Faculty of Resource Science and Technology QUALITY AND LONGEVITY DURING STORAGE... · Faculty of...

Geffry Ujie Anak Joshua

Master of Science

(Agronomy)

2013

Faculty of Resource Science and Technology

SEED QUALITY AND LONGEVITY DURING STORAGE OF FOUR

JATROPHA CURCAS L. ACCESSIONS FOUND IN SARAWAK

I

ACKNOWLEDGEMENTS

Praise the Almighty God, my Lord Jesus Christ and Holy Spirit for it was by His divine

grace and mercy alone that knowledge was perceived. I wish to express my utmost

gratitude and heartfelt appreciation to my major supervisor, Associate Professor Dr. Petrus

Bulan for his dedicated supervision, guidance, concerns, assistance, supports, comments

and friendship in the execution and completion of this study. I would also wish to express

my sincere gratitude to my co-supervisor, Dr. Siti Rubiah Bt Zainudin for her guidance and

encouragement throughout the study. My honest thanks and gratitude extended to the

Faculty of Resource Science and Technology for providing the facilities for this study and

the Centre for Graduate Studies for awarding me a full scholarship of UNIMAS

Postgraduate Zamalah. I also would like to express my thankfulness to the farmers (Mrs.

Gunang Ak Agot, Mr. Jonathan Ak Tinggi, Mr. Majing Ak Rimbau and Mr. Tapa Ak

Santap) for their generous assistance and co-operation to permit me to collect samples

from their farms. My deepest gratitude and heartfelt gratefulness also go to my beloved

parents, Mr. Joshua Ak Jalak and Mrs. Ulat Ak Dobi for their great supports, prayer, love

and cares, and unequivocal sacrifices to ascertain better life and education for me. I am

sincerely indebted and earnest thankful to my beloved wife, Mrs. Nancy Ak Majing for her

tremendous love, endless encouragement and assistance, intuition, trust, prayer and cares

which are the strength for me to move on and enable me to accomplish this study and

dissertation successfully. Lastly, my sincere appreciation also goes to my other family

members including my sisters, parents-in-law, brothers-in-laws and my cousin, Mr. Eder

Ak Dari (scholarship guarantor), for their moral support, understanding, prayer, generosity

and trust which gave me strength to complete my master’s degree study successfully.

II

DECLARATION

I hereby, declare that no portion of the work referred to in this thesis has been submitted in

support of an application for another degree of qualification of this to any other university

or institution of higher learning.

......................................................... .........................................................

Date (GEFFRY UJIE ANAK JOSHUA)

Student no.: 09021462

14 October 2013

III

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS I

DECLARATION II

TABLE OF CONTENT III

LIST OF ABBREVIATIONS VIII

LIST OF TABLES XI

LIST OF FIGURES XIII

ABSTRACT / ABSTRAK XV

INTRODUCTION 1

LITERATURE REVIEW 6

Jatropha curcas L. 6

J. curcas oil 9

Orthodox and recalcitrant seeds 10

Storage of seeds 11

Seed moisture content 13

Seed germination 15

Seed viability and vigor 17

Seedling vigor 18

Soil chemical properties and texture 19

Plant nutrient 20

Seed deterioration during storage 21

Seed maturation 23

IV

MATERIALS AND METHODS 26

Materials 26

J. curcas seeds 26

Silica gel desiccation 26

Solution of 2, 3, 5-Triphenyl Tetrazolium Chloride (C19H15CIN4) 26

Transparent ziplock and conventional polyethylene bag and sealed plastic

container

28

Germination tray 28

River sand 28

Ethanol solution (70% v/v) 28

Fungicide solution (0.2 % w/v) 29

Paper envelope 29

Aluminum foil 29

Soil auger 29

Methods 29

Seed treatment 29

Initial evaluation 30

Preliminary test 30

Seed moisture content test 30

Viability (tetrazolium) test 31

Live seed (stained embryo) 32

Analyses of live seeds 32

Standard germination test 35

V

Seedling vigor classification test (SVC) 35

Seedling growth rate test (SGR) 36

Biomass of normal seedling 37

Determination of seed oil content 38

Soil and plant nutrient analysis 38

Soil analysis 38

Plant nutrient analysis 39

Data analysis 39

RESULTS AND DISCUSSION 40

Study 1: Initial Assessment of Fresh Seed Lots 40

Seed quality of fresh seeds 40

Moisture content and standard germination 40

Seed viability 44

Live seed 44

Analyses of live seeds 46

Seedling vigor 48

Seedling growth and biomass allocation 50

Discussion 54

Study 2: Seed Quality at Post-storage 62

Quality of seed at post-storage 62

Seeds weight loss 62

Seed deterioration 66

Fungi 67

VI

Flaccid kernel 68

Shriveled kernel 69

Discussion 71

Study 3: Post-Storage 77

Post-storage seeds quality 77

Moisture content 77

Moisture content for 30 days stored seed lots 77



Seed germination 81

Germination for 30 days stored seed lots 81



Seed viability 85

Viability of 30 days stored seed lots 85



Analyses of viable seeds 89

Seed lots stored for 30 days 89



Seedling morphogenesis 97

Seedling vigor 97

VII

Strong seedling 101

Weak seedling 101

Abnormal seedling 105

Dead seedling 107

Anomalous emergence of seedling 108

Seedling growth 110

Seedling biomass allocation 114

Discussion 118

Study 4: Agrobiology and Seed Oil Content 125

Ecology of studied sites 125

Seed oil content, analysis of soil and plant nutrient 128

Discussion 131

CONCLUSIONS 133

REFERENCES 135

APPENDIXES 144

VIII

LIST OF ABBREVIATIONS

Abbreviation Terminology

Ac Accession

ABA Abscisic acid

CEC Cation Exchange Capacity

CSMC Critical Seed Moisture Content

CKs Cytokinins

CPO Crude Palm Oil

Dp Desiccation period

G Germination

GAs Gibberellins

G-0 Standard germination test for undesiccated seed lot

G-24 Standard germination test for 24 hours desiccated seed lot



IAA Indoleacetic Acid

LEA Late embryogenesis abundant

IX

Mt Maturity

M0 Maturity 0

(selective ratio 1:1:1 combination of seed from M1, M2 and M3)

M1 Maturity 1

M2 Maturity 2

M3 Maturity 3

mg milligram

RH Relative Humidity

SD Storage and desiccation

SMC Seed Moisture Content

SMCL Seed Moisture Content Loss

SMC-0 Seed moisture content test for undesiccated seed lot

SMC-24 Seed moisture content test for 24 hours desiccated seed lot



SVC Seedling Vigor Classification

SVC_Ss Strong seedling obtained from Seedling Vigor Classification Test

Tt Seed test

TZ-0 Tetrazolium test for undesiccated seed lot

X

TZ-24 Tetrazolium test for 24 hours desiccated seed lot



V Viability

Viability_Gv Germinable and viable seed obtained from Viability Test

XI

LIST OF TABLES

Table Page

1 Staining intensity of embryo. 34

2 Initial germination (%) and moisture content (%) of J. curcas seeds as

illustrated in Figure 1. 145

3 Mean value of initial germination (%) of J. curcas seeds. 145

4 Initial viability (%) of J. curcas seeds as illustrated in Figure 2. 145

5 Mean value of initial viability (%) of J. curcas seeds. 44

6 Analyses of viability of J. curcas seeds for four accessions and

maturity indexes. 46

7 Classification of stained seeds of J. curcas as illustrated in Figure 3. 47

8.1 Seedling vigor classification for four accessions. 49

8.2

Initial seedling vigor classification of four different accessions and

maturity indexes for germinated J. curcas seeds as extracted in Table

8.1.

146

9 J. curcas seedling lot group classification based on performance of

seedlings. 53

10 Deterioration of J. curcas seed in storage after desiccation up to 168

hours. 67

11.1 Critical seed moisture content (%) at 24 hours desiccation. 150



12.1 Seed moisture content (%) for 30 days stored seed lots. 79



13.1 Germination (%) for 30 days stored seed lots. 83

XII



14.0 Classification of stained seeds of J. curcas as illustrated in Figure 12. 153 -154

14.1 Analyses of viability of 30 days stored J. curcas seed lots for four

accessions, desiccation periods and maturity indexes. 91 - 92





15.1 Seedling vigor classification for 30 days stored seed lots. 98



16 Anomalous emergence of seedling. 109

17.1 Seed lot group classification based on performance of seedling growth

for 30 days stored seed lots. 111





18 Ratio of seedling growth and biomass allocation. 124

19 Localities and ecology of the studied sites. 126 - 127

20 Seed oil content, soil and plant nutrient. 130

21 Statistical analyses. 159 - 168

22.1 Viability (%) of live J. curcas seed stored for 30 days as illustrated in

Figure 11.1. 169


Figure 11.2. 169


Figure 11.3. 170

XIII

LIST OF FIGURES

Figure Page

1 Initial germination and moisture content of J. curcas seeds. 43

2 Initial viability of J. curcas seeds. 45

3 Staining pattern and deterioration in fresh embryos of J. curcas

seeds as manifested in tetrazolium reaction. 47

4 Initial seedling growth and biomass allocation of J. curcas. 51

5 Estimated marginal mean for maturity of J. curcas. 55

6.1 Quality of different J. curcas seeds base on performance of

accession from Bintulu. 61


accession from Miri. 61


accession from Samarahan. 61


accession from Sri Aman. 61

7 Weight loss of J. curcas seed lots stored for 30, 60 and 90 days. 63

8.1 Accessions: Weight loss of J. curcas seed stored for 30, 60 and 90

days. 64

8.2 Seed maturity index: Weight loss of J. curcas seed stored for 30, 60

and 90 days. 64

9.1 Weight loss of J. curcas seeds from four maturities in accession

from Bintulu stored for 30, 60 and 90 days. 65


from Miri stored for 30, 60 and 90 days. 65


from Samarahan stored for 30, 60 and 90 days. 65


from Sri Aman stored for 30, 60 and 90 days. 65

XIV

10 Types of deterioration in J. curcas after desiccation up to 168 hours

and stored to 90 days. 66

11.1 Viability of live J. curcas seeds for 30 days stored seed lots. 86



12 Pattern of staining and deterioration in embryos as manifested in

tetrazolium test reactions. 155-158

13.1 Seedling biomass allocation for 30 days stored seed lots. 115



14 Estimated marginal means of J. curcas seeds for desiccation prior to

storage. 119

15 Estimated marginal mean of J. curcas seeds for different accessions

prior to storage. 121

16 Estimated marginal mean for J. curcas seeds following desiccation

and storage. 122

17 Estimated marginal mean for J. curcas seeds of different maturity

index. 123

18.1 Plant tissue macronutrient uptake levels. 129

18.2 Plant tissue micronutrient uptake levels. 129

XV

Seed quality and longevity during storage of four Jatropha curcas L. accessions found in

Sarawak


ABSTRACT

Seeds of Jatropha curcas L. collected from four accessions from Bintulu, Miri, Samarahan

and Sri Aman were studied to determine moisture content, germination, viability and other

related properties (seedling morphogenesis and seed oil content). Outcomes of this

research were crucial for developing and innovating the suitability of seed storage

methodology in retaining quality for future precedent. This study emphasized the effects of

desiccation and storage of seed from three different level of fruit maturity. In addition, an

environmental influence on the mother tree was also studied to determine the relatedness

yield of seed oil content by ecological features and soil property. This study revealed that

the maturity indexes was significant (p = 0.009) to determine the quality of the fresh seed.

However, at post-storage the outcome was vice-versa (p = 0.055) due to seed aging. The

best criterion to determine seed quality at post-storage was by accession where it showed a

significant outcome of p < 0.001 compared to fresh seed (p = 0.051). Desiccation period of

96 hours reduced seed moisture content up to 70% for 60 days of storage was optimal in

minimizing the risk of deterioration caused by fungi and seed biochemical reaction, as well

as retained seed viability. Dry microclimate and ample content of phosphorus, magnesium,

sulfur, copper and zinc elements available in soil were believed as a factor for which

contributed to high oil yield in J. curcas seed.

Key words: Jatropha curcas, accession, desiccation, storage, maturity indexes.

XVI

Kualiti dan jangka hayat semasa penyimpan biji benih Jatropha curcas L. dari empat

aksesi yang ditemui di Sarawak


ABSTRAK

Biji benih Jatropha curcas L. dari empat aksesi iaitu Bintulu, Miri, Samarahan dan Sri Aman

telah dikaji untuk mengenalpasti kandungan kelembapan, percambahan, kebolehidupan dan

lain-lain sifat berkaitan (morfogenesis anak benih dan kandungan minyak biji). Hasil kajian ini

sangat penting untuk rujukan masa depan demi pembangunan dan inovasi kesesuaian kaedah

penyimpanan biji benih dalam mengekalkan kualiti. Kajian ini memfokuskan kesan

pengeringan dan penyimpanan ke atas biji benih dari tiga tahap kematangan buah. Pengaruh

persekitaran ke atas pokok ibu turut dikaji untuk mengenalpasti perkaitan kandungan minyak

biji yang terhasil dengan ciri-ciri ekologi dan sifat tanah. Hasil kajian menunjukkan bahawa

indeks kematangan adalah signifikan (p = 0.009) dalam penentuan kualiti biji benih segar.

Namun, di peringkat selepas penyimpanan hasil kajian adalah sebaliknya (p = 0.055)

disebabkan proses penuaan biji. Aksesi merupakan kriteria yang paling sesuai digunakan untuk

menentukan kualiti biji benih selepas penyimpanan kerana hasil yang signifikan iaitu p < 0.001

berbanding biji benih segar (p = 0.051). Pengeringan selama 96 jam mengurangkan kandungan

kelembapan biji benih sehingga 70% untuk 60 hari penyimpanan adalah optimum dalam

meminimakan risiko kerosakan disebabkan oleh kulat dan tindakbalas biokimia, serta

memelihara kebolehidupan biji benih. Iklim mikro yang kering dan kandungan elemen

fosforus, magnesium, sulphur, kuprum dan zink yang mencukupi tersedia di dalam tanah

merupakan faktor penyumbang penghasilan minyak tinggi di dalam biji J. curcas.

Kata kunci: Jatropha curcas, aksesi, pengeringan, penyimpanan, indeks kematangan.

1

INTRODUCTION

Currently, rocketing price of world petroleum due to gradual depletion reserves and great

demand for transportation and industries activities has increased the cost of living globally.

Furthermore, the impact of environmental pollution of increasing exhaust emissions

significantly induces notion that mineral oil is irrelevant for future use. Therefore, the

usage of mineral oil should be replaced into alternative energy base on crop. In the context

of growing interest for alternative, renewable and inexpensive energy sources, liquid

bioenergy production from vegetable oils is proposed as one of the possible options to

reduce these concerns.

Biodiesel is monoalkyl ester of fatty acids derived from vegetable oils or animal fats

produced by transesterification with methanol or ethanol (Knothe et al. (2006) cited in

Akbar et al., 2009; Erliza et al., 2007; Veny et al., 2009). Biodiesel has many advantages

such as it is renewable, safe for use in all conventional diesel engines, offer the same

performance and engine durability as petroleum diesel fuel, biodegradable, non-flammable,

non-toxic, less visible smoke, non-noxious fumes and odors, and cost-effective (Akbar et

al., 2009; Erliza et al., 2007; Patil & Deng, 2009). This reason made biodiesel production

from Jatropha curcas L. has become a booming business.

J. curcas is an important and promising source of diesel. Its extracted oil from seed can be

used as it is crushed without being refined, blended with normal diesel and used in car, and

refined and sold as pure diesel (CJP, 2007). This type of biodiesel is the most valuable

form of renewable energy that can be used directly in any existing and unmodified diesel

2

engine (CJP, 2007; Rintos, 2008). The jatropha oil is not characterized as edible oil and its

usage as biodiesel feedstock will not disturb the supply of edible oil and the usage of the

palm oil for oleo chemical industries or CPO export purpose (Erliza et al., 2007). The oil

produced by J. curcas can be easily converted to liquid bio-fuel, which meets the

American and European standards (Achten et al., 2008). Apart from its use as a liquid fuel,

the oil also has been used to produce biocides such as insecticide, molluscicide, fungicide

and nematicide (Achten et al., 2008).

The oil contain of J. curcas is 10 to 50 percent of the seed (CJP, 2007; Erliza et al., 2007;

Fang, 2008; Glicerio, 2007; Rintos, 2008) and 52 to 67 percent of the kernel (Fang, 2008),

which can be processed into Jatropha Methyl Ester (JME) or Jatropha Biodiesel by

transesterification. Due higher viscosity (16 to 18 hydro-carbon atoms per molecule

content), transesterification is performed to reduce viscosity and increasing burning power

in order to use the oil as diesel fuel for vehicles (Erliza et al., 2007). This process changed

triglyceride molecule or a complex fatty acid into methyl ester (biodiesel) and glycerol

using sodium methoxide (mixture of methanol with sodium hydroxide). The reaction

resulting glycerin (glycerol) is left on the bottom and methyl esters (biodiesel) is left on top

(CJP, 2007; Rintos, 2008).

The J. curcas contain toxin such as phorbol esters, curcin, trypsin inhibitors, lectins and

phytates (Achten et al., 2008). This toxin composition makes J. curcas plant is non-edible

either by man or animal, thus it is suitable for a hedge (living fence) by farmer because it is

not browse by animals like cattle or goats (Achten et al., 2008; Reinhard, 2004; Srivastava,

1999 cited in Ginwal et al., 2004). Furthermore, the plant is a valuable multi-purpose crop

3

to alleviate soil degradation, desertification and deforestation (Abdrabbo & Nahed, 2008),

prevent and control soil erosion, and reclaim wasteland (Achten et al., 2008), use as a

source of shade for coffee plants in Cuba and support plant for vanilla plants in Comore

Islands, Papua New Guinea and Uganda (Reinhard, 2004), and as ornamental plant.

In medicinal purpose, it is used for diseases like cancer, piles, snakebite, paralysis, dropsy,

etc. The seeds of J. curcas can be used against constipation, ulcer and tumor. Latex for

wound healing and its alkaloid known as jatrophine, is believed to have anti-cancerous

properties, Leaves as tea against malaria. Root for rheumatism and bark for snakebite

(BATCD, 2007; CJP, 2007; Reinhard, 2004; Satish, n.d.). Furthermore, an extracted curcin

is useful for anti-virus and anti-fungi (Fang, 2008).

Instead of biodiesel as it main product, the by-product, ‘glycerin’ has many uses in soap,

detergent and cosmetic production (Abdrabbo & Nahed, 2008; Rintos, 2008). The pressed

seed residues or press cake which rich in nitrogen, phosphorous and potassium (Abdrabbo

& Nahed, 2008) can be used as a fertilizer, for electricity and heat production and the

organic waste products can be digested to produce biogas (CH4) (Achten et al., 2008;

Rintos, 2008).

To obtain good quality of J. curcas seeds at post-storage, some factors should be taken into

consideration, for instance, the level of fruit maturity, safe seed moisture content,

appropriate storage condition and pre-storage treatment, duration of storage, good viability

and vigor of seeds. Rantje et al. (2008) reported that storage duration gave very significant

differences on the percentages of germination and vigor, total fungal population and lipid

4

contents of J. curcas. Therefore, the increment of storage duration caused the parameters

(germination, vigor, fungal dan lipid content) decreased but for free fatty acid contents and

lipase activity were vice versa. This finding also concluded that the moisture content of J.

curcas was in equilibrium with the relative humidity of the storage. In Christensen and

Kaufmann (1969) finding, during storage seeds or grains could be infected by fungi which

cause a decrease in viability, discolouration, various biochemical changes, heating and

mustiness, loss in weight, and production of toxins when it is consumed may be injurious

to human and domestic animals.

In recent years, there are many studies about J. curcas has been published and mostly

focused on cultivation, propagation, oil utilization, molecularity, genetics and economics

importance. However, a study on storability of J. curcas seed is less reported or published.

Although reported studies are little but the scope of research is aimed on one seed source

(accession) and one seed maturity (physiological or harvest maturity). Hence,

determination for high quality seed could not be ascertained due to lack of comparison

criterion. Moreover, the quality of seedling from post-storage seed is slightly published.

Despite an extensive search, no published literature regarding J. curcas planted in Sarawak

was found on the detailed physical properties and oil content of seed and their dependency

on agronomical parameters, which would be useful for the design of agricultural systems in

future. Therefore this research is essential in order to identify the suitability of seed

storage methodology and documenting the agronomical properties and distribution of J.

curcas crop.

5

The main objectives of this study were to (1) assess viability, vigor, germination and oil

content of four accessions of J. curcas seeds; (2) determine the effect of storage on

viability and germination of J. curcas seeds; (3) determine suitable period of storage for J.

curcas seeds; (4) analyze quality of J. curcas seed based on different fruit maturity index

and accession.

6

LITERATURE REVIEW

Jatropha curcas L.

The J. curcas is belonging to the Euphorbiaceae family. It is a drought-tolerant perennial

and able to survive in very dry soils in condition considered marginal for agriculture

(Erliza et al., 2007; FAO, 2010; Rao et al., 2008). The life expectancy and fruit production

of J. curcas can up to 50 years (Achten et al., 2008; CJP, 2007). However, the economic

life is expected from 30 to 50 years (FAO, 2010). J. curcas grows in tropical and sub

tropical regions, with cultivation limits at 30oN and 35

oS has its native distributional range

in Mexico, Central America, Brazil, Bolivia, Peru, Argentina and Paraguay (Achten et al.,

2008; FAO, 2010). Now it has been domesticated in a widespread manner in Africa and

Asia due to its adaptive ability (Rao et al., 2008; Srivastava, 1999 cited in Ginwal et al.,

2004). It also grows in lower altitudes of 0 to 1000 meters above sea level with optimum

rainfall range of 600 to 800 mm/year (Abdrabbo & Nahed, 2008; Erliza et al., 2007; FAO,

2010; Ginwal et al., 2004). It grows well in optimum temperatures between 20oC to 28

oC

(Erliza et al., 2007; FAO, 2010) and well adapted to conditions of high light intensity

(Jongschaap, 2007 cited in FAO, 2010).

J. curcas is a small tree or large shrub with irregular branch, wooden cylindrical stem and

smooth gray bark, which exudes whitish coloured, watery, latex when cut (Erliza et al.,

2007; CJP, 2007). It can reach a height up to 1-7 meter tall or more under favourable

condition. J. curcas is deciduous, shedding the leaves in the dry season. Flowering occurs

throughout the year (Heller, 1996 cited in FAO, 2010) and at peak during wet season (CJP,

7

2007). Normally, the flowers are pollinated by insects especially honey bees. Below are the

morphological characteristics of J. curcas:

i. Leaf

The leaf is broad (10 to 15 cm length and width) with 4 to 6 lobed, smooth, colored

green (adaxial) to pale-green (abaxial) and spiral phyllotaxis with alternate to sub-

opposite position. It is hollow, stripes and pointed at the end. The petiole is about 4

to 15 cm long. (Achten et al., 2008; CJP, 2007; Erliza et al., 2007)

ii. Flowers

The plant is monoecious and the terminal inflorescences which are formed in the

leaf axil contain unisexual flowers. The stalk length ranges between 6 to 23 mm.

Flowers are formed terminally, individually, with female flowers usually slightly

larger and occurs in the hot seasons. The ratio of male to female flowers ranges

from 13:1 to 29:1 and decreases with the age of the plant (Achten et al., 2008). In

conditions where continuous growth occurs, an unbalance of pistillate or staminate

flower production results in a higher number of female flowers (CJP, 2008).

iii. Fruits

After pollination, the inflorescences form a bunch of green ellipsoidal fruits. Each

inflorescence yields a bunch of approximately 10 or more fruits. A three, bi-valved

cocci is formed after the seeds mature and the fleshy exocarp dries (Achten et al.,

2008; CJP, 2007).

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