Plant Biology and Biotechnology
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Forward genetic approach is the classical
phenotype- based approach for screening mutants
in a biological pathway or process of interest.
Large-scale forward genetic screens have provided a basis for the discovery of a multitude of
new genes and pathways fundamental to various
aspects of plant biology. Gene disruption is the
most robust and direct approach to address the
biological function of a gene. Various libraries of
mutants for forward genetics generated in A.
thaliana are available as a public resource and are
discussed below.
1.3.1.1 EMS Mutagenesis
Ethyl methane sulfonate (EMS), a known and
commonly used chemical mutagen (alkylating
agent), induces point mutations which vary from
complete knockouts to hypomorphic mutations,
thus allowing isolation of a series of allelic variants of a given gene (Bowman et al. 1991 ).
Isolation of weak alleles is advantageous especially when characterizing genes involved in
essential cellular functions. EMS treatment has
been successfully used for generating a high
frequency of irreversible, randomly distributed
mutations across the Arabidopsis genome
(Greene et al. 2003 ). To dissect any biological
process, a saturated mutagenized population is
screened for a desired phenotype, and the classical positional cloning approach is used for identifying the causal mutation/gene (Fig. 1.3 ). This
approach requires the mutant to be crossed to an
Arabidopsis accession signifi cantly polymorphic
at the DNA level to generate a segregating F 2
mapping population. The mapping is done in a
biphasic manner,
1.3.1.2 Insertional Mutagenesis and Its
Modifi cation
Since the classical positional cloning is an extensive and a long-drawn exercise for identifying a
corresponding gene responsible for a phenotype
of interest, insertional mutagenesis – an alternate
tool for gene disruption – was developed.
Integration/insertion of T-DNA/transposable element (TE) into the genic region causes disruption
of the gene. Since the mutant genes are tagged
with T-DNA inserts, the gene can be easily identifi ed by isolating the sequences fl anking the
insertion sites. Success of this approach depends
upon (i) an easy and effi cient transformation
system and (ii) the ability of T-DNA and transposable elements to integrate randomly into the
host genome (Galbiati et al. 2000 ). Effi cient and
simplifi ed Agrobacterium -mediated fl oral dip
transformation method has helped in generating
exceptionally large numbers of insertional
mutants and a near-saturation mutagenesis of the
Arabidopsis genome (Clough and Bent 1998 ).
Four major T-DNA mutant collections available
at TAIR collectively encompass 95 % of predicted Arabidopsis genes: (a) SALK lines
(Alonso et al. 2003 ), (b) GABI-Kat lines (Rosso
et al. 2003 ), (c) Syngenta Arabidopsis Insertion
Library (SAIL) (Sessions et al. 2002 ), and (d)
INRA/Versailles lines (Samson et al. 2002 ).
These stocks are in the public domain and are
available from ABRC and NASC stock centers.
These are popular resources for both forward and
reverse genetic approaches for functional analysis. Similar to EMS mutants, insertion mutants
too have been used for unraveling molecular
mechanisms underlying various biological processes, viz., meiotic recombination in plants
(Reddy et al. 2003 ; Kerzendorfer et al. 2006 ),
embryo development (Stacey et al. 2002 ), organ
development (Dievart et al. 2003 ), and systemic
acquired resistance signaling (Maldonado et al.
2002 ).
1.3.1.2.1 Trap lines
Traditionally, gene identifi cation relied on disruption of a gene function leading to a recognizable phenotype. But most of the genes in
Arabidopsis and other crop plants are members
of multigene families and can act redundantly,
which makes it diffi cult to characterize them
using the classical approach. In addition, some
phenotypic characters are hard to be detected
unless the mutated gene is studied in a certain
mutant background which reveals its loss-offunction phenotype. Yet another class of genes
not amenable to classical genetic studies is the
ones that function at multiple developmental
stages and whose loss of function may lead to
lethality at early developmental stages.
Modifi cation of the insertional mutagenesis tool
kit has led to the development of an alternate
powerful strategy that permits gene identifi cation
based on their expression pattern, thus eliminating the need for a mutant phenotype (Sundaresan
et al. 1995 ; Springer 2000 ). The basic principle
underlying this strategy is to randomly integrate
into the genome a promoterless reporter construct (gene/promoter trap) or a reporter construct
with a minimal promoter (enhancer trap) close to
1 Arabidopsis thaliana: A Model for Plant Research
8
the end of the insertional element (T-DNA or
TE). The expression of the reporter gene is activated when an endogenous cis-acting promoter
or transcriptional enhancer is present at the site of
integration. Bacterial uidA encoding for
β-glucuronidase (GUS) is a commonly used
reporter since endogenous β-glucuronidase activity in plants is absent (Jefferson et al. 1987 ).
Alternatively, light emitting bacterial protein
(lux) and luciferase (luc) enzyme from the fi re fl y
have been used as reporters for nondestructive
screens. Gene traps have been extensively used to
unravel genes involved in various developmental
processes like lateral root formation (Malamy
and Benfey 1997 ), female gametophyte development (Springer et al. 1995 ), embryo development
(Topping and Lindsey 1997 ), and fl oral organ
development (Nakayama et al. 2005 ). Trap lines
have also been used to identify stress responsive
genes (Alvarado et al. 2004 ). Besides gene identifi cation, several organ-, tissue-, cell-, and stagespecifi c markers have been identifi ed which are
useful tools in developmental biology studies.
Additionally, the promoter traplines provide a
direct access to highly specifi c promoters. For
example, to tackle the problem of drought stress
in crop plants engineering stomatal activity is an
attractive idea. Guard cells control the infl ux of
CO 2 for photosynthesis and water loss during
transpiration, and the signaling cascade involved
in these responses are well dissected (Schroeder
et al. 2001 ). Francia et al. ( 2008 ) screened gene
trap and promoter trap lines to isolate stomataspecifi c genes and promoters for biotechnological applications. This approach has also been
extended to other crops like rice to identify celltype-/tissue-, stage-, and/or conditionally specifi c
regulatory elements (Yang et al. 2004 ).
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