This document describes a proposed study to produce insect-resistant transgenic plants through chloroplast transformation. Researchers will construct a chloroplast transformation vector containing insecticidal genes and a selectable marker gene driven by a chloroplast-specific promoter. This will allow expression of the insecticidal proteins to be confined to the green parts of plants. Explants will be biolistically transformed with the vector and transgenic lines containing and expressing the insecticidal genes will be selected and analyzed. The goal is to develop transplastomic plants for use in integrated pest management programs to control major insect pests in an environmentally sustainable way.

1. INTERNATIONAL AGRICULTURAL SCIENCE CONFERENCE
Van, Turkey, May 09-12, 2018
Production of Transplastomic Insect Resistant Plants; A way
Towards Better Integrated Pest Management
Md Jakir HOSSAIN, Emre AKSOY, Zahide Neslihan ÖZTÜRK ĞÖKÇE, Allah BAKHSH
Department of Agricultural Genetic Engineering , Faculty of Agricultural
Sciences and Technologies,
Nigde Omer Halisdemir University, Nigde, Turkey.
Correspondence: mjakirbotru@gmail.com
ABSTRACT:
In 21st century, integrated pest management (IPM) strategies have been developed worldwide and the use of Plants with
transgenic plastid genomes (‘‘Transplastomic’’ plants) ensures a broad spectrum insect-resistance that can offer a unique solution
to combat major devastating insect pest. Transplastomic plants offer an attractive alternative to conventional transgenic plants
and becoming more popular means of insect resistence because of various advantages like high protein levels, the feasibility of
expressing multiple proteins from polycistronic mRNAs, transgene stacking in operons and a lack of epigenetic interference
allowing stable transgene expression and gene containment through the lack of pollen transmission. Chloroplast transformation
vector can be constructed containing insectcidal genes and these genes can be flanked by DNA sequences of plant chloroplast
genome. Biolistic transformation of explants can be carried out using gene gun. IPM is a durable, environmentally and
economically justifiable system, in which damage caused by pests, diseases and weeds is prevented through the use of natural
factors which limit the population growth of these organisms, if needed supplemented with appropriate control measures. The
transplastomic lines can be regenerated and selected containing selective agent. Analysis of Putative transplastomic lines,
measurement of existence of toxin proteins, insect bioassays will be carried out to authenticate the results of this study that will
assure the IPM.
 OUR AIM AND PROPOSAL:
The overall aim of the study is to produce insect resistant transplastomic plant lines having confined insecticidal gene expression
in only green parts of the plant and further to incorporate these lines in an efficient plant breeding programme to ensure the
integrated pest management. We are aim to produce transplastomic plant by expressing insecticidal genes in chloroplast genome
only in green parts of plants that will mitigate two imperative concerns against GMOs:
(a)presence of toxic proteins in edible plant parts that will be resolved by using green tissue specific promoter (pRCA)
ensuring transgene expression only to green parts of plants
b) Emergence of resistance in insects due to low levels of toxin protein. specific promoter (pRCA) ensuring transgene
expression only to green parts of plants. Expression of multiple genes in the form of an operon in plant chloroplast genome
will solve this problem.
Acknowledgement: This research work is a part of project supported by The Scientific and Technological
Research Council of Turkey (TUBITAK) . Project No-216O027
CONSTRUCTION OF TRANSFORMATION VECTOR:
In order to develop transplastomic plants expressing both single and hybrid insecticidal genes, chloroplast transformation vector
can be constructed by PCR amplification and subsequent ligation of plastome sequences as flanks, using of pBluescript vector,
amplification of two specific chloroplast genome regions from chloroplast. Later it can ligate insecticidal genes and suitable
selectable marker gene (Chimeric gene aadA-aminoglycoside 3′ adenylyltransferase to confer resistance to streptomycin and
spectinomycin) under same or different UTRs in between flanking regions. Gene ligation may conduct under the control of green
tissue specific promoter pRCA which is a chloroplast-localized enzyme that catalyzes the activation of ribulose-1,5-bisphosphate
carboxylase/oxygenase (Rubisco).Consistent with the transcriptional pattern of the RCA gene, both promoters are only active in
the green part of plants and pRCA promoters can confine expression of insecticidal genes in green parts of the plant. Species
specific or heterologous chloroplast transformation vectors are usually develop in a manner that flanks the foreign genes and
insert them through homologous recombination at predetermined and precise location in the plastome.
1.
2
Figure: 1. a. Basic design of a typical vector for transforming the plastid genome. Both the expression cassette and the
selection cassette are placed between the two plastid regions. These flanking regions are taken from the wild-type plastid
genome of a plant species whose plastome is to be manipulated, to allow a crossover event take place to integrate DNA
sequences between them. Green arrows in the chloroplast expression vector represent promoters (P) and the direction of
transcription, whereas terminators (T) are indicated by red rectangles. The untranslated regions are represented by white
circles. The thin dotted lines with arrows indicate homologous recombination b. Chloroplast transformation vector showing
marker gene, cry3A and SN-19 gene under the control of tissue specific promoter pRCA.
Figure: 2. Sorting of ptDNA at the organelle and cellular levels yields homoplastomic plants.
A. Chloroplast (CHL) to proplastid (PP) dedifferentiation accelerates ptDNA sorting. Transformed and nontransformed
nucleoids (N) in plastids are symbolized with red and blue circles, respectively. Transformed and nontransformed ptDNA in
enlarged nucleoids are red and blue circles, respectively. ptDNA are anchored to membranes by proteins (black dots).
Nucleoid 1 is heteroplastomic and is the progenitor of homoplastomic transgenic (1a) and wild-type (1b) proplastids.
Homoplastomic proplastid differentiates into chloroplast (bottom). Wild-type proplastids (2, 1b) are antibiotic sensitive and
divide more slowly.
B. Reduction in plastid number during chloroplasts (~100 per leaf cell; green, elongated) to proplastid (~10–14 per
meristematic cell; greenish, oval) transition and lack of exact duplication of the cytoplasm accelerates formation of
homoplastomic cells. On top is a leaf mesophyll cell with one transformed chloroplast (red) and nucleus (Nu). Meristematic
cell 1 is heteroplastomic. Cleavage of the cytoplasm yields one meristematic cell with transformed chloroplasts only (1a) and
one with wild-type plastids (1b). Cell 1a is the progenitor of homoplastomic mesophyll cells (1c).
 REFERENCES:
Ralph Bock: Engineering Plastid Genomes: Methods, Tools, and Applications in Basic Research and Biotechnology, 10.1146/annurev-arplant-050213-040212
 Niaz Ahmad, Franck Michoux, Andreas G. Lössl, and Peter J. Nixon: Challenges and perspectives in commercializing plastid transformation technology, 10.1093/jxb/erw360
 Pal Maliga: PLASTID TRANSFORMATION IN HIGHER PLANTS, 10.1146/annurev.arplant.55.031903.141633
M. Manuela Rigano, Nunzia Scotti & Teodoro Cardi:Unsolved problems in plastid transformation, https://doi.org/10.4161/bioe.21452
b How Plastids are Transformed