Yam cultivars of Tropical D. rotundata (TDr) 2579 and 2436 were obtained as plantlets from in vitro germplasm collection at International Institute of Tropical Agriculture (IITA)-Ibadan, Nigeria. All the cultivars were maintained in vitro and multiplied as shoot cultures on yam basic medium (YBM) containing Murashige and Skoog medium (MS) salts and vitamins, 0.05 mg/l 6-Benzylaminopurine (BAP), 0.02 mg/l Naphthaleneacetic acid (NAA), 25 mg/l Ascorbic acid, 30 g/l sucrose, 2.4 g/l gelrite. The pH of the medium was adjusted to 5.8 prior to autoclaving. The cultures were incubated in growth room at 28^∘C with 16/8 h photoperiod. The nodal explants (3–5 mm) containing axillary buds were excised from young in vitro shoots.

Prior to transformation experiments, the sensitivity tests to selective agents (hygromycin and kanamycin) were carried out in order to find an effective inhibitory concentration, which arrests the formation of shoot buds and shoots from nodal explants. The sensitivity to antibiotics was determined by culturing nodal explants having axillary buds on shoot bud induction medium (SBM; MS salts and vitamins, 1 mg/l BAP, 0.318 mg/l Copper sulfate, 20 g/l sucrose, 2.4 g/l gelrite) supplemented with different concentrations of hygromycin (0–15 mg/l) or kanamycin (0–250 mg/l). The cultures were transferred to a fresh medium containing the same level of antibiotic every 2 weeks and then scored for the frequency of regeneration after 8 weeks. The minimal inhibitory concentration of antibiotics was used in all the transformation experiments.

Agrobacterium tumefaciens strains LBA4404 and EHA105 were used in this study. The binary vectors pCAMBIA1301, pCAMBIA2301 (CAMBIA Company, Australia) and pCAMBIA2300-gfp were used for transformation (Figure 1). The pCAMBIA1301 contained hygromycin phosphotransferase(hpt) gene as selection marker, while pCAMBIA2301 and pCAMBIA2300-gfp contained neomycin phosphotransferase II (nptII) as selectable marker. Plasmids pCAMBIA1301 and pCAMBIA2301 contained the intron-containing gusA reporter gene, while plasmid pCAMBIA2300-gfp contained gfp as reporter gene. The binary vectors were transformed into A. tumefaciens strains LBA4404 and EHA105 by electroporation. Single colonies from Luria Bertani (LB) agar (10 g/l Tryptone, 5 g/l Yeast extract, 10 g/l Nacl, 15 g/l Agar, pH 7.5) plates containing kanamycin (50 mg/l), rifampicin (50 mg/l) and streptomycin (100 mg/l) were used to initiate 2 ml LB medium starter cultures. After 48 h shaking at 150 rpm at 28^∘C, this suspension was used to inoculate a 20 ml LB medium containing the same antibiotics, and grown overnight on a shaking platform at 150 rpm to reach an OD₆₀₀ of 1.0. Bacterial culture was centrifuged at 3500 rpm for 15 min and pellet was re-suspended in liquid SBM medium supplemented with 200 μM acetosyringone (Sigma Chemical Co.) and grown further for 2–3 h at 25^∘C with shaking at 100 rpm. The optical density (OD₆₀₀) of culture was checked and adjusted to 0.5. The bacterium suspension was used for transformation experiments.

The explants of D. rotundata were immersed in bacterial suspension and vacuum infiltrated for 5 min followed by gentle shaking at 45 rpm for 30 min at room temperature. After inoculation, explants were blotted on sterile paper towels and co-cultivated for 3 days under dark condition at 28^∘C, in petri dishes containing SBM medium supplemented with 100 μM acetosyringone. Fifty explants were used in each experiment and transformation efficiency was compared with two Agrobacterium strains (LBA4404 and EHA105) and plasmids with different selection marker genes (hpt and nptII). Experiments were repeated twice.

Following co-cultivation, the explants were rinsed three to four times with liquid SBM medium supplemented with 500 mg/l carbenicillin and blotted dry on sterile filter paper and placed onto SBM supplemented with 250 mg/l carbenicillin for 1 week of recovery at 28^∘C 16/8 h photoperiod. After 1 week of incubation, the explants were transferred to fresh SBM medium supplemented with 250 mg/l carbenicillin and 7.5 mg/l hygromycin or 100 mg/l kanamycin depending on the plasmid used and incubated for 14 days at 28^∘C under 16/8 h photoperiod. This step was repeated twice with gradually increasing the antibiotic selection to 10 and 15 mg/l for hygromycin selection or 125 and 150 mg/l for kanamycin selection. The elongated shoots were separated and transferred to YBM containing 250 mg/l carbenicillin and 15 mg/l for hygromycin or 150 mg/l for kanamycin for 1 month for rooting. The well rooted plantlets were transferred to peat pellets, covered with transparent polythene bag and placed in a glasshouse at 28^∘C. After 3–4 weeks, each peat pellet is fragmented and plantlets transferred into pots containing sterile soil and covered with plastic bags. When plants have reached 30–50 cm in height the plastic bags were opened to allow further growth. The putative transgenic plants regenerated on selective medium were subjected to β-glucuronidase (GUS) histochemical assay or fluorescent microscopy and molecular analysis.

Transient and stable histochemical GUS assay was carried out in different tissues as described by Jefferson et al. (1987) with modifications. Tissues were immersed in a buffer containing 2 mM X-Gluc, 50 mM phosphate, 50 mM potassium ferrocyanide and 5% Trition X-100 at pH 7.0 and vacuum infiltered for 10 min, and then incubated overnight at 37^∘C for 24 h. Tissues containing chlorophyll were repeatedly soaked in 95% ethanol until chlorophyll was removed. Transient expression of gusA gene was examined in Agro-infected explants after 3 days of co-cultivation, while stable expression of the reporter gene was analyzed in leaves, shoots and roots isolated from putative transgenic plants regenerated on selective medium.

Transient and stable GFP expression was analyzed using a Nikon SMZ1500 stereomicroscope with GFP-Plus fluorescence module. The images were recorded in TIFF format using a digital camera. All plants putatively transformed with pCAMBIA2300-gfp were tested for GFP expression.

Plant genomic DNA for polymerase chain reaction (PCR) was extracted from regenerated putative transgenic young leaves using a DNeasy kit (Qiagen, GmbH, Germany). Specific primers used for gusA were: forward 5′-TTTAACTATGCCGGGATCCATCGC-3′ and reverse 5′-CCAGTCGAGCATCTCTTCAGCGTA-3′. Specific primers for hpt were: forward 5′-CCACTATCGGCGAGTACTTCTACACAGC-3′ and 5′-GCCTGAACTCACCGCGACGTCTGTC-3′. PCR was conducted in a total volume of 20 μl, containing 100 ng template DNA, 2 μl 10 × buffer, 0.5 μl of 10 mM dNTP, 0.5 μl of 10 μM primers, 1 unit of Taq DNA polymerase (Qiagen, GmbH, Germany). The PCR conditions were: initial denaturation at 94^∘C for 10 min, 35 cycles of denaturation at 94^∘C for 15 s, annealing at 62^∘C for 40 s for the gusA gene, 58^∘C for 40 s for the hpt gene, and extension at 72^∘C for 50 s, followed by final extension at 72^∘C for 7 min and holding at 4^∘C. The amplified PCR products were separated by electrophoresis on 0.8% (w/v) agarose gel stained with GelRedTM (Biotium) and visualized under a UV transilluminator and photographs were taken by the gel documentation system.

Total RNA was extracted from 100 mg young leaf tissue of 10 transgenic lines and non-transgenic control plants using the RNeasy plant mini kit (Qiagen, GmbH, Hilden, Germany) and treated with DNase (RNeasy Plant Mini kit, Qiagen). The quantity and quality (A_260/230 and A_260/280) of total RNA were determined using the Nanodrop 2000. RNA was checked with PCR for absence of genomic DNA. Complementary DNA (cDNA) was synthesized using 1 μg of total RNA and reverse transcriptase of the Maxima H Minus First Strand cDNA synthesis kit with oligoDT primers (Thermo scientific). For reverse transcriptase polymerase chain reaction (RT-PCR), 2 μl of each cDNA synthesized was used. PCR cycling conditions included initial denaturation of 94^∘C for 10 min, followed by 35 cycles of 94^∘C for 15 s, 62^∘C for 40 s and 72^∘C for 50 s and final extension for 7 min. RT-PCR was performed with primers specific to the gusA gene as described above and housekeeping gene actin primers forward 5′- ACCGAAGCCCCTCTTAACCC-3′ and reverse 5′-GTATGGCTGACACCATCACC-3′. The amplified RT-PCR products were separated and visualized as described in the PCR section above.

The integration of the transgene into the genome of yam was analyzed using dot blot and Southern hybridization. Genomic DNA for dot blot analysis was extracted from twelve PCR positive transgenic lines using a DNeasy kit (Qiagen, GmbH, Germany). About 200 ng of genomic DNA in triplicate for each transgenic line was denatured at 98^∘C for 10 min, immediately chilled on ice for 5 min and immobilized onto a positively charged nylon membrane (Roche Applied Sciences, Mannheim, Germany) using a BIORAD Bio-Dot Microfiltration apparatus following the manufacturer’s protocols and recommendations. The DNA samples were fixed on the membrane by cross-linking in a STRATA-LINKTM UV cross-linker. A gusA-specific probe was labeled with DIG-dUTP using PCR DIG Probe Synthesis Kit (Roche Applied Sciences, Mannheim, Germany). Hybridization, stringency washes and detection was carried out using a DIG Luminescent Detection Kit for Nucleic Acids (Roche Diagnostics, UK) according to the manufacturer’s instructions.

For Southern blot analysis, genomic DNA was isolated from in vitro grown plants using cetyltrimethylammonium bromide (CTAB) method developed by Sharma et al. (2008) with modifications. The genomic DNA (20 μg) of transgenic lines and non-transgenic control plant was digested with HindIII (New England Biolabs, USA) for overnight at 37^∘C. The plasmid DNA digested with HindIII was used as positive control. Restricted DNA was separated on a 0.8% (w/v) agarose gel at 40 V for 6 h and transferred to a positively charged nylon membrane (Roche Applied Sciences, Mannheim, Germany) by capillary transfer method and fixed by cross-linking in a STRATA-LINKTM UV cross-linker. Hybridization and detection was performed as described above.

Data were subjected to significance by analysis of variance (ANOVA) and mean separation by Duncan’s multiple range tests (DMRTs; p < 0.05) using SPSS 11.09 software for Windows.

An effective selection strategy is very important for developing an efficient genetic transformation procedure. This can be achieved by the use of a selective agent which prevents non-transformed tissues from regenerating, while permitting the development of transformed cells into shoots without any lethality of the explant tissues (Song et al., 2012). The choice of selection agent depends on the plant nature and each plant species responds differently to the selection agent. The bacterial nptII and hpt are the most frequently used selectable marker gene used for generating transgenic plants. These enzymes detoxify aminoglycoside antibiotics by phosphorylation, thereby permitting cell growth and development of transformed plant cells into shoots in the presence of antibiotics. Therefore, optimization of the dose of selection pressure using hygromycin or kanamycin is important, as a suboptimal dose results in high frequency of escapes (Datta et al., 1990). On the other hand unnecessary high antibiotic doses not only kill untransformed tissues, but also inhibit growth of transformed cells, leading to delay in the regeneration process (Wilmink and Dons, 1993). Therefore, optimization of the aminoglycoside concentration was based on the minimal antibiotic concentration sufficient to prevent regeneration of untransformed tissues. The effective antibiotic concentration is another important factor for selection and regeneration of transgenic plant cells. Antibiotics decay in the plant tissue media due to various factors such as light, pH, temperature (Padilla and Burgos, 2010) as well as the antibiotic degradation in the vicinity of transgenic cells able to inactivate the antibiotic (Rosellini et al., 2007). Regular subculturing on fresh selection media as described in our study increases the effective inhibitory action of the antibiotic used.

It has been reported that monocotyledonous plants are sensitive to hygromycin, but not to kanamycin (Hauptmann et al., 1988; Eady and Lister, 1998; Chin et al., 2007). Tör et al. (1993) also reported that the suspension cells of the D. alata showed a high tolerance to kanamycin and no growth inhibition even at a concentration of 500 ug/ml. However, our results indicated that shoot induction of D. rotundata is sensitive to both hygromycin and kanamycin (Table 1, Figure 2). Shoot bud induction and plant regeneration from axillary buds of nodal explants were completely inhibited on a medium containing 10 mg/l of hygromycin or 150 mg/l kanamycin. No study has been performed so far on the hygromycin-based selection for the transformation of yam. The effective inhibitory concentration of hygromycin and kanamycin determined through this study will assist with the design of selection conditions for both hpt and nptII gene-based plasmids in the future for effective transformation of yam and will also be useful to engineer yam with multiple T-DNA insertion.

Efficient selection of transformed tissues was accomplished by increasing the selection pressure in a step wise manner from 7.5 to 15 mg/l and 100 to 150 mg/l for hygromycin and kanamycin, respectively. This process allows transformed explants to express effectively the antibiotic-resistance gene and initiate cell division, thus improving regeneration of explants to produce plants (Bull et al., 2009). Final selection on higher concentration of antibiotics also eliminates generation of false positive or escape plants. The use of low antibiotic concentrations in regeneration medium at early stages promotes transformed cell recovery, while a subsequent gradual increase of antibiotic concentration effectively eliminates non-transformed cells (Burgos and Alburquerque, 2003). Such pattern of selection was previously reported to be effective for cassava (Zhang and Puonti-Kaerlas, 2000), castor (Sujatha and Sailaja, 2005), dendrobium (Suwanaketchanatit et al., 2007), Lotus corniculatus (Nikolicć et al., 2007), grapevine (Fan et al., 2008), rapeseed (Liu et al., 2011), and spinach (Milojevicć et al., 2012) and also for jute, under kanamycin selection (Sarker et al., 2008).

After co-cultivation, the explants were subjected to a resting period of 5–7 days in carbenicillin supplemented medium lacking selection agent to improve the regeneration of Agro-infected explants. It is reported that direct transfer to selective medium after co-cultivation could result in tissue necrosis of the explants (Khanna et al., 2007). Agro-infected nodal explants began to form axillary buds after 7 days on selective medium (Figure 3). The induced buds started producing shoots 4–6 weeks after Agro-infection on selective regeneration medium supplemented with gradual increase of antibiotics every 2 weeks (Figure 3). In 8–10 weeks some of the shoots elongated and turned green and other shoots turn white or chimeric. In this study, a clear difference was observed during kanamycin selection process of transformed (green) and non-transformed (bleached) developing shoot buds. In order to eliminate possible chimeric plants, the shoots produced were sub-cultured several times with the same level of selection pressure (15 mg/l or 150 mg/l kanamycin). After three subcultures, the chimeric shoots completely bleached and died while the transgenic shoots continued to survive and grow normally. All the shoots generated on selective medium produced roots when transferred onto YBM containing 15 mg/l hygromycin or 150 mg/l of kanamycin. In this rooting assay, only transformed shoots survived to rooting, whereas the escaping shoots did not produce roots. The putative transgenic plants generated were validated by PCR and proved to be transgenic by GUS assay and GFP fluorescence. A generalized scheme for stable genetic transformation protocol is shown in Figure 4.

The effect of cultivars, Agrobacterium strains and selection marker genes was examined on transformation efficiency using axillary buds as explants. Significant variation in transformation efficiency was observed among different cultivars based on Agrobacterium strains and antibiotic selection marker used (Table 2). We observed transformation efficiency of 9.4–18.2% depending on different transformation factors including the yam cultivars, Agrobacterium strains and antibiotic selection marker. Differences are known to exist between transformation efficiencies of different genotypes, expression vectors, selection marker genes, and the strain of Agrobacterium as well as the tissue culture conditions (Cheng et al., 2004). Among these factors, the cultivar of the explants is considered as a crucial one that can hardly be overcome or complemented through optimizing other external factors, for example by manipulating highly virulent strains (Hansen et al., 1994) or by optimizing plant culture conditions (Zuo et al., 2002).

In the initial experiment we compared the effect of two Agrobacterium strains (EHA105 and LBA4404) harboring plasmid pCAMBIA1301 on the transformation efficiency of two cultivars of D. rotundata. No significant difference was observed in transformation efficiency due to various Agrobacterium strains used in the transformation (Table 2). However, there was a significant difference (p < 0.05) in transformation efficiency among the two cultivars transformed. The transformation efficiency was higher (16–18%) for cultivar TDr 2436 in comparison to cultivar 2579 (12–14%). As there is no significant effect of Agrobacterium strain used on transformation efficiencies, only one strain EHA105 was used for further studies. Our results suggest that different cultivars of the same species may differ remarkably in their susceptibility to Agrobacterium infection. The biochemical basis of these variations involves more complex mechanisms, as has been extensively reviewed by many authors (McCullen and Binns, 2006; Citovsky et al., 2007; Gelvin, 2010), that the transfer of DNA from A. tumefaciens to plant genome is a complex process involving a number of discrete, essential steps. The difference in the susceptibility of cultivars to Agrobacterium could be due to the presence of inhibitory metabolites to Agrobacterium sensory machinery (Liu and Nester, 2006; Maresh et al., 2006). Plant host defense response stimulated by Agrobacterium infection may be another factor influencing the susceptibility of plant cells to Agrobacterium (Ditt et al., 2005; Zipfel et al., 2006; Anand et al., 2008).

Selectable marker genes are required for establishment of efficient transformation in plants. In most cases, selection is based on antibiotic (kanamycin or hygromycin) or herbicide (phosphinothricin) resistance (Miki and McHugh, 2004). Selectable marker genes allow the plant cells that carry them to regenerate in media containing selective agents, while non-transformed cells die. The choice of a selectable marker gene depends on its efficiency, applicability to a wide range of plants, availability for researchers and its market acceptance (Kraus, 2010). In this study, we compared two selection marker genes (hpt and nptII) for transformation efficiency of two cultivars of D. rotundata. There was significant difference (p < 0.05) in transformation efficiency using hpt and nptII as selectable marker genes (Table 2). Although there was a significant difference between hpt and nptII selectable markers, our study demonstrates that hygromycin as well as kanamycin selection are efficient and can be used for the recovery of transgenic yam tissues and plants. No escape plants were obtained with any of the selection agents used. The hpt-hygromycin system has been reported to be more efficient than the nptII-kanamycin and the phosphinothricin acetyl transferase (PAT)-phosphinothricin systems (Song et al., 2012). However, the availability of multiple resistance gene/antibiotic selection systems that allow for efficient selection of transgenic yam is essential to generate multiple improved traits from independent T-DNA cassettes. From a regulatory perspective, nptII is particularly interesting since it is present in a large proportion of commercialized genetically modified crops (Miki and McHugh, 2004) and several independent studies have demonstrated its safe use in transgenic crops (Fuchs et al., 1993; Ramessar et al., 2007).

Other selection strategies that are free of antibiotic and/or herbicide-resistance genes can also be used to select transgenic plants. One of these strategies is the use of visual markers such as GUS (Jefferson et al., 1987) and green fluorescent protein (GFP; Prasher et al., 1992). In this study, we also compared two reporter genes (gusA and gfp) for the transformation efficiency of two cultivars of D. rotundata. There was no significant difference in transformation efficiency using gusA and gfp reporter genes for both cultivars (Table 2). Our results demonstrate that both gusA and gfp can be used as reporter genes for developing transgenic yam. The use of the gusA gene as a marker for transformation is effective and is widely applied in many species, including monocots such as rice (Wakasa et al., 2012) and orchard grass (Lee et al., 2006), and dicots such as common bean (Mukeshimana et al., 2013) and alfalfa (Duque et al., 2007). The presence of an intron in the gusA gene guards against false positives that may result from expression of the gene in A. tumefaciens (http://www.cambia.org). However, the GUS assay involves destruction of the tested tissues of transgenic explants; therefore, it should be performed at a later stage of the transformation study and presents a bottleneck for verification strategies in large-scale plant transformation protocols. GFP fluorescence visualization is also a useful tool for selecting transgenic yam plants. It has been widely used in the transformation of many plant species such as pepper (Jung et al., 2011), Petunia hybrida (Muβmann et al., 2011), and alfalfa (Duque et al., 2007). The fluorescent marker GFP is a highly versatile reporter gene, because the gfp gene expression can be monitored any time in living cells under a fluorescence microscope in a non-destructive manner (Chalfie et al., 1994). The same tissues may then be used for regeneration of stable transformants, which is not possible with other marker genes requiring destructive or toxic enzyme assays. Visual marker genes like gfp can even be used for yam transformation without using antibiotics or herbicides as the selection agents.

Complete transgenic plantlets ready for transfer to the greenhouse were produced within 3–4 months after Agro-infection. The rooted plants grew normally after transplanting to soil in the glasshouse (Figure 3) confirming that selection marker (hpt or nptII) or/and the reporter genes (gusA or gfp) does not have any apparent adverse effect on the normal development and morphology of the transgenic yam plants.

The use of both gfp and gusA genes as visual markers provides a useful method for confirming putative transformed plants (Padilla et al., 2006; Duque et al., 2007). The putative transgenic lines were verified by GUS histochemical assay or GFP fluorescence at different levels of plant development. The GUS staining was observed in nodal explants and emerging axillary buds indicating successful Agro-infection and transient expression of gusA gene (Figure 5). Transgenic plants displayed intense blue coloration in the leaf, stem, root, in contrast with non-transformed plant tissues, indicating the stable integration of the gusA gene into the genome and its expression (Figure 5).

In this study, we examined explants for GFP fluorescence at different stages, including axillary bud induction and in vitro plantlet (Figure 5). GFP expression was observed in both the axillary buds and leaves of transgenic plantlets (Figure 5). An advantage of visualizing GFP expression in our system was to enable us to select transformation events at an early stage thus avoiding the transfer of non-transgenic shoot that survived the kanamycin selection, saving both time and labor.

To confirm the presence of foreign genes into the genome of transgenic plants, antibiotic-resistant plants were analyzed by PCR and RT-PCR (Figure 6). PCR analysis was performed with genomic DNA of putative transgenic and control non-transgenic plants in order to confirm the presence of transgene. The amplified product of about 500 base pairs corresponding to the internal fragment of gusA gene was observed from genomic DNA of all the transgenic plants tested using gusA gene-specific primers confirming the presence gusA transgene in transgenic plants. An amplified fragment of 958 base pairs was also observed from all tested transgenic plants using hpt specific primers confirming the presence of hpt gene. The amplified products were observed in all the plants tested, confirming the presence of both transgenes gusA and hpt, without any escape plant. No amplified product was observed in case of non-transgenic control plants.

The transgenic lines were analyzed using RT-PCR in order to verify the expression of gusA gene. The gusA transcript amplification of the expected fragment size (∼500 base pair) was observed from samples of all the transgenic lines tested (Figure 6). Specific Actin transcript amplification was detected from all plants as an internal control for cDNA synthesis. A gDNA control was included in the assay with actin primers and showed the larger unspliced fragments, indicating that DNA contamination was below PCR detection levels in RNA samples. The results indicated that target genes were successfully incorporated into plant genome and were expressed in transgenic plants.

Polymerase chain reaction positive transgenic lines were further analyzed by dot blot and Southern blot hybridization using gusA probe to confirm integration of the transgene into the genome of yam. Genomic DNA of all the 12 transgenic lines tested by dot blot analysis were confirmed to contain the gusA gene (Figure 7A). Three transgenic lines were further tested with Southern blot hybridization. Their unique hybridization patterns indicated that each transgenic line resulted from an independent transformation event (Figure 7B). No hybridization signal was detected in the non-transgenic control plant.

Pests and diseases are among the most important of the many factors that have deleterious effects on yam tuber yield and quality and over time these constraints have become more severe (Amusa et al., 2003; Baimey et al., 2006; Aidoo et al., 2011). The progress made in this study in establishing an efficient genetic transformation system of D. rotundata could open up many avenues to produce disease resistant yams, through pathogen-derived resistance strategies, that would not be possible using conventional breeding approaches. Host plant resistance to anthracnose has been proposed as a viable alternative to the use of chemical fungicides in controlling the disease. However, studies have shown that there are no genotypes tolerant or resistant to the disease (Abang et al., 2002). Therefore, the most attractive strategy for anthracnose control in yam is probably the production of disease resistant plants through the transgenic approach. These approaches could include the expression of genes encoding plant, fungal or bacterial hydrolytic enzymes (Lorito et al., 1998), genes encoding elicitors of defense response (Keller et al., 1999) and antimicrobial peptides (AMPs; Broekaert et al., 1997). AMPs have a broad-spectrum antimicrobial activity against fungi as well as bacteria and most are non-toxic to plant and mammalian cells.

Use of resistant varieties can be an effective strategy in controlling yam nematodes, but there are no varieties known to be tolerant to nematodes. The use of transgenic plants will be an alternative approach to improve the nematode resistance of yam. Several transgenes confer plant resistance to both tropical and temperate plant parasitic nematodes (Atkinson et al., 2003). Cystatins inhibit nematode digestive cysteine proteinase activity, suppressing the growth and multiplication of these pests (Urwin et al., 1997) and is one of the transgenes that has been successfully used to control plant nematodes. It has been found that the cystatins confers the improved resistance to a range of nematodes in different crops like potato, sweet potato, rice, tomato, and plantain (Atkinson et al., 1996; Vain et al., 1998; Urwin et al., 2001; Chan et al., 2010; Gao et al., 2011; Roderick et al., 2012) and have proven efficacy under field conditions (Urwin et al., 2001, 2003). Such an approach could also be used to enhance resistance of yam against nematodes in the near future.