Principles of Plant Genetics and Breeding

1 Efficient plant regeneration system. Without an
efficient regeneration system to grow full plants from
cells, it is futile to undertake any transformation. In
effect, if may be said that “thou shall not transform
unless thou can regenerate”! A small number of cells
in a plant are competent for both transformation and
regeneration.
2 Determination that the cells are susceptible to
Agrobacterium transformation. The Agrobacteriummediated
transfer system should be efficient and be
able to transform most of the cells at the target site.
3 An efficient and sensitive selection method.
Similarly, an efficient selection system should be
available to readily identify and select the transformed
cells from among untransformed cells.
4 Stable transformation. Transformation is successful
if the cells regenerate, and the transgene is expressed
in subsequent generations. The fewer the number
of cells producing the regenerated plant the better,
otherwise a chimeric product will result.
Procedure of Agrobacterium transformation
Agrobacterium cells containing the DNA inserts are
co-cultivated (co-cultured) (e.g., with leaf disks, cotyledons,
or other materials) on a regeneration medium
for 2–3 days. The bacteria bind to the wounded sites
and subsequently transfer the insert DNA into the host
genome. The explant is transferred to a regeneration
medium containing carbenicillin (to kill Agrobacterium)
and an antibiotic (e.g., kanamycin) to inhibit growth
of untransformed cells. Successful transformants are
cultured to full plants. The bacterium gains entrance
into the plant material through the wounds. Thus, gene
delivery is more successful with dicots than monocots
because the former are more wound responsive than
cereals.
Transient versus stable transformation
As previously stated, stable expression of a transgene is
desired so that the transgene would be heritable and
expressed from one generation to the next. However,
the breeder may need to have an earlier indication of
successful transformation and hence may evaluate the
expression of the transgene at an intermediate stage in
the transformation process. For example, protoplasts
may be transformed with the target DNA and the protoplasts
isolated after only 1–2 hours for the evaluation
of expression of encoded genes. This preliminary
expression of the transgene is described as transient
expression. Transient expression occurs when the insert
BIOTECHNOLOGY IN PLANT BREEDING 235
gene is not integrated into the host genome. Such an
expression dissipates with time.
Tissue culture and selection of transformation events
Tissue culture is a critical part of a genetic engineering
project. This is because transgenes are usually delivered
into cells before they differentiate. Common explants
used are protoplasts, cell suspension, immature embryos,
shoot meristem, and immature inflorescences. Because
a transgene may not be successfully delivered into all
cells in a mass of callus, it is important to have a selection
system to discriminate among cells to identify and
isolate only genuine transformants.
Cloning vectors, as previously discussed, are designed
for specific purposes. Selectable marker systems built
into vectors may be grouped as follows: antibiotic
selection (e.g., kanamycin), herbicide selection (e.g.,
acetolactate synthase genes are also used in herbicide
selection (for glyphosate or sulfonylurea herbicides)),
scorable gene-mediated selection, and positive selection.
The selection agent is included in the tissue culture
medium. Selection systems differ in their ease of use and
efficiency.
Antibiotic selection
This is the first generation selection marker system
in biotechnology. Like antibiotic markers, herbicide
markers are used for establishing preferential growth of
transgenic cells as in other marker systems.
Scorable gene-mediated selection
Reporter genes are expressed in cells without integration
into the genome and are assayed in a variety of
ways. In a transformation project, it is always desirable,
whenever researchers can, to have a preliminary evaluation
of success of transformation. Scorable marker
genes (reporter genes) are used for rapid visual confirmation
for transient expression following transformation.
Reporter gene assay is a transient assay and can
be conducted within 24 hours after transformation of
the cells. The commonly used scorable marker genes
encode enzymes that have distinct substrate specificities,
thereby enabling researchers to monitor changes with
various visualization systems. Examples of scorable marker
genes are GUS (β-glucuronidase), which is visualized
by a histochemical or fluorometric protocol, and LUC
(luciferase), which is visualized by a luminescence system
and a green fluorescent protein (GFP) system that allows