Sugarcane (Saccharum officinarum L.) belongs to genus Saccharum, family Poaceae and characterized by high levels of polyploidy (2n=80~270) and frequently aneupolidy (Heinz and Mee, 1969). It accounts for approximately 80% of world sugar production FAO, (2009). This grass is the most suitable promising crop which could be utilized mainly for sugar production and then for power generation, paper making, live stock feed, chipboard, cane wax, fertilizer, bioethanol, syrup and mulch (Chaudhry and Naseer, 2008). Sugarcane is the second major cash crop in Pakistan after cotton. Many factors are involved in low cane and sugar yields in which drought or low rainfall, salinity, insect pests and diseases are remarkable especially whip smut (Ustilago scitaminea) a fungal disease which causes 30%~100% economic damage (Rangashawami, 1996; Nasir et al., 2000; Gururaj, 2001; Khaliq et al., 2005; Ajit, 2006). Gene introduction by conventional breeding becomes more difficult due to limited flower production, environmental interactions, large complex genome, slow breeding advances, back crossing, low fertility, susceptibility to insect, pest and diseases especially whip smut (Gururaj, 2001). Flowering is a major constraint for sugarcane improvement by adopting breeding tool Khan et al (2004). In Pakistan, flowering and viability are still a major problematic issue due to lack of favorable environmental conditions (Khan et al., 2007). Thus, unavailability of viable fuzz makes this crop unsuitable under the umbrella of conventional breeding in Pakistan. In conventional breeding and selection system, 10 to 15 years are tentative time span for commercial release of variety with improved characters James (2004).

Moreover, during vegetative propagation, the pathogens keep on accumulating generation after generation, which ultimately results in the decline of the variety. Preservation of germplasm collections is an integral part of all breeding programmes. Current methods for this purpose include conservation stands and greenhouse collections, requiring land and facilities, which are labor intensive and expensive to maintain. Furthermore, under such conditions, there is a high risk of germplasm loss through natural disasters, pest and disease infestations. Traditional plant breeding techniques have been widely used to enhance important economic traits in agronomic crops, but this approach is laborious and time-consuming, especially in vegetatively propagated species like sugarcane. Moreover, various important traits such as resistance to insects, viruses, and herbicides are often absent from the normal sugarcane germplasm. DNA-mediated plant transformation can serve an important function to introduce useful genes into sugarcane that otherwise would be difficult or impossible by standard procedures.

Tissue culture plays an important role in crop improvement. Sugarcane in vitro regenerants have higher yield potential in terms of excellent sugar recovery, high tiller ratio, more weight and excellent ratooning performance (Comstock and Miller, 2004). Through tissue culture successful attempts were made to eliminate diseases in sugarcane. Sugarcane yellow leaf virus (SCYLV) and sugarcane yellows phytoplasma (SCYP) were eliminated in nineteen cultivars and showed no disease attack for one year in green house (Parmessur et al., 2002; Ramgareeb et al., 2010). In vitro culture system is also used for screening of diseases viz., eye spot disease, fiji disease and downy mildew and whip smut in sugarcane (Singh et al., 2005). One advantage of the use of sugarcane rolled leaf and sheath tissue for embryogenic callus initiation is that it is relatively easy to arrange year-round availability of this explant type from field-grown plants, so that fresh callus batches can be regularly initiated to minimize time in culture for gene transfer. By comparison with more recalcitrant related species such as sweet sorghum (Raghuwanshi and Birch, 2010), the surface layers of sugarcane embryogenic callus evidently have a higher proportion of cells that are able to proliferate and regenerate under conditions that permit the selection of transformed plantlets.

The application of plant biotechnology approaches like genetic transformation of foreign genes into the plant genome, it is very crucial to be the optimization of efficient regeneration system in terms of homozygous plantlet formation through tissue culture with 100% purity to the mother plant. Therefore, assessment of the genetic stability of in vitro regenerated planets is an important step in the application of biotechnology. For the application of in vitro culture system and clonal propagation, it is important to determine genetic purity (Rani et al., 2000). Hence testing the genetic homogeneity of in vitro regenerated plants is very essential and important. The use of molecular markers is becoming widespread for the identification of somaclonal variants and the assessment of in vitro regeneration protocols (Taylor et al., 1995). Different molecular markers (ISSR, RAPD, Trap, RFLP, AFLP and microsatellites etc) are used to detect and characterize somaclonal variation at the genomic DNA level by Ford-Lloyd et al (1992) and Cloutier and Landry (1994).

Among the various molecular marker techniques, RAPD marker is found to be the most useful one to detect genetic changes at DNA level by Taylor et al (1995); Soliman et al (2003) and Anand (2003). RAPD analysis technique is the quick, simple, easy to perform, require small amount of DNA for analysis and the most important advantage of this marker is that the independence of prior information requirement to detect genetic stability of in vitro regenerated plants by Williams et al (1990). These benefits justify the frequent application of the technique in genetic variability studies by Mondal and Chand (2002); Bennici et al (2003) and Feuser et al (2003).

Biotic stresses such as insect, pests and diseases are the alarming threats for sugarcane grass. Whip smut, Shoot borer, giant borer, Red rot, Leaf scald, Eye spot, Mosaic virus, Pineapple disease, Ratoon stunting disease are the major threats for sugarcane. Whip smut is a serious threat for sugarcane and occurs in almost all sugarcane growing countries Comstock (2000). Few attempts have been made to develop resistance against the most devastating disease. These are pre-plant heat therapy of planting sets of sugarcane; pre-plant fungicidal dips of planting sets and screening of sugarcane clones for identification of resistant varieties against the pathogen. Genetic mapping with SSR marker was done for sugarcane smut resistance by Raboin et al (2001). A recent report describes the use of cDNA-AFLP and suppression subtractive hybridization (SSH) to identify differentially expressed sugarcane genes upon inoculation with the sugarcane smut fungus U. scitaminea. Using a Restriction Fragment length Polymorphism (RFLP) approach, markers were developed and used on a population of 78 well characterized sugarcane genotypes that are used in a breeding program. In this study, 59 polymorphisms showing correlation with smut resistance were identified. PCR and microscopy were used for smut disease assessment in sugarcane (Singh et al., 2004). Amplified Fragment length Polymorphism (AFLP) analysis of cDNA was used to identify sugarcane genes differentially expressed in disease-resistant but not in susceptible sugarcane somaclones (Hidalgo et al., 2005).

Several strategies have been used to improve plant defense against insects and pathogens. The activation of stress-response transcription factors was found to enhance plant tolerance to fungal and bacterial pathogens in transgenic plants by Gu et al (2002). However, little is known about the function of other components of the plant transcription machinery during stress. The identification and characterization of agronomically interesting genes related to herbivores and pathogens are a major challenge for sugarcane functional genomics. One of the most promising areas is to improve insect control through the use of proteinase inhibitor genes Allsopp et al (1997) and Nutt et al (1999) or Bt genes Arencibia et al (1997) against the sugarcane borer (Diatraea saccharalis), responsible for considerable losses in the field. For over ten years now, the directed genetic modification of sugarcane has been a reality in laboratories and field trials has been conducted (Bower and Birch, 1992; Gallo-Meagher and Irvine, 1996; Leibbrandt and Snyman, 2003; Manickavasagam et al., 2004). Genes can be silenced or over expressed to study their function and to produce new phenotypes not possible through conventional breeding. But genetic manipulation through biotechnology such as marker assisted tool (Butterfield, 2005), DNA mapping (Grivet et al., 1996) and genetic transformation have emerged as novel approaches. Successful genetic transformation of cry1Ab gene for shoot borer (Chilo infuscatellus) (Arvinth et al., 2010), Chitinase and Chitosanase genes against Colletotrichum falcatum which cause Red rot disease in sugarcane crop (Ijaz, 2012).

1 Results
1.1 Sugarcane in vitro studies
Disease free planting material is a necessary component for propagation of next generation for getting higher yield. Tissue culture is an ideal technique for the production of problem free plants in a short time from small amount of planting material. In vitro culture of sugarcane provides the planting material throughout the year for the formation of stable transformant lines. Callus formation followed by regeneration varies among genotypes which depicts that genotype plays a significant role in callus formation and regeneration. Therefore, it is significant for every genotype to optimize media with different levels of plant growth regulators along with growing conditions so that optimum regeneration could be attained. Yield is a major constraint to make it a green profitable crop which mainly attributed to insect pest and diseases. Whip smut is a major hurdle for getting good yield. Therefore, under these circumstances this study was conducted to develop stable in vitro regeneration system for genetic transformation experiment in selected genotypes.

1.2 Callus formation
Two whip smut susceptible genotypes S-2003-us-127 and S-2003-us-371 with good agronomic features and an excellent sugar recovery were selected for this study. Good callus mass is required for efficient regeneration. Embryogenic callus also have significant role in effective regeneration. In the present study, immature young leaves of selected sugarcane genotypes were surface sterilized and cultured on callus formation media (CFM). Combinations of different plant growth regulators (2,4-D, BAP and Kinetin) were used in CFM. For callus induction study, four levels of 2,4-D (1 mg/L, 2 mg/L, 3 mg/L and 4 mg/L); two levels of BAP (0 mg/L and 0.1 mg/L) and two levels of Kinetin (0 mg/L and 0.1 mg/L) were used for both genotypes. Data for callus formation response of both genotypes were scored on the basis of callus proliferation rate. Analysis of variance of data revealed significant variability between genotypes as well as among CFM. Interaction between genotypes and CFM was also highly significant (Table 1). Data over five weeks of culturing, revealed that callus formation response of both genotypes was good at all 2,4-D levels, alone or in combination with kinetin, but less response was observed when BAP was used in combination with different levels of 2,4-D. In genotype, S-2003-us-127, when BAP was used in combination with 2,4-D no callus mass formation was observed but swallowing of ex-plant was noted (Figure 1). But in genotype S-2003-us-371, BAP in combination with 2,4-D gave very little callus formation with mean value of 1.00.

Figure 1 Ex-plant swelling in genotype S-2003-us-127

Table 1 Analysis of variance (ANOVA): Statistical analysis (ANOVA) for callus formation in different sugarcane genotypes on different CFM

Genotype S-2003-us-127 showed an excellent callus mass proliferation by scoring the mean value of 5.00 on CFM3, CFM4, CFM11 and CFM12. Genotype S-2003-us-371 gave highest mass of calli on CFM3, CFM11 and CFM12 with mean value 4.00. Comparison among callus formation media (CFM) revealed that the response of CFM3, CFM11 and CFM12 was the best with mean of 4.50, followed by the response of CFM4 and CFM10 by scoring the mean value of 3.50. As for as, genotypes are concerned, the response of genotype S-2003-us-127 was overall good with mean score of 2.67 followed by the genotype S-2003-us-371 with mean value of 2.33 (Table 2; Figure 2).

Figure 2 Callus formation response of sugarcane genotypes on different callus formation media (CFM)

Table 2 Mean interaction of Genotypes and CFM

These both genotypes gave embryogenic calli on callus induction media. Different stages of calli of both genotypes can be seen in Figure 3 and Figure 4.

Figure 3 Stages of calli of genotype S-2003-us-127

Figure 4 Stages of calli of genotype S-2003-us-371

1.3 Regeneration Response in sugarcane genotypes
In order to obtain an efficient regeneration, plant growth regulators (Auxin and cytokinin) as well as amino acid play a key role for in vitro regeneration. CFM3 and CFM11 were selected for in vitro regeneration study, because on these media, both genotypes produced maximum callus. Despite of an excellent callus formation response of both genotypes also on CFM12, this medium was rejected because high level of 2,4-D might disrupt genetic stability of in vitro regenerants which is significantly not desired for genetic transformation approaches. For regeneration study, four regeneration media (RM) were used in which different plant growth regulators viz., 2,4-D (0.1 mg/L), BAP (0.5 mg/L and 1.0 mg/L), kinetin (0.25 mg/L) and proline (250 mg/L) were used. Calli of 3 different ages, viz., 21 days, 28 days and 35 days were used for regeneration experiment. Calli of both genotypes, those were induced on CFM3 and CFM11, shifted on four regeneration media (RM). Data were collected in the form of total number of shoots per explants. Analysis of variance for regeneration showed that the interaction between days, genotype, CFM and RM is highly significant (Table 3).

Table 3 Analysis of variance table for regeneration: Statistical analysis (ANOVA) for Regeneration in different sugarcane genotypes on different RM

Among genotypes, the genotype S-2003-us-127 produced more number of shoots per explant. Calli of 28 days from CFM11 produced 380 shoots per explant in genotype S-2003-us-127 on RM2 (Figure 5). This proved to be the best combination for regeneration; followed by 35 days old calli of this genotype on the same medium (RM2) gave 136.67 shoots per explant. Calli of age 35 days from CFM3 gave 73 shoots per explants on RM1, in genotype S-2003-us-127 followed by 28 days old calli of this genotype on the same regeneration media (RM1) produced 31.67 shoots per explants (Table 4; Figure 5). Contrary to this, the genotype S-2003-us-371, gave maximum number of shoots on RM4. Thirty five (35) days old calli from CFM11 produced 46 shoots per explant on RM4 followed by 28 days old calli from CFM11 gave 36.67 shoots per explant on the same regeneration medium (RM4).