Genomics and Applied Biology, 2017, Vol.8, No.1, 1-7
3
Sahoo, 2009; Biradar et al., 2009; Gopitha, 2010; Satpal et al., 2011; Godheja et al., 2014; Tuan et al., 2015;
Dinesh et al., 2015). High concentration of NAA (5.0 mg/l) or a combination of NAA and IAA was reported to
promote good rooting (Anbalagan et al., 2000). Recently, Tesfa et al. (2016) obtained profuse rooting in half-MS
medium fortified with 3 to 5 mg/l NAA and 50 g/l sucrose (Yadav et al., 2012; Tesfa et al., 2016). Many workers
also reported that 5 mg/l NAA was good for rooting. More than 5 mg/l NAA could inhibit rooting and the most
efficient auxin for root initiation was NAA and IBA at 3mg/l with half MS media (Yadav. et al., 2012; Godheja et
al., 2014).
Substrate mixture for acclimatization comprising sand and soil substrate in 1:1 ratio is suitable for plant
establishment. However, a mixture of alluvial soil, clean humus, ¼ micro organic fertilizer and sand (1:1:1/ 4:1)
was reported to be ideal for best ex vitro acclimatization with higher plantlet survival rate for sugarcane genotypes
(Tuan et al., 2015; Tesfa et al., 2016).
2 Transgenic Approaches in Sugarcane
Success of the genetic transformation depends on stable integration of the transgene into the genome of the target
tissue, expression of the transgene and selection of transformed cells (Pillay, 2013; Singh, 2013; Rashid and
Lateef, 2016). Expression of transgenes requires suitable constitutive promoter sequence. A number of researchers
used promoters e.g., Emu, Maize Adh 1, CaMV 35S, Maize ubiquitin promoter, TMV 35S and Rab17 for
construction of gene cascade in sugarcane genetic transformation (Pillay, 2013; Kumar et al., 2013; Reis et al.,
2014).
In planta genetic transformation using sugarcane seeds (Mayavan et al., 2013), shoot tip explants (Khan et al.,
2013), axillary bud explants from 6-month-old plants (Manickavasagam et al., 2004) and young leaf whorl
have
been reported. Besides, production of transgenic plants via in vitro culture
of somatic embryogenic callus (Kumar
et al., 2014; de Alcantara et al., 2014) or cell aggregates of suspension culture (Efendi and Matsuoka, 2011) is a
method of choice. Embryogenic calli (Taparia et al., 2012), protoplasts (Arencibia et al., 1995), and apical
meristems have been used in sugarcane transformation studies. Among these, embryogenic calluses are the
preferred explant for transformation owing to their high regeneration response (Taparia et al., 2012). The genetic
transformation using polyethylene glycol (Aftab and Iqbal, 2001) microprojectile delivery system (Rani, 2012)
and electroporation (Rakoczy-Trojanowska, 2002) are best suited to protoplasts and cell suspension cultures.
Bower and Birch (1992) reported production of transgenic plants by bombardment of embryogenic callus with
high velocity DNA-coated micro-projectiles.
3
Agrobacterium
-mediated Genetic Transformation
The Agrobacterium-mediated transformation has the potential advantages over biolistic method owing to its
simple methodology and a high efficiency of transgene integration. The selection system and co-cultivation
medium were the most important factors determining the success of genetic transformation and transgenic plant
regeneration (Joyce et al., 2010). The most important and widely used selectable marker is npt II (neomycin
phosphotransferase) gene conferring resistance to phytotoxic amino-glycoside antibiotics, kanamycin and
geneticin (Bower and Birch, 1992; Fitch et al., 1995). Inhibitory effect of selective agents is tissue and species
specific (Cai et al., 1999; Yu et al., 2003). Therefore, it is necessary to know the minimal inhibitory concentration
of selective agent for different sugarcane cultivars before attempting genetic transformation. Genetic
transformation in sugarcane also involves use of reporter genes to establish the stability of transgene expression
and any other effect of gene transfer process (Hansom et al., 1999).
A reproducible method for transformation of sugarcane using various strains of
Agrobacterium tumefaciens
such
as AGL0, AGL1, EHA105 and LBA4404 carring vectors like pAHC27, pEmuKN, pR11F- (Pillay, 2013),
pGreen0029, (Kumar et al., 2013), pBract 302 (Reis et al., 2014), pMLH7133 (Efendi and Matsuoka, 2011),
Pu912 (McQualter and Dookun-Saumtally, 2007), pGFP35S (Rasul et al., 2014), pWBvec10a (Joyce et al., 2010),
pKYLX80 (Gilbert et al., 2005) has been developed. Kumar et al. (2013) employed EHA105 strain of
