In vitro Propagation Studies and Partial Biochemical ...
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In vitro Propagation Studies and Partial Biochemical Characterization for Drought Stress in Jatropha curcas L. Sadia Basharat Department of Botany University of the Punjab Lahore, Pakistan
Transcript of In vitro Propagation Studies and Partial Biochemical ...
In vitro Propagation Studies and Partial Biochemical
Characterization for Drought Stress in Jatropha
curcas L.
Sadia Basharat
Department of Botany
University of the Punjab
Lahore, Pakistan
In vitro Propagation Studies and Partial Biochemical
Characterization for Drought Stress in Jatropha
curcas L.
A Thesis Submitted to the University of the Punjab in Partial
Fulfillment of the Requirements for the Degree of Doctor of
Philosophy
In Botany
By
Sadia Basharat
Department of Botany
University of the Punjab
Lahore, Pakistan
September, 2018
i
DEDICATED TO
My Family
Who is everything for me
My consolation in sorrow,
My hope in misery,
My strength in weakness,
And
Who sacrificed a lot for me
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CERTIFICATE
This is to certify that the research work entitled “In vitro Propagation Studies and
Partial Biochemical Characterization for Drought Stress in Jatropha curcas L.”
described in this thesis by Ms. Sadia Rizwan is an original work of the author and has
been carried out under my direct supervision. I have personally gone through all the
data, results, materials reported in the manuscript and certify their correctness and
authenticity. I further certify that the material included in this thesis has not been used
in part or full in a manuscript already submitted or in the process of submission in
partial or complete fulfillment of the award of any other degree from any institution. I
also certify that the thesis has been prepared under my supervision according to the
prescribed format and I endorse its evaluation for the award of PhD degree through
the official procedures of the University of the Punjab, Lahore.
________________________________
Supervisor
Prof. Dr. Faheem Aftab
Department of Botany
University of the Punjab, Lahore
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ACKNOWLEDGEMENTS
All praises and glory belongs to Almighty Allah Who had blessed me with
the opportunities and potential to complete this research work and compile this thesis.
I would like to record my sentiments of indebtedness to my respected
supervisor Prof. Dr. Faheem Aftab, Department of Botany, University of the Punjab,
Lahore, for his scholarly guidance, illustration, constructive criticism, keen interest,
cooperation and encouragement which was the real source of inspiration for me
during my research work.
Special thanks are due to Prof. Dr. Firdaus e Bareen, Chairperson,
Department of Botany for providing necessary help during this research work.
I am also grateful to Prof. Dr. Khan Rass Masood and Prof. Dr.
Muhammad Saleem (Ex-Chairmen, Department of Botany), Prof. Dr. Shahida
Husnain (Ex-Chairperson, Doctoral Programme Coordination Committee, University
of the Punjab) and Prof. Dr. Anjum Nasim Sabri (Chairperson, Doctoral Programme
Coordination Committee, University of the Punjab).
I also appreciate and gratefully acknowledge Dr. Humera Afrasiab and Prof.
Dr. Abdul Nasir Khalid (Co-ordinator PhD programme, Department of Botany) for
their valuable guidance throughout the study.
I would like to extend my gratitude to Higher Education Commission (HEC)
for providing financial support during my research work (Indigenous 5000 Fellowship
Program PIN No. 063-121479-Bm3-027).
I am also thankful to all my lab fellows and colleagues; Dr. Neelma Munir,
Dr. Muhammed Akram, Dr. Zahoor Ahmed Sajid, Dr. Adeela Haroon, Farrah,
Shehla, Samina, Madiha and Dr. Arifa Khalid for their cooperation and moral
support.
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I also feel great pleasure to say a lot of thanks to my parents, parents in law,
brothers, sisters and especially my husband Rizwan Hussain and kids Ahmed,
Hamza and Moaaz for their patience, encouragement, love and countless prayers.
Sadia Basharat
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ABSTRACT
Development of rapid and efficient propagation methods for Jatropha curcas
are highly desirable since its seed oil can be used as biofuel and hence of high
economic value around the world. In this study, tissue culture techniques were
employed to resolve conventional propagation issues. In vitro seed germination
experiments in soil and on half or full strength MS medium, specifically during the
dormant periods by using some pretreatments were performed. Pretreatments included
presoaking of seeds in water overnight, scarification, stratification, removal of seed
coats (before/after disinfection) and combination of these treatments. It was observed
that the orientation of the seeds on the culture media also had significant effect on its
germination rate. Disinfection of naked seeds could not support subsequent
germination so the seeds were disinfected before removing the seed coats. It was
observed that the removal of seed coats only could break the dormancy of seeds to get
100% in vitro germination on full strength MS medium kept in the dark at 25 ± 2˚C in
the months of December to January. Such seedlings were shifted in light conditions
(16 h photoperiod) after the root emergence at the same temperature to support
chlorophyll development. Seedlings were successfully acclimatized by shifting to the
soil containing a mixture of peat, clay and silt (1:1:1 v/v) in greenhouse.
Efficient callus-mediated regeneration system was developed using various
explants of Jatropha curcas like young/mature/cotyledonary leaf and hypocotyl.
Different growth regulators including TDZ, Kin, BAP, NAA, IAA, 2, 4-D were
supplemented in MS medium either singly or in combinations of different
concentrations for callus induction and its proliferation. Cultures were kept in either
darkness or 16/8 h photoperiod. It was observed that 22.17 µM BAP + 5.35 µM NAA
supplemented in medium gave 100% embryogenic callus induction with all the
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explants used except mature leaf, when kept in 16/8 h photoperiod. Cultures kept in
complete darkness also give good callus induction frequency (90%) but calluses were
white friable and non-embryogenic. Developed calluses were shifted to shoot bud
induction medium. Shoot bud induction medium was also MS medium supplemented
with different plant growth regulators both auxins and cytokinins (BAP, NAA, GA3,
TDZ, Kin, IBA) in combinations of two or three. Calluses developed on medium
containing 22.17 µM BAP + 5.35 µM NAA, shifted to same combination of growth
regulators have shown maximum number of shoot buds per culture vessel (17).
However, frequency of shoot bud induction was low. Addition of GA3 or Kin in the
medium having BAP and NAA have enhanced the frequency of shoot bud induction.
However, when both GA3 and Kin were used together, they did not show any
significant effect on shoot bud induction frequency. TDZ supplemented in the
medium having BAP and NAA, have shown negative effect on regeneration potential.
Maximum shoot bud induction frequency (37%) was achieved on MS medium with
6.65 µM BAP + 2.45 µM IBA added.
Direct shoot regeneration from young leaf explant of Jatropha currcas was
also achieved on MS medium supplemented with 6.65 µM BAP + 2.45 µM IBA.
Developed and elongated shoots of average 2 cm length were shifted to another
medium for root development. Maximum root induction frequency was achieved on
MS medium supplemented with 4.9 µM IBA. Rooting was not very successful in
recent experiments because of the callus formation at the base of shoots shifted to the
rooting medium.
Effect of water/osmotic stress (synonymously referred as drought stress in
literature) on morphological and biochemical activities of Jatropha curcas plants
were elucidated in the present experiments. The experiments were performed both
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under in vitro (seed germination, early growth of seedlings and callus cultures) and
field conditions (pot-grown 5-month old plants). Different sorbitol treatment levels (0,
0.05, 0.1, 0.15, 0.20, 0.25, 0.3, 0.35, 0.40, 0.45 and 0.5 M) were supplemented to MS
medium in order to increase the osmotic stress for in vitro studies. Five-month-old
greenhouse plants were subjected to different field capacities of water (100, 75, 50, 25
and 0%). Results have shown that increased osmotic stress in the medium resulted in
decreased germination along with its delayed onset. However above 0.3 M sorbitol
concentration, germination process was stopped. Similarly fresh/dry weights and
shoot lengths of germinating seedlings were also influenced significantly with
increase in osmotic stress. Among the biochemical parameters of germinating
seedlings studied, it was observed that there was trend towards significant increase in
SOD and peroxidase activities with an increase in osmotic stress. However, the
soluble protein contents were not affected significantly. Callus cultures were not
influenced physiologically and biochemically with increased osmotic stress however,
higher osmotic stress lead to reduction in fresh weight and water content and slight
enhancement in soluble protein and peroxidase activity. Five-month-old plants
subjected to different field capacities of water for 30 days have not shown any visual
symptoms of stress like necrosis or chlorosis. However, minimum fresh weight per
unit area of leaves was observed in lowest field capacity (0%). Similarly minimum
SOD activity was observed in plants subjected to 50% field capacity and there was
trend towards increase in SOD activity both in lower and higher field capacities.
Peroxidase activities remained unaffected. However, slight increase in soluble protein
contents was observed in 0% field capacity. Hence it can be concluded that
germination and early seedling growth are influenced by drought stress to a great
extent as compared to mature plants where no remarkable changes were observed in
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both physical and biochemical activities except in extreme stress condition. Same was
the case with callus cultures derived from mature leaf explants. Hence Jatropha
curcas plants may be planted in areas of low water availability if irrigated properly at
seed germination stage.
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TABLE OF CONTENT
Page No.
Chapter 1: Introduction ..........................................................................................1
Chapter 2: Review of Literature ............................................................................7
2.1 Propagation of Jatropha curcas ...........................................................................8
2.2 Tissue culture techniques for Jatropha curcas propagation ................................11
2.2.1 In vitro seed germination ......................................................................12
2.2.2 Callus induction and callus-mediated regeneration .............................13
2.2.3 Direct regeneration................................................................................15
2.2.4 Somatic embryogenesis ........................................................................17
2.2.5 Rooting .................................................................................................19
2.3 Abiotic stress tolerance ........................................................................................19
2.3.1 Role of antioxidant enzymes under abiotic stress .................................19
2.3.2 Role of soluble proteins under abiotic stress ........................................22
2.3.3 Drought stress studies on Jatropha curcas ...........................................24
2.3.4 Studies on drought inducing substances ...............................................27
Chapter 3: Methodology..........................................................................................29
3.1 Preparation of Media ...........................................................................................29
3.1.1 Stock Solutions .....................................................................................29
3.1.2 Growth Regulators ................................................................................29
3.1.3 Preparation of Medium using Stocks ....................................................29
3.2 Sterilization ..........................................................................................................30
3.2.1 Sterilization of Glassware .....................................................................30
3.2.2 Sterilization of the Media ......................................................................30
3.2.3 Sterilization of Laminar Airflow Cabinet .............................................30
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3.2.4 Sterilization of Surgical Tools .............................................................30
3.3 Plant Material .......................................................................................................31
3.3.1 Source of Plant Material .......................................................................31
3.3.2 Explant Disinfection ..........................................................................31
3.4 Culture Conditions ...............................................................................................32
3.5 Biochemical Studies.............................................................................................32
3.5.1 Extraction of Soluble Proteins and Enzymes .......................................33
3.5.2 Estimation of Soluble Protein Contents ................................................33
3.5.3 Peroxidase Estimation ...........................................................................34
3.5.4 Superoxide Dismutase Estimation ........................................................35
3.6 Experimental Plan ................................................................................................35
3.6.1 Seed germination ..................................................................................35
3.6.2 Callus induction and its proliferation ....................................................36
3.6.3 Regeneration from callus cultures ........................................................37
3.6.4 Direct regeneration from young leaf explant ........................................37
3.6.5 Effect of various sorbitol concentrations on in vitro seed germination 37
3.6.6 Effect of sorbitol concentrations on callus cultures ..............................38
3.6.7 Effect of different field capacities of water on pot-grown plants .........38
3.7 Statistical Analysis ...............................................................................................39
Chapter 4: Breaking dormancy and in vitro germination of seeds of Jatropha
curcas L. ....................................................................................................................40
Chapter 5: Callus induction, maintenance and in vitro regeneration using
different explants of Jatropha curcas L. .................................................................46
5.1 Standardization of medium for callus induction and maintenance for Jatropha
curcas .........................................................................................................................46
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5.1.1 Callus induction from younger leaf explants ........................................46
5.1.2 Callus induction from older leaf explants .............................................50
5.1.3 Callus induction from cotyledonary leaf explants ................................53
5.1.4 Calluses induced from hypocotyl explants ...........................................56
5.1.5 Comparison of different growth regulators supplemented media and
explant type ....................................................................................................59
5.1.6 Callus induction from leaf explants of Jatropha curcas L. kept in dark.
....................................................................................................................................60
5.2 Standardization of medium for callus mediated regeneration and subsequent
elongation of shoots of Jatropha curcas ...................................................................62
5.3 Standardization of medium for direct regeneration from young leaf explant of
Jatropha curcas .........................................................................................................67
5.4 Rooting of Regenerated Shoots ...........................................................................68
Chapter 6A: Effect of sorbitol induced osmotic stress on seed germination, early
growth of seedlings and callus cultures in Jatropha curcas L. .............................76
6.1 Effect of different sorbitol concentrations on seed germination of Jatropha curcas
L. ................................................................................................................................76
6.2 Effect of different sorbitol concentrations on fresh/dry weight, shoot/root length of
germinating seedlings of Jatropha curcas L. ............................................................78
6.3 Effect of different sorbitol concentrations on protein contents, peroxidase and
superoxide dismutase activities in germinating seedlings of Jatropha curcas L. .....80
6.4 Effect of different sorbitol concentrations on fresh /dry weight and water contents
of Jatropha curcas callus cultures .............................................................................82
6.5 Effect of different sorbitol concentrations on SOD, POX and soluble protein
contents of Jatropha curcas callus cultures ...............................................................84
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Chapter 6B: Effect of different field capacities of water in pot soil on five-month-
old plants of Jatropha curcas L. ..............................................................................92
6.6 Effect of different field capacities of water on morphological features of Jatropha
curcas ........................................................................................................................92
6.7 Effect of different field capacities of water on biochemical activities in leaves of
Jatropha curcas .........................................................................................................94
Conclusion ................................................................................................................99
References .................................................................................................................101
Annexure 1: Formulation of MS Medium (Murashige and Skoog, 1962) for the
Preparation of Stock Solutions...................................................................................132
Annexure 2: Preparation of Stock Solutions for MS (Murashige and Skoog, 1962)
Medium .....................................................................................................................133
Annexure 3: Preparation of Stock Solutions of Growth Regulators .........................135
Annexure 4: Preparation of 1 liter MS Medium .......................................................136
Annexure 5: Composition of Different Media Used for Callus induction/Maintenance
from different explants of Jatropha curcas .................................................................137
Annexure 6: Composition of Different Media Used for Plant Regeneration from
Callus Cultures/ young leaf explants of Jatropha curcas ..........................................138
Annexure 7: Composition of Different Media used for Rooting of Regenerated Shoots
of Jatropha curcas .....................................................................................................139
Annexure 8: Composition of Different MS Media Used in Osmotic Stress
Experiments for Callus Cultures of Jatropha curcas ................................................140
Annexure 9: Composition of different MS Media for In Vitro Germination of
Jatropha curcas Seeds ...............................................................................................141
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Annexure 10: Details of different Treatments Given to Jatropha curcas Seeds for
Germination ...............................................................................................................142
Annexure 11: Composition of different MS Medium to Study the Effect of Osmotic
Stress on In Vitro Seed Germination of Jatropha curcas ..........................................143
Annexure 12: 0.1 M Phosphate Buffer (pH 7.2) for Extraction of Proteins and
Enzymes .....................................................................................................................144
Annexure 13: Composition of Biuret Reagent for Protein Estimation. .....................145
Annexure 14: Reagents for Peroxidase Estimation ...................................................146
Annexure 15: A. Reagents for Superoxide Dismutase Estimation ............................147
Annexure 16: Solutions for sterilization of explants .................................................148
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LIST OF TABLE
Page No.
Table 4.1: Effect of various pretreatments and MS medium strength on in vitro seed
germination of Jatropha curcas L. ............................................................................41
Table 4.2: Effect of orientation of S5 seeds on germination behavior on full strength
MS medium ................................................................................................................41
Table 5.1 Effect of different growth regulators on callus induction from younger leaf
explant of Jatropha curcas. .......................................................................................48
Table 5.2 Effect of different growth regulators on callus induction from older leaf
explant of Jatropha curcas. ......................................................................................51
Table 5.3 Effect of different growth regulators on callus induction from cotyledon ary
leaf explants of Jatropha curcas. ...............................................................................54
Table 5.4 Effect of different growth regulators on callus induction from hypocotyls
explants of Jatropha curcas .......................................................................................57
Table 5.5 Effect of growth regulators added in MS medium for Regeneration from
Callus Cultures of Jatropha curcas ...........................................................................66
Table 5.6 Effect of different growth regulators on root induction in regenerated shoots
of Jatropha curcas .....................................................................................................68
Table 6.1 Effect of different field capacities of water in the pots soil on biochemical
parameters of Jatropha curcas leaves after one month of treatment. ........................94
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LIST OF FIGRUE
Page No.
Fig 4.1 Root induction (arrow) after 2 days from a Jatropha carcus seed sown
dorsally on full strength MS medium (a), after 3 days of sowing (b), root emergence
in ventrally oriented seed (c), Developed shoot after 7-8 days of sowing (d), further
shoot elongation after 15 days of sowing (e), seedling taken out of the culture tube for
acclimatization in glasshouse after 15 days of sowing (f), withdrawal of cotyledonary
leaves and emergence of a new leaf 7-8 days after acclimatization (g). ....................42
Fig. 5.1 Callus induced in MS medium with 9.3 µM Kin .......................................49
Fig. 5.2 Callus induced in MS medium with 22.17 µM BAP + 5.35 µM NAA ........49
Fig. 5.3 Callus induced in MS medium with 2.21 µM BAP + 5.71 µM IAA + 4.52
µM 2, 4-D .................................................................................................................49
Fig.5.4 Callus induced in MS medium with 9.3 µM Kin ........................................52
Fig.5.5 Callus induced in MS medium with 22.17 µM BAP + 5.35 µM NAA.........52
Fig.5.6 Callus induced in MS medium with 2.21 µM BAP + 5.71 µM IAA + 4.52 µM
2, 4-D .........................................................................................................................52
Fig 5.7 Callus induced in MS medium with 22.17 µM BAP + 5.35 µM NAA.........55
Fig 5.8 Callus induced in MS medium with 2.21 µM BAP + 5.71 µM IAA + 4.52 µM
2, 4-D .........................................................................................................................55
Fig.5.9 Callus induced in MS medium with 22.17 µM BAP + 5.35 µM NAA.........58
Fig.5.10 Callus induced in MS medium with 2.21 µM BAP + 5.71 µM IAA + 4.52
µM 2, 4-D ..................................................................................................................58
Fig.5.11 Callus induced in MS medium with 9.3 µM Kin ........................................58
Fig. 5.12 Effect of different growth regulators supplemented in MS medium on callus
induction using different explants ..............................................................................59
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Fig.5.13 Callus from leaf explant when kept in dark conditions ...............................60
Fig 5.14 Seven month old callus culture developed and maintained on the same
medium having 22.17 µM BAP + 5.35 µM NAA. ....................................................61
Fig 5.15 Callus culture developed on MS medium having 22.17 µM BAP + 5.35 µM
NAA after 35 days of sub-culturing on the same medium ........................................61
Fig. 5.16 Shoot bud initiation after 15 days of sub-culturing on R-14 medium .......65
Fig. 5.17 Shoot bud initiation after 15 days of sub-culturing on R-1 medium ..........65
Fig. 5.18 Shoot elongation after 30 days of sub-culturing on R-1 medium ..............65
Fig. 5.19 Shoot elongation after 60 days of sub-culturing on R-1 medium ..............65
Fig. 5.20 Shoot bud initiation (arrows) after 20 days of sub-culturing on R-2
medium ......................................................................................................................65
Fig. 5.21 Shoot elongation (arrows) after 30 days of sub-culturing on R-2 medium 65
Fig. 5.22 Shoot elongation along with shoot primordia (arrows) after 30 days of sub-
culturing on R-15 medium .........................................................................................65
Fig.5.23 Multiple shoot regeneration (arrows) directly from surface of young leaf
explant of Jatropha curcas, bar= 4.3mm ....................................................................67
Fig.5.24 Regenerated shoots shifted to rooting medium ...........................................69
Fig.5.25 Regenerated shoot shifted to rooting medium showing the formation of
callus (arrow) at the base of shoot .............................................................................69
Fig. 6.1 Effect of different sorbitol concentrations on in vitro seed germination of
Jatropha curcas .........................................................................................................77
Fig. 6.2 Effect of different sorbitol concentrations on germination energy of Jatropha
curcas .........................................................................................................................77
Fig. 6.3 Effect of different sorbitol concentrations on shoot/root length of
germinating seedlings of Jatropha curcas .................................................................78
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Fig. 6.4 Effect of different sorbitol concentrations on fresh/dry weight of germinating
seedlings of Jatropha curcas .....................................................................................79
Fig. 6.5 Effect of different Sorbitol concentrations on physical appearance of in vitro
germinating seedlings of Jatropha curcas ................................................................79
Fig. 6.6 (a, b, c) Effect of different sorbitol concentrations on biochemical parameters
of in vitro germinating seedlings ...............................................................................81
Fig. 6.7 (a, b) Effect of different sorbitol concentrations on fresh/dry weight and
water contents of callus cultures derived from Jatropha curcas leaf explants ..........83
Fig. 6.8 (a, b, c) Effect of different sorbitol concentrations on SOD/POX activities
and protein contents of Jatropha curcas callus cultures ............................................85
Fig. 6.9 Effect of different field capacities of water in pot soil (after one month) on
physical appearance of 5 month old plants of Jatropha curcas ......................................92
Fig. 6.10 Effect of different field capacities of water in the pot soil om fresh/dry
weight and water contents per unit area of Jatropha curcas leaves after one month of
treatment ....................................................................................................................93
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LIST OF ABBREVIATIONS
% Percent
≤ Less than or equal to
µM Micro molar
µmol m-2 s-1 Micromole per meter square per second
½ MS Half strength Murashige and Skoog (1962) basal medium
2, 4-D 2, 4- dichloro phenoxy acetic acid
AFLP Amplified Fragment length Polymorphism
APX Ascorbate peroxidase
BA/BAP 6-Benzyladenine/ aminopurine
BADH Betaine Aldehyde Dehydrogenase
CAT Catalase
cm Centimeters
Conc. Concentration
CPPU Forchlorfenuron
CuSO4 Copper sulphate
Dist.H2O Distilled Water
DK Dikegulac
FFr Florescence on far red
Fr Florescence on red
g Gram
GA3 Gibberellic acid
GR Glutathione Reductase
h Hour
H2O Water
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H2O2 Hydrogen per oxide
HCl Hydrochloric acid
HgCl2 Mercuric chloride
hrs Hours
IAA Indol acetic acid
IBA Indol butyric acid
IEA International Energy Agency
ISSR Inter Simple Sequence Repeats
KN Kinetin
L. Linnaeus
lbs inch-2 Pounds per square inches
Lux Unit of illuminance
M Molar
m Meters
mb/d Millions of barrels per day
MDA Malondialdehyde
mg l-1 Milligram per liter
mg/g protein/ min Milligram per gram protein per minute
mg/l Milligram per liter
mgl-1 Milligram per liter
MH Maleic hydrazide
min. Minutes
MIPs Major Intrinsic Proteins
ml Milliliter
ml/l Milliliter per liter
xx
mM Milli molar
MS Murashige and Skoog (1962) Basal medium
N Normal
Na2HPO4 Disodium hydrogen phosphate
NAA Naphthalene acetic acid
NaCl Sodium chloride
NaClO Sodium hypochlorite
NaOH Sodium hydrooxide
NBT Nitroblue tetrazolium
N-fertilizers Nitrogen-fertilizers
nm Nanometer
ºC Degree Celsius
PAL Phenylalanine ammonia lyase
Pb Lead
PEG Polyethylene glycol
PGRs Plant Growth Regulators
pH Power of Hydrogen ion concentration
PIP Plasma Membrane Intrinsic Proteins
pM Pico Molar
POX Peroxidase
PVP Polyvinyl Polypyrrolidone
RAPD Random Amplification of Polymorphic DNA
ROS Reactive Oxygen Species
rpm Revolutions per minute
SIP Small Basic Intrinsic Proteins
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SOD Superoxide dismutase
SPSS Statistical Package for Social Sciences
TDZ Thidiazuron
TIBA 2, 3, 5-triiodobenzoic acid
TIP Tonoplast Intrinsic Proteins
UV Ultra violet
v/v Volume per Volume
W Watt
w/v Weight per Volume
1
Introduction
Worldwide energy demand in all life forms is rapidly growing. There is 2%
increase in energy demand every year from last three decads. According to IEA-2017
world oil demand has increased from 91.2 mb/d in 2013 to 96.54 mb/d in 2017. Fossil
fuel is basically the primary source of energy but being non-renewable it is depleting
with the passage of time. According to some workers approximate estimated time for
depletion of oil, coal and gas are 2044, 2112 and 2144 respectively (Shafiee and
Topal, 2009). If this situation continues to happen as such then it will not only affect
the economic growth of industries directly but also affect the basic necessities of
common people. So, renewable energy resources are considered as better options to
meet an ever-increasing energy demand. Biofuels are gaining more and more attention
in this regard as they are not only renewable but also eco-friendly. Many edible
vegetable oils and animal fats have been used as biofuel such as rapeseed oil,
soybean, sunflower, coconut, palm etc (Demirba, 2003; Meher et al., 2006) but value
of these oils as food purposes is also high. Also their use as biofuel is more expensive
(Demirba, 2003). Hence non-edible oil plants are gaining more attention as they do
not compete with food crops. Jatropha curcas L. (physic nut) is one such plant that
contains variable amount of non-edible oil (containing toxic agents like curcin and
phorbol esters) in their seeds. Its seed oil and press cake is considered as potential
source of biofuel (Martínez-Herrera et al., 2006; Kamel et al., 2018) and its chemical
specifications also match with international biodiesel standards (Azam et al., 2005).
Due to such properties of its seed oil, Rashid et al., (2010) also recommended its
cultivation in Pakistan on large scale for biodiesel production. Another advantage of
Jatropha curcas is that it can be cultivated on waste lands after a little amendment
2
(Juwarkar et al., 2008). Thus the possibility of land use in competition with food
plants is quite low.
Jatropha curcas L. belongs to Euphorbiaceae family. It is a valuable
multipurpose deciduous shrub. Mature plants can obtain the height of 5m. It is an eco-
friendly plant as its plantation on waste land can reduce soil erosion and make it
fertile. Its plantation also proved helpful in improvement of soil heath in terms of
microbial and biochemical properties (Mahmoud et al., 2018). It can solve energy
shortage problems, reduce carbon emission and can increase the income of farmers
(Banerji et al., 1985; Martin and Mayeux, 1985; Gubitz et al., 1999; Keith, 2000;
Zhou et al., 2006). Jatropha curcas is not only a source of biofuel but also provides
many other benefits to the farmers. Like, if the toxins are removed then Jatropha
curcas could also serve as a very nutritious protein source for animals (Becker and
Makkar, 1998). Other parts of the plant including press cake after oil extraction have
many other medicinal and industrial applications. This press cake is rich in important
components like nitrogen, phosphorous and potassium. Hence can be used as a
manure.
Jatropha curcas is originated basically from tropical America, but today is
widely distributed throughout the world (Cano-Asseleih et al., 1989). Jatropha curcas
thrives well on gravelly, sandy or saline soils. Regarding climate, Jatropha curcas is
thermophilic mean it likes high temperatures. However, it can also withstand in lower
temperatures and even a light frost. Water requirements of Jatropha curcas are also
considered to be low and it was reported that it can withstand prolonged drought
conditions by shedding the leaves to reduce the loss of water through transpiration.
Propagation of Jatropha curcas is gaining serious attention all over the world
since last 10-15 years. It was cultivated on thousands of hectares of land in many
3
regions of China, India and other tropical and subtropical countries (Bueso et al.,
2016; Freitas et al., 2016; Montes and Melchinger, 2016). However, the profits earned
by the farmers are less than expectations because of the lower seed production and
lacking other high value-added products (Tian et al., 2017). Actually Jatropha curcas
is not a self-propagating plant. It is propagated normally by seeds or through
vegetative cuttings. Problems associated with plants propagated by stem cuttings are
lower tolerance to stresses as compared to seed propagated plants. At the same time,
in vegetatively-propagated plants seed set has also been reported to be lower (Sujatha
et al., 2005). Problems associated with Jatropha curcas propagation through seeds are
also many like poor viability of its seeds, variable germination rate, and very little and
delayed rooting (Openshaw, 2000). Variation in seed germination ranging from 10-
90% may be due to different cultural practices or difference in genotype, cultivar,
location etc (Islam et al., 2009; Ayadi et al., 2011). Considering all pieces of
evidence, it becomes quite apparent that a large quantity of better propagating
material of Jatropha curcas is essential to meet demands for its mass plantation in the
future. Tissue culture techniques can be applied to solve this problem. At the same
time Wang et al., (2011) suggested that callus induction and micropropagation
protocols should be standardized for each plant species to improve the plants through
various technologies including genetic transformation. A comparison of tissue-
culture-propagated plants and seed-propagated plants of Jatropha curcas in terms of
their yield shown that there is no remarkable difference between the two (Sujatha and
Dhingra, 1993; Sujatha and Mukta, 1996).
Efficient regeneration system using various explants of Jatropha curcas like
stem, nodal segments, shoot tips, epicotyl, hypocotyl and leaf have been worked out
by many researchers (Singh et al., 2010; Misra et al., 2010; Datta et al., 2007; Rajore
4
and Batra, 2005; Sujatha et al., 2005; Qin et al., 2004). Somatic embryogenesis in
Jatropha curcas have also been reported by Jha et al., (2007). Reproducibility of
these protocols for enhanced propagation of Jatropha curcas however is limited.
Mostly plants have to undergo various stress conditions that cause
considerable amount of reduction in growth and development. Some Plants have
ability to cope with stresses by certain changes in their physiological and
developmental processes (Kazuo and Kazuko, 1996). Proline accumulation, for
instance, is reported in certain plants under drought stress (Choudhary et al., 2005).
Similarly seed germination in Jatropha was shown to be reduced under various
abiotic stresses (Shakirova and Sahabutdinove, 2003). Soil water shortage or drought
stress (also synonymously referred to as osmotic stress in the literature) can also
reduce the plant growth and yield (Boyer, 1982). It is a general concept that water
requirement of Jatropha curcas is very low. However, there are contrasting views
about drought tolerance ability of Jatropha curcas. Some workers considered it as a
drought and salinity tolerant plant that have ability to grow in areas with limited
availability of water without any significant effect on its growth and physiology
(Maes et al., 2009; Silva et al., 2010; Sapeta et al., 2013; Hishida et al., 2014; Kheira
and Atta, 2009). Others reported a remarkable influence on the yield of Jatropha
curcas with fluctuations in available amount of water (Rao et al., 2012; Singh and
Saxena, 2010; Phiwngam et al., 2016).
Responses of plants to various stresses are hence usually determined by
certain biochemical characteristics like protein, proline contents and some stress-
related antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POX)
etc. Enhanced activity of JcNAC1 (a ribosome inactivating protein) responsible for
stress tolerance in Jatropha curcas was reported by Qin et al., (2014). Previously
5
some stress proteins produced as a result of stress applied to plants are also reported
by some workers in Jatropha curcas (Zhang et al., 2007; Qin et al., 2005). Later,
Lama et al., (2016) reported that drought causes oxidative stress in Jatropha curcas.
However, information regarding the extent of drought tolerance in quantitative terms
is limited. Drought tolerance ability of Jatropha curcas could be estimated by
employing biochemical characterization.
Most of the experiments for studying drought tolerance ability were performed
in green house conditions with controlled watering to develop water deficit conditions
(Gimeno et al., 2012; Niu et al., 2012; Santos et al., 2013 etc). To the best of our
knowledge, studies on early seedlings of Jatropha curcas under osmotic stress in in
vitro conditions are limited. Different osmotic agents like Sorbitol, Manitol and
Polyethylene glycol (PEG) have already been used by various workers to induce
osmotic stress in in vitro culture media (Almansouri et al., 2001; Frank et al., 2005;
Wang et al., 2011a). Polyethylene glycol (PEG) has mostly been used in such studies
on Jatropha curcas (Qin et al., 2005; Silva et al., 2010; Wang et al., 2011a; Wang et
al., 2011b, Qin et al., 2014). However, reports on Manitol and Sorbitol being used as
a drought inducing substance for Jatropha curcas are scanty. Sorbitol is metabolically
more inert than other saccharides and is considered as a non-metabolite (Lambers et
al., 1981). Addition of sorbitol in medium resulted in decreased water potential thus
inducing osmotic stress (Abu-Romman, 2010). In a similar manner, response of
callus cultures and/or whole plants to various abiotic stresses have also been studied
and compared for many plant species (Smith and McComb, 1981; Rus et al., 1999;
Wang et al., 1999; Al-kaaby and Abdul-Qadir, 2011). There is hardly any such study
involving Jatropha curcas though its growth on marginalized, water deficit soil is
reported.
6
On the bases of above information, the need was felt to initiate work in order
to explore and standardize in vitro propagation in Jatropha curcas. The present work
hence was aimed at standardization of in vitro approaches involving in vitro seed
germination, callus induction, callus-mediated regeneration and direct regeneration
from various explants for enhanced propagation of elite germplasm of Jatropha
curcas. At the same time water stress tolerance ability of Jatropha curcas was also
planned to be estimated by employing partial biochemical characterization involving
soluble protein contents and activities of SOD and POX. Different sorbitol
concentrations were used in the present study to create an osmotic stress in the culture
medium. The experiments were performed both at the tissue level using callus
cultures and at whole plant level under in vitro and pot conditions. At the same time
effect of water stress on responses of callus cultures and whole plants were also
compared. This information will be helpful for providing quality planting material and
at the same time will enable us not only to better understand general mechanism for
drought tolerance in Jatropha curcas but may also be of applied significance in future
endeavors involving Jatropha curcas propagation at mass scale on marginal lands.
.
7
Review of Literature
Jatropha curcas L. is considered as a beneficial energy crop throughout the
world because of its seed oil and press cake being used as biofuel. This biofuel is eco-
friendly and economic. In conventional agriculture practices, seeds and cuttings of
Jatropha curcas are mostly used for its plantation. However, the seeds are
heterozygous with lower germination rates. Similarly the cuttings are seasonal and do
not form true taproot system (Sujatha et al., 2005). Hence tissue culture techniques
can be applied to get continuous supply of good quality Jatropha curcas planting
material. It is recalcitrant to tissue culture techniques because of the latex-producing
nature of this plant (Sardana et al., 1998; Shrivastava and Banerjee, 2008). Endopytic
bacterial contaminations are also great issues in Jatropha curcas tissue culture. Misra
et al., (2010) and Toppo et al., (2012) described the use of antibiotics (Augmentin) in
the culture medium to resolve that issue. In vitro regeneration and further
multiplication of shoots using various explants of Jatropha curcas with a variety of
plant growth regulators have been worked out by several researchers (Misra et al.,
2010; Purkayastha et al., 2010; Kumar et al., 2011; Biradar et al., 2012; Maharana et
al., 2012). Further research work is necessary to enhance the regeneration potential,
rooting and acclimatization.
Tissue culture techniques can also be applied for crop improvement through
advance technologies like genetic transformation. Previously Agrobacterium-
mediated gene transfer protocols were reported by several workers in Jatropha curcas
(Li et al., 2008; Kumar et al., 2010; Mazumdar et al., 2010 and Pan et al., 2010).
Gene transfer through particle bombardments have also been reported by some
workers in Jatropha curcas (Purkayastha et al., 2010; Joshi et al., 2011). These
techniques are useful for the production of improved varieties of Jatropha curcas.
8
However, genetic diversity amongst the Jatropha curcas species was also assessed by
using marker techniques like RAPD, AFLP and ISSR markers by several workers
(Basha and Sujatha, 2007; Tatikonda et al., 2009; Cai et al., 2010 and Tanya et al.,
2011 etc). Recently, Xia et al., (2018) have constructed genetic linkage map that
would be proved helpful for enhancing fruit yield and in turn seed yield of Jatropha
curcas.
Soil water shortage or water stress (synonymously referred to as osmotic stress
in the literature) can also reduce the plant growth and yield (Boyer, 1982). However,
there are contrasting views about drought tolerance level of Jatropha curcas. Some
workers considered it to be a drought and salinity tolerant plant capable of growing in
areas with limited availability of water without any significant effect on its growth
and physiology (Maes et al., 2009; Silva et al., 2010; Sapeta et al., 2013; Hishida et
al., 2014). Others reported a great influence on the yield of Jatropha curcas with
fluctuations in available amount of water (Singh and Saxena 2010; Rao et al., 2012;
Phiwngam et al., 2016). Still more information is needed to explore water use
efficiency of this crop. Responses of different stages of life cycle of Jatropha curcas
plant to drought stress are also unexplored. A brief overview of work related to
conventional propagation of Jatropha curcas, propagation through tissue culture
techniques, effect of drought stress etc have been cited below:
2.1 Propagation of Jatropha curcas
Jatropha curcas propagation is of high value around the world. It is not a self-
propagating plant species. Conventionally, it is propagated by seeds, roots or stem
cuttings. Vegetatively propagated plants by stem cuttings show lower longevity, lower
resistance to stresses and poor seed set (Heller, 1996; Sujatha et al., 2006). Some
9
problems associated with Jatropha curcas’s propagation by seeds are poor viability of
seeds and lower germination rates (Openshaw, 2000).
Poor germination of seeds is due to water impermeable testa that exerts
physical exogenous dormancy in seeds (Holmes et al., 1987). Jatropha curcas seeds
germinate best only in the month of October or in March-April and during rest of the
year are usually dormant. Being an oil crop, it cannot be stored for long as it loses up
to 50 percent of its viability upon 15 months storage (Kobilke, 1989). Maturity of
capsule also affected germination and vigor of seedling in Jatropha curcas as reported
by Kaushik, (2003). He also observed that maximum percent germination was
achieved from seeds harvested at maturity and sown at 30˚ C but contrary to the
previous reports he suggested that the storage period did not affect the % germination
and seedling vigour. Seed source used for propagation of Jatropha curcas not only
affected the growth performance (Ginwal et al., 2004) but also affected morphology
of seeds and germination percentage (Ginwal et al., 2005).
Jatropha curcas seeds are usually unpredictable in terms of germination that
varies from 10-95 % (Niranjan et al., 2010). Different pretreatment techniques were
applied to halt seed dormancy and to increase rate of germination. Some
pretreatments include presoaking of seeds in water and some include certain
phytohormones or chemicals. Kureel, (2006) revealed that soaking of Jatropha curcas
seeds in GA3 solutions for 24 h have speeded up the germination process. Similarly
Kumari et al., (2011) also used pre-soaking of seeds in GA3 (100 ppm) for that
purpose and attained 67.38% germination. Soaking Jatropha curcas seeds in di-
sodium hydrogen orthophosphate (Na2HPO4) for six hours had also improved
germination rate and seedling vigour (Srimathi and Paramathma, 2006). Later, some
workers reported that seeds soaked in water showed the maximum germination % as
10
well as survival rate (Feike et al., 2008). Influence of plant growth hormones and seed
soaking time individually or in combination on seed germination have also been
reported (Idu et al., 2007; Koorneef et al., 2007; Pascual et al., 2009). Islam et al.,
(2009) also reported that pre-sowing seed treatments have potential to enhance
germination in Jatropha curcas. .
There are also some reports available on vegetative propagation from stem
cuttings of Jatropha curcas in different areas of the world (Kaushik 2003; Kochhar et
al., 2008). One year old plants can be used for vegetative propagation through stem
cuttings (Jones and Miller, 1991). Some workers suggested the pretreatments of stem
cuttings and foliar applications with plant growth regulators for enhanced growth.
Like, Kochhar et al., (2008) reported that rooting and sprouting of buds on stem
cuttings of Jatropha curcas were increased by pretreatment with auxins and further
investigated that indol-3-butyric acid (IBA) showed better results as compared to
naphthalene acetic acid (NAA). Effect of foliar application of PGRs for promoting
branching has been reported by Abdelgadir et al., (2009). According to them, PGRs
showed better result in promoting branching as compared to manual pruning.
Dikegulac (DK) 2.0 mM was the best treatment in this regard followed by 2,3,5-
triiodobenzoic acid (TIBA) 1.0 mM, BA 12 mM and Maleic hydrazide (MH) 3.0
mM respectively. Later they also suggested that PGRs application on Jatropha curcas
flowers had greatly improved the quality of fruits, enhanced the production of seeds
and oil contents (Abdelgadir et al., 2010). Some other workers (Joshi et al., 2011)
also studied the effect of PGRs (ethrel, IAA, NAA) on Jatropha curcas. They
reported that the higher concentrations of ethrel and IAA had significantly enhanced
the production of fruits and seeds. Fertilizers, especially N-fertilizers were also
11
proved beneficial for better growth and enhanced yield of Jatropha curcas plants
(Mohapatra and Panda, 2011).
Numbers of seeds produced per plant of Jatropha curcas are not sufficient for
biofuel industry. Exogenous application of benzyladenine (160 mg/l) on
inflorescences of Jatropha curcas plants has significantly increased the seed yield of
this plant (Pan and Xu, 2011). They further observed that there was an increase in
female to male ratio of flowers and at the same time total number of flowers. That in
turn resulted in increased number of fruits and at the end total number of seeds.
Similarly, Froschle et al., (2017) also reported that exogenous application of BA at
early flowering stage resulted in increased number of flowers however, application of
forchlorfenuron (CPPU) have increased the ratio of female-to-male flowers.
Jha and Saraf, (2012) worked on the influence of plant growth promoting
bacteria on Jatropha curcas. They reported that all the four bacterial strains used in
their experiments had significantly enhanced the growth parameters and further the
effect was more pronounced when three of them were applied together.
Recently Singh and Agarwal, (2017) suggested a novel method of
micrografting for Jatropha curcas propagation by treating scion and rootstock with
0.05 mg/l BAP and 0.05 mg/l zeatin. This method had not only modified the
architecture of plant (making it dwarf) and also enhanced the crop yield and abiotic
stress tolerance ability of the plants.
2.2 Tissue culture techniques for Jatropha curcas propagation
Tissue culture techniques are mostly used for plants that are difficult to
propagate conventionally, require longer time for propagation or give poor yield.
Jatropha curcas is one of such plants. These techniques can offer continuous supply
of the quality plant material for rapid propagation (Thepsamran et al., 2006). Sujatha
12
et al., (2005) revealed that there is no significant difference in in vitro developed
plants and seed propagated plants of Jatropha curcas in terms of their yield.
However, these techniques produce pathogen free plants. Wang et al., (2011)
suggested that callus induction and micropropagation protocols should be
standardized for each plant species to improve the plants through various technologies
including genetic transformation. Protocols for better shoot regeneration using
different explants of Jatropha curcas with different growth regulators have been
reported by several workers (Sujatha and Dhingra, 1993; Sujatha and Mukta, 1996;
Sujatha and Reddy, 2000; Sujatha and Prabakaran, 2003; Li et al., 2012; Soong et al.,
2016 etc).
2.2.1 In vitro seed germination
Problems associated with Jatropha curcas propagation through seeds are also
many such as poor viability of its seed, lower germination rates, scanty and delayed
rooting of its seedlings (Heller, 1996; Openshaw, 2000). There are few reports on in
vitro seed germination of Jatropha curcas. Qin et al., (2004) used uncoated seeds for
in vitro germination on MS basal medium. Sterilization of seeds was done with 70%
ethanol for 1 min and with 0.15% HgCl2 for 25 min. Thorough rinsing was done with
sterile distilled water after every step. Embryos were extracted carefully in sterile
environment with scalpel and placed in culture tubes with radicles in contact with the
medium.
Pre-soaked Jatropha curcas seeds for four hours in autoclaved distilled water
were sterilized for 3 minutes with 0.1% HgCl2. After that seed coats were removed in
aseptic conditions and cultured on 0.9% plain agar medium and kept in dark at
27±2ºC for radical emergence (2 days) and then transferred to light (2000 Lux 16/8
hrs photoperiod). This protocol was reported by Soomro and Memon, (2007).
13
However Deore and Johnson, (2008) followed another protocol where they removed
the seed coat before sterilization procedures. Decoated seeds were soaked in distilled
water for 48 hours and sterilized with 1% Bavistine for 30 minutes, followed by 0.1%
HgCl2 for 6 minutes. Rinsing with sterile distilled water was done after every step.
Cotyledons were excised carefully and placed on MS medium. Cultures were kept at
26± 2ºC under16 h photoperiod. Li et al., (2008) also followed almost the same
protocol except that they used 70% ethanol for 30 s instead of Bavistine and ½ MS
medium for culturing.
Li et al., (2012) followed a method in which they did not remove the seed
coats for germination. They surface-sterilized the Jatropha curcas seeds in 1% CuSO4
for 15 minutes. After thorough rinsing, seeds were soaked in distilled water for 24 hrs.
That seeds were sown in trays with covers containing six layers of wet filter papers
and placed for germination in dark controlled environment at 26°C for 8 days.
Shrivastava and Banerjee, (2009) developed a unique protocol for in vitro
germination of embryo in Jatropha curcas. In place of chemical hormones, they used
cyanobacterial (Aulosira fertilissima) culture filtrate for in vitro propagation.
Cynobacterial filterate was obtained by centrifugation of 2-month old cynobacterial
cultures and 10 ml/l of that filtrate added in MS medium showed best results for
germination of embryos. They also reported that this filtrate can induce shoot and root
in comparatively lesser time as to chemical hormones.
2.2.2 Callus induction and callus-mediated regeneration
Callus is an unorganized mass of proliferating parenchyma cells.
Callus induction and its proliferation are very important not only for further
regeneration but also for gene transformation procedures. It has also gained
14
importance in many plant species for studying various physiological phenomenon
including resistance against abiotic stresses.
A wide variety of explants, plant growth regulators and culture conditions are
reported for callus induction and its proliferation in Jatropha curcas. Like, Monacelli
et al., (1995) revealed that callus was induced from hypocotyl explants of Jatropha
curcas when inoculated on MS basal medium with 0.5 mg/l 2, 4-D supplemented in it.
They further reported that these calluses have faster growth rate during the first seven
to thirty days and after that maintain their constant growth rate.
A combination of auxins and cytokinines was suggested for callus induction
by many workers. Like, 1 mg/l NAA (auxin) and 5 mg/l BAP (cytokinine)
supplemented in MS medium were proved best for organogenic callus induction when
Jatropha curcas leaf was used as explant (Rajore and Batra, 2007). However, at the
same time, they suggested BAP (1.5 mg/l) and IBA (0.5 mg/l) fortified in MS
medium as the best combination for shoot regeneration from those induced
organogenic calluses. Previously Qin et al., (2004) also suggested that epicotyls
explants of Jatropha curcas inoculated on MS basal medium supplemented with
different IBA and BA concentrations were developed into organogenic callus which
by further sub-culturing on the same medium produced shoots. Similarly Sharma et
al., (2006) also revealed that embryo inoculated on MS medium with NAA (1.25
mg/l) and zeatin (0.06 mg/l) were developed into healthy, green and friable calluses.
Those calluses were developed into shoot buds by further sub-culturing on NAA (0.5
mg/l) and zeatin (0.06 mg/l) added medium. They further observed that shoot bud
elongation was done by sub-culturing on the same medium. Later, Mazumdar et al.,
(2010) while working on the same line also reported callus mediated regeneration
from cotyledonary leaf explant of Jatropha curcas. According to them calluses were
15
induced on MS medium with 6.66 µM BAP + 0.24 µM IBA. They further revealed
the addition of 1.44 µM GA3 in the same medium for multiple shoot induction from
calluses. At the same time they also reported that younger the explants, higher the
callus induction and regeneration frequency.
Similarly, indirect shoot organogenesis in Jatropha curcas was obtained by
inoculation of explants in the medium fortified with 0.5 mg/l BA and 1.0 mg/l each of
2, 4-D and IAA (Misra et al., 2010). Later, Khemkladngoen et al., (2011) while
working on callus-mediated regeneration from young cotyledonary leaf of Jatropha
curcas revealed that developed micro shoots were further elongated on medium
having only cytokinin (2 mg/l BA) as it continued to form callus in combination with
auxin (IBA). Li et al., (2012) suggested the addition of 1 mg/l NAA + 0.1 mg/l Kin in
MS medium for callus induction when epicotyls, hypocotyls, petioles and cotyledons
of 8 days old Jatropha curcas seedlings were used as explants. For good regeneration
from those calluses they proved a combination of TDZ (1 mg/l) and IBA (0.1 mg/l) as
best. However, at the same time BA (8 µM) and IBA (2 µM) fortified medium was
also reported as the best for callus mediated regeneration and elongation of shoots
(Maharana et al., 2012). Similarly, 6 mg/l BAP with 3 mg/l IAA in MS medium was
suggested as best medium for callus induction when apical shoot tips and axillary
leaves of Jatropha curcas were used as explants (Biradar et al., 2012). Along with
this they also reported that quantity of callus was increased on further subcultures
with addition of ascorbic acid and charcoal in the medium. Recently, Soares et al.,
(2016) also used cotyledonary leaf explants for callus-mediated regeneration on MS
medium with 1.5 mg/l BAP and 0.05 mg/l of IBA added in it.
2.2.3 Direct regeneration
Sometimes shoots were regenerated directly from the surfaces of explants.
This process is called direct regeneration and was observed in many plant species
16
including Jatropha curcas. Epicotyl explants of Jatropha curcas inoculated on MS
medium with IBA 0.1 mg/l and BA 0.5 mg/l were regenerated directly into
adventitious shoot buds (Qin et al., 2004).
Nodal segments of Jatropha curcas inoculated on MS medium with addition
of different growth regulators in it (1.5 mg/l BAP, 0.5 mg/l Kin and 0.1 mg/l IAA)
resulted in direct shoot induction as revealed by Kalimuthu et al., (2007). Later,
Shrivastava and Banerjee, (2008) studied the influence of additives like adenine
sulphate, glutamine, L-arginine and citric acid for micropropagation of Jatropha
curcas. They reported that a single initial nodal explant could generate 100 shoots
within a period of three subcultures.
Direct shoot organogenesis was achieved from 2nd to 3rd node Jatropha curcas
leaf explants when incubated in MS medium fortified with BA and IBA. Maximum
shoot buds/explant (7) were differentiated within 6 weeks of initial culture incubation
(Misra et al., 2010). At the same time addition of 0.90 µM TDZ along with 0.98 µM
IBA in MS medium for direct shoot bud induction from leaf explants of Jatropha
curcas was reported by Khurana-Kaul et al., (2010). At the same time they also
suggested that an increase in CuSO4 concentration 10 times as compared to normal
MS level significantly enhanced the shoot bud induction. Stem explants inoculated on
MS medium with 1.0 mg/l BAP and 1.0 mg/l Kin were also developed directly into
10-15 shoot buds per explants (Singh et al., 2010).
Microshoots were also developed directly from cotyledonary leaves of
Jatropha curcas by Kumar et al., (2010). Horizontal placement of explants on MS
medium fortified with 2.27 µM TDZ showed maximum shoot bud induction
frequency per explant. Kumar and Reddy, (2010) followed the same protocol with
17
different explants. They used petiol either from in vitro or field plants and reported
that in vitro petiole explants showed better results.
MS medium supplemented with different concentrations of BAP, Kin and IAA
was proved good for direct shoot induction from apical shoots (Biradar et al., 2012).
They also suggested that NAA did not show any promising effect on shoot
proliferation as compared to other auxins.
Nodal segments were proved better for direct regeneration as compared to leaf
explants (Maharana et al., 2012). Like previous workers they also suggested BAP and
Kin fortified medium for direct shoot induction.
Along with growth regulators, role of some other components was also studied
by some researchers. Like, The addition of coconut water (15 mg/l) from green
coconuts in the MS medium supplemented with L-glutamine (15 mg/l), L-arginine (15
mg/l), Augmentin (50 mg/l) along with BA (0.5 mg/l), IBA (0.1 mg/l) and adenine
sulphate (10 mg/l) produced healthy shoots from nodal explants (Toppo et al., 2012).
Similarly the role of nickel concentrations in culture medium on regeneration
potential of Jatropha curcas leaf explants was studied by Sarkar et al., (2010). They
suggested that lower nickel concentrations in the culture medium (≤ 0.01 mM)
stimulate growth and regeneration from leaf explants but higher concentrations
showed negative effect on the regeneration potential.
Later, Soong et al., (2016) also investigated that the addition of 50 µM ADS
(adenine sulphate) in MS medium along with 15 µM TDZ and 0.5 µM IBA was the
best combination for shoot bud induction from petiole and leaf explants of Jatropha
curcas.
2.2.4 Somatic embryogenesis
Somatic embryogenesis is the production of embryos from somatic plant cells.
It is also a well-known process for micropropagation. An efficient method for somatic
18
embryo development in Jatropha curcas and their regeneration have been proposed
by Sardana et al., (2000). They reported a 2-stage method using leaf explants. Stage 1
was induction of embryogenic callus using MS-Gamborg medium (Gamborg et al.,
1968) with MS basal salts + Gamborg’s vitamins, fortified with BA (3 mg/l) and IAA
(3 mg/l). Stage 2 was induction of plantlet on MS medium containing GA3 (3 mg/l)
and IAA (1 mg/l). These plants after transfer to soil grew well and showed 100%
survival rate.
Somatic embryogenesis in Jatropha curcas was also investigated by Jha and
his colleagues (2007). They reported the use of leaf as an explant on MS basal
medium having 2.3 µM Kin, 1.0 µM IBA and an additive (13.6 µM adenine sulphate)
for embryogenic callus induction. Matured somatic embryos were developed to
plantlets on ½ MS medium. Those plantlets showed very good survival rate (90%)
when shifted to field conditions.
Kalimuthu et al., (2007) also suggested that somatic embryos could be directly
induced using green cotyledonary leaf explants on MS medium fortified with BAP (2
mg/l). Somatic Embryos were also induced on MS medium containing 0.8 mg/l
dicamba from cotyledon explants (Siang et al., 2012). Somatic embryos were
regenerated by shifting the callus to WPM medium fortified with 0.3 mg/l BAP and
0.4 mg/l GA3. They also reported that maturation of somatic embryos prior to
regeneration was not preferable.
Saxena et al., (2012) also suggested cotyledonary leaf explants for
embryogenic callus induction. MS medium with IAA (0.2 mg/l) was suggested as the
best for that purpose. Somatic embryos were further developed into plantlets on MS
medium containing 1.5 mg/l BAP along with 0.2 mg/l IAA. Developed somatic
19
embryos were germinated to produce shoot and root primordia and ultimately into
complete plantlets.
2.2.5 Rooting
In vitro root development on regenerated micoshoots is of great value for
acclimatization and further successful propagation of planlets in soil conditions.
Several efforts were made for this purpose in case of Jatropha curcas. Microshoots
could be rooted well on MS medium (Qin et al., 2004), MS medium with 3 mg/l IBA
(Rajore and Batra, 2007), 1.0 mg/l IAA (Kalimuthu et al., 2007), with 0.1 mg/l IBA in
MS medium (Singh et al., 2010), 0.5 mg/l IBA + 342 mg/l trehalose (Varshney and
Johnson, 2010), ½ MS with 0.2 mg/l IBA (Khemkladngoen et al., 2011) or on ½ MS
medium with 0.1 mg/l IBA (Li et al., 2012). But the rooting percentage is low in
Jatropha curcas (Datta et al., 2007). Further research work is required to improve the
rooting process in Jatropha curcas.
2.3 Abiotic stress tolerance
Abiotic stress means harsh environmental situations like salinity, extreme
temperature, drought, heavy metal toxicity and as a result oxidative damage that limits
plant growth and developmental process. Understanding plant responses to these
stresses is of great importance to develop improved varieties that have the ability to
tolerate them (Yoshida, 2002). Although Jatropha curcas is considered as tolerant
crop to adverse environmental conditions (Juwarkar et al., 2008) but due to its low
production on marginal and sub-marginal lands, efforts were made by several workers
to improve its tolerance ability (Johnson et al., 2011).
2.3.1 Role of antioxidant enzymes under abiotic stress
During abiotic stress, disruption of cellular homeostasis is accompanied by the
generation of reactive oxygen species (ROS), and the extent of stress-induced damage
20
can be attenuated by the action of the cell’s antioxidant systems (Zhang et al., 2010).
Antioxidant enzymes play important roles in scavenging the overproduction of ROS
in plants exposed to water stress (Tanaka et al., 1990; Kubo et al., 1999). These
antioxidant enzymes include superoxide dismutase (SOD), peroxidase (POX),
ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) etc
(Mittler, 2002). Activities of these enzymes correlated with drought tolerance ability
of plants were also observed by Turkan et al., (2005). SOD is an important
antioxidant enzyme which has the ability to overcome oxidative damage caused by
the reactive oxygen species (ROS). Peroxidase (POD) is involved in different
processes like metabolism of growth regulators, lignification and abiotic stress
tolerance. Similarly catalase (CAT) is also an effective antioxidant enzyme, which
degrades the hydrogen peroxide molecules into water and oxygen to lessen the
oxidative damage during stress conditions.
Zhang and Kirkham, (1994) reported that when wheat seedlings were
subjected to water stress, an increase in POX activity was observed. However, SOD
and CAT showed different behavior with increased levels at early phases of growth
and then decreased with increase in water stress. Decrease in SOD activities and
increase in other antioxidant enzymes was also reported on the same plant by Bartoli
et al., (1999). But an increase in antioxidant enzyme activities with PEG-induced
water stress was observed in drought tolerant genotype of the same plant (Gupta et al.,
2005). Several studies have been carried out to compare the biochemical
modifications in stress tolerant and sensitive genotypes of different plant species in
response to stress. Simova-Stoilova et al., (2009) reported enhanced antioxidant
enzyme activities in drought tolerant varieties as compared to sensitive ones. Wang et
al., (2009) also reported low production of H2O2 and increased activities of
21
antioxidant enzymes in stress-tolerant alfalfa cultivars. Previously transgenic tobacco
plants showing enhanced SOD activities also showed tolerance to salt and PEG-
induced water stresses confirming their role in regulating the stress mechanisms
(Badawi et al., 2004).
Role of antioxidant enzymes in salt stress, drought stress, extreme temperature
and heavy metal toxicity were conducted on seedlings of Jatropha curcas by several
workers. Kumar et al., (2008) reported an enhancement in POX and SOD activity of
salt stressed Jatropha curcas callus cultures. Jatropha curcas seedlings showed
increased activities of POD, SOD and CAT in cotyledons, hypocotyls and radicals
under salt stress conditions (Gao et al., 2008a). Hence it can be concluded that
antioxidant enzymes are involved in salt stress tolerance ability of Jatropha curcas.
They also reported an increased activity of phenylalanine ammonia lyase (PAL)
enzyme in seedlings grown under salt stress. Later the same group working with
Jatropha curcas seedlings exposed to high copper and lead concentrations (heavy
metal stresses) reported an enhancement in activities of antioxidant enzymes like
SOD, POD, CAT and PAL (Gao et al., 2008b; Gao et al., 2009). Increased
antioxidant enzyme activities at lower nickel concentrations were reported by Yan et
al., (2008). The authors suggested that those activities are involved in protective
mechanism against heavy metal stress. Another study by Luo et al., (2010) also
suggested that CAT, SOD, POD and PAL enzymes play important roles in defense
mechanism of Jatropha curcas under high Zinc concentrations (excess heavy metal).
Role of antioxidant enzymes in alleviating lead toxicity was also studied by Shu et al.,
(2012). SOD, POD and CAT activities were reported to be enhanced with increasing
Pb concentrations.
22
Protective mechanisms involved during heat and drought stresses against
oxidative damage in Jatropha curcas plants were studied by Silva et al., (2010a).
Yin et al., (2012) also concluded that antioxidant enzymes (H2O2 and
malondialdehyde (MDA) contents) production was enhanced in stress conditions.
Enhanced catalase activity in Jatropha curcas plants was also observed by Santos et
al., (2013) under water stress. Thus authors suggested that Jatropha curcas have a
protection mechanism against water stress to survive.
Silva et al., (2013), while working on combined stresses of heat and salinity,
also reported the stimulation of antioxidant enzymes of Jatropha curcas plants. They
concluded that APX, CAT, SOD activities were enhanced in salt and heat + salt
treatment but heat treatment alone did not show any significant influence on
antioxidant enzymes.
2.3.2 Role of soluble proteins under abiotic stress
Several workers have reported that abiotic stress results in varying levels of
soluble protein contents in plant cells and tissues (Levine et al., 1990; Niknam et al.,
2006 etc). Shankhadar et al., (2000) revealed a decrease in total protein contents
under NaCl-induced salt stress while decrease in protein contents with sorbitol-
induced osmotic stress was reported by Brito et al., (2003).
However, some workers indicated in their studies that abiotic stresses are
responsible for accumulation of stress-associated proteins. Close, (1997) has observed
that dehydrin (a protein) accumulate in plants in response to water as well as
temperature stress. The possible reason for accumulation of stress proteins may be the
de novo synthesis of proteins under stress (Pareek et al., 1997). Ashraf and O’Leary,
(1999) while working on abiotic stress studies in wheat also reported that total soluble
protein contents were increased in stress as compared to control. A significant
23
increase in protein contents in salt stressed plants was also observed by Radic et al.,
(2006). At the same time they also found that protein contents were not significantly
affected with manitol-induced osmotic stress treatment. Change in quality and
quantity of proteins under stress conditions in Jatropa curcas was also observed by
Kumar et al., (2008).
Aquaporins are basically major intrinsic proteins (MIPs) or membrane
proteins. These proteins form pores in biological cell membranes called water
channels. These pores are involved in controlling water movement across the cell
membranes and thus play important role under osmotic/water stress conditions. These
membrane proteins are distributed in cells of all life forms (Amiry-Moghaddam et al.,
2005). Plant MIPs are of four different types on the basis of its location: TIP
(tonoplast intrinsic proteins); PIP (plasma membrane intrinsic proteins); nodulin-26
and SIP (small basic intrinsic proteins). On the basis of amino acid sequence, PIPs are
further classified into subgroups PIP1 and PIP2. PIP2 proteins possess a shorter N-
terminal as compared to PIP1 and at the same time a longer C-terminal end (Johanson
and Gustavsson, 2002; Fetter et al., 2004). The work of Zhang et al., (2007) indicated
that in Jatropha curcas seedlings, some aquaporine (Jc PIP2) proteins are involved in
drought resistance. Different concentrations of PEG6000 (10, 20, 30 and 40%) were
used to impose drought conditions. Significant increase in the level of JcPIP2 in
higher levels of drought stress has indicated that it plays important role in drought
resistance ability of the plant. Hence, in the growth of Jatropha curcas plants under
water stress, aquaporins are considered to play important role. A novel betaine
aldehyde dehydrogenase gene (BADH) also called JcBD1 has also been isolated in
higher concentration from Jatropha curcas plants exposed to heat, salt and drought
stresses (Zhang et al., 2008). Hence this gene is involved in abiotic stress tolerance. A
24
ribosome inactivating protein (cucine 2) was produced by the Jatropha curcas
seedlings under both abiotic and biotic stress conditions (Qin et al., 2005). Authors
conclude that this protein was involved in stress tolerance ability of Jatropha curcas.
2.3.3 Drought stress studies on Jatropha curcas
Responses of plants to water stress vary from species to species. Strategies for
drought tolerance in plants involve both physiological and biochemical mechanisms
for better growth of the plants (Galle et al., 2010). Yin et al., (2012) suggested that
under poor N availability conditions Jatropha curcas plants are affected
physiologically and biochemically in excess soil water contents. However, in a
greenhouse experiment by Niu et al., (2012), water deficit have remarkably reduced
the growth. However, growth was not stopped and plants continue to grow even at
30% daily water use.
Different techniques and methods were employed to study the effect of
drought stress. Silva et al., (2012a) used led induced chlorophyll florescence analysis
to reveal the effect of drought and salinity on growth of Jatropha curcas plants. Their
results indicated that florescence ratio between red (Fr) and far red (FFr) chlorophyll
florescence around 685 nm and 735 nm respectively are early warning indicators of
drought stress.
Maes et al., (2009) suggested that newly formed leaves of dry treatment plants
have developed stomata on both sides and make their orientation vertical to receive
equal radiations on both sides. That’s why Jatropha curcas plants as compared to
other stem succulent plants, have the ability to withstand drought stress without any
leaf damage.
Infra-red thermography technique was also employed to study the relationship
between stomatal conductance and drought stress under sub-optimal weather
25
conditions in a plastered green house in autumn season (Maes et al., 2011). This study
has supported their previous observations.
Reduction in stomatal conductance under different drought stress treatments
on Jatropha curcas plants was also revealed by Díaz-López et al., (2012). They
further studied that stem and leaf growths were more reduced in drought stress
treatment as compared to root growth. Carbon dioxide assimilation was also reduced
with decreasing water supply. Hence they concluded that stomatal conductance and
biomass of aerial parts of the plant are involved in drought tolerance ability of this
plant. Role of stomatal conductance in drought tolerant ability of Jatropha curcas was
also studied by Sapeta et al., (2013). However, they concluded that net photosynthetic
activity was not influenced in moderate water stress but was reduced in severe stress.
Like previous workers they also reported the reduced growth of aerial parts to
minimize water loss in water deficit conditions.
Closure of stomata during dry season resulted in reduction of net
photosynthetic rate in Jatropha curcas (Santos et al., 2013). Chronic photoinhibition
was also observed during severe water deficit conditions. Accumulation of organic
solutes and enhanced CAT activity under drought stress was also observed.
Root system of Jatropha curcas was studied to investigate the drought
tolerance by Krishnamurthy et al., (2012). It has shallow root system with fine roots
distributed at the surface. It has narrow xylem vessels with a very small uptake of
water and nutrients which is actually a drought avoidance strategy. Kenan et al.,
(2012) used a geometric modeling technology based on computer simulation system
to study the effect of water stress at different levels. They investigated that root space
structure, dry mass of root and water use efficiency was higher when water stress
coefficient was 0.7 as compared to 0.9.
26
Water stress has no effect on the composition of fatty acid in seed oil of
Jatropha curcas (Kheira and Atta, 2009). Effect of water stress (using different PEG
concentrations) on photosynthetic activities and water relations was studied by Silva
et al., (2010b). Authors revealed that drought-stress showed more restricted leaf
growth as compared to salt-stress in Jatropha curcas plants.
Effects of drought stress on photosynthetic activities and growth parameters of
Jatropha curcas seedlings under different levels of nitrogen fertilization (low,
medium and high) were studied by Yin et al., (2010). They reported that with increase
in drought stress leaf relative water content, height, diameter, leaf area, photosynthetic
rate, stomatal conductance and transpiration rate decreased significantly irrespective
of nitrogen fertilization level. On the other hand under adequate water conditions
growth and photosynthesis increased with increasing nitrogen fertilization level.
Singh and Saxena, (2010) reported that increase in drought stress from -4 to -
20 bars not only delayed the onset of germination but also reduced % germination of
seeds of Jatropha curcas. At the same time they also indicated that effect of drought
stress on germination behavior was more pronounced beyond -8 bars, hence Jatropha
curcas plants could be better adapted in moderate water deficit areas. However,
Windauer et al., (2012) reported that Jatropha curcas seeds show high sensitivity to
water shortage.
While working on four different wild populations of Jatropha curcas to
investigate the causes of leaf senesces of Jatropha curcas, Matos et al., (2012)
reported that water deficit was not involved in the process.
Wen et al., (2012) suggested humid environment and fertile land for better
seed weight and oil content in Jatropha curcas plants. However, this plant was
considered as sensitive to flooding with either freshwater or saltwater as revealed by
27
Gimeno et al., (2012). Leaf gas exchange and chlorophyll fluorescence parameters are
also reduced in both freshwater and saltwater flooding treatments in the same ratio
indicating that Jatropha curcas plants are sensitive to flooding but tolerant to salinity
under flooding conditions.
2.3.4 Studies on drought inducing substances
Plants grown under abiotic stresses accumulate compatible solutes like
sorbitol, mannitol, proline etc. These substances are found to function as osmotic
buffers, so are termed as osmolytes. It has been reported that these osmolytes also
work as reactive oxygen species (ROS) scavengers (Xiong and Zhu, 2002). Different
concentrations of sorbitol/mannitol and polyethylene glycol (PEG) have been used for
inducing osmotic stress in many plants.
Several workers have reported that in many plants, osmotic adjustment under
drought stress is facilitated by sorbitol accumulation. It has been widely used to
induce osmotic stress in plant tissue culture experiments (Jovanovic et al. 2000; Frank
et al., 2005 etc). Similarly mannitol is also one of the end products of photosynthesis
and is involved in osmotic adjustment of plants as a response to water deficit.
Manitol induced stress has also been studied by several workers (Meena et al., 2016;
Możdżeń et al., 2015).
Polyethylene glycol (PEG) is a long chain of polymers which is highly soluble
in water and is available in different molecular weights like PEG 4000, PEG 6000 and
PEG 8000. PEG of high molecular weight decreases the water potential of nutrient
solutions thus inducing water stress in plants (Krizek, 1985). Jatropha curcas is
considered as drought tolerant plant as described by several workers previously. Most
of the experiments for studying drought tolerance ability were performed in green
house conditions with controlled watering to develop water deficit conditions
28
(Gimeno et al., 2012; Niu et al., 2012; Santos et al., 2013 etc). In case of in vitro
studies on Jatropha curcas, PEG was mostly used by workers (Qin et al., 2005; Silva
et al., 2010b; Wang et al., 2011a) as a drought stress inducing substance. To our
knowledge there are no reports on mannitol/sorbitol being used as drought inducing
substance in Jatropha curcas. Hence further research work is seriously needed using
manitol, sorbitol or sucrose as drought stress inducing substance for this plant.
29
Methodology
3.1 Preparation of Media
3.1.1 Stock Solutions
Formulation of MS medium (Murashige and Skoog, 1962) contains many
minerals, vitamins and iron whose formulations are listed in Annexure 1. Entire
solutions were prepared in double-distilled water with high quality chemicals. Stock
solutions of all components were prepared in advance as detailed in Annexure 2 and
kept in refrigerator at 4℃. Preparation of stock solutions is not only convenient but
also provides accuracy during the experiments.
3.1.2 Growth Regulators
Solutions of plant growth regulators in the form of stocks were also prepared
in advance as detailed in Annexure 3. Stock solutions of PGRs were prepared in either
milli molar or micro molar depending upon the requirement of experimental plan.
3.1.3 Preparation of Medium using Stocks
MS basal medium (One liter) was prepared using the appropriate quantities of
stock solutions as detailed in Annexure 4. Medium was supplemented with growth
regulators (singly or in combinations) for callus induction (Annexure 5), for
regeneration, multiplication and elongation of shoots (Annexure 6), rooting of
regenerated shoots (Annexure 7). Sorbitol was also added to create an osmotic stress
in the medium for some in vitro experiments as detailed in Annexure 8 and 11. Half
strength MS medium the requirement of some of our experiments (Annexure 7 and 9)
was prepared following the same protocol except macro and micronutrients that were
added in half amount as compared to MS basal. After adding and mixing all the
components in volumetric flask, double-distilled-water was used to mark up the total
volume. Then pH of prepared medium was closely adjusted to 5.8. For solidification
30
of the medium, agar (8g; Oxoid, Hampshire, England) was added after pH adjustment.
After melting the agar by heating, medium was immediately poured in culture vessels
of different sizes according to the requirement of the undergoing experiments.
Amount of medium in each culture vessel also depend upon the size of culture
vessels.
3.2 Sterilization
3.2.1 Sterilization of Glassware
The glassware to be used was washed with house hold detergent (Karachi,
Pakistan) and rinsed with water before sterilization. In the present experiments, dry
heat sterilization method was used for glass ware. Cleaned glassware was kept in an
oven at high temperature (180º) C for approximately two to three hours and then
stored in dust proof area to avoid contaminations.
3.2.2 Sterilization of the Media
Medium was sterilized by autoclaving at high temperature (121℃) and
pressure (15 lbs inch-2) for a time period of 15-20 minutes. After autoclaving the
medium was kept at room temperature to cool down before explant inoculation.
3.2.3 Sterilization of Laminar Airflow Cabinet
Careful aseptic manipulations are strongly needed for laminar airflow cabinet
because it is the main working area. Whole area of laminar airflow cabinet (ESCO,
Singapore; Model 1750) was first scrubbed with cotton plug dipped in 70% ethanol
and then sprayed with 70 % ethanol. After that UV lamp was turned on in laminar
airflow cabinet for one hour and was turned off at least 15 minutes before start
working there.
3.2.4 Sterilization of Surgical Tools
The surgical tools were sterilized by putting them in a glass-bead sterilizer
(Steri 350, Swiss made) at 250 ºC. The surgical tools were cooled by dipping in 70%
31
ethanol and allowed to dry out for some time before their use in culture
manipulations.
3.3 Plant Material
3.3.1 Source of Plant Material
Seeds used in the present experiments were supplied by Jatropha Pakistan
Four Friends Group Multan, Pakistan. Plants were developed from these seeds in pot
soil in the wire house. In vitro seed germination was also carried out. Cotyledonary
leaves and hypocotyl of in vitro germinating seedlings and young and mature leaves
of pot-grown 2-3 years old plants have been used for callus induction, its proliferation
and subsequent regeneration experiments.
3.3.2 Explant Disinfection
Leaves: young leaves from 1st, 2nd or 3rd node and mature leaves from 4th, 5th or lower
nodes excised from 2-3 year old plants were used as explants for callus induction, its
proliferation and also for regeneration experiments. Freshly excised leaves were
washed thoroughly with house-hold detergent (Lemon max Unilever Karachi,
Pakistan) and tap water. Washed leaves were cut into small pieces and dipped in 0.1%
mercuric chloride solution (composition given in Annexure 16. a) in conical flask.
They were wrapped with polypropylene sheet, shacked well for 5 minutes and HgCl2
solution was drained and leaf pieces were rinsed 4-5 times with distilled-autoclaved
water. Then 10% sodium hypochlorite (NaClO) solution (composition given in
Annexure 16. b) was added in conical flasks having leaf pieces, wrapped with
polypropylene sheet and shacked well for 5 minutes. After that NaClO solution was
decanted and leaves were rinsed 5- 6 times with distilled autoclaved water. All the
steps of decantation and rinsing with distilled autoclaved water were done in pre-
sterilized airflow cabinet. Leaves were then ready for inoculation in culture tubes.
32
Seeds: Seeds after thorough washing with tap water were dipped in mixture of tween
20 (Daejung Chemicals) and detergent and kept on magnetic stirrer at 50˚C for 10-15
minutes. Then seeds were rinsed several times with tap water and dipped in 0.1%
HgCl2 solution (composition given in Annexure 16a) for 10 minutes, rinsed three to
four times with distilled autoclaved water and then dipped in 20 % NaClO
(composition given in Annexure 16c ) solution for 15-20 minutes and rinsed again 4-5
times with distilled autoclaved water. Rinsing procedures with distilled autoclaved
water were done in pre-sterilized airflow cabinet. The same method was used for
naked seeds (seeds with removed seed coats) except that tween 20 was not used with
detergent and also kept on magnetic stirrer at room temperature for 5-10 minutes.
Instead of 20% NaClO, seeds were dipped in 10% NaClO solution for 10 minutes.
3.4 Culture Conditions
Callus induction and its proliferation experiments were studied both in light
(16/8 hour photoperiod) and in complete darkness. For regeneration experiments,
cultures were placed in light conditions. For in vitro germination of seeds, cultures
were kept in dark for first 2- 3 days and then shifted in light for chlorophyll
development and further elongation of seedlings. Cultures for drought stress
experiments were all kept in light. All the cultures in present experiments were kept at
25 ± 2º C. Culture room was already standardized to maintain light and temperature
conditions with least fluctuations.
3.5 Biochemical Studies
Total soluble proteins and antioxidant enzymes were analyzed quantitatively
and details are given below:
33
3.5.1 Extraction of Soluble Proteins and Enzymes
Weighed amount (1.0 gm) of plant material (germinating seedling, callus or
leaf) was crushed in pestle and mortar (ice-chilled overnight) with 0.1gm polyvinyl
polypyrrolidone and then added 2 ml of 0.1 M phosphate buffer (composition given in
Annexure 12). It was then centrifuged at 15,400 g for 15 minutes at 4ºC. The
supernatant so obtained was stored at 0ºC in refrigerator and used for further protein
quantification, peroxidase activity and superoxide dismutase estimation.
3.5.2 Estimation of Soluble Protein Contents
Protein was estimated following Biuret method proposed by Racusen and
Johnstone, (1961).
The following two samples (experimental and the control) were prepared:
Constituents Experimental Control
Protein extract 0.2 ml _
Biuret reagent 2.0 ml 2.0 ml
(Composition given in Annexure 13)
Distilled H2O _ 0.2 ml
Both set of tubes were kept at room temperature for 10 minutes. The optical
densities of experimental samples were measured spectrophotometrically (Hitachi
U1100 spectrophotometer) at 560 nm against control which was considered as zero. A
standard curve for proteins was developed in advance using bovine serum albumin (E
Merck Ag Darmstadt Germany) that was used to calculate the amount of protein.
Protein contents were determined using this formula:
Protein content = CV×TE / EU×Wt × 1000
Where
CV= Curve Value
34
TE = Total Extract
EU = Extract Used
Wt = Fresh weight of sample tissue
3.5.3 Peroxidase Estimation
Peroxidase activity was analyzed by using the modified Guaiacol-H2O2
method proposed by Racusen and Foote, (1965). Solutions of guaiacol (1%) and H2O2
(0.3%) were prepared freshly for analysis (Composition given in Annexure 14 a, b).
Two sets of tubes (sample and blank) for peroxidase estimation were prepared
as follows:
Constituents Experimental Control
Enzyme extract 10 µl 10 µl
0.1 M phosphate buffer 2.5 ml 2.5 ml
(Composition given in Annexure 12)
1 % Guaiacol 0.2 ml _
Dist.H2O _ 0.2 ml
Both sets of tubes tubes were kept at room temperature for 30 minutes and
then 0.1 ml H2O2 was added to both tubes and immediately kept in spectrophotometer
to observe increase in optical density in one minute at 470 nm.
Enzyme activity was then determined as follows:
Where,
A = Increase in optical density in one minute
df = dilution factor
EU = Extract Used
Wt = Fresh weight of the sample tissue
35
3.5.4 Superoxide Dismutase Estimation
Superoxide dismutase (SOD) activity was determined by the slightly modified
method given by Maral et al., (1977) during the present study. It was measured in
terms of its ability to inhibit photochemical reduction of nitroblue tetrazolium (NBT).
Two sets of tubes covered initially with black paper were prepared as follows:
Constituents Experimental Control
Reaction mixture 2 ml 2 ml
(Composition given in Annexure 15)
Enzyme extract 5 µl ----
Both sets of tubes were placed below two 30-W fluorescent tube lights for
about 15 min. The absorbance of the sample and control were taken at 560 nm by
using a spectrophotometer.
Percentage inhibition of NBT was determined by using the following formula:
The SOD activity was then calculated based on the fact that one unit of SOD
caused 50% inhibition. SOD activity was expressed as units per milligram of protein.
3.6 Experimental Plan
3.6.1 Seed germination
The first step in the presented work was germination of seeds of Jatropha
curcas in order to obtain plant material to be used as explants in further experimental
works. The source of seeds used in the present experimental work were ‘Jatropha
Pakistan Four Friends Group, Multan’. As Jatropha curcas is a deciduous plant in
nature and its leaves fall off during winter season from November till mid-March.
36
Seeds also remain dormant during that period. So availability of explants for
experimental work was a serious issue in those days. To resolve the problem different
techniques were used to break the seed dormancy for its germination in both in vitro
and soil conditions in the months of December and January. Different pretreatments
given to seeds for the germination were listed in Annexure 10. Different media used
for in vitro germination were detailed in Annexure 9. Culture tubes used here were
slightly larger in size (30 × 250 mm). Sterilized, pretreated seeds were cultured on
different media for in vitro germination and kept initially in dark (2-3 days) for root
emergence and then shifted in light (16/8 photoperiod) for chlorophyll development.
Pretreated seeds were also sown in pot soil (mixture of peat, clay and silt; 1:1:1 v/v)
for soil germination studies and kept in glass house at 25 ± 2ºC. Percentage
germination results were recorded on daily basis. Each treatment was given to 20
seeds and experiments were repeated three times.
Developed seedlings after 10-15 days were taken out from agar medium,
washed with tab water to remove traces of agar from the roots and shifted to the soil
mix (peat, clay and silt 1:1:1 v/v). After watering, plantlets were covered with glass
jars for 24 hours at 25 ± 2˚C and 16 h photoperiod. Seedlings were then shifted in
glasshouse conditions after 3-4 days.
3.6.2 Callus induction and its proliferation
The recent study was carried to sort out the best combination of hormones and
explants for callus induction of Jatropha curcas. Maintenance of callus cultures by
subsequent sub-cultures and regeneration of shoots were also aimed here. Medium
used for callus induction and its proliferation in present experiments was MS basal
medium (Murashige and Skoog, 1962) fortified with a variety of growth regulators.
Details of different media (C-1 to C-10) with different growth regulators are given in
Annexure 5. Different explants used are detailed in section 3.3.1. Twenty four culture
37
vessels (150 × 25 mm) were used for each medium and experiment was repeated three
times. Sterilized leaf explants were cut into small pieces of 2-3 mm with sterilized
scalpel in pre-sterilized airflow cabinet and inoculated on autoclaved medium
containing culture vessels. Half culture vessels were placed in dark and half in light
(16/8 h photoperiod) at 25 ± 2ºC. Data were recorded on weekly basis and sub
cultured (shifted to fresh medium) after every 15 days to avoid browning and also for
continuous supply of nutrients.
3.6.3 Regeneration from callus cultures
Plant growth regulators in different combinations (supplemented in MS
medium) were tested to study the regeneration behavior of Jatropha curcas callus
cultures. Details of various combinations (R-1 to R-15) to check the regeneration
behavior were given in Annexure 6. Calluses (30 days old) were sub cultured for
regeneration. Twelve culture vessels were used for each combination and experiment
was repeated three times. Regeneration frequencies and number of shoots and average
shoot length per culture vessel were noted after every 15 days. Regenerated shoots
were then sub cultured on rooting medium. Details of different rooting media (T-1 to
T-6) were given in Annexure 7. Data for percentage root regeneration were also
recorded after 30 days.
3.6.4 Direct regeneration from young leaf explant
Different combinations of plant growth regulators detailed in Annexure 6 were
also tested to study the regeneration behavior directly from young leaf explant of
Jatropha curcas. Twelve culture vessels were used for each combination and
experiment was repeated thrice.
3.6.5 Effect of various sorbitol concentrations on in vitro seed germination
Sorbitol was added in culture media to create an osmotic stress. Different
sorbitol concentrations used in the recent experiments for in vitro germination are
38
given in Annexure 11. Pretreated S5 seeds were used here. Seeds were oriented
dorsally on media with different sorbitol concentrations and kept in dark initially (2-3
days) for root emergence and then shifted in light for chlorophyll development. Rate
of germination was observed after 2, 4, 6 and 10 days of inoculation. Germination
percentage was measured by number of seeds from which radicle emerged out to
develop root out of total number of seeds. Rate of germination was observed after 2,
4, 6 and 10 days of inoculation. Germination energy was calculated by the method
proposed by Afzal et al., (2017). It was calculated by counting the number of
germinated seedlings at fourth days after the start of germination. Data on root/shoot
length, fresh/dry weight and biochemical parameters were collected after 10 days of
inoculation.
3.6.6 Effect of sorbitol concentrations on callus cultures
Different sorbitol concentrations were used to study the effect of osmotic
stress (or drought stress) on Jatropha curcas callus cultures. Addition of sorbitol in
the medium reduced its water potential thus inducing water stress or osmotic stress
(Abu-Romman, 2010). Details of ten different sorbitol concentrations along with
callus inducing growth regulators tested in the present study are given in Annexure 8.
Ten culture vessels of each concentration were used and the experiments were
repeated three times. Freshly excised and sterilized leaves were cut into small pieces
of 2-3 mm and inoculated in culture vessels and placed in 16/8 h photoperiod at 25 ±
2ºC. Data were recorded after 22 days of inoculation. Fresh/dry weight, water
contents, soluble proteins and antioxidant enzymes were analyzed.
3.6.7 Effect of different field capacities of water on pot-grown plants
Stem cuttings of Jatropha curcas plants were obtained from Jatropha Pakistan
Four Friends Group Multan and were sown in pots (14" × 16") containing an equal
39
amount (by volume) of a mixture of sand, soil and peat (1:1:1) during the month of
May. Pots were irrigated regularly on weekly basis until the plants reached almost at
the age of four months. These plants were then shifted in a greenhouse covered with
polythene sheet (at a height of approximately 14′) one week before exposing to
different levels of water stress.
Effect of drought stress on five-months-old soil-grown-plants developed from
stem cuttings was also studied. A wide range of water availability was developed.
One group of plants was irrigated with 100% field capacity. That means pots were
irrigated to the extent that water start leaking from the bottom of the pot. The amount
of water given to each pot was already measured. Second group of plants with 75%
field capacity were irrigated with ¾ the amount of water given to first group of
plants. Likewise third group with 50% field capacity were given ½ and fourth group
with 25% field capacity with ¼ amount of that water. Field capacities were
maintained by irrigating the pots regularly using the same methodology. While fifth
group with 0% field capacity were not irrigated at all during the experiment period.
Each experimental group consisted of three plants. Plants were subjected to this
treatment for 30 days (from 3rd September to 3rd October). Fresh/dry weight, water
content per unit area of leaves and biochemical parameters of leaves were then
assessed.
3.7 Statistical Analysis
Data was analyzed statistically using one way ANOVA with the help of
computer software (SPSS Version 16). Mean values and the standard errors were
calculated for each experiment. Significant difference between the mean values were
also compared using Duncan’s multiple range tests.
40
Breaking dormancy and in vitro germination of seeds of
Jatropha curcas L.
Results
No seed germination was obtained in soil by using all the seven pretreatments.
However, in vitro seed germination was observed in only one treatment where seed
coats were removed after sterilization of seeds (S5) as shown in Table 4.1 on both MS
and ½ MS medium but germination percentage was higher on full strength as
compared to half strength MS medium. Orientation of seeds in culture tubes also
effect germination. Maximum germination (100%) was achieved when seeds were
oriented dorsally in culture tubes as shown in Table 4.2. Radical emerges out after 2
days of sowing and then hypocotyls enlarged to the full length of culture tubes within
15 days. Seedlings were then taken out of the medium washed with water and shifted
to the soil mix (peat, clay and silt 1:1:1 v/v). After watering, plantlets were covered
with glass jars for 24 hours at 25±2˚C and 16 h photoperiod. Seedlings were then
shifted in glasshouse conditions after 3-4 days. All the process described is shown
diagrammatically in the Figure 4.1 (a, b, c, d, e, f and g).
41
Table 4.1: Effect of various pretreatments and MS medium strength on in vitro seed
germination of Jatropha curcas L.
Pretreatments* Full strength MS medium Half strength MS medium
Root
induction
Shoot
induction
%
germination
Root
induction
Shoot
induction
%
germination
S 0 0 0 0 0 0 0
S1 0 0 0 0 0 0
S2 0 0 0 0 0 0
S3 0 0 0 0 0 0
S4 0 0 0 0 0 0
S5 30 24 80 14 4 13.3
S6 0 0 0 0 0 0
S7 0 0 0 0 0 0
*S0 non treated, S1 scarified, S2 stratified, S3 scarified+ stratified, S4 seeds soaked in water, S5 seed
coats removed after sterilization, S6 seed coats removed before sterilization, S7 seeds flamed on
Bunsen burner. Each treatment given to 30 seeds in 3 replicates
Table 4.2: Effect of orientation of S5 seeds on germination behavior on full strength
MS medium
Orientation Root induction Shoot induction % germination
Dorsal 15 15 100
Ventral 15 9 60
Out of total 30 S5 seed, half were oriented dorsally while the other half ventrally.
42
Fig. 4.1: Root induction (arrow) after 2 days from a J. carcus seed sown dorsally on
full strength MS medium (a), after 3 days of sowing (b), root emergence in
ventrally oriented seed (c), Developed shoot after 7-8 days of sowing (d),
further shoot elongation after 15 days of sowing (e), seedling taken out of the
culture tube for acclimatization in glasshouse after 15 days of sowing (f),
withdrawal of cotyledonary leaves and emergence of a new leaf 7-8 days
after acclimatization (g).
a b
c d
e f g
6mm 10mm
5mm 9mm
20mm 10mm 30mm
43
Discussion
There was no seed germination in the soil by using any pretreatment method.
Even the removal of seed coats in December to January was of no help in this study.
Our results therefore are indirectly in line with a previous study from Thailand in
which the author suggested October to be the most suitable month for Jatropha
curcas plantation (Ratree, 2004). Rahman et al., (2009) have suggested March to
early April as best time for Jatropha curcas plantation in Bangladesh. There was, on
the other hand, 100% in vitro germination using coatless seeds oriented dorsally in
full strength MS medium (Table 1). This result agrees with Abdelgadir et al., (2012).
They reported that seed coat removal enhanced water-imbibition and seeds
germinated within 48 h. Different pretreatments to enhance germination of seeds were
reported by several workers (Idu et al., 2007; Koorneef et al., 2007 and Pascual et al.,
2009) but most pretreatments in the present research work did not help support even
in vitro seed germination in Jatropha curcas. This may be due to the prevailing
dormancy in the months of December and January. Poor germination of seeds is
perhaps also because of water impermeable testa which exerts physical exogenous
dormancy (Holmes et al., 1987). Low germination rate of Jatropha curcas is also
possibly due to seed coats that form mucilage surrounding the seeds which prevents
diffusion of oxygen to the embryo and hence inhibits germination (Kumari et al.,
2011). Jatropha curcas seeds are usually dark brown to blakish-brown in colour. Dark
colour of seeds is also considered to be a factor for enhancing dormancy (Duran and
Retamal, 1989). Therefore removal of seed coats in the present investigation might
have been helpful in breaking dormancy and enhancing seed germination. Rodrigues
and Rodrigues, (2014) in their studies on Macaranga peltata seed germination
confirmed that seed coat dormancy is responsible for inhibited germination.
44
Mwang`Ingo et al., (2004) in their investigation also suggested the removal of seed
coats and keeping for some time in hot water for enhanced germination and better
seedling growth. Islam et al., (2009) also reported that pre-sowing treatments to
Jatropha curcas seeds influenced germination parameters. Some workers used pre-
soaking of seeds in GA3 (100 ppm) for that purpose and attained 67.38% germination
(Kumari et al., 2011).
Germination of Jatropha curcas seeds was attained in just 2-3 days (Fig1 a, b)
as compared to previous studies where seeds germinated normally within 8-9 days
(Kumari et al., 2011). Germination in Jatropha curcas is epigean (Fig. 1 a-g) in
nature (cotyledons emerge above ground). Cotyledonary leaves soon wither away or
fall-off after new leaves are developed (Fig1 g). This fact was also supported by
Becker and Francis, (2003). Sterilization of seeds after removing the seed coats did
not favor germination process. This might be attributed to the effect of strong
sterilizing agents on delicate embryo tissues.
Seeds soaked in water overnight could not germinate even after 15-20 days of
sowing. These results do not agree with those of Feike et al., (2008) who reported that
seeds soaked in water showed highest survival and germination rate. This
contradiction was perhaps due to dormant season in which the present investigation
was carried out. Mechanical scarification and stratification both increase germination
but the germination rate was highest and fastest when both treatments were applied
together (Kaye and Kuykendall, 2001) but presently both the treatments either
separately or in combination had no effect on germination. Our results also disagree
with a recent work by Geisler et al., (2017) who reported that wet shocks of 40 to
50oC proved helpful in breaking physical dormancy of seeds. Seeds oriented dorsally
in the culture medium resulted in better germination and seedling development as
45
compared to ventral orientation (Table 2; Fig.1c). This may be due to an easy
approach of radical to the growth medium in dorsally-placed seeds. Zewdie and
Welka, (2015) also suggested that orientation played important role in germination of
seeds. They further reported that sowing of seeds in such a way that micropyle was
directed downwards showed best results in terms of germination. Same was the case
in our experiments where micropyle was directed downwards in dorsally-placed
seeds. Developed seedlings (30%) from both the treatments were successfully
acclimatized in the soil.
Thus the study highlighted that removal of seed coat could help germinate
seeds of Jatropha curcas even in dormant season (December, January ) on simple full
strength MS medium. This method takes least time for germination (2-3 days) as
compared to previous studies and will be of help for future propagation of this plant at
mass scale level. This study also opens new horizon to explore more suitable methods
in the near future for acclimatization and hardening of these in vitro-developed
plantlets.
46
Callus induction, maintenance and in vitro regeneration using
different explants of Jatropha curcas L.
Results
5.1 Standardization of medium for callus induction and maintenance
for Jatropha curcas
The recent study was carried to sort out the best combination of hormones in
MS medium as well as explants source for callus induction of Jatropha curcas.
Maintenance of callus cultures by subsequent sub-cultures and regeneration of shoots
were also aimed here. Different growth regulators studied for callus induction in the
present investigation were BAP, NAA, TDZ, IAA, Kin and 2,4-D either separately or
in combinations at various levels. Different explants used were younger leaves (upper
part of the plant second or third node), older leaves (mature leaf from lower part of
the plant), cotyledonary leaves (first leaves that emerge out of the seed) and
hypocotyls.
5.1.1 Callus induction from younger leaf explants
Different concentrations of TDZ (1.0, 1.5, 2.0 pM) supplemented in MS
medium could not support callus induction from younger leaf explant (Table 5.1).
There was just swelling of leaf explants even after 30 days of inoculation. Swelling of
explant frequency was also reduced from 10 to 7% with increasing TDZ
concentrations indicating that TDZ could not support callus formation in Jatropha
curcas.
However, at the same time with different concentrations of Kin (4.65, 9.3,
13.95 µM) added medium, callus induction started within 22-25 days of inoculation
(Table 5.1). Although callus was green and compact with granular surface but had a
47
little growth (Fig. 5.1). Likewise with increasing Kin concentration there was little
increase in callus induction frequency from 12 to 17% as shown in Table 5.1.
Maximum callus induction frequency from younger leaf explants was
recorded on medium with 22.17 µM BAP + 5.35 µM NAA added (C-8). The process
of callus induction started approximately within five days of inoculation as shown in
Table 5.1. Callus formed was lush green in color with white granular surface and
compact in nature having extensive growth (Fig. 5.2). Further sub-culturing to
different media showed that they were having embryogenic potential.
BAP in combination with IAA (2.21 µM BAP + 5.71 µM IAA) also proved
better for callus induction (42%) within 15-20 days. Callus had moderate growth and
was whitish translucent and friable in nature. Addition of 2, 4-D (4.52 µM) in same
combination of BAP and IAA resulted in development of calluses with same
morphology and growth (Fig. 5.3). However, callus induction started early (within 10-
12 days) and callus induction frequency was also increased. At the same time it was
also observed that those calluses had not shown embryogenic response on further sub-
culturing to different media for regeneration.
48
Table 5.1: Effect of different growth regulators on callus induction from younger
leaf explant of Jatropha curcas L.
Medium Medium
composition
No. of days
for callus
induction
Callus
induction %age
Callus Morphology
C-1 MS No callus
induction
ND ND
C-2 MS + 1 pM TDZ 29.6±0.54a 10.0±1.41ef Just swelling of explant
C-3 MS + 1.5 pM TDZ 30.4±1.14a 8.0±0.70fg Just swelling of explant
C-4 MS + 2.0 pM TDZ 30.6±2.5a 6.8±0.83g Just swelling of explant
C-5 MS + 4.65 µM Kin 25.2±1.48b 12.4±1.81e Lush Green, with white
granular surface, compact,
having minute growth,
embryogenic
C-6 MS + 9.3 µM Kin 22.00 ±1.58c 16.8±2.04d Lush Green, with white
granular surface, compact,
having minute growth,
embryogenic
C-7 MS + 13.95 µM Kin 21.6 ±1.81c 18.4±1.51d Lush Green, with white
granular surface, compact,
having minute growth,
embryogenic
C-8 MS + 22.17 µM
BAP + 5.35 µM
NAA
5.2±0.83f 100±00a Lush Green, with white
granular surface, compact,
having extensive growth,
embryogenic
C-9 MS + 2.21 µM BAP
+ 5.71 µM IAA
18.0±2.12d 43.2±3.83c Creamy, translucent, white,
friable, moderate growth,
non-embryogenic
C-10 MS + 2.21 µM BAP
+ 5.71 µM IAA +
4.52 µM 2, 4-D
10.4±1.14e 60.0±3.53b Creamy, translucent, white,
friable, moderate growth,
non-embryogenic
Data presented here are means of 30 values per treatment
Different letters within a column represent significant difference at P=0.05 according to Duncan’s
multiple range test
49
Figure 5.1- 5.3: Callus induction from young leaf explants cultured in MS medium
and placed under 16/8 hours photoperiod at 25±2ºC after 25-30 days
of inoculation
Fig. 5.1 bar=3mm Fig. 5.2 bar=2.5mm
Fig. 5.3 bar=3mm
Fig. 5.1: Callus induced in MS medium with 9.3 µM Kin
Fig. 5.2: Callus induced in MS medium with 22.17 µM BAP + 5.35 µM NAA
Fig. 5.3: Callus induced in MS medium with 2.21 µM BAP + 5.71 µM IAA +
4.52 µM 2, 4-D
50
5.1.2 Callus induction from older leaf explants
Older leaf explants inoculated on MS medium without any growth regulators
could not support callus induction (Table 5.2). TDZ (1.0, 1.5, 2.0 pM) added medium
also could not support callus induction even after 30 to 40 days of inoculation.
Morphology of callus induced on other growth regulators supplemented medium with
older leaf explants were almost the same as with younger leaf explants (Fig. 5.4- Fig.
5.6) but callus induction frequency was reduced. There was only four to five percent
callus induction in Kin added medium as shown in Table 5.2. A combination of BAP
and NAA (22.17 + 5.35 µM) was proved best for maximum callus induction (60%)
from older leaf explant after 18-20 days of inoculation. It was not only reduced but
also delayed as compared to younger leaf explants where there was 100% callus
induction frequency and process of callus formation started within first five days of
inoculation. Same was the case with BAP and IAA combination. However, addition
of 2, 4-D in this combination had not shown any significant effect on time period and
frequency of callus induction.
51
Table 5.2: Effect of different growth regulators on callus induction from older leaf
explant of Jatropha curcas
Medium Growth
Regulators
No. of days
for callus
induction
Callus
induction%
Callus Morphology
C-1 MS - ND -
C-2 MS + 1 pM TDZ - - -
C-3 MS + 1.5 pM
TDZ
- - -
C-4 MS + 2.0 pM
TDZ
- - -
C-5 MS + 4.65 µM
Kin
24.4±1.94a 5.0±0.70c Lush Green, with white
granular surface, compact,
having minute growth,
embryogenic
C-6 MS + 9.3 µM Kin 27.0±1.41a 4.8±0.83c Lush Green, with white
granular surface, compact,
having minute growth,
embryogenic
C-7 MS + 13.95 µM
Kin
23.6±1.67a 4.2±0.83c Lush Green, with white
granular surface, compact,
having minute growth,
embryogenic
C-8 MS + 22.17 µM
BAP + 5.35 µM
NAA
15.0±7.87b 60.0±3.53a Lush Green, with white
granular surface, compact,
having moderate growth,
embryogenic
C-9 MS + 2.21 µM
BAP + 5.71 µM
IAA
28.0±1.22a 28.0±2.73b Creamy, translucent, white,
friable, moderate growth,
non-embryogenic
C-10 MS + 2.21 µM
BAP + 5.71 µM
IAA + 4.52 µM
2, 4-D
27.6±1.51a 30.0±3.53b Creamy, translucent, white,
friable, moderate growth,
non-embryogenic
Data presented here are means of 30 values per treatment
Different letters within a column represent significant difference at P=0.05 according to Duncan’s
multiple range test
52
Figure 5.4- 5.6: Callus induction from older leaf explants cultured in MS
medium placed under 16/8 hours photoperiod at 25±2ºC after
25-30 days of inoculation
Fig.5.4 bar=3mm Fig.5.5 bar=4.5mm
Fig.5.6 bar=4.5mm
Fig.5.4: Callus induced in MS medium with 9.3 µM Kin
Fig.5.5: Callus induced in MS medium with 22.17 µM BAP + 5.35 µM NAA
Fig.5.6: Callus induced in MS medium with 2.21 µM BAP + 5.71 µM IAA +
4.52 µM 2, 4-D
53
5.1.3 Callus induction from cotyledonary leaf explants
Cotyledonary leaf explants also did not show any callus induction response on
growth regulator free medium. With different concentrations of TDZ used, only one
(1 pM TDZ) showed a little (2%) callus induction response after 30 days (Table 5.3).
kinetin supplemented in medium (4.65, 9.3, 13.95 µM) also could not support callus
induction. There was just swelling of explants in few culture tubes after 22 days of
inoculation (Table 5.3).
Callus induction frequency and morphology of callus induced on medium
containing 22.17 µM BAP + 5.35 µM NAA from cotyledonary leaf explants were
almost the same as with young leaf explants (Fig. 5.7). There was 100% callus
induction frequency and process of callus induction started within 5 days of
inoculation. Calluses were having vigorous growth and also embryogenic in nature.
At the same time, a good callus induction frequency (60%) was also obtained
on medium added with 2.21 µM BAP + 5.71 µM IAA. Developed calluses were light
green to translucent white with loose surface. These calluses show restricted growth.
Addition of 4.52 µM 2, 4-D in this combination produced the same type of calluses.
However, the callus induction frequency was enhanced (65%) as shown in Table 5.3.
54
Table 5.3: Effect of different growth regulators on callus induction from
cotyledonary leaf explants of Jatropha curcas L.
Medium Medium
composition
No. of days
for callus
induction
Callus
induction %a
ge
Callus Morphology
C-1 Nil No callus
induction
ND ND
C-2 MS + 1 pM TDZ 30.6±2.50a 1.4±2.19f Just swelling of explant
C-3 MS + 1.5 pM
TDZ
- - -
C-4 MS + 2.0 pM
TDZ
- - -
C-5 MS+4.65 µM Kin 25.2±1.48b 9.4±1.94e Just swelling of explant
C-6 MS + 9.3 µM Kin 21.6±1.81c 14.4±3.78d Just swelling of explant
C-7 MS + 13.95 µM
Kin
21.6±1.14c 14.4±4.39d Just swelling of explant
C-8 MS + 22.17 µM
BAP + 5.35 µM
NAA
5.4±1.14f 100±0.00a Lush green, compact in
the center and
superficially loose friable,
highly vigorous growth,
embryogenic in nature
C-9 MS + 2.21 µM
BAP + 5.71 µM
IAA
18.0±2.12d 60.0±3.53c Light green to translucent
white in color, partially
loose having little growth
C-10 MS + 2.21 µM
BAP + 5.71 µM
IAA + 4.52 µM
2, 4-D
10.4±1.14e 64.8±3.56b Light green to translucent
white in color, partially
loose having little growth
Data presented here are means of 30 values per treatment
Different letters within a column represent significant difference at P=0.05 according to Duncan’s
multiple range test
55
Figure 5.7- 5.8: Callus induction from cotyledonary leaf explants in MS medium
placed under 16/8 hours photoperiod at 25±2ºC after 25-30 days
of inoculation
Fig. 5.7 bar=3.1mm
Fig 5.8 bar=3.5mm
Fig 5.7: Callus induced in MS medium with 22.17 µM BAP + 5.35 µM NAA
Fig 5.8: Callus induced in MS medium with 2.21 µM BAP + 5.71 µM IAA +
4.52 µM 2, 4-D
56
5.1.4 Calluses induced from hypocotyl explants
Like other explants used in the present investigation, hypocotyls also could not
develop callus without any growth regulators added in the medium. TDZ
supplemented (1.0, 1.5, 2.0 pM) medium also did not support callus induction when
hypocotyl was used as an explant (Table 5.4). However, creamy white translucent
callus was formed on medium with Kin (Fig. 5.11). Callus induction frequency was
low (15%) with 4.35 µM Kin supplemented medium which was enhanced slightly by
increasing the concentration of Kin to 9.3 µM. At the same time further enhancement
of Kin to 13.95 µM could not enhance frequency of callus induction (Table 5.4).
Calluses developed on Kin supplemented medium were compact in center and loose
on periphery and had very slow growth.
Calluses induced on medium with 22.17 µM BAP + 5.35 µM NAA had
vigorous growth and maximum callus induction frequency like young and
embryogenic leaf explants (Table 5.4). Callus formed on that medium was lush green
and compact and embryogenic in nature (Fig. 5.9).
Medium fortified with 2.21 µM BAP + 5.71 µM IAA also had good callus
induction frequency (60%) (Table 5.4) but callus could not be regenerated on further
sub-culturing. These calluses were light green to translucent in color and loose, friable
in texture (Fig. 5.10). Addition of 4.52 µM 2, 4-D in same medium produced the
calluses with same structure within same period of time. However, callus induction %
was slightly enhanced (Table 5.4).
57
Table 5.4: Effect of different growth regulators on callus induction from hypocotyls
explants of Jatropha curcas L
Medium Growth
Regulators
No. of days
for callus
induction
Callus
induction %age
Callus Morphology
C-1 Nil - ND ND
C-2 MS + 1 pM
TDZ
- - -
C-3 MS + 1.5 pM
TDZ
- - -
C-4 MS + 2.0 pM
TDZ
- - -
C-5 MS + 4.65 µM
Kin
28.4±1.51a 13.6±2.50e Creamy white
translucent, compact in
center and loose on
periphery
C-6 MS + 9.3 µM
Kin
27.8±1.92a 18.4±1.67d Creamy white
translucent, compact in
center and loose on
periphery
C-7 MS + 13.95 µM
Kin
23.2±2.86b 17.8±1.48d Creamy white
translucent, compact in
center and loose on
periphery
C-8 MS + 22.17 µM
BAP + 5.35 µM
NAA
18.0±2.12c 100±0.00a Lush green, compact,
vigorous growth,
embryogenic
C-9 MS + 2.21 µM
BAP + 5.71 µM
IAA
28.8±1.30a 62.0±2.73c Light green to
translucent, loose and
friable
C-10 MS + 2.21 µM
BAP + 5.71 µM
IAA + 4.52 µM
2, 4-D
28.2±2.16a 69.0±4.18b Light green to
translucent, loose and
friable
Data presented here are means of 30 values per treatment
Different letters within a column represent significant difference at P=0.05 according to Duncan’s
multiple range test
58
Figure 5.9- 5.11: Callus induction from hypocotyls explants in MS medium
placed under 16/8 hours photoperiod at 25±2ºC after 25-30 days
of inoculation
Fig. 5.9 bar=2.2mm Fig. 5.10 bar=1.8mm
Fig. 5.11 bar=1.6mm
Fig.5.9: Callus induced in MS medium with 22.17 µM BAP + 5.35 µM NAA
Fig.5.10: Callus induced in MS medium with 2.21 µM BAP + 5.71 µM IAA +
4.52 µM 2, 4-D
Fig.5.11: Callus induced in MS medium with 9.3 µM Kin
59
5.1.5 Comparison of different growth regulators supplemented media and
explant type
Fig. 5.12: Effect of different growth regulators supplemented in MS medium on
callus induction using different explants
It is evident from above mentioned results that C-8 is the best combination of
hormones added in MS medium for callus induction as also shown in Fig. 5.12. It
gave 100% callus induction with all different explants used. However, older leaf
explants gave 60% callus induction. Other combinations like C-9 and C-10 also gave
good callus induction. It is also clear in Fig. 5.12 that young leaf explants had shown
more potential of callus induction with different combinations (from C-2 to C-10) as
compared to other explants.
0
20
40
60
80
100
120
C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10
Cal
lus
ind
uct
ion
%
MS medium with different growth regulators
Younger leaf older leaf Cotyledonary leaf Hypocotyl
60
5.1.6 Callus induction from leaf explants of Jatropha curcas L. kept in dark.
Callus induction from leaf explants cultured on MS medium with 22.17 µM
BAP + 5.35 µM NAA was also observed by keeping the cultures in dark at 25±2ºC.
Number of days for callus induction and callus induction frequency were same as in
16 h photoperiod except that calluses were white in color and loose friable (Fig. 5.13)
instead of green and compact. Callus developed in dark had also vigorous growth.
However, these calluses were non-embryogenic as they had not shown any
regeneration potential when sub-cultured to regeneration medium.
Fig.5.13 bar=2.4mm
Fig.5.13. Callus from leaf explant when kept in dark conditions
5.1.7 Maintenance of callus cultures
Callus cultures of Jatropha curcas developed on MS medium with 22.17 µM
BAP + 5.35 µM NAA could be maintained for 11-12 months if sub-cultured after
every 22 days on the same medium. With passage of time calluses lose their green
color and compactness and became whitish and friable. Fig. 5.14 is showing the
morphology of a seven month old callus that have been sub-cultured regularly at
intervals of 22 days. Callus cultures become brown and necrotic if not sub-cultures
within 30 days (Fig. 5.15).
61
Fig 5.14 bar=2.5mm
Fig 5.14: Seven month old callus culture developed and maintained on the same
medium having 22.17 µM BAP + 5.35 µM NAA.
Fig 5.15 bar=3mm
Fig 5.15: Callus culture developed on MS medium having 22.17 µM BAP + 5.35
µM NAA after 35 days of sub-culturing on the same medium
62
5.2 Standardization of medium for callus mediated regeneration and
subsequent elongation of shoots of Jatropha curcas
MS medium fortified with 22.17 µM BAP + 5.35 µM NAA showed maximum
callus induction frequency and maximum growth. These calluses showed maximum
regeneration potential when sub-cultured and hence were used for further regeneration
experiments. Different concentrations of growth regulators like GA3, TDZ and Kin
along with BAP, NAA and IBA were used in the present investigation. Different
combinations and concentrations of these growth regulators used were named as R-1
to R-15. Proliferated calluses (22 days old) were shifted to these media for
regeneration. Maximum regeneration frequency (38%) was achieved on R-15 medium
that was MS medium with 6.65 µM BAP + 2.45 µM IBA but maximum number of
shoot bud per culture vessel (16) were produced by sub-culturing on the R-1 medium
that was same medium on which callus was developed having 22.17 µM BAP + 5.35
µM NAA. Lowering the concentrations of both BAP and NAA in the medium (6.65 +
2.67 µM respectively) resulted in enhanced regeneration frequency. Addition of GA3
and Kin in the medium have not shown any significant effect on regeneration
frequency, however TDZ added in the medium had shown negative effect on
regeneration as shown in Table 5.5. The replacement of NAA with IBA in the
medium along with BAP had significantly enhanced regeneration frequency (R-14).
Lowering the concentrations of both BAP and IBA further enhanced the regeneration
frequency (R-15) as is clear in Table 5.5.
63
Fig 5.16- 5.22: Shoot bud initiation and regeneration from callus cultures of
Jatropha curcas
Fig.5.16 bar=3.2mm
Fig.5.17 bar=3mm
Fig.5.18 bar=3mm
64
Fig.5.19 bar=2.5mm
Fig.5.20 bar=3mm
Fig.5.21 bar=3mm
65
Fig.5.22 bar=6mm
Fig. 5.16: Shoot bud initiation after 15 days of sub-culturing on R-14 medium
(Arrow showing the cluster of multiple shoot buds)
Fig. 5.17: Shoot bud initiation after 15 days of sub-culturing on R-1 medium
(Arrows indicating multiple shoot buds development)
Fig. 5.18: Shoot elongation after 30 days of sub-culturing on R-1 medium (Arrow
is indicating elongation of shoot developed from shoot buds)
Fig. 5.19: Shoot elongation after 60 days of sub-culturing on R-1 medium
(Arrows are indicating further elongation of shoots)
Fig. 5.20: Shoot bud initiation (arrows) after 20 days of sub-culturing on R-2
medium
Fig. 5.21: Shoot elongation (arrows) after 30 days of sub-culturing on R-2
medium
Fig. 5.22: Shoot elongation along with shoot primordia (arrow) after 30 days of
sub-culturing on R-15 medium
66
Table 5.5: Effect of growth regulators added in MS medium for Regeneration from
Callus Cultures of Jatropha curcas
Mediu
m
Concentrations of growth
regulators in MS medium
Regeneration
frequency
(%)
No. of
shoots per
culture
vessel
Average
shoot
length (cm)
R-1 22.17 µM BAP + 5.35 µM
NAA
10.6±2.60g 16.33±1.15a 0.96±0.21a
R-2 6.65 µM BAP + 2.67 µM
NAA
26.2±2.16c 8.0±1.00bc 0.8±0.20ab
R-3 6.65 µM BAP + 2.67 µM
NAA + 0.72 µM GA3
23.6±2.07cd 6.67±0.57d 0.5±0.20b
R-4 22.17 µM BAP + 5.35 µM
NAA + 0.72 µM GA3
11.8±2.04fg 8.0±0.00bc 0.56±0.21b
R-5 22.17 µM BAP + 5.35 µM
NAA + 0.72 µM GA3 + 1.5
µM Kin
10.0±3.53g 5.33±0.57e 0.73±0.35ab
R-6 22.17 µM BAP + 5.35 µM
NAA + 0.5 µM TDZ
0h 0g 0c
R-7 22.17 µM BAP + 5.35 µM
NAA +1.0 µM TDZ
0h 0g 0c
R-8 22.17 µM BAP + 5.35 µM
NAA + 2.0 µM TDZ
0h 0g 0c
R-9 22.17 µM BAP + 5.35 µM
NAA + 3.0 µM TDZ
0h 0g 0c
R-10 22.17 µM BAP +10.70 µM
NAA + 1.0 µM TDZ
0h 0g 0c
R-11 22.17 µM BAP + 5.35 µM
NAA + 0.5 µM Kin
14.0±4.18f 4.0±1.00f 0.5±0.26b
R-12 22.17 µM BAP + 5.35 µM
NAA + 1.0 µM Kin
18±2.73e 8.66±1.53b 1.06±0.15a
R-13 22.17 µM BAP + 5.35 µM
NAA + 1.5 µM Kin
23.0±2.73d 7.33±0.57cd 0.76±0.30ab
R-14 22.17 µM BAP + 14.7 µM
IBA
30.0±3.53b 5.33±0.57e 0.96±0.15a
R-15 6.65 µM BAP + 2.45 µM
IBA
38.0±2.73a 5.33±0.57e 1.0±0.26a
Data presented here are means of 10 values per treatment
Different letters within a column represent significant difference at P=0.05 according to Duncan’s
multiple range test
67
5.3 Standardization of medium for direct regeneration from young
leaf explant of Jatropha curcas
Direct regeneration means shoot buds induction directly from the explants
without an intervening callus formation. Most of the growth regulators supplemented
in MS medium for regeneration from young leaf explant studied during the present
experiments resulted in callus induction. Only the MS medium with 6.65 µM BAP +
2.45 µM IBA resulted in direct initiation of shoot buds from young leaf explants as
shown in Fig. 5.23. The regeneration frequency attained here was 30%. There was
also swelling of explant and minute callus formation along with shoot bud induction
after 25 to 30 days of inoculation. Developed shoot buds were elongated and then
shifted to rooting medium when reached the length of 2 to 3 cm.
Fig.5.23: Multiple shoot regeneration (arrows) directly from surface of young
leaf explant of Jatropha curcas, bar= 4.3mm
68
5.4 Rooting of Regenerated Shoots
Regenerated shoots (2-3 cm length) were shifted to rooting medium for the
completion of regeneration process (Fig. 5.24 and 5.25). Different growth regulators
(IBA, NAA and IAA) were added in ½ MS and MS medium for root induction. Root
induction was not very successful in our experiments. MS medium with 4.9 µM IBA
have shown 6.67% root induction as is clear from Table 5.6. Other growth regulators
NAA and IAA supplemented in either full or ½ MS medium could not support root
induction in developed shoots. However, callus formation was noticed at the base of
shoots which is clear in Fig 5.25.
Table 5.6: Effect of different growth regulators on root induction in regenerated
shoots of Jatropha curcas
Medium Medium Composition Rooting frequency
T-1 MS 0b
T-2 MS + 4.9 µM IBA 6.67±5.39a
T-3 ½ MS 0b
T-4 ½ MS + 4.9 µM IBA 0b
T-5 MS + 5.35 µM NAA 0b
T-6 MS + 5.71 µM IAA 0b
Data presented here are means of 10 values per treatment
Different letters within a column represent significant difference at P=0.05 according to Duncan’s
multiple range test
69
Fig. 5.24 bar=6.5mm
Fig. 5.25 bar=6.5mm
Fig.5.24: Regenerated shoots shifted to rooting medium
Fig.5.25: Regenerated shoot shifted to rooting medium showing the formation of
callus (arrow) at the base of shoot
70
Discussion
Jatropha curcas have been investigated previously for micropropagation by
many workers (Khemkladngoen et al., 2011; Li et al., 2012; Biradar et al., 2012;
Maharana et al., 2012; Soong et al., 2016; Soares et al., 2016; Liu et al., 2016). Role
of different growth regulators using variety of explants have been studied for this
purpose. However reproducibility of the suggested protocols is still limited. Thus
there is wide scope for further improvement of methods.
In the present experimental work, in vitro regeneration of Jatropha curcas has
been studied systematically including explant types, leaf age, different growth
regulators (their concentrations and combinations) and light conditions (16/8 hours
photoperiod or complete darkness). It was evident from results that the best hormone
composition for callus induction was 22.17 µM BAP + 5.35 µM NAA kept in 16/8
hours photoperiod. It gave 100% callus induction frequency with younger leaf,
embryogenic leaf and hypocotyl explants and 60% with older mature leaf explants
(Fig.5.12). These calluses had also shown good regeneration potential when sub-
cultured on regeneration media. However, same cultures kept in complete darkness
not only have a different morphology of callus but also did not show any regeneration
potential. The same combination of hormones (1.0 mg/l NAA and 5.0 mg/l BAP)
supplemented in MS medium were proved best for organogenic callus induction when
leaf was used as explant by Rajore and Batra, (2007). However in the present
experiments the combination was also proved best for cotyledonary leaf and
hypocotyl explants.
Callus cultures of Jatropha curcas developed in the present experiments were
maintained for more than one year by repeated sub-culturing on the same medium
after every 22 days. Morphology of callus was changed with every sub-culture. They
71
become more loose and friable and also lose their green color and embryogenic
response with successive sub-cultures. However, calluses started browning or necrotic
if not sub-cultured upto 30 to 35 days. That browning of calluses may be the result of
accumulation and oxidation of phenolic compounds if not sub-cultured within specific
time period (Dubravina et al., 2005). Thus it can be concluded that successive sub-
cultures on fresh medium is necessary to maintain the growth of calluses.
Plant regeneration is mostly influenced by characteristics of explant, like age
of plant, its genotype, and part of plant from where explant is excised (Gamborg et
al., 1976). Here it was also observed that young leaves of 1st, 2nd, or 3rd node gave
much better results as compared to mature older leaves. Seeni and Latha, (1992) were
also reported that young leaves of red Vanda showed regeneration potential on
medium containing 44.4 µM BA and 10.7 µM NAA while mature leaves did not show
any response. In another study on peanut plantlets by Mroginski et al., (1981), it was
observed that maximum regeneration was achieved on medium with 1 mg/l each of
BA and NAA with young leaves while no regeneration at all with mature leaves. Phua
et al., (2016) also investigated that young leaf explants of Clinacanthus nutans had
shown vigorous growth of callus and had the tendency to develop somatic embryos
than mature leaf. Similarly Zhang et al., (2013) also observed that young Jatropha
curcas leaves have more potential of regeneration as compared to mature leaves.
Effect of variable amount of light on callus induction in different plant species
was also reported previously by some workers (Jaramillo and Summers, 1991; Khan
et al., 2006; Rikiishi et al., 2008; Afshari et al., 2011). In the present experiments
callus cultures developed in light conditions only have regeneration potential while
cultures developed in dark conditions are white, friable and also did not show
regeneration potential. Our results are in line with Rikiishi et al., (2008) who also
72
suggested that when cultures were incubated in continuous dark for callus induction,
they lose the subsequent regeneration ability in one variety of barley however other
verities showed opposite results. Bordon et al., (2000) and Liu et al., (2001) are of the
view that effect of light conditions on callus induction and subsequent regeneration
are species-dependent while Afshari et al., (2011) observed that incubation of
cultures in light conditions stimulated the callus growth in rapeseed (Brasica napus
L.) irrespective of genotype or explant. Khan et al., (2006) also investigated that yield
of callus under dark incubation is greater as compared to light incubation in one
genotype of Citrus reticulata and vice versa in other genotype hence concluding that
effect of light or dark conditions was genotypic dependent.
TDZ is a substance that possesses the characteristics of both auxins and
cytokinins and is commonly used in tissue culture of woody plants. Our cultures did
not show any response of callus induction or organogenesis when medium was
fortified with various levels of TDZ. However, Soong et al., (2016) while working on
organogenesis in Jatropha curcas reported more callus induction and shoot formation
in TDZ supplemented medium than others. They have used higher TDZ
concentrations (15µM) compared to our experiments. The positive effect of TDZ in
the medium as basic plant growth regulator for direct organogenesis using various
explants like leaves, cotyledons, petioles, hypocotyl and radical have also been
reported previously (Deore and Johnson, 2008; Khurana-Kaul et al., 2010; Kumar et
al., 2010; kumar et al., 2011; Sharma et al., 2011; Zhang et al., 2013). However, Liu
et al., (2016) concluded that use of TDZ in the medium resulted in induction of lower
quality shoot buds with lesser regeneration potential. They further suggested that the
pretreated explants in higher concentrations of TDZ have shown much better results.
73
Kinetin is another plant growth regulator frequently used in tissue culture for
micropropagation. Role of kinetin in embryogenic callus induction using Jatropha
curcas leaf as explants was reported by Jha et al., (2007). In the present experiments,
embryogenic callus was formed on MS medium supplemented with Kin in different
concentrations but time required was high and callus induction frequency was lower.
However, Kin also plays important role for shoot induction from embryogenic callus
when used in the medium in combination with BAP and NAA. These results agree
with that of Zhang et al., (2013) who also investigated that Kin played important role
in shoot bud induction in Jatropha curcas. Previously Deore and Johnson, (2008) and
Kumar et al., (2010) have also identified the role of Kin in shoot elongation. Jeevan et
al., (2013) also reported the BAP and Kin as best combination for maximum shoot
bud induction from nodal segments.
Kin and GA3 added together in the culture medium have enhanced the shoot
bud induction frequency than they were added independently (Zhang et al., 2013).
However, our results were not matched with them as frequency of shoot bud induction
was higher when Kin was added with BAP and NAA than with addition of GA3.
Addition of BAP and NAA in MS medium has also shown supportive role in
shoot bud induction on further sub-culturing, along with callus induction. However,
callus induction frequency was lower on that combination of growth regulators.
Lowering the levels of both (BAP and NAA) was helpful in increasing the shoot bud
induction frequency. Our results match with that of Imtiaz et al., (2014). They also
reported that by increasing BAP concentration in MS medium, shoot length and
number of shoots (per explant) were reduced. In the present experiment maximum
number of shoots (16- 17 per explant) were also produced on MS medium having
22.17 µM BAP + 5.35 µM NAA. Elias et al., (2015) while working on
74
micropropagation of Echinocereus cinerascens, also proved the same combination of
hormones as best for maximum shoot bud induction per explant.
Maximum shoot bud induction frequency from callus cultures was achieved
on medium with 6.65 µM BAP + 2.45 µM IBA (table 5.5). Previously Qin et al.,
(2004) also observed maximum shoot bud induction frequency with the same
combination of growth regulators (0.5 mg/l BAP, 0.1 mg/l IBA) using epicotyl as an
explant. Same combination of growth regulators was also proved successful for direct
shoot induction from young leaf explants without an intervening callus induction.
These results also agree with Misra et al., (2010).
Various growth regulators like TDZ, Kin, GA3, IAA have been studied by
workers and proved their role in direct shoot induction in Jatropha curcas explants
(Khurana-Kaul et al., 2010; Singh et al., 2010; Purkayastha et al., 2010; Kumar et al.,
2010; Kumar and Reddy, 2010). However, in our experiments these growth regulators
when used in different combinations resulted in either callus induction or drying of
explants even after 30 to 40 days of inoculation.
Regenerated and elongated shoots (2-3 cm length) were sub-cultured for root
induction on ½ MS or MS medium with auxins (IBA, NAA or IAA). MS medium
with 4.9 µM IBA was the only medium favoring root induction. Different
concentrations of IBA supplemented to full or ½ MS medium were also shown to be
helpful by many workers (Rajore and Batra, 2007; Khemkladngoen et al., 2011; Li et
al., 2012). However, rooting frequency was low in our experiments. It was due to
callus formation at the base of regenerated shoots when shifted to rooting medium.
The same problem rooting of regenerated shoots of Jatropha curcas was also faced
previously by some workers (Daud et al., 2013).
75
Hence, it can be concluded from the present investigations that 22.17 µM BAP
+ 5.35 µM NAA supplemented in MS medium is the best combination for
embryogenic callus induction within 5 days with all the explants of Jatropha curcas
used in the present experiments except with older leaf explants where it take 18-20
days for callus induction. It was also observed that 16/8 h photoperiod is more
suitable for embryogenic callus induction than complete darkness. Developed calluses
were having potential to survive for more than one year if sub-cultured repeatedly on
fresh medium every 22 days. Later, these calluses were shifted to fresh medium after
25-30 days of initial inoculation for shoot bud induction. It was clearly observed that
same combination of growth regulators (22.17 µM BAP + 5.35 µM NAA) produced
the maximum shoot buds per culture tube. However, lowering the concentration of
both was helpful in enhancing the frequency of callus induction and lowering the
number of shoot buds. At the same time maximum shoot bud induction frequency was
achieved on medium with 6.65 µM BAP + 2.45 µM IBA supplemented. Direct shoot-
organogenesis from young leaf explant was also achieved on the same combination
(MS + 6.65 µM BAP + 2.45 µM IBA). Developed shoots (2-3 length) were shifted to
rooting medium which was not very successful in our experiments and only 6-7% root
induction was observed with 4.9 µM IBA. However developed shoots were
successfully acclimatized in field conditions. Hence, the developed protocol of callus
induction and its proliferation has large application in further improvement of this
plant using different techniques including genetic transformation and also for the
production of bioactive compounds. At the same time callus mediated and direct
regeneration is helpful in terms of mass propagation of this plant.
76
Effect of sorbitol induced osmotic stress on seed germination, early
growth of seedlings and callus cultures in Jatropha curcas L.
Results
6.1 Effect of different sorbitol concentrations on seed germination of
Jatropha curcas
Germination percent was not only decreased but also delayed with an increase
in sorbitol concentration from 0 to 0.3 M as shown in Fig. 6.1. After 2 days of in vitro
sowing of seeds most of seeds start germination in control and 0.05 M sorbitol while
no remarkable germination was observed in higher concentrations. After 4 days, most
of the seeds also germinated in 0.1 M and after 6 days in 0.15 M. After 10 days there
was also some increase in percentage germination in higher sorbitol concentrations. It
is clear from the data that increased sorbitol concentration not only decreased the rate
of germination but also delayed its onset. At the same time it is also clear that after 10
days sorbitol concentrations more than 0.15 M significantly affect percentage
germination. However, at the same time germination energy was also reduced
significantly with increasing osmotic stress (Fig. 6.2).
77
Fig. 6.1: Effect of different sorbitol concentrations on in vitro seed germination of
Jatropha curcas
Data presented here are means of 30 values per treatment
Different alphabetical letters on same colour bars are showing that they are significantly different
(P=0.05) according to Duncan’s multiple range test
Fig. 6.2: Effect of different sorbitol concentrations on germination energy of Jatropha
curcas
Data presented here are means of 30 values per treatment
Different alphabetical letters on bars are showing that they are significantly different (P=0.05)
according to Duncan’s multiple range test
78
6.2. Effect of different sorbitol concentrations on fresh/dry weight,
shoot/root length of germinating seedlings of Jatropha curcas L.
Fresh/dry weight and shoot/root length of germinating seedlings under
different treatment levels were measured after 10 days of inoculation. Fresh, dry
weight and shoot length of germinating seedlings were decreased with enhanced
sorbitol treatment as is clear from Fig. 6.3, 6.4 and 6.5. However, root length was not
affected significantly up to 0.1 M sorbitol concentration and then afterward decreased
with increasing sorbitol concentration as shown in Fig. 6.3.
Fig. 6.3: Effect of different sorbitol concentrations on shoot/root length of
germinating seedlings of Jatropha curcas
Data presented here are means of 30 values per treatment
Different alphabetical letters on same colour bars are showing that they are significantly different
(P=0.05) according to Duncan’s multiple range test
79
Fig. 6.4 Effect of different sorbitol concentrations on fresh/dry weight of germinating
seedlings of Jatropha curcas
Data presented here are means of 10 values per treatment
Different alphabetical letters on same colour bars are showing that they are significantly different
(P=0.05) according to Duncan’s multiple range test
Fig. 6.5 Effect of different Sorbitol concentrations on physical appearance of in vitro
germinating seedlings of Jatropha curcas bar=10mm
80
6.3 Effect of different sorbitol concentrations on protein contents,
peroxidase and superoxide dismutase activities in germinating
seedlings of Jatropha curcas L.
After 10 days, in vitro germinating seedlings growing under different sorbitol
treatment levels were taken out of culture tubes and crushed following the procedures
mentioned in methodology section for extraction of soluble protein contents and
antioxidant enzymes. When they were quantitatively analyzed, it became clear that
soluble protein contents were not affected significantly with increase in sorbitol
treatment as compared to control. However, activities of SOD and Peroxidases of in
vitro germinating seedlings were enhanced with increase in sorbitol concentrations in
culture medium compared to control as can be seen in Fig. 6.6 (a, b, c).
81
Fig. 6.6 (a, b, c): Effect of different sorbitol concentrations on biochemical
parameters of in vitro germinating seedlings
Data presented here are means of 6 values per treatment
Different alphabetical letters on same colour bars are showing that they are significantly different
(P=0.05) according to Duncan’s multiple range test
82
6.4 Effect of different sorbitol concentrations on fresh /dry weight
and water contents of Jatropha curcas callus cultures
Effect of sorbitol-induced osmotic stress on callus cultures of Jatropha curcas
was studied. It was observed that frequency of callus induction was not influenced
even at 0.5 M sorbitol concentration. Fresh weight of callus cultures first increased in
lower sorbitol concentrations and then decreased with further increase in sorbitol
concentrations as shown in Fig. 6.7(a). It was also clear from data presented in Fig.
6.7(a) that dry weight of callus cultures were not affected significantly as compared to
control but water contents (Fig. 6.7b) followed the same pattern as fresh weight of
callus cultures.
83
Fig. 6.7 (a, b): Effect of different sorbitol concentrations on fresh/dry weight and
water contents of callus cultures derived from Jatropha curcas leaf explants
Data presented here are means of 15 values per treatments
Different alphabetical letters on same color bars are showing that they are significantly different
(P=0.05) according to Duncan’s multiple range test
84
6.5: Effect of different sorbitol concentrations on SOD, POX and
soluble protein contents of Jatropha curcas callus cultures
There was no significant effect on peroxidase activity shown by callus
cultures upto 0.35 M sorbitol and then increased with further increase in sorbitol
concentration (Fig. 6.8a). Osmotic stress also had not shown any remarkable effect on
SOD activity of callus cultures (Fig. 6.8b).
Soluble protein contents in callus cultures were also remained
unaffected with increase in osmotic potential of the medium. However there was
slight increase in soluble protein contents of calluses in highest sorbitol concentration
(Fig. 6.8c).
85
Fig. 6.8 (a, b, c): Effect of different sorbitol concentrations on SOD/POX activities
and protein contents of Jatropha curcas callus cultures
Data presented here are means of 6 values per treatments
Different alphabetical letters on same color bars are showing that they are significantly different
(P=0.05) according to Duncan’s multiple range test
86
Discussion
Seed germination is the first step towards adult phase during which it utilizes
the stored food to develop into an active autotrophic seedling. This process cannot be
completed in the absence of water (Belin and Lopez-Molina, 2008). The most
sensitive stages in life cycles of many crop plants to environmental stresses are seed
germination and germinating seedlings (Jones, 1986). Under water stress, cell
development is suppressed because of turgor pressure lose and osmotic imbalance
which results in reduced growth and metabolic activity of the plants and finally leads
to their death (Jaleel et al., 2008). To maintain the osmotic equilibrium, plants
produce several osmotica such as mannitol, sorbitol, sucrose etc. These compounds
not only increase the solute concentration but also play a major role to protect the
cells from dehydration damage (Rontein et al., 2002). However, higher accumulation
of these compounds cause impaired growth in plants (Maggio et al., 2002) and behave
as stress agents (Da-Silva, 2004). During the present investigation, sorbitol was used
to induce osmotic stress for in vitro seed germination and callus cultures. Sorbitol is a
sugar alcohol which is used in various in vitro experiments to create osmotic stress in
culture medium (Jovanovic et al., 2000; Brito et al., 2003; Frank et al., 2005 etc).
Increased sorbitol concentrations mean an increase in osmotic potential which can
create a drought like condition. Increase in osmotic stress had a significant effect on
in vitro germination of seeds in the recent experiments. It has not only reduced the
percentage germination but also delayed it. A decreased germination percentage with
increased osmotic stress was also observed by Pratap and Sharma, (2010). Similar
decreases in seed germination of different plants have also been reported by several
workers ((Khan and Ungar, 1984; Woodell, 1985; Gupta et al., 1993; Singh et al.,
1996; Ungar, 1996). Reduced percentage germination and delayed onset of
87
germination of Jatropha curcas seeds by an increase in drought stress from -4 to -20
bars was also reported by Singh and Saxena, (2010). Decreased germination by
abiotic stresses was also reported by Shakirova and Sahabutdinove, (2003). Windauer
et al., (2012) also reported that Jatropha curcas seeds show high sensitivity to water
shortage in terms of germination. Reduced germination of seeds under osmotic stress
or water stress is related to the development of an osmotically enforced dormancy (an
adaptive strategy) under stressful conditions (Singh et al., 1996; Prado et al., 2000).
However at the same time, reduced water potential and water contents in embryos and
endospermic tissues under osmotic stress was observed by several workers in
different plants (Siddique et al., 2000; Prado et al., 2000; Gill et al., 2001). Hence it
can be concluded that tissues were in stressful situation thus preventing germination
process.
There was a remarkable decrease in fresh/dry weight, shoot/root length of in
vitro germinating seedlings with increase in osmotic stress. Severe reduction in in
vitro developed plantlets of potato on culture medium with 0.3 to 0.4 M sorbitol was
observed by Gopal and Iwama, (2007). Reduced plantlet growth may be due to high
osmotic stress caused by high sorbitol concentration that exceed the plantlet capacity
of osmotic adjustment which resulted in severe water deficit conditions for the
plantlets in the culture medium. The reduced vegetative growth of plants in stress
conditions may be attributed to cyclin-dependent kinase-activity reduction. That
causes slower cell division under low water conditions (Schuppler et al., 1998).
Decrease in shoot length was more pronounced as compared to root length. Díaz-
López et al., (2012) were also of the same view that aerial parts of Jatropha curcas
plants show more reduced growth in drought stress as compared to roots. They also
investigated the decreased growth under water deficit conditions was the result of
88
decreased carbon dioxide assimilation. Decreased carbon fixation in stress conditions
resulted in affected plant growth was also reported by Delfine et al., (2001). These
results are in line with that of Yin et al., (2010) who reported that with increase in
drought stress leaf relative water content, height, diameter, leaf area, photosynthesis,
transpiration and stomatal conductance of Jatropha curcas plants decreased
significantly. Decreased leaf growth of Jatropha curcas during water stress conditions
was also investigated by Silva et al., (2010b).
Response of callus cultures and/or whole plants to various abiotic stresses
have also been studied and compared for many plant species (Smith and McComb,
1981; Rus et al., 1999; Wang et al., 1999; Al-kaaby and Abdul-Qadir, 2011). Some
are of the view that their response to stresses are very similar to each other while
others have contrasting views depending upon the plant species and their genotypes
under study. Like Smith and McComb, (1981) while working on different plant
species under stress conditions reported that Beta Vulgaris showed same response at
whole plant level and at tissue level (callus culture) in terms of growth. On the other
hand, they also reported that Phaseolus vulgaris showed very different responses to
osmotic-stress at whole plant level and at tissue level in terms of growth.
Increase in callus fresh weight and water contents in lower sorbitol
concentrations may be due to increased accumulation of carbohydrates for turgor
maintenance as also reported by Javed and Ikram, (2008). Plants which cannot
maintain turgor are having poor growth with the increasing stress (Newton et al.,
1987). Decrease in callus fresh weight and water contents at higher sorbitol
concentrations can be correlated with loss of cell turgor (Rhodes and Samaras, 1994).
Abu-Romman, (2010) also reported decrease in fresh weight of cucumber callus
cultures at higher sorbitol concentrations. The in vitro growth retardation at higher
89
concentrations of sorbitol might be the result of phenolic compounds accumulation
(Hilae and Te-Chato, 2005). The other possibility may be due to the fact that drought
can lead to changes in cell structure, metabolic disruption and ultimately results in the
termination of enzymatic reactions of the plants (Smirnoff, 1993).
Exposure of plants to various abiotic stresses including drought stress leads to
production of ROS that include superoxide radicals (O2-), hydroxyl radicals (OH),
hydrogen peroxide (H2O2) and singlet oxygen (Munne-Bosch and Penuelas, 2003). In
order to remove these ROS, plants have developed certain defense systems out of
which antioxidant enzymes are most efficient (Polle and Rannenberg, 1994).
Antioxidant enzymes play significant role in abiotic stress tolerance ability of plants.
Activities of antioxidant enzymes correlated with drought tolerance abilility of plants
were observed by a number of workers (Tanaka et al., 1990; Kubo et al., 1999;
Turkan et al., 2005). Peroxidase and SOD activities of in vitro germinating seedlings
were enhanced in sorbitol added medium significantly as compared to control in
present investigation. Zhang and Kirkham, (1994) also reported enhanced SOD and
POX activities of wheat seedlings in early stages of growth under drought stress
conditions. Enhanced antioxidant enzyme activities of plants in PEG-induced water
stress were also detected by Gupta et al., (2005). Simova-Stoilova et al., 2009
reported enhanced antioxidant enzyme activities in drought tolerant varieties
compared to sensitive ones. Badawi et al., (2004) while working on transgenic
tobacco plants investigated that over-expressing SOD activity plants were showing
more tolerance to PEG-induced water stress. It has confirmed the role of SOD in
stress tolerance. Role of antioxidant enzymes in stress-tolerance ability of Jatropha
curcas seedlings was also studied by several workers (Kumar et al., 2008; Luo et al.,
2010; Shu et al., 2012; Yin et al., 2012).
90
In case of callus cultures, POX and SOD activities were not affected up to
0.35 M sorbitol added medium and then enhanced significantly above that
concentration. This indicated that callus cultures showed different behavior as
compared to in vitro germinated seedlings. Neumann, (1997) also reported such
contrasting behavior at cellular level and whole plant level. They concluded that
isolated cells are much more tolerant to osmotic stress as compared to whole plant.
Apart from SOD and POX, xanthophyll cycle, photorespiration and antioxidants
present in cell organelles (chloroplasts and peroxisomes) also serve to prevent
oxidative damage (Coue´e et al., 2006). This could be the reason for unaffected POX
and SOD activities up to 0.35 M sorbitol concentration.
Increase in protein contents of Jatropha curcas germinating seedlings at 0.05
M and 0.1 M in the present experiment may be due to production of stress induced
proteins (Cherian and Reddy, 2003) while decrease in soluble protein content at
higher osmotic potential may be due to decreased production of proteins under stress
conditions (Vogel et al., 2011). Decrease in protein contents with sorbitol-induced
osmotic stress was also reported by Brito et al., (2003). Some stress proteins produced
as a result of stress applied to plants were also reported by some workers in Jatropha
curcas (Zhang et al., 2007; Qin et al., 2005). Protein contents of Jatropha curcas
callus cultures were not affected in sorbitol added medium except at extreme stress
conditions.
Hence it is clear in the recent study that sorbitol-induced osmotic stress have
reduced as well as delayed the germination of Jatropha curcas seeds. At the same
time germinating seedlings also showed sensitivity to osmotic stress as it is evident
from reduced growth and enhanced antioxidant enzyme activities. Later, callus
cultures developed under different osmotic stress conditions from leaves of mature
91
plants did not show any significant effect on their growth and biochemical
parameters.
92
Effect of different field capacities of water in pot soil on five-
month-old plants of Jatropha curcas L.
Results
6.6 Effect of different field capacities of water on morphological
features of Jatropha curcas plants
Leaves from five month old plants with water deficit treatment did not show
any visual symptoms of stress like necrosis, chlorosis or even drying as shown in Fig.
6.9. Only the plants that were not watered at all (0% F.C.) showed slight decrease in
fresh/dry weight of leaves per unit area. Maximum fresh/dry weight and water
contents (per unit area) of leaves were shown by plants with moderate water supplies
(50% and 75% F.C.) as shown in Fig. 6.10.
Figure 6.9: Effect of different field capacities of water in pot soil (after one month)
on physical appearance of 5 month old plants of Jatropha curcas
(Picture was taken on 3rd October) bar=6 inch
93
Fig. 6.10: Effect of different field capacities of water in the pot soil om fresh/dry
weight and water contents per unit area of Jatropha curcas leaves after
one month of treatment
Data presented here are means of 6 values per treatments
Different alphabetical letters on same color bars are showing that they are significantly different
(P=0.05) according to Duncan’s multiple range test
94
6.7 Effect of different field capacities of water on biochemical
activities in leaves of Jatropha curcas
There is no significant effect on peroxidase activity shown by leaves in all
levels of water stresses applied. Minimum SOD activity was shown by leaves of
plants in 50% field capacity and there was trend towards increase in SOD activity
both in lower (0 and 25%) and higher (75 and 100%) field capacities. Maximum
soluble protein contents were detected in leaves that were not watered at all during the
experimental period (0% field capacity) as is clear in Table 6.1.
Table 6.1: Effect of different field capacities of water in the pots soil on
biochemical parameters of Jatropha curcas leaves after one month of
treatment.
Treatments ( %
Field Capacity)
Peroxidase
activityA
(mg/g/min)
SOD activityA Soluble proteinsA
µg/g fresh wt. of
sample
0 0.062±0.003a 60.72±0.43ab 0.214±0.002a
25 0.051±0.01a 59.64±0.72b 0.181±0.013b
50 0.079±0.006a 48.62±1.16d 0.166±0.006bc
75 0.077±0.02a 54.07±1.63c 0.142±0.005c
100 0.072±0.004a 62.99±0.19a 0.185±0.011b
AData presented here are means of 6 values per treatments
Different alphabetical letters on same color bars are showing that they are significantly different
(P=0.05) according to Duncan’s multiple range test
95
Discussion
Availability of water is an important factor influencing plant development and
its yield. Responses of plants to drought stress vary considerably among different
plant species (Silva et al., 2012b). Different field capacities of water in pot soil were
applied on 5-month-old Jatropha curcas plants in present experiments that could be
categorized as water deficit (0% F.C), moderate water supplies (25%, 50% and 75%
F.C) and excess water supplies (100% F.C). It was observed that there was no visual
symptom of chlorosis; necrosis or even drying in all field capacities of water under
observation even at 0% field capacity (did not watered at all during the whole month
of experiment). These observations are in line with previous workers (Silva et al.,
2012a) who indicated that Jatropha curcas could withstand drought conditions
without any visual symptoms. But Gimeno et al, (2012) observed symptoms of
necrosis on defoliated leaves of flooded Jatropha curcas plants. Yin et al., (2012) also
suggested that Jatropha curcas plants are affected physiologically and biochemically
in excess soil water contents.
There was slight decrease in fresh weight and water contents per unit area of
leaves of plants that were not watered at all during the experiment period but dry
weight per unit area of leaves remain unaffected in the present investigation. Hence it
can be said that imposed time period of drought stress was not long enough for any
remarkable difference in biomass. However maximum dry weight was shown by
plants with moderate water supplies. That might be due to the reason that carbon
dioxide assimilation was reduced with decreasing water supplies as also reported by
Díaz-López et al., (2012). Our results disagreed with that of some of the previous
workers. Like, in a greenhouse experiment done by Niu et al., (2012), it was reported
that water deficit have remarkably affected the growth and development of Jatropha
96
curcas plants but growth was not stopped and plants continue to grow even at 30%
daily water use. However, in the present experiments it was witnessed that Jatropha
curcas plants continue to grow even without any water supply for one month
indicating that it is a drought tolerant plant. Maes et al., (2009) suggested that newly
formed leaves of dry treatment plants have developed stomata on both sides and make
their orientation vertical to receive equal radiations on both sides. That’s why
Jatropha curcas as compared to other stem succulent plants, have ability to withstand
drought stress without any leaf damage. While Silva et al., (2010b) were also of the
view that drought stressed plants showed restricted leaf growth.
Water stress can create oxidative damage in leaves due to an imbalance
between the light capture and electron fixation (Guerfel et al., 2009) which results in
an increased generation of reactive oxygen species. This oxidative stress can affect
peroxidation of membrane lipids, degradation of photosynthetic pigments and
inactivation of photosynthetic enzymes (Guerfel et al., 2009). Many plant species
have evolved multiple photoprotective and antioxidant mechanisms to withstand
drought-induced oxidative stress. Like in Jatropha curcas drought stress increased the
activities of SOD and POX as reported by Silva et al., (2012b) to avoid accumulation
of reactive oxygen species. In the present investigation minimum SOD activity was
shown by plant leaves with 50% field capacity and increase in SOD activity was
observed in both higher and lower field capacities.
Enhanced antioxidant enzyme activities in limited water supplies were also
reported by several workers. Pompelli et al., (2010) also reported that the activities of
antioxidant enzymes in leaves of Jatropha curcas were significantly higher in the
water-stressed plants compared to well-watered plants. Yin et al., (2012) also
concluded that antioxidant enzymes production was enhanced in stress conditions.
97
Enhanced catalase activity in Jatropha curcas plants was also observed by Santos et
al., (2013) during periods of low water availability in the soil.
Jatropha curcas plants were considered as flood sensitive by Gimeno et al.,
(2012). Excess water supplies in soil (75% and 100% F. C) were also proved to be
stress for Jatropha curcas plants and there was an increase in SOD activities as
compared to moderate water supplies (50%). There are no reports on activities of
antioxidant enzymes in Jatropha curcas plants under excessive water supplies but
there are certain reports available on other plants. Monk et al., (1987) reported
enhanced superoxide dismutase (SOD) activity when the rhizomes of Iris
pseudacorus were flooded. Reduced leaf water potential was reported by Gimeno et
al., (2012). This reduction was due to reduced permeability of plants to water uptake
in flooding conditions resulting in dehydration (Nicolas et al., 2005).
Enhanced soluble protein contents were observed in water deficit situation in
the present investigation. These results are supported by several workers in the
literature where it was indicated that abiotic stresses are responsible for accumulation
of stress proteins (Pareek et al., 1997; Ashraf and O’Leary, 1999; Radic et al., 2006).
Close, (1997) had observed that dehydrin (a protein) accumulate in plants in response
to water stress. Production of ribosome inactivating protein, curcin2 in Jatropha
curcas plants under stress conditions was also investigated by Qin et al., (2005). It
was proved to be involved in stress-tolerance ability of plants.
Our results indicated that five-months-old pot-plants were tolerant to water
stress, though exhibited better growth and development with moderate water supplies.
This tolerance to water stress is correlated with enhanced antioxidant enzyme
activities and soluble protein contents. Enhanced antioxidant enzyme activities in
98
excess water supplies in present experiments indicated that it is also a stressful
condition for Jatropha curcas plants.
99
Conclusion
The present study was focused on propagation of Jatropha curcas using tissue
culture techniques and on elucidation of effect of water/osmotic stress on
morphological and biochemical aspects of the plant. To achieve the goals, first step
was to break the dormancy of seeds using different pretreatments for a good
germination frequency either in soil or under in vitro conditions. It was concluded that
during dormant periods, the removal of seed coats from pre-sterilized Jatropha curcas
seeds only could break the dormancy of seeds to get 100% in vitro germination on full
strength MS medium kept in the dark at 25 ± 2˚C. However, seeds could not
germinate in soil. At the same time orientation of the seeds on the culture media also
had significant effect on its germination rate. In vitro germinating Seedlings were
successfully acclimatized by shifting to a mixture of peat, clay and silt (1:1:1 v/v) in
greenhouse.
In vitro techniques for Jatropha curcas propagation were also included callus
induction, callus-mediated regeneration and direct regeneration from different
explants using variety of growth regulators. However, it was concluded that 22.17 µM
BAP + 5.35 µM NAA supplemented in MS medium gave 100% embryogenic callus
induction with young leaf, embryogenic leaf and hypocotyl explants used and 60%
with mature leaf. 16/8 h photoperiod was also proved best as cultures kept in
complete darkness also give good callus induction frequency (90%) but calluses were
white friable and non-embryogenic.
Calluses were sub-cultured for shoot induction to MS medium having different
combinations of growth regulators and it was clearly noticed that maximum shoot
buds per culture vessel (17) were attained on medium having 22.17 µM BAP + 5.35
µM NAA and maximum shoot bud induction frequency (37%) was observed on
100
medium with 6.65 µM BAP + 2.45 µM IBA. Direct regeneration or development of
shoot buds on the surface of young leaf explant of Jatropha curcas was also obtained
on same medium with 6.65 µM BAP + 2.45 µM IBA. Rooting of regenerated shoots
was not very successful due to formation of callus at the base of shoots when shifted
to rooting medium.
Sorbitol-induced osmotic stress has reduced as well as delayed germination of
Jatropha curcas seeds. At the same time seedlings also showed sensitivity to osmotic
stress as it is evident from reduced growth and enhanced antioxidant enzyme
activities. Later, callus cultures developed under different osmotic stress conditions
from leaves of mature plants did not show any significant effect on their growth. Our
results indicated that five months old pot plants were tolerant to water stress, though
exhibited better growth and development with moderate water supply. Callus cultures
were mostly used in experiments for studying physical and biochemical parameters as
the response of individual cell was considered equivalent to the whole plant. We
might say that while the early seedling growth was affected by drought stress, mature
plants would have developed mechanism for combating the stress conditions. Callus
cultures were developed from young leaves of matured plants and their responses to
osmotic stress are almost the same as those of matured plants. Hence the mechanism
of stress tolerance perhaps works at cellular level in Jatropha curcas. Hence the use
of callus cultures could be suggested for further investigations of Jatropha curcas
under abiotic stresses. From this study, it could also be suggested that if provided with
adequate amount of water for germination and early seedling growth, Jatropha curcas
may be propagated in marginal wastelands in future
101
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Annexure 1
Formulation of MS Medium (Murashige and Skoog, 1962) for the Preparation of
Stock Solutions Ingredients Concentration in
stocks
Final Concentration in MS
medium
(a) Macronutrients g/L (20X) g/L
NH4NO3 1.65 x 20 = 33 1.65
KNO3 38 1.90
MgSO4. 6H2O 7.4 0.37
KH2PO4 3.4 0.17
CaCl2. 2H2O 8.8 0.44
(b) Micronutrients g/L (100X) g/L
MnSO4. 4H2O 0.0223 x 100 = 2.23 0.0223
ZnSO4. 4H2O 0.86 0.0086
H3BO3 0.62 0.0062
KI 0.082 0.00083
Na2MoO4. 2H2O 0.025 0.00025
CuSO4. 5H2O 0.0025 0.000025
CoCl2. 6H2O 0.0025 0.000025
(c) Vitamins g/L (100X) g/L
Glycine 0.002 x 100 = 0.2 0.002
Nicotinic acid 0.05 0.0005
Pyridoxine HCl 0.05 0.0005
Thiamine HCl 0.01 0.0001
(d) Myo-inositol g/L (100X) g/L
0.1 x 100 =10 0.1
(e) Iron g/L (200X) g/L
Na2 EDTA 0.0336 x 200 = 6.72 0.0336
FeSO4. 7H2O 5.56 0.0278
132
Annexure 2
Preparation of Stock Solutions for MS (Murashige and Skoog, 1962) Medium
a) Macronutrients
Macronutrients stock for MS medium was prepared at the final concentration of 20X
(Annexure 1, section a). All the salts were weighed individually and dissolved
separately in distilled water. Separately dissolved salts were mixed together in a
conical flask already containing an appropriate amount of distilled water so as to
avoid precipitation. The solution was then transferred to a 1000 ml capacity
volumetric flask to make up the final volume.
b) Micronutrients
Stock solution of micronutrients was prepared 100 times more concentrated than the
final volume (100X). All the salts of micronutrients as given in Annexure 1, under
section “b” were weighed and dissolved separately and made up to the final volume as
described above in section a.
c) Fe EDTA
Iron EDTA stock solution was prepared at a concentration of 200X. The salts for this
stock solution are given in Annexure 1, section c. The prepared 200X stock was
poured in an amber-colored bottle and stored in refrigerator. For the preparation of
1liter of MS medium, 5 ml of this stock solution was used.
d) Vitamins
Vitamins of MS medium were prepared as 200X. Separately dissolved vitamins (as
given in Annexure 1, section d) were transferred to a 500 ml volumetric flask and
final volume was made with distilled water. For the preparation of 1 liter medium, 5
ml of vitamin stock was used.
133
e) Myo-inositol
Stock solution of myo-inositol was prepared separately as 100X. It was prepared by
dissolving 10 g of myo-inositol in 1000 ml of distilled water and 10 ml of this stock
was taken for one litre MS medium.
134
Annexure 3
Preparation of Stock Solutions of Growth Regulators
Auxins (2,4-D, NAA, IBA etc.) were dissolved initially in a little quantity of 0.1 N
NaOH while the initial solvent for cytokinins (BAP, TDZ, Kinetin etc.) was 0.1 N
HCl. Gibberelic acid (GA3) also dissolve initially in 0.1 N NaOH. Once dissolved, the
final volume was made up with distilled water in an appropriate volumetric flask and
stored at 4℃ in refrigerator till use.
135
Annexure 4
Preparation of 1 liter MS Medium
One liter MS medium for callus induction and proliferation was prepared in a manner
given below.
Medium Components Volume of Stock solution
1) Macronutrients 50 ml l-l
2) Micronutrients 10 ml l -l
3) Vitamins 05 ml l-l
4) Myo-inositol 10 ml l-l
5) Iron-EDTA 05 ml l-l
6) Sugar 30 g l-l
7) Agar (Oxoid, Hampshire, England) 8 g l-l
8) pH 5.8
9) Growth regulators According to the requirement of a
specific medium
136
Annexure 5
Composition of Different Media Used for Callus induction/Maintenance from
different explants of Jatropha curcas
Medium Medium Composition
C-1 MS basal
C-2 MS + 1 pM TDZ
C-3 MS + 1.5 pM TDZ
C-4 MS + 2.0 pM TDZ
C-5 MS + 4.65 µM Kn
C-6 MS + 9.3 µM Kn
C-7 MS + 13.95 µM Kn
C-8 MS + 22.17 µM BAP + 5.35 µM NAA
C-9 MS + 2.21 µM BAP + 5.71 µM IAA
C-10 MS + 2.21 µM BAP + 5.71 µM IAA + 4.52 µM 2, 4-D
137
Annexure 6
Composition of Different Media Used for Plant Regeneration from Callus
Cultures/ young leaf explants of Jatropha curcas
Medium Medium Composition
R-1 MS + 22.17 µM BAP + 5.35 µM NAA
R-2 MS + 6.65 µM BAP + 2.67 µM NAA
R-3 MS + 6.65 µM BAP + 2.67 µM NAA + 0.72 µM GA3
R-4 MS + 22.17 µM BAP + 5.35 µM NAA + 0.72 µM GA3
R-5 MS + 22.17 µM BAP + 5.35 µM NAA + 0.72 µM GA3 +
1.5 µM Kn
R-6 MS + 22.17 µM BAP + 5.35 µM NAA + 0.5 µM TDZ
R-7 MS + 22.17 µM BAP + 5.35 µM NAA +1.0 µM TDZ
R-8 MS + 22.17 µM BAP + 5.35 µM NAA + 2.0 µM TDZ
R-9 MS + 22.17 µM BAP + 5.35 µM NAA + 3.0 µM TDZ
R-10 MS + 22.17 µM BAP +10.70 µM NAA + 1.0 µM TDZ
R-11 MS + 22.17 µM BAP + 5.35 µM NAA + 0.5 µM Kn
R-12 MS + 22.17 µM BAP + 5.35 µM NAA + 1.0 µM Kn
R-13 MS + 22.17 µM BAP + 5.35 µM NAA + 1.5 µM Kn
R-14 MS + 22.17 µM BAP + 14.7 µM IBA
R-15 MS + 6.65 µM BAP + 2.45 µM IBA
138
Annexure 7
Composition of Different Media used for Rooting of Regenerated Shoots of
Jatropha curcas
Medium Medium Composition
T-1 MS
T-2 MS + 4.9 µM IBA
T-3 ½ strength MS
T-4 ½ strength MS + 4.9 µM IBA
T-5 MS + 5.35 µM NAA
T-6 MS + 5.71 µM IAA
139
Annexure 8
Composition of Different MS Media Used in Osmotic Stress Experiments for
Callus Cultures of Jatropha curcas
Medium Medium Composition
Control MS + 22.17 µM BAP + 5.35 µM NAA
DC-1 MS + 22.17 µM BAP + 5.35 µM NAA + 0.05 M sorbitol
DC-2 MS + 22.17 µM BAP + 5.35 µM NAA+ 0.1 M sorbitol
DC-3 MS + 22.17 µM BAP + 5.35 µM NAA+ 0.15 M sorbitol
DC-4 MS + 22.17 µM BAP + 5.35 µM NAA+ 0.20 M sorbitol
DC-5 MS + 22.17 µM BAP + 5.35 µM NAA+ 0.25 M sorbitol
DC-6 MS + 22.17 µM BAP + 5.35 µM NAA+ 0.30 M sorbitol
DC-7 MS + 22.17 µM BAP + 5.35 µM NAA+ 0.35 M sorbitol
DC-8 MS + 22.17 µM BAP + 5.35 µM NAA+ 0.40 M sorbitol
DC-9 MS + 22.17 µM BAP + 5.35 µM NAA+ 0.45 M sorbitol
DC-10 MS + 22.17 µM BAP + 5.35 µM NAA+ 0.50 M sorbitol
140
Annexure 9
Composition of different MS Media for In Vitro Germination of Jatropha curcas
Seeds
Medium Medium Composition
G-1 MS medium
G-2 ½ strength MS medium
141
Annexure 10
Details of different Treatments Given to Jatropha curcas Seeds for Germination
Treatment Treatment details
S0 Non treated
S1 Scarification (seeds were scratched with emery sand paper to the
extent that black coating of seed coats was removed)
S2 Stratification (Cold shock was given to the seeds by keeping the seeds
at 4-5˚C for 24 hours).
S3 Scarification + Stratification
S4 Seeds were soaked in water overnight for 24 hours at room
temperature
S5 Seed coats were removed after sterilization in air blow cabinet
S6 Seed coats were removed before sterilization
S7 Seeds (with seed coat) were flamed on Bunsen burner after dipping in
ethyl alcohol
142
Annexure 11
Composition of different MS Medium to Study the Effect of Osmotic Stress on In
Vitro Seed Germination of Jatropha curcas
Medium Medium Composition
Control MS + 0M Sorbitol
D1 MS + 0.05M Sorbitol
D2 MS + 0.1M Sorbitol
D3 MS + 0.15M Sorbitol
D4 MS + 0.2M Sorbitol
D5 MS + 0.25M Sorbitol
D6 MS + 0.3M Sorbitol
D7 MS + 0.35M Sorbitol
D8 MS + 0.4M Sorbitol
D9 MS + 0.45M Sorbitol
D10 MS + 0.5M Sorbitol
143
Annexure 12
0.1 M Phosphate Buffer (pH 7.2) for Extraction of Proteins and Enzymes
Components Amount
KH2PO4 13.61 g
K2HPO4 17.42 g
Distilled water was added to make up final volume, i.e., 1000 ml.
144
Annexure 13
Composition of Biuret Reagent for Protein Estimation.
Components Amount
CuSO4.5H2O 3.8 g
KI 1.0 g
Na-EDTA 6.7 g
5N NaOH 200 ml
Distilled water 700 ml
145
Annexure 14
Reagents for Peroxidase Estimation
a) 1 % Guaiacol
Components Amount
Guaiacol 1 ml
Distilled H2O 99 ml
b) 0.3 % H2O2
Components Amount
H2O2 (35 %) 0.86 ml
Distilled H2O 99.14 ml
146
Annexure 15
A. Reagents for Superoxide Dismutase Estimation
1. Phosphate buffer (pH 7.8):
Dissolved 6.9 g NaH2PO4.H2O in 900 ml distilled water and adjusted to pH 7.8 by
10 % NaOH. Final volume was made up to 1 liter with distilled water.
2. Riboflavin solution:
(prepared fresh and kept in darkness)
Dissolved 7.5 mg of riboflavin in 100 ml distilled water.
3. Sodium cyanide:
Dissolved 13 g sodium cyanide in 1 liter distilled water
4. Nitroblue tetrazolium (NBT):
(prepared fresh and kept in darkness).
Dissolved 137 mg NBT in 10 ml distilled water
5. Methionine:
(Prepared fresh and kept in darkness).
14.9 mg methionine dissolved in 10 ml phosphate buffer.
6. EDTA
245 mg of di-sodium salt of EDTA dissolved in 10 ml buffer solution.
B. Preparation of Reaction Mixture
The reaction mixture was prepared as follows.
1. 1 ml NaCN
2. 10 ml methionine
3. 10 ml EDTA
4. 1 ml NBT
5. 1 ml Riboflavin
The final volume was made up to 100 ml with buffer solution. This mixture was
prepared away from a direct light source and kept in a dark bottle.
147
Annexure 16
Solutions for sterilization of explants
a). 0.1 % HgCl2
Components Amount
HgCl2 0.1 g
Distilled water 100ml
b). 10% NaClO
Components Amount
NaClO 10 ml
Distilled water 90 ml
c). 20% NaClO
Components Amount
NaClO 20 ml
Distilled water 80 ml
