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

 Lack

of quality seed Several abiotic and biotic stresses Poor management practices

Negative impact of non-living

factors on the living organisms in a

specific environment

 Drought
Salinity Extreme temperature Chemical toxicity and oxidative stress

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

Conventional breeding methods time consuming and laborious (Cullins 1991). Genetic modification of plants using recombinant DNA techniques holds the promise of increased crop productivity, product quality and reduced dependence on chemical inputs for pest control (Asano et al. 1991) Modern plant genetic engineering involves the transfer of desired genes into the plant genome and then regeneration of a whole plant from the transformed tissue High frequency regeneration of plant from in vitro cultured tissues and cells a pre-requisite for successful application of tissue culture and genetic engineering technologies for crop improvement.

Several protocols for genetic transformation using leaf discs success for biotic stresses unsatisfactory achievements in potato

(Bencheckroun et al. 1995). Regeneration and transformation processes depend on optimum growth condition

Purpose:

to standardize the callus induction potentiality

to develop abiotic stress tolerant potato through Agrobacterium-mediated genetic transformation.

January-June 2009 Potato cv. Granola

containing pBI121 CIPK sense gene encoding calcineurin Blike protein conferring abiotic stress tolerance

Agrobacterium tumefaciens strain LBA4404

The experiments were laid in Completely Randomized 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)

Agrobacterium-mediated genetic transformation of potato cv. Granola factors:

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)

Supplement Media YMB & LB MS Medium MS Medium MS Medium MS Medium MS Medium Function Grow modified bacteria strain Co-cultivation Post-cultivation Low selection medium High selection medium Regeneration

NAA (mg/L) 3.0 3.0 3.0 3.0

BAP Cefotaxime Kanamycin (mg/L) (mg/L) (mg/L) 2.5 2.5 2.5 2.5

200 100 100 100

Not specified 20 40 40

Flow

Callus initiation was started after 8 days of explants incubation callus initiated explant producing callus

explant induced calli

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

explants incubated

leaf and callus with + response to GUS assay

CALLUS INDUCTION

Leaf explant of potato cv. Granola was taken from sub-cultured micro plants % of callus induction, days to callusing & weight of callus varied to phytohormon concentration No callus in MS medium without phytohormone Calli were induced in medium supplemented with plant hormone combination

Effect of different concentrations of NAA & BAP was significant on days to callus initiation
Higher callus weight higher higher callus induction potentiality

Transformation Potentiality of potato cv. Granola

GUS histochemical assay result positive responses towards transformation (blue color)

Innoculation time and co-cultivation period important factors of Agrobacterium-mediated gene transformation

Results Callus Induction

callus per vial

Medium spec.
Highest
94.44% NAA :3.0 mg/L BAP :2.0 mg/L 22.67 days NAA :0.0 mg/L BAP :2.0 mg/L 2.66 g NAA :3.0 mg/L BAP :2.5 mg/L 27.78% 20 mg/L Kanamycin 83.33% 7 mnt inoculation 5 d co-cultivation 88.89% 3 mnt inoculation 5 d co-cultivation

Lowest
0.00% NAA :0.0 mg/L BAP :0.0 mg/L 12.67 days NAA :2.5 mg/L BAP :0.0 mg/L 1.21 g NAA :2.5 mg/L BAP :2.0 mg/L 22.22% 40 mg/L Kanamycin 0.00% 9 mnt inoculation 7 d co-cultivation 27.78% 9 mnt inoculation 7 d co-cultivation

Days to callus initiation

Callus weight

Transformation Potentiality

% of survived calli after antibiotic treatment GUS positive response leaf disc GUS positive response Callus

Presence of blue colour preliminary indication of GUS gene transfer from bacterial plasmid into plant cell Potato cv. Granola showed positive reponses toward transformation (indicate CPIK 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

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 against

Gram positive and Gram negative bacteria

GUS gene: -glucuronidase blue marker for transgenic gene Kanamycin : Antibiotic to grow the strain of genetically engineered