agrobacterium-mediated gene transfer in potato for

8/3/2019 Agrobacterium-mediated Gene Transfer in Potato For

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POTATO(Solanum tuberosum L.) Important tuber crop

Grows well both in temperate and tropical

countries

1st rank vegetable both in area &

production in Bangladesh (BBS 2008). Average yield in Bangladesh is 14.83 t ha-1

compared to 45-52 t ha-1 in european

country


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Lack of quality seed

Several abiotic and biotic

stressesPoor management practices


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Negative impact of non-living

factors on the living organisms in a

specific environment


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Drought Salinity

Extreme temperature

Chemical toxicity

and oxidative stress

It has been claimed by one study that abiotic stresscauses the most crop loss of any other factor andthat most major crops are reduced in their yield bymore than 50% from their potential yield.


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Conventional breeding methods time consumingand laborious (Cullins 1991).

Genetic modification of plants using recombinantDNA techniques holds the promise of increasedcrop productivity, product quality and reduceddependence on chemical inputs for pest control(Asano et al. 1991)

Modern plant genetic engineering involves thetransfer of desired genes into the plant genome andthen regeneration of a whole plant from thetransformed tissue

High frequency regeneration of plant from in vitrocultured tissues and cells a pre-requisite forsuccessful application of tissue culture and geneticengineering technologies for crop improvement.


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Several protocols for genetic transformationusing leaf discs

success for biotic stresses

unsatisfactory achievements in potato

(Bencheckroun et al. 1995).

Regeneration and transformation processesdepend on optimum growth condition


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Purpose:

to standardize the callus inductionpotentiality

to develop abiotic stress tolerant potatothrough Agrobacterium-mediatedgenetic transformation.


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January-June 2009

Potato cv. Granola

Agrobacterium tumefaciens strain LBA4404containing pBI121

CIPK sense gene encoding calcineurin B-

like protein conferring abiotic stresstolerance


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The experiments were laid in CompletelyRandomized Design (CRD) with four

replications.

Callus induction factors:

i) Different concentrations of NAA (0.0, 2.5,3.0 and 3.5 mg/L)

ii) Different concentrations of BAP (0.0, 2.0,2.5 and 3.0 mg/L)


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Agrobacterium-mediated genetictransformation of potato cv. Granolafactors:

i) Different concentrations of cefotaxime(100, 200 and 500 mg/L)

ii) Different concentrations of kanamycin (20,40 and 50 mg/L)

iii) Inoculation time (3, 5, 7 and 9 minutes)

iv) Co-cultivation period (3, 5 and 7 days)


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Media FunctionSupplement

NAA(mg/L)

BAP(mg/L)

Cefotaxime(mg/L)

Kanamycin(mg/L)

YMB & LB Grow modifiedbacteria strain

- - -Not

specified

MSMedium

Co-cultivation- - - -

MSMedium

Post-cultivation3.0 2.5 200 -

MS

Medium

Low selection

medium 3.0 2.5 100 20

MSMedium

High selectionmedium

3.0 2.5 100 40

MSMedium

Regeneration3.0 2.5 100 40


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Flow
http://flow%20diagram.doc/http://flow%20diagram.doc/


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Callus initiation was started after 8 daysof explants incubation

callus initiated

explant producing callus explant induced calli

% callus induction = ---------------------- X 100

explants incubated

leaf and callus with + response to GUSassay


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CALLUS INDUCTION

Leaf explant of potato cv. Granola was taken fromsub-cultured micro plants

% of callus induction, days to callusing & weight ofcallus varied to phytohormon concentration

No callus in MS medium without phytohormone

Calli were induced in medium supplemented withplant hormone combination


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Effect of different concentrations of NAA &BAP was significant on days to callus initiation

Higher callus weight higher higher callusinduction potentiality

Transformation Potentiality of potato cv.

Granola

GUS histochemical assay result positiveresponses towards transformation (blue color)

Innoculation time and co-cultivation periodimportant factors ofAgrobacterium-mediatedgene transformation


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ResultsMedium spec.

Highest Lowest

CallusInduction

callus per vial 94.44%NAA :3.0 mg/L

BAP :2.0 mg/L

0.00%NAA :0.0 mg/L

BAP :0.0 mg/L

Days to callus initiation 22.67 daysNAA :0.0 mg/L

BAP :2.0 mg/L

12.67 daysNAA :2.5 mg/L

BAP :0.0 mg/L

Callus weight 2.66 gNAA :3.0 mg/L

BAP :2.5 mg/L

1.21 gNAA :2.5 mg/L

BAP :2.0 mg/L

Transformation

Potentiality

% of survived calli afterantibiotic treatment

27.78%20 mg/L Kanamycin

22.22%40 mg/L Kanamycin

GUS positive response leaf disc

83.33%7 mnt inoculation

5 d co-cultivation


7 d co-cultivation

GUS positive response Callus


5 d co-cultivation


7 d co-cultivation


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Presence of blue colour

preliminaryindication of GUS gene transfer frombacterial plasmid into plant cell

Potato cv. Granola showed positivereponses toward transformation (indicateCPIK gene was transferred in potato)

A.tumefaciens strain LBA4404 vestor pBI121

with CPIK sense gene used for infection

CPIK give defense against drought, light,cold and wounding


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NAA is an auxin

BAP is a cytokinine

Both are a class of plant growth substance(often called phytohormone or plant hormone).They work together.

Cefotaxime:

antibiotics with broad spectrum activity againstGram positive and Gram negative bacteria

GUS gene: -glucuronidase blue marker fortransgenic gene

Kanamycin : Antibiotic to grow the strain of

genetically engineered

agrobacterium-mediated gene transfer in potato for

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