Agrobacterium-mediated genetic transformation of Fonio ( Digitaria exilis ( L . ) Stapf )

Fonio (Digitaria exilis) is a crop grown in Africa for its excellent culinary and nutritional properties and is a valuable source of income for smallholder farmers. Its improvement has long been neglected by scientists because the crop was one of the lost crops of Africa. Recent advances in Fonio research are centered on its evolution, nutritional and economic values. The lack of a transformation and other biotechnology protocols developed for Fonio has greatly hindered genetic improvement of this crop. In this study, Agrobacterium-mediated genetic transformation was developed for two genotypes of D. exilis („Agyong‟ and „Churiwe‟). Three-weeks-old callus derived from stem segments were co-cultivated with Agrobacterium tumefaciens for 3 days. After selection on Hygromycin B medium, plants were regenerated on Murashige and Skoog (MS) medium supplemented with 0.5 mg l -1 BA and 0.1 mg l -1


INTRODUCTION
Fonio (Digitaria exilis) is a C4 annual herb that tillers freely.It is a native of West Africa and belongs to the family Poaceae, sub-family Panicoideae and tribe Paniceae.There are two important cultivated species in the genus, namely, D. exilis and Digitaria iburua.The inflorescence in both species is a panicle with finger-like projections, 2 to 5 in D. exilis and 4 to 10 in D. iburua.
The grains are extraordinarily tiny, measuring 0.5 to 1 mm in diameter and 0.75 to 2 mm in length.The fruit is a caryopsis firmly secured between two husks (lemma and palea); in D. exilis the husk is white, hence the name white Fonio, in contrast to D. iburua that has dark-brown husk and is commonly called black Fonio.The most variable of all the species is D. exilis which is cultivated in *Corresponding author.E-mail: ntuival@yahoo.com.Tel: +2347066987008.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License many West African countries where they are called by different names (Ayo and Nkama, 2006;Adoukonou-Sagbadja, 2010).
Fonio seeds have excellent culinary and nutritional properties and are sometimes regarded as the "grain of life" because they supply food to several millions of people, when the major crops are yet to mature and food is generally scarce.The crop is also used as a valuable source of income, especially for small-scale farmers (Adoukonou-Sagbadja et al., 2007a).According to Kuta et al. (2003), the significant contribution that Fonio could make in resolving food crisis in Africa is expected to rekindle interest in the improvement of this crop.Available literature on the crop centers around genetic characterization of the plant using Random Amplified Polymorphic DNA (RAPD) markers and flow cytometry (Adoukonou-Sagbadja et al., 2007a, b, Adoukonou-Sagbadja, 2010) as well as microsatellite markers (Barnaud et al., 2012).Information is also available on the nutritional and physiochemical property of the crop (Temple and Bassa, 1991;Jideani and Akingbala, 1993;Jideani et al., 1994Jideani et al., , 1996;;Chukwu and Abdul-Kadir, 2008;Ballogou et al., 2013) as well as cultivation and uses of Fonio (Jideani, 1999).
Although, the use of genetic transformation as a tool for crop improvement and for the development of functional genomics is well documented, literature on in vitro cultures or genetic engineering of Fonio is lacking.There is therefore the need to explore the possibility of using genetic transformation for improvement of Fonio so as to increase its productivity.In our previous study, an efficient regeneration protocol for Fonio was developed (Ntui et al., 2010).In the present report, a protocol is given for transformation of D. exilis based on Agrobacterium-mediated technology.

Seed germination and callus induction
Seeds of two genotypes of D. exilis, "Agyong" and "Churiwe" were surface sterilized in 70% ethanol for 5 min and 1.5% (v/v) sodium hypochlorite solution for 20 min before rinsing them thrice in sterile distilled water.The seeds were then germinated and maintained for one month on Murashige and Skoog (1962) medium, containing 10 g l -1 sucrose and 8 g/l agar in a growth room at 25±1°C, 16 h photoperiod, 30 to 40 µmol m -2 s -1 cool white fluorescent light.The pH of the medium was adjusted to 5.8 prior to the addition of agar and autoclaving at 121°C for 15 min.
Stem segments (culm) about 5 mm in length were cut from the four week-old seedlings and cultured on callus induction medium (CIM, which was Murashige and Skoog (MS) medium containing 2 mg/l 2,4-D, 1 g/l casamino acid, pH 5.8 and 8 g /l agar).Cultures were placed at 26 ± 1°C in the dark for three weeks.Stem segments were subcultured to fresh medium after two weeks.
Three-week-old calli were dipped in the bacterial suspension for 10 min with gentle shaking.Thereafter, the calli were dried on a Whatman filter paper and co-cultivated in the dark on CIM, pH 5.2 and supplemented with 100 µM acetosyringone for three days.
After co-cultivation, the calli were washed three times with sterilized distilled water containing 10 mg/l meropenem trihydrate (Meropen; Dainippon Sumitomo Pharma, Osaka, Japan) (Ogawa and Mii, 2007) and cultured for 4 weeks on CIM supplemented with 20 mg/l meropenem to kill bacterial carryover and 50 mg/l  Values in brackets are percentages.
hygromycin B for selecting transformed tissues in the dark.The calli were subcultured bi-weekly into fresh medium of same composition.
After selection, brown or black calli were removed and only creamish healthy calli were transferred to MS medium containing 30 g/l sucrose, 0.5 mg/l BA, 0.1 mg/l gibberellic acid (GA3), 20 mg/l hygromycin B and 10 mg/l meropenem for shoot regeneration.Regenerated shoots were transferred to hormone-free MS medium containing 20 mg/l hygromycin B and 10 mg/l meropenem.

Gus assay and polymerase chain reaction
Histochemical gus assay (Jefferson et al., 1987) was used to assess gus staining of putative transformed plants using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide (X-Gluc) as the substrate.After selection, a few creamish healthy calli were subjected to gus assay by incubation in sodium phosphate buffer containing X-Gluc for 18 h at 37°C.
Transformation efficiency was calculated as the number of PCR positive plants × 100/number of calli inoculated.

Overview of the transformation and regeneration protocol
Two genotypes, "Agyong" and "Churiwe" were selected to develop transformation protocol for Fonio.These genotypes were selected because of their high regeneration efficiency based on our previous study (Ntui et al., 2010).Culm segments were used as the explants, as these were the only tissues which developed calli based on our previous study (Ntui et al., 2010).
Approximately, 80% of the culms developed calli within 3 weeks of culture on callus induction medium.After inoculation with Agrobacterium and upon selection on hygromycin medium, some calli turned brownish and died while others remained creamish and produced friable callus (Figure 2).These friable calli together with other hygromycin resistant calli were transferred to regeneration medium.Of the calli that were transferred to regeneration medium, only 13.25 and 11.87%, respectively for Agyong and Churiwe, produced shoots (Table 1).This regeneration frequency was very low and was produced only by friable calli.Regenerated shoots were successfully rooted in hormone-free medium (Figure 2).

Gus assay and PCR analysis
Gus activity was only detected in transgenic tissues but not in the untransformed tissues after using X-Gluc (Figure 3A).From PCR analysis done, it was found that a DNA fragment corresponding to the gus gene was amplified from genomic DNA isolated from leaves of putative transformed plants (Figure 3B).The transformation efficiencies obtained were 2.14 and 2.74, respectively for cultivars "Agyong" and "Churiwe" (Table 1), which are quite low.The low transformation efficiencies may have resulted from the high number of escapes probably because of insufficient selection regime and also due to the low concentration of hygromycin used in the regeneration medium.In some cereals, hygromycin concentration of up to 100 mg/l has been used to select transgenic plants.In some rice genotypes, 50 mg/l hygromycin is sufficient to select transgenic tissues (Sahoo et al., 2011).Since the sensitivity of callus to hygromycin B was not checked, possibly the right concentration of the antibiotic was not used for the selection.Checking the sensitivity of callus to hygromycin is important to minimize escape.However, as this is not the only factor that affects transformation efficiency of plants, other factors such as Agrobacterium strain and concentration, pH, inoculation and co-cultivation time amongst others should be optimized to increase the transformation efficiency of this plant and subsequently facilitate its functional genomics.
Genetic transformation provides a greater potential for overcoming breeding barriers in crop improvement (Wambugu, 1999(Wambugu, , 2001;;Machuka, 2001).The transformation protocol developed above could prove useful for genetic engineering of Fonio for increased grain size, pest and disease resistance, herbicide tolerance and ability to withstand lodging among others.A number of gene constructs for many of these traits and which are routinely used for cereal transformation are available and can be explored for Fonio genetic engineering by adopting the transformation protocol developed in this study.

Conclusion
In this protocol, it was observed that it took about 10 weeks (the essential steps given in Figure 4) to produce transgenic plants from co-cultivation with Agrobacterium.Although, the transformation efficiency is low, this is however, the first report of Agrobacterium-mediated genetic transformation of Digitaria.The protocol described here will provide useful platform for developing high efficient transformation protocol and for genetic improvement of this important cereal.

Figure 2 .
Figure 2. Production of transgenic plants from three month old callus after inoculation with Agrobacterium strain EHA 101pIG121-Hm.

Figure 4 .
Figure 4. Flow chart for the transformation of Fonio.

Table 1 .
Transformation efficiencies of two cultivars of D. exilis inoculated with Agrobacterium tumafaciens EHA101plG121-Hm.