Transcript Nanoparticle Mediated Genetic Transformation in Plants

Nanoparticle Mediated
Genetic Transformation in
Plants
PBIO 4500/5500 BIOTECHNOLOGY AND GENETIC ENGINEERING
 Presented by :
ISHA SHRIVASTAVA
How small is a nano?
A nanometer is one billionth of a meter
The thickness of an
individual page is 100,000
nms
A fine human hair is 10,000
nms
Our finger nails grow at the
rate of 1nm per second
Source: http://www.umt.edu/ethics/Debating%20Science%20Program/ODC/NanoODC/Intro/Properties/Size.aspx
Vehicles for Nuclear Transformation in Plants
Agrobacterium mediated:
Most extensively used, wide host range (mostly
dicotyledonous)
Incompatibility between tissues of plant species
Microparticle Bombardment:
Capable of delivering DNA into nucleus, mitochondria
Cell Damage, high copy number of transgene,
expensive equipment
Electroporation:
Generate transgenic plants by protoplast
transformation
Cell damage by electric pulses of wrong length, ion
imbalance and cell death
Comparative study of different delivery systems
Source: Rai,M., Deshmukh, S. , Gade, A., Elsalam, K.A. 2012 . Current Nanoscience .8 : 170-179
Nanoparticle Mediated Genetic Transformation
 Nanoparticles combined with chemical
compounds deliver genes into target cells
 Decreasing the particle size from micro
to nano scale, hindrance due to cell wall
can be removed
 Cell Damage can be minimized
 The particle can reach the chloroplast
and mitochondria easily
Different NPs used are calcium
phosphate, Carbon materials, silica, gold
magnetite, strontium phosphate.
Enable controlled release conditions
Figure: Synthesis of mesoporous silica
Source: http://www.rmat.ceram.titech.ac.jp/research-e/mesoporus-e.html
Experiment with MSNs – Genetic Transformation
Purpose: To investigate interaction of MSN with plant cells
Synthesize series of MSNs with different surface
functional groups/caps
Investigation of MSN in protoplasts (plant cells with cell
wall removed)
Protoplasts incubated with Type-I MSN didn’t take up
nanoparticles, Type-II MSN ( Type-I functionalized with
triethylene glycol) entered the protoplasts
Conclusion:
 MSN system can serve as a new and versatile tool for
plant endocytosis and cell biology studies
Figure : Type-I and Type-II
MSNs
Purpose: To prove MSNs can function as DNA delivery agents
Plasmid containing a green fluorescent protein (GFP) gene under the control of
constitutive promoter is used
Optimal coating ratio for DNA/Type-II MSNs was 1/10
Type-II MSN bound DNA not digested by restriction enzyme
Transient GFP expression observed 36 hr after protoplasts were incubated with
DNA coated Type-II MSN
Conclusion:
 Type-II MSN system can serve as efficient delivery system for
protoplasts and make DNA accessible to transcription machinery
Representation of Nanoparticle Mediated Gene Transfer
Source: Rai,M., Deshmukh, S. , Gade, A., Elsalam, K.A. 2012 . Current Nanoscience .8 : 170-179
Purpose: To introduce MSN into plants with gene gun system
• Attempts to bombard Type-I and
Type –II MSNs didn’t lead to
successful transformation
•Use of Type-III MSNs, where
mesopores are capped by surface
functionalized gold nanoparticles
•In comparison to traditional system,
allows to load biogenic moeitiesincluding chemicals that are
membrane impermeable or
incompatible with cell growth media
into pores
Figure: Gene gun system for bombarding
micro/nano particles
Source: http://swineflucaretips.blogspot.com/2012/06/dna-vaccines-and-gene-gun.html
Purpose : To deliver different biogenic species simultaneously
• Release the encapsulated chemicals in a controlled fashion
•Generate transgenic tobacco containing inducible promoter controlled GFP gene
•Expression of GFP observed only when chemical β-oestradiol is
present
•Transgenic plantlets bombarded with Type-IV MSNs ( filled with β-oestradiol) , and
pores capped with gold nanopartcles
•Release of β-oestradiol is triggered by DTT ( Dithiotheritol)
Mesoporous silica Nanoparticles for plant cell internalization
Conclusion
 Nanobiotechnology could take the genetic engineering of
agriculture to the next level down – atomic engineering
 Further developments such as pore enlargement and
multifunctionalization of these MSNs may offer new possibilites
in target specific delivery of proteins, nucleotides and chemicals.
 Opposition is mounting from civil society, unions and world
leading scientists who point to ecological, health and socioeconomic risks associated with nanogenetics.
References:
 Husaini,A.M., Abdin, M.Z. , Parray, G.A. , Sanghera, G. S. , Murtaza, I. , Alam,
T. , Srivastava, D. K. , Farooqi, H. , and Khan, H. N. 2010 . Vehicles and ways
for efficient nuclear transformation in plants. Landes Bioscience . 1(5) : 276287
 Nair, R. , Varghese, S. H. , Nair, B. G. , Maekawa , T. , Yoshida , Y. , and Kumar ,
D. S. 2010. Nanoparticulate material delivery to plants. Plant Science. 179 :
154-163
 Rai,M., Deshmukh, S. , Gade, A., Elsalam, K.A. 2012 . Current Nanoscience .8 :
170-179
 Torney, F. , Trewyn, B. G. , Lin, V.S. , and Wang, K. 2007. Mesoporous silica
nanoparticles deliver DNA and chemicals into plants. Nature Nanotechnology .
2 : 295-300