Biotechnology approaches to overcome biotic and abiotic stress constraints in legumes - Legumes Under Environmental Stress: Yield, Improvement and Adaptations - page 258

have been genetically manipulated using the transgen-
esis process and have successfully acquired tolerance
against pests, salinity, drought and chilling (Chen et al.,
2010; Coba de la Peña et al., 2010; Hanafy et al., 2013;
Lüthi et al., 2013).

is introduced into a particular part of the plant. This and
other techniques for suppressing viral replication are
highly dependent upon the localization of the virus
within a plant organ. This may be achieved by the
physical barriers between the cells, inactivation of trans-
port proteins or by the secretion of natural chemicals
that might neutralize the virus and decrease cell-to-cell
transfer. Once the virus has been isolated in a particular
region, the chances for cell-to-cell transmission and
resulting disease signs are considerably reduced. Other
biotechnological techniques employed for virus control
include the increased production of antiviral agents like
the inhibitors of virus replication (IVRs). IVRs, for in-
stance, have been indicated in the suppression of
replication of a number of plant viruses including
tobacco mosaic virus (TMV) and begomoviruses
(Jeffries, 2008).
Another technique that has proved successful in
defending plants against biotic stress factors is by the
induction of secondary metabolites that may help in
retarding the growth of the infesting agent. The method
requires the isolation of disease resistance genes
involved in the detoxification process, in response to a
particular threat. After isolating the gene, it is cloned in
a suitable vector and the plasmid is transformed through
either biological or physicochemical methods into a
susceptible plant variety. The plant is then subjected to
evaluation for the degree of tolerance developed to a
particular stress condition. The genetic variability of
various plant species has provided a means to isolate the
detoxification-catalysing systems.
Genetic engineering has, therefore, helped in address-
ing the environmental and toxicological problems
associated with earlier pest control techniques. Tabtoxin
and proteinase inhibitors, for instance, are involved
in the neutralization of various biotic stress factors in
legumes (Hirel et al., 1997; Ramírez-Suero et al., 2010).
The introduction of small interfering RNAs (siRNAs)
into plant systems can help in the provision of broader
cover against a number of pesticides. This method,
however, is still under test and needs to be evaluated
against a relatively larger number of microorganisms.
Apart from these metabolites that are directly involved
in neutralizing a biological attack, certain chemical
entities are involved in the enhancement of disease
resistance in leguminous plants. One such group
includes the surface active peptides, which help not
only in signal transduction but also in complementing

15.7.4 Screening methods
Confirmation of the insertion of a transgene into a
vector is imperative for determining the success of the
genetic manipulation process. The screening methods
are based either upon the analysis of a foreign gene in
the vector or on the final product obtained as a result of
expression. PCR provides one such easy method for
confirmation of a gene in a vector. Once the gene incor-
poration has been confirmed, the steps leading to
expression must also be considered to achieve an effec-
tive transgene. However, the cloning and expression
procedures do not necessarily result in attainment of
tolerance against the stress factor. Confirmation needs
to be made by performing certain functional assays. In
case the introduced transgene encodes for a protein, the
final product needs to be screened and analysed.
However, if any morphological or physiological change
is desired, the progeny are evaluated for the appearance
of the desired characteristics.

15.7.5 Specific induction of stress response
mechanisms
Finding the means to inhibit viral growth in susceptible
legumes has not been very successful. Viruses, like other
biological stress factors, initiate a series of cellular and
molecular mechanisms in the host plant. Moreover,
many resistance genes isolated from plants as a result of
virus infection have demonstrated their ability to
neutralize the signs of the virus infection, but they do
not inhibit the viral replication altogether. However,
expressing some of the viral resistance genes in plants
has been successful. This strategy may include the incor-
poration of genetic sequences that bear homology or
complementarity to the viral genes. Utilization of
satellite RNA, defective interfering DNA, antisense RNA,
all or part of the viral replicon or the use of subunits like
viral capsid protein, have shown to be effective in
decreasing viral replication and disease severity. The use
of viral subunits ensures the activation of a monogenic
resistance trait, in comparison to other methods of
developing resistance. The method involves the use of a
chimaeric assembly incorporating the viral gene, which