Biotechnology approaches to overcome biotic and abiotic stress constraints in legumes - Legumes Under Environmental Stress: Yield, Improvement and Adaptations - page 256

Table 15.3 Biotechnology tools applied to leguminous plants
for stress resistance.

has been applied to identify many resistant genes.
Moreover, genetic markers have been isolated by the
technique known as restriction fragment length poly-
morphism (RFLP) mapping. It involves the comparison
of enzyme-digested restriction fragments to identify the
different genes observed in stress conditions. Randomly
amplified polymorphic DNA analysis makes use of a
similar strategy. However, in the latter case short nucle-
otide sequences are utilized to differentiate between the
amplification patterns of genetic material in different
growth conditions. The polymerase chain reaction
(PCR) forms the basis of all these techniques by
providing a means to rapidly amplify large genetic
sequences. Once the individual genes have been identi-
fied, a walk along the genome length is carried out to
identify other genes that might have taken part in
providing resistance to the plant. Consequently, the
whole genome may be screened from one end to the
other, avoiding any chances of misreading any gene
sequence. These marker genes are usually cloned in
yeast artificial chromosomes (YACs), bacterial artificial
chromosomes (BACs) or other cloning vectors.
Moreover, complementary DNA libraries may also be
developed to keep a record of the candidate gene. In
certain cases, chromosome jumping may be performed
to study different segments one by one, with relatively
larger spanning distances. The stress resistance candi-
date genes once identified need to be evaluated and
confirmed by observing the phenotypic presentation of
the plants as well.
Another method for successful identification of a can-
didate gene is the gene tagging technique. It involves
the establishment of a plant line that contains a molec-
ularly characterized transposable element. Based upon
the principle of gene transposition, screening is done by
hybridizing the gene of interest with a particular marker.
This method helps in the rapid identification of trans-
posable elements, so that the gene involved in providing
disease resistance can successfully be identified and
isolated. However, high rates of transposition or the
availability of transposable sites in close proximity can
hinder the isolation of effective stress resistance genes.
The method requires the successful inculcation of the
resistance gene into the progeny. A number of tech-
niques have been adapted to increase the frequency of
transposition including the incorporation of foreign
promoter elements, selection of transposable elements
close to the gene of interest and decreasing the

Biotechnology tool Purpose

Gene-phenotype relation,
polymerase chain reaction

Identification of diseased plant

Culturing techniques, sampling
of growth conditions

Confirmation of aetiological agent

Restriction fragment length
polymorphism, amplified fragment
length polymorphism, quantitative
trait loci

Isolation of candidate gene

Transgenesis, site-directed
mutagenesis

Introduction of a transgene into a
host

Expressed sequence tags,
metabolic assays, functional
assays

Confirmation of expression of the
gene in the target host

are dependent upon protein-degrading enzymes to gain
entry into the legume body. These enzymes tend to help
the prey gain easy access to the plant's internal systems,
thereby causing serious harm to the plant's health;
the plant responds by producing certain 'anti-stress'
elements. These elements are generally classified as the
pathogenesis-related (PR) proteins and antifungal
proteins, on the basis of the pathogenic agents they
target. Many PR proteins are activated against bacterial
and fungal species in legumes, for example chitinase,
thaumatin-like protein and Nod factor perception
protein (Pearce et al., 2010; Gough & Jacquet, 2013; Rey
et al., 2013). Knowledge of these PR proteins and their
pathogenesis mechanisms can help in the development
of agents to help tackle infection at the plant-to-plant
transmission level, while not interfering with any meta-
bolic process. Additionally, genetic cassettes can be
introduced into bacteria or fungi to produce heterolo-
gous proteins that can be of biotechnological utility.

15.7.2 Isolation of disease resistance genes
A number of techniques may be employed in identi-
fying the disease resistance genes. The more resistance
genes available, the more chances there are of devel-
oping and employing effective defence mechanisms
against the susceptible pathological factors. Chromosome
walking is one such technique employed by plant
biotechnologists to identify the resistance genes. Based
upon the polymorphic nature of the genetic material of
normal plants and plants under stress, this technique