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

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

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 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

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

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: 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 socio-economic 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) : 276-287 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