Agriculture Reference
In-Depth Information
Institute (IARI) in New Delhi and National Botanical Research Institute (NBRI)
in Lucknow. Besides, some State Universities like The University of Burdwan
(BU), Punjab Agricultural University (PAU), Tamil Nadu Agricultural University
(TNAU) and crop specific National Research Centers have contributed a lot in mu-
tation breeding programme in India.
Functional Genomics Approach
In plants, the two most common methods for producing reduction-of-function
mutations are antisense RNA suppression (Finnegan et al. 1996 ) and insertional
mutagenesis (van Houwelingen et al. 1998 ; Speulman et al. 1999 ). However, anti-
sense RNA suppression requires considerable effort for any given target gene be-
fore knowing whether it will work, and insertional mutagenesis occurs at a low
frequency per genome. However, its efficacy is not yet clear; for example, epigen-
etic phenotypes can be variegated and unpredictable (Que and Jorgensen 1998 ).
Because these techniques rely either on Agrobacterium T-DNA vectors for trans-
mission or on an endogenous tagging system, their usefulness as general reverse
genetics methods is limited to very few plant species. Moreover, these techniques
produce a very limited range of allele types. Therefore, as the amount of sequence
data grows for Arabidopsis and other organisms, it is important to develop genome-
scale reverse genetic strategies that are automated, broadly applicable, and capable
of creating the wide range of mutant alleles that is needed for functional analysis.
Targeting specific loci is especially attractive when only a few genes of interest
exist. Of the targeting methods for plants, posttranscriptional gene silencing (PTGS)
is becoming increasingly popular (Waterhouse et al. 1998 ), replacing the less re-
liable antisense suppression methods that have been used for years. PTGS takes
advantage of the innate RNAi system that is found in most eukaryotes, in which
double-stranded RNAs are processed into 22-25-bp pieces (Baulcombe 2002 ) that
can diffuse out of cells through plasmodesmata and the vascular system and into
other cells. These pieces then target homologous transcripts for degradation and
even can target genes for DNA methylation (Matzke et al. 2001 ). Reports of suc-
cess using this method are encouraging (Chuang and Meyerowitz 2000 ), although
the efficiency of silencing can vary, so results may be unpredictable. Furthermore,
throughput is limited by the needs to engineer a construct for each gene of interest
and to individually transform plants with each one. PTGS may be the best way to
simultaneously target multiple closely related genes in a family.
Homologous recombination may be the most desirable strategy for targeted mu-
tagenesis and has been routine in some microbial organisms such as E. coli and
yeast for decades (Struhl et al. 1979 ). However, this technique has thus far been dif-
ficult or infeasible in multicellular eukaryotes, which have less-active homologous
recombination systems. In a few model organisms, including mammals (Capecchi
2000 ), flies (Rong and Golic 2000 ), and the moss Physcomytrella (Schaefer 2001 ),
there has been substantial progress in developing targeting by homologous recom-
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