Cassava (Manihot esculenta Crantz) is a major food security crop providing about 500 calories per day for 800 million people in sub-Saharan Africa and other regions of the world (Nassar et al., 2009). It is the third most important source of calories, after rice and maize for many populations in the humid tropics (FAO, 2015). Currently, cassava is cultivated by millions of small-scale farmers in more than 100 countries in the world (FAO, 2013). It is widely grown by farmers because of its remarkable characteristics such as reliability and cheap source of available year-round food, reasonable yields on marginal soils, tolerant to major pests, diseases and drought (Dawit, 2010).

The global production of cassava was estimated at 152 MT per year, of which, 50% was cultivated by small scale farmers in Africa, 30% in Asia and 20% in America (CIAT, 2001; FAO, 2013). Sub-Saharan Africa is major contributor to the global trends in the development of the cassava value chain. However, new uses of cassava are emerging as commercial livestock feed, partial substitute for wheat flour in bread making; and as an industrial raw material such as starch (FAO, 2013). The role of cassava as a traditional food for human consumption is rapidly changing to that of an efficient industrial crop in some parts of Africa including Sierra Leone, where it is considered as the second most important staple food (Nweke, 2004; NARCC/MAFFS, 2005). The crop plays a significant role in the drive to national economic recovery in Sierra Leone with potential spill over effects to neighbouring countries (FAO, 2008). As a major carbohydrate crop, it is a versatile resource with potential for creating diverse products such as chips, broken dried roots meal, starch, flour and ethanol (Kuiper et al., 2007; Heuzé et al., 2015). Dried cassava roots and meal are used as raw material for animal feed, while cassava starch is used for industrial purposes (NARCC/MAFFS, 2005).

Despite its importance, the traditional stem cutting method used for its propagation is one of the bottlenecks for production of disease free planting materials (Ogero et al., 2012). Cassava seeds are normally dormant and germinate very slowly; consequently, farmers utilize the conventional stem cutting propagation method. This method leads to viral, fungal and bacterial diseases which may contribute to decreasing cassava productivity and loss of superior genotypes (Nassar and Ortiz, 2007). The cassava mosaic disease alone has been reported to account for an overall loss of 15-24% in Africa, which is equivalent to 12-23 MT of cassava (Acheremu, ‎2011). Furthermore, Zhang et al. (2005) noted a yield loss of 19.6 to 27.8% of the actual cassava production in Africa.

Viral diseases lack effective chemical control strategy compared to bacterial and fungal diseases (Jones et al., 2012). Severely infected plants incur heavy yield losses of over 25%, which necessitate the development and use of efficient micro-propagation protocols for healthy planting materials (Acheremu, 2011). However, thermotherapy and chemotherapy techniques can also be used for production of healthy planting materials (Panattoni et al., 2013).

Plant tissue culture is used for the growth and multiplication of cells, tissues and organs under an aseptic and controlled environment, and for rapid clonal propagation culture, somatic embryogenesis, embryo rescue and germplasm exchange (Rao, 1996). The process depends on the fact that cells are totipotent, with each cell capable of expressing the full genome and reproducing the whole plant (Loyola-Vargas and Vázques-Flota, 2006). Micro-propagation techniques are used for rapid clonal multiplication of selected genotypes of diverse plant species (Rani and Raina, 2000).

In vitro culture has contributed significantly to crop improvement by overcoming certain limitations associated with conventional techniques (Garcia-Gonzales et al., 2010). Culturing of an organized tissue in the form of very small shoots or meristem has allowed the most valuable application of plant tissue culture in order to eliminate virus from infected mother plant (Dawit, 2010). For instance, meristem-tip culture has been widely used to remove yield-limiting pathogens from plant cuttings (Acheremu, 2011). The major merits of tissue culture include the elimination of pathogens in donor plants and maintenance of stability in genetic inheritance (Dawit, 2010). The full potential of cassava will not be realized until production constraints are reduced in high-yielding varieties and cassava producers have access to disease-free planting materials. Therefore, the aim of this study was to optimize and screen efficient in vitro protocols for rapid multiplication and production of disease-free cassava planting materials through tissue culture techniques.

Nodal explants were initiated in test tubes containing MS medium supplemented with 30 g sucrose, 100 mg/L myo-inositol, 8500 mg/L agar, 1 mg/L thiamine, 1.6 mg/L GA₃ and 0.01 g/L NAA at 5.8% pH. The cultures were incubated in an air conditioned room at a temperature of 25±1°C, light intensity of 4000 lux provided by fluorescent bulbs and photoperiod of 16 h light and 8 h dark cycles. Sub-culturing was done at an interval of every four weeks.

Acclimatization of plantlets in the screen house

After root initiation within four weeks, plantlets were carefully removed from test tubes and washed in distilled water to remove the MS medium from the roots. The roots were dipped into antifungal redomil solution and transferred into large plastic bag in a screen-house to minimize shock from the environment. The plastic bag was gradually punched every three days to allow gentle aeration and acclimatization of tissue-cultured plants. The plants were finally weaned after six weeks and transplanted into 2 L volume plastic pots in the nursery.

Treatments, experimental design, phenotypic data collection and analysis

The treatments consisted of five varieties (Warima, Munafa, Coco cassada, SLICASS 6 and SLICASS 7) and five levels of BAP (0, 0.02, 0.10, 1.00 and 2.00 mg/L). The BAP supplemented MS medium trial was laid out in a 5 x 5 factorial arrangement in Completely Randomized Design (CRD) with three replications. Phenotypic data collected were: number of leaves, number of shoots and height of BAP supplemented plantlets.

Data were subjected to analysis of variance (ANOVA) using the GENSTAT statistical program (GENSTAT, 15th release, Rothampstead, UK). The Least Significance Difference (LSD) was used to compare between treatment means using a significance level of α = 0.05.

Extraction of genomic DNA

Genomic cassava DNA was extracted from leaves of tissue-cultured plantlets and those of uncultured sprouted shoot of explant exhibiting various degrees of severity symptoms. The DNA samples were extracted using a slightly modified cetyltrimethylammonium bromide (CTAB) method by Aldrich and Cullis (1993). About 50 mg each of fresh young healthy and diseased leaf samples were grinded separately in liquid nitrogen to a fine powder with 500 μl of 2% CTAB and 0.1% (0.5 μl) of mercaptoethanol. The content was intermittently vortexed in 1.5 ml Eppendorf tube. This was followed by addition of 300 μl of 2% CTAB and 0.5 μl (0.1 %) of ϐ-mercaptoethanol and incubated in a water bath at 65ºC for 60 min. The contents were cooled at room temperature, and equal volume of chloroform: isoamyl alcohol (24:1) was added and mixed by inversion of the tube to wash DNA from debris. Samples were then centrifuged at 10,000 rpm for 5 min and the supernatant phase of the sample was transferred into a clean 1.5 mL tube. The chloroform: isoamyl alcohol cleaning step was repeated. The precipitation of DNA was done by addition of 200 μl of ice cold isopropanol to the supernatant, kept on ice for 30 min and centrifuged at 10,000 rpm for 5 min. The isopropanol was decanted and the DNA pellet washed with 350 μl washing buffer (70% ethanol) for 15 min and again centrifuged at 6000 rpm for 4 min. The ethanol was decanted and DNA was dried in a vacuum for 60 min and suspended in 100 μl TE buffer (1 mM Tris HCI, pH 8, 0.1 mM EDTA, pH 8) and stored at -20°C. About 10 μl DNA sample and 5 μl sample buffer (Bromophenol blue) from each extract was run simultaneously with λ Hind III molecular marker on 0.8% Agarose gel containing Ethidium bromide.

Preparation of agarose gel and loading of DNA into wells

A 1.5% agarose gel was prepared by dissolving 3.0 g agarose gel (Sigma company) in 100 ml of TAE (242 g Tris base, 57.1 mL glacial acetic, 100 mL 0.5 M EDTA) buffer. The agarose gel was melted in a microwave oven and cooled to 65°C by constant stirring. After cooling, 4 μl of ethidium bromide was added to 100 ml agar and poured into gel plate with comb already set for loading of sample. The agarose gel was allowed to solidify for about 30 min before removing comb and immersing gel into the electrophoresis tank containing 1xTAE buffer. A 2 μl loading buffer was added to 10 μl of each DNA sample and loaded into the wells. The first well was loaded with 10 μl of 100 bp ladder and run at 100 volts for 1.5 h. After electrophoresis, the gel was visualized on a UV light transilluminator and photographed.

Virus indexing using Polymerase Chain Reaction (PCR) method

The ACMV and EACMV strains causing the mosaic symptoms in leaves of cassava genotypes grown in screen house and in vitro culture were detected using PCR method described by Fondong et al. (2000). The DNA samples of the cassava genotypes were tested for presence or absence of CMD using specific primers that could detect the strains of ACMV and EACMV. Two pairs of primer sequences designed by Mills et al. (1992) were used (Table 1).

The PCR analysis of the DNA of each of the 10 samples was performed in a BIO-RAD PTC-220 thermocycler (Dyad MJ Research) for 30 cycles of 35 s at 94°C, 35 s at 55°C, and 1 min at 72°C, and an additional 10 min cycle at 72°C. Reactions were carried out in a total volume of 25 µL containing 0.2 µM dNTPs, 0.5 µM primers, 2.5 µM 1x PCR amplification buffer, 0.5 mM MgCl₂, 0.4U Taq DNA polymerase (Invitrogen), 0.4 µM DNA template and 50 ng ultrapure water. Reactions were done twice to evaluate the consistency of the banding patterns for all isolates studied. Products were separated on a 2% (w/v) agarose gel in 1x TAE buffer (40 mMTris acetate, 1 mM EDTA, pH 8.0) at 125 V for 3 h. The gels were stained with ethidium bromide and visualized with UV light using a Cole Palmer FLUO-LINK FLX apparatus and photographed for later assessment.

The effect of various BAP amended culture media on the mean plantlet height is presented in Table 4 and Figure 2. Addition of BAP at 0.02, 0.1 and 1.0 mg/L induced taller plantlets compared to 2.0 mg/L BAP concentration. The tallest mean plantlet height (3.7 cm) was obtained in SLICASS 6 amended at 0.1 mg/L BAP, followed by SLICASS 7 (3.2 cm) and Coco cassada (2.6 cm) amended at 0.1 mg/L, whereas Warima (0.8 cm) had the shortest mean plantlet height at 0.0 mg/L (Table 4). These findings implied that, cassava genotypes responded differently to various concentrations of BAP; thus, genotype Warima was consistently among genotypes that exhibited shortest plantlets across the various BAP media.

Virus indexing

Amplification of the EACMV with the species specific primer pair (OjaRep/EACVMRep) did not generate any PCR products at 650 bp for healthy cassava plantlets from the growth room (lanes 1, 3 and 4), whereas lanes 6, 8 and 10 from the screen house and lanes 2 and 5 from the growth room were +ve for EACMV (Figure 3). Unlike the negative control (-ve), which did not produce PCR product, the positive control (+ve) (infected plants) produced an amplicon at approximately 650 bp (Figure 3). Similar trends were observed for OjaRep/ACMVRep specific primers in which the positive control (+ve) and lanes 2, 5, 6, 8 and 10 exhibited an amplicon at approximately 400 bp (Figure 4).

Results on plantlet height agree with Mushiyimana et al. (2011) who observed that culturing nodal explants harvested from in vitro grown plantlets on different MS formulations supplemented with 5, 10 20 and 40 μM/L GA₃ produce significant shoot elongation in cassava. Mushiyimana et al. (2011) suggested that, the best regeneration medium for cassava explants is full strength MS medium supplemented with 40 μM/L GA₃ provided that, the highest mean shoot length of 8.93±2.67 mm. Acedo (2006) worked on improvement of in vitro techniques for rapid meristem development and mass propagation of Philippine cassava using both liquid and solid MS media supplemented with GA₃, BAP and NAA. The present study partly agree with his report that plant regeneration was most effectively promoted by liquid MS medium supplemented with GA₃, BAP and NAA produced 2-nodal stages complete plantlet with callus after three weeks from inoculation. However, when the hormones were used singly, GA₃ did not show growth response whereas BAP caused shoot formation.

BAP is the most widely used phytohormone/cytokinin and it is effective at 0.01 – 10 mg/L for plantlet height. However, all the plantlet heights considered were significantly different at various concentrations. The interaction between BAP and genotype was also significantly (p<0.01) different on plantlet leaves and shoots evaluated. This could be associated with the effectiveness of cytokinin even at low concentration in cassava which was consistent with previous findings (Onuoch and Onwubiku, 2007; Staden et al., 2008).

Viral indexing

The success of virus indexing depends on an effective method of virus detection. A good test is needed to ensure that, plantlets regenerated from meristem culture are disease-free (Milošević et al., ‎2012). The DNA extracted from infected cassava leaves was used as positive control and it revealed that cassava genotypes studied were infected with both ACMV and EACMV diseases. The DNA of virus indexed genotypes in lanes 2, 5, 6, 8 and 10 exhibited infection of the diseases possibly due to contamination of samples. The DNA of the untreated positive control exhibited bands on the gel electrophoresis visualized, implying the presence of disease on infected cassava leaves that had not been virus indexed (Figures 6 and 7). The PCR results of treated or virus indexed plantlets from growth room (non-acclimatized) and screen-house (acclimatized) confirmed that, some plants were virus-free. Sseruwagi et al. (2004) noted known occurrence of ACMV in most cassava growing regions of Africa including Sierra Leone, whereas the EACMV has not widely spread (Neuenschwander et al., 2002; Freddy et al., 2015). The discovery of EACMV in genotypes of cassava from Sierra Leone is the first report of the disease. However, the strain(s) in the country are yet to be investigated. The PCR technique used in this study distinguished between virus infected and non-infected plants similar to those noted by (Ogbe et al., 2003).

This study showed that growth architecture of cassava depends on genotype, growing medium and interaction of growing environment with genotype. The cytokinin, BAP, was effective in enhancing growth of cassava. The nodal culture technique applied was adequate and effective in eliminating viruses in some genotypes from Sierra Leone. The PCR technique confirmed the presence of ACMV and EACMV in some cassava indicating the need for thorough and routine cleaning of cassava planting materials against viruses. Future studies will target the molecular characterization of different strains of viruses existing within cassava germplasm in the country, multiplication and/or conservation of virus indexed or virus free materials for utilization in breeding and crop production.

The authors have not declared any conflict of interests.

The authors are grateful for the financial aid by the World Bank through the West Africa Agricultural Productivity Programme (WAAPP 1C).