Morphological and molecular analysis of jatropha curcas l. related to aluminum stress as a preliminary study to develop transgenic plant
MORPHOLOGICAL AND MOLECULAR ANALYSIS OF
Jatropha curcas L. RELATED TO ALUMINUM STRESS AS A
PRELIMINARY STUDY TO DEVELOP TRANSGENIC PLANT
RATNA YUNIATI
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2012
DECLARATION
I hereby declare that this dissertation entitled “Morphological and Molecular
Analysis of Jatropha curcas L. Related to Aluminum Stress as a Preliminary Study
to Develop Transgenic Plant” is a true record of the original research work of my
supervisor and me except for quotations and citations which have been duly
acknowledged. I also declare that it has not been previously or concurrently
submitted to any other University either in whole or in part for the award of any
degree, fellowship or any other similar titles.
Bogor, August 2012
Ratna Yuniati
G363070031
ABSTRACT
RATNA YUNIATI. Morphological and Molecular Analysis of Jatropha curcas L.
Related to Aluminum Stress as a Preliminary Study to Develop Transgenic lant.
Supervised by SUHARSONO, UTUT WIDYASTUTI SUHARSONO, DIDY
SOPANDIE, and AKIHO YOKOTA.
As a response to a progressive price increase of fossil based oil in the world,
various initiatives to develop a reliable source of renewable energy become a major
global attention. One potentially promising option is biofuel since these are derived
from biomass. Biofuel crops should be planted on land that is considered marginal. A
crop that is often cited as ideal for growing on marginal land including acid soil with
high Al solubility, is the physic nut, Jatropha curcas. However, the major problem of
the plant cultivation in acid soil is the decreasing productivity. Jatropha plants are
not, by nature, tolerant to Al stress. A promising approach to overcome this
limitation is to develop a transgenic Jatropha plant that can withstand to severe Alstress. Thus it is possible to use TaALMT (Triticum aestivum Al-activated malate
transporter 1) gene which encodes the member of a novel membrane protein family
that functions as an anion channel to mediate malate efflux from plant roots cells in
the presence of extracellular Al3+. Investigating the physiology and molecular basis
of Al-stress tolerance during seedling development is an essential prerequisite before
developing Al-tolerant Jatropha plant by genetic engineering. Among six
populations of Jatropha curcas L., IP-2P population could be categorized as slightly
tolerant to Al stress, but it had uniform seed viability, and high productivity character
which became the reason to use it as a plant material to develop transgenic Jatropha
plant. For preliminary study, J. curcas IP-2P was choosen to be used for gene
expression analysis by Quantitative Real-Time Polymerase Chain Reaction (qRT
PCR). To normalize mRNA expression in the gene expression analysis we used actin
gene. Because there is no information of actin gene nucleotide sequence from J.
curcas we isolated Jatropha actin (JcACT) by PCR. Three cDNA of actin genes from
J. curcas L. IP-2P had been isolated. The analysis revealed that Al stress induced
ALMT transcript accumulation both in roots and leaves of non transgenic J. curcas
IP-2P. Low pH treatment alone could also induce transcript accumulation both in the
root apices and leaf but at lower rates than that observed for Al. This result indicated
that response of Jatropha seedlings were not specific to Al stress but also low-pH
stress. The TaALMT gene was successfully introduced into Jatropha curcas L. IP-2P
via Agrobacterium tumefaciens-mediated transformation. Two putative transformant
shoots were regenerated following selection on the bispyribac-containing medium.
The presence of the transgene in the genome of transgenic plants was confirmed by
PCR. The results indicated that the exogenous TaALMT1 gene was successfully
integrated into the genome of J. curcas IP-2P plants.
Key words: actin gene, Agrobacterium tumefaciens, aluminum stress, ALMT gene,
Jatropha curcas L., Quantitative Real Time PCR
SUMMARY
RATNA YUNIATI. Morphological and Molecular Analysis of Jatropha curcas L.
Related to Aluminum Stress as a Preliminary Study to Develop Transgenic Plant.
Supervised by SUHARSONO, UTUT WIDYASTUTI SUHARSONO, DIDY
SOPANDIE, and AKIHO YOKOTA.
Aluminum toxicity is the primary factor limiting crop production on strongly
acidic soils. Plants have evolved different mechanisms to overcome Al stress. In a
number of plant species it has been shown that Al tolerance appears to be mediated
by Al-activated release of organic acid anions such as malate, oxalate, or citrate,
which chelate Al3+ in the rhizosphere and prevent its entry into the root apex. A gene,
TaALMT1 (Triticum aestivum Al-activated malate transporter 1), which is
responsible for malate release, has been identified in wheat. ALMT gene encodes the
member of a novel membrane protein family that functions as an anion channel to
mediate malate efflux from plant roots cells in the presence of extracellular Al3+.
Therefore it has an important role in the detoxification of Al3+ ion to increase Al
tolerance in plants.
Jatropha is a large perennial shrub, which produces non-edible seeds that
contain 30-40% oil that ideal for biodiesel production. Jatropha plants can grow in
marginal lands such as acid soils, and therefore do not compete with the existing
agricultural resources. However, the major problem of the plant cultivation in acid
soil is the decreasing productivity.
One promising approach to overcome this limitation is to develop a
transgenic Jatropha plant that can withstand to severe Al-stress. In most plant species
there is considerable genotypic variation for the ability to withstand to Al toxicity.
Investigating the physiology and molecular basis of Al-stress tolerance during
seedling development is an essential prerequisite before developing Al-tolerant
Jatropha plant by genetic engineering. Six populations of Jatropha curcas L. were
different under low pH and Al-stress. The Al treatment affected all growth
parameters. There was no single population that showed the best performance on
every growth parameter. This information suggests that the adaptability and tolerance
to Al stress among the six populations were nearly the same following the one month
treatment duration. Although based on the growth parameter, IP-2P could be
categorized as slightly tolerant to Al stress, it has high productivity and those became
the reason to use it as plant material to develop transgenic Jatropha plant.
For preliminary study, J. curcas L. IP-2P was choosen to be used for gene
expression analysis by Quantitative Real-Time Polymerase Chain Reaction (qRT
PCR). Quantitative RT-PCR analysis revealed that Al stress induce ALMT transcript
accumulation both in roots and leaves of the wild type J. curcas L. IP-2P. The gene
expression was higher in the 24 h exposure rather than 7 days. Low pH treatment
alone could also induce transcript accumulation both in the root apices and leaf but at
significantly lower rates than that observed for Al. This result indicated that response
of Jatropha seedling were not specific to Al stress but also the response occurred to
other factors, such as low-pH stress.
Internal control is used to normalize mRNA expression in the experiments
by qRT PCR, usually one of the so-called housekeeping genes. In the current study,
we used actin gene which was constitutively expressed and involved in basic
housekeeping functions required for cell maintenance. Because the information of
nucleotide sequence of actin gene of J. curcas L. population IP-2P from Indonesia
has not been available yet, we isolated Jatropha actin (JcACT) by PCR, using total
cDNA as template and actin primer designed from conserved region of Arabidopsis
thaliana actin gene sequences. Three cDNA of actin genes from J. curcas IP-2P had
been isolated, cloned and characterized. Those cDNA clones which were 610, 534,
and 701 nucleotides in length, shares 98% similarity with actin mRNA of Hevea
brasiliensis and 99% with actin mRNA of Ricinus communis. Those three sequences
had already registered in the GenBank databases under the following accession
numbers: HM587793 for JcACT1, HM587794 for JcACT2, and HM587795 for
JcACT3. These actin cDNA nucleotide sequences are the first J. curcas actin
information from Indonesia reported.
The TaALMT1 gene from wheat Triticum aestivum L. was introduced into
Jatropha curcas L. IP-2P. The binary vector pA2-ALMT construction carrying the
ALMT gene had been introduced to Jatropha via Agrobacterium tumefaciensmediated transformation. This method involved the use of cotyledon explants that
co-cultivated with disarmed Agrobacterium strain LBA4404, which on the T-DNA
region carries CaMV 35S promoter driven the ALMT coding region, and AtALS
(Arabidopsis thaliana Acetolactate synthase) encoding bispyribac resistance. Two
putative transformant shoots were regenerated following selection on the bispyribac
containing medium. The presence of the transgene in the genome of the three months
old transgenic plants was confirmed by PCR. The results indicated that the
exogenous TaALMT1 gene was successfully integrated into the genome of J. curcas
L. IP-2P plants.
Key words: actin gene, Agrobacterium tumefaciens, aluminum stress, ALMT gene,
Jatropha curcas, Quantitative Real Time PCR.
© Copyright 2012, Bogor Agricultural University
All rights reserved
Neither this paper nor any part may be reproduced or transmitted in any
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Quotation only for educational purposes, research, writing papers, preparing
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damage the interest of Bogor Agricultural University.
MORPHOLOGICAL AND MOLECULAR ANALYSIS OF
Jatropha curcas L. RELATED TO ALUMINUM STRESS AS A
PRELIMINARY STUDY TO DEVELOP TRANSGENIC PLANT
A dissertation submitted to The Graduate School of
Bogor Agricultural University, in partial fulfillment of the requirements for the
degree of Doctor of Philosophy in the field of plant biology
By
RATNA YUNIATI
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2012
External Examiner on Closed Examination July, 12th 2012:
Dr. Ir. Ence Darmo Jaya Supena
`
Dr. Ulfah J. Siregar
External Examiner on Open Examination July, 19th 2012
Prof. Dr. Ir. Iskandar Zulkarnain Siregar, M.Sc. For.
Dr. Susiani Purbaningsih, DEA.
Title of dissertation
Name of student
Registered number
: Morphological and Molecular Analysis of Jatropha curcas L.
Related to Aluminum Stress as a Preliminary Study to
Develop Transgenic Plant
: Ratna Yuniati
: G363070031
Approved by
1. Advisory committee
_________________________
Prof. Dr. Ir. Suharsono, DEA
(Chairman)
_________________
_____________________
Dr. Ir. Utut Widyastuti, M.Si.
(Member)
Prof. Dr. Ir. Didy Sopandie, M.Agr
(Member)
_________________
Prof. Akiho Yokota
(Member)
2. Plant Biology Study Program
Graduate School
__________________
__________________
Dr. Ir. Miftahudin, M.Si.
(Head)
Dr. Ir. Dahrul Syah, M.Sc.Agr.
(Dean)
Date of Examination
July, 19th 2012
Date of graduation:
ACKNOWLEDGEMENTS
This dissertation entitled “Morphological and Molecular Analysis of
Jatropha curcas L. Related to Aluminum Stress as a Preliminary Study to Develop
Transgenic Plant” consists of the research results on Jatropha transgenic for
marginal land. It is presented as a partial fulfillment of the requirements for the
Doctoral Degree in the Graduate School of Bogor Agricultural University.
This dissertation is the “stop by” of my long journey in obtaining my degree
in Plant Biotechnology. This dissertation would not have been possible without the
guidance and the help of several individuals who in one way or another contributed
and extended their valuable assistance in the preparation and completion of my
study.
All Praises only to Allah, The Lord of the universe. Shalawat and salam
always devote to our Prophet Muhammad (Peace be upon Him), all of His families
and His disciples, who bring the world hijrah from jahiliah period to akhlakul
karimah period. A figure whom teaches us the meaning of life.
First of all, I am expressing my deepest gratitute to ALLAH S.W.T., the
Almighty for answering my prayers, for giving me the strength to carry out this work
with sound health and mind.
I would like to express my deep and sincere gratitude to my supervisor,
Professor Suharsono. His wide knowledge and his logical way of thinking have been
of great value for me. His understanding, encouraging and personal guidance have
provided a good basis for the present work.
I also express my sincere thanks to members of my thesis committee Dr. Ir.
Utut Widyastuti, and Prof. Dr. Ir. Didy Sopandie, M.Agr. for their valuable
suggestions during the research preparation, lab works, and writing of this
dissertation.
I owe my most sincere gratitude to Professor Akiho Yokota, and Dr. Kinya
Akashi who gave me a valuable opportunity to conduct some part of my research in
Graduate School of Biological Sciences in Nara Institute of Science and Technology
(NAIST), Nara, Japan and gave me untiring help during my stay in Japan.
All my deep respect and appreciation to the Indonesian Ministry of
Education, Dikti, Bogor Agricultural University and University of Indonesia for the
scholarship and financial support. I also thank Sandwich-like Program, The Bilateral
Exchange Program JSPS-DGHE Joint Research projects 2009, Hibah Kompetensi
2010, International Research Collaboration and Scientific Publication under contract
number 203/SP2H/PL/Dit.Litabmas/IV/2012 on behalf of Prof. Suharsono who
financially supported this research.
I thank Dr. Sasaki (Okayama University, Japan) who provided the ALMT
gene to the research group of Prof. Suharsono. I warmly thank Dr. Kajikawa
Masataka and Dr. Saki Hoshiyasu for their valuable advice and friendly help. Their
extensive discussions around my work have been very helpful for this study.
My sincere thanks are due to Dr. Ir. Ence Darmo Jaya Supena, Dr. Ir. Ulfah
Siregar, Prof. Dr. Ir. Iskandar Zulkarnain Siregar, M.For., and Dr. Susiani
Purbaningsih, DEA as the examiner for their detailed review, constructive criticism
and excellent advice for the revision of this dissertation. I also wish to thank
Dr. Ir. Miftahudin, M.Si. as the Head of
Plant Biology Study Program who
contributed to the revision of this dissertation, as well as for his continuous support
in the Ph.D. program.
Also my sincere thank to all post graduate student in IPB, for their help in all
my experiments. Thank you to the numerous dear friends who gave me a second
home in Bogor, to all of my co-workers in the BIORIN (Biotechnology Research
Indonesia-The Netherland) lab, Research Center for Bioresources and Biotechnology
(RC Bio), Bogor Agricultural University for their invaluable support, help and
friendship throughout the study.
I highly appreciate to all my colleagues at the Department of Biology
University of Indonesia, for sharing their expertise in different fields.
My special gratitude is due to my parents, H. Muhammad Noer Burhanuddin,
Hj. Suwartini, H. Muchsin and Hj. Masrifah for teaching me that even the largest
task can be accomplished if it is done one step at a time. They have been an
inspiration throughout my life. They have never failed to give me financial and moral
support. This dissertation is dedicated to them. To my brother, sisters and their
families for their loving support, I cannot find words to express my gratitude to you
all.
Finally, to my lovely husband Zakaria and my lovely sons Taufiqi Akmal and
Atariki Naufal, thank a million for all the prayers, for your sincere understanding,
moral support, and being patience during the period of my study. Certainly, without
their support, and constant encouragements, I would not be able to accomplish this
study.
This research could not have been accomplished without a strong belief in the
ideas being tested. To all the scientist whose challenging and educational comments
inspired me and greatly contributed to my way of scientific thinking, I owe you a
great deal.
Bogor, August 2012
Ratna Yuniati
CURRICULUM VITAE
Ratna Yuniati was born on June, 24, 1967 in Banjarmasin, South Kalimantan.
She spent her childhood with her parent H. Muhammad Noer Burhanuddin, B.A.E
and Hj. Suwartini in Jakarta until she graduated from senior high school in 1986. She
is married with Ir. Zakaria and having two son, Taufiqi Akmal and Atariki Naufal
She pursued her study at University of Indonesia and she graduated from
Department of Biology, The Faculty of Mathematics and Natural Sciences in 1992.
Since 1993 she has been working as teaching staff in the Laboratory of Plant
Physiology, Department of Biology, Faculty of Mathematics and Natural Sciences,
University of Indonesia. In 1999, she received her Master of Science degree in
Biotechnology from Graduate School of Bogor Agricultural University sponsored by
Tim Manajemen Program Doktor (TMPD).
In 2007 she admitted as a Doctoral Student in the Plant Biology Study
Program, Graduate School Bogor Agricultural University sponsored by Beasiswa
Pendidikan Pascasarjana (BPPS). In May to December 2009 she was a recipient of a
Postdoctoral Fellowship from the Japan Society for the Promotion of Science (JSPS)
in the frame of the Bilateral Exchange Program, JSPS – DGHE Joint Research
Project entitled: “Molecular adaptation of Jatropha curcas to acid soil for
reforestation of tropical wastelands”. In September 2010 to December 2010 she got
a grant scholarship for Sandwich Program from DGHE. During those time, she
carried out research in the Graduate School of Biological Sciences at Nara Institute
of Science and Technology, Nara, Japan.
The research topic III entitled “Isolation, Cloning, and Characterization of
Actin-encoding cDNAs from Jatropha curcas L. IP-2P were already published in
Makara Sains Journal VOL. 15, NOVEMBER 2011:168-172.
TABLE OF CONTENTS
LIST OF TABLES ……………………………………………………………..
LIST OF FIGURES ……………………………………………………………..
CHAPTER
I
INTRODUCTION ……………………………................
Background ……………………………………………………………
Objectives………………………………………………………………
Approach……………………………………………………………….
Novelty ……………………………………………………………….
II
LITERATURE REVIEW ……………………………………………..
Jatropha curcas L. …………………………………………………….
Acid Soils ……………………………………………………………...
Aluminum Toxicity ……………………………………………………
General Effects and Symptoms of Al Toxicity in Plants ……………...
Uptake and Distribution of Al at the Whole Plant and Root Level ……
Aluminum Tolerance Mechanisms ……………………………………
Effect of Al on Organic Acid Anions Exudation ……………………..
Aluminum Stress-induced Genes ……………………………………..
Aluminum-activated Malate Transporter (ALMT) Gene ……………...
ALMT Function ……………………………………………………….
The ALMT Family …………………………………………………….
Structure of Membrane Topology of ALMT1 Protein ………………..
Quantitative Real Time Polymerase Chain Reaction ………………….
Actin …………………………………………………………………...
Agrobacterium-mediated Plant Transformation ………………………
Agrobacterium tumefaciens T-DNA Transfer Process ………………..
III
TOLERANCE ANALYSIS AND EXPRESSION ANALYSIS OF
Jatropha curcas L. ALMT GENES UNDER LOW pH AND
ALUMINUM STRESS ………………………………………………..
Abstract ………………………………………………………………..
Introduction ……………………………………………………………
Materials and Methods ………………………………………………...
Results and Discussion ………………………………………………...
Conclusion ……………………………………………………………..
IV
ISOLATION, CLONING AND CHARACTERIZATION OF
ACTIN-ENCODING cDNAs FROM Jatropha curcas L. IP-2P ……..
Abstract ………………………………………………………………..
Introduction ……………………………………………………………
Materials and Methods ………………………………………………..
Results and Discussion ………………………………………………..
Conclusion …………………………………………………………….
Page
xvi
xvii
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38
43
43
45
49
60
63
63
63
64
67
72
V
VI
VII
VECTOR CONSTRUCTION AND INTRODUCTION THE ALMT1
GENE FROM WHEAT INTO Jatropha curcas L. IP-2P BY
Agrobacterium-MEDIATED GENETIC TRANSFORMATION ……
Abstract ………………………………………………………………..
Introduction ……………………………………………………………
Materials and Methods ………………………………………………..
Results and Discussion ………………………………………………...
Conclusion ……………………………………………………………..
GENERAL DISCUSSION …………………………………………….
GENERAL CONCLUSION AND SUGGESTION ………………..
REFERENCES ………………………………………………………..
APPENDICES …………………………………………………………
73
73
73
76
83
92
93
99
101
127
LIST OF TABLES
Page
1.
Effect of Al treatment on growth parameters of six Jatropha
populations………………………………………………………….
54
2.
Abbreviations of various media used for Jatropha regeneration
and transformation …………………………………………………
81
LIST OF FIGURES
Page
1.
Research flow chart …………………………………………………….
5
2.
Important parts of the physic nut: a - flowering branch, b - bark, c - leaf
veinature, d – pistillate flower, e - staminate flower, f - cross-cut of
immature fruit, g - fruits, h - longitudinal cut of fruits (Source: Heller
1996) ……………………………………………………………………
9
3.
Models for the Al-stimulated secretion of organic acid anions (OA)
from plant roots (Source: Delhaize et al. 2007) ………………………
20
4.
Diagrammatic representation of carbon pathways in plant cells related to
malate and citrate metabolism. Aco, aconitase; CS, citrate synthase;
MDH, malate dehydrogenase; NAD-ICDH, NAD specific isocitrate
dehydrogenase; NADP-ICDH, NADP specific isocitratedehydrogenase;
OAA, oxaloacetate; PEPC, phosphoenolpyruvate carboxylase; PK,
pyruvate kinase; PM, plasma membrane. Hatched ellipses on the plasma
membrane and mitochondria denote membrane transporter (Source:
Mariano et al. 2005) ……………………………………………………
21
5.
Hypothetical models (a) Pattern I and (b) Pattern II for the Al3+activated efflux of organic anions by members of the ALMT and MATE
families of proteins (Source: Delhaize et al. 2007)……………………..
26
6.
Topological model of ALMT1. The model depicts the membrane
topology of ALMT1 (Source: Motoda et al. 2007) …………………….
29
7.
Secondary structure of ALMT proteins. Most ALMT proteins are predicted to have 5–7 membrane spanning regions in the amino terminal
half of the protein (Source: Motoda et al. 2007).......................................
30
8.
Agrobacterium-plant cell interactions. This diagram summarizes all
major cellular reactions involved in T-DNA transport. Steps 1 through 7
indicate sequential processes that occur during Agrobacterium infection.
Step 1, binding of Agrobacterium to the host cell surface receptors; Step
2, recognition of plant signal molecules by the bacterial VirA/VirG
sensor-transducer system; Step 3, activation of the bacterial vir genes;
step 4, production of the transferable T-strand; step 5, formation of the
T-complex and its transport into the host plant cell; step 6, nuclear
import of the T-complex; and step 7, T-DNA integration. IM, bacterial
inner membrane; NPC, nuclear pore complex; OM, bacterial outer
membrane; PP, bacterial periplasm (Source: Sheng & Citovsky 1996)..
39
9.
The inhibitory percentages of root growth six Jatropha population
seedlings after one month exposure to Al (A= Asembagus, M=
Muktiharjo, P=Pakuwon) ………………………………………………..
50
10.
The reduction percentages of plant height six Jatropha population
seedlings after one month exposure to Al (A= Asembagus, M=
Muktiharjo, P=Pakuwon) ………………………………………………..
51
11.
The reduction percentages of leaf number six Jatropha population
seedlings after one month exposure to Al (A= Asembagus, M=
Muktiharjo, P=Pakuwon) ………………………………………………..
52
12.
The reduction percentages of plant dry weight six Jatropha population
seedlings after one month exposure to Al (A= Asembagus,
M=Muktiharjo, P=Pakuwon) ……………………………………………
53
13.
Total RNA of J.curcas L.IP-2P from (A) root tips and (B) leaves .……
55
14.
Expression level of JcALMT transcript relative to actin in the root and
leaf tissues of J.curcas treated by Al for (A) 24 hour and (B) 7 days……
57
15.
Expression level of JcALMT transcript relative to actin in the root and
leaf tissues of J.curcas treated by pH for (A) 24 hour and (B) 7 days…...
59
16.
pGEM®T Easy vector for carrying actin gene fragment from J. curcas
L. IP-2P......................................................................................................
66
17.
Total RNA from J. curcas root …………………………………………
68
18.
PCR amplification to isolate actin fragments using cDNA as a template
and degenerate primer for actin genes, P1Ac12S and P1Ac284N. Lane
1, 2 = actin fragments from root, Lane 3= actin fragments from leaf, M
= 1 kb DNA ladder markers ……………………………………………..
68
19.
Location of Jatropha actin fragment inside the general structure of the
A. thaliana actin genes. (Source: McDowell et al. 1996) …………….
69
20.
Restriction analysis of three positive recombinant clones with EcoRI,
700 bp (1), 600 bp (2,3), M = 1 kb DNA ladder marker ........................
70
21.
Phylogenetic tree describes relationship among Jatropha actin with
another actins. Tree built with the neighbor-joining method, formed
well-defined three separate groups: I = dicotyledonous-woody plants, II
= monocotyledonous plants, III = dicotyledon-herbaceous plants. The
bootstrap values shown…………………………………………………..
71
22.
pGEM-ALMT1-1 plasmid which carried the ALMT1-1 coding region
(Source: Sasaki et al. 2004) …………………………………………….
76
23.
Schematic representation of pMSH1 vector. RB=Right Border, LB=Left
Border of T-DNA; NPTII=neomycin phosphotransferase II gene;
HPT=hygromycin phosphotransferase gene, P35S =cauliflower mosaic
virus 35S promoter; PNOS=promoter of the nopalin synthase gene;
T=terminator of the nopalin synthase gene ……………………
77
24.
The T-DNA región of pA2. AtALS gene = Arabidopsis thaliana
W574L/S653IALS gene, ALS P = ALS promoter, ALS T = ALS
terminator, LB = Left Border, RB = Right Border ………………………
78
25.
Agarose gel profile of the PCR product the 1500 bp fragment for the
ALMT1 gene (Lane 1) M = 1 kb DNA ladder…………………………..
84
26.
Agarose gel profile of pMSH1 (lane 1), digested pMSH1 (lane 2), 1500
bp ALMT1 insert (lane 3 and 4), digested-pA2 (lane 5). (M1= λ Sty
marker, M2 = 1 kb DNA ladder).……………………………………….
84
27.
PCR amplification of the cloned 1500-bp fragment of the ALMT gene
inside (A) pMSH1 and (B) pA2 vector was performed by direct colony
PCR using specific primer for ALMT. (M = 1 kb DNA ladder, Lane 1-3
transformed E. coli carrying ALMT inside the binary vector) …………
Agarose gel electrophoresis of M = 1 kb DNA ladder, lane 1, positive
control ALMT fragment; lane 2, E.coli colonies PCR harboring pA2ALMT construct, lane 3-4, A. tumefaciens LBA4404 colonies PCR
transformed with pA2-ALMT construct. Arrows indicate a 1500 bp
fragment of ALMT gene. Resolved on 1% agarose gel…………………
(A) Calli induced on callus-inducing medium containing bispyribac.and
(B) Non-infected explants. Bar equals 1.0 cm ……………………..
85
28.
29.
30.
31.
32.
86
87
Three months-old selection of regenerated adventitious shoots on
bispyribac containing medium. Bar equals 1.0 cm.. ……………………
(A) Non-transformed shoot on root elongation media. (B) Arrow points
to root of non-transformed plant after two weeks on root elongation
media (Inset: picture taken from the bottom side of the culture bottle).
Bar equals 1.0 cm. ……………………………………………………….
89
PCR confirmation of four bispyribac-resistant regenerated shoots.
Arrows indicates the amplification of ALMT gene (1500 bp). M= 1 kb
DNA ladder. Lane 2 and 3 were putative transgenic shoots…………….
91
90
LIST OF APPENDICES
Page
1.
Area and distribution of acid soils based on soil type in Indonesia ……..
127
2.
Description of J. curcas L.IP-2A population ……………………………
128
3.
Description of J. curcas L.IP-2M population …………………………...
129
4.
Description of J. curcas L.IP-2P population ……………………………
130
CHAPTER I
INTRODUCTION
Background
Global energy supply is currently mainly based on fossil fuels such as coal,
oil and gas. Increased use of fossil fuels causes environmental problems both locally
and globally. Developing a reliable source of renewable energy is therefore attracting
a major global attention (Jain & Sharma 2010). One potentially promising option is
biofuel, since these are derived from biomass and do not contribute to the greenhouse
effect (Eijck & Romijn 2008; Jain & Sharma 2010).
Indonesia started to develop the biofuel industry in 2006 as a response to a
progressive price increase of fossil based oil in the world, declining domestic crude
oil production and considerable progressive increase in domestic oil consumption. In
promoting the production of biofuel, the Indonesian government already had a
number of legal instruments, including the Presidential Decree No. 5/2006 on
national policies for optimizing energy use and the Presidential Instruction No.
1/2006 on the use of biofuel. Since then, various initiatives both from the
government and private sectors underline efforts to develop biofuel industry in
Indonesia.
Indonesia has the potential to become a major producer of biofuel. There are
many crops that could be used as raw materials, including palm, Jatropha curcas,
sweet sorghum, sugar cane and cassava. Crude Palm Oil (CPO) can be used to
produce biodiesel, a replacement for diesel, while sugar and cassava can be used to
produce bioethanol to replace gasoline. CPO and molasses are the current primary
feedstock used in Indonesia’s biofuel production. Cassava, Jatropha and sweet
sorghum are other potential feedstocks. However, the development of those
commodities is still in the early stage. To increase biofuel production, the
government encourages private companies to build more processing plants.
Partly in order to respond to accusations that biofuel compete with food
production, some policy makers have proposed that biofuel crops should be planted
on land that is considered marginal or idle. There are millions of hectares of such
land around the world, which play no role in food production. In Indonesia marginal
lands is about 32.0 million hectares which are potential to be developed, scattered in
2
all provinces (Adimihardja et al. 2004; Directorate of Land Rehabilitation and Soil
Conservation 2000). According to the Indonesian government, there are three types
of land not used for agriculture: marginal land, critical land and sleeping land. In
Indonesia, “marginal” land is considered to be less productive land, whether dry or
wetland with high acidity due its formation process and its nature and properties.
Indonesian marginal land therefore includes swampland, wetlands and peat forests,
as well as dry land on acidic soil. “Critical” land is land that has been ecologically
degraded as a result of intensive agricultural practices, and which is no longer
suitable for farming. These degraded lands were once important for maintaining food
security in the country, and priority should be given to improving food security for
example through better paddy field irrigation. “Sleeping” land is temporarily
uncultivated or neglected land, that does not match its previously allocated land use
planning classification such as for agriculture, housing, industry and public services
(Anonim 2008).
Acid soils cover a large area of cultivated land in Indonesia. The pH of these
soils ranges from 4 to 5. On highly acidic soils (pH 4.0), the rhizotoxic aluminum
species, Al3+, is solubilized to ionic form, which is toxic to all living cells at low
concentrations. In plants, ionic Al rapidly inhibits root elongation by targeting
multiple cellular sites and subsequently the uptake of water and nutrients (Kochian et
al. 2005; Ma 2007), resulting in poor growth. Al toxicity has therefore been
recognized as a major factor limiting crop production on acid soils, which account
for 30% to 40% of the world’s arable soils (von Uexküll & Mutert 1995).
However, some plant species have developed mechanisms to detoxify Al,
both internally and externally (Ryan et al. 2001; Ma et al. 2001; Kochian et al.
2005). The most-documented and general mechanism of Al tolerance in both
monocots and dicots is release of organic acid anions, including malate, citrate, and
oxalate, from the roots in response to Al (Kochian et al. 2005; Ma 2007). The Aldependent stimulation of organic acid efflux from roots has been associated with an
increase in Al resistance. Those anions of organic acids released by roots are thought
to chelate the toxic Al cations, to form non-phytotoxic Al form, and thus prevent
them from interacting with the root apices. Genes responsible for the secretion of Al-
3
induced malate in wheat, citrate in barley and sorghum have been identified (Sasaki
et al. 2004; Magalhaes et al. 2007).
One crop that is often cited as ideal for growing on marginal land including
acid soil with high Al solubility, is the physic nut, Jatropha curcas. The problem of
great concern regarding the Jatropha plant is these plants are not, by nature, tolerant
to Al stress. One promising approach to overcome this limitation is to develop a
transgenic Jatropha plant that can withstand severe Al-stress. Thus it is possible to
use TaALMT (Triticum aestivum Aluminum-activated Malate Transporter) cDNA to
generate transgenic Jatropha with enhanced tolerance to Al-stress.
Jatropha is one of such renewable oils, an important multipurpose plant
belonging to the family Euphorbiaceae. Jatropha is grown as a boundary fence to
protect field from the grazing animals and as a hedge to prevent soil erosion. It is a
native of the central America and occurs mainly at low altitudes in areas with annual
temperature of well above 30°C. Among the various oil seeds, Jatropha has been
found more suitable for biodiesel production on the basis of various characteristics.
This oil-bearing tree produces seeds that contain 30-40% oil which ideal for
biodiesel production. Jatropha oil has higher cetane number (51) compared to other
oils, which is compared to diesel (46–50) and make it an ideal alternative fuel (Jain
& Sharma 2010).
In 2006 The Plantation Research and Development Center (Puslitbangbun),
Indonesian Ministry of Agriculture through selection mechanism, has obtained six
lines/ population of Jatropha i.e. IP-1A, IP-2A, IP-1M, IP-2M, IP-1P and IP-2P.
Those plant materials were a composite result of superior individuals which selected
from different provenances. From those IP-1 populations the next selected were IP-2
populations which is the derivation of plant material IP-1 (Hasnam 2007a). There is
wide genetic variation, both within and between species, in the resistance of plants to
Al. Such variation provides breeders with a strategy for improving the ability of crop
plants to cope with Al toxicity.
One of the requirements in developing Al-tolerant Jatropha transgenic that
can adapt to acid soil with high level of soluble Al is the availability of a good
selection tool that is able to find parental resources based on Al-tolerant and Alsensitive plant characteristics. Plant morphological characteristics are considered as
4
typical traits and could be used as a criterion to evaluate Al-tolerance of plant. It may
be expected that the higher tolerance may associate with higher plant morphological
traits values. Besides investigating morphological characteristics, plant stress study
based on gene expression is also required. Up until now, there has been no data about
ALMT gene expression analysis on J. curcas under Al-stress. The analysis of gene
expression requires sensitive, precise, and reproducible measurements for specific
mRNA sequences. Quantitative Real-Time PCR (qRT PCR) is, at present, the most
sensitive method. To avoid bias, qRT-PCR is typically referenced to an internal
control gene which should not fluctuate during treatments. Mainly housekeeping
genes were used for the quantification of mRNA expression. Currently, at least nine
housekeeping genes are well described for the normalization of expression signals.
One of the most common are actin (Sturzenbaum & Kille 2001). Until recently the
actin gene has never been isolated from J. curcas.
Objectives
For all the above reasons, the general objective of the present study was
to introduce the TaALMT gene from wheat into J. curcas L. IP-2P plants. While the
specific objectives were:
1. To evaluate the low pH and Al-tolerance of six Jatropha population in the acid
soil with Al-stress treatment.
2. To examine the ALMT gene expression from the roots and leaves of wild type
J. curcas L. IP-2P during Al-stress treatment.
3. To isolate, clone and characterize actin gene from J.curcas L. IP-2P.
To achieve these objectives, the research was conducted as described by Figure 1.
Approach
Transgenic plant generating experiments was initiated with selected
population. Six populations of Jatropha are used for starting materials of this study.
The required data include information on plant tolerance under low pH and Al-stress
and Al-induce gene expression. Analysis of these data allows distinguishing more or
less promising population in relation to their ability to tolerate Al-stress. Gene
5
expression study of the Jatropha population was done as the preliminary research
before developing transgenic Jatropha plants carrying ALMT gene. The gene
expression study by using Quantitative RT PCR needs actin gene as an internal
control, thus it needed to isolate from Jatropha cDNA.
Jatropha curcas L. plants
TaALMT gene
from wheat
Construction of
expression vector
harboring TaALMT
Introduction of
TaALMT gene into J.
curcas L. IP-2P via
Agrobacterium
J. curcas IP-2P use as
material study
J. curcas L.
cDNA synthesis
Actin gene isolation
from J. curcas L as a
qRT PCR reference gene
Tolerance analysis
of six J. curcas
population under
low pH and Al
stress
Expression analysis
of J. curcas IP-2P
under low pH and Al
stress
Plant regeneration
and selection
J. curcas L
transgenic plant
carrying TaALMT
Research Topic I
Research Topic III
Research Topic II
Figure 1. Research flow chart
Novelty
This dissertation involved developing a transgenic J. curcas L. IP-2P plants
capable of growing in acid soil with high aluminum solubility. This dissertation is
the first report on J. curcas transgenic plants carried the ALMT gene. The second
novelty of this dissertation is the isolation of cDNA encoding actin (JcACT) from J.
6
curcas L. IP-2P as internal control in the gene expression analysis by using qRT
PCR. The three actin cDNA nucleotide sequences included in this dissertation are the
first J. curcas actin information from Indonesia reported.
7
CHAPTER II
LITERATURE REVIEW
Jatropha curcas L.
Jatropha curcas L. is a deciduous, multipurpose shrub belonging to the
family Euphorbiaceae. It is distributed naturally in the equatorial Americas, from
where it has been introduced and become naturalized in many parts of the tropical
and subtropical regions of the world (Heller 1996). J. curcas plant has medicinal
values and is commonly grown as hedges to protect gardens and fields from animals.
However, in the recent years, the species has gained tremendous significance as a
potential biodiesel plant, which is characterized as a safe, healthy environment and
renewable resource, alternative to petrodiesel.
J. curcas is very adaptable to a wide range of soils and climates and grows
well without any special nutrition regime (Patil & Singh 1991). It can be grown in
arid zones (20 cm rainfall) as well as in higher rainfall zones. It is a quick yielding
species even in adverse land situations, viz., degraded and barren lands under forest
and non-forest use, dry and drought prone area, marginal lands even an alkaline soils
and also as agro forestry crops. Jatropha can be a good plant material for ecorestoration in all types of wasteland. It can be propagated from seed or cuttings of
stem or branch, and produces large quantity of oil-seed within 2–3 years after
planting.
Jatropha, the physic nut, by definition, is a small tree or large shrub which
can reach a height of up to 5 m, but can attain a height of 8 or 10 m under favorable
conditions. The plant shows articulated growth (Kumar & Sharma 2008) straight
trunk, thick branchlets with a soft wood and a life expectancy of up to 50 years
(Achten et al. 2008). The branches contain latex. Normally, five roots are formed
from seedlings, one central and four peripheral. A tap root is not usually formed by
vegetatively propagated plants (Kobilke 1989). Jatropha has 5 to 7 shallow lobed
leaves with a length and width of 6 to 15 cm, which are arranged alternately. The
plant produces flowers in racemose inflorescences. Inflorescences are formed
terminally. The flowers are unisexual, male and female flowers are produced in the
same inflorescence; occasionally hermaphrodite flowers occur (Dehgan & Webster
1979). Normally, the inflorescences produce a central female flower surrounded by a
8
group of male flowers. In a few, the expected places of
female flowers are
substituted by male flowers. Numerically, 1–5 female flowers and 25–93 male
flowers are produced per inflorescence. The average male to female flower ratio is 29
: 1. Both sexes have five nectar glands produced nectar located at the base of flower.
Male flower has ten stamens performed a circle patterns. Pollen is yellow, globular.
The female flowers has three cell ovary that terminated by three styles. Each
inflorescence, once it begins flowering, flowers daily, and the flowering lasts for 11
days. Jatropha flowers during the rainy season with concentrated flowering from late
July to late October. Pollination is by insects. The rare hermaphrodite flowers can be
self-pollinating.
During pollination, sepal and petal will protect the fruit
development. After pollination, a trilocular ellipsoidal fruit is formed. The exocarp
remains fleshy until the seeds are mature. A fruit contain 3-4 black seed which 2 cm
long and 1 cm thick. Fruits will ripe after 40-50 days after pollination follows with
fruit color change from green to yellow (Raju & Ezradanam 2002; Bhattacharya et
al. 2005). Gupta (1985) investigated the anatomy of other plant parts. Relevant parts
of the plant are shown in Figure 2.
Carvalho et al. (2008) found that the genome of J. curcas is relatively small
(C = 416 Mb) and in the same size range as that of rice and an average base
composition of 38.7% GC. The karyotype of J. curcas is made up of 22 relatively
small metacentric and submetacentric chromosomes whose size range from 1.71 to
1.24 mm. In addition, the morphometric similarities observed between chromosomes
from heterologous pairs suggest that J. curcas is an autotetraploid species.
Seed storage behaviour of Euphorbiaceae is generally orthodox according to
Ellis et al. (1985) (one exception, Hevea). Orthodox seed storage behaviour means
“mature whole seeds not only survive considerable desiccation (to at least 5%
moisture content) but their longevity in air-dry storage is increased by reduction in
seed storage moisture content and temperature (e.g. to those values employed in
longterm seed stores). Jatropha also has orthodox seeds.
9
Figure 2. Important parts of the physic nut: a. flowering branch; b. bark; c. leaf
veinature; d. pistillate flower; e. staminate flower; f. cross-cut of immature
fruit; g. fruits; h. longitudinal cut of fruits (Source: Heller 1996).
Two- or sixmonth-old seeds stored in unsealed plastic bags at ambient temperatures
(approximately 20°C) for 5 months and germinated on average by 62% (ranging
from 19 to 79%) after having been seeded in soil.
10
Kobilke (1989) investigated the viability of seeds of different ages (1 to 24
months) that were collected directly from the sites or stored for a certain time. Seeds
older than 15 months showed viabilities below 50%. One explanation for this rapid
decrease is that these seeds remained at the site, having been exposed for long
periods to extreme changes in levels of humidity and temperature. High levels of
viability and low levels of germination shortly after harvest indicate innate (=
primary) dormancy. This behaviour has also been reported for other Euphorbiaceae
(Ellis et al. 1985).
Acid Soils
Acid soils, which are soils with a pH of 5.5 or lower, are one of the most
important limitations to agricultural production worldwide. Approximately 30% of
the world’s total land area consists of acid soils and as much as 50% of the world’s
potentially arable lands are acidic (von Uexkull & Mutert 1995). Furthermore, the
large areas of acidic soils in the tropics and subtropics are critical food-producing
regions for developing countries. Thus, acid soils limit crop yields in many
developing countries (Kochian et al. 2004).
Most of the total area land available in Indonesia is 190 million ha, for
agricultural land is classified as ultisols (47 million ha) (See Appendix 1). Those
soils are considered acid with high Al content, low cation exchange capacity and low
nutrient content (Mulyadi & Soepraptohardjo 1975; Santoso 1991). The most
widespread dry acid soil in Indonesia is in Sumatera, Borneo, and Papua (WidjajaAdi et al.1992). Acid soils in Indonesia are characterized by low pH (4.0-5.50),
cation-exchange capacity (CEC) 10.17 - 10.89 me/100 g (low), base saturation 19 21 % (very low); and Al exchangeable 3.43 - 3.80 me/100 gram (Pusat Penelitian
Tanah 1983).
Mineral acid soils result from parent materials that are acidic and naturally
low in the basic cations (Ca, Mg, K and Na), or because these elements are leached
from the soil, reducing pH and the buffering capacity of the soil. As soil pH
decreases, aluminum (Al) is solubilized and the proportion of phytotoxic aluminum
ions increases in the soil solution. In most mineral soils there is sufficient Al present
to buffer the soil to around pH 4. Organic acid soils, consisting of large amounts of
humic acids and partially decomposed plant matter, typically have little Al buffering
11
and the pH of these soils can fall below pH 4 (Kidd & Proctor 2001). Superimposing
agricultural production on an ecosystem, especially ammonium fertilization,
accelerates soil acidification due to nitrification. Furthermore, acid rain, containing
nitric and sulfuric acids, is increasing the rate of soil acidification.
Slightly acidic soils (pH of 6.5) are considered most favorable for overall
nutrient uptake. Such soils are also optimal for nitrogen-fixing legumes and nitrogenfixing soil bacteria. Some plants are adapted to acidic or basic soils due to natural
selection of species in these conditions. Potatoes grow well in soils with pH 7.5). Root
growth in spinach was significantly inhibited at pH 4.5 compared with growth at
more neutral conditions. Soil pH also affects the soil in other ways. For example, soil
microbe activity; particularly nitrogen-fixing bacteria may be reduced in acid soil. In
the case of nitrogen-fixing plants, soil acidity is even more problematic since their
symbiotic bacteria are also sensitive to Al and acidity (Hungria & Vargas 2000).
A number of factors contribute to acid soil toxicity depending on soil
composition. In acid soils with a high mineral content, the primary factor limiting
plant growth is Al toxicity (Kinraide 1991). But Al toxicity is not the only stress in
acid soils; among others, proton and manganese toxicity as well as phosphorus
deficiency are also common (Marschner 1995).
Plants in acid soils also suffer from
Jatropha curcas L. RELATED TO ALUMINUM STRESS AS A
PRELIMINARY STUDY TO DEVELOP TRANSGENIC PLANT
RATNA YUNIATI
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2012
DECLARATION
I hereby declare that this dissertation entitled “Morphological and Molecular
Analysis of Jatropha curcas L. Related to Aluminum Stress as a Preliminary Study
to Develop Transgenic Plant” is a true record of the original research work of my
supervisor and me except for quotations and citations which have been duly
acknowledged. I also declare that it has not been previously or concurrently
submitted to any other University either in whole or in part for the award of any
degree, fellowship or any other similar titles.
Bogor, August 2012
Ratna Yuniati
G363070031
ABSTRACT
RATNA YUNIATI. Morphological and Molecular Analysis of Jatropha curcas L.
Related to Aluminum Stress as a Preliminary Study to Develop Transgenic lant.
Supervised by SUHARSONO, UTUT WIDYASTUTI SUHARSONO, DIDY
SOPANDIE, and AKIHO YOKOTA.
As a response to a progressive price increase of fossil based oil in the world,
various initiatives to develop a reliable source of renewable energy become a major
global attention. One potentially promising option is biofuel since these are derived
from biomass. Biofuel crops should be planted on land that is considered marginal. A
crop that is often cited as ideal for growing on marginal land including acid soil with
high Al solubility, is the physic nut, Jatropha curcas. However, the major problem of
the plant cultivation in acid soil is the decreasing productivity. Jatropha plants are
not, by nature, tolerant to Al stress. A promising approach to overcome this
limitation is to develop a transgenic Jatropha plant that can withstand to severe Alstress. Thus it is possible to use TaALMT (Triticum aestivum Al-activated malate
transporter 1) gene which encodes the member of a novel membrane protein family
that functions as an anion channel to mediate malate efflux from plant roots cells in
the presence of extracellular Al3+. Investigating the physiology and molecular basis
of Al-stress tolerance during seedling development is an essential prerequisite before
developing Al-tolerant Jatropha plant by genetic engineering. Among six
populations of Jatropha curcas L., IP-2P population could be categorized as slightly
tolerant to Al stress, but it had uniform seed viability, and high productivity character
which became the reason to use it as a plant material to develop transgenic Jatropha
plant. For preliminary study, J. curcas IP-2P was choosen to be used for gene
expression analysis by Quantitative Real-Time Polymerase Chain Reaction (qRT
PCR). To normalize mRNA expression in the gene expression analysis we used actin
gene. Because there is no information of actin gene nucleotide sequence from J.
curcas we isolated Jatropha actin (JcACT) by PCR. Three cDNA of actin genes from
J. curcas L. IP-2P had been isolated. The analysis revealed that Al stress induced
ALMT transcript accumulation both in roots and leaves of non transgenic J. curcas
IP-2P. Low pH treatment alone could also induce transcript accumulation both in the
root apices and leaf but at lower rates than that observed for Al. This result indicated
that response of Jatropha seedlings were not specific to Al stress but also low-pH
stress. The TaALMT gene was successfully introduced into Jatropha curcas L. IP-2P
via Agrobacterium tumefaciens-mediated transformation. Two putative transformant
shoots were regenerated following selection on the bispyribac-containing medium.
The presence of the transgene in the genome of transgenic plants was confirmed by
PCR. The results indicated that the exogenous TaALMT1 gene was successfully
integrated into the genome of J. curcas IP-2P plants.
Key words: actin gene, Agrobacterium tumefaciens, aluminum stress, ALMT gene,
Jatropha curcas L., Quantitative Real Time PCR
SUMMARY
RATNA YUNIATI. Morphological and Molecular Analysis of Jatropha curcas L.
Related to Aluminum Stress as a Preliminary Study to Develop Transgenic Plant.
Supervised by SUHARSONO, UTUT WIDYASTUTI SUHARSONO, DIDY
SOPANDIE, and AKIHO YOKOTA.
Aluminum toxicity is the primary factor limiting crop production on strongly
acidic soils. Plants have evolved different mechanisms to overcome Al stress. In a
number of plant species it has been shown that Al tolerance appears to be mediated
by Al-activated release of organic acid anions such as malate, oxalate, or citrate,
which chelate Al3+ in the rhizosphere and prevent its entry into the root apex. A gene,
TaALMT1 (Triticum aestivum Al-activated malate transporter 1), which is
responsible for malate release, has been identified in wheat. ALMT gene encodes the
member of a novel membrane protein family that functions as an anion channel to
mediate malate efflux from plant roots cells in the presence of extracellular Al3+.
Therefore it has an important role in the detoxification of Al3+ ion to increase Al
tolerance in plants.
Jatropha is a large perennial shrub, which produces non-edible seeds that
contain 30-40% oil that ideal for biodiesel production. Jatropha plants can grow in
marginal lands such as acid soils, and therefore do not compete with the existing
agricultural resources. However, the major problem of the plant cultivation in acid
soil is the decreasing productivity.
One promising approach to overcome this limitation is to develop a
transgenic Jatropha plant that can withstand to severe Al-stress. In most plant species
there is considerable genotypic variation for the ability to withstand to Al toxicity.
Investigating the physiology and molecular basis of Al-stress tolerance during
seedling development is an essential prerequisite before developing Al-tolerant
Jatropha plant by genetic engineering. Six populations of Jatropha curcas L. were
different under low pH and Al-stress. The Al treatment affected all growth
parameters. There was no single population that showed the best performance on
every growth parameter. This information suggests that the adaptability and tolerance
to Al stress among the six populations were nearly the same following the one month
treatment duration. Although based on the growth parameter, IP-2P could be
categorized as slightly tolerant to Al stress, it has high productivity and those became
the reason to use it as plant material to develop transgenic Jatropha plant.
For preliminary study, J. curcas L. IP-2P was choosen to be used for gene
expression analysis by Quantitative Real-Time Polymerase Chain Reaction (qRT
PCR). Quantitative RT-PCR analysis revealed that Al stress induce ALMT transcript
accumulation both in roots and leaves of the wild type J. curcas L. IP-2P. The gene
expression was higher in the 24 h exposure rather than 7 days. Low pH treatment
alone could also induce transcript accumulation both in the root apices and leaf but at
significantly lower rates than that observed for Al. This result indicated that response
of Jatropha seedling were not specific to Al stress but also the response occurred to
other factors, such as low-pH stress.
Internal control is used to normalize mRNA expression in the experiments
by qRT PCR, usually one of the so-called housekeeping genes. In the current study,
we used actin gene which was constitutively expressed and involved in basic
housekeeping functions required for cell maintenance. Because the information of
nucleotide sequence of actin gene of J. curcas L. population IP-2P from Indonesia
has not been available yet, we isolated Jatropha actin (JcACT) by PCR, using total
cDNA as template and actin primer designed from conserved region of Arabidopsis
thaliana actin gene sequences. Three cDNA of actin genes from J. curcas IP-2P had
been isolated, cloned and characterized. Those cDNA clones which were 610, 534,
and 701 nucleotides in length, shares 98% similarity with actin mRNA of Hevea
brasiliensis and 99% with actin mRNA of Ricinus communis. Those three sequences
had already registered in the GenBank databases under the following accession
numbers: HM587793 for JcACT1, HM587794 for JcACT2, and HM587795 for
JcACT3. These actin cDNA nucleotide sequences are the first J. curcas actin
information from Indonesia reported.
The TaALMT1 gene from wheat Triticum aestivum L. was introduced into
Jatropha curcas L. IP-2P. The binary vector pA2-ALMT construction carrying the
ALMT gene had been introduced to Jatropha via Agrobacterium tumefaciensmediated transformation. This method involved the use of cotyledon explants that
co-cultivated with disarmed Agrobacterium strain LBA4404, which on the T-DNA
region carries CaMV 35S promoter driven the ALMT coding region, and AtALS
(Arabidopsis thaliana Acetolactate synthase) encoding bispyribac resistance. Two
putative transformant shoots were regenerated following selection on the bispyribac
containing medium. The presence of the transgene in the genome of the three months
old transgenic plants was confirmed by PCR. The results indicated that the
exogenous TaALMT1 gene was successfully integrated into the genome of J. curcas
L. IP-2P plants.
Key words: actin gene, Agrobacterium tumefaciens, aluminum stress, ALMT gene,
Jatropha curcas, Quantitative Real Time PCR.
© Copyright 2012, Bogor Agricultural University
All rights reserved
Neither this paper nor any part may be reproduced or transmitted in any
form or by any means, including electronic, mechanical, photocopying,
translating, recording, or by any information storage and retrieval system
without including or mentioning the source and without the prior written
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Quotation only for educational purposes, research, writing papers, preparing
reports, writing criticism or review of a problem; and citations will not
damage the interest of Bogor Agricultural University.
MORPHOLOGICAL AND MOLECULAR ANALYSIS OF
Jatropha curcas L. RELATED TO ALUMINUM STRESS AS A
PRELIMINARY STUDY TO DEVELOP TRANSGENIC PLANT
A dissertation submitted to The Graduate School of
Bogor Agricultural University, in partial fulfillment of the requirements for the
degree of Doctor of Philosophy in the field of plant biology
By
RATNA YUNIATI
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2012
External Examiner on Closed Examination July, 12th 2012:
Dr. Ir. Ence Darmo Jaya Supena
`
Dr. Ulfah J. Siregar
External Examiner on Open Examination July, 19th 2012
Prof. Dr. Ir. Iskandar Zulkarnain Siregar, M.Sc. For.
Dr. Susiani Purbaningsih, DEA.
Title of dissertation
Name of student
Registered number
: Morphological and Molecular Analysis of Jatropha curcas L.
Related to Aluminum Stress as a Preliminary Study to
Develop Transgenic Plant
: Ratna Yuniati
: G363070031
Approved by
1. Advisory committee
_________________________
Prof. Dr. Ir. Suharsono, DEA
(Chairman)
_________________
_____________________
Dr. Ir. Utut Widyastuti, M.Si.
(Member)
Prof. Dr. Ir. Didy Sopandie, M.Agr
(Member)
_________________
Prof. Akiho Yokota
(Member)
2. Plant Biology Study Program
Graduate School
__________________
__________________
Dr. Ir. Miftahudin, M.Si.
(Head)
Dr. Ir. Dahrul Syah, M.Sc.Agr.
(Dean)
Date of Examination
July, 19th 2012
Date of graduation:
ACKNOWLEDGEMENTS
This dissertation entitled “Morphological and Molecular Analysis of
Jatropha curcas L. Related to Aluminum Stress as a Preliminary Study to Develop
Transgenic Plant” consists of the research results on Jatropha transgenic for
marginal land. It is presented as a partial fulfillment of the requirements for the
Doctoral Degree in the Graduate School of Bogor Agricultural University.
This dissertation is the “stop by” of my long journey in obtaining my degree
in Plant Biotechnology. This dissertation would not have been possible without the
guidance and the help of several individuals who in one way or another contributed
and extended their valuable assistance in the preparation and completion of my
study.
All Praises only to Allah, The Lord of the universe. Shalawat and salam
always devote to our Prophet Muhammad (Peace be upon Him), all of His families
and His disciples, who bring the world hijrah from jahiliah period to akhlakul
karimah period. A figure whom teaches us the meaning of life.
First of all, I am expressing my deepest gratitute to ALLAH S.W.T., the
Almighty for answering my prayers, for giving me the strength to carry out this work
with sound health and mind.
I would like to express my deep and sincere gratitude to my supervisor,
Professor Suharsono. His wide knowledge and his logical way of thinking have been
of great value for me. His understanding, encouraging and personal guidance have
provided a good basis for the present work.
I also express my sincere thanks to members of my thesis committee Dr. Ir.
Utut Widyastuti, and Prof. Dr. Ir. Didy Sopandie, M.Agr. for their valuable
suggestions during the research preparation, lab works, and writing of this
dissertation.
I owe my most sincere gratitude to Professor Akiho Yokota, and Dr. Kinya
Akashi who gave me a valuable opportunity to conduct some part of my research in
Graduate School of Biological Sciences in Nara Institute of Science and Technology
(NAIST), Nara, Japan and gave me untiring help during my stay in Japan.
All my deep respect and appreciation to the Indonesian Ministry of
Education, Dikti, Bogor Agricultural University and University of Indonesia for the
scholarship and financial support. I also thank Sandwich-like Program, The Bilateral
Exchange Program JSPS-DGHE Joint Research projects 2009, Hibah Kompetensi
2010, International Research Collaboration and Scientific Publication under contract
number 203/SP2H/PL/Dit.Litabmas/IV/2012 on behalf of Prof. Suharsono who
financially supported this research.
I thank Dr. Sasaki (Okayama University, Japan) who provided the ALMT
gene to the research group of Prof. Suharsono. I warmly thank Dr. Kajikawa
Masataka and Dr. Saki Hoshiyasu for their valuable advice and friendly help. Their
extensive discussions around my work have been very helpful for this study.
My sincere thanks are due to Dr. Ir. Ence Darmo Jaya Supena, Dr. Ir. Ulfah
Siregar, Prof. Dr. Ir. Iskandar Zulkarnain Siregar, M.For., and Dr. Susiani
Purbaningsih, DEA as the examiner for their detailed review, constructive criticism
and excellent advice for the revision of this dissertation. I also wish to thank
Dr. Ir. Miftahudin, M.Si. as the Head of
Plant Biology Study Program who
contributed to the revision of this dissertation, as well as for his continuous support
in the Ph.D. program.
Also my sincere thank to all post graduate student in IPB, for their help in all
my experiments. Thank you to the numerous dear friends who gave me a second
home in Bogor, to all of my co-workers in the BIORIN (Biotechnology Research
Indonesia-The Netherland) lab, Research Center for Bioresources and Biotechnology
(RC Bio), Bogor Agricultural University for their invaluable support, help and
friendship throughout the study.
I highly appreciate to all my colleagues at the Department of Biology
University of Indonesia, for sharing their expertise in different fields.
My special gratitude is due to my parents, H. Muhammad Noer Burhanuddin,
Hj. Suwartini, H. Muchsin and Hj. Masrifah for teaching me that even the largest
task can be accomplished if it is done one step at a time. They have been an
inspiration throughout my life. They have never failed to give me financial and moral
support. This dissertation is dedicated to them. To my brother, sisters and their
families for their loving support, I cannot find words to express my gratitude to you
all.
Finally, to my lovely husband Zakaria and my lovely sons Taufiqi Akmal and
Atariki Naufal, thank a million for all the prayers, for your sincere understanding,
moral support, and being patience during the period of my study. Certainly, without
their support, and constant encouragements, I would not be able to accomplish this
study.
This research could not have been accomplished without a strong belief in the
ideas being tested. To all the scientist whose challenging and educational comments
inspired me and greatly contributed to my way of scientific thinking, I owe you a
great deal.
Bogor, August 2012
Ratna Yuniati
CURRICULUM VITAE
Ratna Yuniati was born on June, 24, 1967 in Banjarmasin, South Kalimantan.
She spent her childhood with her parent H. Muhammad Noer Burhanuddin, B.A.E
and Hj. Suwartini in Jakarta until she graduated from senior high school in 1986. She
is married with Ir. Zakaria and having two son, Taufiqi Akmal and Atariki Naufal
She pursued her study at University of Indonesia and she graduated from
Department of Biology, The Faculty of Mathematics and Natural Sciences in 1992.
Since 1993 she has been working as teaching staff in the Laboratory of Plant
Physiology, Department of Biology, Faculty of Mathematics and Natural Sciences,
University of Indonesia. In 1999, she received her Master of Science degree in
Biotechnology from Graduate School of Bogor Agricultural University sponsored by
Tim Manajemen Program Doktor (TMPD).
In 2007 she admitted as a Doctoral Student in the Plant Biology Study
Program, Graduate School Bogor Agricultural University sponsored by Beasiswa
Pendidikan Pascasarjana (BPPS). In May to December 2009 she was a recipient of a
Postdoctoral Fellowship from the Japan Society for the Promotion of Science (JSPS)
in the frame of the Bilateral Exchange Program, JSPS – DGHE Joint Research
Project entitled: “Molecular adaptation of Jatropha curcas to acid soil for
reforestation of tropical wastelands”. In September 2010 to December 2010 she got
a grant scholarship for Sandwich Program from DGHE. During those time, she
carried out research in the Graduate School of Biological Sciences at Nara Institute
of Science and Technology, Nara, Japan.
The research topic III entitled “Isolation, Cloning, and Characterization of
Actin-encoding cDNAs from Jatropha curcas L. IP-2P were already published in
Makara Sains Journal VOL. 15, NOVEMBER 2011:168-172.
TABLE OF CONTENTS
LIST OF TABLES ……………………………………………………………..
LIST OF FIGURES ……………………………………………………………..
CHAPTER
I
INTRODUCTION ……………………………................
Background ……………………………………………………………
Objectives………………………………………………………………
Approach……………………………………………………………….
Novelty ……………………………………………………………….
II
LITERATURE REVIEW ……………………………………………..
Jatropha curcas L. …………………………………………………….
Acid Soils ……………………………………………………………...
Aluminum Toxicity ……………………………………………………
General Effects and Symptoms of Al Toxicity in Plants ……………...
Uptake and Distribution of Al at the Whole Plant and Root Level ……
Aluminum Tolerance Mechanisms ……………………………………
Effect of Al on Organic Acid Anions Exudation ……………………..
Aluminum Stress-induced Genes ……………………………………..
Aluminum-activated Malate Transporter (ALMT) Gene ……………...
ALMT Function ……………………………………………………….
The ALMT Family …………………………………………………….
Structure of Membrane Topology of ALMT1 Protein ………………..
Quantitative Real Time Polymerase Chain Reaction ………………….
Actin …………………………………………………………………...
Agrobacterium-mediated Plant Transformation ………………………
Agrobacterium tumefaciens T-DNA Transfer Process ………………..
III
TOLERANCE ANALYSIS AND EXPRESSION ANALYSIS OF
Jatropha curcas L. ALMT GENES UNDER LOW pH AND
ALUMINUM STRESS ………………………………………………..
Abstract ………………………………………………………………..
Introduction ……………………………………………………………
Materials and Methods ………………………………………………...
Results and Discussion ………………………………………………...
Conclusion ……………………………………………………………..
IV
ISOLATION, CLONING AND CHARACTERIZATION OF
ACTIN-ENCODING cDNAs FROM Jatropha curcas L. IP-2P ……..
Abstract ………………………………………………………………..
Introduction ……………………………………………………………
Materials and Methods ………………………………………………..
Results and Discussion ………………………………………………..
Conclusion …………………………………………………………….
Page
xvi
xvii
1
1
4
5
6
7
7
10
11
13
14
16
17
22
24
26
27
28
30
32
33
38
43
43
45
49
60
63
63
63
64
67
72
V
VI
VII
VECTOR CONSTRUCTION AND INTRODUCTION THE ALMT1
GENE FROM WHEAT INTO Jatropha curcas L. IP-2P BY
Agrobacterium-MEDIATED GENETIC TRANSFORMATION ……
Abstract ………………………………………………………………..
Introduction ……………………………………………………………
Materials and Methods ………………………………………………..
Results and Discussion ………………………………………………...
Conclusion ……………………………………………………………..
GENERAL DISCUSSION …………………………………………….
GENERAL CONCLUSION AND SUGGESTION ………………..
REFERENCES ………………………………………………………..
APPENDICES …………………………………………………………
73
73
73
76
83
92
93
99
101
127
LIST OF TABLES
Page
1.
Effect of Al treatment on growth parameters of six Jatropha
populations………………………………………………………….
54
2.
Abbreviations of various media used for Jatropha regeneration
and transformation …………………………………………………
81
LIST OF FIGURES
Page
1.
Research flow chart …………………………………………………….
5
2.
Important parts of the physic nut: a - flowering branch, b - bark, c - leaf
veinature, d – pistillate flower, e - staminate flower, f - cross-cut of
immature fruit, g - fruits, h - longitudinal cut of fruits (Source: Heller
1996) ……………………………………………………………………
9
3.
Models for the Al-stimulated secretion of organic acid anions (OA)
from plant roots (Source: Delhaize et al. 2007) ………………………
20
4.
Diagrammatic representation of carbon pathways in plant cells related to
malate and citrate metabolism. Aco, aconitase; CS, citrate synthase;
MDH, malate dehydrogenase; NAD-ICDH, NAD specific isocitrate
dehydrogenase; NADP-ICDH, NADP specific isocitratedehydrogenase;
OAA, oxaloacetate; PEPC, phosphoenolpyruvate carboxylase; PK,
pyruvate kinase; PM, plasma membrane. Hatched ellipses on the plasma
membrane and mitochondria denote membrane transporter (Source:
Mariano et al. 2005) ……………………………………………………
21
5.
Hypothetical models (a) Pattern I and (b) Pattern II for the Al3+activated efflux of organic anions by members of the ALMT and MATE
families of proteins (Source: Delhaize et al. 2007)……………………..
26
6.
Topological model of ALMT1. The model depicts the membrane
topology of ALMT1 (Source: Motoda et al. 2007) …………………….
29
7.
Secondary structure of ALMT proteins. Most ALMT proteins are predicted to have 5–7 membrane spanning regions in the amino terminal
half of the protein (Source: Motoda et al. 2007).......................................
30
8.
Agrobacterium-plant cell interactions. This diagram summarizes all
major cellular reactions involved in T-DNA transport. Steps 1 through 7
indicate sequential processes that occur during Agrobacterium infection.
Step 1, binding of Agrobacterium to the host cell surface receptors; Step
2, recognition of plant signal molecules by the bacterial VirA/VirG
sensor-transducer system; Step 3, activation of the bacterial vir genes;
step 4, production of the transferable T-strand; step 5, formation of the
T-complex and its transport into the host plant cell; step 6, nuclear
import of the T-complex; and step 7, T-DNA integration. IM, bacterial
inner membrane; NPC, nuclear pore complex; OM, bacterial outer
membrane; PP, bacterial periplasm (Source: Sheng & Citovsky 1996)..
39
9.
The inhibitory percentages of root growth six Jatropha population
seedlings after one month exposure to Al (A= Asembagus, M=
Muktiharjo, P=Pakuwon) ………………………………………………..
50
10.
The reduction percentages of plant height six Jatropha population
seedlings after one month exposure to Al (A= Asembagus, M=
Muktiharjo, P=Pakuwon) ………………………………………………..
51
11.
The reduction percentages of leaf number six Jatropha population
seedlings after one month exposure to Al (A= Asembagus, M=
Muktiharjo, P=Pakuwon) ………………………………………………..
52
12.
The reduction percentages of plant dry weight six Jatropha population
seedlings after one month exposure to Al (A= Asembagus,
M=Muktiharjo, P=Pakuwon) ……………………………………………
53
13.
Total RNA of J.curcas L.IP-2P from (A) root tips and (B) leaves .……
55
14.
Expression level of JcALMT transcript relative to actin in the root and
leaf tissues of J.curcas treated by Al for (A) 24 hour and (B) 7 days……
57
15.
Expression level of JcALMT transcript relative to actin in the root and
leaf tissues of J.curcas treated by pH for (A) 24 hour and (B) 7 days…...
59
16.
pGEM®T Easy vector for carrying actin gene fragment from J. curcas
L. IP-2P......................................................................................................
66
17.
Total RNA from J. curcas root …………………………………………
68
18.
PCR amplification to isolate actin fragments using cDNA as a template
and degenerate primer for actin genes, P1Ac12S and P1Ac284N. Lane
1, 2 = actin fragments from root, Lane 3= actin fragments from leaf, M
= 1 kb DNA ladder markers ……………………………………………..
68
19.
Location of Jatropha actin fragment inside the general structure of the
A. thaliana actin genes. (Source: McDowell et al. 1996) …………….
69
20.
Restriction analysis of three positive recombinant clones with EcoRI,
700 bp (1), 600 bp (2,3), M = 1 kb DNA ladder marker ........................
70
21.
Phylogenetic tree describes relationship among Jatropha actin with
another actins. Tree built with the neighbor-joining method, formed
well-defined three separate groups: I = dicotyledonous-woody plants, II
= monocotyledonous plants, III = dicotyledon-herbaceous plants. The
bootstrap values shown…………………………………………………..
71
22.
pGEM-ALMT1-1 plasmid which carried the ALMT1-1 coding region
(Source: Sasaki et al. 2004) …………………………………………….
76
23.
Schematic representation of pMSH1 vector. RB=Right Border, LB=Left
Border of T-DNA; NPTII=neomycin phosphotransferase II gene;
HPT=hygromycin phosphotransferase gene, P35S =cauliflower mosaic
virus 35S promoter; PNOS=promoter of the nopalin synthase gene;
T=terminator of the nopalin synthase gene ……………………
77
24.
The T-DNA región of pA2. AtALS gene = Arabidopsis thaliana
W574L/S653IALS gene, ALS P = ALS promoter, ALS T = ALS
terminator, LB = Left Border, RB = Right Border ………………………
78
25.
Agarose gel profile of the PCR product the 1500 bp fragment for the
ALMT1 gene (Lane 1) M = 1 kb DNA ladder…………………………..
84
26.
Agarose gel profile of pMSH1 (lane 1), digested pMSH1 (lane 2), 1500
bp ALMT1 insert (lane 3 and 4), digested-pA2 (lane 5). (M1= λ Sty
marker, M2 = 1 kb DNA ladder).……………………………………….
84
27.
PCR amplification of the cloned 1500-bp fragment of the ALMT gene
inside (A) pMSH1 and (B) pA2 vector was performed by direct colony
PCR using specific primer for ALMT. (M = 1 kb DNA ladder, Lane 1-3
transformed E. coli carrying ALMT inside the binary vector) …………
Agarose gel electrophoresis of M = 1 kb DNA ladder, lane 1, positive
control ALMT fragment; lane 2, E.coli colonies PCR harboring pA2ALMT construct, lane 3-4, A. tumefaciens LBA4404 colonies PCR
transformed with pA2-ALMT construct. Arrows indicate a 1500 bp
fragment of ALMT gene. Resolved on 1% agarose gel…………………
(A) Calli induced on callus-inducing medium containing bispyribac.and
(B) Non-infected explants. Bar equals 1.0 cm ……………………..
85
28.
29.
30.
31.
32.
86
87
Three months-old selection of regenerated adventitious shoots on
bispyribac containing medium. Bar equals 1.0 cm.. ……………………
(A) Non-transformed shoot on root elongation media. (B) Arrow points
to root of non-transformed plant after two weeks on root elongation
media (Inset: picture taken from the bottom side of the culture bottle).
Bar equals 1.0 cm. ……………………………………………………….
89
PCR confirmation of four bispyribac-resistant regenerated shoots.
Arrows indicates the amplification of ALMT gene (1500 bp). M= 1 kb
DNA ladder. Lane 2 and 3 were putative transgenic shoots…………….
91
90
LIST OF APPENDICES
Page
1.
Area and distribution of acid soils based on soil type in Indonesia ……..
127
2.
Description of J. curcas L.IP-2A population ……………………………
128
3.
Description of J. curcas L.IP-2M population …………………………...
129
4.
Description of J. curcas L.IP-2P population ……………………………
130
CHAPTER I
INTRODUCTION
Background
Global energy supply is currently mainly based on fossil fuels such as coal,
oil and gas. Increased use of fossil fuels causes environmental problems both locally
and globally. Developing a reliable source of renewable energy is therefore attracting
a major global attention (Jain & Sharma 2010). One potentially promising option is
biofuel, since these are derived from biomass and do not contribute to the greenhouse
effect (Eijck & Romijn 2008; Jain & Sharma 2010).
Indonesia started to develop the biofuel industry in 2006 as a response to a
progressive price increase of fossil based oil in the world, declining domestic crude
oil production and considerable progressive increase in domestic oil consumption. In
promoting the production of biofuel, the Indonesian government already had a
number of legal instruments, including the Presidential Decree No. 5/2006 on
national policies for optimizing energy use and the Presidential Instruction No.
1/2006 on the use of biofuel. Since then, various initiatives both from the
government and private sectors underline efforts to develop biofuel industry in
Indonesia.
Indonesia has the potential to become a major producer of biofuel. There are
many crops that could be used as raw materials, including palm, Jatropha curcas,
sweet sorghum, sugar cane and cassava. Crude Palm Oil (CPO) can be used to
produce biodiesel, a replacement for diesel, while sugar and cassava can be used to
produce bioethanol to replace gasoline. CPO and molasses are the current primary
feedstock used in Indonesia’s biofuel production. Cassava, Jatropha and sweet
sorghum are other potential feedstocks. However, the development of those
commodities is still in the early stage. To increase biofuel production, the
government encourages private companies to build more processing plants.
Partly in order to respond to accusations that biofuel compete with food
production, some policy makers have proposed that biofuel crops should be planted
on land that is considered marginal or idle. There are millions of hectares of such
land around the world, which play no role in food production. In Indonesia marginal
lands is about 32.0 million hectares which are potential to be developed, scattered in
2
all provinces (Adimihardja et al. 2004; Directorate of Land Rehabilitation and Soil
Conservation 2000). According to the Indonesian government, there are three types
of land not used for agriculture: marginal land, critical land and sleeping land. In
Indonesia, “marginal” land is considered to be less productive land, whether dry or
wetland with high acidity due its formation process and its nature and properties.
Indonesian marginal land therefore includes swampland, wetlands and peat forests,
as well as dry land on acidic soil. “Critical” land is land that has been ecologically
degraded as a result of intensive agricultural practices, and which is no longer
suitable for farming. These degraded lands were once important for maintaining food
security in the country, and priority should be given to improving food security for
example through better paddy field irrigation. “Sleeping” land is temporarily
uncultivated or neglected land, that does not match its previously allocated land use
planning classification such as for agriculture, housing, industry and public services
(Anonim 2008).
Acid soils cover a large area of cultivated land in Indonesia. The pH of these
soils ranges from 4 to 5. On highly acidic soils (pH 4.0), the rhizotoxic aluminum
species, Al3+, is solubilized to ionic form, which is toxic to all living cells at low
concentrations. In plants, ionic Al rapidly inhibits root elongation by targeting
multiple cellular sites and subsequently the uptake of water and nutrients (Kochian et
al. 2005; Ma 2007), resulting in poor growth. Al toxicity has therefore been
recognized as a major factor limiting crop production on acid soils, which account
for 30% to 40% of the world’s arable soils (von Uexküll & Mutert 1995).
However, some plant species have developed mechanisms to detoxify Al,
both internally and externally (Ryan et al. 2001; Ma et al. 2001; Kochian et al.
2005). The most-documented and general mechanism of Al tolerance in both
monocots and dicots is release of organic acid anions, including malate, citrate, and
oxalate, from the roots in response to Al (Kochian et al. 2005; Ma 2007). The Aldependent stimulation of organic acid efflux from roots has been associated with an
increase in Al resistance. Those anions of organic acids released by roots are thought
to chelate the toxic Al cations, to form non-phytotoxic Al form, and thus prevent
them from interacting with the root apices. Genes responsible for the secretion of Al-
3
induced malate in wheat, citrate in barley and sorghum have been identified (Sasaki
et al. 2004; Magalhaes et al. 2007).
One crop that is often cited as ideal for growing on marginal land including
acid soil with high Al solubility, is the physic nut, Jatropha curcas. The problem of
great concern regarding the Jatropha plant is these plants are not, by nature, tolerant
to Al stress. One promising approach to overcome this limitation is to develop a
transgenic Jatropha plant that can withstand severe Al-stress. Thus it is possible to
use TaALMT (Triticum aestivum Aluminum-activated Malate Transporter) cDNA to
generate transgenic Jatropha with enhanced tolerance to Al-stress.
Jatropha is one of such renewable oils, an important multipurpose plant
belonging to the family Euphorbiaceae. Jatropha is grown as a boundary fence to
protect field from the grazing animals and as a hedge to prevent soil erosion. It is a
native of the central America and occurs mainly at low altitudes in areas with annual
temperature of well above 30°C. Among the various oil seeds, Jatropha has been
found more suitable for biodiesel production on the basis of various characteristics.
This oil-bearing tree produces seeds that contain 30-40% oil which ideal for
biodiesel production. Jatropha oil has higher cetane number (51) compared to other
oils, which is compared to diesel (46–50) and make it an ideal alternative fuel (Jain
& Sharma 2010).
In 2006 The Plantation Research and Development Center (Puslitbangbun),
Indonesian Ministry of Agriculture through selection mechanism, has obtained six
lines/ population of Jatropha i.e. IP-1A, IP-2A, IP-1M, IP-2M, IP-1P and IP-2P.
Those plant materials were a composite result of superior individuals which selected
from different provenances. From those IP-1 populations the next selected were IP-2
populations which is the derivation of plant material IP-1 (Hasnam 2007a). There is
wide genetic variation, both within and between species, in the resistance of plants to
Al. Such variation provides breeders with a strategy for improving the ability of crop
plants to cope with Al toxicity.
One of the requirements in developing Al-tolerant Jatropha transgenic that
can adapt to acid soil with high level of soluble Al is the availability of a good
selection tool that is able to find parental resources based on Al-tolerant and Alsensitive plant characteristics. Plant morphological characteristics are considered as
4
typical traits and could be used as a criterion to evaluate Al-tolerance of plant. It may
be expected that the higher tolerance may associate with higher plant morphological
traits values. Besides investigating morphological characteristics, plant stress study
based on gene expression is also required. Up until now, there has been no data about
ALMT gene expression analysis on J. curcas under Al-stress. The analysis of gene
expression requires sensitive, precise, and reproducible measurements for specific
mRNA sequences. Quantitative Real-Time PCR (qRT PCR) is, at present, the most
sensitive method. To avoid bias, qRT-PCR is typically referenced to an internal
control gene which should not fluctuate during treatments. Mainly housekeeping
genes were used for the quantification of mRNA expression. Currently, at least nine
housekeeping genes are well described for the normalization of expression signals.
One of the most common are actin (Sturzenbaum & Kille 2001). Until recently the
actin gene has never been isolated from J. curcas.
Objectives
For all the above reasons, the general objective of the present study was
to introduce the TaALMT gene from wheat into J. curcas L. IP-2P plants. While the
specific objectives were:
1. To evaluate the low pH and Al-tolerance of six Jatropha population in the acid
soil with Al-stress treatment.
2. To examine the ALMT gene expression from the roots and leaves of wild type
J. curcas L. IP-2P during Al-stress treatment.
3. To isolate, clone and characterize actin gene from J.curcas L. IP-2P.
To achieve these objectives, the research was conducted as described by Figure 1.
Approach
Transgenic plant generating experiments was initiated with selected
population. Six populations of Jatropha are used for starting materials of this study.
The required data include information on plant tolerance under low pH and Al-stress
and Al-induce gene expression. Analysis of these data allows distinguishing more or
less promising population in relation to their ability to tolerate Al-stress. Gene
5
expression study of the Jatropha population was done as the preliminary research
before developing transgenic Jatropha plants carrying ALMT gene. The gene
expression study by using Quantitative RT PCR needs actin gene as an internal
control, thus it needed to isolate from Jatropha cDNA.
Jatropha curcas L. plants
TaALMT gene
from wheat
Construction of
expression vector
harboring TaALMT
Introduction of
TaALMT gene into J.
curcas L. IP-2P via
Agrobacterium
J. curcas IP-2P use as
material study
J. curcas L.
cDNA synthesis
Actin gene isolation
from J. curcas L as a
qRT PCR reference gene
Tolerance analysis
of six J. curcas
population under
low pH and Al
stress
Expression analysis
of J. curcas IP-2P
under low pH and Al
stress
Plant regeneration
and selection
J. curcas L
transgenic plant
carrying TaALMT
Research Topic I
Research Topic III
Research Topic II
Figure 1. Research flow chart
Novelty
This dissertation involved developing a transgenic J. curcas L. IP-2P plants
capable of growing in acid soil with high aluminum solubility. This dissertation is
the first report on J. curcas transgenic plants carried the ALMT gene. The second
novelty of this dissertation is the isolation of cDNA encoding actin (JcACT) from J.
6
curcas L. IP-2P as internal control in the gene expression analysis by using qRT
PCR. The three actin cDNA nucleotide sequences included in this dissertation are the
first J. curcas actin information from Indonesia reported.
7
CHAPTER II
LITERATURE REVIEW
Jatropha curcas L.
Jatropha curcas L. is a deciduous, multipurpose shrub belonging to the
family Euphorbiaceae. It is distributed naturally in the equatorial Americas, from
where it has been introduced and become naturalized in many parts of the tropical
and subtropical regions of the world (Heller 1996). J. curcas plant has medicinal
values and is commonly grown as hedges to protect gardens and fields from animals.
However, in the recent years, the species has gained tremendous significance as a
potential biodiesel plant, which is characterized as a safe, healthy environment and
renewable resource, alternative to petrodiesel.
J. curcas is very adaptable to a wide range of soils and climates and grows
well without any special nutrition regime (Patil & Singh 1991). It can be grown in
arid zones (20 cm rainfall) as well as in higher rainfall zones. It is a quick yielding
species even in adverse land situations, viz., degraded and barren lands under forest
and non-forest use, dry and drought prone area, marginal lands even an alkaline soils
and also as agro forestry crops. Jatropha can be a good plant material for ecorestoration in all types of wasteland. It can be propagated from seed or cuttings of
stem or branch, and produces large quantity of oil-seed within 2–3 years after
planting.
Jatropha, the physic nut, by definition, is a small tree or large shrub which
can reach a height of up to 5 m, but can attain a height of 8 or 10 m under favorable
conditions. The plant shows articulated growth (Kumar & Sharma 2008) straight
trunk, thick branchlets with a soft wood and a life expectancy of up to 50 years
(Achten et al. 2008). The branches contain latex. Normally, five roots are formed
from seedlings, one central and four peripheral. A tap root is not usually formed by
vegetatively propagated plants (Kobilke 1989). Jatropha has 5 to 7 shallow lobed
leaves with a length and width of 6 to 15 cm, which are arranged alternately. The
plant produces flowers in racemose inflorescences. Inflorescences are formed
terminally. The flowers are unisexual, male and female flowers are produced in the
same inflorescence; occasionally hermaphrodite flowers occur (Dehgan & Webster
1979). Normally, the inflorescences produce a central female flower surrounded by a
8
group of male flowers. In a few, the expected places of
female flowers are
substituted by male flowers. Numerically, 1–5 female flowers and 25–93 male
flowers are produced per inflorescence. The average male to female flower ratio is 29
: 1. Both sexes have five nectar glands produced nectar located at the base of flower.
Male flower has ten stamens performed a circle patterns. Pollen is yellow, globular.
The female flowers has three cell ovary that terminated by three styles. Each
inflorescence, once it begins flowering, flowers daily, and the flowering lasts for 11
days. Jatropha flowers during the rainy season with concentrated flowering from late
July to late October. Pollination is by insects. The rare hermaphrodite flowers can be
self-pollinating.
During pollination, sepal and petal will protect the fruit
development. After pollination, a trilocular ellipsoidal fruit is formed. The exocarp
remains fleshy until the seeds are mature. A fruit contain 3-4 black seed which 2 cm
long and 1 cm thick. Fruits will ripe after 40-50 days after pollination follows with
fruit color change from green to yellow (Raju & Ezradanam 2002; Bhattacharya et
al. 2005). Gupta (1985) investigated the anatomy of other plant parts. Relevant parts
of the plant are shown in Figure 2.
Carvalho et al. (2008) found that the genome of J. curcas is relatively small
(C = 416 Mb) and in the same size range as that of rice and an average base
composition of 38.7% GC. The karyotype of J. curcas is made up of 22 relatively
small metacentric and submetacentric chromosomes whose size range from 1.71 to
1.24 mm. In addition, the morphometric similarities observed between chromosomes
from heterologous pairs suggest that J. curcas is an autotetraploid species.
Seed storage behaviour of Euphorbiaceae is generally orthodox according to
Ellis et al. (1985) (one exception, Hevea). Orthodox seed storage behaviour means
“mature whole seeds not only survive considerable desiccation (to at least 5%
moisture content) but their longevity in air-dry storage is increased by reduction in
seed storage moisture content and temperature (e.g. to those values employed in
longterm seed stores). Jatropha also has orthodox seeds.
9
Figure 2. Important parts of the physic nut: a. flowering branch; b. bark; c. leaf
veinature; d. pistillate flower; e. staminate flower; f. cross-cut of immature
fruit; g. fruits; h. longitudinal cut of fruits (Source: Heller 1996).
Two- or sixmonth-old seeds stored in unsealed plastic bags at ambient temperatures
(approximately 20°C) for 5 months and germinated on average by 62% (ranging
from 19 to 79%) after having been seeded in soil.
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Kobilke (1989) investigated the viability of seeds of different ages (1 to 24
months) that were collected directly from the sites or stored for a certain time. Seeds
older than 15 months showed viabilities below 50%. One explanation for this rapid
decrease is that these seeds remained at the site, having been exposed for long
periods to extreme changes in levels of humidity and temperature. High levels of
viability and low levels of germination shortly after harvest indicate innate (=
primary) dormancy. This behaviour has also been reported for other Euphorbiaceae
(Ellis et al. 1985).
Acid Soils
Acid soils, which are soils with a pH of 5.5 or lower, are one of the most
important limitations to agricultural production worldwide. Approximately 30% of
the world’s total land area consists of acid soils and as much as 50% of the world’s
potentially arable lands are acidic (von Uexkull & Mutert 1995). Furthermore, the
large areas of acidic soils in the tropics and subtropics are critical food-producing
regions for developing countries. Thus, acid soils limit crop yields in many
developing countries (Kochian et al. 2004).
Most of the total area land available in Indonesia is 190 million ha, for
agricultural land is classified as ultisols (47 million ha) (See Appendix 1). Those
soils are considered acid with high Al content, low cation exchange capacity and low
nutrient content (Mulyadi & Soepraptohardjo 1975; Santoso 1991). The most
widespread dry acid soil in Indonesia is in Sumatera, Borneo, and Papua (WidjajaAdi et al.1992). Acid soils in Indonesia are characterized by low pH (4.0-5.50),
cation-exchange capacity (CEC) 10.17 - 10.89 me/100 g (low), base saturation 19 21 % (very low); and Al exchangeable 3.43 - 3.80 me/100 gram (Pusat Penelitian
Tanah 1983).
Mineral acid soils result from parent materials that are acidic and naturally
low in the basic cations (Ca, Mg, K and Na), or because these elements are leached
from the soil, reducing pH and the buffering capacity of the soil. As soil pH
decreases, aluminum (Al) is solubilized and the proportion of phytotoxic aluminum
ions increases in the soil solution. In most mineral soils there is sufficient Al present
to buffer the soil to around pH 4. Organic acid soils, consisting of large amounts of
humic acids and partially decomposed plant matter, typically have little Al buffering
11
and the pH of these soils can fall below pH 4 (Kidd & Proctor 2001). Superimposing
agricultural production on an ecosystem, especially ammonium fertilization,
accelerates soil acidification due to nitrification. Furthermore, acid rain, containing
nitric and sulfuric acids, is increasing the rate of soil acidification.
Slightly acidic soils (pH of 6.5) are considered most favorable for overall
nutrient uptake. Such soils are also optimal for nitrogen-fixing legumes and nitrogenfixing soil bacteria. Some plants are adapted to acidic or basic soils due to natural
selection of species in these conditions. Potatoes grow well in soils with pH 7.5). Root
growth in spinach was significantly inhibited at pH 4.5 compared with growth at
more neutral conditions. Soil pH also affects the soil in other ways. For example, soil
microbe activity; particularly nitrogen-fixing bacteria may be reduced in acid soil. In
the case of nitrogen-fixing plants, soil acidity is even more problematic since their
symbiotic bacteria are also sensitive to Al and acidity (Hungria & Vargas 2000).
A number of factors contribute to acid soil toxicity depending on soil
composition. In acid soils with a high mineral content, the primary factor limiting
plant growth is Al toxicity (Kinraide 1991). But Al toxicity is not the only stress in
acid soils; among others, proton and manganese toxicity as well as phosphorus
deficiency are also common (Marschner 1995).
Plants in acid soils also suffer from