Agrobacterium Mediated Transformation Of Spinach With Chitanase Gene

Muhammad Rameez KhanClass No 07

Introduction

Spinach (Spinacia Oleracea) is an edible flowering plant in the family of Amaranthaceae.

Native to central & Southwestern Asia. It is an annual plant. Spinach has a high nutritional value & is

extremely rich in antioxidants, especially when fresh, steamed, or quickly boiled.

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It is a rich source of vitamin A,C,E,K, B2, B6, magnesium, manganese, folate, iron, calcium, potassium, folic acid, copper, protein, phosphorus, zinc, niacin, selenium & omega-3 fatty acids.

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Agrobacterium Mediated Transformation is preferred because:

1) Easiest way of transformation. 2) Less expensive. 3) Highly efficient. One of the defense strategies is the deployment

of small antimicrobial proteins for defense against pathogens (Boman, 1995; Nicolas & Mor, 1995; Hoffmann, 1995).

Plant produce a number of antimicrobial peptides to combat the pathogen attack (Cammue et al.,1994).

Cysteine-rich peptides play a role in plant defense against pathogens (Cammue et al.,1994).

Chitanases are glycosyl hydrolases catalizing the degradation of chitin; a polymer of N-acetylglucosamine.

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They are produced by a wide range of organisms including bacteria (Lee et al.,2007),fungi ( Terakawa et al.,1997),insects (Ding et al.,1998),plants (Collinge et al.,1993) & have been shown to play important physiological roles (Gooday, 1990).

Review Of Literature

Transgenic plants harbouring chitanase ChiC genes have already been produced in several species.

In carrot, Punja & Raharjo (1996) reported that the tobacco class I ChiC gene showing resistance to Botrytis cinirea.

Transgenic tobacco transformed with bean class I ChiC exhibited enhanced resistance Rhizoctonia solani (Broglie et al.,1991).

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The rice chitinase gene exhibited increased resistance to sphaerotheca humule in transgenic strawberry (Asao et al.,1997)

Objectives

To produce transgenic spinach resistant to fungal pathogen, by incorporating a chitinase genes ChiC.

Methodology

The present research study will be conducted at Institute Of Biotechnology And Genetic Engineering (IBGE) Khyber Pakhtunkhwa Agricultural University Peshawar during 2011-12.

Plant Material

Spinach leaf disc will be used for regeneration and transfomation.

Surface Sterilization

Fresh leaves of spinach variety Desi will be washed with tap water & then subjected to surface sterilization with 25% bleach solution.

Medium For Regeneration

Full strength MS medium will be used for callus formation.

A.tumefaciens Strain In Binary Vector

A. tumefaciens strain EHA101 harbouring the plasmid pEKH1 which will be used to infect spinach leaf discs will be imported from Japan.

Agrobacterium Transformation By Freeze/Thaw Method

For agrobacterium transformation they will be melted on ice & then a brief heat shock at 37C for 5 minutes.

The plates will be checked for colonies after 2-3 days.

Conformation Of Transformation

Transformation will be confirmed by extracting plasmid DNA from grown colonies of agrobacterium & presence of desired gene will be checked through PCR.

Literature Cited

Broglie, K.E., Chet.I., Holliday.M., Cressman.R., Biddle.O., Knowlton.S., Mauvais.C.J & Broglie.R.(1991). Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani.Sci 254:1194-1197.

Graham,L.S & Sticklen.M.B.(1993).Plant chitanases.Can.J.Bot.72:1057-1083.

Hoffmann, J.A.(1995).Innate immunity of insects.Corr.Opin.Immunol 7:4-10.

Budget:

Fixed Budget:

800000Grand total

100,000 -Incubator2.

700000 - -700000 PCR1.

TotalCostYear 2CostYear 1S.No

Recurrent budget

30,50,000 - - - -Grand Total

600,000300,000Miscellaneous300,000 Miscellaneous 8..

400,000200,000Conferences200,000 Conferences7.

200,000100,000Travelling100,000 Travelling 6.

200,000100,000Honraria100,000 Honraria 5.

12,0000600,000 Salary For Researchers

600,000 Salary For Researchers

4.

50,00025,000Stationary25,000 Stationary 3.

100,00050,000Glasswares50,000Glasswares2.

300,000 150,000 Chemicals150,000 Chemicals1.

TotalCostYear 2CostYear 1S.No

Total Budget

Total Budget = Fixed Budget + Recurrent Budget

= 800000 + 30,50000

= 38,50000