**1. Introduction**

Yam is the colloquial name for some species in the genus *Dioscorea*, members of the family *Dioscoreaceae*. Yams are perennial monocotyledonous plants with a vine and underground tubers. There are about 700 species within the family, nine of which are medicinal plants that accumulate steroid saponins in their rhizomes and six species namely *D*. *bulbifera*, *D*. *cayenensis*, *D*. *dumentorum*, *D*. *prahensilis*, *D. alata* and *D. rotundata* are edible. The cultiva‐ tion of the edible tuber is mostly in Africa, Asia, Latin America and the Oceania regions. In West and Central Africa, especially Ghana the underground yam edible tuber is very important as a staple providing food security and income for some 26.2% of the popula‐ tion [6]. The daily average yam consumption is approximately 300 kcal per capita [4]. Being the third most important energy source in the Ghanaian diet, yam accounts for 20% of total caloric intake [4]. It is a versatile root crop which has various derivative products after process as it can be barbecued, roasted, fried, grilled, boiled, smoked and when grated it is processed into a dessert recipe. At present farmers are only getting about 20% of the potential yield of yams. In Ghana, the consumer has developed preference for a particu‐ lar *Dioscorea rotundata* variety locally called "Pona" because of its peculiar taste and texture. It is with this in mind that the CSIR-CRI Yam breeding program evaluated and selected for release three new yam varieties (CRI-Pona, CRI-Kukrupa, and Mankrong Pona). These varieties were officially released in May 2005 and are all high and stable yielding, pest and

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disease tolerant and have good culinary characteristics [14]. These released yam varieties are in high demand by farmers and all possibilities are being explored to enhance the production of clean planting materials which could be achieved using tissue culture techniques.

#### **1.1. Yam production and associated challenges**

Traditionally, yams are grown from the edible tuber in the form of whole tuber or sections, or setts on mounds and as they produce vines, they are staked with sticks to permit the vines to climb up the stake (Figure 1). The growth period is between 6 and 10 months, depending on the variety, after which they produce the edible underground tubers (Figure 2). Annually production of yam stands at about 48 million tonnes in the sub-saharan West African region and this represents 93% of the global production indicating that the region is the hub for production of the tuber. The countries involved in the production are Benin, Cote d'Ivoire, Ghana, Nigeria and Togo. Globally, Ghana is the leading yam exporter, having exported 20,841 metric tons of yams in 2008, but with the increasing global demand for yam from Europe, the U.S and neighbouring African countries, there is a potential for higher production and export volumes [10]. Research conducted in Council for Scientific and Industrial Research-Crops Research Institute (CSIR-CRI) showed there is a potential to increase the yields of yam from the average of 12 tons/ha to between 65-70 tons/ha. The major challenge in meeting the production targets are inadequate access and high cost of seed yam, hence despite the availability of fertile land and demand for yam domestically and abroad cultivation is low. Typically, cost of seed covers about 58% of production cost. This challenge is due to the fact that yams are vegetatively propagated from the edible tuber, and there is the general lack of high quality disease-free seed yam as the planting materials are usually infested with Fungi, Bacteria and Viruses. In order for farmers to maintain the clonal materials yams have been propagated vegetatively. This means of propagation has the potential of contributing im‐ mensely to the spread of diseases and pathogens endemic in the planting materials since same planting materials are used for propagation year after year which leads to accumulation when clean materials are not used. Currently the use of tissue culture techniques along with cryotherapy [7], chemotherapy and thermotherapy are the sure means of producing clean virusfree planting materials of vegetatively propagated crops. The presence of several viruses in West African yam was reported in 1992 [20] and these are supposed to have led to significant losses in crop production [9]. *Yam mosaic virus, a Potyvirus* is the most important infection in West African yams. It was first detected in *D. cayenensis* in Côte d'Ivoire [21]. The symptoms include mosaic, shoestring, green vein banding and stunting of the yam plant [17; 5]. Other viruses reported in yams include Badnavirus and Cucumber mosaic virus. It has been reported that sequences of badnaviruses have been integrated into the yam genome [18]. This has culminated from years of cropping yam with infected planting material. It is therefore important that tissue culture techniques be developed to facilitate the production and mass propagation of clean planting material. Other pathogens commonly associated with yams are nematodes and anthracnose.

Molecular Fingerprinting and Selection of Appropriate Media for Rapid *In Vitro* Multiplication of Three Yam Varieties http://dx.doi.org/10.5772/60411 35

**Figure 1.** Yam plant vines growing on a stake in the field

disease tolerant and have good culinary characteristics [14]. These released yam varieties are in high demand by farmers and all possibilities are being explored to enhance the production of clean planting materials which could be achieved using tissue culture

Traditionally, yams are grown from the edible tuber in the form of whole tuber or sections, or setts on mounds and as they produce vines, they are staked with sticks to permit the vines to climb up the stake (Figure 1). The growth period is between 6 and 10 months, depending on the variety, after which they produce the edible underground tubers (Figure 2). Annually production of yam stands at about 48 million tonnes in the sub-saharan West African region and this represents 93% of the global production indicating that the region is the hub for production of the tuber. The countries involved in the production are Benin, Cote d'Ivoire, Ghana, Nigeria and Togo. Globally, Ghana is the leading yam exporter, having exported 20,841 metric tons of yams in 2008, but with the increasing global demand for yam from Europe, the U.S and neighbouring African countries, there is a potential for higher production and export volumes [10]. Research conducted in Council for Scientific and Industrial Research-Crops Research Institute (CSIR-CRI) showed there is a potential to increase the yields of yam from the average of 12 tons/ha to between 65-70 tons/ha. The major challenge in meeting the production targets are inadequate access and high cost of seed yam, hence despite the availability of fertile land and demand for yam domestically and abroad cultivation is low. Typically, cost of seed covers about 58% of production cost. This challenge is due to the fact that yams are vegetatively propagated from the edible tuber, and there is the general lack of high quality disease-free seed yam as the planting materials are usually infested with Fungi, Bacteria and Viruses. In order for farmers to maintain the clonal materials yams have been propagated vegetatively. This means of propagation has the potential of contributing im‐ mensely to the spread of diseases and pathogens endemic in the planting materials since same planting materials are used for propagation year after year which leads to accumulation when clean materials are not used. Currently the use of tissue culture techniques along with cryotherapy [7], chemotherapy and thermotherapy are the sure means of producing clean virusfree planting materials of vegetatively propagated crops. The presence of several viruses in West African yam was reported in 1992 [20] and these are supposed to have led to significant losses in crop production [9]. *Yam mosaic virus, a Potyvirus* is the most important infection in West African yams. It was first detected in *D. cayenensis* in Côte d'Ivoire [21]. The symptoms include mosaic, shoestring, green vein banding and stunting of the yam plant [17; 5]. Other viruses reported in yams include Badnavirus and Cucumber mosaic virus. It has been reported that sequences of badnaviruses have been integrated into the yam genome [18]. This has culminated from years of cropping yam with infected planting material. It is therefore important that tissue culture techniques be developed to facilitate the production and mass propagation of clean planting material. Other pathogens commonly associated with yams are

techniques.

34 Biotechnology

**1.1. Yam production and associated challenges**

nematodes and anthracnose.

**Figure 2.** Edible yam tubers harvested from underground

#### **1.2. Tissue culture production**

Crop propagation through *in vitro* approaches offers a scope for improving root and tuber crops with desirable traits in larger quantities. The technique allows for rapid mass propaga‐ tion of clean planting materials all year round in a limited space and is ideal for the conserva‐ tion of germplasm. This notwithstanding, the widespread application of tissue culture has few limitations such as high initial cost of production, choice of crops restricted to species with acceptable propagation protocols and reproducibility of protocols.

Propagation of plants through tissue culture has become an important and popular technique to reproduce crops that are otherwise difficult to propagate conventionally by seed and/ or vegetative means. Specialised and matured cells are manipulated to give rise to multiple copies of the parent plant under optimum aseptic environmental conditions and appropriate stimuli. It offers many unique advantages over conventional propagation methods such as rapid clonal multiplication of valuable genotypes, expedition release of improved varieties, production of disease free plantlets, non-seasonal production, germ‐ plasm conservation and facilitating their easy international exchange. The application of tissue culture techniques towards the production of clean planting material is critical for vegetatively propagated crops. On the field, tissue culture produced plants were found to establish more quickly, grow more vigorously and produce higher yields than convention‐ al propagules with approximately 30% higher yield [15].

A number of factors come into play when establishing crops *in vitro*, and these include the plant part used (explant), its developmental stage, conditions under which it was grown, and the choice of growth conditions. Success of most tissue culture works depend much on the levels and kinds of plant growth regulators included in the medium. Root and shoot initiation, callus formation and differentiation are closely regulated by the relative concentration of auxin and cytokinin in the medium [3]. Plant growth regulators are critical media component in determining the developmental pathway of the plant cell. When establishing yam cultures *in vitro*, it has been shown that the age of the explant is critical [2] and also there seem to be a good reserve of endogenous auxins hence cultures do not require the supplementation of auxins [2]. Cytokinins such as benzylaminopurine (BAP) and kinetin are generally known to reduce apical meristem dominance as well as enhance both axillary and adventitious shoots formation from meristematic explants [8]. BAP has a marked effect in stimulating the growth of axillary and adventitious buds, and foliar development of shoot tip cultures [1]. In *Dioscor‐ ea* it has been shown to enhance the development of multiple buds and shoots under high concentrations [2]. Efficient growth and development is achieved when *in vitro* growth media and conditions are determined for the various growth stages namely culture establishment, mass propagation and plantlet development prior to transferring crops to the field. In yams, nodal culture establishment requires the addition of NAA and BAP to the medium, whereas meristem establishment requires the inclusion of GA3 and adenine sulphate to the medium. The mass propagation state requires only cytokinin be it Kinetin or BAP, whereas the rooting and plantlet establishment stage does not require any growth regulator in the medium [2]. *In vitro* manipulations when established are very important for germplasm maintenance.

#### **1.3. DNA fingerprinting**

In Ghana, crops developed, evaluated and selected for utilisation are given to the farmers through the agriculture extension agents. Most of the time as much as each crop variety has a name, they are all referred to as agriculture varieties. There are even incidents where different settlements give different names to the same crop variety, although the selection of the name at the variety developmental stages is done with the farmer. This makes it very difficult for the researcher after releasing a variety to track the extent of spread and adoption. Fingerprint‐ ing and documentation of genetic make up at the DNA level is thus vital to facilitate the researchers efforts. Fingerprinting can also be referred to as genotyping; which is the process of determining the genetic constitution – the genotype – of an individual by examining their DNA sequence. This provides information necessary to characterise germplasm and is a vital tool for identification of germplasm, as well as ensuring the genetic integrity with time. Genotyping can be applied to a broad range of organisms, including microorganisms. The genotype of an individual provides the fingerprint and comparing fingerprints allows you to determine the similarity between two individual, to find matches. This kind of information is vital to document the identity of crop varieties released to facilitate the ability to trace individual at any point in time.

Currently, methods of characterization used by breeders include morphological, agronomic, and biochemical systems. Characterization based on morphologic characteristics alone may be limited since the expression of quantitative traits is subjective to strong environmental influence. Alternatively, molecular characterization techniques are capable of identifying polymorphism represented by differences in DNA sequences. This has the ability of analyzing variation at the DNA level during any stage of the development of the plant, where environ‐ mental influences are excluded. The PCR-based methods constituted a new milestone in the field of DNA fingerprinting that has to be included in the requirements for varietal release in Ghana.

#### **1.4. Study objective**

limitations such as high initial cost of production, choice of crops restricted to species with

Propagation of plants through tissue culture has become an important and popular technique to reproduce crops that are otherwise difficult to propagate conventionally by seed and/ or vegetative means. Specialised and matured cells are manipulated to give rise to multiple copies of the parent plant under optimum aseptic environmental conditions and appropriate stimuli. It offers many unique advantages over conventional propagation methods such as rapid clonal multiplication of valuable genotypes, expedition release of improved varieties, production of disease free plantlets, non-seasonal production, germ‐ plasm conservation and facilitating their easy international exchange. The application of tissue culture techniques towards the production of clean planting material is critical for vegetatively propagated crops. On the field, tissue culture produced plants were found to establish more quickly, grow more vigorously and produce higher yields than convention‐

A number of factors come into play when establishing crops *in vitro*, and these include the plant part used (explant), its developmental stage, conditions under which it was grown, and the choice of growth conditions. Success of most tissue culture works depend much on the levels and kinds of plant growth regulators included in the medium. Root and shoot initiation, callus formation and differentiation are closely regulated by the relative concentration of auxin and cytokinin in the medium [3]. Plant growth regulators are critical media component in determining the developmental pathway of the plant cell. When establishing yam cultures *in vitro*, it has been shown that the age of the explant is critical [2] and also there seem to be a good reserve of endogenous auxins hence cultures do not require the supplementation of auxins [2]. Cytokinins such as benzylaminopurine (BAP) and kinetin are generally known to reduce apical meristem dominance as well as enhance both axillary and adventitious shoots formation from meristematic explants [8]. BAP has a marked effect in stimulating the growth of axillary and adventitious buds, and foliar development of shoot tip cultures [1]. In *Dioscor‐ ea* it has been shown to enhance the development of multiple buds and shoots under high concentrations [2]. Efficient growth and development is achieved when *in vitro* growth media and conditions are determined for the various growth stages namely culture establishment, mass propagation and plantlet development prior to transferring crops to the field. In yams, nodal culture establishment requires the addition of NAA and BAP to the medium, whereas meristem establishment requires the inclusion of GA3 and adenine sulphate to the medium. The mass propagation state requires only cytokinin be it Kinetin or BAP, whereas the rooting and plantlet establishment stage does not require any growth regulator in the medium [2]. *In vitro* manipulations when established are very important for germplasm maintenance.

In Ghana, crops developed, evaluated and selected for utilisation are given to the farmers through the agriculture extension agents. Most of the time as much as each crop variety has a

acceptable propagation protocols and reproducibility of protocols.

36 Biotechnology

al propagules with approximately 30% higher yield [15].

**1.3. DNA fingerprinting**

The need for clean planting materials on mass production scale is crucial to complement limitations of seed production in yam industry. *In vitro* rapid multiplication offers the best system to be used in efforts to meet seed yam targets in the dissemination of clean planting material of released root and tuber varieties. This system is not in existence in Ghana, therefore first objective of this paper is to establish appropriate medium for each of the three released yam varieties to enhance *in vitro* rapid multiplication, and document *in vitro* production scheme for the released yam varieties.

Following the evaluation and selection of a crop variety for dissemination, it is vital to have a system that will permit the ability to trace the product. Genomic fingerprinting is one such tools that when used can facilitate variety identification. This study uses SSR Microsatellites to establish the molecular identity of three released yam varieties alongside 21 other yam accessions comprising species *Dioscorea rotundata, D. cayenensis, D. bulbifera, D. alata, D. dumentorum,* and *D. esculenta.* The documentation of this information will enable the researcher to identify their samples at any point in time and also provide genetic relatedness information that is vital for breeding.
