Plant transformation methods

Abhishek thakur

Plant Agriculture

Sustain life on Earth

Oxygen Food, Fiber & Shelter Habitats for animals Preserve soil Plants beautify

Ensure with five food groups

Cereals Vegetables & fruits Milk & milk products Pulses / non-veg protein Fat, oils & sugars Good breakfast, moderate lunch & light dinner

Sufficient, nutritionally adequate & culturally acceptable food for an active, healthy life.

Conventional breeding

Genetic engineering

Tissue culture

Plant Transformation Methods

Biological In planta
A. Tumefaciens A. Rhizogenes Virus-mediated

Physical
Microinjection Pressure Biolistics - gene gun/ particle bombardment Electroporation Microinjection Silica/carbon fibers Lazer mediated SAT

Chemical
PEG DEAE-dextran Calcium phosphate Artificial lipids Proteins Dendrimers

1) Plants - physical methods Transformation

Microinjection Electroporation Biolistics - gene gun Silica/carbon fibers Lazer mediated SAT

Microinjection of GOI
The microinjection method uses a fine needle to inject a solution of DNA into a cell.

Biolistic / Gene Gun

Sonication & SAAT

2)Virus as cloning vector

Caulimovirus vectors Geminivirus

3) Vector mediated gene transfer method3

Agrobacterium tumefaciens a natural genetic engineer

Plant Tumor

Agrobacterium
Agrobacterium (disease symptomology and host
range)

A. radiobacter - avirulent species

A. tumefaciens - crown gall disease A. rhizogenes - hairy root disease

A. rubi A. vitis
- cane gall disease - galls on grape and a few other plant species

Ti Plasmid
DNA introduced into the plant resides on a plasmid (200 kb) Ti plasmid (Tumor-inducing). The DNA introduced into the plants lies between a left and right border on the Ti plasmid. This T-DNA harbors genes encoding novel amino acids called opines (for use by the bacterium) genes encoding biosynthetic enzymes for auxin and cytokinin.

 Removed and replaced with A selectable marker (usually NPTII - neomycin phosphotransferase confers resistance to Kanamycin). Gene Of Interest. Elsewhere on the Ti plasmid are a group of genes called the vir genes (virulence genes) which are initially activated by wounding of the plant tissue which inititiates a cascade of vir gene activation.

Ri Plasmid
A. rhizogenes - casuse hairy root disease in plants

Gene transfer methods in plants

Use of Ti plasmid to in plants

1) binary vector stretegyVIR REGION T-DNA

Unique restriction site

HOST SPECIFICITY REGION

2)Co-integration strategy
Small pBR-type T-DNA Normal Ti-plasmid

Host specificity

Gene to be cloned

virulence

Recombinant Tiplasmid

T- DNA
Nopaline type ti plasmid- 23kb T-DNA Octopine type ti plasmid- 13kb left hand piece + 8kb right hand piece T-DNA- carry gene for opine synthesis + phytochrome biosynthesis Oncogenic region- 1) rooty locus 2) shooty locus)

Ri plasmid
25bp highly conserved flanked between TDNA Left & right border

Virulence gene
40kb vir region of octopine Regulated by 8 operon virA-virH

CHROMOSOMAL GENE Constitute number of genes coded by A.tumifaciens chvA, chvB, - exopolysaccharide production help in attachment to cell wall

 chvE- glucose/galactose transporter Vir gene coinducer Opine catabolism by noc & occ genes

Isolate and clone gene of interest

Add DNA segments to initiate or enhance gene expression

Add selectable markers

Introduce gene construct into plant cells (transformation)

Select transformed cells or tissues

Regenerate whole plants

Selection
Transformation frequency is low (Max 3% of all cells) and unless there is a selective advantage for transformed cells, these will be overgrown by non-transformed. Usual to use a positive selective agent like antibiotic resistance. The NptII gene encoding Neomycin phospho-transferase II phosphorylates kanamycin group antibiotics and is commonly used.

Selection
Transgenic Nicotiana cells cultivated on selective

 Visible markers -glucuronidase (GUS) .

Gives a blue colour from a colourless substrate (Xglu) for a qualitative assay.

Green Fluorescent Protein (GFP) Fluoresces green under UV illumination Non-destructive

Also causes fluorescence from Methyl Umbelliferyl Glucuronide (MUG) for a quantitative assay

B-glucuronidase (GUS) based selection

Selection based on GFP reporter gene (green fluorescent protein)

Rice embryo

advantages
Natural mean of gene transfer Agrobacterium can infect intect plant hence overcome tissue culture problems Agrobacterium can transfer large DNA fragments easily Integration of T-DNA quite precise Stabilty of gene transfer excellent

limitations
Limitation in host range Emgryogenic cells difficult to transformed- are in deep layers

sharmamasddy32@gmail.com