GENETIC ENGINEERING OF

CHLOROPLASTS
EXP RESSION SYSTE MS

Tran sg en ic
– Gene spliced directly into the plant’s DNA

– Stable transgenic line is reproduced

– New seeds are produced that will grow into plants

that produce the protein

Tran si en t

– Gene is spliced into a vector such as a virus

– A normal plant (no genetic modification) is infected

with the vector and then produces the protein
 Ex am ples of Tran sge ni c
Ex pre ssion Sys tem s
Nucle ar tran sf orma tio n

– Gene is inserted into nuclear DNA

Ch loro pl ast tran sf orma tio n

– Gene is inserted into chloroplast

– Each cell contains multiple chloroplasts

Pl ant s Used i n Trans geni c
Sys te ms
 Toba cco

• Corn

• Ri ce

• Fl ax See d

• Alf alf a

• Toba cc o Cel l Cultu re

• Carrot Cel l Cult ure

• Alga e
 ECOL OGICAL C ONC ERNS
REG ARDI NG GE NE TI CALLY
EN GI NEER ED C ROP S
TRAN SG EN E ESCAP E
• Out crossing
• Intergenic gene flow-Brassica napus and Brassica rapa
• Hybrid seed generation and dispersal

GENE POLLU TI ON AN D CRE ATIO N OF SU PE RW EE DS AN D

RE SI STAN T I NSE CTS

• Herbicide resistant weeds

• Insects resistant to toxins
• INCREASED GENETIC HOMOGENITY AND SELECTION PRESSURE
• Example->non engineered cotton,strawberry,canola
 Multiple
Gene expression

No position Maternal
effect WHY inheritance

GENETICALY
ENGINEER A CHOROPLAST
High
NO Transgene
Transgenic
Escape
expression
High
stability
Ho w ar e ch loro plasts
tran sfo rme d?

Bio li st ics
Po lye thyl eneglyc ol (PEG )-m edia ted
Gali sta n Exp ans io n
Femtos yr inge
BIOLIST ICS
 PE G-med iat ed
tran sforma ti on

Thi s approach requ ire s p rep ara ti on of

protop lasts an d th e targ et spec ies us ed must
be regenerab le fr om pr oto plas ts . PE G-
me diated t ra nsformation is l ess effi ci ent th an
the b ioli sti c ap proa ch .
Protop lasts tak e up D NA in th e pres ence of
PEG and chang es i n th e p lasma memb rane
allow DN A to penetra te and mov e in to th e
cytop las m. Th e fore ign D NA is tr ansported b y
som e unk nown mea ns from the cytop lasm in to
th e chl orop las t where i t may be integ rat ed
in to the the gen ome
Ga listan exp ans ion
fem to syring e
 Molecu la r Biolog y of
Ch lorop la st
Tran sform ation
VE CTO RS USE D
Flanking sequences homologous to
chloroplast genome resulting in
homologous recombination and non
random integration
Transcribed by chloroplast specific
promoters and use chloroplast
specific termination signal
Two open reading frames
 Ty pes of Sele ct able
Markers Av ail ab le F or
Ch loropl ast
Tr ansfor ma tion
• Domi na nt a nt ib iotic resi st an ce g ene s
• Rec essi ve ant ib iotic-r esis ta nce g enes
encodi ng ant ib ioti c-i nsensi ti ve a llel es
of ribos om al R NA gene s
• Rece ssi ve ma rkers t ha t rest ore
photoa ut otr ophi c gr owth b y
comp leme nti ng non- phot osynt he tic
mut ants
 Ap pli cat ion s F or
Ch lorop la st G en etic
En gin eer in g
Gl yph osat e re sista nce
broad spectrum herbicide
affects the aromatic amino acid
biosynthetic pathway in plants and
microorganisms
not toxic to animals
inactivated rapidly in the soil
Longer survival of tobacco plants
In sect res ista nce v ia B t
tox in
Bacillus thuringensis (Bt) toxins are harmful to
insects when ingested.
Kota et al. (1999) observed hyper-expression
of Bt toxins in transformed tobacco chloroplasts
high insect mortality rate.

toxins are located in green leaf tissue which is

the most likely part of the plant to be consumed
by insects.
insecticidal proteins are not produced in fruit
OTHE R AP PLI CA TIONS
OF C TT
CTT facilitates production of
enzymes,biopolymers,biopharmaceuticals(insulin
like growth factors,antimicrobial peptide,
monoclonal antibodies,etc) in plants.

CTT is developing cure for GENETIC DISEASES

(diabetes), and VACCINES for infectious
diseases (anthrax , plaque
,tetanus).
CTT produces low cost enzymes to make
BIOFUELS (etanol,etc)
 LIMITAT IO NS OF CTT
 Bipar ent al inhe rit ance in
gym no sperm s
 Rye , ki wi gen ome is p at ernall y
inh eri ted
 Not ye t avail abl e for cereals or
monoc ots
 Se ed media ted di spe rs al is no t
pr even ted