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Triticeae Genomics and Genetics
TGG 2010, Vol.1, No.2
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methods were also tried but no fertile transgenic plants
were obtained. Production of transgenic wheat by
pollen tube pathway method (Chong et al., 1998)
seems very attractive but little success has been
reported so far.
1.2.1 Bilolistic/microprojectile bombardment
Until 1988 there was no gene delivery procedure that
could provide an alternative to the
Agrobacterium
transformation
. Although the success of
Agrobacterium
vector was paramount, its inability to infect monocots
and difficulties involved in protoplast culture lead to
the invention of so called "biolistic" procedure,
particle bombardment or gene gun. The main
advantage of this method is to deliver DNA into intact
regenerable plant cells eliminating the need for
protoplast culture. This also minimized the
abnormalities resulting from long-term protoplast
cultures. Particle bombardment is a physical method
of cell transformation in which high-density
sub-cellular sized particles coated with DNA
molecules are accelerated to high velocity in order to
deliver DNA molecules to living cells. This method
efficiently overcomes physical barriers and does not
need long tissue culture and DNA can be introduced in
all living tissues. Relatively higher number of
transgenic plants can be obtained by this method.
Despite its versatile nature, tissue damage and the
relatively low yield of stable transformants (1%~5%),
limited size of the DNA construct and inconsistency
of bombardment replications are its limitations;
consumable items are expensive and the method is
sometimes laborious (http://ss.jircas.affrc.go.jp/engpa-
ge/jarq/32-4/hagio/hagio.htm)
The direct delivery of DNA by micro projectile is the
most reliable and satisfactory method for the
production of fertile transgenic wheat plants. There are
two major requirements for efficient transformation; the
efficient delivery of DNA-coated particles into larger
number of target cells and high level of division and
regeneration into seedlings. In most of the studies
PDS-1000/He (BioRad) was used for delivery of
transgene into primary explants or proliferating wheat
callus. The cells are then induced to become
embryogenic and regenerate. The influence of particle
size and type, different procedures for coating DNA
onto particles and distance of target tissue were
studied in relation to the transformation efficiency.
Vasil and others (Vasil et al., 1992) first time
developed fertile transgenic wheat plants using particle
bombardment. Efficiency of wheat transformation was
commonly reported at around 1% (Alpeter et al., 1996).
Instability of transgene after successful transformation
was a major problem in addition to partial suppression
of transgene. Optimum expression of genes in the
target cells is important for achieving a high frequency
of stable transformation. Considerable efforts were
made to develop suitable gene expression vectors for
transformation of wheat. The inclusion of intron
between the promoters and coding region enhanced
transient gene expression in wheat (Chibbar et al.,
1991). Experiments were also conducted to optimize
the culture conditions and DNA microprojectile delivery
procedures for plant regeneration and gene expression in
wheat calli. Osmotic treatment prior to bombardment,
elimination of spermidine, replacement of CaCl
2
by
Ca(NO
3
)
2
and addition of thiosulfate enhanced the gus
expression in the transformed calli. Development of
improved protocol (Vasil et al., 1993) reduced the time
to produce transformed plants. Need of establishing
long term callus, cell suspension or protoplast culture
was eliminated by introduction of a system for
enhanced induction of somatic embryogenesis and
regeneration of plants from isolated scutellum tissue
of wheat (Becker et al., 1994). Modification in the
particle bombardment and tissue culture procedures
helped to increase the transformation efficiency up to
7% (Rasco-Gaunt et al., 2001; Zhang et al., 2000),
facilitated quantitative production of multiple
transgenic plants and significantly reduced the cost
and labor. Low 2,4-D concentrations, increased
sucrose content in callus induction and incorporation
of silver nitrate in regeneration media were found to
increase embryogenesis and shoot regeneration.
Efficient genetic transformation demands high
regeneration capability of wheat tissues used for DNA
delivery. Genotype is the primary factor for successful
transformation. Several wheat genotypes were tested
for callus induction and regeneration capability and a
variety of explants were used in attempts to establish
regenerable tissue cultures of wheat including