Principles of Plant Genetics and Breeding

234 CHAPTER 14
Gene identification
With a library constructed, the breeder may search it to
identify a specific gene of interest using one of several
techniques based on two approaches:
1 Based on gene product. The breeder may purify
the protein expressed by the gene and from that
obtain the amino acid sequence (e.g., by 2D gel
electrophoresis). The information is used to design
synthetic oligonucleotides corresponding to the
sequence. This is labeled and used in a Southern
hybridization to identify the corresponding gene.
The method of antibodies is also effective.
2 Based on sequence characteristics. The breeder
may use nucleotide sequence information from
studies in other species. The probes designed from
such information are called heterologous probes
and represent a complete or partial gene sequence.
Genes may also be isolated by techniques such as
chromosome walking (map-based cloning), molecular
tagging (transposon tagging), or differential
screening.
Gene transfer
Once the desired gene has been identified from the
library, it is ready to be transferred into a host cell, a
process called genetic transformation. There are two
categories of transgene transfer or delivery procedures –
direct and mediated transfer.
Direct gene transfer
By particle acceleration or bombardment
One of the commonly used direct gene transfer method
is microprojectile bombardment (or biolistic). A
biolistic device (called a gene or particle gun) is used to
literally shoot the target DNA into intact cells (hence
the nickname of shotgun transformation). The widely
used particle delivery system is marketed by DuPont
(called the DuPont Biolistic® PDS1000/He device)
and uses helium as the propellant gas. Small amounts
(about 50 µg) of micron-size (1–5 µm diameter) carrier
particles (tungsten or gold) are coated with the target
DNA and propelled in the barrel of the gene gun
at energies high enough to penetrate plant cells. The
rate of acceleration may be up to 430 m/s in a partial
vacuum. The carrier particles pass through a mesh, hitting
the target tissue (e.g., callus) in a Petri dish below the
biolistic device. A low penetration number of projectiles
(1–5 per cell) is desirable. More than 80% of bombarded
cells may die if particle penetration reaches 21 projectiles
per cell.
Electroporation
Callus culture (or explants such as immature embryos
of protoplasts) is placed in a cuvette and inserted into
a piece of equipment called an electroporator, for electroporation.
This procedure widens the pores of the
protoplast membrane by means of electrical impulses.
The widened pores allow DNA to enter through them
to be integrated with nuclear DNA.
Other methods
Other direct methods are available, including microinjection
and silicon carbide procedures.
Mediated gene transfer
In directed or mediated gene transfer, intermediary
agents are used to act as couriers (vectors) for carrying
the target DNA into recipient cells. In plants, the
Agrobacterium-mediated gene transfer is a common
practice. The bacterium causes crown gall tumors in
dicots. Upon infection, the bacterium transfers a part
of its DNA into the host. The tumor-inducing part
of the specially designed bacterium is deleted. It has
been determined that the oncogenic (tumor-inducing)
properties of A. tumifaciens reside on a large tumorinducing
(Ti) plasmid (called Ri plasmid in A. rhizogenes).
The Ti plasmid has two regions, the T-region
(called T-DNA or transferred DNA) is what is transferred
into the plant cell and integrated into the host
chromosome (i.e., natural genetic engineering!). A
second region on the Ti/Ri plasmid, called the virulence
(vir) region, carries the genes for tumor induction and
is also involved in the transfer of T-DNA. T-DNA also
carries genes for synthesizing metabolic substrates for
bacteria called opines. Further, the T-DNA is flanked or
bordered by short (25 bp) direct repeats of DNA. The
deletion of the tumor-inducing segment of the DNA
does not prevent the transfer of T-DNA.
Requirements for transformation
The following are some key considerations for a successful
Agrobacterium-mediated transformation project: