Contents - Faperta

210 Biotechnological Approaches for Pest Management and Ecological Sustainability
Genetic Transformation: The Protocols
The effi ciency of tissue culture and transformation protocols is one of the most important
components for successful generation of transgenic crops (Birch, 1997). The major components
for developing transgenic plants are: (1) reliable tissue culture and regeneration systems,
(2) preparation of gene constructs and transformation with suitable vectors, (3) effi cient
transformation techniques to introduce genes into the crop plants, (4) recovery and multiplication
of transgenic plants, (5) molecular and genetic characterization of transgenic
plants, (6) transferring genes of interest into elite cultivars, and (7) evaluation of transgenic
plants for their effectiveness in controlling the target pests without being a hazard to the
environment. Although several approaches have been tried successfully for genetic transformation
of crop plants, only the following three approaches have been used widely to
introduce genes into a wide range of crop plants (Potrykus, 1991; Dale, Irwin, and Scheffer,
1993; Seetharam et al., 2002).
• Agrobacterium-mediated
gene transfer
• Microprojectile bombardment with DNA or biolistics
• Direct DNA transfer into isolated protoplasts
Agrobacterium-Mediated Gene Transfer
Agrobacterium tumefaciens (Smith and Townsend) has been used widely to transfer novel
genes into crop plants. It is a soil-inhabiting bacterium that results in gall formation at the
wound site in many dicotyledonous plants. This tumor-inducing capability is due to the
presence of a large Ti (tumor-inducing) plasmid in virulent strains of Agrobacterium.
Likewise, Ri (root-inducing) megaplasmids are found in virulent strains of Agrobacterium
rhizogenes (Ricker et al.) Conn., the causative agent of “hairy root” disease. The Ti and Ri
plasmids, and the molecular biology of crown gall and hairy root induction, have been
studied in considerable detail (Zambryski et al., 1983; Zambryski, 1992). Agrobacteriummediated
transformation is brought about by incorporation of genes of interest from an
independently replicating Ti plasmid within the A. tumefaciens cell, which then infects the
plant cell and transfers the T-DNA containing the gene of interest into the chromosomes of
the actively dividing cells of the host plant.
Microprojectile Bombardment with DNA or Biolistics
In the particle bombardment (biolistics) method, tungsten or gold microprojectiles are
coated with the DNA to be inserted, and bombarded into cells or tissues capable of subsequent
plant regeneration. Acceleration of heavy microprojectiles (0.5 to 5.0 μm diameter
tungsten or gold particles) coated with DNA carry the genes into the cell and tissue (Klein
et al., 1987; Sanford, 1990). The DNA-coated particles enter the plant cells and the DNA is
incorporated in a small proportion of the treated cells. The transformed cells are then
selected for plant regeneration.
Direct DNA Transfer into Isolated Protoplasts
Genetically engineered DNA can also be directly injected into nuclei of embryogenic single
cells, which can be induced to regenerate plants in cell culture (Neuhaus et al., 1987).