Sugarcane - Bioenergy Crop Plants - page 538

first genetically modified clones were obtained by biolistics (Bower and Birch 1992). Transgenic
plants have since then been obtained that incorporate agronomic traits of interest (Gallo-Meagher
and Irvine 1996; Arencibia et al. 1997, 1999; Enriquez-Obregon et al. 1998; Ingelbrecht et al.1999;
Zhang et al. 1999; Falco et al. 2000; Butterfield et al. 2002; Falco and Silva-Filho 2003; Leibbrandt
and Snyman 2003; McQualter et al. 2004; McQualter et al. 2005; Vickers et al. 2005a, b; Wang
et al. 2005; Snyman et al. 2006) including drought tolerance (Zhang et al. 2006; Molinari et al.
2007). Several plant tissues can be used to produce callus (Liu 1981 1993; Irvine 1987). Somatic
embryogenesis is the most studied (Guiderdoni et al. 1995; Manickavasagam and Ganapathi 1998)
and regeneration can be direct (Manickavasagam and Ganapathi 1998) or through the induction of
embryogenic callus from immature leaf explants (Guiderdoni 1988). Embryogenic callus can be
maintained for months without loss of regeneration capacity (Fitch and Moore 1993). Regeneration
efficiency can be optimized for transformation as shown recently (Lakshmanan et al. 2006; Snyman
et al. 2006).
Agrobacterium tumefaciens -mediated transformation has also been used in sugarcane (Arencibia
1998; Enriquez-Obregon et al. 1998; Elliott et al. 1998; Manickavasagam et al. 2004). With adequate
manipulation of in vitro culture conditions and adequate A. tumefaciens cell lines the method can
lead to the transference of relatively long DNA fragments, little rearrangements, low copy number
and low costs. Sugarcane cultivars differ in their regeneration capacity and the methods must be
optimized. Many cultivars that regenerate have already been described (Falco et al. 2000; Lima et
al. 2001; Falco and Silva-Filho 2003; Cidade et al. 2006). Although the methods are available, so
far there are no genetically modified cultivars released for trading. Transgene expression is largely
unstable and many groups are searching for gene promoters that may lead to stable expression in
mature plants. Examples of plant promoters useful for transformation but that vary in their efficacy
are CaMV35S , nopaline, and octopine from A. tumefaciens , Ubi1, Emu , Act1, but there is no guaran-
tee of tissue specificity (Last 1991; McElroy et al. 1991; Zheng et al. 1993; Green 2002; Neuteboom
2002; Christensen and Quail 1996). Ubi-1 is the most used in sugarcane for constitutive expression
(Lakshmanan 2005) and some studies point to more adequate promoters (Liu 2003; Braithwaite
2004) but still there is little guarantee of targeting the gene expression for specific tissues (Benfey
1989; Neuteboom 2002). It is important to note that constitutive expression of a transgene can lead
to phenotypic anomalies and that in the case of drought tolerance a drought inducible promoter can
greatly ameliorate the effects (Liu et al. 1998; Huang 2001). Sugarcane promoters active in culms
have been described recently (Hansom 1999). Research is also focusing on post-transcriptional
silencing events that may render transgene expression unstable.
21.8 suGarcane FunctIonal GenomIcs and BIoInFormatIcs
No matter which route one chooses for sugarcane improvement, target genes for genetic manipula-
tion or use as markers need to be identified. The analysis of the sugarcane transcriptome has been
extensively used for many years in a search for genes associated to agronomic traits of interest
(Menossi et al. 2008). Transcriptomics complements gene marker identification and either tech-
nique can be excellent tools for breeders that wish to improve sugarcane through transgenics or
classic breeding. Also, the use of the transcriptome can aid in the identification of markers for
QTL mapping and expression-QTL (eQTL), which in sugarcane is difficult until statistical genetic
tools are available for highly polyploid genomes. Techniques that have been used for transcrip-
tomics in sugarcane include EST sequencing, nylon-based cDNA macroarrays, cDNA microarrays,
long oligonucleotide microarrays (Agilent Technologies®) and short oligonucleotide microarrays
(Affymetr ix®).
Transcriptome studies in sugarcane were first undertaken by EST sequencing (Carson 2000;
Carson and Botha 2002; Casu et al. 2003, 2004; Ma et al. 2004; Vettore et al. 2003; Bowers et al.
2005). The largest collection of ESTs was generated by the SUCEST Project (http://sucest-fun.
org), a consortium of over 100 Brazilian laboratories that generated approximately 238,000 ESTs