The presentation describes the advantages of plastid transformation over 'conventional' nuclear transformation, hurdles to plastid transformation, its advantages. The presentation also covers some successful plastid engineering and its potential.

1. For a greener future
Sachin S Rawat
School of Biotech, GGS IP University

2. A Look at the Plastid

3. A Plastid is a….
 Major organelle of plant and algal cells
 Site of manufacture and storage of important
chemical compounds
 Has circular, dsDNA copies
 Replicates autonomously of the cell
 Thought to have been originated from
endosymbiotic bacteria
 Plastid genes show maternal inheritance

4. Derived from proplastids in meristem

5. Have diverse functions
 Chloroplasts – green plastids – for
photosynthesis
 Chromoplasts – coloured plastids – for pigment
synthesis and storage
 Gerontoplasts – control dismantling of
photosynthetic apparatus during senescence
 Leucoplasts – colourless plastids – monoterpene
synthesis
 Leucoplasts include amyloplasts (starch),
elaioplasts (fats), proteinoplasts (proteins) and
tannosomes (tannins)

6. 120-130 plastid genes
Are densely packed and fall into 2 categories:
 Photosynthesis-related genes
 Genetic system genes - genes for rRNAs, tRNAs,
ribosomal proteins and RNA polymerase subunits

7. A Fresher Look at the Plastid

8. Why plastid transformation?
 High protein expression levels
 Absence of epigenetic effects
 Uniparental inheritance is commercially favoured
 Easy transgene stacking in operons
 Increased biosafety – Since plastids are
maternally inherited, they aren’t transmitted by
pollen

9. Hurdles to ‘transplastomic’ plants
 Difficulty in delivering foreign DNA through double
membrane of the plastid
 The enormous copy number (polyploidy) of the
plastid genome
 The desired genetic modification must be in each
copy of plastid genome in each cell
 Failure to achieve homoplasmy results in rapid
somatic segregation and genetic instability
 Repeated rounds of selection and regeneration
are required

10. DNA delivery into plastids
 2 successful methods include 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
 Homologous recombination operates in plastids
at a high efficiency

11. Biolistic chloroplast transformation and transgene integration into the
plastid genome via homologous recombination

12. Recent success
 Expression of Bt toxin gene from the tobacco plastid
genome
 High accumulation levels of Bt toxin protein (3-5 % of
TSP)
 Plants with high-level resistance to herbivorous insects
 Co-expression with upstream ORFs further increased
Bt toxin accumulation and even resulted in its
crystallization in chloroplast
 Production of somatotropin (7% TSP) in tobacco
plastids

13. Case Study I – Lactuca sativa

14. Protoplast isolation
 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
 Protoplasts were collected at surface after
centrifugation at 70g for 8min

15. Transformation and culture
 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
 The suspension was plated onto Petri dishes and
cultured at 25ºC in the dark
 Selection was initiated on the 7th day by fresh
medium containing spectinomycin dihydrochloride

16. Analyses
 PCR – specific primers were used to assess the
presence of aadA gene in resistant cell lines
 Immunoblot analysis – using HRP-conjugated
secondary antibodies
 Southern and Northern blots were performed to
look for target genes and their transcripts
 After 2 weeks, non-transformants were yellow
while spectinomycin-resistant seedlings were
green and growing vigorously

17.  100% of spectinomycin-resistant lettuce cell lines
were true plastid transformants
 A limitation was the high frequency of polyploid
cell lines

18. Production of human
therapeutic proteins
Why lettuce is favoured over tobacco?
 Most of the plant is leaf tissue and this tissue
contains the greatest number of plastids per cell
 Unlike tobacco, lettuce has no toxic alkaloids that
need to be removed - low purification and
downstream processing costs
 Lettuce is a relevant human foodstuff that can be
consumed without cooking

19. Case Study II – Petunia hybrida

20. Plastid transformation
 Leaf pieces were placed on MS medium
supplemented with 1 mg/l 6-benzylaminopurine, 0.1
mg/l IAA, 30 g/l sucrose and 0.8% agar (MSB30)
 Leaves were bombarded with 1µm, vector-coated
gold particles from distance of 6cm
 Incubated in dim light for 48h at 25ºC
 Leaves were transferred to MSB30 medium with
200mg/l each of streptomycin sulfate and
spectinomycin dihydrochloride pentahydrate
 Resistant shoots first appeared after 8 weeks

21. Vector design

22. Analyses
 DNA blot – gene specific primers were used
 GUS assay – 5-Bromo-4-chloro-3-indolylbeta-D-
glucuronic acid was used to compare the protein
expression levels between the wild type and the
transformants by detecting fluorescence
 Selection on two antibiotics overcomes the
problem of spontaneous resistant mutants
associated with using spectinomycin alone

23. Comparing plastid
transformants with
non-transformants

24. Good model to study plastid biology
 N. tabacum is amphi-diploid
 A. thaliana doesn’t give rise to fertile
transplastomes
These limitations are overcome in Petunia as:
 P. hybrida is diploid
 Suitable for mutation screening to identify nuclear
loci affecting the maintenance and expression of
plastid transgenes

25. A Look at the Future

26. Metabolic pathways into plastids?
 Cost-effective production platform for
pharmaceuticals and nutraceuticals
 Production of trehalose in tobacco chloroplasts
 Tryptophan overproduction by feedback-
insensitive synthesis of α-subunit of anthranilate
synthase
 Simplifying technology, extending crop range

27. Can we engineer
photosynthesis?
 Efficiency of photosynthesis
 The most abundant protein in the world
 Its CO2:O2 specificity that matters
 Getting a better RubisCO for your plant
 Equally precise tools for nuclear genome required

28. Plastids for Synthetic Biology
 A compact, minimal genome
 High transgene expression and low cost ideal for
synthetic biology
 Designing totally new plastids

29. References
 Bock and Khan; Taming plastids for a green
future; Trends in Biotechnology
 Lelivelt et al.; Stable plastid transformation in
lettuce; Plant Molecular Biology
 Zuilen et al.; Stable transformation of Petunia
plastids; Transgenic Research

30. Thank you 