It is one of the blooming and emerging plant breeding technique

1. Student
J.DELINCE
2015603402
Transplastomics in Vegetable Crops
Chairman
Dr. T. ARUMUGAM
Professor and Head,
Dept. of Vegetable Crops
Dr. S. GANESHRAM
Professor and Head,
Department of Plant Genetic Resources
Dr. V. PREMALAKSHMI
Assistant Professor,
Department of Vegetable Crops
Members

2. Introduction
Plastids and Plastomes
Milestones in Plastid engineering
Methods of gene transfer
Selection
Validation
Case studies
Drawbacks

3. Transplastomics
• Plastome – Genome of the plastids
• Transplastomics – transfer of gene into the
genome of plastids
• A technology for biological containment of
genes
(Bock and Khan, 2004)

4. Plastids and Plastomes (ptDNA)
• Major organelle of plant and algal cells
• Site of manufacture and storage of important chemical compounds
• Replicates autonomously of the cell
• Thought to have been originated from endosymbiotic bacteria
• Plastid genes show maternal inheritance
• Has circular, dsDNA with size of 120 – 160kb
• Present in numbers upto 10,000 copies per plant cell
• It is tetra partite and consists of
1. Large single copy region (LSC)
2. Small single copy region (SSC)
3. 2 inverted repeats (IRs)
(Herrmann and Possingham, 1980)

5. Aspinall et al., 2006

6. Why Plastids?
• High yield of target protein,
• Removing the risk of outcrossing with weeds,
• lack of silencing mechanisms,
• Absence of epigenetic effects
• Ability to engineer the entire metabolic pathways
rather than single gene traits.
(Łojewska et al, 2016)

7. Milestones
Year Milestone DNA delivery Approach Selection Reference
1988 Chlamydomonas
reinhardtii
1st stable plastid
transformation
Biolistic
Homologous
targeting
Photosynthetic
competence
Boynton &
Gillham , 1988
1990 Nicotiana tabacum
1st stable plastid
transformation
Biolistic Spectinomycin
(rrn16)
Svab et al., 1990
1993 Nicotiana tabacum
1st high level
foreign protein
(2.5% GUS)
PEG Spectinomycin
Kanamycin
Golds et al., 1993
1999 Solanum tuberosum
(potato)
Biolistic Spectinomycin Sidorov et al., 1999
2001 Lycopersicon
esculentum (tomato)
1st foreign protein
in fruitMarker gene
elimination: CRE-
lox
Biolistic Spectinomycin Ruf et al., 2001
2003 Brassicacea (oil
seeds)
Phytoremediatio
n: Mercury
Biolistic Spectinomycin Ruiz et al., 2003
Rigano et al., 2012

8. Year Milestone DNA
delivery
Approach Selection Reference
2004 Linum usitatissimum L.
(flax): PHB polymer
expression
Daucus carota
Abiotic stress tolerance
Biolistic
Homologous
targeting
aph A-6
npt II
Spectinomycin
Wrobel et al.,
2004
Kumar, Dhingra
et al, 2004
2007 Brassica oleracea var.
capitata (Cabbage)
Biolistics Spectinomycin
and streptomycin
Liu et al., 2004
2008 Lycopersicon
esculentum (tomato)
HIV antigens
Biolisic Spectinomycin Zhou,
Badillo‐Corona et
al, 2008
2009 Lycopersicon esculentum
(tomato)
Enhanced β-carotene
Biolisic Spectinomycin Apel and Bock,
2009
2010 Lettuce
Fructose-
1,6/sedoheptulose-1,
7-bisphosphatase
Biolistics Spectinomycin Ichikawa et al.,
2010
2013 Lettuce
hG-CSF
Biolistics Spectinomycin Tabar, Habashi et
al.,2013

9. Homologous recombination
• Genetic recombination in which nucleotide sequences are exchanged between two
similar or identical molecules of DNA
• Chloroplast expression has flanking regions of genes present in the plastome
(Day and Goldschmidt‐Clermont, 2011)

10. Method of Chloroplast transformation
(Haider et al., 2010)

11. Designing the expression vectors
Navaneeth et al , 2012

12. Methods of transferring gene
• Biolistics and polyethylene glycol-mediated transfer
• Biolistics is preferred as it is less time-consuming and
demanding
• Integration of foreign DNA into plastid genome occurs via
homologous recombination

13. Biolistics
• Direct gene transfer methods
• Expression vectors are coated with gold nanoparticles (carriers)
• And they are delivered to the plant sample at high speeds
• Costlier method but with quicker transformation
No of
plates
Rupture
disc
Distance
(cm)
Independen
t events
Events per
plate
Efficiency
30 650psi 6 0 0 0
24 9 1 1/24 4.2
26 12 0 0 0
36 900psi 6 0 0 0
34 9 1 1/34 2.9
36 12 0 0 0
38 1100psi 6 0 0 0
34 9 3 1/10 6.7
30 12 4 1/7.5 13.3
( Dhingra et al,
2004)
Biolistics in Carrot calli

14. Polyethylene Gylcol (PEG) Method
• Lettuce seeds were sterilized and sown on MS medium with 2%
sucrose
• Shoot tips from leaves obtained were transferred to MS medium with
3% sucrose
• The leaves were cut into pieces and incubated in PG solution, followed
by enzyme solution consisting of 1% cellulase and 25% macerozyme
• Protoplast suspension was filtered through nylon mesh
(McCabe et al, 2005)

15. Contd..,
• Protoplasts were collected at surface after centrifugation at 70g for
8min
• 10µl transforming DNA and 0.6ml PEG solution was added to
protoplast suspension and incubated at 25ºC for 10min
• Protoplasts were mixed with 1:1 solution of B5 and 2% agarose to a
density of 3.6 X 104 protoplasts per ml
• Needs more standardization of procedures but with less equipments.

16. Homoplasmy and Heteroplasmy
• Heteroplasmy refers to the presence of more than one type of organelle genome
• In transplastomics, after bombardment we get some pt DNA transformed some may not.
• After repeated cycles of multiplication and selection we reach homoplasmy.
• Homoplasmic plastids are more stable than Heteroplasmic plastids.
(Day and Goldschmidt‐Clermont, 2011)

18. Selection and Validation
• Selection by presence of antibiotic resistance gene or herbicide
tolerance gene.
• aadA gene - aminoglycoside-3″-adenylyltransferases –resistance to
streptomycin and Spectinomycin
• Npt II – Neomycin phosphotransferase – Neomycin
Validation:
• Gene – PCR
• Northern blotting
• Western blotting

19. Plastid Expressed Betaine Aldehyde Dehydrogenase
gene in carrot cultured cells, root, and leaves,
confers salt tolerance
• Glycine Betaine – osmoprotectant
• Over expression of betaine by manipulation of badh gene.
• Insertion at 16S/trnI- trnA/23S region of chloroplast
• Transformation through particle bombardment
• Selection in spectinomycin containing media
• Regeneration through somatic embryogenesis
(Dhingra et al, 2004)

20. Visual Selection & PCR validation
A: Transformed
B: Non- transformed
C & D : Heteroplasmic calli
Lane 1 – ladder
Lane 2 – untransformed
Lane 3 – 9 : Transformed lines

21. T – Transformed
U - Untransformed

22. Enhancement of Carotenoid Biosynthesis in
Transplastomic Tomatoes by Induced Lycopene-
to-Provitamin A Conversion
• Lycopene β- Cyclase genes from Erwinia herbicola and
daffodil (Narcissus pseudonarcissus).
• Raised till homoplasmy condition along with antibiotic
selection
• Screened with herbicide CPTA (2-(4- chlorophenylthio)-
trimethylamine).
(Apel and Bock, 2009)

24. Enzyme activity assay using herbicide CPTA

26. Human Granulocyte Colony-Stimulating Factor
(hG-CSF)Expression in Plastids of Lactuca sativa
• hG – CSF – a valuable biopharmaceutical for cancer treatment and
research
• hG-CSF gene was cloned into pCL vector between prrn16S promoter
and TpsbA terminator by replacing the GUS gene
• Vector was coated with gold nanoparticles and transformed using
biolistics method
(Habashi et al, 2013)

27. Verification by PCR
Lane M – 1Kb plus ladder
Lanes 1 -5 – Transplastomic plants
Lane w – Wild type

28. Immunoblot analysis
Immunoblot analysis
1- 4 transgenic lines
W –Wilf type

29. Generation of transplastomic lettuce
with enhanced growth and high yield
• The lettuce chloroplasts were transformed with genes of
cyanobacterial fbp/sbp encoding fructose-1,6-/sedoheptulose-1,7-
bisphosphatase (FBP/SBPase)
• pLD6 transformation vector is used.
• Screened with antibiotic spectinomycin
• Increased photosynthetic ability
(Tamoi et al, 2010)

32. Expression of a Bacillus thuringiensis toxin (cry1Ab)
gene in cabbage (Brassica oleracea L. var. capitata L.)
chloroplasts confers high insecticidal efficacy against
Plutella xylostella
• K–Y cross and Summer Summit are the varieties used
• Cry 1Ab gene is transformed into the chloroplast genome
• Usage pASCC201 chloroplast vector.
(Lin et al, 2008)

33. Cry 1Ab expression
Cabbage
Cultivars
Bombarded leaf
samples
Antibiotic
resistant calli
Southern
positive
PCR positive Northern
positive
K –Y Cross 100 8 8 5 4
Summer
Summit
100 8 8 5 5
Regeneration and genetic analysis of chloroplast-transformed cabbage

34. % insect damage
Cultivar Insect death (%)
K – Y Cross
WT – 1 0
A1 67
A3 100
A5 93
A6 91
Summer Summit
WT 2 0
B2 67
B4 80
B6 100
B7 100
B9 67

35. Transformation of Solanum tuberosum plastids
allows high expression levels of β-glucuronidase
both in leaves and microtubers developed in vitro
• Vector - pBSW-utr
• Trangenes –uidA and aadA
• Transformed between genes 16S and trnI.
• Transformation done on S. tuberosum cv. Desiree.
• β-glucuronidase (GUS) accumulation levels up to 41% of TSP in
mature leaves
(Lentz et al, 2012)

37. High-level expression of human
immunodeficiency virus antigens from the
tobacco and tomato plastid genomes
• HIV contains three important retroviral genes gag, env and pol.
• Also some regulatory and accessory proteins (Tat, Rev, Vpu, Vpr, Vif
and Nef).
• Gag encodes for structural components (p24 –conical core) and an
important target for T-cell mediated immunity
• P24 is key component of vaccine
• In tomato plastid it is expressed in the plastome by selecting good
combination of gene cassete with p24-Nef gene in the tobacco earlier
(Badillo‐Corona et al, 2008)

40. Improvement of vitamin E quality and quantity
in tobacco and lettuce by chloroplast genetic
engineering
• Lactuca sativa L. cv Green Wave
• The Tocopherol synthesis pathway is engineered by introducing the
gene for Toc cyclase (TC) and c-Toc methyltransferase (c-TMT)
• The TC gene inserted in pTTC gene cassette
• The TMT gene inserted in pTMT gene cassette
• Both TC & TMT gene inserted as operon in pTTC –TMT gene
cassette
(Tanaka et al, 2013)

41. Tocopherol synthesis
RT – PCR Analysis

43. Drawbacks
• Difficult to deliver foreign DNA through double membrane
• Repeated number of selection is required to reach homoplasmy
• Not all plant species have plastome sequenced
• Monocots are recalcitrant to the plastid transformation
( Scotti et al., 2012)

44. Thank you 