agrobacterium mediated gene transfer-130709035850-phpapp01

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m Mediated Plant Tr

30th April’2012

Presented by,Prabhu Thirusangu,Research Scholar,


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Transformation – the process of obtaining transgenic plants

Transgenic plant – a plant with a foreign gene (or genes) from another plant/animal that is incorporated into its chromosome

Marc Van Montagu and Jeff Schell, discovered the gene transfer mechanism between Agrobacterium and plants, which resulted in the development of methods to alter the bacterium into

an efficient deliver sstem for genetic engineering in plants.

Most common genes (and traits) in transgenic or biotech crops

herbicide resistance

!nsecticide resistance

"t genes in field corn (mai#e)

virus$resistance (coat$protein) genes

Transgenic plants – An introduction


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Plant Transformation Methods

Physical Chemical Biological

MicroinjectionPressureBiolistics - gene gun/particle bobardent!lectroporationSilica/carbon "bers#a$er ediatedSAT

P!%&!A!-de'tran(alciuphosphate

Arti"cial lipidsProteins&endriers

A. Tumefaciens A. Rhizogenes)irus-ediated


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*

Introduction to Agrobacteria…

A rod shaped, Gram

negative soil bacterium

A natural genetic engineer

2 species A.tumefaciens (produces

a gall)

A. rhizogenes (produces

roots)

Oncogenes (for auxin and

cytokinin synthesis) +

Opines

• isease producing

Agrobacterium are higly

pathogenic and do not

benefit the plant

• !n the presence ofexudates (e"g"

acetosyringone) from

#ounded plants, $irulence

($!%) genes are activatedand cause the t&'A to be

transferred to plants"

verything bet#een the

left and right border is

transferred"


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Scientic classication

• +ingdo Bacteria

• Phylu Proteobacteria

•

(lass Alproteobacthaperia• rder Rhi$obiales

• aily Rhi$obiaceae

•%enus Agrobacterium

• Species A. tumefaciens


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Ti plasmid and virulence genes

• A Ti plasid is a circular piece o&0A ound in alost all Agrobacteria

• Three ain regions

T-&0A region1Bet2een right and

let T-&0A border3 ncogene

pine

)irulence region

pine catabolis region


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(ellular process o Agrobacterium–host interaction

Tzvi Tzfira and Vitaly Citovsky, 2002, Trends in Cell Biol. 12(!, 121"12#


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Molecular in4ol4eent in t-&0A Transer5


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mages of !ro"n #all Tumor


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Appro4ed Traits.....

• %lufosinater herbicide

• Setho&dimr herbicide

• "romo&nilr herbicide

• %lphosater herbicide

• Sulfonlurear herbicide

• "t gene

• Male$sterilit

• Modified fatt acid

•

'lower colour

• 'lower life

• elaed fruit ripening

• Virus resistance


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Binary 4ector

t-&0A

)6R genes

Plasid &0A

Bacterial

(hroosoe Bacterial R6

Apicillinresistance

(onstruction o 4ector 2ith disired genes


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Production of transgenic plants

solate and clone gene of interest

Add $%A segments to initiate orenhance gene e&pression

Add selectable mar'ers

ntroduce gene construct intoplant cells (transformation)

Select transformed cells ortissues

*egenerate "hole plants


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Procedure or plant transoration

6MA%! Mol bio o the cell by Albert 1pg no7883


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Regeneration, Selection And &etection...

Regeneration or shoot organogenesis, cyto9inin1lo2er aounts o au'in3 are re:uired

Selection t2o antibiotics are re:uired• an antibiotic to ill the *grobacterium, while not affecting the plant+s cell

growth and division

• a second antibiotic allows growth of transformed shoots (w/selectablemarer) but inhibits growth of untranformed plant cells

&etection o the ;trait; gene -. methods can detect the presence of the 0trait0 1*

protein detection methods are used where a gene product is produced thatdefines the trait

verification of the incorporation of the trait gene into the plant+s

chromosome

• b Southern hbridi#ation

• b demonstrating transfer of the trait to the original transformant+s

progen


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Appro4ed Transgenic plants.....

• Sobean

• .orn

• .otton

• 2il Seed rape

• Sugarbeet

• S3uash

•Tomato

• Tobacco

• .arnations

• -otato

• 'la&

• -apaa

• .hicor

• ice

• Melon


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Types o genetic odi"cation

4556 of all

transgenic crops

are either herbicide

or insect resistant

786 have other

traits


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Probles and potential


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9 Scientists can insert an gene the want into the plasmid in place of the tumorcausing genes and subse3uentl into the plant cell genome

9 " varing e&perimental materials, culture conditions, bacterial strains, etc

scientists have successfull used A. tumefaciens %ene Transfer to produce "T .orn

9 This method of gene transfer enables large 1* strands to be transferred into

the plant cell without ris of rearrangement whereas other methods lie the %ene %un

have trouble doing this

9 The vast ma:orit of approved geneticall engineered agriculture has been

transformed b means of Agrobacterium tumefaciens Mediated %ene Transfer

9 2riginal problems e&isted in that Agrobacterium tumefaciens onl affects

dicotledonous plants

9 Monocotledon plants are not ver susceptible to the bacterial infection

+enets and Problems "ithAgrabacteria,-


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Transformed plants will help the world to meet the human demands for food,

energ, medicine, shelter, clothing, and a cleaner environment

%ene transfer technolog is being used to produce plants capable of hper

accumulating to&ic metals from soil and water, thus contributing to a cleanerenvironment

!ncreases in plant production are needed to meet growing demands for food

Transformed -lants offers significant advantages over traditional plant breeding

in developing improved crops

(onclusion5..


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Reerences

ntroduction to plant biotechnolog. b. /-S!ha"la

Molecular biolog. of the cell b. Albert et

alnline Reerenceshttp//222.nepadbiosaety.net/or-regulators/resources/subjects/biotechnology/ plant-transoration-agro

http//222.bio.da4idson.edu/people/9abernd/seinar/


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Than' .ou all

By, T.Prabhu,

@=th April

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