1. Nanoparticle Mediated Genetic Transformation in Plants
PBIO 4500/5500 BIOTECHNOLOGY AND GENETIC ENGINEERING
Presented by :
ISHA SHRIVASTAVA

2. 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:

3. Why do we need to combine nanotechnology and genetic engineering?
Nano Farming
Syngenta, BASF, Bayer, Monsanto using nano-scale materials to produce nanopesticides “gutbuster”
Recent breakthrough includes replacing the genetic material of one bacteria from another
Nano Food
“Smart Foods” which respond to allergies, dietary needs and food preferences
Alter the properties and traits of food including its nutrition, flavor, texture, heat tolerance & shelf life

4. 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

5. Comparative study of different delivery systems
Source: Rai,M., Deshmukh, S. , Gade, A., Elsalam, K.A Current Nanoscience .8 :

6. 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:

7. 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
Figure : Type-I and Type-II MSNs
Conclusion:
MSN system can serve as a new and versatile tool for plant endocytosis and cell biology studies

8. 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

9. Representation of Nanoparticle Mediated Gene Transfer
Source: Rai,M., Deshmukh, S. , Gade, A., Elsalam, K.A Current Nanoscience .8 :

10. Figure: Gene gun system for bombarding micro/nano particles
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 moeities-including chemicals that are membrane impermeable or incompatible with cell growth media into pores
Figure: Gene gun system for bombarding micro/nano particles
Source:

11. 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)

12. Mesoporous silica Nanoparticles for plant cell internalization

13. 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 socio-economic risks associated with nanogenetics.

14. 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 Vehicles and ways for efficient nuclear transformation in plants. Landes Bioscience . 1(5) :
Nair, R. , Varghese, S. H. , Nair, B. G. , Maekawa , T. , Yoshida , Y. , and Kumar , D. S Nanoparticulate material delivery to plants. Plant Science. 179 :
Rai,M., Deshmukh, S. , Gade, A., Elsalam, K.A Current Nanoscience .8 :
Torney, F. , Trewyn, B. G. , Lin, V.S. , and Wang, K Mesoporous silica nanoparticles deliver DNA and chemicals into plants. Nature Nanotechnology . 2 :