The field experiments were conducted at two sites; Kenya Agricultural Research Institute (KARI)-Kiboko, and KARI-Thika, while the greenhouse experiment was conducted in KARI- Muguga. The KARI-Kiboko research station is located in Makueni county, 187 km east of Nairobi, at a latitude of 2o15’ south and longitude of 37o45’E and altitude of 993 m asl. The research station is classified under agro-ecological zone five and receives mean annual rainfall of 560 mm with long-term annual average rainfall of 615 mm and is ideal for dry land research. The station receives bimodal rainfall with the more reliable short rains falling in late October to December (330 mm) and the poor long rains from March to May (230 mm). The annual mean maximum temperature is 30.6oC, while the annual mean minimum temperature is 17.4oC which translates to an annual mean temperature of 24oC. The soils are of rhodic ferrasols to ferric luvisols on the old peneplain and eutric fluvisol at the bottom of the river valley (Mwacharo et al., 2004). KARI-Thika is located 75 km north east of Nairobi, latitude 0o59’ South, longitude 37o04’ East, altitude 1548 m asl. The area receives bimodal mean rainfall of about 1000 mm annually with long rains of 142 mm falling March to May and the
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short rains of 116 mm falling in October to December. The areas has minimum temperature of 13.7°C, maximum of 25.1°C, and mean annual temperature of 19.4°C (Ndegwa et al., 2009). The soils at Thika are well drained ferric luvisols.
KARI-Muguga is located 27 km north west of Nairobi, latitude 1o13’ South, longitude 36o38’
East, altitude 2096 m asl. The area receives bimodal mean rainfall of 900 to 1000 mm annually with long rains of 550 mm falling mid-March to June and the short rains of 400 mm falling in mid-October to December. The area has minimum temperature of 7°C and maximum of 20°C, which translate to 15°C. The area is classified as sub humid with a well- drained, very deep, dark reddish brown to dark red, friable clay soil classified as humic nitisols (UNESCO, 1977) or an oxicpaleustaf (USDA, 1975).
3.2.2 Sweetpotato germplasm
The germplasm consisted of 84 sweetpotato genotypes sourced from the gene bank of Kenya, the International Potato Centre (CIP), Nairobi and farmers’ fields. The genotypes were selected on the basis of high yield, orange fleshed sweetpotato (OFSP), high dry matter, early maturity, drought tolerance, and sweetpotato virus disease (SPVD) resistance.
The germplasm was multiplied in the greenhouse at KARI Muguga to increase the planting materials. The genotypes Marooko and Gatumbi were obtained from the National Gene Bank of Kenya (NGBK) and used as local checks.
3.2.3 Field evaluation
At KARI-Kiboko, the trial was planted in October 2010 when the rains had subsided and repeated for another season in April 2011. At KARI Thika the trial was planted in May 2011 after the rains had subsided and repeated in December 2011 (Fig. 3.1). A split plot design was used with irrigation as the main plot and clones as subplots arranged in a 14 x 6 alpha lattice replicated twice. The genotypes were planted on 25 cm high beds in single rows of 10 plants per plot at a spacing of 30 cm between plants and 90 cm between rows. Each row was separated by one meter planted with two plants of a clone with different vine and root skin colour, to ensure uniform competition within the rows. Distinct clones with coloured vines and roots were planted in four guard rows surrounding the outer rows of the experiment. Six tensiometers were installed in each replication, two at each of the following soil depths; 20 cm, 40 cm and 60 cm to monitor soil moisture content and determine when to irrigate. Vines used for planting were about 25 cm long and had 4-6 nodes. The vines were planted in a 600 slanting position and kept at field capacity soil moisture levels for four weeks until they were established. Thereafter, the drought stress environment received no irrigation water until harvesting while the irrigated environment received 35 mm equivalent irrigation water every time the tensiometer read 15 centibars. The amount of irrigation water was
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collected and measured using artificial rain gauges and cumulative irrigation water quantified to mm of rainfall.
Soil analysis was conducted before planting to determine nutrient status of the trial site and moisture content (Table 3.1). Double ammonium phosphate fertiliser was applied during planting at the rate of 50 kg P2O5 ha-1,while weeding was done by hand hoe twice in each experiment. The centrally placed six hills were used to collect data leaving two hills each side of the row.
Data on morphological traits were collected following IPGRI (bioversity) descriptors (CIP et al., 1991). Storage root harvesting was done 150 DAP using hand hoes and data collected on the six plants centrally placed in the rows. Data collected included:
• Number of storage roots (NSR) - a count of storage roots plant-1
• Number of marketable storage roots (MSR) - a count of storage roots weighing between 100 – 500 g plant-1
• Fresh root weight (FSR) - the weight of all storage roots at harvesting plant-1
• Fresh vine weight plant-1 (FVW) - the weight above ground biomass at harvesting plant-1
• Fresh biomass (FB) - the sum of storage root and vine weight plant-1
• Leaf chlorophyll content (CC)
• Harvest index (HI) - fresh storage root weight/fresh biomass
• Days to permanent wilting point (DPWP) under rapid box drought screening experiments.
Fresh root samples of 200 g and fresh vine samples of 300 g or the available were taken for determining dry matter. Drying of the samples was done in a forced air drying oven set at 40oC until a constant mass was attained and percent storage root dry matter content (%RDM), percent vine dry matter content (%VDM) were calculated. Drought sensitivity index (DSI) according to Fischer and Maurer (1978) rating the clones as, DSI 1=neutral, >1 sensitive, <1 tolerant, was determined.
3.2.4 Chlorophyll content determination
The chlorophyll content was determined using a chlorophyll meter (SPAD 502 plus:
STSPAD502PLUS, Konica Minolta, Tokyo, Japan). The SPAD was calibrated by analysing the chlorophyll content of samples collected from some of the genotypes used. Three leaves were randomly sampled in each of the following randomly selected genotypes for calibration;
194515.15, A2, 441725, Resisto, and 1990621. For each leaf, two SPAD readings were taken at mid left and mid right of the selected leaves and 9 mm diameter discs cut using a
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cork borer at each point. The chlorophyll in the discs was extracted in 4 ml of 0.1N HCl in Methanol (BDH) at 21oC in a dark room for 24 hours in a test tube. A blank sample with only the extraction solution was also included as a control. Absorbance of the extracts was measured using a WPA S105 spectrophotometer (Griffin and George Ltd. – WPA Scientific Instrument, located at Saffron Walden, England). The absorbance of the chlorophyll extracts was measured at 653 nm and chlorophyll content in mg cm-2 calculated as absorbance at 653 nm (A653)*24.88 (Mutui et al., 2001). A correlation scatter graph was generated using the SPAD measurements as x-axis and the chlorophyll extract absorbance at y-axis. The calibration equation fitted with the y-intercept set at zero was Y = 0.0168X, with R2= 0.935.
Thus, chlorophyll content (mg cm-2) was calculated as Y = 0.168X *24.88. Where Y = chlorophyll content in mg cm-2, X is the SPAD reading and 24.88 is a constant.
3.2.5 Greenhouse evaluation
This experiment was conducted in the greenhouse at KARI Muguga between January and December 2011 (Fig. 3.1). Each of the 84 genotypes was planted in a box filled with sterilized soil comprised of forest soil, cattle manure, gravel of size one eighth of an inch, and NPK (17:17:17) fertiliser all at a ratio of 5:2:1:0.25. The soil was moistened and sterilized at 82.5oC in drums of 210 kg for 20 minutes, cooled for 21 days before use. The soil media was filled in to the boxes made of 15 x 0.45 x 0.45 m high density black polythene sheet supported by wooden pegs to a height of 0.4m (Fig. 3.1 b). The experiment was conducted using a RCBD design replicated four times and repeated two times. Prior to the beginning of the experiment, the growth media was watered to field capacity. Data on the number of days from establishment to permanent wilting point were counted. The growth media was watered to field capacity, indicated by a tensiometer reading of zero. Shoots of 10 cm were cut from each experimental genotype using a sterilized scalpel. Each cutting was planted to a depth of five cm, in upright position. The soil moisture was maintained at field capacity by putting more water every time the tensiometer reading dropped to 15 centibars, until it read zero again, carefully avoiding flooding. Water application was stopped 15 days after planting and observation of the plants done. Data on number of days from planting to permanent wilting point were taken for each plantlet (Laurie et al., 2004). Data on soil moisture stress using tensiometers and relative humidity, light intensity, maximum and minimum temperature were collected using a data logger (HOBO U12-012 – data logger temp/RH/Light/ext channel, Micro Precision Calibration Inc., 22835 Industrial Place Grass Valley, CA USA 95949).
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Figure 3.1: (a) Field evaluation of 84 clones – drought and irrigated environments (b) Greenhouse evaluation of the 84 clones in box drought screening
3.2.6 Data analysis
The data was subjected to analysis of variance (ANOVA) to determine differences among genotypes and environments using SAS (2002), and means separated using least significant difference (LSD) at p≤0.05. The clones were ranked as follows; rank y= ∑x , where; y is the rank, x is the sum of genotype ranks from trait k to trait n for the traits measured, where k
= rank in trait 1 on merit, and n = rank of the last trait. Thus, the best genotype in each trait scored one, while the worst scored 84, which indicates that, considering more than one trait, the best genotype had the least cumulative scores while the worst had the most cumulative scores.