Contents - Faperta

Genetic Transformation of Crops for Resistance to Insect Pests 211
This requires micromanipulation of single cells or small colonies of cells under the microscope,
and precise injection of small amounts of DNA with a thin glass micropippette.
Injected cells or clumps of cells are subsequently raised in in vitro culture, and regenerated
into plants. In the protoplast transformation, the cell wall of the target cells is removed by
enzymatic treatment, and the cells are covered by a plasma membrane (Zhang and Wu,
1988). The DNA can be added into cell suspension, which can be introduced by affecting
the plasma membrane by polyethylene glycol or by passing an electric current through the
protoplast suspension. The DNA gets incorporated into the genome of a few cells. A suitable
marker may be inserted to select the transformed protoplasts and the cell colonies that
develop from them (Shimamoto et al., 1989).
DNA transfer into the protoplast has resulted in high levels of expression of the transgene.
The Bacillus thuringiensis (Bt) (Berliner) toxin gene cry2Aa2 operon expressed in
tobacco chloroplasts resulted in Bt protein content of up to 45.3% of the total protein in
mature leaves, which resulted in 100% mortality of cotton bollworm, Heliothis virescens (F.),
and the beet armyworm, Spodoptera exigua (Hubner) (Cosa et al., 2002). Under rice chloroplast
transcription elements, the CryIIa5 Bt toxin accumulated up to 3% of total soluble
protein in leaf tissue, which was 300 times more compared to expression of the same
protein in nuclear transformed plants (Leelavathi et al., 2002). In another study, chloroplast
transformed tobacco plants with the cryIIa5 gene under the control of rice psbA transcriptional
elements showed high levels of expression of Bt toxin without imposing a yield
penalty. The transgenic plants with up to 3% toxin protein of total soluble protein with
high levels of resistance to the noctuid, Helicvoerpa armigera (Hubner), have also been developed
(Reddy et al., 2002). Analysis of T 0, T 1, and T 2 generation plants revealed site-specifi c
integration, maternal inheritance, and uniform expression of the transgenes. The chloroplast
transformation vector pNRAB carrying two expression cassettes for the spectinomycin
resistance gene aadA and the insect resistance gene cry1Aa10 (sited between the rps7
and ndhB) resulted in chloroplast transformants at a frequency of four in 1,000 bombarded
cotyledon petioles (Hou et al., 2003). Chloroplast transformation of oilseed rape with the
cry1Aa10 gene resulted in 47% mortality of the second instar larvae of the diamond back
moth, Plutella xylostella (L.). Chloroplasts of transplastomic tobacco expressing the cry1C
gene have also shown high levels of Bt protein (about 1% of total protein) and the plastid
transgenes were not transmitted through pollen (C.H. Lin et al., 2003). This will facilitate
not only improvement in breeding for insect-resistant plants, but also the prevention of
contamination of transgenes among crop plants. The results suggested that overexpression
of insecticidal toxin coding genes in chloroplasts would be an effective strategy to
produce transgenic plants with high effi ciency for controlling the target pests, and delay
the emergence of resistance among phytophagous pests. This also prevents the inadvertent
movement of the transgene into closely related wild relatives of the crops due to
maternal inheritance of the chloroplasts (Bansal and Sharma, 2003).
Gene Expression
Effi cient genetic engineering relies on being able to generate a specifi c gene product at the
desired level of expression, in appropriate tissues, at the right time. This can be accomplished
by creating gene constructs that include promoters and/or transcription regulation
elements that control the level, location, and timing of gene expression. A major