El documento detalla la transformación mediada por Agrobacterium, destacando su uso en la modificación genética de plantas. Explica el mecanismo de transferencia de ADN T desde Agrobacterium tumefaciens hacia las células vegetales, incluyendo la estructura del plásmido Ti y el sistema de vectores binarios. Se discuten también varios métodos de transformación para generar plantas genéticamente modificadas.

1. Agrobacterium mediated
Transformation
Dr. Varsha C Mohanan
Assistant Professor
Department of Biotechnology
St. Mary’s College Thrissur

2. Agrobacterium mediated Transformation, Dr. Varsha C Mohanan, St.Mary’s CollegeSt.Mary’s College
Agrobacterium - a natural genetic engineer
 Agrobacterium- Crown Gall
and root hair disease in plants,
characterised by tumor and hairy
root morphology.
Induces disease in dicot
angiosperms, some monocots
and gymnosperms
 Disease- Genetic transfer of
disease causing genes from the
Agrobacterium to the host plant.
Delivery of transferred (T)-DNA
from bacterial cells into host
plant cells through a bacterial
type IV secretion system (T4SS).
Courtesy: www.rhs.org.uk
Most well known
Agrobacterium sps
•Agrobacterium
tumefaciens
•Agrobacterium
rhizogenes

3. Agrobacterium mediated Transformation, Dr. Varsha C Mohanan,St.Mary’s CollegeSt.Mary’s College
Infects wounded parts of
the plant
Agrobacterium infection
Courtesy: B. Hohn, Encyclopedia of Genetics, 2001
Sciencephoto.com

4. Ti-plasmid
DNA sequence regions in
Ti plasmid -Left border,
ORFs for Auxin, Cytokinin
and Opine synthesis, Right
border, Conjugal plasmid
transfer between bacteria,
Octopine catabolism,
mannityl opine catabolism,
Ori/inc regions and Vir
genes
 Chromosomal and Vir Genes
 T-DNA Transfer
 T-DNA Integration
Mechanism of gene transfer
Agrobacterium mediated Transformation, Dr. Varsha C Mohanan, St.Mary’s CollegeSt.Mary’s College
M. Ashraf et al. (eds.), Crop Production for Agricultural
Improvement, 2012
Tumor inducing genes

5. Acetosyringone is likely
perceived by the VirA
protein encoded on the
Ti plasmid
VirA and VirG induce other
vir genes in response to
plant signals
Plant-derived small
molecules such as
acetosyringone induce
Agrobacterium vir genes
Virulence induction in presence of Plant
secondary metabolites
www.plantcell.org
Agrobacterium mediated Transformation, Dr. Varsha C Mohanan,St.Mary’s CollegeSt.Mary’s College

6. Chromosomal genes and Vir genes
Chromosomal and vir genes -involved in T-DNA transfer
Chromosomal genes: Attachment to plant cell, vir gene regulation
Vir Genes:
• VirA- Chemoreceptor, responds to chemicals like acetosyringone
and activates Vir G
• VirB- Transmembrane complex
• VirC- Host-range specificity
• VirD2- Site-specific endonuclease, cuts the T-DNA at the borders
on single strand, also have NLSs, guides T- DNA into plant nucleus.
• VirE1- T-DNA processing and protection, acts as a chaperon
VirE2- Form channels in the membrane to facilitate DNA
transport by binding to Ss of T-DNA
• VirF- Host range specificity
• VirG- Positively regulates transcription of Vir B, C, D, E, F
Agrobacterium mediated Transformation, Dr. Varsha C Mohanan, St.Mary’s CollegeSt.Mary’s College

7. Agrobacterium mediated Transformation, Dr. Varsha C Mohanan, St.Mary’s CollegeSt.Mary’s College
Agrobacterium tumefaciens – pathogen
and useful tool for genetic engineering
The features of Ti plasmid can be modified to use it for genetic
engineering
A modified Ti or Ri plasmid can be used. The plasmid is
'disarmed' by deletion of the tumor inducing genes; the only
essential parts of the T-DNA are its two small (25 base pair)
border repeats, at least one of which is needed for plant
transformation.

8. Binary Vectors system for Transformation
A transfer DNA (T-DNA) binary system is a pair of plasmids
consisting of a binary plasmid and a helper plasmid. The two
plasmids are used together (thusbinary) to produce genetically
modified plants.
Agrobacterium mediated Transformation, Dr. Varsha C Mohanan, St.Mary’s CollegeSt.Mary’s College
Lee L-Y & Gelvin S. B, 2008 Plant Physiology

9. •Binary Vector construction
•Remove the aux and cyt genes and disarm Ti Plasmid.
• Move it into a small vector plasmid
•Insert a selection marker for bacterial selection (eg.
Kanamycin resistance) and eukaryotic selection (eg.
Hygromycin resistance) within the T-DNA regions.
• Insert the gene of interest between the T- DNA borders
without disrupting the selection marker
•Retain Vir genes on different plasmid (Helper plasmid)
•Transconjugant generation
•Co-transform Agrobacterium with both plasmids
•Plant Transformation
•Infect plant with transformed bacteria
Agrobacterium mediated Transformation, Dr. Varsha C Mohanan, St.Mary’s CollegeSt.Mary’s College

10. Plant Transformation methods
•Callus transformation
• Leaf Disc Transformation
•Floral dip Transformation
•Root Transformation
Agrobacterium mediated Transformation, Dr. Varsha C Mohanan, St.Mary’s CollegeSt.Mary’s College
www.Biology Discussion.com

11. REFERENCES
•Hohn, B. (2001). Transfer of Genetic Information from Agrobacterium
tumefaciens to Plants. Encyclopedia of Genetics, 1984–
1986.doi:10.1006/rwgn.2001.1638
•Ashraf, M., Öztürk, M., Ahmad, M. S. A., & Aksoy, A. (Eds.). (2012). Crop
Production for Agricultural Improvement. doi:10.1007/978-94-007-4116-4
•Stachel, S.E., Messens, E., Van Montagu, M. and Zambryski, P. (1985).
Identification of the signal molecules produced by wounded plant cells
that activate T-DNA transfer in Agrobacterium tumefaciens. Nature. 318:
624-629
•Stachel, S.E., Nester, E.W. and Zambryski, P.C. (1986). A plant cell factor
induces Agrobacterium tumefaciens vir gene expression. Proc. Natl. Acad.
Sci. USA. 83: 379-383.
• Lee, L.-Y., & Gelvin, S. B. (2007). T-DNA Binary Vectors and Systems. Plant
physiology, 146(2), 325–332. doi:10.1104/pp.107.113001
•http://www.biologydiscussion.com/gene/gene-transfer/top-4-methods-
of-gene-transfer-with-diagrams/11898
Agrobacterium mediated Transformation, Dr. Varsha C Mohanan,St.Mary’s CollegeSt.Mary’s College