Biotechnology approaches to overcome biotic and abiotic stress constraints in legumes - Legumes Under Environmental Stress: Yield, Improvement and Adaptations - page 257

background mutation rates. Additionally, transposition-
independent methods, generally relying on vector-based
gene delivery, have been employed for performing gene
tagging procedures with some success.
Quantitative trait loci (QTL) mapping is a similar
mechanism that involves the study of genes in association
with various polygenic traits that are involved in the
phenotypic expression of a character. The method uses
various molecular tags, like amplified fragment length
polymorphisms (AFLPs) or single nucleotide polymor-
phisms (SNPs), to provide valuable information related
to the genetic basis of any stress response.
Subtractive hybridization cloning, another method
for isolating a gene of interest of unknown biochemical
origin, has also been successful in leguminous plants.
The procedure relies on the comparative genetic anal-
ysis between two plants, one grown under normal
conditions and another subjected to the stressful envi-
ronment. Biotin is linked to the DNA obtained from the
plant lacking the gene of interest and then the genetic
material from both the species is combined. Through
repeated cycles of association and subtraction, pure
genetic material containing the gene of interest can be
isolated. The product can then be amplified using PCR
and cloned into suitable complementary DNA (cDNA)
libraries. The chief advantage of this method is the
universality and comparative ease in comparison to
other methods (Figure 15.3).

Two other methods employed for the isolation of
stress resistance genes are the isolation of resistance
gene proteins and the functional cloning approach.
The former depends upon determining the interaction
of an external stress stimulus with the plant cell's
receptors. The receptor may, hence, be identified, iso-
lated and cloned (Postel & Kemmerling, 2009). The
sensory units of legumes involved in the interaction
with microbes include Lysine K and M (Lys K/M) and
other protein kinases (Boller & Felix, 2009; Gimenez-
Ibanez et al., 2009). Similarly, a number of molecular
entities, like Nod factor receptor (Nfr) proteins and
beta-glucan-binding protein, are involved in assisting
the legume-rhizobium interaction (Choudhury &
Pandey, 2013; Monaghan & Zipfel, 2012). Functional
cloning, however, involves the molecular dissection of
the entire genome. The genetic material is isolated,
cleaved and cloned into suitable vectors to produce
libraries. This DNA library is then introduced into the
plant line lacking the gene and the transformants
thereby produced are teffective in isolation of many
stress resistance genes, one major challenge these
methods present is the development of a functional
assay for the final evaluation and screening purposes.

15.7.3 In vitro mutagenesis and
transgenesis
Once the target gene has been isolated, its introduction
into the target cell helps to provide resistance against a
specific stress factor. The method involves the introduc-
tion of specific genes or gene segments into a target
tissue to successfully carry out the protein engineering
process. This requires the introduction of a synthetic
primer encoding the necessary mutation. Once the
primer anneals with the target genetic material, the rep-
lication process helps in the incorporation of the desired
mutation or target gene at a specific site. This method
has been used for neutralizing a gene or introducing a
resistance gene for a particular stress factor. Another
method, transgenesis, employs various biological or
physicochemical methods to introduce a transgene
(foreign stress-response gene) into a target cell. The
gene of interest is cloned in a suitable vector, which is
then introduced into a particular cell by means of
vectors, liposomes, gene gun or related physicochemical
methods. Once inside the cell, the gene is expressed in
order to obtain the product in the form of protein or
other secondary metabolites. A number of legumes

Biotic and abiotic stress factors

Legumes

Upregulation of stress resistance

Isolation of candidate genes

Molecular breeding Genetic engineering

Induction of stress tolerance or adaptation

Figure 15.3 Biotechnology intervention pathway for stress
resistance in legumes.