Introduction

Sugarcane (Saccharum officinarum L.) is a commercial cash crop. Brazil tops sugarcane production followed by India which contribute nearly 15% of world sugar production. Besides, it has immense potential for production of many diversified products. Its enormous potential for ethanol production has been recognized which is used as a fuel blend with petrol for running automobiles. Besides, green top of sugarcane is in vogue used as fodder and cattle feed. Dried filter cake is used as animal feed supplement, fertilizer and source of sugarcane wax. While, the filter mud resulted from sugar processing is utilized as manure. Bagasse is used as bio-sorbent for waste water purification and also for manufacture of paper, paper board products, hardboard as well as fuel to run boilers for boiling the juice.

In India, sugarcane is constrained with low productivity owing to sensitivity to salt, drought and biotic stresses (Nasir et al., 2000; Khaliq et al., 2005). Drought alone accounts about 17% potential yield loss. Being a typical glycophyte, its growth is severely affected leading to significant reduction in yield potential under salt stress (Suprasanna, 2010). Narrow gene pool, higher ploidy (2n=100-120), rare flowering, low fertility, large genome size, long breeding cycle and complex environmental interactions seem to be major hindrances for breeding of sugarcane. However, production of transgenic sugarcane can be a better alternative to integrate desired gene(s) related to diseases, abiotic stresses, set yield and quality traits. The approach avoids the problem of linkage drag besides shortcutting the period of breeding in sugarcane.

1 Response to in vitro Culture

In vitro culture is an integral part of plant genetic transformation (Tiel Kenia et al., 2006). 2,4-D is in vogue reported to produce white creamy embryogenic (nodular) callus from leaf whorl explant which is ideal for genetic transformation. Khamrit et al. (2012) got profused and best callusing response in MS with 3 mg/l 2,4-D + 15% coconut water. Khan et al. (2004) revealed best callus induction and proliferation on MS medium containing 2.0 mg/I 2, 4-D, while combination of 2, 4-D (2 mg/l) with kn (0.5 mg/l) proved to be best for rapid callus growth in all sugarcane genotypes (Srivong et al., 2015). Similarly, combination of 3.0 mg/l of 2,4-D with 0.2 mg/l Kinetin gives best callus from young leaves with callusing response up to 83% in 13-15 days (Satpal et al., 2011).

Direct morphogenesis minimizes somaclonal variation and it can be amenable for genetic transformation. Shoot tip explant (Yadav et al., 2012) and Leaf discs from young leaf whorl is reported be a quick, effective and reproducible direct regeneration system (Ali et al., 2012). Direct regeneration was achieved on MS medium with MS + BAP+NAA (1.5+0.5 mg/L) + casein hydrolysate (500 mg/L) (Ali et al., 2012), MS + 5 mg/l NAA + 0.5 mg/l Kn (Gill et al., 2006) and MS with 2 mg/l BAP (Biradar et al., 2009). MS with paclobutrazol (40 mg/l) increases the production of plantlets and reduce the number of dead leaves and height of shoots (Panti, 2016). Besides, thiodizuron at lower concentration (0.01 and 0.1 mM) can be used as alternative for BAP and Kinetin (Vinayak et al., 2009).

Somatic embryos (SEs) can serve as excellent material for genetic transformation in sugarcane as these avoid chimerism. Creamy, nodular and friable calli resulted due to somatic embryogenesis may be considered ideal for rapid plantlet regeneration. Gandonou et al. (2005) revealed varied embryogenic callusing response (60 to 100%) in nine elite sugarcane genotypes. The plants derived through direct somatic embryogenesis have been found to be uniform in growth pattern with more vigour compared to plants derived through indirect somatic embryogenesis pathway (Suprasanna, 2010). Somatic embryogenesis has been reported in this crop by several workers (Asad et al., 2009, Ming et al., 2006). MS with 3 mg/L 2,4-D alone revealed highest embryogenic calli (Ijaz et al., 2012; Jahangir and Nasir, 2010). Khamrit et al. (2012) reported maximum percentage of somatic embryogenic callus induction in MS medium supplemented with 3 mg/l 2,4-D and 15% (v/v) coconut water. In contrast, somatic embryos were reported to be induced at lower concentrations of 2,4-D (1 mg/l), whereas higher concentrations induced non-embryogenic calli (Zamir et al., 2014). Sequential removal of 2,4-D followed by sub-culture on MS supplemented with 2mg/l BAP induced 98% shoot induction (after 3 weeks of culture) with maximum shoot elongation (9.4 cm) (Zamir et al., 2014). Similarly, callus induced in MS + 3.0 mg/l 2,4-D and subsequently incubated on 2,4-D-free media was found to be commercially viable for plantlet regeneration on MS + 1.0-1.5 mg/l BAP + 0.2 mg/l NAA in elite sugarcane genotypes (Abdu et al., 2012). Besides, Naz et al. (2008) revealed maximum callusing response from young leaf explants in MS + 3 mg/l 2,4-D; maximum somatic embryogenesis in sub-culture by sequential removal of 2,4-D (up to 2 mg/l) and higher frequency of plantlet formation in hormone-free media. MS with BAP as low as 0.25 mg/l was also reported to be efficient for shoot induction in 80% of embryogenic masses and the hormone free-MS medium proved to be appropriate for elongation and rooting of shoots (Dibax et al., 2011).

Media composition along with hormonal recipes also influenced somatic embryogenesis. Desai et al. (2004) reported high frequency of somatic embryo development in MS with 0.5 mg/l NAA, 2.5 mg/l kinetin, 100 mg/l L-glutamine and 4% sucrose. Besides, MS with 1.5 mg/l 2,4 D + glycine (0.75 mM), arginine (0.5 mM) and cysteine (0.25 mM) showed significant effect on somatic embryogenesis (94%) and shoot production as compared to medium without any amino acid. Among different amino acids, glycine seems to be most effective to promote somatic embryogenesis and maximum shoot regeneration.

A good regeneration system is a pre-requisite for effective exploitation of genetic transformation (Ijaz et al., 2012). The regeneration potential of callus seems to be genotype-specific and dependent on hormonal concentration and combinations. Addition of BAP (2-2.5 mg/l) alone caused an increase in percentage of shoot formation, number of shoot per callus clumps, and average shoot length (Hapsoro et al., 2012). Regeneration response as high as 82.32% was revealed on MS medium supplemented with 0.5 mg/l BAP and activated charcoal (2.0 g/l) (Mittal et al., 2016). Cefotaxime has been found to be a growth promoting substance in sugarcane tissue culture. Therefore, it is used in the medium subsequent to co-cultivation (Kaur et al., 2008). Patel et al. (2015) revealed emergence of microtillering on the media with 1.0mg/l BAP, 0.25 mg/l GA3, 20 g/l sucrose and 7 g/l agar. Whereas, MS with 3.0 mg/l GA3 alone resulted maximum shoot elongation (10.52±1.88) along with the highest number of root emergence (6.51±2.41).

Many workers tried with different types of auxins at different concentrations and combinations to regenerate adventitious roots. In this context; NAA, IAA and IBA seem to have good response and comparatively better response in case of NAA combination with IBA than IAA for profuse rooting (Behera and Sahoo, 2009). Some variety gives normal and healthy rooting within two week in MS or ½ MS medium supplemented with 2.5 to 4 mg/l NAA followed by 0.5 mg/l NAA+2.5 to 3 mg/l IBA with good root length (Mamun et al., 2004; Behera and 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 Agrobacterium harboring pGreen0029 vector containing AVP1 (Arabidopsis Vacuolar Pyrophosphatase-1) gene driven under 35S CaMV promoter for genetic transformation against drought and salinity tolerance in sugarcane. Bax Inhibitor-1 gene from Arabidopsis thaliana (AtBI-1) into sugarcane offers suppression of ER (endoplasmic reticulum) stress in C4 grasses which can be an effective means of conferring improved tolerance to long-term water deficit (Ramiro et al., 2016).

In recent years, development of transgenic plants is increasing rapidly in sugarcane. Sugarcane has been also genetically modified for sugar yield and quality traits (Botha and Groenewald 2001; Vickers et al., 2005), pharmaceuticals (Wang et al., 2005), novel sugars with potential benefits to consumer (OGTR, 2004). Besides, many biotic and abiotic stresses related to physiological characters have been studied in transgenic sugarcane. These include resistance to sugarcane mosaic virus (SCMV) (Gilbert et al., 2005), yellow leaf virus (Gilbert et al., 2009), sugarcane borer (Gao et al., 2016) and leaf scald resistance, herbicide tolerance, antibiotic resistance, drought and salinity tolerance (Kumar 2013; Reis et al., 2014). Production of naturally occurring compounds for use in bioplastics, altered plant growth, enhanced nitrogen use efficiency, improved sucrose accumulation, improved cellulosic ethanol production from sugarcane biomass, altered plant architecture, enhanced water use efficiency, incorporation of green fluorescent reporter gene, altered juice colour (Manickavasagam 2004; Mitchell 2011) are the outcome of transgenic technology. Further, genetic engineering of sugarcane varieties that can produce high-value compounds e.g., pharmaceutically important proteins, functional foods, nutraceuticals, biopolymers, precursors, enzymes and biopigments are paving ways to launch sugarcane as a biofactory in coming years (Grice et al., 2003; Suprasanna 2010).

The expression of G. frondosa TSase gene under the control of a promoter CaMV35S improve drought tolerance in sugarcane (Zhang et al., 2006) compared with non-transgenic plants. Similarly, Wang et al. (2005) developed the transgenic sugarcane plants harboring Grifola frondosa synthase gene which improved tolerance to osmotic stress. In another study, over-expression of heterologous P5CS gene under stress inducible promoter (AIPC) was also reported to enhance drought tolerance in sugarcane (Molinari et al., 2007). The Arabidopsis CBF4 gene transferred to sugarcane under the control of the maize ubiquitin promoter and the nos terminator was reported to improve drought tolerance (McQualter and Dookun-Saumtally, 2007). Besides, drought tolerance has been attempted in sugarcane by using Arabidopsis Vacuolar Pyrophosphatase (AVP1) gene (Kumar et al., 2014) and induced over-expression of AtDREB2A CA (a transcriptional factor) (Reis et al., 2014), SodERF3 (a novel sugarcane ethylene responsive factor) and Arabidopsis bax inhibitor-1 gene.

4 Conclusion

A ready-in highly regeneration protocol is the pre-requisite for successful genetic transformation in any crop. Several researchers developed cost effective, rapid and efficient regeneration system in elite sugarcane genotypes using varied hormonal recipes, other media supplements and culture conditions. Recently, a number of transgenic techniques have been used for transfer of useful genes from diversified genetic background. However, Agrobacterium-mediated genetic transformation proved to have several advantages over direct gene transfer techniques in sugarcane. The present review reveals successful development of genetically modified genotypes with improved quality features and resistance to biotic and abiotic stresses in sugarcane.

Acknowledgement

We sincerely acknowledge and thank all researchers for their valuable contributions included in this pursuit.

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