1. Introduction

Sorghum (Sorghum bicolor L.) it is the fifth important cereal crop grown in the world (US Grains Council). In the years 2012/2013, production of sorghum was reported to be 59.3 million metric tons worldwide (USDA Grain: World Markets and Trade, Feb. 2012/2013).

Sorghum used as staple food for over 500 million people in the world, especially in African and Asian countries. Sorghum is rich in antioxidants and it is grain free of gluten and for this reason provides an attractive grain replacement for people suffer from celiac disease. Sorghum is drought-resistant crop addresses global climate change and limited water resources issues, sorghum used as source of food, feed and fiber, is also (Kargi et al., 1985; Gnansounou et al., 2005; Laopaiboon et al., 2007; Rooney et al., 2007). There are several reports indicating the tolerance of sorghum to drought and high temperature than corn, wheat and other cereal crops (Gnansounou et al., 2005). Sorghum has the unique ability to grow under a wide range of harsh environmental conditions (House, 1995).

Modifications of tissue culture protocols of sorghum have been reported (2010). In these reports, based on callus induction experiments, the highest callus induction reported were 4.5% (Shridhar et al., 2010). Transformation of sorghum via Agrobacterium-mediated method was reported unsuccessful due to it is recalcitrant for tissue culture and genetic engineering (Shrawat and Lorz, 2006). Regeneration of transgenic sorghum was successfully reported using Agrobacterium-mediated transformation (Zhao et al., 2000). This was followed by reports from other researchers using Agrobacterium-mediated transformation method (Gurel et al., 2009; Howeet et al., 2006; Lu et al., 2009; Nguyen et al., 2007). There are several factors that affect transformation efficiency, including the sensitivity of sorghum explants to Agrobacterium infection, type of explant, composition of the culture media. Addition of coconut water to the co-cultivation medium together with the use of vigorous and actively growing immature embryos as explants for infection and the removal of excess Agrobacterium significantly improved the survival rate of explants and were critical for the success of transformation (Hiei et al., 1997).

2. Material and Methods

Plant materials

Seeds of four sorghum varieties (Tabat, Elwafer, Yrwasha and Dwarf White Milo (D.W. Milo)) were sterilized by first soaking in 70% ethanol for 30 seconds. The seeds were then soaked in 2.5% sodium hypochlorite for one hour before washing with sterile water three times under sterilized conditions. The sterile seeds were used as sources of mature embryos as explants for callus induction.

Callus induction and culture conditions

All callus induction experiments were performed on callus induction medium (CIM). The CIM comprised of MS salts and vitamins supplemented with 0.7 g/l L-Proline, 0.5 g/l MES, 0.5 g/l casein hydrolysate, 0.2 mg/L Kinetin and 3% (w/v) sucrose. The pH of the medium was adjusted to 5.8 with 1M NaOH or 0.1M HCL. 0.65% (w/v) gelrite was added to solidify the medium. CIM was sterilized by autoclaving. Five levels of 2,4-D (0, 1, 2, 4, and 6 mg/l) were tested to establish their efficacy in establishing callus from four different varieties of sorghum via mature embryos.

The Mature embryo explants were obtained from sterile seeds washed in distilled and autoclaved water. Mature embryos were cultured using a sterile forceps on CIM containing different 2,4-D levels (0, 1, 2, 4, 6, mg/L). The embryos were orientated with the embryo axis in contact with the medium. Twenty embryos were cultured in a 90 x 15 mm Petri dish for each 2,4-D level. The cultures were completely covered with aluminium foil and incubated in the dark at 25±1°C for two weeks. Callus induction frequency was recorded after four weeks of culture on the CIM.

After six weeks of callus induction all calli were transferred to MS medium for 14 days to initiate shoots. For induction of shoots the embryogenic calli were transferred to shoot induction medium (RII) comprising MS basal salts and vitamins (Murashige and Skoog, 1962), supplemented with 30 g/L sucrose, casein hydrolysate and proline. The number of regenerated plantlets per calli was evaluated.

Statistical analysis

The experiments were designed in randomized complete block design (RCBD) with four replications per treatment. Callus induction frequency were calculated on number callus induced from the total number of explants were cultured, these data were used to compute callus induction frequency. Analyses of variance (ANOVA) were done by using statview statistical software to test the statistical significance of differences among explants source and 2,4-D levels. Mean separation was done using least significance difference (LSD) test at 5% probability level.

3. Results and Discussion

Clear differences in callus induction frequencies were observed among the four varieties used in this project, the variety Elwafer gave the lowest callus induction frequency among all the varieties. While the variety Tabat gave the highest callus induction frequency among all the varieties. Callus induction frequency from variety Tabat was highest at 2,4-D concentrations of 4 and 6 mg/L with callus induction frequencies of 85.0 and 93.8 respectively (Table 1). This was significantly higher than at other concentrations but there was no significant difference in callus induction between the concentrations 2 and 4 mg/L (Figure 2C). There was no significant difference in callus induction frequency for all the treatments for the variety Elwafer the concentrations 2 and 4 mg/L gave the same and the highest callus induction (1.250 and 1.250) (Table 1) with no significant differences between them. The variety Yrwasha gave the highest callus induction frequency at concentrations 2, 4 and 6 mg/L of 2,4-D and there were no significant differences in callus induction for those three concentrations but there were significant differences between the other 2,4-D level (Figure 2B). For the variety Dwarf Milo the highest callus induction frequency was obtained at 2,4-D concentration of 2 and 4mg/L with means of 8.750 and 6.250. This was not significant than that of other 2,4-D concentrations but there was significant differences between 0 mg/Land 2 mg/L of 2,4-D (Table 1).

Table 1. Callus induction frequency from mature embryos

Mature embryos gave the lowest callus induction frequencies from the variety Elwafer while the highest callus induction from mature embryos was observed from the variety Tabat because Elwafer produced phenolic compound on callus induction medium which affect tissue culture response.

The hormone 2,4-D is important for cereal callus induction and callus formation from mature and immature embryos. Mature embryos from dry seeds are more readily available and can be used as an effective and alternative explants source in tissue culture (Figure 2A).

The variety Tabat gave the highest embryogenic callus induction from mature embryos at 2,4-D concentrations of 4 mg/L and 6 mg/L (Figure 1A and 1B). The varieties Yrwasha and D.W. Milo were producing non embyogenic callus non friable, compact callus which was not able to regenerate (Figure 1C and 1D).

Figure 1. Callus induction from mature embryos of sorghum achieved using 4 mg/l of 2,4,D. A and B: Embryogenic callus induced from the variety Tabat at 4 mg/L2,4-D. C: non embryogenic callus induced from mature embryos of Yrwasha. D: Callus contain phenolic compound induced from mature embryos of Elwafer

The highest shoot induction from mature embryos was observed in the variety Tabat only, the variety Tabat was able to produce higher number of shoots at 4 mg/L of 2,4-D it gave shoots on shoot induction medium (Figure 2D) while the lowest number of shoots was observed at 2 and 6 mg/L of 2,4-D (Table 3). All the other varieties used in this study they were not able to produce shoots. The variety Tababt it is the most promising variety for tissue culture among all the varieties used in this study.

Table 2. Embryogenic callus induction frequency from mature embryos of Tabat

Table 3. Number of shoot from mature embryos of Tabat

Figure 2. Callus induction and regeneration from mature embryos of sorghum achieved using different concentrations of 2,4,D

This research focuses on developing protocols for callus induction and regeneration of sorghum through tissue culture techniques. There are many factors that affect the response of crops to tissue culture, including the source of explants, composition of the culture media and size of explants were critical for the success of tissue culture techniques.

Tropical germplasm have been reported to be recalcitrant to tissue culture manipulation and plant regeneration methods, making the regeneration of sorghum a challenging issue. Tissue culture is a popular techniques for improvement of germplasm for tolerance to drought and salinity through somaclonal variation (Matheka et al., 2008).

There are several reports indicating that the responses of sweet sorghum to callus induction depending on the concentrations of Auxin used in the callus induction medium ( Liming et al., 2010) in our experiments we indicated that callus formation was highly stimulated by addition of 4 or 6 mg/L of 2,4-D for all the four verities. The auxin 2,4-D is important for callus induction and embryogenic callus induction from mature and immature embryos in monocotyledon plants Rakshit 2010 and Jogeswar et al., 2007). The output of this research has indicated that the presence of 2,4-D could induce callus formation from seeds. However, high concentrations of 2,4-D made callus subculture easy and reduce regeneration frequency for the other three varieties (Mendoza and Kaeppler, 2002).

Mature embryos are ready and available all the season and for this reasons can be used as an effective and alternative source of explants for tissue culture of crops (Jia et al., 2008). The varieties Yrwasha and Elwafer induced callus which become watery and non-embryogenic it is reported that callus induction and regeneration of cereal crops is genotypic dependent (Armstrong and Green, 1985). Also there was report indicated that the addition of proline promoted the embryogenic callus production and enhancement of plant regeneration during culture of maize and sorghum (Rao et al., 1995) and for this reason we used proline in our tissue culture medium. Induction of callus from mature embryos of Tabat and Yrwasha increased with increasing the concentrations of 2,4-D in the range of 4 and 6 mg/L with addition of 0.2 mg/L Kinetin, the induction percentage of embryogenic callus has negative effect of KT on callus culture was noted in sorghum and wheat (Lazar et al., 1983).

In conclusion, in this research we have optimized a protocol for tissue culture and regeneration from mature embryos of sorghum. This report shows that it possible to improve tissue culture conditions by optimizing the compositions of callus induction and plant regeneration media for specific varieties, because of reproducibility and the easy accessibility of mature seeds, the optimization of tissue culture protocols of sorghum provides foundation for genetic transformation of for improving important traits.

4. Conclusions

This work has formed the base for transformation of sorghum there for transformation can be achieved because we have the infrastructure and protocols for sorghum at Agricultural Research Corporation of Sudan.

The variety Tabat it is the most promising sorghum genotype and shall be used for tissue culture and transformation of sorghum.

Tissue culture research has been optimized in sorghum there for protocols for tissue culture of millet and finger millet can be establish for enhancement of cereal crops.

ACKNOWLEDGEMENTS

This work was achieved through funding provided by African Bioscience Challenge Fund Fellowship, the work was carried out in the Tissue Culture Unit in the Biotechnology and Biosaftey Research Center at agricultural research corporation of Sudan.

Appendices

Appendix 1. Analysis of varieance for callus induction from mature embryos for the varieties Tabat, Elwafer, Yrwasha and D.W Milo