

(Kropff and van Laar, 1993).

Sufficient mineral nutrient reserves in the seed are necessary to

maintain seedling growth until root uptake starts supplying soil nutrients.

This is particularly important for crops grown on soils with low nutrient

availability (Asher, 1987) as demonstrated by numerous studies. Rice

plants obtained from seeds with high Zn contents could better cope with

limited Zn availability in soils as compared with those grown from seeds

with low seed reserves (Rengel and Graham 1995; Hacisalihoglu and

Kochian 2003; Tehrani et al., 2003). Zhu and Smith (2001) have shown

that wheat plants grown from seeds with high P contents were able to

accumulate more P in the shoot compared to plants from low P seeds.

Also, vigor and normal seedling development is strongly determined by

mineral nutrient seed reserves. Seeds with low Mn contents produced

low-vigor wheat seedlings and low yields at harvest (Marcar and

Graham, 1986; Singh and Bharti, 1985). Delayed germination and

seedling development was observed in barley and lupin with low Mn

seed contents (Genc et al., 2000; Longenecker et al., 1991; Crosbie et

al., 1994). It has been shown that percentage of abnormal seedlings

increased with decreasing level of B in the soybean seeds (Rerkasem

et al., 1997) and in green and black grams (Rerkasem et al., 1990; Bell

et al., 1989). High seed P contents have improved early seedling growth

and dry matter production in clover (Thomson and Bolger, 1993), barley

(Zhang et al., 1990), spring wheat (Bolland and Baker, 1988) and rice

(Ros et al., 1997). Seed P contents also affected the number and


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biomass of nodules in common bean (Teixeira et al., 1999).

Various techniques are employed to improve seed quality, to reduce the

time between sowing and seedling emergence and also to minimize

biotic and abiotic stresses during germination, e.g. use of

biotechnology, hybrid seeds, mechanical seed treatments such as

polishing off or removal of seed coat (testa), sorting the seeds into

different seed size classes and various seed treatments like seed

coating, pelleting or soaking in different solutions (seed priming)

(Farooq et al., 2012).

In general, seed priming is a pre-sowing seed treatment in which seeds

are soaked in water and dried back to storage moisture contents until

further use. Seed priming in water has been shown to decrease time

between sowing and emergence and to improve seedling vigour (Harris,

1996; Parera and Cantliffe, 1994). ‘Nutrient seed priming’ is a technique

in which seeds are soaked in nutrient solution instead of simple water.

Nutrient seed priming increases the seed nutrient contents along with

the priming effect to improve seed quality for better germination and

seedling establishment. Priming seeds in solutions of macro- or micro-

nutrients has been shown to improve yield of rice (Peeran and

Natanasabapathy, 1980), wheat (Khalid and Malik, 1982; Marcar and

Graham, 1986; Wilhelm et al., 1988), and forage legumes (Sherrell,

1984), but a risk of seed damage and impairment of germination by

priming at high nutrient concentrations has been also reported (Roberts,

1948; Ajouri et al., 2004).

The present study investigates perspectives for seed priming with water

and various nutrients (P, Zn, Mn, and B) in maize. After establishing and

optimising the priming conditions, hydroponic experiments were

conducted to characterise the nutritional potential and the persistence of

priming effects. Perspectives of seed priming to overcome problems of

limited soil nutrient availability were investigated in pot experiments and


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a field experiment was conducted to investigate the effects of seed

priming on maize yield formation under temperate climatic conditions in

South West Germany.

3.3. Materials and methods

3.3.1. Water priming

To determine the optimal time duration for maize seed priming, 100

seeds were soaked in 200 ml distilled water for 0, 8, 16, 24 and 32

hours (h). Soaking treatments were started from longer to shorter

incubation periods and seeds were finally removed from water at the

same time. Thereafter, seeds were rinsed with distilled water for 1

minute to remove any leached compounds from the seed surface, and

dried for 2 hours at room temperature before starting the germination

test. For germination, 10 seeds were incubated in rolls of 4 layers moist

filter paper (MN 710, Macchery & Nagel, Düren, Grmany) for 5 days at

24°C, using 6 replicates per treatment. Germination speed, shoot and

root lengths of the seedlings were recorded.

3.3.2. Nutrient seed priming

Nutrient seed priming is a technique, in which seeds are soaked in

mineral nutrient solution for a particular time duration, dried back to

initial moisture contents and stored for further use. Maize seeds were

primed for 24 h (pre-determined for water priming) by soaking in distilled

water and nutrient solutions containing Zn+Mn, B and P, respectively.

Based on the seed water absorbing capacity (approx. 0.08 ml seed-1),

nutrient solution concentrations were calculated to double the contents

of Zn, Mn, B and 50% of the seed P reserves. Concentrations of

nutrients in the priming solutions are shown in Table 3.1. The priming

and germination procedure was further conducted as described for

water priming. Un-primed seeds and water-primed seeds were used as

control treatments.


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Table 3.1: Concentrations and salts used for maize nutrient seed priming treatments.

3.3.3.Plant culture

Pot experiments were conducted in a growth chamber under controlled

environmental conditions with average air temperatures of 25 + 3 °C,

15/9 h light/dark periods, light intensity 400 mol m-2 s-1, and a relative air

humidity of 60-80%. For the solution culture experiment, seeds were

germinated for 7 days in filter papers moistened with distilled water as

described for the germination test. Thereafter, seedlings were

transferred into pots filled with 2.5 liters (L) continuously aerated

nutrient solution (6 plants pot-1). As a positive control treatment, plants

were grown in complete nutrient solution, while plants from nutrient

primed seeds were grown in nutrient solution lacking the respective

nutrients. Two plants per pot were harvested 1, 2 and 3 weeks (first

(1st), second (2nd) and third (3rd) harvest) after transfer to nutrient

solution. The nutrient solution was renewed every 2-3 days and had the

following composition: 2 mM Ca(NO3)2, 0.7 mM K2SO4, 0.1mM KCl, 0.5

mM MgSO4, 0.2 mM KH2PO4, 1 µM H3BO3, 0.5 µM MnSO4, 0.2 µM

CuSO4, 0.01 µM (NH4)6Mo7O24, 0.5 µM ZnSO4 and 20 µM Fe-EDTA.

For soil culture, seeds were sown directly at a depth of 2cm and 4

plants were grown in each pot with 1 kg of a highly-buffered calcareous

Loess sub-soil (pH (CaCl2) 7.6; with extremely low nutrient availability

with organic carbon (Corg. <0.3%), CaCO3 -23.3%, available P (CAL

Treatment Seed priming

Control Un-primed

Water Water primed

Zn+Mn 4 mM Zn + 2.5mM Mn solution

as ZnSO4 .H2O and MnSO4

B 5 mM B as H3BO3

P 0.2 M P as K2HPO4


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extraction) 5 mg kg-1 soil, water- extractable Ca2+ and Mg2+ (mg kg−1

soil) 59.9 and 11.3, respectively. Calcium chloride–diethylenetriamine

penta-acetic acid (CAT)-extractable micronutrient concentrations (mg

kg−1 soil): Mn=7.4, Fe=369, Zn=0.8, B=0.9, and Cu 0.5 (VDLUFA,

2004). The soil was fertilized once at the start of the experiment with

nitrogen (N), phosphorus (P), potassium (K) and magnesium (Mg).

Nitrogen was applied as NH4NO3 at 100 mg kg-1 soil, P as Ca(H2PO4)2

at 100 mg kg-1 soil, K as K2SO4 at 150 mg kg-1 soil and Mg as MgSO4

at 100 mg kg-1 soil. The different fertilizers were mixed with the soil

substrate and sieved through a 2 mm mesh size before filling the pots.

Soil moisture content was adjusted to 18% on weight (w/w) basis and

kept constant throughout the experiment by gravimetric control.

The field experiment was conducted at the University of Hohenheim,

research station farm “Heidfeldhof”, Stuttgart, Germany. For each of 4

replicates, 80 seeds were sown in 2 rows on an area of 7.5 m2 (20th of

April 2010). Field soil is a silty-clay Luvisol with organic matter 0.97%

and pH (CaCl2) 7.3. After seedling emergence, the number of plants

was reduced to34 in each row (68 plants per replicate). Final harvest

was performed in October 2010. Fertilization was conducted according

to farmers practice with N 167 kg ha-1, P2O5 42 kg ha-1, K2O 42 kg ha-1,

MgO 23 kg ha-1 and sulpher (S) 63 kg ha-1.

3.3.4. Plant analysis

At harvest, shoot and root dry biomass were determined for plants

grown in nutrient solution and soil culture. In the nutrient solution

experiment, three sequential harvests of each 2 plants were performed

at 2, 3 and 4 weeks after sowing. In field-grown plants, final grain yield

was determined. Plant mineral nutrient analysis was conducted for whole shoots of plants

grown in solution culture. In soil-grown plants, 2 young leaves from the

top and rest of the shoot were analyzed separately. For the extraction of


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mineral elements of plant and seed samples, microwave wet digestion

in a microwave (MLS 1200 Mega microwave system, Milestonl,

Leutkirch, Germany) was performed. For digestion, 500 mg of dried

plant material from each replicate were transferred into a microwave

digestion vessel (50 mL Teflon bottles). Five mL concentrated HNO3

and 4 mL 30 % H2O2 solution were used as digestion mixture. After

digestion the final volume was adjusted to 25 ml. After addition of 0.1

mL Cs/La buffer to 5 mL of the digested solution, Mn andZn

concentrations were measured by atomic absorption spectrometry

(UNICAM 939, Offenbach/Main Germany) and mass spectrometry (ICP

- ELAN 6000, Perkin Elmer, USA) was used for B analysis. Phosphate

in the ash solution was determined spectrophotometrically

(Spectrophotometer, Hitachi, Japan) by the method of Gericke and

Kurmis (1952). 3.3.5. Statistical analysis

A complete randomized block design with 4 replicates was used in all

experiments. Analysis of variance was performed at a significance level

of p < 0.05 using ‘Sigma Stat version 3.1 software (SYSTAT Software

Inc., Erkrath, Germany).

3.4 Results 3.4.1. Effect of water priming on seed germination

Water priming of maize seeds for 24 and 36 h accelerated germination

and growth of young seedlings. Germination rate of 100 percent (%)

was detectable already at 3 days after sowing, - more than 2 days

before seeds of the remaining treatments which reached approximately

90 % germination (Fig. 3.1a). Shoot and root lengths of 5 days old

maize seedlings were also increased by more than 300% by 24 and 32

h water priming as compared with unprimed control (Fig. 3.1b). Similar

results were obtained when nutrient solutions (Table 3.1) instead of


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distilled water were used as priming media (data not shown). Therefore,

a 24 h priming period was used for the experiments.

Fig. 3.1: Effect of water priming for different time periods on germination rate (a) and

seedling growth (b) of maize. Data points represent means and SE of 4 replicates. Different characters indicate significant differences between treatments

3.4.2. Nutrient seed priming

Nutrient priming of maize seeds for 24 hours increased the contents of

the primed nutrient in the maize seeds (Table 3.2) by 20 % for P, 600%

for Zn 900 % for Mn and 1000 % for B after nutrient priming as


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compared to the natural seed reserves, corresponding with an absolute

increase of .185, 45, 16 and 6.5 µg seed-1, respectively. Differences in

nutrient concentrations (not shown) and contents (Table 3.2) between

un-primed and water primed seeds were negligible for the measured

nutrients. Table 3.2: Mineral nutrient contents of maize seeds after nutrient seed priming. Data

represent means of 4 replicates. Different characters indicate significant differences

between treatments.

Nutrient Seed nutrients contents (µg sesd-1)

Treatments Zn Mn B P

Unprimed 8.5 +0.4 b 2.0 +0.1 b 0.72 +0.2 b 965 +23 b

Water 7.9 +0.4 b 1.7 +0.1 b 0.63 +0.11 b 970 +27 b

Zinc + Mn 52.7 +2.5 a 18.1 +1.0 a 0.71 +0.07 b 961 +31 b

Boron 7.8 +0.6 b 1.7 +0.1 b 7.08 +0.07 a 960 +21 b

Phosphorus 7.8 +0.6 b 1.7 +0.1 b 0.62 +0.09 b 1155 +37 a

3.4.3. Hydroponic culture experiment

Effects on biomass production were most expressed after Zn+Mn

priming, particularly at final harvest at 4 weeks after sowing. Plants

primed with Zn+Mn produced ≈50% more shoot biomass and ≈100%

more root biomass as compared with control plants supplied with

complete nutrient solution (Fig. 3.2), despite of the fact that the nutrient

status of control plants supplied with complete NS was always sufficient

throughout the culture period (Fig. 3.3).


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Fig. 3.2: Shoot (a) and root (b) dry biomass of maize plants, grown in nutrient solution after nutrient seed priming with sequential harvests at 2, 3 and 4 weeks after sowing. Data represent means and SE of 4 replicates at each harvest. Different characters indicate significant differences between treatments By contrast, in Zn+Mn priming treatments a critical Zn status was

reached at final harvest with 67 % of the Zn seed reserves recovered in

the shoot.

Manganese deficiency was detectable already after the 2nd harvest and

50% of the seed reserves were detected in the shoot tissue. However,

both, Mn and Zn shoot contents increased continuously during the


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Fig. 3.3: Nutrient concentrations in shoots of maize plants, grown in nutrient solution

after nutrient seed priming with sequential harvests at 2, 3 and 4 weeks after sowing. Data represent means and SE of 4 replicates at each harvest. Different characters indicate significant differences between treatments.

Figure 3.4: Nutrient contents in shoots of maize plants, grown in nutrient solution

after nutrient seed priming with sequential harvests at 2, 3 and 4 weeks after sowing. Data represent means and SE of 4 replicates at each harvest. Different characters indicate significant differences between treatments


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Fig. 3.4: Nutrient contents in shoots of maize plants, grown in nutrient solution after

nutrient seed priming with sequential harvests at 2, 3 and 4 weeks after sowing. Data represent means and SE of 4 replicates at each harvest. Different characters indicate significant differences between treatments


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culture period of Zn+Mn-primed plants. Moreover, priming treatments

with Zn+Mn also increased shoot P contents (Fig. 3.4) in shoot by 40%

over the control with complete nutrient solution as soon as Zn shoot

concentrations reached the deficiency threshold.

Shoot growth of plants obtained from B and P-primed seeds did not

reach the biomass level of control plants with complete NS but root

biomass production after P priming exceeded the control treatment at all

harvest dates. This coincided with P limitation already at the 2nd harvest

in P-primed plants.

Boron deficiency in B primed plants was detectable at the 2nd harvest

with marginal effects on biomass production. At the 1st harvest root

biomass of B-primed plants was higher as the control in complete NS.

In contrast to Zn +Mn priming, plants obtained from P and B-primed

seeds did not continuously increase B and P shoot contents during the

culture period. About 50 % of the P reserves in P primed seeds were

translocated to the shoot compared with only 7 % in case of B-primed

plants.

Phosphate priming increased the Zn and Mn contents at the 1st harvest

as compared to control plants supplied with complete nutrient solution.

In contrast, at later stages of plant development P riming reduced Zn

and Mn contents. Only at the 1st harvest, B priming had a positive effect

on Zn and P contents of the plants.

3.4.4. Soil culture experiment

In soil-grown plants, seed priming with Zn+Mn induced only a moderate

stimulation of shoot biomass production (+18%) as compared with the

unprimed and water-primed control, while root biomass was not affected

(Fig. 3.5).

Plants suffered from Zn deficiency in all treatments, although Zn priming

improved the Zn status, particularly, in young leaves. However, even in

Zn-primed plants, only15% of the Zn seed reserves reached the shoot


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tissue (= 8µg) and this level was even lower in water-primed and

unprimed controls (= 4 µg). ln contrast to Zn, Mn supply was sufficient in

all treatments and the shoot contents of Mn primed seeds exceeded the

seed reserves (Table 3.3).

Fig. 3.5: Shoot (a) and root (b) dry biomass of 4 weeks old maize plants grown in soil culture after nutrient seed priming. Data represent means and SE of 4 replicates. Different characters indicate significant differences between treatments. Also P priming increased shoot growth by, approximately, 20 % and

root growth by 30 % as compared with the unprimed and water primed

control plants. However, the P status was deficient in all treatments and

P priming did not significantly increase P shoot concentrations. About

50 % of the P seed reserves were found in the shoot.


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Table 3.3: Nutrient concentrations and contents in young leaves and the remaining shoot of maize plants grown in soil culture. Data represent means of 4 replicates +SE. Different characters indicate significant differences between treatments in similar shoot portion.

Mineral nutrient contents and concentrations in maize shoots

Contents (plant-1) Concentration (g-1)

Treatments P (mg) Mn (µg) Zn(µg) P (mg) Mn (µg) Zn (µg)

Control

Young leaves 0.26+0.02 a 3.9+0.2 a 1.3+0.1 c 1.6+0.01a 24.7+0.7b 8.1+0.4c

Rest of stem 0.29+0.01 a 13.0+1.2 a 3.1+0.1 b 1.2+0.01a 54.9+5.5a 13.4+0.6b

Total 0.55+0.03 b 16.9+1.4 b 4.4+0.3 c

Water

Young leaves 0.23+0.01ab 4.9+0.3 a 2.2+0.2 b 1.4+0.01ab 29.3+2.1a 13.5+0.6b

Rest of stem 0.29+0.0 1a 14.0+1.4 a 3.3+0.2 b 1.2+0.01a 54.9+2.44a 12.8+0.7b

Total 0.52+0.02 b 18.7+1.3 b 5.5+0.3 b

Zn+Mn

Young leaves 0.31+0.02 a 5.8+0.6 a 3.3+0.3 a 1.7+0.01a 32.1+1.0a 17.9+0.4a

Rest of stem 0.35+0.01 a 17.9+1.8 a 5.2+0.6 a 1.2+0.01a 61.9+4.9a 18.0+1.1a

Total 0.66+0.04 a 23.7+1.4 a 8.5+0.5 a

Boron

Young leaves 0.20+0.02 b 4.7+0.6 a 2.2+0.2 b 1.3+0.01b 29.8+1.1a 14.2+0.7b


Total 0.55+0.07 b 22.5+0.9 a 5.9+0.5 b

Phosphorus

Young leaves 0.23+0.03 b 5.5+0.3 a 2.4+0.2 b 1.3+0.01b 31.6+1.1a 13.6+0.5b


Total 0.58+0.05 b 24.3+1.6 a 5.9+0.5 b

Phosphate priming increased Mn by, approximately, 30% as compared

to control plants but had no effect on Zn shoots contents.Boron priming

had no significant effects on plant growth but Mn contents were


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increased by approximately 20 % as compared with the unprimed

control.

3.4.5. Field experiment

In the field experiment, a significant yield increase was observed in

Zn+Mn-primed plants as compared with unprimed control plants, with

+15 % for fresh, dry and marketable grain yield. Also all water, B and P

priming resulted in slightly higher yield values. However, the differences

were not significant (Table 3.4).

Table 3.4: Final grain yield of maize grown (kg ha-1). Data represent means +SE of 4 replicates. Different characters indicate significant difference between treatments.

Grain yields (kg ha-1)

Treatments Fresh grain yield Dry grain yield Marketable grain yield

Control 11026+341 b 6920+200 b 7889+229 b

Water 11281+184 b 7069+118 b 8059+135 b

Zn+Mn 12700+173 a 7967+144 a 9083+164 a

Boron 11691+260 b 7358+180 b 8388+205 b

Phosphorus 11626+121 b 7295+95 b 8317+108 b

3.5. Discussion

3.5.1. Seed priming

Water priming and nutrient seed priming of maize seeds for 24 hours

accelerated germination and early growth of young seedlings (Fig. 3.1

ab) as previously reported also for other crops, such as rice, sorghum,

pearl millet, finger millet, cotton, beans, and maize (Harris, 1996; Harris

and Jones, 1997; Harris et al., 1999). This has been attributed to pre-

mobilization of seed reserves during the priming period. (Bewley, 1997;

Gallardo et al., 2001; Job et al., 1997), leaching of germination inhibitors

into the priming solution (Hopkins, 1995), hydrolysis of ABA (Ajouri et


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al., 2004) and pre-organization of membrane structures (Bewley and

Black, 1982).

Nutrient seed priming increased the seed contents of all applied

micronutrients with the highest levels for P (+195 µg seed-1), Zn (+45

µg seed-1) followed by Mn (+16 µg seed-1) and B (+6 µg seed-1),

corresponding to increases of 20%, 530 %, 800 % and 600% of the

natural seed reserves, respectively (Table 3.2). Similar increases have

been reported in earlier studies after Zn priming of barley (Ajouri et al.,

2004) and maize (Harris et al., 2007). Nutrient concentrations in the

priming solution were calculated to double the natural seed reserves

during imbibition. Much higher uptake rates recorded in case of Zn, Mn

and B suggest that during priming substantial amounts of the primed

nutrients were removed from the priming solution by adsorption to seed

structures and replacement of the removed nutrients via diffusion.

Earlier studies on Zn priming in Vicia faba revealed that nutrient

adsorption to the seed coat may substantially contribute to this process

and only 20.6 % of the primed nutrient entered the cotyledons and the

embryo, while the rest was retained by the seed coat (Polar, 1970). In

contrast, P uptake via seed priming was lower than expected. This may

be attributed to limited uptake capacity in face of P seed contents

exceeding the contents of the investigated micronutrients by 2-3 orders

of magnitude.

3.5.2. Seed priming effects in hydroponic culture

In hydroponic culture, Zn+Mn priming stimulated shoot (+50%) and root

(+100 %) biomass production (Fig. 3.2 ab) as compared with plants

supplied with complete nutrient solution particularly at final harvest at 4

weeks after sowing. This was, particularly, surprising since the nutrient

status of control plants supplied with complete nutrient solution was

always sufficient throughout the culture period.


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In Zn+Mn priming treatments, a critical Zn deficiency status was

reached after the 3rd harvest (Fig. 3.3) with 67 % of the Zn seed

reserves recovered in the shoot (Fig. 3.4). Manganese deficiency was

detectable already after the 2nd harvest at 3 weeks after sowing and

50% of the seed reserves were detected in the shoot tissue. However,

both, Mn and Zn shoot contents increased continuously during the

culture period of Zn+Mn primed plants, indicating an intense root to

shoot translocation of these nutrients with increasing Zn and Mn

limitation of the shoot tissue. The mobility was particularly high for Mn,

where more than 80% of the seed reserves were detected in the shoot

at final harvest.

Stimulation of root biomass production in the Zn+Mn primd plants may

be a reaction to Zn limitation (Fig. 3.3) and has been similarly observed

in soybean plants (Imran et al., unpublished). Zinc limitation can induce

inhibition of P re-translocation to the roots, thereby triggering P

deficiency responses in the root tissue, such as increased root/shoot

ratio and up-regulation of high affinity root P uptake systems even in

presence of high external P concentrations (Zhang et al., 1991; Huang

et al., 2000). Accordingly, due to stimulation of P uptake, shoot P

contents in Zn+Mn primed plants increased by 40% over the control

with complete nutrient solution as soon as shoot concentrations

reached the Zn deficiency threshold. However, the large stimulation of

shoot growth even above the control supplied with complete nutrient

solution, despite of critical levels of Zn and Mn, is a yet unexplained

phenomenon.

Shoot growth of plants obtained from B and P-primed seeds did not

reach the biomass level of control plants with complete nutrient solution

(Fig. 3.2a), but root biomass production after P priming exceeded the

control treatment at all harvests (Fig. 3.2b). This coincided with P

limitation already at the 2nd harvest in P-primed plants suggesting that P

deficiency stimulated root growth and inhibited shoot biomass


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production. Boron deficiency in B primed plants was detectable at the

2nd harvest but without pronounced effects on biomass production.

In contrast to Zn+Mn priming, plants obtained from P and B-primed

seeds did not continuously increase B and P shoot contents (Fig. 3.4)

during the culture period, suggesting, only a limited distribution from

seed or root reserves to the shoot. About 50 % of the P reserves in P

primed seeds were translocated to the shoot. Since P is a macronutrient

this was obviously not sufficient to cover the P demand of the shoot

tissue at later stages of plant development (2nd and 3rd harvest). Even

more extremely, only 7 % of the B reserves in B-primed seeds reached

the shoot tissue during the culture period. Thus, the success of P and B

seed priming is limited mainly by the high demand in case of P and by

limited mobility in case of B.

3.5.3. Seed priming effects in soil culture

In soil culture, plants suffered from P and Zn deficiency in all treatments

(Table 3.3), despite of additional P fertilisation, demonstrating the high

P fixation capacity of the calcareous subsoil. In contrast to plants grown

in nutrient solution, in Zn+Mn primed soil-grown plants only 15% of the

Zn seed reserves reached the shoot tissue (≈ 8µg) and this level was

even lower in water-primed and unprimed plants (≈4 µg) (Table 3).

These findings suggest also a very low Zn availability of the respective

soil. Similarly, Cenc et al., (2000) reported that plants grown on low Zn

soils from seeds with high Zn contents have shown greater Zn shoot

concentrations than those grown from low Zn seeds.

The soil culture system, however, obviously also affected the Zn

distribution within the plants leading to much lower Zn supply to the

shoots as compared with the hydroponic culture system. In calcareous

soils, plants are facing various stress factors such as P and Fe

limitation, high pH and bicarbonate levels with the potential to induce

the accumulation of carboxylates and phenolics in the root tissue


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(Neumann and Römheld, 2007). The chelating properties of these

compounds may promote Zn sequestration in the root tissue and limit

translocation to the shoot as similarly reported by Forno et al., (1975)

and Dogar and Hai, (1980). Therefore, the marginal surplus of Zn

supply to the shoot tissue by Zn+Mn priming in soil-grown plants also

induced only a moderate stimulation of shoot biomass production

(+18%) as compared to plants grown in hydroponics (+50%). An intense

stimulation of root growth as observed in hydroponics was absent in soil

culture. This may be also attributed to additional stress factors in

calcareous soils, e.g. high pH, high bicarbonate levels (Neumann and

Römheld, 2007).

In contrast to Zn, Mn supply was sufficient in all treatments and the

shoot contents of Mn primed seeds exceeded the seed reserves (Table

3.3), demonstrating efficient additional Mn acquisition via the roots. Also

P priming increased shoot growth by approximately 20 % and root

growth by 30 % as compared with the unprimed and water primed

control and similar to nutrient solution culture about 50 % of the P seed

reserves were found in the shoot (Table 3.3). Obviously, on the highly P

fixing soil, P priming had a certain potential to mitigate P limitation,

which was even present after additional P fertilisation. However, the

moderate surplus of primed P was immediately transformed into plant

growth and the P nutritional status at final harvest was not improved.

Similarly Derrick and Ryan, (1998) and Zhu and Smith, (2001) reported

that high P seed contents improved seedling growth under P-deficient

soil conditions and also enhanced the utilization efficiency of fertilizer P.

Phosphorus priming increased Mn, but not Zn shoot contents, by

approximately 30% as compared to control plants. This may be a

consequence of improved Mn acquisition due to the observed

improvement of root growth in P-primed plants. Zinc availability in the

respective soil was obviously too low to observe a significant effect on

Zn acquisition in this case.


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3.5.4. Seed priming effects under field conditions

In a field experiment, conducted with maize in 2010 on a silty-clay

Luvisol with pH (CaCl2) 7.3 fertilized according to farmer’s practice,

micronutrient (Zn+Mn) seed priming was efficient enough to produce a

15 % surplus in final grain yield (Table 3.4). The may be attributed to

the stimulation of vegetative plant growth (Fig. 3.2 and 3.5) observed in

the model experiments. On the field soil with higher nutrient availability

as compared to the extremely nutrient-deficient soil used in the model

experiment, stimulatory effects on root growth (Fig. 3.5) may promote

nutrient acquisition. This may act as an advantage not only during early

seedling development but also during reproductive and grain filling

stages. Similar long-lasting effects of seed priming treatments on yield

formation have been reported for maize (Harris et al., (2007), chickpea

and wheat (Arif et al., 2007).

Although fertilisation and sowing was performed according to good

farmers practice, field-grown maize plants in the present study were

facing low temperature stress (Fig. 3.6) during germination and early

growth, a stress factor which can severely impair root growth and

nutrient acquisition of young maize plants in temperate climates (Engels

and Marschner, 1996).

In face of the great importance of Zn and Mn in stress tolerance of

higher plants (Cakmak and Marschner, 1988 a, b, 1992; Cakmak,

2000), it remains to be established whether Zn and Mn seed priming

can exert positive effects to mitigate low temperature stress during

germination and early growth.


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Fig. 3.6: Minimum and maximum temperature at research farm (°C) recorded in

April-May, 2010.

3.6. Conclusion

The present study revealed that nutrient seed priming with Zn and Mn

offers perspectives to improve early seedling establishment and growth

of maize. Shoot mineral contents of maize plants grown in solution

culture indicate that the primed nutrients, particularly, Zn, Mn and P are

efficiently translocated within the plant during early seedling

development and can supply the Zn demand for up to 4 weeks.

Beneficial effects on plant growth have been detected under limited

micronutrient conditions. Phosphorus did not show promising effect due

to high demands for P at early growth stages. Up to 15% increase in

grain yields suggests that nutrient priming can be useful strategy to

improve low temperature stress tolerance in maize. However, in face of

the general importance of Zn and Mn in biotic and abiotic sress

adaptations, further investigations are needed in this context for a more

comprehensive evaluation of perspectives for micronutrient seed

priming in maize culture, also in the view of physiological background of

the nutrient priming effects as well as their expression under different


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environmental conditions (e.g. soil type, soil-water and redox status,

fertilisation management, variation in priming treatments and

genotypes).

Chapter(4((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((Maize(at(Low(RZT(

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Chapter 4

Nutrient seed priming improves seedling development of maize

exposed to low root zone temperatures during early growth

(European Journal of Agronomy. (2013) 49: 141-148)

Authors

Muhammad Imran, Asim Mahmood, Volker Römheld, Günter Neumann



Corresponding author: Muhammad Imran



Phone: 0049 152 11325368 Fax: 0049 711 459 23295



Planning of experiments, Preparation of experimental materials, Nutrient

analysis, Root length measurements, Conducting field trials, Statistical

analysis, Manuscript writing.


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4.1. Abstract

Low root zone temperature (RZT) in early spring is a major constraint for

maize production in Central and Northern Europe. Nutrient acquisition,

nutrient uptake and particularly root growth are severely reduced at low

RZT and the consequences of these growth depressions are frequently

not completely compensated until final harvest. Perspectives to overcome

these limitations by seed priming treatments with different micronutrients

(Fe, Zn, Mn) were studied with maize seedlings exposed to low RZT

(12°C).

Model experiments were performed in nutrient solution and soil culture

using rhizo-boxes with root observation windows under green house

conditions. To observe effects on final grain yield, additionally two field

experiments were conducted in 2010 and 2011. Nutrient seed priming

resulted in a significant increase in seed contents of the respective

nutrients i.e. Fe (25 %), Zn (500%) and Mn (800%). At low RZT, biomass

production and total root length of maize plants were significantly

increased after Fe and Zn+Mn priming treatments, both in nutrient

solution and in rhizo-box culture. There was no prominent difference in

shoot Fe, Zn, Mn and P concentrations but total shoot contents per plant

were significantly increased after nutrient seed priming. Plant growth

promotion and improved micronutrient status was detectable also under

field conditions at 5 weeks after sowing. This offers perspectives for using

micronutrient seed priming for improving early seedling development and

plant nutrient status of maize under low temperature climatic conditions.

Keywords: Maize, nutrient seed priming, low root zone temperature, root

growth, grain yield.


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4.2. Introduction

Low temperature at early spring is a major yield-limiting factor for maize

grown in Central and Northern Europe. Temperature between 25-30°C is

optimal for maize growth i.e. germination, shoot and root growth, leaf

development and expansion (Miedema et al., 1987; Duncan and Hesketh,

1968; Muldoon, et al., 1984). Foyer et al., (2002) discussed limited

capacity of maize plants to acclimatize to low temperature during early

growth. Maize seed germination is significantly reduced at low soil

temperature (Miedema, 1982). Seedling development largely depends on

soil temperature from the very beginning as the shoot meristem remains

belowground up to the V6 stage (Fortin and Pierce, 1991; Ritchie et al.,

1986; Stone et al., 1999). Maize growth is severely impaired below 15°C

(Miedema, 1982; Verheul et al., 1996). Mozafer et al. (1993) reported

stagnant growth of maize plants at low root zone temperature (9°C) at

varying photoperiods (6, 12 and 18 hours). Air temperature below 10°C

increases necrosis of maize leaves and reduces plant survival drastically

(Janowiak and Markowski, 1994). At temperatures below 15°C

development of secondary root branches and root dry biomass production

significantly declines (Cutforth et al., 1986) but root dry biomass seems to

be less affected as compared to root length (Kaspar and Boland, 1992).

Nutrient acquisition at early developmental stages is particularly limited by

low temperature. Low RZT involves two major problems with respect to

nutrient uptake: (i) limited solubility and availability of certain nutrients

(Mengel and Kirkby, 1987; Salisbury and Ross, 1992) and (ii) limited root

activity (Sowinski and Maleszewski, 1989; Pregitzer and King, 2005). Low

soil temperature is associated with declining solubility of nutrients in the

soil solution and reduction in the speed of diffusion. Therefore, sparingly

soluble nutrients such as P, K, NH4+, Fe, Zn, Mn, and Cu mainly delivered

by diffusion (Barber, 1984; Jungk, 1991; Martin et al., 1965; Giordano and

Mortvedt, 1978) are more strongly affected. Viscosity of water is inversely

related to soil temperature (Brown and LeMay, 1981). This increase in


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water viscosity reduces the speed of transport processes of mineral

nutrients from soil to the roots also via mass flow (Kramer and Boyer,

1995; Wan et al., 2001). Additionally, the root itself is affected by low RZT

by inhibition of root growth for spatial nutrient acquisition (Chassot and

Richner, 2002; Hund et al., 2007), inhibition of adaptive root exudation for

nutrient mobilisation in soils, e.g. release of phytosiderophores for Fe and

Zn acquisition (Marschner et al., 1987) and inhibition of nutrient uptake

and root to shoot translocation of nutrients. According to Ehdaie et al.

(2010) there is a positive correlation between root biomass production and

uptake of P and K. At low root zone temperature, also Zn uptake and

translocation from root to shoot is impaired, which may result in elevated

Zn root concentrations (Ellis et al., 1964; Sharma et al., 1968; Schwartz

et al., 1987; Moraghan and Mascagni, 1991). Engels and Marschner

(1996) reported decreased net translocation rates of Zn and Mn. Also Fe

uptake is affected under low temperature conditions. Iron concentration in

maize shoots and roots significantly declined with decreasing root zone

temperature, and shoot Fe concentration was further decreased when

plants were exposed to longer photoperiods (Mozafar et al., 1993).

According to Engels and Marschner (1996), the net root to shoot

translocation rate of Fe is markedly enhanced when shoot is grown at

24°C even in presence of low root zone temperature and similar results

have been reported for Cu, Ca, K and N, suggesting a regulation of the

translocation by the shoot demand. By contrast, translocation of Zn, Mn

and P was affected by low RZT but not by the shoot temperature,

indicating a general limitation of acquisition and uptake of these nutrients

under conditions of low RZT (Engels and Marschner, 1996, 1991).

Only strong and vigorous maize seedlings can cope with cold and wet

weather conditions, frequently observed in Central and Northern Europe.

Well-developed shoot and root systems are key features for genotypic

differences in cold tolerance (Stamp, 1984; Richner et al., 1997; Lee et

al., 2002; Hund et al., 2007). According to Cooper and McDonald (1970)


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maize seed reserves are exhausted, at the 3-4 leaf stage. After depletion

of seed reserves, the seedling relies on nutrient uptake from the soil. A

high density of long lateral roots can improve early vigour of young

seedlings under chilling stress conditions (Hund et al., 2007). Availability

and uptake of the limiting nutrients can be increased by starter

fertilization. In soils with limited P availability, relative efficiency of P

fertilizer was increased when placed near the seed (Fiedler et al., 1983).

Seed dressing or coating of limiting nutrients is a cost effective and very

useful approach to improve early plant growth (Robert, 1973; Ros et al.,

2000). Zinc seed dressing of wheat even increased final grain yield

(Yilmaz et al., 1998). In barley, nutrient priming of seeds with P and Zn

enhanced germination, seedling growth and uptake of these elements

(Ajouri et al., 2004).

In general, seed priming is a pre-sowing seed treatment in which seeds

are soaked in water and dried back to storage moisture levels until further

use. This can help crop plants to cope with stress factors, such as

drought, and pest damage and can increase crop yield (Harris et al.,

1999; Harris et al., 2000). ‘Nutrient seed priming’ is a technique in which

seeds are soaked in nutrient solution instead of pure water as an

approach to increase seed nutrient contents along with the priming effect

to improve seed quality for better germination and seedling establishment.

Maize seed priming with 1% ZnSO4 not only enhanced plant growth but

also increased the final grain yield and seed Zn contents in plants grown

on soil with limited Zn availability (Harris et al., 2007). Recently, Guan et

al. (2009) reported that maize seed priming with ‘chitosan’ improved

germination and seedling growth under low temperature stress conditions.

In face of the multitude of functions reported for micronutrients, such as

Zn, Mn and Fe in stress resistance of higher plants (Marschner, 2012), the

aim of the present study was to investigate the effects of micronutrient

seed priming (seed priming with water, Zn+Mn and Fe) on seedling

development, early growth and yield formation in maize exposed to low


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root zone temperature during early growth. For this purpose three different

experimental approaches were employed. Model experiments were

performed in nutrient solution (hydroponic study) and as rhizo-box

experiments in soil culture under greenhouse conditions. Additionally two

field experiments were conduced in 2010 and 2011 to evaluate effects on

grain yield.


4.3.1. Seed priming

Seeds of Zea mays L. cv F030xF047 (provided by Institute of Plant

Breeding, Seed Science and Population Genetics) were used for all

experiments. Maize seeds were primed for a pre-determined time of 24

hours for water and nutrient seed priming by soaking in distilled water and

nutrient solutions, respectively. Water absorbing capacity of maize seeds

was 0.08 ml seeds-1, about 60% of the absorbed water during priming

was removed during drying the seeds. For water priming, 60 seeds were

soaked in 200 ml of distilled water. Iron, Zn and Mn were used for nutrient

seed priming. Iron was used alone (8.5 mM Fe as Fe-EDTA), while Zn

and Mn were combined together (4mM Zn + 2.5mM Mn solution as ZnSO4

.H2O and as MnSO4 .7H2O). 60 seeds were soaked in 200 ml of

respective solutions in the dark for 24 hours. Thereafter, seeds were

taken out, rinsed with running distilled water for 1 minute to remove the

priming solutions. Subsequently, seeds were air dried at room

temperature for a minimum time period of 1 hour for easy handling and to

facilitate clump-free sowing according to on-farm priming method adopted

by Harris et al. (2007). Optimum priming conditions (priming duration and

nutrient concentrations) were determined in a separate set of experiments

(Imran et al., 2012). Seeds for all model and field experiments were

primed with the same method. Un-primed seeds were used for control

treatments. Table 4.1 summarises the various priming treatments

employed for the experiments.


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Table 4.1: Seed priming treatments and root zone temperature regimes of maize

seedlings grown in nutrient solution and rhizo-boxes.

4.3.2. Plant culture

The plants were grown under greenhouse conditions with an average air

temperature of 20°C, light intensities during the growing period ranged

between 250- 400 µmol m-2 s-1 PAR and relative humidity from 60-80%.

For the nutrient solution experiment, primed seeds were germinated for 7

days in rolls of filter papers moistened with distilled water before transfer

into pots containing 400 mL of aerated nutrient solution. (1 plant per pot

with 4 replicates per treatment).

Composition of the nutrient solution used in hydroponic study was: 2 mM

Ca(NO3)2, 0.7 mM K2SO4, 0.1mM KCl, 0.5 mM MgSO4, 0.1 mM KH2PO4,

10 µM H3BO3, 0.5 µM MnSO4, 0.2 µM CuSO4, 0.01 µM (NH4)6Mo7O24, 0.5

µM ZnSO4, and 20 µM Fe-EDTA. The pH varied between 6.0 and 6.5. The

nutrient solution was renewed every 2-3 days to avoid nutrient depletion.

For soil culture, plants were grown in rhizo-boxes (35 x 10 x 2 cm)

equipped with root observation windows and filled with 500 g of a silty-

clay Luvisol (organic matter 0.97% and pH (CaCl2) 7.3). The soil was

fertilized once with N, P, K and Mg. Nitrogen was applied as NH4NO3 at

100 mg N kg-1 soil, P as Ca(H2PO4)2 at 100 mg P kg-1 soil, K as K2SO4 at

150 mg K kg-1 soil and Mg as MgSO4 at 50 mg Mg kg-1 soil. Before filling

Treatment Seed priming

Root zone temperature

Control Un-primed 12 + 2°C

Water Water primed 12 + 2°C

Fe Fe priming 12 + 2°C

Zn+Mn Zn+Mn priming 12 + 2°C

Control (20°C) Un-primed 20 + 2°C


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the rhizo-boxes, the different fertilizers were mixed with the soil substrate

and the soil was sieved through 2 mm mesh size. The soil moisture

content was kept at 18% (w/w) by gravimetric determination. Into each

box, three seeds were sown at a depth of 2 cm and finally one plant was

kept for further cultivation. To stimulate gravitropic root growth along the

root observation window, the boxes were fixed at an angle of 60° with the

observation window on the lower side of the rhizo-box and covered with

black plastic foil.

Two field trials were conducted at the University of Hohenheim, research

station farm “Heid-feldhof”, Stuttgart, Germany in 2010 and 2011. The

field soil had the following properties: pH 6.9, Ctot 1.5%, Ntot 0.14%,

mineral N 14.6 mg kg–1, SOM 2.2%. Concentrations of plant-available

nutrients (CAL: Ca-acetate-lactate extract) were 250 mg kg–1 K, 160

mg kg–1 Mg, 225 mg kg–1 P. The soil texture was silty–loam (FAO-

UNESCO, 1997) consisting of 22.6% clay, 62.9% silt, 14.5% sand.

Mineral fertilizers were applied according to farmers practice with N 167

kg ha-1, P2O5 42 kg ha-1, K2O 42 kg ha-1, MgO 23 kg ha-1 and S 63 kg ha-1

in 2010 and N 147 kg ha-1, P2O5 38 kg ha-1, K2O 33 kg ha-1, MgO 28 kg

ha-1 and S 61 kg ha-1 in 2011. In both years, winter wheat was grown in

experimental fields before setting maize experiments. The experiment

was laid out as a randomized complete block design with four replicates.

Each plot consisted of two rows, and each row was 5 m length with a

distance of 0.75 m between rows (Bhosle et al., 2007). Sowing was

performed in the last week of April in both years. After seedling

emergence thinning was conducted to obtain a plant density of 88 000

plants ha-1. Final harvest was performed in October.

4.3.3. Low RZT stress treatment

After transferring seven days old seedlings into hydroponics, the pots

were transferred to a temperature controlled water bath and cultivated for

4 weeks with a RZT of 12°C in the greenhouse. Similarly, after a culture


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period of 7 days, rhizo-boxes were sealed in plastic bags and cultivated in

the water bath for 3 weeks at a RZT of 12°C. One set of 4 replicates for

both, solution and soil culture was grown at ambient temperature (Control

20°C).

4.3.4. Plant analysis

At harvest, fresh biomass of shoots and roots, and dry matter of shoots

was determined for plants grown in nutrient solution and soil culture. The

field experiment 2010 was designed as a first pilot trial. In the field

experiment (2011) intermediate sampling was performed. Shoots of 4

plants from each replicate of treatments were collected at random in an

intermediate harvest at 5 weeks after sowing to determine fresh and dry

biomass and mineral analysis. Plant dry matter (500 mg) was ashed at

500 °C for 5 h in a muffle oven. After cooling, the samples were extracted

twice with 2 mL of 3.4 M HNO3 until dryness to precipitate SiO2. The ash

was dissolved in 2 mL of 4 M HCl, subsequently diluted ten times with hot

deionized water, and boiled for 2 min. After addition of 0.1 mL Cs/La

buffer to 4.9 mL ash solution, Fe, Mn and Zn concentrations were

measured by atomic absorption spectrometry (UNICAM 939,

Offenbach/Main Germany). Phosphate in the ash solution was determined

spectro-photpmetrically by the method of Gericke and Kurmis (1952).

Total root lengths of plants grown in the nutrient solution and rhizo-boxes

were calculated. For this purpose, washed roots were stored in 30% (v/v)

ethanol solution until further analysis. For length determination of the

roots grown along the root windows in rhizo-boxes, root sketches were

drawn on transparent plastic foil, before and after the exposure to low

RZT. After digitalization with a flatbed scanner, the root analysis software

WinRhizo (WinRHIZO, Pro V. 2009, Regent Instrument Canada Inc.) was

used to determine the total root length, root diameter of the root samples

washed out from soil and root growth along the root observation windows


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4.3.5. Statistical analysis

A complete randomized block design with 4 replicates for each treatment

was used in all experiments. Significant differences were tested by

analysis of variance (p < 0.05), using the Sigma Stat version 3.1’software

(SYSTAT Software Inc., Erkrath, Germany).

4.4. Results 4.4.1. Nutrient seed priming effects on seed nutrient levels

Nutrient seed priming of maize seeds with Fe and particularly with Zn+Mn

significantly increased the concentrations and contents of these elements

within the seeds (Table 4.2). No significant differences in nutrient

concentrations and contents were detected between un-primed and water

primed seeds.

4.4.2. Priming effects on seedling growth at low RZT

The effects of priming treatments on seedling growth (shoot and root

fresh biomass production and root length) under low RZT conditions were

studied in nutrient solution and rhizo-box experiments conducted under

green house conditions. Plants grown in nutrient solutions at 20 °C were

more healthy and vigorous as compared to those at low RZT, irrespective

of the priming treatments. Water priming and particularly, Fe and Zn+Mn

seed priming improved seedling growth compared to unprimed seeds at

low RZT (Figure 4.1), both in nutrient solution and rhizo-box experiments

(Table 4.3).


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Table 4.2: Seed mineral nutrient concentrations and contents after nutrient seed priming. Data represent means and SE of 4 replicates.

Different characters indicate significant differences between treatments.

Seed nutrient concentrations and contents

Zinc Manganese Iron Priming Treatments

µg g-1 µg seed-1 µg g-1 µg seed-1 µg g-1 µg seed-1

Unprimed 28 +1 b 8.5 +0.4 b 7 +0.3 b 2.0 +0.1 b 39 +1.5 b 11.8 +0.4 b

Water 26 +1 b 7.9 +0.4 b 6 +0.2 b 1.7 +0.1 b 37 +1.3 b 11.2 +0.4 b

Iron 24 +1 b 7.2 +0.3 b 6 +0.3 b 1.6 +0.1 b 49 +2.0 a 14.7 +0.3 a

Zinc + Mn 176 +2 a 52.7 +2.5 a 60 +2.5 a 18.1 +1.0 b 29 +2.0 c 8.7 +0.5 c


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Table 4.3: Shoot and root biomass production, and root/shoot ratio of maize plants grown in nutrient solution and rhizo-boxes after nutrient

seed priming. Data represent means and SE of 4 replicates. Different characters indicate significant differences between treatments.

Seedling growth

Nutrient solution Rhizo-boxes

Experiment Treatment

Shoot fresh biomass

g plant-1

Root fresh biomass

g plant-1

Shoot dry biomass

g plant-1

Root: shoot

ratio Shoot

fresh biomass g plant-1

Root fresh biomass

g plant-1

Shoot dry biomass

g plant-1

Root: shoot

ratio

Unprimed 0.6 +0.1 c 0.5 +0.1 c 0.08+0.03 c 0.76 +0.01 a 2.9 +0.2 c 2.6 +0.3 c 0.34+0.05 c 0.78+0.01 b

Water 1.3 +0.2 c 0.7 +0.1 c 0.11+0.01 c 0.56 +0.05 b 3.3 +0.4 c 2.6 +0.3 c 0.41+0.06 c 0.89+0.08 a

Iron 2.2 +0.1 b 1.3 +0.1 b 0.24+0.01 b 0.60 +0.04 b 4.4 +0.1 b 3.2 +0.1 b 0.52+0.02 b 0.79+0.01 b

Zinc + Mn 2.1 +0.1 b 1.3 +0.1 b 0.22+0.02 b 0.60 +0.01 b 4.7 +0.5 b 3.7 +0.3 b 0.56+0.06 ab 0.75+0.08 ab

Control (20°C) 4.4 +0.2 a 2.1 +0.1 a 0.38+0.03 a 0.46 +0.01 c 5.9 +0.5 a 4.3 +0.3 a 0.64+0.06 a 0.72+0.02 c

(


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(Fig. 4.1: Five weeks old maize plants grown in nutrient solution (hydroponic

experiment) under low RZT stress.

1Under low RZT conditions, seedlings grown in rhizo-boxes were higher in

shoot and root fresh biomass and root length than the seedlings grown in

nutrient solution. In the nutrient solution experiment, there was almost 60-

70% increase in shoot and root fresh biomass and root length with Fe and

Zn+Mn priming as compared to water priming alone, whereas for the

seedlings grown in the rhizo-boxes, this difference was reduced to 30%

for shoot and root fresh biomass production (Table 4.3). Root to shoot

ratio was significantly increased for the plants grown at low RZT

compared to 20°C control plants, both, in solution and soil culture (Table

4.3). In both culture systems, Fe and Zn+Mn priming resulted in an

approximately 100% increase in total root length after 3-4 weeks exposure

to low RZT (Table 4.4). Root length in different diameter classes (0.2, 0.4

and 0.6 mm) revealed that nutrient seed priming particularly promoted the

growth of fine roots, as compared to un-primed and water primed

treatments at low RZT. There was more than 80% increase in root length

of the diameter class ≤ 0.2 mm after nutrient seed priming (Table 4.4). In ((((((((((((((((((((((((((((((((((((((((((((((((((((((((1((


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the rhizo-box experiment, lengths for the roots grown along the

transparent root window (Figure 4.2) were measured at the end of 1 and 3

weeks after exposure to low RZT stress (Figure 3). At low RZT, there was

considerable increase in root growth for Fe and Zn+Mn priming compared

to water-, and un-primed seedlings. Root length in the 20°C control was

doubled as compared to un-primed seedlings under low RZT stress.

(

Fig. 4.2: Roots of maize plants grown along the root windows in rhizo-boxes after 3

weeks of low RZT stress.

4.4.3. Priming effects on the nutrient status of the seedlings

Control plants grown without exposure to low RZT stress were higher in

shoot Zn, Mn, Fe and P concentrations and total contents per plant, both

in nutrient solution and rhizobox experiments (Table 4.5 and 4.6).

Concentrations of Zn and Fe were almost doubled for plants grown in


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Table 4.4: Total root lengths (cm plant-1) and root lengths in different diameter classes of maize plants grown in nutrient solution and rhizo-

boxes after nutrient seed priming. Data represent means and SE of 4 replicates. Different characters indicate significant differences between

treatments.

Total root length and diameter classes (Nutrient Solution) cm plant-1

Total root length and diameter classes (Rhizoboxes) cm plant-1

Diameter Diameter Priming

Tsreatment Total

0.2 mm 0.4 mm 0.6 mm Total

0.2 mm 0.4 mm 0.6 mm

Unprimed 43 +6 d 9+5 c 6+4 c 7+2 c 602 +115 c 246+43 c 185+61 bc 87+22 b

Water 75 +8 c 16+9 c 12+3 c 11+4 bc 679 +21 c 247+43 c 111+26 c 34+5 c

Fe 119 +14 b 38+9 b 24+7 b 12+2 b 1506 +220 b 606+95 b 210+8 b 71+9 b

Zn+Mn 122 +8 b 34+8 b 27+7 b 17+1 b 1372 +236 b 582+68 b 188+36 b 65+11 b

Control (20°C) 247 +4 a 71+15 a 104+8 a 35+6 a 2641 +245 a 1788+224 a 446+71 a 144+46 a


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nutrient solution as compared to those grown in the rhizo-boxes under low

RZT stress. Fe concentration was considerably higher in un-primed

seedlings as compared to all other treatments. Zinc concentration of

plants grown in rhizo-boxes at low RZT was close to the critical limit of Zn

deficiency for optimal plant growth. Shoot concentrations of P were in the

critical range even in plants grown at ambient RZT and further declined at

low RZT.

(

(Fig. 4.3: Root lengths of maize plant roots grown along the root windows after 1 and 3

weeks of low RZT stress. Data represent means and SE of 4 replicates.

Different characters indicate significant differences between treatments.

Seedlings with Fe and Zn+Mn priming had significantly higher contents of

these elements compared to water and the unprimed control in both

experiments under low RZT stress (Table 4.6). Shoot Mn, Fe and P

contents of seedlings grown in rhizo-boxes were considerably higher

compared to the seedlings grown in nutrient solution, but shoot Zn

contents were almost similar in both cases. Shoot P contents were

significantly increased by nutrient seed priming compared with the

unprimed control in both experiments.


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4.4.4. Plant growth and mineral nutrient status in field trails In Germany, at the time of maize sowing in early spring (April-May),

temperature frequently drops markedly below the optimum range for

maize germination and seedling development. This is also reflected by

daily minimum temperature recordings of the research station area for

April-May ranging between 1-12 °C (Figure 4.4).

Fig. 4.4: Minimum temperature in Hohenheim (°C) recorded in April-May, 2010 and

2011.

Maize seedlings were harvested, five weeks after sowing and analyzed for

shoot fresh and dry biomass production. Iron and Zn+Mn priming

significantly increased shoot dry matter as compared to water priming and

the unprimed control (Figure 4.5). There was no statistical difference in

shoot Zn and Mn concentrations in all treatments, and the Zn status was

critical. The Fe concentration was significantly higher in all priming

treatments as compared to unprimed plants (Table 7) and was generally

in the range of luxury supply. Shoot Zn and Fe contents were significantly

higher in plants after Fe and Zn+Mn seed priming compared to control

and water primed, while Mn content was higher in plants with Fe priming

compared to the other treatments.

��

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Table 4.5: Shoot mineral concentrations of maize plants grown in nutrient solution and rhizo-boxes under low RZT stress. Data represent means and SE of 4 replicates. Different characters indicate significant differences between treatments.

Shoot nutrient concentrations Nutrient solution Rhizo-boxes Priming

Treatments Zn (µg g-1)

Mn (µg g-1)

Fe (µg g-1)

P (mg g-1)

Zn (µg g-1)

Mn (µg g-1)

Fe (µg g-1)

P (mg g-1)

Unprimed 65 +16b 46 +9b 195 +8a 1.09+0.14 b 21 +2c 36 +2b 73 +5b 0.85+0.11 b

Water 57 +7b 36 +4b 117 +16bc 1.12+0.10 b 19 +1c 37 +2b 62 +5b 0.87+0.08 b

Iron 63 +4b 50 +3b 124 +7b 0.92+0.11 b 24 +1b 36 +2b 74 +3b 0.83+0.04 b

Zinc + Mn 62 +1b 50 +3b 141 +16b 1.12+0.08 b 23 +1b 42 +2b 61 +3b 0.76+0.10 b

Control (20°C) 114 +16a 84 +9b 99 +8c 1.52+0.06 a 30 +1a 73 +2a 100 +2b 1.48+0.07 a

Table 4.6: Shoot mineral contents of maize plants grown in nutrient solution and rhizo-boxes under low RZT stress. Data represent means and SE of 4 replicates. Different characters indicate significant differences between treatments.

Shoot nutrient contents (µg plant-1)

Nutrient solution Rhizo-boxes Priming Treatments

Zn (µg plant-1)

Mn (µg plant-1)

Fe (µg plant-1)

P (mg plant-1)

Zn (µg plant-1)

Mn (µg plant-1)

Fe (µg plant-1)

P (mg plant-1)

Unprimed 7 +1c 3 +0c 13 +1c 0.08+0.02 c 7 +1c 12 +2c 24 +5c 0.28+0.05 c

Water 6 +0c 4 +0c 12 +0c 0.10+0.01 c 8 +1c 15 +2c 26 +4c 0.36+0.05 bc

Iron 15 +1b 11 +1b 29 +2b 0.24+0.01 b 13 +1b 18 +2b 37 +1b 0.42+0.01 b

Zinc + Mn 13 +1b 12 +1b 30 +2b 0.26+0.02 b 13 +2b 23 +2b 34 +3b 0.43+0.05 b

Control (20°C) 42 +4a 31 +2a 38 +2a 0.56+0.02 a 19 +1a 45 +2a 64 +2a 0.93+0.07 a

��

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Fig. 4.5: Shoot fresh and dry biomass of maize plants harvested in the field 5 weeks after sowing (year 2011). Data represent means and SE of

4 replicates. Different characters indicate significant differences between treatments. �


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Table 4.7: Shoot mineral concentrations and contents of maize plants grown in the field for 5

weeks (year 2011). Data represent means of 4 replicates and SE Different characters indicate

significant differences between treatments.

4.4.5. Grain Yield

Final grain yield was calculated as total fresh grain yield just after harvest, total

dry grain yield after drying back at 65 °C to constant seed weight and marketable

grain yield at 12% moisture contents (Table 4.8). There was a significant 10-15%

increase in total fresh, dry and marketable yields per hectare with Fe and Zn+Mn

seed priming relative to unprimed seeds and water priming. The highest increase

for all investigated yield parameters was obtained in Zn+Mn primed seeds.

Table 4.8: Final grain yields of maize grown in the field (2010-2011). Different characters

indicate significant differences between treatments.

4.5. Discussion

Nutrient seed priming increased the seed contents of all applied micronutrients

with the highest levels for Zn (+45 µg seed-1) followed by Mn (+16 µg seed-1)

and Fe (+3 µg seed-1), corresponding to increases of 530 %, 800 % and 25% of

the natural seed reserves, respectively (Table 4.2). The large variation in uptake

Nutrient concentrations (µg g-1)

Nutrient contents (µg plant-1) Priming

treatments Zn Mn Fe Zn Mn Fe

Unprimed 21 +1a 55 +11a 327 +12b 69 +1b 182 +4b 1082 +22b

Water 21 +1a 65 +10a 387 +40a 64 +2b 195 +7b 1163 +40b

Iron 20 +1a 56 +10a 378 +37a 81 +7a 224 +9a 1517 +129a

Zinc + Mn 21 +2a 55 +14a 413 +52a 77 +3a 198 +9ab 1479 +66a

Yield (kg ha-1), 2010 Yield (kg ha-1), 2011 Treatments Fresh grain Dry grain Marketable Fresh grain Dry grain Marketable

Unprimed 11026+341 b 6920+200 b 7889+229 b 14267 +145b 10237 +97b 11670 +110b

Water 11281+184 b 7069+118 b 8059+135 b 15210 +143b 10915 +121b 12443 +138b

Iron 12565+359 a 7913+257 a 9021+293 a 15887 +179a 11559 +124a 13177 +141a

Zinc + Mn 12700+173 a 7967+144 a 9083+164 a 16370 +202a 11736 +84a 13379 +96a


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within the different applied nutrients despite of similar concentrations of

micronutrients in the priming solutions (2.5 - 8.5 mM) requires further

investigations on uptake kinetics and compartmentation within the seeds. Based

on calculations of nutrient concentrations (Table 2), similar increases have been

reported in earlier studies after Zn priming of barley (Ajouri et al., 2004) and

maize (Harris et al., 2007).

Both, in hydroponics and in soil culture, plant exposure to low RZT without

priming treatments resulted in a severe reduction of biomass production by 75-

85% and 40-50% respectively (Table 4.3). Particularly root length was reduced

by approximately 80% in both culture systems. Due to stronger inhibition of shoot

growth as compared to root growth, the root/shoot biomass ratio increased under

low RZT in comparison with plants gown under ambient temperature. Probably

due to a generally better nutrient availability in hydroponics, the root/shoot ratio

was generally lower than in soil culture.

The strong inhibition of shoot growth has been related to hormonal imbalances,

such as reduced biosynthesis and root to shoot translocation of cytokinins (Atkin

et al., 1973), as a typical response also to N and P deficiency. Accordingly,

Marschner and Engels (1991) reported reduced root to shoot translocation of N

and particularly of P in maize plants exposed to low RZT. However, cytokinin

biosynthesis may be also impaired by a general reduction of metabolic activity in

the root meristems at lower soil temperatures (Covey-Crump et al., 2002). At the

same time, root to shoot translocation of the cytokinin antagonist abscisic acid

(ABA), counteracting the biosynthesis of cytokinins and vice versa, increased

with inhibitory effects on shoot growth (Atkin et al.1973). Increased ABA

translocation to the shoots is also a common response to water limitation and

may therefore also be associated with a reduced hydraulic conductivity of the

roots and limited water supply to the shoots frequently reported in response to

low RZT (Huang et al., 1991).

All priming treatments, but particularly micronutrient seed priming alleviated the

detrimental effects of low RZT on plant growth. Biomass production of primed

seedlings reached 50 % of the control plants grown at ambient temperature in


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the hydroponic culture system and 75-85 % in soil culture (Table 4.3). Particularly

in the soil culture system, root length development was much stronger stimulated

by micronutrient seed priming (120-150 %, Table 4.4) than root biomass

production (20-40%, Table 3). This indicates the formation of longer and finer

roots in response to the priming treatments (Table 4.4), a feature promoting

nutrient acquisition under low RZT (Hundt et al., 2007) but also after recovery

from stress conditions. Figure 2 shows that this was particularly attributed to

stimulation of lateral root formation.

Inhibition of plant growth due to low RZT, associated with reduced uptake and

root to shoot translocation of micronutrients has been previously reported by

Engels and Marschner (1996a) for Zn, Mn, Cu and Fe. At the same time also

assimilate translocation to the roots was inhibited (Engels and Marschner,

1996b). This may indicate a limitation of photosynthesis, which strongly depends

on the supply with micronutrients, such as Fe, Mn, Zn. and Cu (Marschner, 1995)

with growth inhibition as a final consequence. Accordingly, Strauss et al. (2007)

reported a functional limitation of the photosystem II (PSII) in plants exposed to

low RZT, while Nagasuga et al. (2011) stated that reduced growth and biomass

production of rice plants, grown under low RZT was mainly attributed to the

reduction of shoot growth and leaf area.

Almost all types of plant stresses, including suboptimal temperatures, involve

oxidative stress as one of the rapid responses (Blokhina et al., 2003). This is

associated with increased production of active oxygen species (AOS), such as

superoxide, hydrogen peroxide and the hydroxyl radical, involved in membrane

damage by lipid peroxidation, protein degradation, enzyme inactivation and

disruption of DNA strands (Allen 1995). Micronutrients, such as Zn and Mn are

important co-factors of various enzymes involved in the detoxification of AOS,

such as superoxide dismutases (SODs, Cakmak and Marschner, 1988 a, b,

1992; Cakmak, 2000). Deficiency of Zn, Mn and Cu alters the activities of SOD

forms and these changes depend on the type and the duration of the deficiency

stress (Yu and Rengel, 1999). Moreover, Mn is a cofactor of various enzymes

involved in the biosynthesis of phenolic compounds with anti-oxidative properties


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(Thompson and Huber, 2007). Zinc has functions in direct membrane

stabilization, in biosynthesis of auxins (Salami and Kenefick, 1970) and

gibberellins (Sekimoto et al., 1997) involved in plant growth regulation and in

protein synthesis in general. Therefore, limited supply of these micronutrients

under conditions of low RZT (Engels, 1994) may increase oxidative damage of

root cells and induce disturbances in plant growth, which may be alleviated by

supplementation via micronutrient seed priming.

Both in hydroponics and in soil culture, micronutrient shoot concentrations were

not substantially elevated by the priming treatments (Table 4.5). However

significant increases were observed for the contents of the respective nutrients

(Table 4.6). This increase was still detectable even in field grown plants at 5

weeks after sowing (Table 4.7). Obviously, the surplus in micronutrient supply in

the priming treatments was immediately transformed into plant growth. For Mn

and particularly for Zn, the amount of micronutrients supplemented by seed

priming (Table 4.2) was sufficiently high to explain the increase in micronutrient

contents in the plants at 3-5 weeks after sowing (Tables 4.6 and 4.7). However,

the amount of Fe taken up by the seeds (3 µg seed-1, Table 2) was much lower

than the observed increases in Fe shoot contents (Table 4) of up to 500 µg

seedling-1 in field grown plants (Table 4.7). This finding demonstrates that

improvements of the micronutrient status by seed priming are not only a direct

consequence of nutrient supplementation but most probably also of improved

nutrient acquisition from the soil via stimulation of root growth (Table 4.4). This is

further underlined by the observation that iron seed priming simultaneously

increased the Zn and Mn status of the plants and vice versa (Table 6) and that

also P content, as an important sparingly soluble macronutrient, was increased

by the priming treatments.

Micronutrient seed priming treatments were efficient enough to produce a 10-15

% increase in final grain yield in two independent field experiments conducted in

2010 and 2011 (Table 4.8). The extraordinary persistence of the priming effects

may be attributed particularly to the enormous stimulation of root growth

observed in the model experiments (Tables 4.3 and 4.4). This may act as an


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advantage not only during the stress treatments but may further promote plant

growth during and even after the recovery phase. Moreover, several other

studies have demonstrated long-lasting positive effect of micronutrient seed

priming also on the alleviation of other stress factors. Similarly, Harris et al.,

(2007) have shown beneficial effects of Zn seed priming on maize by 27%

increase in grain yield and almost 3 mg kg-1 increase in seed Zn concentration on

Zn deficient soils in Pakistan.

4.6. Conclusion

Many previous investigations have demonstrated declining nutrient supply to

sensitive plant species exposed to low RZT during germination and early growth.

However, this is the first report showing that micronutrient (Fe, Zn, Mn) supply

acts as a specific growth limiting factor under these conditions, which can be

supplemented by micronutrient seed priming with long-lasting effects even until

final harvest. The physiological background of these effects as well as their

expression under different environmental conditions (e.g. soil type, soil-water and

redox status, fertilisation management, variation in priming treatments and

genotypes etc.) need to be further elucidated. Nevertheless, the present study

demonstrates that micronutrient seed priming may offer a realistic perspective to

mitigate problems of plant performance under low RZT. The seed priming

technique is simple, inexpensive and can be conducted directly prior to sowing

e.g. by small-scale farmers but allows also combinations with industrial seed

dressing techniques due to persistence of the effects after drying-back the

primed seeds.

Chapter(5((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((Soybean(Nutrient(Seed(Priming(

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Chapter 5

Accumulation and distribution of Zn and Mn in soybean seeds after

nutrient seed priming and its contribution to plant growth under Zn

and Mn deficient conditions (Journal of Plant Nutrition (submitted))

Authors

Imran Muhammad, Römheld Volker, Neumann Günter



Corresponding author: Imran Muhammad



Phone: 0049 152 13172241 Fax: 0049 711 459 23295



Planning of experiments, Preparation of experimental materials, Nutrient

analysis, Root length measurements, Conducting field trials, Statistical

analysis, Manuscript writing.


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5.1. Abstract

Nutrient seed priming is a strategy to increase the seed reserves of mineral

nutrients as primary source for mineral nutrition during seedling

development and early growth. The present study investigates effects of

zinc (Zn) and manganese (Mn) seed priming on growth and nutritional

status of soybean under conditions of Zn and Mn limitation. Soybean

seeds were primed with distilled water and nutrient solutions containing Zn

(10mM), Mn (5mM) and Zn+Mn (10mM + 5mM) for 12 hours between

layers of moist filter paper to minimize imbibition damage. Nutrient seed

priming increased the natural seed reserves for Zn by, approximately 6 fold

and by 5 fold for Mn. However, 40-60 % of the primed nutrients were

adsorbed to the seed coat.

Soybean plants were grown for 5 weeks in nutrient solution under Zn and

Mn deficiency and on a calcareous Loess subsoil low in plant available Zn

and Mn. Zinc and Zn+Mn priming improved plant growth and increased

shoot biomass production both, in solution culture and in the soil

experiment. Zinc seed priming was able to maintain plant growth for five

weeks in the same way as Zn supply via the nutrient solution, while the

amount of primed Mn was not sufficient to overcome Mn deficiency

symptoms. Antagonistic effects were observed after combined priming with

Zn and Mn.

It is concluded that particularly nutrient seed priming with Zn offers

perspectives to improve seed quality of soybean for early seedling

development under limited nutrient supply or availability and needs further

investigation on performance under various stress conditions.

Keywords: Soybean, nutrient seed priming, Zn and Mn localization,

nutrient uptake


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5.2. Introduction

Seed priming is a pre-sowing seed treatment in which seeds are soaked

in water and dried back to storage moisture contents for further use. In

the recent past, the ‘on-farm seed priming’ method according to Harris et

al., (1997) has become very popular in developing countries. For ‘on-

farm seed priming’, seeds are soaked in water durng a certain time

period and only shortly air-dried (not to initial moisture contents) before

sowing. This can speed up germination, improve the tolerance to various

stress conditions and can increase crop yield (Harris et al., 2000).

‘Nutrient seed priming’ is a technique in which seeds are soaked in

nutrient solutions instead of water. This increases seed nutrient contents

along with the priming effects to improve seed quality for better

germination and seedling growth. Maize seed priming with Zn stimulated

plant growth and increased final grain yield and seed Zn contents on

soils with limited Zn supply (Harris et al., 2007) and under stress of low

root-zone temperature (Imran et al., 2013). In barley, nutrient priming of

seeds with P and Zn stimulated germination, seedling growth and plant

uptake of these elements (Ajouri et al., 2004). In contrast, Zn and B

seed priming had shown no effects on germination rate and grain yield

of chickpea, lentil and cowpea grown in Zn and B deficient soils in Nepal

(Johnson et al., 2005).

Under stress conditions during the sensitive stages of germination and

early seedling development, micronutrients are of outstanding

importance. Zinc and manganese are co-factors of various enzymes

(Superoxide dismutase, SOD) involved in the detoxification of reactive

oxygen species such as O2- (superoxide radical) and H2O2 (hydrogen

peroxide) (Cakmak, 2000). Moreover, Mn is a cofactor of many

enzymes involved in the biosynthesis of phenolic compounds with anti-

oxidative and antibiotic properties (Thompson and Huber, 2007). Zinc

has functions in direct membrane stabilization, in biosynthesis of auxins

(Salami and Kenefick, 1970) and gibberellins (Sekimoto et al., 1997)


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involved in the plant growth regulation and in protein synthesis in

general.

In the recent past, soybean (Glycine max L.) cropping has been largely

extended and its cultivation is rapidly increasing even in countries with

temperate climates, with a shorter growth period for crop development

(from germination to maturity) (Dashti et el., 1998). Under these

conditions, speeding up germination, and early seedling development is

vital for on-time crop maturity and optimal grain yields. Moreover,

soybean cultivation in temperate climates implies additional abiotic and

biotic stress factors, such as sub-optimal air and soil temperature

leading to reduced water uptake, decreased nutrient availability and

short photoperiods as well as disease problems (Ogle et al., 1979;

James, 1998; Osborne, 2006). Based on the promising results of Zn

and Mn seed priming with respect to improvement of plant growth and

stress tolerance in various crop species (Ajouri et al., 2004; Imran et al.,

2012b), the present study investigates perspectives for seed priming

with Zn and Mn on growth of soybean under conditions of limited

nutrient availability, with the major aims:

(i) To develop an optimised priming protocol for soybean seeds.

(ii) To estimate the time frame for a direct nutritional impact of

micronutrient seed priming during plant development.

(iii) Testing the effects of micronutrient seed priming on early

growth of soybean on a soil with limited availability of Zn and

Mn.


5.2.1. Seed priming methods

Soybean (Glycine max L. cv. ‘Conquista’) seeds were obtained from

Adriana Farms (Brazil). Seeds were primed using the ‘on-farm seed

priming method’ according to Harris et al., (1997), in which seeds were


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surface dried for 2 hours after the priming treatment to avoid soil

clogging on seed surface during sowing. Optimisation tests for priming

treatments were performed with each 100 seeds in 4 replicates using

distilled water for 6, 12 and 24 hours with three different methods:

(i) Priming according to water uptake capacity of the seeds (absorption

capacity - (AC) - method)

Seeds were soaked in a pre-determined quantity of distilled water

(equal to the water absorption capacity of the seeds) for each time

treatment in glass beakers. To facilitate uniform absorption, seeds were

evenly immersed in distilled water by placing them in a single layer in

the beakers. Beakers were covered with parafilm to avoid evaporation

and incubated at room temperature (22 °C) in the dark.

(ii) Submerged priming (SP-Method)

In this method, seeds were soaked into 100 ml of distilled water in glass

beakers. All seeds were submerged during the priming period and were

incubated at room temperature in the dark in beakers closed with

Parafilm..

(iii) Priming between filter paper layers (FP-Method)

Seeds were incubated in petri dishes between 3 layers of filter paper

(MN 710, Macchery & Nagel, Düren, Germany) with three layers below

and two layers on top of the seeds. A calculated amount of priming

solution equal to the absorbing capacity of the seeds and filter papers

was applied. Petri dishes were covered with lids to minimize

evaporation losses, and incubated at room temperature in the dark.

After the priming treatments, the seeds were rinsed with distilled water

to remove any adhering compounds and nutrients from the seed

surface. Rinsed seeds were air dried at room temperature for 2 hours

before further use .


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5.2.2 Germination test

Primed soybean seeds were germinated in olls prepared of 4 layers of

germination paper (MN 710, Macchery & Nagel, Düren. Germany)

moistened with 60 mL of distilled water in 4 replicates (10 seeds per

replicate) for 7 days. Seed germination was evaluated according to

ISTA (International Seed Testing Association) rules, 1983.

Additionally, early seedling development up to 7 days was also

monitored in peat culture substrate (TKS®1 Instant Plus, Floragard,

Germany). For this purpose, four water primed seeds were sown in 0.5

liter pots filled with moistened peat culture substrate . Development of

normal seedlings was assessed according to ISTA rules.

5.2.3 Nutrient seed priming

To determine the suitable concentrations of Zn and Mn for optimal

effects of nutrient seed priming, soybean seeds were primed with 5, 10

and 15 mM ZnSO4 and MnSO4 solutions. Zinc and Mn concentrations

for nutrient priming were selected based on the development of

abnormal seedlings in the germination test described in section 2.2..

Seeds were primed according to the FP-Method described in section

2.1.

5.2.4 Plant culture

Experiments were conducted in a climate chamber under controlled

environmental conditions. Average air temperature was adjusted to 25+

3 °C with a light period of 15 h, a light intensity of 200 mol m-2 s-1, and a

relative air humidity of 60-80%.

Nutrient solution experiment

For hydroponic culture, primed seeds were germinated in rolls with 4

layers of moist germination paper (MN 710, Macchery & Nagel, Düren.


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Germany). After 7 days, homogenously developed seedlings were

transferred to pots containing 500 mL aerated nutrient solution (one

plant per pot in 4 replicates).

Seedlings obtained from water primed seeds (positive control) were

grown in complete nutrient solution, while plants from nutrient primed

seeds and their respective water primed controls were grown in a

nutrient solution lacking the primed nutrients. Table 5.1 shows the

treatment scheme of the nutrient solution experiment. The complete

nutrient solution had the following composition: 2 mM Ca(NO3)2, 0.7

mM K2SO4, 0.1mM KCl, 0.5 mM MgSO4, 250 µM KH2PO4, 10 µM

H3BO3, 0.5 µM MnSO4, 0.2 µM CuSO4, 0.01 µM (NH4)6Mo7O24, 0.5 µM

ZnSO4, and 20 µM Fe-EDTA. The pH varied between 6.0 and 6.5.

Nutrient solution was replaced every 2-3 days to avoid nutrient

depletion.

Table 5.1: Nutrient seed priming treatments and composition of nutrient solution for plant growth in hydroponics.

Priming treatment

Priming solution Nutrient solution for plant growth

Water primed (WP)

Distilled water Complete nutrient solution (CNS)

Zinc (Zn) 10 mM Zn as ZnSO4.7H2O

Nutrient solution (NS) without Zn (NS – Zn)

WP Distilled water Nutrient solution without Zn (NS – Zn)

Manganese (Mn)

5 mM Mn as MnSO4.H2O

Nutrient solution without Mn (NS – Mn)

WP Distilled water Nutrient solution without Mn (NS – Mn)

Zn + Mn 10 mM Zn + 5 mM Mn

Nutrient solution without Zn and Mn ((NS – (Zn + Mn))

WP Distilled water Nutrient solution without Zn and Mn ((NS – (Zn + Mn))


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Soil culture experiment

For the soil culture experiment, four primed soybean seeds (FP-

Method) were sown directly at a depth of 2 cm into pots filled with 500

grams of a calcareous Loess subsoil (pH (CaCl2) 7.6; Corg.<0.3%;

CaCO3 23.3%; water- extractable Ca2+ and Mg2+ (mg kg−1 soil) 59.9 and

11.3, respectively). Calcium chloride–diethylenetriamine penta-acetic

acid (CAT)-extractable micronutrient concentrations (mg kg−1 soil):

Mn=7.4, Fe=369, Zn=0.8, B=0.9, Cu= 0.5 and P (CAL) was 5 mg kg-1

(VDLUFA, 2004).

Before sowing, the soil was fertilized once with N, P, K and Mg.

Nitrogen was supplied as NH4NO3 at 100 mg kg-1 soil, P as Ca(H2PO4)2

at 100 mg kg-1 soil, K as K2SO4 at 150 mg kg-1 soil and Mg as MgSO4

at 100 mg kg-1 soil. The different fertilizers mixed homogenously with

the soil substrate. Before filling the pots, the soil was sieved through 2

mm mesh size. Soil moisture was daily adjusted to 18% (w/w). After 7

days, in each pot two seedlings were removed and two well-developed

seedlings were further cultivated for 4 weeks. Control treatments

comprised unprimed and water-primed seeds.

5.2.5 Plant analysis

Soybean plants grown in hydroponics and in soil culture were

harvested after a growth period of five weeks. Fresh biomass of plant

shoots and roots was determined and the plant material was

subsequently oven-dried at 65°C for determination of dry matter and

mineral nutrient analysis.

For seed mineral nutrient analysis after the priming treatments, seeds

were rinsed with de-ionized water for 1 min to remove any compounds

and nutrients adhering to the seed coat. Afterwards the seed coat was

removed carefully to avoid damage of the remaining parts.

Mineral nutrients were determined in complete unwashed and washed

seeds, in the seed coat, the remaining seed and in shoots of soybean


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plants. After drying at 65 °C, ground samples were ashed in a muffle

furnace at 500°C for 5h. After cooling, the samples were extracted

twice with 2 ml of 3.4 M HNO3 (v/v) and subsequently evaporated to

dryness. The ash was dissolved in 2 mL of 4 M HCl, subsequently

diluted 10-fold with hot de-ionized water, and boiled for 2 min. After

adding 0.1 ml of Cs/La buffer to 4.9 ml ash solution, Zn and Mn

concentrations were measured by atomic absorption spectrometry

(UNICAM 939, Offenbach/Main, Germany).

2.6 Statistics

A completely randomised block design with 4 replicates was used in all

experiments. The data were analysed by analysis of variance with a

significance level of p < 0.05. For mean separation Duncan’s Multiple

Range Test (DMRT) were used. The software packages Microsoft

Excel Professional 2007 and interaction between priming methods and

durations was analysed using the ‘Sigma Stat version 3.1’ software

(SYSTAT Software Inc., Erkrath, Germany).

5.3 Results

5.3.1 Optimisation of seed priming treatments

Soybean seeds, water primed for 6, 12 and 24 hours with three

different methods (submergence in water = SP-Method, submergence

in water according to the water uptake capacity of the seeds = AC-

Method) incubation between layers of moist filter papers = FP-Method))

were tested for germination and seedling development.

Figure 5.1 shows the time course of radicle emergence (germination

speed) after seed water priming between filter paper layers. Water

priming significantly increased germination speed in 12 h priming

treatment but not after priming durations of 6 h and 24 h. (Fig 5.1a).

Within all tested priming treatments, seed priming for 12 h between


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(Figure 5.1: (a) Radicle emergence of soybean seeds, water-primed between layers of moist filter paper for different time periods. Data points represent means and standard errors of four replicates. (b) Effect of different priming methods and durations on development of abnormal seedlings. Bars show means and standard errors of four replicates. Significant differences (Duncan's Multiple Range Test, p= 0.05) are indicated by different characters. AC-Method = volume of priming solution according to water absorption capacity of seeds; FP-Method = priming between moist filter paper; SP-Method = submerged priming.


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layers of moist filter paper resulted in the lowest proportion of abnormal

seedlings (Fig. 5.1b) and the most homogenous seedling emergence

on peat culture substrate (Fig. 5.2) All three methods and priming

durations were significantly different from each other with p values of

0.001 and 0.008, respectively, but the interaction between methods and

priming durations was found to be non-significant with a p value of

0.066.

Figure 5.2: Soybean seedlings grown for 7 days on peat culture substrate after water priming with different methods and priming durations. AC-Method = volume of priming solution according to water absorption capacity of seeds; FP-Method = priming between moist filter paper; SP-Method = submerged priming. Based on these findings, priming treatments for all other experiments

were routinely performed for 12 h between moist layers of filter paper.

In germination tests, the development of abnormal seedlings was

significantly reduced by Zn and Mn seed priming in all treatments

compared to the unprimed control, but no significant differences was

found between different concentration levels for both Zn and Mn (Table

5.2). Therefore, based on the lowest means determined for abnormal

seedling development (Table. 5.2), 10 mM Zn and 5 mM Mn

concentrations and their combination (10 mM Zn + 5mM Mn) were

selected for soybean seed nutrient priming.


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Table 5.2: Effect of seed priming with different Zn and Mn concentrations on abnormal seedling development. Data represent means and standard errors of four replicates. Significant differences (Duncan's Multiple Range Test, p= 0.05) are indicated by different characters.

Development of abnormal seedlings (%)

Concentrations of priming solution

Seed treatments Unprimed 5 mM 10 mM 15 mM

Zn priming 31 + 2 a 13 + 5 b 10 + 4 b 19 + 5 b

Mn priming 31 + 2 a 13 + 2 b 19 + 5 b 15 + 4 b

5.3.2 Nutrient uptake into primed seeds

Soybean seeds primed with Zn, Mn and Zn+Mn were analysed for the

change in seed nutrient contents and accumulation of Zn and Mn in

different parts of the seed. Natural Zn and Mn contents (15.6 mg per

seed and 9.4 mg per seed) in soybean seeds were significantly

increased after seed priming by approximately 6 fold and 5 fold

respectively, when primed separately with Zn and Mn as compared to

un-primed and water primed seeds (Table 5.3). Combined seed priming

with Zn+Mn also increased contents of these nutrients in the seeds, but

the increase was only half as compared to those primed separately with

Zn and Mn (Table 5.3).

Mineral analysis of the seed coat and the remaining seed after water

priming showed that less than 10% of the total seed Zn and Mn content

is situated in the seed coat. Zinc and Mn priming, significantly

increased Zn and Mn contents in the seed coat (Zn ≈ 20 fold, Mn ≈ 6

fold) and in the remaining seed (Zn ≈3 fold, Mn ≈ 4 fold). Based on Zn

and Mn contents in washed seeds after nutrient priming, about 40 % of

the Zn content and 50-60 % of the Mn content were detected on the

seed coat of nutrient-primed seeds. There was a negligible difference in

seed Zn and Mn contents between unwashed and washed seeds,

which indicates tight adsorption of primed nutrients to the seed coat.


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Table 5.3: Distribution of Zn and Mn in soybean seeds after nutrient seed priming. Data represent means and standard errors of four replicates. Column values followed by different letter are significantly different by Duncan's Multiple Range Test, p= 0.05).

5.3.3 Effects of micronutrient seed priming in hydroponic culture

5.3.3.1 Effects of Zn seed priming

Zinc deficiency in the nutrient solution inhibited hoot biomass

production of 5-weeks old soybean plants grown from water primed

(WP) control seeds (Fig. 5.3), while root biomass production was not

affected. The absence of Zn in the nutrient solution induced a moderate

leaf intervenial chlorosis. Zinc seed priming reverted the detrimental

effects on plant growth and Zn-deficiency chlorosis.

Zn µg seed-1 Priming treatment Unwashed

Complete seed Washed

complete seed Seed coat Remaining seed

Water 15.6+0.2 c 13.7+0.5 c 1.4+0.1 c 14.7+0.5 b

Zn 104.6+0.2 a 101.7+2.2 a 28.3+1.1 a 46.9+2.1 a

Mn 15.7+0.2 c 16.4+1.1 c 2.5+0.2 b 15.2+0.2 b

Zn+Mn 57.1+1.4 b 57.3+2.0 b 31.4+0.5 a 45.1+0.3 a

Mn µg seed-1 Priming treatment Unwashed

Complete seed Washed

complete seed Seed coat Remaining seed

Water 9.4+0.1 c 9.5+0.5 c 0.6+0.1 c 7.2+0.6 c

Zn 9.5+0.1 c 9.5+0.4 c 0.8+0.1 c 8.5+0.3 c

Mn 52.3+0.8 a 48.1+4.2 a 37.2+2.1 a 21.8+0.3 b

Zn+Mn 32.3+0.7 b 33.6+0.4 b 30.4+0.5 b 26.3+0.5 a


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(Figure 5.3: (a) Shoot and (b) root dry biomass production of 5 weeks old soybean plants grown in nutrient solution. Bars show means and standard errors of four replicates. Significant differences (Duncan's Multiple Range Test, p= 0.05) are indicated by different characters. (NS = Nutrient solution; WP = water priming; NS – Zn = nutrient solution without Zn; NS – Mn = nutrient solution without Mn; NS – (Zn+Mn) = nutrient solution without Zn and Mn).

Although Zn priming significantly increased total Zn shoot contents, at

final harvest (5 weeks after sowing), Zn shoot concentrations were in

the deficiency range and not significantly different from the WP

treatment without any Zn supply. Apart from Zn levels, Zn deficiency

also affected the Mn nutritional status but the Zn status was not

affected by Mn deficiency. In the variants supplied with Zn via the

nutrient solution, the Zn status was always sufficient (Fig. 5.4).

5.3.3.2 Effects of Mn seed priming

Without Mn supply in the nutrient solution, both shoot and root biomass

production was significantly reduced with or without Mn priming

treatments compared to plants grown in complete nutrient solution.


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Figure 5.4: Shoot Zn and Mn (a) concentrations and (b) contents in 5 weeks old soybean plants grown in nutrient solution. Deficiency thresholds for Zn and Mn are marked by horizontal lines..Bars show means and standard errors of four replicates. Significant differences (Duncan's Multiple Range Test., p= 0.05) are indicated by different characters. (NS = Nutrient solution; WP = water priming; NS – Zn = nutrient solution without Zn; NS – Mn = nutrient solution without Mn; NS – (Zn+Mn) = nutrient solution without Zn and Mn).


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(Both, Mn contents and Mn concentrations in the shoot tissue were

significantly increased after Mn seed priming. However, at final harvest

(5 weeks after sowing), Mn concentrations were still below the Mn

deficiency threshold of 20 µg g-1 dry matter, while the Mn status was

sufficient when Mn was supplied via the nutrient solution (Fig. 5.4).

3.3.3 Effects of combined seed priming with Zn and Mn

Surprisingly, in contrast to plants grown under sole Mn deficiency, root

biomass production of plants grown under combined deficiency of Zn

and Mn in the nutrient solution was almost similar to the plants grown in

complete nutrient solution. Shoot biomass was reduced but shoot

elongation was not affected and the leaves showed typical Mn

deficiency chlorosis. Shoot Zn and Mn status was in the deficiency

range but higher shoot concentrations of Zn were detected under

combined deficiency of Zn and Mn as compared to Zn deficiency alone.

The beneficial effect of Zn supply on shoot growth was less expressed

in the Zn+Mn priming treatment as compared with Zn priming alone and

Mn deficiency chlorosis was not reverted by the priming treatment.

Seed priming with Zn+Mn increased both Mn and Zn shoot contents but

concentrations did not increase above the deficiency thresholds.

5.3.4 Effects of micronutrient seed priming in soil culture

In the soil culture experiment on a calcareous Loess sub-soil, both, Mn

and Zn seed priming had a positive effect on shoot elongation and

slightly increased shoot biomass production by approximately 15 - 20

%, while root biomass was not affected (Fig. 5.5).


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Figure 5.5: (a) Shoot and (b) root dry biomass of 5 weeks old soybean plants grown in soil culture. Deficiency thresholds for Zn and Mn are marked by horizontal lines..Bars show means and standard errors of four replicates. Significant differences (Duncan's Multiple Range Test., p= 0.05) are indicated by different characters. In all treatments, Mn supply was sufficient. In variants without Zn

priming, Zn shoot concentrations were in the deficiency range but Zn

deficiency chlorosis was only slightly expressed. Seed priming with Zn

and Zn+Mn significantly increased Zn shoot contents but only in the Zn

priming treatment Zn concentrations were increased above the

deficiency threshold. By contrast, water priming alone had no effects

on plant growth or the plant-nutritional status (Fig. 5.6).


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(((((((((((((((((((((

Figure 5.6: Shoot Zn and Mn (a) concentrations and (b) contents in 5 weeks old soybean plants grown in soil culture. A line across the bars indicates critical deficient levels of Zn and Mn. Deficiency thresholds for Zn and Mn are marked by horizontal lines. Bars show means and standard errors of four replicates. Significant differences (Duncan's Multiple Range Test., p= 0.05) are indicated by different characters.


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5.4 Discussion

5.4.1 Seed priming effects on seedling vigor and micronutrient

status

In the seed priming treatment optimized in this study for soybean

seeds, germination was accelerated during the first 24 h but already

after two days the number of germinated seeds was similar in unprimed

and all water priming treatments (Fig. 5.1a). Accelerated germination

and early seedling development have been similarly reported in maize

and barley as a result of water priming (Ajouri et al., 2004; Harris et al.,

2007). This has been attributed to pre-mobilisation of seed reserves

during the priming period (Bewley, 1997), leaching of germination

inhibitors into the priming solution (Hopkins, 1995), hydrolysis of ABA

(Ajouri et al., 2004) and pre-organisation of membrane structures

(Bewley and Black, 1982). The comparatively moderate effect of seed

priming treatments on germination speed observed in the present study

may be attributed to the fact that priming was performed between

layers of moist filter paper, slowing down the speed of imbibition. This

treatment may also reduce the induction speed for the priming

reactions described above.

However, imbibition and rehydration of dry seeds is a critical process

during germination and particularly large-seeded leguminous plants are

sensitive to imbibition damage. Rapid absorption of water can cause

severe membrane damage leading to leakage of electrolytes, sugars,

and amino acids (Powell and Matthews, 1978). Wuebker et al. (2001).

Bochicchio et al. (1990) reported embryo membrane damage, directly

linked with the speed of imbibition at low seed moisture. Accordingly,

pre-emergence exposure to flooding for 24 hours reduced emergence

of soybean by 50% (Wuebker et al., 2001). Also in the present study,

seed priming by slow imbibition between filter papers for 12 h resulted

in the highest germination rate and the smallest proportion of abnormal


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seedlings (Fig. 5.1b), which was reduced by up to 75 % as compared

with seed soaking directly in priming solutions. Alternatively, osmotic

seed priming has been reported to improve germination rate in Brassica

spp. (Brassica oleracea L.), cabbage (Khan, 1992; Zheng et al., 1994).

During osmotic priming, seeds are slowly moisturized by adjusting the

osmotic potential of the priming solution by using chemically inert but

osmotically active compounds like polyethylene glycol (PEG) for

reducing imbibition damage to seeds during soaking (Khan, 1992).

Nutrient seed priming treatments with Mn and Zn substantially

increased the natural seed content of Zn and Mn by 6 fold and 5 fold,

respectively (Table 5.3). However, 40 – 60 % of the primed nutrients

were found attached to the seed coat as compared to only 10 % in

unprimed seeds. Seed washings with distilled water did not significantly

change Zn and Mn contents suggesting a tight adsorption at the

surface of the seed coat. Similarly an isotope study by Polar (1970)

revealed that only 20.6 % of the total (Zn65) radionuclide absorbed by

the whole seed entered the cotyledons and the embryo of Vicia faba,

while the rest was retained by the seed coat.

Interestingly, the increase of Zn and Mn seed content after combined

seed priming with Zn+Mn was only half as compared with Zn or Mn

priming alone. This finding suggests a competitive interaction of both

nutrients during the priming treatment. However, the differences in seed

contents disappeared when only the remaining seed was analysed after

removal of the seed coat, indicating that competition between Zn and

Mn was mainly restricted to the outer parts of the seeds. Finally,

nutrient seed priming increased Zn and Mn contents in the inner part of

the seeds (embryo + endosperm + cotyledons) by 300 - 400%. Total

seed contents obtained after micronutrient seed priming of approx. 210

mg kg-1 seeds are similar to values reported in earlier studies of Ajouri

et al. (2004) on barley and Harris et al. (2007) on maize.


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5.4.2 Seed priming effects on plant growth

In hydroponics, Zn deficiency reduced shoot growth of 5 weeks old

soybean plants and inhibited shoot-, but not root biomass production

(Fig. 5.3). Inhibition of shoot growth in Zn-deficient plants may be

attributed to increased oxidative degradation or inhibited synthesis of

IAA, as previously suggested by Cakmak et al. (1989). On the other

hand, Zn deficiency can also induce root P deficiency responses, such

as increased expression of high affinity P uptake (Huang et al., 2000),

even in presence of high external P levels as a consequence of Zn

deficiency-induced inhibition of shoot to root re-translocation of P in the

phloem (Zhang et al., 1991). This phenomenon may also be

responsible for the Zn deficiency-induced increase in root/shoot ratio

observed in the present study, which resembles a typical response to P

limitation (Marschner, 2012).

Detrimental effects on early soybean growth due to Zn limitation were

completely reverted by Zn seed priming. However, at final harvest, the

Zn shoot concentration was in the deficiency range, demonstrating that

any surplus of Zn supply was immediately transformed into plant growth

and Zn priming could cover the Zn demand of the plant for a growth

period of maximum 5 weeks (Fig. 5.4a).

Manganese deficiency in the nutrient solution inhibited both, shoot and

root biomass production (Fig. 5.3) associated with intense leaf

intervenal chlorosis. Obviously, Mn deficiency affected photosynthesis

more severely than Zn deficiency (Shenker et al., 2004) as indicated by

stronger expression of leaf chlorosis in Mn-deficient plants. Limited

photosynthesis and limited carbohydrate supply to the roots have been

also reported in earlier studies as main factors limiting root

development and growth of Mn-deficient plants (Campbell and Nable,

1988).

However, in contrast to shoot biomass production, shoot elongation

was not affected by Mn deficiency. This may be attributed to the


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concomitant Zn supply via the nutrient solution, which was sufficient to

maintain IAA production. (Cakmak et al., 1989).

Manganese seed priming was not able to increase shoot Mn

concentrations above the deficiency threshold and the detrimental

effects of Mn deficiency on plant growth were not reverted by the

priming treatments, although the improvement of Mn nutrition was

much stronger as compared with the effect of Zn priming on the Zn

nutritional status (Fig. 5.4a). This may reflect a particularly high Mn

demand of the soybean plants. Moreover, the Mn status of the plants

was negatively affected even by water priming (Fig. 5.4b), an effect that

was not observed in case of Zn nutritional status.

Under combined deficiency of Zn and Mn, the inhibitory effect of Mn

deficiency on root biomass production was not detectable and growth

responses where similar as observed in Zn-deficient plants but with a

stronger expression of leaf chlorosis. This may indicate that the

observed responses to Zn deficiency, such as reduction of shoot

elongation and shoot biomass production but maintenance of root

growth may be induced earlier in the culture period as compared to the

responses to Mn limitation. Therefore, in the absence of both Zn and

Mn supply, the more rapidly induced growth responses to Zn limitation

may override to some extent the negative Mn effect on root growth..

In Zn+Mn priming treatments, about 47 % of primed Zn and 90 % of

primed Mn was recovered in the shoots of 5 weeks old soybean plants

grown in Zn- and Mn-deficient nutrient solutions. Despite of sufficient

translocation of these nutrients to the growing shoot, physiological

mechanisms involved in the translocation of primed nutrients from the

seed coat and other seed components to growing shoot and root during

early plant growth are still unknown. Similarly, seed priming with Zn+Mn

have shown that 50% and 80% of the seed Zn and Mn contents,

respectively, were found in the shoots 4 weeks old maize seedling

grown under Zn and Mn deficient conditions (Imran et al., 2012a).


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In plants, simultaneously primed with Zn and Mn, the beneficial effect of

Zn priming on shoot biomass production was less expressed as

compared with Zn priming alone (Fig. 5.4). This may indicate some

antagonistic interactions between these micronutrients as already

observed for Zn and Mn uptake during the priming treatments. Similar

Zn/Mn antagonisms have been reported for other plant species such as

rice (Ishizuka and Ando, 1968) and maize (Aref, 2012).

In soil culture, both priming treatments with Zn and Mn had a positive

effect on shoot elongation and slightly increased shoot biomass

production while root biomass was not affected (Fig. 5). In all

treatments without additional Zn supply, Zn shoot concentrations were

in the deficiency range. This may reflect an extremely low Zn availability

of the highly calcareous soil (23.3 % CaCO3) used for the experiments,

leading to Zn immobilisation by adsorption to CaCO3 and clay minerals

(Trehan and Sekhon, 1977).

Manganese supply was sufficient in all treatments and due to the

absence of Mn deficiency root growth was not affected. In accordance

with the observed stimulation of shoot growth, seed priming with Zn

increased the shoot concentrations of Zn even above the deficiency

threshold (Fig. 5.6). However, as already observed in the nutrient

solution experiment, this effect was less expressed after combined

priming with Zn and Mn, suggesting the presence of an antagonistic

interaction

5.5 Conclusion

The present study demonstrates that nutrient seed priming, particularly,

with Zn offers perspectives to improve seedling development and early

growth of soybean. Controlled, slow imbibition of soybean seeds

increased beneficial effects of seed priming, demonstrating the

importance of priming conditions avoiding imbibition damage during

priming process. Shoot Zn and Mn contents of soybean plants grown in


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nutrient solution indicate that the primed nutrients are efficiently

translocated within the plant during early seedling development and

could be sufficient to cover the complete Zn demand up to 5 weeks

under limited Zn supply. Beneficial effects of micronutrient seed priming

comprised improvements of early plant growth and micronutrient status.

However, in face of the general importance of Zn and Mn in biotic and

abiotic stress adaptations, further investigations are required for a more

comprehensive assessment of perspectives for micronutrient seed

priming in soybean culture, considering also the physiological

background of the nutrient priming effects as well as their expression

under variable growth conditions.such as. soil types, soil-water and

redox status, fertilisation management, variation in priming treatments

and genotypes.

Chapter(6((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((General(Discussion(

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Chapter 6

General Discussion

Uniform germination, vigorous seedling development and continuous

nutrient supply to young fast growing seedlings is vital for better crop

establishment and ultimately for maximum yield. A young seedling

passes through various stages for acquiring and utilizing mineral

nutrients during growth, based on the internal and external nutrient

supplies and on crop nutrient requirements (Scaife and Smith 1973). At

initial growth stages, seed mineral reserves are the primary source of

nutrients for young seedling, and only a small part is taken from soil.

During germination process, nutrients stored in the endosperm are

released by the enzymes, synthesized by aleurone and scutellum and

transported to the growing embryo (Fincher, 1989). Therefore, sufficient

mineral nutrient reserves in the seed are crucial to nourish young

seedlings until the development of the root system and its functioning in

supplying the nutrients. This is important for crops, particularly, grown in

soils with low available nutrients for plant growth (Asher, 1987). Seed

mineral contents have been shown to play an important role in the

germination process and early seedling development in different crop

plants (Rengel and Graham, 1995; Hacisalihoglu and Kochian, 2003;

Tehrani et al., 2003, Nadeem et al., 2011).

Application of mineral nutrients to the seeds (seed treatment) prior to

sowing is an efficient and cost-effective approach to improve nutrient

availability to plants at early growth stages (Robert, 1973; Ros et al.,

2000). Farooq et al. (2012) have reported that seed treatment with

different mineral nutrients can improve plant growth and increase grain

yield of field crops. Different approaches are practiced for applying

mineral nutrients to the seed like, such as coating or dressing and

soaking in nutrient solution. Zinc dressing of wheat seeds has shown

significant increase in grain yield (Yilmaz et al., 1998). Zhang et al.


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(1998) have shown that soaking barley seeds in P solution stimulated

seedling growth and plant used the added P as early as three days after

application (Zhang et al., 1998).

In past few years, seed priming has acquired special attention due to its

promising role in improving germination rate and plant growth. Seed

priming is a pre-sowing seed treatment in which seeds are soaked in

water and dried back to storage moisture contents before further use.

Several authors have reported the beneficial affects of seed priming in

improving seed vigour and early seedling development (Taylor et al.,

1998; Harris et al; 1999; McDonald, 2000; Harris, 2006; Farooq et al.,

2009, 2010) for better crop production under optimal and various stress

conditions.

‘Nutrient seed priming’ is a technique in which seeds are soaked in

nutrient solutions instead of simple water. Nutrient seed priming

increases seed nutrient contents along with the priming effects to

improve seed quality for better germination and seedling growth. Maize

seed priming with Zn have shown stimulated plant growth, increase in

grain yield and seed Zn contents on soils with limited Zn supply (Harris

et al., 2007) and under stress of low root-zone temperature (Imran et al.,

2013). In barley, nutrient priming of seeds with P and Zn stimulated

germination, seedling growth and plant uptake of these elements (Ajouri

et al., 2004).

Various abiotic stresses like low or high soil temperature, very low or

high soil pH, flooding and drought conditions are important constraints

for normal seed germination, plant growth and nutrient acquisition. Low

temperature at early spring is a major yield-limiting factor for maize

grown in Central and Northern Europe. Maize seed germination, early

seedling development and plant growth is severely inhibited at low

temperature. Nutrient acquisition, at early developmental stages, is

particularly limited by low temperature due to (i) limited solubility and

availability of certain nutrients and (ii) disrupted root activity.


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Even at optimal environmental conditions, soil properties e.g. soil

structure, pH and soil fertility level play very important role in early

seedling establishment, plant growth, availability and uptake of nutrients

from the soil. Based on the promising results of nutrient seed priming

with respect to improvement of early seedling establishment, plant

growth and stress tolerance, the present thesis investigated the role of

nutrient seed priming in mitigating low root zone temperature stress on

early seedling development and nutrient acquisition in maize. In

addition, effects of nutrient priming were investigated on germination,

plant growth and nutrient uptake in soybean under deficient and limited

nutrient supply. A series of experiments conducted on maize (chapter 3,

4) and soybean (chapter 5) have shown substantial results of nutrient

seed priming in improving germination rate, plant growth and nutrient

acquisition under limited nutrient availability and supply. The following

discussion is focused on the most prominent results of this thesis to a

greater relevance for agricultural crop production under abiotic stress

conditions and use of nutrient seed priming for improvement of plant

growth and nutrient uptake in crop plants.

6.1 Seed water priming improves germination rate and seedling

establishment

Early and healthy seedling establishment is very important for crop

growth in any kind of environmental conditions. Water priming is a

technique in which seeds are soaked in water for a certain time period

and then dried back to storage moisture contents for further use. During

the germination process, initially seed takes up water rapidly to start the

germination process and thereafter, only a small change occurs in seed

water contents until radicle emergence (Bardford, 1986). Seed priming

up to the point, just before the radicle emergence, can be positive for

seed germination, while further extension in priming duration may have

negative effects on seed quality (Ajouri et al., 2004). Water priming has


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been shown to improve germination rate and early seedling

development in some tropical crops, such as rice, chickpea, sorghum

and maize (Harris, 1996). Germination time of different rice cultivars

was reduced up to 50% by water priming in West Africa (Harris and

Jones, 1997). Water priming also showed significant increase in

germination rate and early seedling emergence of barley (Ajour et al.,

2004).

In the present study, water priming of maize seeds for 24 hours by

soaking into distilled water (chapter 4) revealed that germination speed

(Fig. 4.1a) and growth of young seedlings (Fig. 4.1b) was significantly

improved in standard germination tests. The faster germination speed

and improved seedling growth can be attributed to acceleration of a

number of biochemical processes (Fig. 1.2) initiated during controlled

imbibition. It has been shown that water priming of Arabidopsis

(Gallardo et al., 2001) and sugar beet (Job et al., 1997) increased the

polypeptide fraction in the seeds, identified as degradation products of

storage proteins. Abscisic acid (ABA) is involved in maintaining the

seed in the dormant state. Abscisic acid is reported to prevent, radicle

cell wall loosening as a late event during germination (Bewley, 1997),

which leads to the inhibition of radicle extension in the germinating

embryo (Schopfer and Plachy, 1985). Moreover, presence of ABA in the

seed does not affect the osmotic potential and/or water uptake ability of

the embryonic axes (Bewley and Black, 1994). Gibberallin (GA) is

responsible for the synthesis of various hydrolytic enzymes involved in

the mobilization of endosperm reserves (Jacobsen et al., 1995). In

various reports, ABA has shown antagonistic effects on GA activities,

both, in gene expression level and in modulating germination per se

(Constance et al., 2000; Jacobsen et al., 1995; Liu et al., 1994; Steber

et al., 1998). Inhibited activity of GA represses the synthesis of amylase

enzymes in germinating seeds (Gomez-Cadenas et al., 2001). α-

amylase plays an important role in hydrolyzing the endosperm starch


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into sugars as energy source for the newly developing shoot and roots

(Kaneko et al., 2002). Another amylase enzyme, β-1,3-glucanase has

been reported to contribute to loosening of cell wall components for

radicle protrusion (Wu and Bradford, 2003).

It is reported that pre-soaking of seeds causes the hydrolysis of ABA

together with the leaching of other germination inhibitors like coumarin

and phenolic compounds from the seed into solution (Hopkins, 1995).

Therefore, seed priming might result in diminishing the antagonistic

effect of ABA on GA activity during the germination process. Positioning

of short chain fatty acids (C6-C12) is also known to be involved in

inhibition of seed germination. During imbibition, the position of certain

fatty acids associated with seed dormancy is changed and does not

reverse on drying (Bewley and Black, 1982).

Hence, in the process of seed priming, all the necessary physiological

and biochemical changes occurred in the seeds during the priming

period persist upon drying. Therefore, when a primed seed is sown, a

small uptake of water can already stimulates the radicle emergence.

Instead, an unprimed seed has to go through all the phases of seed

germination after sowing under sometimes unfavorable soil conditions,

which is a possible reason for lower germination rate and seedling

growth compared to primed seeds.

In addition, although imbibition is vital for re-constitution of bio-

membranes, activation of enzymes, mobilization of storage compounds

and protein synthesis in the seed, it can also induce imbibition damage,

particularly when water is taken up rapidly.

Powell and Matthews (1978) have reported damages to pea seeds

soaked in free water due to physical damages of cell membranes

leading to impaired cell structure. For soybean seeds cracking of

cotyledons and its vascular bundles has been reported during

uncontrolled imbibition (Kokuryu et al., 2009). Fast absorption of water

increases the leakage of electrolytes, sugars, and amino acids (Powell


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& Matthews, 1978; Egley et al., 1983; Pereira & Andrews, 1985;

Wuebker et al., 2001) and disruption of cell structures affects

germination and seedling growth (Larson, 1968; Bewley and Black,

1978; Hahalis et al., 1996).

Results of the germination tests in soybean (chapter 5) revealed that

water priming between filter paper layers is a suitable method to prime

soybean seeds to reduce imbibition damage to seeds. A significant

decrease in the development of abnormal seedlings (Fig. 5.1) by water

priming between filter paper layers demonstrated that slow uptake of

water during priming not only provides the potential benefits of water

priming, but also decreases the possible damages to the seed during

imbibition. In case of maize, soaking seeds in the water for 24 hours did

not result in any abnormal seedlings, revealing that maize seeds are

more tolerant to imbibition damage as compared with soybean.

In previous studies, it has been reported that various physiological

stress factors including ethanol toxicity, oxygen deprivation, carbon

dioxide accumulation and oxidative damages (Sung, 1995; Sallam and

Scott, 1987; Scott et al., 1989) are affecting seed germination during

and after submerging seeds in water for extended periods of time.

Results of the present study on priming duration suggested that it is

important not only to control the speed of germination but also oxygen

supply and priming duration are important factor in determining the

efficiency of the priming process. Hence, slow germination speed,

development of abnormal seedlings and reduced seedling growth of

soybean and maize occasionally observed in the present study may be

attributed to different reasons (i) disruptive and incomplete activation of

the germination process for shorter priming duration treatments due to

insufficient water uptake in to the seed (ii) anoxic and post anoxic

damages to various seed parts because of longer exposure to

submerged conditions (longer priming durations) in the absence of

oxygen.


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6.2 Increasing seed nutrient contents with nutrient seed priming

Priming maize (chapter 3, 4) and soybean (chapter 5) seeds in nutrient

solutions revealed that nutrient priming is a suitable technique to

increase seed nutrient contents. Concentrations of micronutrients in

priming solutions were calculated to double the amount of seed nutrient

contents based on the seed water absorbing capacity, while for P, it

was calculated to increase 50% in natural seed P content. However,

results of seed nutrient contents in maize (table 3.2, 4.1) and soybean

(Fig. 5.2) showed a relatively higher increase in seed micronutrient

contents than the calculated amount, for example Zn contents were

increased from 500 % (maize) up to 700 % (soybean) in seeds. Uptake

of micronutrients in the seeds higher than the calculated, almost 200 %,

may reflect a high capacity of the seed for micronutrient immobilization

e.g. at the seed coat (since the calculated increase of 200 % refers only

to the soluble micronutrients in the imbibition solution). If micronutrients

were continuously removed from this solution by adsorption to outer

seed structures, this would have induced additional uptake driven by

diffusion. Accordingly, in soybean seeds 40-60 % of primed Zn and Mn

remained at the seed coat. Similarly, in an isotope uptake study (Polar,

1970) about 20.6 % of the total (Zn65) radionuclide absorbed by the

whole seed enters the cotyledons and embryo of Vicia faba, while the

rest was retained by the seed coat. The high binding capacity of seed

coat, may act as filter, for excluding ions occasionally present in

excessive concentrations in the soil solution to avoid toxic effects of

metal ions. This revealed that in case of soybean the seed coat was

the most important sink for removal of micronutrients from the imbibition

solution. In water-primed soybean seed, the natural Zn and Mn contents

were 16 µg seed-1 and 9 µg seed-1, respectively. After Zn and Mn seed

priming, Zn and Mn contents of the remaining seed parts (embryo +

cotyledon) were 47 µg seed-1 and 21 µg seed-1, respectively. Hence,


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seed coat absorbed less than 3% of the total seed water contents,

which reflects that Zn and Mn contents were not much higher than the

calculated amounts based on the seed imbibition capacity, as the

remaining seed parts imbibes most of the water during priming.

After maize seed P priming, seed P contents would have increased by

500 µg seed-1 but increase was 200 µg P seed-1 in natural seed P

contents (965 µg P seed-1), less than 50 % of the calculated amount.

This could be probably due to P uptake already reached the storage

capacity of the seeds or a high osmotic potential of the priming solution

counteracted P uptake. However, the low efficiency of P priming in

terms of plant growth in comparison with micronutrient priming must be

attributed to the high plant demand for P as a macronutrient.

Nutrients stored in the embryo and cotyledons are the primary source of

nutrient supply during germination and early seedling development. As

a result of nutrient seed priming, in addition to an increase in Zn and Mn

contents in the embryo + cotyledons, there was an enormous increase

in seed coat Zn and Mn contents (Table 5.1). The nutrients absorbed in

the seed coat may also be an important source of nutrient supply for

growing plants, as the seed coat remained attached to the cotyledons,

even after seedling emergence. Also shedding and decomposition of

seed coat near the seedling may a be source of nutrient supply as a

localized fertilizer.

Therefore, the results of the present study revealed that increase in

seed nutrient contents via nutrient priming could be a suitable technique

for improving seed quality and nutrient supply for young maize and

soybean seedlings.

6.3 Improving maize and soybean growth under nutrient limitation

via nutrient seed priming.

It is well documented that seed nutrient reserves play an important role

in seedling development and early growth. Increased seed Zn and Mn


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contents via nutrient seed priming have shown pronounced effects on

maize (Chapter 3) and soybean (chapter 5) seedling growth under

nutrient deficient conditions. Particularly, affects of Zn priming have

been enormous on shoot growth, in both maize and soybean under

complete absence of Zn supply.. It has been reported that increased P

uptake and its translocation to the shoot is responsible for Zn deficiency

in crop plants grown, both, in solution or soil cultures (Cakmack and

Marschner, 1987; Loneragan et al. 1982, Cakmak and Marschner

1986). Zhu et al. (2001) reported that highly P-efficient wheat

genotypes inadvertently depress Zn uptake and tissue Zn

concentration. Results of the present study suggests that additional Zn

in the seed via Zn priming was readily available for early plant growth

and was not effected by the antagonistic affects of P due to rapid

translocation to the fast growing shoots. This indicates that Zn priming

might be an appropriate strategy to improve plant Zn status on high P

soils and high lvels of P fertilization.

Stimulation of root biomass production in maize (Fig. 3.2b) and soybean

(Fig. 5.4) may be a reaction to Zn limitation, as Zn deficiency-induced

inhibition of P re-translocation to the roots induces P deficiency

responses in the root tissue, such as increased root/shoot ratio,

increased mycorrhization and up-regulation of high affinity root P uptake

systems (Marschner, 2012).

Root dry biomass was also increased under P deficiency in nutrient

solution and also in soil grown maize plants by P seed priming. The

increase in root-shoot ratio could be attributed to some extent by net re-

translocation of P from shoot to roots in P deficient plants (Smith et al.,

1990). Marschner et al. (1996) have suggested that under P deficient

conditions, a higher proportion of uptaken mineral nutrients from the

substrate is retained in the roots, but also the cycling from shoot to roots

provides additional P to the roots. Even though shoot growth is strongly

reduced under P deficiency but the plant struggles at least to maintain


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chlorophyll concentrations and the photosynthetic activity (Fredeen et

al., 1989; Cakmak, 1994), to ensure the constant supply of

photosynthates to the root as a dominant sink in deficient plants

(Marschner et al., 1996). It is shown that shoot dry biomass markedly

reduced under P deficiency, mainly due to severely inhibited growth of

shoot apex in beans plants (Cakmak, 1994a). However, in both P

deficient and sufficient plants, sucrose concentration in primary leaves

(source) and its export in phloem exudate is maintained, therefore, root

growth is similar in the P deficient and sufficient plants (Cakmak et al.,

1994b). In the present study, maize shoot P concentration (Fig. 3.3) in

P-primed plants showed initial translocation of P to the growing shoot,

thereafter, a continuous decrease in shoot P concentration and increase

in root growth indicates that shoot P was translocated to the growing

roots. Therefore, shoot dry biomass of 4 weeks old maize plants was

severely affected.

In soil culture, maize and soybean plants suffered from Zn deficiency in

all treatments. In calcareous soils, the plant faces various stress factors

such as P, Fe and Zn limitation, high pH and higher levels of

bicarbonates with the potential to induce the accumulation of

carboxylates and phenolics in the root tissue (Neumann and Römheld,

2007). The chelating properties of these compounds may promote Zn

sequestration in root tissues and limit their translocation to the shoot.

Therefore, the marginal surplus of Zn supply to the shoot tissue by

Zn+Mn priming in soil-grown plants also induced only a moderate

stimulation of shoot biomass production (+ 18%) as compared to plants

grown in hydroponics (+ 50%) (Fig. 3.5). An intensive stimulation of root

growth as observed in hydroponics was absent in soil culture. This may

be also attributed to additional stress factors in calcareous soils (e.g.

high pH, high bicarbonate levels (Neumann and Römheld, 2007).

In the present study, maize B priming has not produced the expected

positive results but in a separate study on wheat growth under salt


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stress (8 dSm-1), B seed priming has shown alleviating effects of salt

stress on young wheat seedlings by improving shoot and root growth

(Fig. 6.1, 6.2).

Fig. 6.1: Effect of B seed priming on 2 weeks old wheat seedlings grown under salt

stress (8dSm-1).

Fig. 6.2: Effect of B seed priming on root growth of 4 weeks old wheat seedlings

grown under salt stress (8dSm-1).


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Increase in shoot and root biomass accumulation by B priming of wheat

plants under salt stress (Fig. 6.3) suggested that despite of no effects of

B priming on maize growth in the present study, it might be helpful for

improving tolerance to other abiotic stresses in crop plants. It shows

that effectiveness of seed priming with a certain nutrient could be

variable in different crops and stress conditions. Therefore, priming

efficiency of mineral nutrients might be also linked to plant requirements

growing under different stress conditions.

Fig. 6.3: Effect of B seed priming on shoot and root fresh biomass of 4 weeks old

wheat plants grown under salt stress (8 dSm-1). Bars show the means and SE of 4

replicates. Different characters show the significant differences between the

treatments.

Phytic acid (Myo-inositol 1,2,3,4,5,6-hexakisphosphate) is a major form

of P stored in the seed. In mature seeds, phytic acid contributes from

one to several percent of the dry weight (Lott, 1984; Lin et al., 2005). In

seed tissues, positively charged cations of mineral nutrients including K,

Mg, Ca, Fe, Zn and Mn bind to phytic acid and form phytate (also

known as phytin), which is stored in special protein storage vacuoles

called globoids. (Lott, 1984; Wada and Lott, 1997; Raboy, 2002). Due to

strong chelating of mineral nutrients to phytic acid, their bioavailability is


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reduced or inhibited in the digestive track of monogastric animals (Zhou

and Erdman, 1995; Lönnerdal, 2003). Development of maize and

soybean mutants for low phytic acid in the seeds have been shown to

improve the bioavailability of mineral nutrient to human and other

monogastric animals (Raboy, 2002; Lönnerdal, 2003).

Recently, Doria et al. (2009) have shown that maize low phytic acid

mutant (lpa1-241) defective in the synthesis of phytic acid during seed

maturation have low germination capacity because of increased

oxidative damages to DNA and other seed proteins. Accordingly, due to

low phytic acid synthesis, alteration in the storage form of mineral

nutrients especially Zn and Mn in the seed would have reduced their

availability during the germination process. This could result in limited

activities of Mn and Zn superoxide dismutases. In this thesis, significant

translocation and utilization of additional Zn and Mn in the seed via

nutrient priming both, in maize and soybean seedlings, suggests their

readily availability for plant growth. Therefore, Zn and Mn seed priming

may be an appropriate strategy to improve germination rate of low

phytate mutants.

6.4 Improving nutritional status of maize and soybean plants grwn

under deficient and limited nutrient available condition by nutrient

seed priming.

Seed dressing or coating of mineral nutrient in soils with low nutrient

availability have been shown an economical and easy method

compared to soil or foliar application (Ajouri et al. 2004; Adhikari and

Rattan, 2000; Slaton et al., 2001; Savithri et al.; 1999; Scott et al.,

1989). Zinc coating of rice seeds or immersing the roots of seedlings in

Zn solution is more efficient than surface application of Zn in calcareous

soils (Giordano and Mordvedt, 1973). Application of nutrients by these

methods require in time availability of the nutrients, may need specific

products, techniques, machinery for foliar application and seed coating;


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furthermore, availability of additional labor might be also required for

handling and sowing. In comparable, nutrient seed priming can be

applied at any time prior to sowing and does not involve extra inputs

other than the nutrients (Ajouri et al., 2004).

In the present study, nutrient seed priming improved the plant nutrient

status of both maize and soybean. Results of shoot mineral contents of

maize grown in nutrient deficient solution culture revealed that more

than 50% of the primed Zn and almost 80% of the Mn reserves were

utilized for shoot growth (Fig. 3.6) until final harvest, that can be

attributed to high mobility of these nutrients in plants.

In contrast to maize plants grown in nutrient solution, in soil grown

plants recovery of Zn seed reserves in shoot of Zn and Zn+Mn primed

plants was low and this level was even lower in water-primed and

unprimed plants. These results can be attributed to low availability of Zn

in the soil culture system (see Materials and methods, Chapter

3),which may also affect Zn distribution within the plants leading to

much lower Zn supply to the shoots as compared with the hydroponic

culture system. In calcareous soils, plants are facing various stress

factors such as P and Fe limitation, high pH and bicarbonate levels with

the potential to induce the accumulation of carboxylates and phenolics

in the root tissue (Neumann and Römheld, 2007.

Although translocation of primed nutrients was reduced in soil culture in

the present study but still it had positive effects on plant growth and

nutrient acquisition. However, there is a need for further investigations

to improve priming efficiency under various soil conditions.

6.5 Improving plant growth and nutrient acquisition at low root

zone temperature stress by nutrient seed priming.

In low root zone temperature experiments on maize, a strong

stimulation of root length (Table 4.3) demonstrated promising effects of

nutrient seed priming on formation of longer and finer roots, particularly


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stimulation of lateral root formation (Fig. 4.2). At low temperature,

reduced uptake and translocation of micronutrients (Zn, Mn, Fe and Cu)

from root to shoot inhibits plant growth (Engels and Marschner, 1996a)

and at the same time limits the translocation of assimilates to the roots

(Engels and Marschner, 1996b). This may cause retarded leaf growth

and limitation of photosynthesis, which strongly depends on the supply

with micronutrients, such as Fe, Mn, Zn. and Cu (Marschner, 1995) with

growth inhibition as a final consequence. Accordingly, Strauss et al.

(2007) reported a functional limitation of photosystem II (PSII) in plants

exposed to low RZT, while Nagasuga et al. (2011) stated that reduced

growth and biomass production of rice plants, grown under low RZT

was mainly attributed to the reduction of shoot growth and leaf area.

Accordingly, the results of the solution culture experiment (Chapter 4)

showed a significant increase in plant leaf area, stomatal conductance

(Fig. 6.4) and net photosynthesis rate per leaf but not per unit leaf area

(Fig. 6.5) in maize plants grown at low root zone temperature.

Results of Zn, Mn and Fe contents in the shoots of maize plants grown

in solution culture (Table 4.6) revealed that improved nutrient

acquisition via nutrient seed priming lead to enhanced root and shoot

growth under low root zone temperature.


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Fig. 6.4: Effect of nutrient seed priming on stomatal conductance (left) and leaf area

(right) of 1st fully developed leaf of 5 weeks old maize plants grown in nutrient

solution at low root zone temperature (10°C for water, Fe and Zn). Bars represent the

means and SE of 4 replicates. Different characters show the significant differences

between the treatments.

Fig. 6.5: Effect of nutrient seed priming on rate of photosynthesis (left) and net

photosynthesis (right) in 1st fully developed leaf of 5 weeks old maize plants grown in

nutrient solution at low root zone temperature (10°C for water, Fe and Zn). Bars

represent the means and SE of 4 replicates. Different characters show the significant

differences between the treatments.

In a separate study, Zn priming of wheat seeds also showed an

increase in shoot Zn concentration of 4 weeks old wheat plant grown

under salt stress (Fig. 6.6). It is also reported that in wheat plants,


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higher seed Fe contents increased leaf area and chlorophyll contents in

the leaf under limited Fe supply (Shen et al., 2002).

Fig. 6.6: Effect of nutrient seed priming on shoot Zn concentration of 4 weeks old

wheat plants grown under salt stress (8dSm-1). Bars show the means and SE of 4

replicates. Different characters show the significant differences between the

treatments.

Almost all types stresses to plants including suboptimal temperatures,

involve oxidative stress as one of the rapid responses (Blokhina et al.,

2003). Germination and early seedling development at low temperature

conditions may change the consumption of oxygen, increased

production of reactive oxygen species (ROS), impair and disrupt the

efficiency and the pathways of electron transport chains and reduce

ATP production (Yin et al., 2009). Increased production of active oxygen

species (AOS), such as superoxide, hydrogen peroxide and the

hydroxyl radicals are involved in membrane damage by causing lipid

peroxidation, protein degradation, enzyme inactivation and disruption of


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DNA strands (Stewart and Guinn, 1969; Cohn et al., 1979; Allen 1995;

Prassad, 1996).

Phenolic compounds play an important role in suppressing lipid

peroxidation by scavenging free radicals or destroying peroxides (Rice-

Evans et al., 1997). In plants, proline and other low molecular weight

soluble sugars are produced and accumulated in response to stress

injuries (Hu et al., 2006). Due to their strong hydrophilic character, these

molecules are involved in repairing cell damages by adjusting the water

potential and maintaining the biological activities of other

macromolecules (Rathinasabapathi, 2000; Watanabe et al., 2000).

Recently, Guan et al. (2009) have reported the accumulation of proline

in maize plants in response to chilling stress tolerance.

Micronutrients, such as Zn and Mn are important co-factors of various

enzymes involved in the detoxification of AOS, such as superoxide

dismutases (SODs, Cakmak and Marschner, 1988 a, b, 1992; Cakmak,

2000). Deficiency of Zn, Mn and Cu alters the activities of SOD forms

and these changes depend on the type and the duration of the

deficiency stress (Yu and Rengel, 1999). Moreover, Mn is a cofactor of

various enzymes involved in the biosynthesis of phenolic compounds

with anti-oxidative properties (Thompson and Huber, 2007). Zinc has

functions in direct membrane stabilization, in biosynthesis of auxins

(Salami and Kenefick, 1970) and gibberellins (Sekimoto et al., 1997)

involved in plant growth regulation and in protein synthesis in general.

Therefore, limited supply of these micronutrients under conditions of low

RZT (Marschner and Engels, 1994) may increase oxidative damage to

the root cells and induce disturbances in plant growth. In the present

study, increased concentrations of total phenolics but decreased

concentrations of the stress metabolite proline (Table 6.1) in roots of

Zn/Mn-primed maize plants grown in soil culture at low root zone

temperature, may indicate alleviated root damage by stimulating


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biosynthesis of phenolics with anti-oxidative properties, which was

triggered by micronutrient seed priming.

Table 6.1: Effect of nutrient seed priming on total phenolic compounds and proline in

the roots of 4 weeks old maize plants grown in soil culture at low zone temperature

(10°C for water, Fe and Zn+Mn treatments). Data represents the means and SE of 4

replicates. Different chracters show the significant differences between the

treatments.

At low RZT, both, in hydroponics and soil culture experiments,

significant increases were observed for the respective micronutrient

contents in the shoot of maize plants after micronutrient priming (Table

4.6). This increase was still detectable in field grown plants at 5 weeks

after sowing (Table 4.7). Obviously, the surplus in micronutrient supply

in the priming treatments was immediately transformed into plant

growth. For Mn and particularly for Zn, the amount of micronutrients

supplemented by seed priming (Table 4.2) was sufficiently high to

explain the increase in micronutrient contents in the plants at 3-5 weeks

after sowing (Tables 4.6 and 4.7). However, the amount of Fe taken up

by the seeds (3 µg seed-1, Table 4.2) was much lower than the

observed increases in Fe shoot contents (Table 4.4) of up to 500 µg

seedling-1 in field grown plants (Table 4.7). This finding demonstrates

that improvements of the micronutrient status by seed priming are not

only a direct consequence of nutrient supplementation but most

probably also of improved nutrient acquisition from the soil via

stimulation of root growth (Table 4.4). This was further emphasized by

the observation that Fe seed priming simultaneously increased the Zn


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and Mn status of the plants and vice versa (Table 4.6) and that also P

content, as an important sparingly soluble macronutrient, was increased

by the priming treatments.

Shen et al., (2002) reported that wheat plants derived from seeds with

high Fe contents showed higher release of phytosiderophores in Fe-

deficient growth medium that leads to increased acquisition of Fe.

Therefore, it should be further elucidated whether Fe priming increases

the release of phytosiderophores in maize plants under low RZT.

Micronutrient seed priming treatments produced a 10-15 % increase in

final grain yield in two independent field experiments conducted in 2010

and 2011 (Table 4.8). The persistence of the priming effects may be

particularly attributed to the enormous stimulation of root growth

observed in model experiments (chapter 3 and 4). This may act as an

advantage not only during stress treatments but may further promote

plant growth during and even after the recovery phase. Moreover, other

studies have demonstrated long-lasting positive effect of micronutrient

seed priming also on alleviating other stress factors. Priming of barley

seeds with Zn significantly improved seed germination, plant growth and

water use efficiency by 44% in 4 weeks-old drought stressed barley

plants (Ajouri et al., 2004). Harris et al., (2007) have reported almost

40% increase in grain yield in maize grown on a Zn deficient soil in

Pakistan.

6.6 Commercial application

Different seed and fertilizer distribution companies are formulating and

selling various products for seed treatment (seed dressing and seed

coating) to improve nutrient uptake in crop plants. ‘Nutrient seed

priming’ is a relatively new approach to increase seed micronutrient

contents (not just soaking into fertilizer solution) as described by Al

Mudaris and Jutzi (1999) and Asgedom and Becker (2001). Some


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fertilizer companies are already selling different solution mixtures of

macro and micronutrients for nutrient seed priming. In Australia, ‘RLF®

The trace element company’ is selling BNS Super Strike® containing P

and micronutrients for seed priming.

In the present study, varying affects of priming methods and different

priming nutrients on germination, plant growth and nutrient acquisition

revealed that the selection of appropriate methods and priming duration

is very important to attain potential benefits of seed priming. In addition,

the selection of particular nutrients and their concentrations in the

priming solution also play a critical role in achieving desired goals of

nutrient seed priming. Priming seeds with low or very high nutrient

concentrations may induce negative effects on seed quality resulting in

decreased germination rate (Ajouri et al., 2004).

In this thesis, Zn priming alone has shown enormous results on plant

growth, but effectiveness of primed Zn was reduced when used

together with Mn, both in maize and soybean. Similarly, as discussed

earlier, B priming improved wheat growth under salt stress but had no

stimulating effect on maize growth. Accordingly, priming solution

containing different nutrients suitable for a crop species might be toxic

for other crops. Figure 6.7 shows soybean growth after seed priming

with a mixture of Zn, Mn, B and P nutrient solution suitable for maize

seed priming (chapter 3). Soybean seed priming with Zn, Mn, B and P

(simultaneously applied in the priming solution) caused leaf shape

deformation. The physiological reasons of these effects need further

elucidation.

For seed producing companies and farmers with larger farm areas, it

might not be easy to manage and handle larger quantities of seeds to

prime at the same time. Hence, persistence of the priming effects during

storage and seed treatment with pesticides is also important for

industrial application of nutrient seed priming. The effect of water

priming on germination in barley seeds was maintained until 9 weeks of


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storage (Ajouri et al., 2004). The persistence of positive affects of

nutrient seed priming should be also investigated for different crop

species.

Fig. 6.7: Effect of P and micronutrients (Zn, Mn and B) seed priming on shoot growth

of 4 weeks old soybean plants.

Therefore, to address various problems and limitations in commercial

application of nutrient seed priming, seed and fertilizer industry should

be actively involved for optimizing nutrient priming for commercial

applications.


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6.7 Future outlook

This thesis demonstrated positive effects of nutrient seed priming in

maize and soybean during early seedling establishment and nutrient

uptake under different plant growth conditions. The physiological role of

primed nutrients needs to be investigated more in detail. The following

section summarises some ideas for further research:

1. Study of the physiological and biochemical changes occurring in

the seed during nutrient seed priming and germination to

elucidate the role of primed nutrients in germination process

under optimal and various stress conditions.

2. Synchrotron X-ray fluorescence analysis of seeds to determine

the localization and forms of nutrients in the seeds.

3. Differential expression of genes responsible for nutrient transport

and synthesis of secondary metabolites in plants and seeds

should be analysed under different growth conditions.

4. Investigating the expression of nutrient seed priming effects

under different environmental conditions e.g. soil type, soil-water

and redox status, fertilisation management, variation in priming

treatments and genotypes etc.

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Curriculum Vitae Name: Muhammad Imran Date of birth: 12th July, 1978. Residential Address: Muhammad Imran Snedkervej 5, 1th 4200, Slagelse, Danmark Nationality: Pakistani Phone No.: 0045 7153 6241 E mail : [email protected] Accademics:

Degree Board/university Year of passing Division

PhD Uni. of Hohenheim, Stuttgart, Germany 2013

M.Sc. (Tropen. Master)

Uni. of Hohenheim, Stuttgart, Germany 2006 1st

M.Sc.(Hons.) Soil Science

University of Agriculture, Faisalabad 2004 1st

B.Sc.(Hons.) Soil Science

University of Agriculture, Faisalabad 2001 1st

F.Sc. (Pre-medical) Govt. College Sargodha 1997 1st

Matriculation (Science group)

G.H.S. Chak No. 104 NB, Sargodha 1994 1st

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Phd Thesis Title: Nutrient seed priming improves early seedling establishment, plant growth and

nutritional status in Zea mays L. and Glycine max L. under abiotic stress

conditions

M.Sc. Thesis Title: Nutrient Seed Priming Improves Germination of Low Quality Soybean Seeds

and Induces Tolerance to Low Temperature Submergence and Salt Stress.

(2006, Hohenheim, Germany)

List of Publications:

Imran M, V Römheld, G Neumann. 2013. Impact of nutrient seed priming on germination, seedling development, nutritional status and grain yield of maize. (In press: Journal of Plant Nutrition).

Imran M, A Mehmood, V Römheld, G Neumann. 2013. Nutrient seed priming improves seedling development and increases grain yield of maize exposed to low root zone temperatures during early growth. European Journal of Agronomy. 49: 141-148.

Imran M, V Römheld, G Neumann. 2013. Accumulation and distribution of Zn and Mn in soybean seeds after nutrient seed priming and its contribution to plant growth under Zn and Mn deficient conditions (Submitted: Journal of Plant Nutrition).

Imran, Rahmatullah, M. A. Gill and Tariq Aziz.2004. Differential phosphorus mobilization by two wheat genotypes on a calcareous soil fertilized with ammonium sulphate, potassium nitrate, ammonium nitrate and urea. Pak. J. Soil Sci. Vol. 22(4) 64-67.

Hafiza salma kousar, M. A. Gill, Rahmatullah, Tariq Aziz, M. S. Akhtar and M. Imran. Solublization of tri-calcium phosphate by different wheat genotype. 2002. Pak. J. Agric. Sci. 39(4); 273-277.

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Tahir, M. A., Rahmatullah, M. A. Gill, Tariq Aziz and M. Imran. 2003. Response of wheat and oat crops to potassium application and artificial irrigation with canal water. Pak. J. Agric. Sci. 40(3-4): 114-118.

Oral presentations

M. Imran. Nutrient seed priming improves low temperature stress tolerance in maize. International Plant Nutrition Conference. 2013. Istanbul. Turkey. M. Imran. Seed fertification: an approach to improve low temperature stress tolerance in maize. DanSeed symposium 2013. Denmark.

M. Imran. Seed priming with Fe and micronutrients improves seedling development and increases grain yield of maize exposed to low root zone temperatures during early growth. 16th International Symposium on Iron Nutrition and Interactions in Plants, June 17-20. Massachusetts. USA

Poster presentations:

Imran M., Asim M., Römheld V., Neumann G. Seed priming with micronutrients improves seedling development and increases grain yield of maize exposed to low root zone temperatures during early growth. ISRR 2012. “Roots to the Future”. 26-29 June. Dundee. Scotland.

Imran M., T. Müller, V. Römheld and G. Neumann. Nutrient seed priming enhances soil productivity under abiotic stress conditions. International Conference on Soil Fertility and Productivity.17-20 March 2010. Berlin, Germany.

Imran M., G. Neumann and V. Römheld. Nutrient seed priming improves germination rate and seedling growth under submergence stress at low temperature. Tropentag 2008. October 7-9. Uni. Hohenheim. Stuttgart.

Nora B., D. Sebastian, M. Imran, M. Weinmann, G. Neumann, T. Muller and V. Römheld. Effectiveness of commercial bio-fertilizers for improves phosphorus acquisition: Use of rapid screening test. Tropentag 2008. October 7-9. Uni. Hohenheim. Stuttgart.

Imran M., Rahmatullah, M.A. Gill and T. Aziz. 2004. Ammonium, nitrate and urea-N fertilizers influence P mobilization by wheat genotypes on a

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calcareous soil. (Abst. 75-76). 10th Ints. Cong. Soil Sci., Soc. Pak., March 16-18, 2004, Sindh Agriculture University Tandojam.

Akhtar, M. S., M. A. Gill, T. Aziz, Rahmatullah, and M. Imran. 2002. Phosphorus nutrition of Brassica species /genotypes supplied with deficient and adequate P levels. (Abst. 40, Plant Sciences) 33rd All Pakistan Science Conference, 26-28 December, Uni. of Agric, Faisalabad, Pakistan.

Research projects:

1. Supervised master thesis of 3 M.Sc. students during my PhD studies in University of Hohenheim.

2. Work as a Research Assistant for two years in a project for the measurement of NO3 leaching in the ground water as a consequence of high input of N fertilizers in Germany.

3. Effect of different bio-fertilizers to control Take-all disease of wheat. 4. Effect of Boron seed priming on germination and growth of wheat under

salinity stress. 5. Effect of different biofertilizers on maize growth at low root zone

temperature. Academic supervisors: PD. Dr. Gunter Neumann Prof. Dr. Volker Römheld University of Hohenheim University of Hohenheim Stuttgart, Germany. Stuttgart, Germany. [email protected] [email protected] Prof. Dr. Torsten Müller University of Hohenheim Stuttgart, Germany. [email protected]

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Acknowledgements.. "In the name of God, most Gracious, most Compassionate"

I would like to express my sincere gratitude to my supervisor Prof.. Dr.. Günter.

Neumann. for his continues support, guidance and also for his vast knowledge that

helped me to reach this milestone. I am also very grateful to Prof.. Dr.. Volker.

Römheld.for his worthy ideas, guidance, motivation and enthusiasm that helped me a

lot to accomplish the objectives of my Ph.D research work. I have no hesitation to say

that both of them are wonderful persons.

I would like to extend my sincere regards to my thesis committee members Prof..Dr..

Michael.Kruse.(Institute of Seed Science Technology) and Prof..Dr..Uwe.Ludewig.

(Institute of Crop Science) for their kind guidance and for giving valuable advice.

My sincere thanks also go to the technical staff in the institute Maria.Ruckwied,.

Helene. Ochott,. Heide. Zimmermann,. Charlotte. Haake,. Hinrich. Bremer. for

their help and guidance, and especially to Hans.Buchar,.who always stood with me

in the fields. I will not forget Mrs. Christa. Shöllhammer. and Mrs. Ursla.

Berghammer.for their help to solve administrative problems in Hohenheim.

Words do not count easy to pay heartiest thanks to my uncle Muhammad.Amin.

Salim. and his family, who always encouraged and support me during my stay in

Germany. I am grateful to my brothers Adnan,.Rizwan,.Irfan.and.Naveed.and my

friends Rai Mukkaram Ali Tahir, Shoaib.Nasir.and Muhammad.Ajmal.for their

love, affection, prayers for my success and consistent encouragement. I am also

thankful to all my Pakistani friends in Pakistan, Stuttgart and to all my fellows in the

lab, office and coffee room who always wished me for success. The sweet memories

of my stay at Hohenheim will always be with me.

My wife, Fariha.Tanwir, whose love and companionship has boosted me at every

step during my PhD. Her endless love and indispensable help have made all this

possible. I must say thanks to Baasil Imran, my son, whose smile is always

energizing me to continue-

.

.

Muhammad.Imran.

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Documents

Nutrient seed priming improves abiotic stress tolerance in Zea mays