| dc.description.abstract | Sorghum bicolor (L) Moench is generally sensitive to salt and acid (high aluminium) soil
stresses. As with any stress phenomenon, intra-specific variability exists within the genus. In
Vitro cell selection and somaclonal variation offer an alternative to traditional breeding
methodology for generating improved breeeding lines for hybrid development. Tissue culture
method was developed for induction and maintenance of embryogenic calli established from
young embryo explants of three Sorghum bicolor cultivars namely Mtama I, el Gardam and
Seredo. Embryogenic calli were obtained by culturing young embryos on Linsmaier and Skoog's
(LS) medium containing 2mg/l 2.4 dichlorophenoxyacetic acid (2,40) and 0.5 mg/l Kinetin.
Calli were subjected to 50 mM. 100 mM, 150 mM and 200 mM NaCI to screen them for salinity
tolerance. Calli subjected to 0.0 mM NaCI served as controls. Calli with a tolerance to salinity
stress had a higher activity of succinate dehydrogenase which reduced trimethyl tetrazolium
chloride (TIC) to formazon. The amount of formazon was measured spectrophotometrically and
a graph of NaCI concentration against absorbance was plotted. From the TTC test (viability test)
100 mM NaCI was selected as the optimum concentration which was incorporated into LS media
to initiate tolerance in sorghum calli.
Sorghum shoots were regenerated from embryogenic calli of both NaCI treated and the
controls cultured on LS medium supplemented with 1.0 mg/l Indole Acetic Acid (IAA) and 0.5
mg/l benzyl adenine (BA). Rooting was achieved by supplementing LS medium with 3 mg/l
Indole Butyric Acid (lBA).
By using Random amplified polymorphic DNA (RAPDs) technique it was established
that there was variation between NaCI treated plants and the controls. This was both in the
individual and pooled DNA samples. A genetic distant matrix was calculated and used to
construct a dendrograme as a measure of relatedness which led to the conclusion that somaclonal
intra cultivar variation had resulted in some of the cell lines becoming tolerant to salinity.
To establish whether saliniry tolerance had been achieved in plants regenerated from
NaCI treated calli some parameters conforming to C4 photosynthetic pathway were measured.
These included CO2 assimilation rate, titratable acidity, malate content and water potential. CO2
assimilation rate was measured along with Photosynthetic Active Radiation (PAR), stomata
conductance (SC) and Transpiration Rate (TR) using infra red gas analyser (IRGA). The CO2
assimilation rate was synchronized with SC and TR. The CO2 assimilation rate started increasing
in the morning reached a peak at noon and decreased as the afternoon progressed following
decrease in PAR. This pattern was observed in all the 3 cultivars throughout the sampling period.
The CO2 assimilation rate in the controls followed the same pattern except the mean values were
significantly lower (P<0.05).
The treated plants accumulated significantly (P:::;0.05) higher levels of titratable acidity
and malate than the controls in all the 3 cultivars. The two parameters had a day light pattern of
increasing in the morning reached a peak at noon in parallel with PAR and CO2 assimilation rate
and decreased as the afternoon progressed.
High positive regression correlations (r2 = 0.7 and above) existed between titratable
acidity and malate content; malate and CO2 assimilation rate (r2 = 0.6 and above) illustrating that
these parameters were interdependent.
Water potential (\jJ) was significantly lower (P<0.05) in the treated plants than in the
controls. Although low water potential indicated physiological drought in the treated plants, they
did not show decline in CO2 assimilation rate which accounted for high TR and SC at high PAR.
This might have indicated that they had become salinity tolerant.
No change in ploidy level was achieved by intercultivar or intracultivar protoplast fusion
because internuclear fusion failed due to nuclei repulsion. However, cytoplasmic hybrids
(cybrids) were obtained but no plants were regenerated from the cybrids due to failure to
resynthesize cytoskeletons.
Phosphoenol phyruvate carboxylase (PEPC) enzyme was successfully purified by
ammonium sulphate precipitation and DEAE' - sepharose 6B gel filtration using sorghum and
Kalanchoe extracts. It showed two subunits of molecular weight 95 kDa and 50 kDa common in
both sorghum and Kalanchoe. The enzyme activity amongst the 3 sorghum cultivars; between
the treated and controls, between sorghum and Kalanchoe was not significant. This might have
indicated that the enzyme from the leaves of the two plant species was similar and therefore, had
same properties. Therefore, detection of polymorphism using PEPC protein as a marker
in measurement of relatedness was not successful. | en |
