Documente Academic
Documente Profesional
Documente Cultură
Abhishek thakur
Plant Agriculture
Sufficient, nutritionally adequate & culturally acceptable food for an active, healthy life.
Conventional breeding
Genetic engineering
Tissue culture
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
Microinjection of GOI
The microinjection method uses a fine needle to inject a solution of DNA into a cell.
Plant Tumor
Agrobacterium
Agrobacterium (disease symptomology and host
range)
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
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
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
Also causes fluorescence from Methyl Umbelliferyl Glucuronide (MUG) for a quantitative assay
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
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