**2. Materials and methods**

#### **2.1.** *In vitro* **manipulations**

#### *2.1.1. Establishment of Mother Plant under screenhouse conditions*

Three yam varieties released by CSIR-CRI namely CRI-Pona, Mankrong Pona and Kukrupa were used in the study. Samples of yam tubers were obtained from the CSIR-CRI yam breeding program, sectioned into minisetts and treated with ash and benlate prior to planting in pots at the Screenhouse. Mini setts sprouted after approximately six weeks and vines older than three weeks were harvested for *in vitro* manipulations.

#### *2.1.2. Preparation of explants*

Yam vines were harvested from plants growing in the screenhouse, labelled appropriately and the cut end was dipped in deionised water and sent to the Tissue Culture laboratory. Vine was thoroughly washed under running tap water and the nodal cuttings and shoot tips were excised (Figures 3 and 4) into autoclaved deionised water in labelled beakers for surface sterilisation.

**Figure 3.** Yam Nodal cutting explant freshly harvested from the field for initiation *in vitro*

**Figure 4.** Yam Shoot tips explant freshly harvested from the field for meristem excision and initiation *in vitro*

Surface sterilization was carried out under sterile conditions in the laminar flow cabinet as follows: Explants were transferred into beakers containing 70% ethanol for five minutes and then surface sterilized with 20% sodium hypochlorite solution (with 6% active chlorine) containing 2-3 drops of tween 20 for 15 minutes. They were washed with sterile distilled water three times after which edges of explants were trimmed. Explants were then further surface sterilized with 10% sodium hypochlorite solution containing 2-3 drops of tween 20 for 10 minutes and the edges of the explant trimmed, rinsed three times in autoclaved distilled water and kept in autoclaved water prior to culturing. After sterilization, meristems (approximately 1x1 mm) were excised from the shoot tips using a dissecting microscope. The meristems and nodal cuttings were then labelled appropriately and cultured on appropriate media and labelled accordingly. The individual cultured explants were code labelled for ease of tracing the material used for initiation. This was vital because once a culture is screened for the presence of a virus and it test negative or positive, the implication is that all clonal materials generated from that particular explant are either cleaned or infected respectively. Infected explants can therefore removed from the mass propagation system.

#### *2.1.3. Nutrient media preparation*

**2. Materials and methods**

*2.1.1. Establishment of Mother Plant under screenhouse conditions*

three weeks were harvested for *in vitro* manipulations.

**Figure 3.** Yam Nodal cutting explant freshly harvested from the field for initiation *in vitro*

**Figure 4.** Yam Shoot tips explant freshly harvested from the field for meristem excision and initiation *in vitro*

Three yam varieties released by CSIR-CRI namely CRI-Pona, Mankrong Pona and Kukrupa were used in the study. Samples of yam tubers were obtained from the CSIR-CRI yam breeding program, sectioned into minisetts and treated with ash and benlate prior to planting in pots at the Screenhouse. Mini setts sprouted after approximately six weeks and vines older than

Yam vines were harvested from plants growing in the screenhouse, labelled appropriately and the cut end was dipped in deionised water and sent to the Tissue Culture laboratory. Vine was thoroughly washed under running tap water and the nodal cuttings and shoot tips were excised (Figures 3 and 4) into autoclaved deionised water in labelled beakers for surface

**2.1.** *In vitro* **manipulations**

38 Biotechnology

*2.1.2. Preparation of explants*

sterilisation.

#### *2.1.3.1. Initiation medium for Yam*

Murashige and Skoog [11] basal salts complimented with growth hormones and vitamins were used. The growth regulators used for meristem cultures were BAP, NAA and GA3. Where nodal cuttings were used, GA3 was not included in the medium. The medium was further supplemented with Adenine Sulphate (AdSO₄) (80 mg/l) as a cytokinin additive and Lcysteine (20 mg/l) as an antioxidant, 30 g sucrose and 7 g agar were used as carbon source and gelling agent respectively. The details of the media composition are as in the table 1 below. The following vitamins Myo- inositol, Nicotinic acid amide, Pyridoxine, Thiamine- HCl and Glycine were used as documented by Murashige and Skoog [11]. The pH of the medium was set at 5.7 ± 1 and sterilised in an autoclave at a temperature of 121°C at 15 psi for 15 minutes. Culture vessels used were pyrex test tubes with dimension 16 X 125mm. Medium to be used for meristem cultures were slated after removal from the autoclave prior to allowing them to cool down. This provided a broad surface for the excised meristem to be placed on the upper part as represented in the Figure 5. Meristem cultures were transferred every eight weeks onto fresh medium till shoots differentiated from the explants after ten months. Shoots differenti‐ ating from nodal buds were excised onto the same medium till actively growing shoots were obtained.

#### *2.1.3.2. Rapid multiplication medium for Yams*

Actively growing shoots (Figure 6) from both meristem and nodal bud cultures were subcul‐ tured onto complete MS medium with vitamins supplemented with 2.5 µM kinetin referred to as yam multiplication medium. During subculture, the shoot tips (Figure 7) and nodal cuttings (Figure 8) were excised from a growing shoot and grown on freshly prepared medium. Kinetin concentration in the rapid multiplication medium was manipulated to optimize yam

**Figure 5.** Growing Yam meristem placed on slanted medium

multiplication medium. The complete MS medium with vitamins was supplemented with kinetin at 0, 2.5, 5, and 10 µM concentration. Another medium that was used was the complete MS medium, supplemented with the following vitamins Myo-inositol, Nicotinic acid amide, Pyridoxine, Thiamine-HCl and Glycine and labelled as "mm". The pH of all the media was set at 5.7±1 using 0.1 M NaOH for adjusting it from a lower pH. The media were sterilised at a temperature of 121°C at 15 psi for 15 minutes in an autoclave. The various media used in these experiments are presented in the Table 1.

**Figure 6.** Actively growing yam cultures

#### *2.1.4. Incubation*

All cultures were incubated at a temperature of 26 ± 2°C with a photoperiod of 16 hours of light and 8 hours of darkness.


\*Murashige and Skoog basal salts and vitamins [11]

**Table 1.** Medium composition for yam (1 litre)

multiplication medium. The complete MS medium with vitamins was supplemented with kinetin at 0, 2.5, 5, and 10 µM concentration. Another medium that was used was the complete MS medium, supplemented with the following vitamins Myo-inositol, Nicotinic acid amide, Pyridoxine, Thiamine-HCl and Glycine and labelled as "mm". The pH of all the media was set at 5.7±1 using 0.1 M NaOH for adjusting it from a lower pH. The media were sterilised at a temperature of 121°C at 15 psi for 15 minutes in an autoclave. The various media used in

All cultures were incubated at a temperature of 26 ± 2°C with a photoperiod of 16 hours of

these experiments are presented in the Table 1.

**Figure 5.** Growing Yam meristem placed on slanted medium

40 Biotechnology

**Figure 6.** Actively growing yam cultures

light and 8 hours of darkness.

*2.1.4. Incubation*

#### *2.1.5. Biomass determination*

Dry matter was also estimated as a measure of growth using 5 plantlets per treatment. Fresh weight was estimated by weighing the plantlets while dry weight was estimated by drying the plantlets in an oven at 80o C for 48 hours. Each treatment was replicated thrice.

**Figure 8.** Yam Shoot tips excised from actively Growing cultures for rapid multiplication

#### *2.1.6. Data collection and analysis*

Data was taken after 8 weeks of initiation and subsequent data were taken every 8 weeks by counting the number of leaves, shoots, multiple buds as well as root development and the general performance of the cultures was also noted. Statistical package used to analyse data was SAS 9.1.

#### **2.2. Fingerprinting**

#### *2.2.1. Plant materials*

Sampling of leaves for DNA isolation towards genomic studies was made from the field germplasm holding of the CSIR – Plant Genetic Resources Research Institute, located at Bunso in the Eastern Region of Ghana. For this study, a total of 21 samples were selected at random from six different species of yam grown in Ghana namely *Dioscorea alata, D. dumenterom, D. rotundata, D. cayenensis, D. bulbifera* and *D. esculenta* (Table 2). There were at least three different samples of each species and these were screened alongside the three released yam varieties.

#### *2.2.2. Extraction of genomic DNA*

Genomic DNA was isolated from 100 mg of young tender leaves. They were weighed into 2 ml eppendorf tubes and grounded with liquid nitrogen into fine powder. The genomic DNA was extracted using the following manufacture's instructions of the Qiagen protocol from the DNeasy plant mini kit.

#### *2.2.3. DNA quantification and gel electrophoresis*

The quality of DNA was checked on 0.8% agarose in 1x TAE (Tris-acetic EDTA) buffer by gel electrophoresis with Ethidium bromide (0.5 ug/ml). Electrophoresis of the DNA was carried out at 120 V for 40mins and then visualized with a UV transilluminator. The quality of DNA was ascertained and the concentration was projected by the intensity and compar‐ ison to 1 kb lambda DNA mass ladder (1 kb invitrogen). Quantification of DNA was Molecular Fingerprinting and Selection of Appropriate Media for Rapid *In Vitro* Multiplication of Three Yam Varieties http://dx.doi.org/10.5772/60411 43


**Table 2.** List of accessions used for the study

evaluated by reading absorbance at 260 nm and 280 nm with the spectrophotometer. The DNA was diluted to 10 ng/µl for PCR amplifications.

#### *2.2.4. PCR amplification*

*2.1.6. Data collection and analysis*

*2.2.2. Extraction of genomic DNA*

from the DNeasy plant mini kit.

*2.2.3. DNA quantification and gel electrophoresis*

was SAS 9.1.

42 Biotechnology

**2.2. Fingerprinting**

*2.2.1. Plant materials*

Data was taken after 8 weeks of initiation and subsequent data were taken every 8 weeks by counting the number of leaves, shoots, multiple buds as well as root development and the general performance of the cultures was also noted. Statistical package used to analyse data

**Figure 8.** Yam Shoot tips excised from actively Growing cultures for rapid multiplication

Sampling of leaves for DNA isolation towards genomic studies was made from the field germplasm holding of the CSIR – Plant Genetic Resources Research Institute, located at Bunso in the Eastern Region of Ghana. For this study, a total of 21 samples were selected at random from six different species of yam grown in Ghana namely *Dioscorea alata, D. dumenterom, D. rotundata, D. cayenensis, D. bulbifera* and *D. esculenta* (Table 2). There were at least three different samples of each species and these were screened alongside the three released yam varieties.

Genomic DNA was isolated from 100 mg of young tender leaves. They were weighed into 2 ml eppendorf tubes and grounded with liquid nitrogen into fine powder. The genomic DNA was extracted using the following manufacture's instructions of the Qiagen protocol

The quality of DNA was checked on 0.8% agarose in 1x TAE (Tris-acetic EDTA) buffer by gel electrophoresis with Ethidium bromide (0.5 ug/ml). Electrophoresis of the DNA was carried out at 120 V for 40mins and then visualized with a UV transilluminator. The quality of DNA was ascertained and the concentration was projected by the intensity and compar‐ ison to 1 kb lambda DNA mass ladder (1 kb invitrogen). Quantification of DNA was

A set of 16 set of primer pairs [19] were used in the experiment (Table 3). DNA amplification was carried out with a 96 well plate Bio-RadTM Thermocycler from BIO-RAD. The PCR conditions were optimized for cycling number, concentrations of the primer, MgCl2 and DNA template. The reaction mixture (10 ul) contained 6.075 ul of Nuclease free sterile water (DNA grade water), 1 ul of 10x PCR Buffer, 0.9 ul of MgCl2 (25 mM), 0.4 ul dNTPs (10 mM), 0.25 ul primer (50 ug/ml) of each forward and reverse, 0.125 ul of SuperthermTaq Polymerase (1unit) and 1 ul of 10 ng DNA template. The cycling conditions were as follows: an initial denaturing step of 94o C for 5 mins, 35 cycles of 94o C for 30 secs, 51o C for 1min, 72o C for 1 min and a final elongation step of 72o C for 1 min. In every experiment, a negative control was included where the template DNA was replaced with PCR grade water. Amplification products were exam‐ ined on a 6% polyacrylamide gel (Water, 10x TBE, 4% acryl amide (19:1), 10% APS and TEMED) and stained with silver nitrate. A 100 bp ladder (Gene Ruler TM, Fermentas) was used as a size marker.


Molecular Fingerprinting and Selection of Appropriate Media for Rapid *In Vitro* Multiplication of Three Yam Varieties http://dx.doi.org/10.5772/60411 45


**Table 3.** List of primers, their sequences and melting temperatures

#### *2.2.5. Silver staining*

grade water), 1 ul of 10x PCR Buffer, 0.9 ul of MgCl2 (25 mM), 0.4 ul dNTPs (10 mM), 0.25 ul primer (50 ug/ml) of each forward and reverse, 0.125 ul of SuperthermTaq Polymerase (1unit) and 1 ul of 10 ng DNA template. The cycling conditions were as follows: an initial denaturing

C for 30 secs, 51o

the template DNA was replaced with PCR grade water. Amplification products were exam‐ ined on a 6% polyacrylamide gel (Water, 10x TBE, 4% acryl amide (19:1), 10% APS and TEMED) and stained with silver nitrate. A 100 bp ladder (Gene Ruler TM, Fermentas) was used as a size

**Primers Sequence Tm/oC**

C for 1min, 72o

C for 1 min. In every experiment, a negative control was included where

CTATAAGGAATTGGTGCC 54.4

AATGCTTCGTAATCCAAC 54.9

GATGCTATGAACACAACTAA 52.5

TTTGACAGTGAGAATGGA 54.6

AATCGGCTACACTCATCT 54.4

GCCTTTGTGCGTATCT 54.2

TATAATCGGCCAGAGG 54.1

TGTTGGAAGCATAGAGAA 53.9

ACGCACATAGGGATTG 54.9

TCAAAGGAATGTTGGG 54.8

TCAAGCAAGAGAAGGTG 54.4

TCCCCATAGAAACAAAGT 54.2

TTGAACCTTGACTTTGGT 55.3

GAGTTCCTGTCCTTGGT 54.5

GCCTTGTTACTTTATTC 46.2

AGACTCTTGCTCATGT 46.7

ACCCATCGTCTTACCC 55.3

ATAGGAAGGCAATCAGG 54.8

CCATCACACAATCCATC 54.9

CATCAATCTTTCTGCTT 54.3

GATTTGCTTTGAGTCCTT 54.1

ACAAGAGAACCGACATAGT 53.4

AACATATAAAGAGAGATCA 45.3

ATAACCCTTAACTCCA 46.3

C for 1 min and a final

step of 94o

44 Biotechnology

marker.

elongation step of 72o

Da1F08R 51

Da1F08F 51

Da1D08F 51

Da1D08R 51

Da1C12R 51

Da1c12F 51

Da1A01F 51

Da1A01R 51

Dpr3F10R 51

Dpr3F10F 51

Dpr3F12R 51

Dpr3F12F 51

Dab2E07F 51

Dab2E07R 51

Dpr3F04R 51

Dpr3F04F 51

Dpr3D06R 51

Dpr3D06F 51

Dpr3B12R 51

Dpr3B12F 51

Dab2D08R 51

Dab2D08F 51

Dab2E09F 51

Dab2E09R 51

C for 5 mins, 35 cycles of 94o

The gels were placed on a shaker with a minimal shaking to allow solution flow over gel swiftly. Fixation was done with 10% Glacial Acetic Acid (100 ml acetic acid, 900 ml water) for 15 mins. This was washed off with distilled water and 1.5% Nitric acid (15 ml Nitric acid, 985ml water) solution was added for 5 mins. The silver stain solution (1.0 g Silver nitrate, 1.5 ml 37% formaldehyde, topped it up with water up to 1000 ml) was preceded after washing off the nitric acid solution for 20mins. Finally, the developer (30 g Sodium carbonate, 1.5 ml 37% formaldehyde and 0.25 ml Sodium thiosulphate 10 mg/ml, water up to 1000 ml) was added and allowed to develop the photographic stains/ bands for visualization. This was stopped with 10% acetic acid and then stored in distilled water for photographic capturing and scoring.

#### *2.2.6. Gel scoring and data analysis*

Bands were scored manually as present (1) or absent (0) from the gels. Similarity matrix was calculated using NYSTS software while cluster analyses were also carried out using Genstat and dendrograms were constructed. Similarity matrices from each primer were compared pair wise using a randomization test. POPGENE32 [22] was used for genetic population analysis as well as to test the effectiveness of loci used.

## **3. Results and discussion**

#### **3.1.** *In vitro* **manipulations**

#### *3.1.1. Explant response on initiation medium*

The growth and development of different crop species vary considerably. The data obtained following *In vitro* initiation of nodal cuttings and meristems are presented in Tables 4 and

5 respectively. The nodal cuttings for Kukrupa and Mankrong-Pona when grown in yam initiation medium, had higher success rate than CRI-Pona (Table 4). The success rate was higher in the nodal cuttings (52.9 – 86.6%) than in the apical meristems (46.4 – 53.33%). In both situations, Mankrong-Pona had a higher success rate than CRI-Pona. The variety Kukrupa was however not included in the apical meristem experiment. The measure of percentage success was based on explants that developed to the extent of producing shoots. Well-developed shoots were obtained in all the successful nodal cutting explants after eight weeks in culture although by two weeks, some explants had already started producing shoots (Figure 9). Meristem development was however very slow since it took six months for shoot differentiation to occur and following that, by ten months, multiple shoots had started developing (Figure 10 a&b). Following culture initiation, successful meristems initially expanded due to cell division, turned dark with green clusters of cells, which later differentiated into buds and then further into shoots. Shoot differentiating from most of the Makrong-Pona meristem cultures had more than ten leaves and up to eight shoots per culture however, on the average, there were 4.43 shoots per culture, whereas CRI-Pona had 1.25 shoots per culture (Table 5). Shoots differentiated from nodal bud explants 12 days after culture (Table 4). The extent of success on meristem cultures was expected to be low, mainly due to the minute size of the explant used in initiation. Culture development is slow as it takes up to ten months for shoot to develop. However multiple shoots develop from the meristems and this facilitates rapid *in vitro* development. Hence if only a few meristems are successful *in vitro*, mass propagation is achieved. Development of shoots from nodal cutting explants is notably high and reliable. However, this method cannot be reliable if pathogens especially viruses have to be eliminated from the crop variety.


**Table 4.** Yam Nodal Cutting initiation Success Rate of the three released varieties


**Table 5.** Yam Meristem initiation success Rate of the three released varieties

http://dx.doi.org/10.5772/60411 47

**Figure 9.** Yam nodal cultures sprouting *in vitro* after two weeks in culture

5 respectively. The nodal cuttings for Kukrupa and Mankrong-Pona when grown in yam initiation medium, had higher success rate than CRI-Pona (Table 4). The success rate was higher in the nodal cuttings (52.9 – 86.6%) than in the apical meristems (46.4 – 53.33%). In both situations, Mankrong-Pona had a higher success rate than CRI-Pona. The variety Kukrupa was however not included in the apical meristem experiment. The measure of percentage success was based on explants that developed to the extent of producing shoots. Well-developed shoots were obtained in all the successful nodal cutting explants after eight weeks in culture although by two weeks, some explants had already started producing shoots (Figure 9). Meristem development was however very slow since it took six months for shoot differentiation to occur and following that, by ten months, multiple shoots had started developing (Figure 10 a&b). Following culture initiation, successful meristems initially expanded due to cell division, turned dark with green clusters of cells, which later differentiated into buds and then further into shoots. Shoot differentiating from most of the Makrong-Pona meristem cultures had more than ten leaves and up to eight shoots per culture however, on the average, there were 4.43 shoots per culture, whereas CRI-Pona had 1.25 shoots per culture (Table 5). Shoots differentiated from nodal bud explants 12 days after culture (Table 4). The extent of success on meristem cultures was expected to be low, mainly due to the minute size of the explant used in initiation. Culture development is slow as it takes up to ten months for shoot to develop. However multiple shoots develop from the meristems and this facilitates rapid *in vitro* development. Hence if only a few meristems are successful *in vitro*, mass propagation is achieved. Development of shoots from nodal cutting explants is notably high and reliable. However, this method cannot be reliable if pathogens especially viruses have to be eliminated from the crop variety.

**Variety Total number of explants**

**Variety**

46 Biotechnology

**initiated**

**Table 4.** Yam Nodal Cutting initiation Success Rate of the three released varieties

**Introduction number**

**Table 5.** Yam Meristem initiation success Rate of the three released varieties

Cri-Pona 51 27 52.9 1.3 Kukrupa 69 54 78.3 2.1 Mankrong-Pona 45 39 86.7 1.5

**Number successful % Success**

Mankrong Pona 90 48 53.3 7.4 4.4 Cri-Pona 84 39 46.4 3.3 1.3

**Number successful % Success Average Shoot**

**Mean No. of leaves**

**formation**

**Mean No. of shoots**

**Plate 10:** Four months old yam meristem (A) differentiating into multiple shoots after 10 months in culture Yam meristem explant forming multiple shoots after ten months (B) in culture **Figure 10.** Four months old yam meristem (A) differentiating into multiple shoots after 10 months in culture Yam mer‐ istem explant forming multiple shoots after ten months (B) in culture.

#### **3.1.2. Selection of appropriate Rapid Multiplication medium**  *3.1.2. Selection of appropriate rapid multiplication medium*

be used for rapid multiplication on yam.

will occur [2] and this will not favour mass propagation.

varieties.

growth additives were used in this study. The responses of the three released varieties on different media are presented in Figures 1 and 2 below. The performance of the three released varieties varied on the four different media. Considering shoot development, the mean was highest on medium containing 10 µM kinetin for CRI-Pona Media supplemented with kinetin at different concentrations as well as MS medium enriched

Media supplemented with kinetin at different concentrations as well as MS medium enriched with vitamins and

at 4±1.39, 8.6±1.08 for CRI-Kukrupa on 5 µM kinetin and 8.29±0.7 for Mankrong-Pona on 2.5 µM kinetin. With the exception of CRI-Kukrupa where highest number of leaves (9.33±1.8) occurred on the control medium (no kinetin), CRI-Pona had 4.67±1.8 leaves on the same medium as medium with highest number of shoots (10 µM kinetin), and Mankrong-Pona also had 13±1.18 leaves on medium containing 2.5 µM kinetin. Notably this attempt is to maximise in vitro rapid multiplication. In tissue culture, nodal cutting are used to generate shoots, and the number of leaves generating is the determining factor for the multiplication rates that can be attained, since within each leaf axil is a bud that can develop into a whole shoot. Hence the higher the number of leaves the higher the multiplication rate. Shoots differentia form buds and within each bud are clusters of meristematic cells which are capable of differentiating in shoots when the growth conditions are appropriate. Reports in previous research where BAP was the cytokinin used had a maximum of four shoots developing on the average [2]. Later efforts on generating somatic embryos reported 7-9 shoots developing per culture where kinetin was used [16]. This present study reported 3 to 8 shoots per culture, it is therefore indicative that Kinetin is an appropriate growth regulator to

 Comparing the effect of different media on rapid multiplication of yam nodal culture in terms of shoots and leave (Table 6), medium supplemented with 2.5 uM Kinetin was the best although the difference was not significant. In tissue culture since a lot of clonal materials can be generated from one culture differences in terms of number are significant although statistically it may not be significant. The performance of cultures on the medium labelled "mm" which was not supplemented with Kinetin was very poor and significantly low numbers of leaves and shoots were recorded. This confirms that to achieve rapid multiplication of yam in vitro, the inclusion of kinetin is critical. Although the data obtained indicated that to maximize rapid multiplication *in vitro*, different media have to be used for the different varieties released, medium supplemented with 2.5 M kinetin may be appropriate for all the

Comparing effect of the yam different varieties nodal cultures during rapid multiplication (Table 7) in terms of shoots response of Mankrong Pona and CRI-Kukrupa were similar. Thevariety CRI-Pona had significantly low number of shoots. In terms of leaf development, CRI-Kukrupa had a significantly higher (8.3) number of leaves whereas CRI-Pona had the lowest (3.7). This response is due to varietal differences and Mankrong Pona will be recommended over CRI-Pona where high number are needed to be produced within a limited time. There was significantly very high positive correlation between leave and shoot development during rapid multiplication (Table 9). This indicates that the concentration of Kinetin used promote both leaf and shoot development as high concentrations of some cytokinin (BAP) can inhibit well developed leaf development although shoot development

10

released varieties on different media are presented in Figures 11 and 12 below. The perform‐ ance of the three released varieties varied on the four different media. Considering shoot development, the mean was highest on medium containing 10 µM kinetin for CRI-Pona at 4±1.39, 8.6±1.08 for CRI-Kukrupa on 5 µM kinetin and 8.29±0.7 for Mankrong-Pona on 2.5 µM kinetin. With the exception of CRI-Kukrupa where highest number of leaves (9.33±1.8) occurred on the control medium (no kinetin), CRI-Pona had 4.67±1.8 leaves on the same medium as medium with highest number of shoots (10 µM kinetin), and Mankrong-Pona also had 13±1.18 leaves on medium containing 2.5 µM kinetin. Notably this attempt is to maximise *in vitro* rapid multiplication. In tissue culture, nodal cutting are used to generate shoots, and the number of leaves generating is the determining factor for the multiplication rates that can be attained, since within each leaf axil is a bud that can develop into a whole shoot. Hence the higher the number of leaves the higher the multiplication rate. Shoots differentiate from buds and within each bud are clusters of meristematic cells which are capable of differentiating in shoots when the growth conditions are appropriate. Reports in previous research where BAP was the cytokinin used had a maximum of four shoots developing on the average [2]. Later efforts on generating somatic embryos reported 7-9 shoots developing per culture where kinetin was used [16]. This present study reported 3 to 8 shoots per culture, it is therefore indicative that Kinetin is an appropriate growth regulator to be used for rapid multiplication on yam.

Comparing the effect of different media on rapid multiplication of yam nodal culture in terms of shoots and leave (Table 6), medium supplemented with 2.5 uM Kinetin was the best although the difference was not significant. In tissue culture since a lot of clonal materials can be generated from one culture differences in terms of number are significant although statistically it may not be significant. The performance of cultures on the medium labelled "mm" which was not supplemented with Kinetin was very poor and significantly low numbers of leaves and shoots were recorded. This confirms that to achieve rapid multiplication of yam *in vitro*, the inclusion of kinetin is critical. Although the data obtained indicated that to maximize rapid multiplication *in vitro*, different media have to be used for the different varieties released, medium supplemented with 2.5 µM kinetin may be appropriate for all the varieties.

Comparing effect of the yam different varieties, nodal cultures during rapid multiplication (Table 7) in terms of shoots response of Mankrong Pona and CRI-Kukrupa were similar. The variety CRI-Pona had significantly low number of shoots. In terms of leaf development, CRI-Kukrupa had a significantly higher (8.3) number of leaves whereas CRI-Pona had the lowest (3.7). This response was due to varietal differences and Mankrong Pona will be recommended over CRI-Pona where high numbers are needed to be produced within a limited time. There was significantly very high positive correlation between leave and shoot development during rapid multiplication (Table 9). This indicates that the concentration of Kinetin used promote both leaf and shoot development as high concentrations of some cytokinin (BAP) can inhibit leaf development will occur [2] and this will not favour mass propagation.

Molecular Fingerprinting and Selection of Appropriate Media for Rapid *In Vitro* Multiplication of Three Yam Varieties http://dx.doi.org/10.5772/60411 49

**Figure 11.** Development of Leaves in cultures of the three released varieties during rapid multiplication

released varieties on different media are presented in Figures 11 and 12 below. The perform‐ ance of the three released varieties varied on the four different media. Considering shoot development, the mean was highest on medium containing 10 µM kinetin for CRI-Pona at 4±1.39, 8.6±1.08 for CRI-Kukrupa on 5 µM kinetin and 8.29±0.7 for Mankrong-Pona on 2.5 µM kinetin. With the exception of CRI-Kukrupa where highest number of leaves (9.33±1.8) occurred on the control medium (no kinetin), CRI-Pona had 4.67±1.8 leaves on the same medium as medium with highest number of shoots (10 µM kinetin), and Mankrong-Pona also had 13±1.18 leaves on medium containing 2.5 µM kinetin. Notably this attempt is to maximise *in vitro* rapid multiplication. In tissue culture, nodal cutting are used to generate shoots, and the number of leaves generating is the determining factor for the multiplication rates that can be attained, since within each leaf axil is a bud that can develop into a whole shoot. Hence the higher the number of leaves the higher the multiplication rate. Shoots differentiate from buds and within each bud are clusters of meristematic cells which are capable of differentiating in shoots when the growth conditions are appropriate. Reports in previous research where BAP was the cytokinin used had a maximum of four shoots developing on the average [2]. Later efforts on generating somatic embryos reported 7-9 shoots developing per culture where kinetin was used [16]. This present study reported 3 to 8 shoots per culture, it is therefore indicative that Kinetin is an appropriate growth regulator to be used for rapid multiplication

Comparing the effect of different media on rapid multiplication of yam nodal culture in terms of shoots and leave (Table 6), medium supplemented with 2.5 uM Kinetin was the best although the difference was not significant. In tissue culture since a lot of clonal materials can be generated from one culture differences in terms of number are significant although statistically it may not be significant. The performance of cultures on the medium labelled "mm" which was not supplemented with Kinetin was very poor and significantly low numbers of leaves and shoots were recorded. This confirms that to achieve rapid multiplication of yam *in vitro*, the inclusion of kinetin is critical. Although the data obtained indicated that to maximize rapid multiplication *in vitro*, different media have to be used for the different varieties released, medium supplemented with 2.5 µM kinetin may be appropriate for all the

Comparing effect of the yam different varieties, nodal cultures during rapid multiplication (Table 7) in terms of shoots response of Mankrong Pona and CRI-Kukrupa were similar. The variety CRI-Pona had significantly low number of shoots. In terms of leaf development, CRI-Kukrupa had a significantly higher (8.3) number of leaves whereas CRI-Pona had the lowest (3.7). This response was due to varietal differences and Mankrong Pona will be recommended over CRI-Pona where high numbers are needed to be produced within a limited time. There was significantly very high positive correlation between leave and shoot development during rapid multiplication (Table 9). This indicates that the concentration of Kinetin used promote both leaf and shoot development as high concentrations of some cytokinin (BAP) can inhibit

leaf development will occur [2] and this will not favour mass propagation.

on yam.

48 Biotechnology

varieties.

**Figure 12.** Development of Shoots in cultures of the three released varieties during rapid multiplication

The study considered biomass as an additional measure of growth. The data, as shown in Figure 13 below revealed that plant biomass was consistently low and on the medium labelled "mm". The variety Kukrupa had the highest biomass on the control medium. Considering Mankrong Pona high biomass was on the control medium as well as 2.5 and 5 µM Kinetin supplemented medium. Significantly high biomass was recorded for CRI-Pona cultures growing on 10 µM Kinetin supplemented medium, it is the same medium on which highest number of leaves and shoots were observed for that variety. It is therefore possible that CRI-Pona accumulated biomass at the expense of sacrificing organ differentiation.


Similar letters are not significant according to Tukey test (p<0.05); Stder – Standard error

**Table 6.** Comparison of effect of different media on nodal culture of Yam (Number of Shoots and Number of Leaves)


Similar letters are not significant according to Tukey test (p<0.05); Stder – Standard error

**Table 7.** Comparison of effect of different varieties on nodal culture of Yam (Number of Shoots and Number of Leaves)


\*\*. Correlation is significant at the 0.01 level (2-tailed).

**Table 8.** Correlations

Molecular Fingerprinting and Selection of Appropriate Media for Rapid *In Vitro* Multiplication of Three Yam Varieties http://dx.doi.org/10.5772/60411 51

**Figure 13.** Dry weight and moisture content among the different treatments of the three released varieties

#### **3.2. Fingerprinting results and discussion**

**Media Mean(±Stder) Shoots Leaves 0** Mean 6.000ab 7.857a

**2.5** Mean 6.421a 8.895a

**5** Mean 6.000ab 7.211a

**10** Mean 5.809ab 7.333a

**mm** Mean 3.952b 2.476b

**Media Mean(±Stder) Shoots Leaves CRI Pona** Mean 3.389b 3.778c

**Kukrupa** Mean 6.000a 5.875b

**Mankrong** Mean 6.182a 8.318a

**Table 7.** Comparison of effect of different varieties on nodal culture of Yam (Number of Shoots and Number of

Sig. (2-tailed) .000

Similar letters are not significant according to Tukey test (p<0.05); Stder – Standard error

Leaves)

50 Biotechnology

**Leaves**

**Shoot**

**Table 8.** Correlations

\*\*. Correlation is significant at the 0.01 level (2-tailed).

Similar letters are not significant according to Tukey test (p<0.05); Stder – Standard error

Stder 0.839 0.988

Stder 0.788 0.1.149

Stder 0.658 0.801

Stder 0.519 0.773

Stder 0.355 0.423

Stder 0.617 0.919

Stder 0.462 0.689

Stder 0.394 0.588

Pearson Correlation 1 .816\*\* Sig. (2-tailed) .000 N 94 94

Pearson Correlation .816\*\* 1

N 94 94

**Leaves Shoot**

**Table 6.** Comparison of effect of different media on nodal culture of Yam (Number of Shoots and Number of Leaves)

Microsatellite in DNA represents repetitive DNA based on very shortrepeats such as dinucleo‐ tides,trinucleotidesortetranucleotides,consistingofrepeatsofamotif,andisotherwisereferred toasSimpleSequenceRepeats (SSR).These repeats serveasmolecularmarkersbywhichgenetic identity can be documented. Although there are several molecular marker systems namely: Amplified fragment length polymorphism (AFLP), DNA amplification fingerprinting (DAF), Inter-simple sequence repeat (ISSR), and Random amplified polymorphic DNA (RAPD) just to nameafew,microsatellitesorSSRs (SimpleSequenceRepeats)hasbeenreportedasbeinguseful for genotyping due to its high polymorphic information content (PIC). It is a codominantly inherited marker with locus specificity and genomic coverage is extensive. Also, systems provide simple PCR amplification detection methods. This present study used a set of yam simple sequence repeat (SSR) markers developed in different species of yam (*Dioscorea* sp.), where microsatellite-enriched bank was created from *Dioscorea alata*, *Dioscorea abyssinica* and *Dioscorea praehensilis*. That study identified and characterized 16 polymorphic loci, which were found to be transferable to species of other *Dioscorea* sections [19].

In this present study, when the 16 SSR primers were screened, 15 produced scorable bands. These 15 primers were used to screen a total of 25 yam accessions (including the three released varieties). Following amplification of PCR products, there were a total of 94 alleles and an average of 6.26 alleles per loci. Similarly when used to screen 22 *D. rotundata* accessions from Benin, 117 alleles were observed with and average of 7.3 alleles per loci [19]. The data obtained in this study was subjected to NTSYS analysis and this revealed the three released varieties clustering into one group (Figure 14). One *D. rotundata* accession from the CSIR-PGRRI collections was grouped with the released varieties. A similarity matrix (Table 9) established the percentage similarity between Mankrong-Pona and CRI-Pona to be 97%. The data obtained indicated that the released yam varieties are distinct from other *D. rotundata* accessions being conserved at the CSIR-PGRRI. It is argued that similarity above 95% is indicative that the two samples are duplicates. This has to be investigated further since *in vitro* growth rates of the two released varieties (Mankrong Pona and CRI Pona) are distinctively different in this current study. It is possible that the set of loci used to conduct molecular diversity assessment are unable to detect much differences in the released *Dioscorea* varieties as the SSR libraries used to design the primers were generated from *Dioscorea alata*, *Dioscorea abyssinica* and *Dioscorea praehensilis* and not *D. rotundata*. The investigator who developed the primers however demonstrated that they are transferable to *D. rotundata*, although only 3 accessions of rotundata were used in that particular study [19].

**Figure 14.** Dendrogram of 25 accessions of yam based on unweighted neighbour joining cluster analysis


**Table 9.** Jaccard's coefficient similarity matrix of 25 yam genotypes using 15 SSR primers

Data analysis using population genetic analysis software PopGen32 [22], revealed 100% overall polymorphic loci overall the populations. According to the genetic variation statistics at all loci [12] presented in Table 10, the mean number of effective alleles (ne) was 4.05±1.43, loci DalA01 and Dab2C05 had the highest value at 6.82±1.2, however, locus Dab2E07 had the lowest number at 1.73±0.7. The mean observed number of alleles was 5.33±1.45. Again, loci DalA01 and Dab2C05 recorded the highest number of observed alleles (8), however, locus Dab2E07 had the lowest number of alleles (3). With an average allele sample size (n) of 38, locus Dpr3F04 had a high sample size of 42 whereas a value of 26 was recorded in loci Dpr3F10 and Dab2E07. The mean, highest (loci-DalA01) and lowest (locus Dab2E07) Shannon index (I) were 1.46±0.37, 1.99, and 0.74 overall respectively, indicating that locus DalA01 estimated the highest level of genetic diversity among the samples used in this study. The overall allele frequency (Table 11) revealed that Allele A of locus Dpr3F04 was the least frequent allele (0.022) among the samples as this allele was unique to only one sample in the *D. alata* population. However allele A in locus Dab2E07 was the most frequent allele (0.73) as this allele was present in *D. rotundata, alata, cayenensis* and the released varieties, however it was absent in *D. alata* sample, *D. bulbifera*, *D. dumentorum* and *D. esculenta*. The Nei's original measures of genetic identity and genetic distance [13] as shown in Table 12 revealed that the released varieties population are closer to *D. cayenensis* accessions (0.57) than *D. rotundata* population (0.89) used in this study. However they were very distantly related to *D. bulbifera* (2.39). had a high sample size of 42 whereas a value of 26 was recorded in loci Dpr3F10 and Dab2E07. The mean highest (loci-DalA01) and lowest (locus Dab2E07) Shannon index (I) were 1.46±0.37, 1.99, and 0.74 overall respectively, indicating that locus DalA01 estimated the highest level of genetic diversity among the samples used in this study. The overall allele frequency (Table 11) revealed that Allele A of locus Dpr3F04 was the least frequent allele (0.022) among the samples as this allele was unique to only one sample in the *D. alata* population. However allele A in locus Dab2E07 was the most frequent allele (0.73) as this allele was present in *D. rotundata, alata, cayenensis* and the released varieties, however it was absent in *D. alata* sample, *D. bulbifera*, *D. dumentorum* and *D. esculenta*. The Nei's original measures of genetic identity and genetic distance genetic [13] as shown in Table 12 revealed that the released varieties population are closer to *D. cayenensis* accessions (0.57) than *D. rotundata* population (0.89) used in this study. However they were very distantly related to *D. bulbifera* (2.39).


=================================================================================

=================================================================================

=========================================================================== Allele \ Locus Dab2D06 Dab2D08 Dpr3F04 Dpr3F10 Dpr3F12 Dab2E07 Dab2E09 =========================================================================== Allele A 0.0625 0.0588 0.0217 0.0385 0.1667 0.7308 0.1875 Allele B 0.1875 0.0588 0.1957 0.0769 0.1429 0.1923 0.5000 Allele C 0.2188 0.5294 0.3478 0.3077 0.5000 0.0769 0.1250

Allele A 0.1818 0.0500 0.2857 0.2955 0.1429 0.0455 0.3409 0.4333 Allele B 0.2273 0.3750 0.1190 0.1667 0.1136 0.3864 0.3000 Allele C 0.1136 0.2000 0.1905 0.2619 0.1591 0.0909 0.1333 Allele D 0.0909 0.1000 0.2857 0.2955 0.3571 0.1364 0.1591 0.1333

Allele F 0.1136 0.0500 0.1136 0.2273 0.0227

Allele E 0.1136 0.1190 0.2955 0.0714 0.1136

Allele H 0.1136 0.1364 Allele I 0.0682

Allele D 0.2500 0.1765 0.3261 0.0769 0.1667

\* na = Observed number of alleles

Allele G 0.0455 0.2250

14

\* ne = Effective number of alleles \* na = Observed number of alleles

to design the primers were generated from *Dioscorea alata*, *Dioscorea abyssinica* and *Dioscorea praehensilis* and not *D. rotundata*. The investigator who developed the primers however demonstrated that they are transferable to *D. rotundata*, although only 3 accessions of rotundata

0.95 0.90 0.85 0.80

*Dr 013 Dr 026 Dr 089 Dr 052 Da 110 Da 130 Da 137 Da 143 Dd 187 Dd 183 Dd 186 Db 187 Db 193 Db 201 De 202 De 208 De 215 Dc 109 Dc 221 Dc 249 Dc 251 Cri- pona Mankrong Kukrapa*

Data analysis using population genetic analysis software PopGen32 [22], revealed 100% overall polymorphic loci overall the populations. According to the genetic variation statistics at all

**Figure 14.** Dendrogram of 25 accessions of yam based on unweighted neighbour joining cluster analysis

**Table 9.** Jaccard's coefficient similarity matrix of 25 yam genotypes using 15 SSR primers

0.75 0.65

0.70

were used in that particular study [19].

SCJ/89/001

TA/97/057 TA/97/071

UWR/97/085

C 82/129

UWR/97/059

KT/01/015

SO/89/093

UWR/97/101

Kurapa

Cri- pona

Mankrong

AGA/97/211

BD/96/023 AGA/97/202 82/430

BD/96/026

TA/97/141 TA/97/093

1.00

AGA/97/173

Dr 013 1 Dr 026 0.10 1.00 Dr 089 0.66 0.21 1.00 Dr 052 0.40 0.42 0.69 1.00 Da 110 0.24 0.16 0.27 0.46 1.00 Da 130 0.16 0.14 0.23 0.47 0.85 1.00 Da 137 0.16 0.20 0.28 0.47 0.85 0.85 1.00 Da 143 0.23 0.27 0.25 0.44 0.72 0.58 0.68 1.00 Dd 187 0.02 0.21 -0.13 0.04 -0.01 -0.03 0.07 0.24 1.00 Dd 183 -0.01 0.24 -0.10 0.08 0.02 0.00 0.11 0.23 0.89 1.00 Dd 186 0.02 0.27 -0.07 0.12 0.00 -0.02 0.09 0.16 0.89 0.94 1.00 Db 187 -0.02 -0.02 -0.02 0.04 0.15 0.12 0.22 0.14 0.28 0.37 0.36 1.00 Db 193 -0.11 -0.01 -0.09 -0.10 0.02 -0.02 0.03 -0.09 -0.05 0.04 0.04 0.37 1.00 Db 201 -0.11 -0.01 0.00 -0.06 -0.03 -0.07 -0.07 -0.14 -0.15 -0.06 -0.07 0.23 0.78 1.00 De 202 0.08 -0.08 0.10 0.12 0.17 0.20 0.20 0.05 -0.07 0.02 -0.02 0.24 0.20 0.20 1.00 De 208 0.05 -0.15 0.14 0.04 -0.07 -0.09 -0.09 -0.16 -0.04 -0.03 -0.01 0.09 -0.01 0.06 0.33 1.00 De 215 0.23 0.03 0.23 0.13 -0.03 0.00 0.00 -0.10 -0.09 -0.07 -0.04 0.04 0.04 -0.06 0.20 0.38 1.00 Dc 109 -0.01 0.33 0.20 0.24 0.17 0.18 0.23 0.06 -0.05 -0.01 0.03 0.05 0.07 -0.02 0.08 0.16 0.56 1.00 Dc 221 0.02 0.36 0.22 0.25 0.14 0.15 0.20 0.03 -0.07 -0.03 0.01 0.02 0.07 -0.02 0.06 0.15 0.63 0.96 1.00 Dc 249 0.04 0.33 0.20 0.19 0.12 0.13 0.18 0.01 -0.10 -0.06 -0.02 0.00 0.07 -0.07 0.03 0.09 0.56 0.91 0.96 1.00 Dc 251 0.03 0.10 0.16 0.31 0.08 0.07 0.07 -0.01 0.11 0.13 0.15 0.27 0.17 0.09 0.15 0.43 0.32 0.28 0.27 0.12 1.00 Cri- pona 0.02 0.34 0.19 0.20 -0.06 -0.03 -0.03 -0.02 -0.01 -0.04 -0.01 -0.05 0.00 0.05 -0.01 -0.04 0.25 0.31 0.38 0.31 0.30 1.00 Mankrong 0.04 0.36 0.21 0.22 -0.05 -0.02 -0.02 -0.01 0.01 -0.02 0.01 -0.03 0.02 0.07 0.01 -0.04 0.27 0.33 0.40 0.33 0.31 0.97 1.00 Kukrapa -0.01 0.31 0.07 0.08 -0.09 -0.06 -0.06 -0.04 -0.04 -0.07 -0.04 -0.02 0.10 0.10 0.08 -0.11 0.17 0.25 0.33 0.30 0.13 0.89 0.85 1.00

52 Biotechnology

TA/97/013

FA/89/026 82/326 FA/89/039

> \* I = Shannon's Information index Table 10. Summary of Genic Variation Statistics for All Loci \* ne = Effective number of alleles

\* I = Shannon's Information index

Allele \ Locus DaIAO1 DaIC12 DaID08 DaIF08 Da3G04 Dab2CO5 Dab2C12 Dpr3D06 ================================================================================ **Table 10.** Summary of Genetic Variation Statistics for All Loci


========================================================================================== Allele \ Locus DaIAO1 DaIC12 DaID08 DaIF08 Da3G04 Dab2CO5 Dab2C12 Dpr3D06 ============================================================================================

**Table 11.** Overall Allele Frequency :

Table 11. Overall Allele Frequency :


Table 12. Nei's original measures of genetic identity and genetic distance genetic

**Table 12.** Nei's original measures of genetic identity and genetic distance

**Acknowledgement** 

 **4. Conclusion** 

#### This screened the in vitro performance of the three released yam varieties in Ghana and revealed that they respond differently to the same medium. Maximising the used of tissue culture for mass propagation of clean **4. Conclusion**

support.

 **4. Conclusion** 

**Acknowledgement** 

appropriate medium. Other growth regulators may have to be screened to further optimise the performance of CRI-Pona. The study has made fingerprint information available to monitor the integrity of the released varieties. healthy planting materials is crucial and to achieve this the individual varieties have to be micropropagated in appropriate medium. Other growth regulators may have to be screened to further optimise the performance of CRI-Pona. The study has made fingerprint information available to monitor the integrity of the released varieties. In this study, *in vitro* performance of the three released yam varieties revealed that they respond differently to the same medium. Maximising the used of tissue culture for mass

healthy planting materials is crucial and to achieve this the individual varieties have to be micropropagated in

This screened the in vitro performance of the three released yam varieties in Ghana and revealed that they respond differently to the same medium. Maximising the used of tissue culture for mass propagation of clean

The team would first like to thank the Dr. E. Otoo the CSIR-CRI yam breeder for the making available the released yam varieties for this study. We would like to also express our sincere gratitude to the WAAPP project

Table 12. Nei's original measures of genetic identity and genetic distance genetic

The team would first like to thank the Dr. E. Otoo the CSIR-CRI yam breeder for the making available the released yam varieties for this study. We would like to also express our sincere gratitude to the WAAPP project for the provision of funds. We are also most grateful to other members of our research team for their treasured

15

15

propagation of clean healthy planting materials is crucial and to achieve this individual varieties have to be micropropagated in appropriate medium. Other growth regulators may have to be screened to further optimise the performance of CRI-Pona. The study has made fingerprint information available to monitor the integrity of the released varieties.
