Agrobacterium Method

Introduction

Researchers are actively making progress in introducing new plant trait by using recombinant DNA technology and one of it is through transgenic technology. Genetic engineering has the potential to fasten the process of plant genetic manipulation and also expand the scope of the field research other than for agriculture purposes. Nowadays, transgenic plant is widely applied in agriculture area to increase the production of food as well as improving the food quality either in its nutritional or physical aspect (Hoque, Mansfield & Bennett, 2005).

Kumar et al. (2005) stated that, the production of transgenic plant generally was done by artificially inserting new gene of interest to the plant sequence instead of gene used in plant

The Agrobacterium method is mainly used for dicots species to produce transgenic tomatoes and soybeans meanwhile, the gene gun technique is mainly applied for the transformation of monocot plant species such as corn and rice (Hiei & Komari, 2008). However, according to Davis et al. (1999), the first transgenic rice plant was produced through micro projectile bombardment in the late 1980s at which the gene transfer was mediated by electroporation. The Agrobacterium method using Agrobacterium tumefaciens became prevalent in producing transgenic plant in mid of 1990s due to its high efficiency of plant transformation compare to other method (Rivera et al., 2012). Rivera et al. (2012) explained that this high plant transformation efficiency is due to large fragments of DNA transferred into the chromosome and it is also due to low transfer DNA (t-DNA) integration into the

The pellet was re-suspended in 5 ml NB liquid and 5 ml of 100 µM acetosyringone in the plate before transfer into Falcon tube. The plate of the Agrobacterium which contain the Ti plasmid that carry the lipase gene of interest was prepared beforehand. Then, calli were picked and immersed in the Agrobacterium solution for 10 minutes using sterile forceps. Next, the calli were drained gently using filter paper and transferred onto sterile petri dish that contains NB medium and 100 µM acetosyringone. The plate was incubated in the dark at 25 oC for 7 days. This process is known as the co-cultivation of the