Abstract:

Sorghum is one of the five most important staple cereals for humans being a source of food,
animal feed and industrial raw material. However, the crop suffers significantly from many
biotic and abiotic stresses. To improve sorghum for these traits, for the most part reliance has
been on both inter- and intra species hybridization also called traditional plant breeding.
Traditional plant breeding can only exploit variation present in species that can be crossed
with sorghum. Genetic transformation offers a direct access outside primary gene pool of
sorghum. Sorghum genetic transformation has been lagging behind other cereals due to its
recalcitrance to tissue culture and genotype dependent regeneration. Therefore, the aim of this
study was two-fold: to develop an in vitro culture system for Ugandan sorghum adapted
genotypes and screen Ugandan elite sorghum genotypes for their amenability to tissue culture
under different test conditions, as well as develop an efficient Agrobacterium mediated
transformation system for genotypes with better response to in vitro culture.
Callus induction medium composition was optimised with emphasis on the levels of 2, 4-
Dichlorophenoxyacetic acid (2, 4-D) and kinetin hormones. Twenty elite sorghum genotypes
were screened for the amenability to tissue culture on two medium types; Murashige and
Skoog basal medium and L3 medium. The genotypes amenable to tissue culture were
advanced to Agrobacterium mediated transformation in which two bacterium strains EHA 105
and AG LI were used.
The results showed that the optimum sorghum regeneration parameters (frequency of somatic
embryo formation, callus growth and number of shoots) were achieved at a level of 2, 4-D of
either 2.0 mg/l or 2.5 mg/1 supplemented with 0.5 mg/1 of kinetin hormone. Inclusion of
kinetin hormone in callus induction medium at low levels of 2, 4-D gradually reduced the
callus induction frequency and higher levels of 2, 4-D exhibited inhibitory effects on callus
induction as well as somatic embryogenesis. The best age of immature zygotic embryo for in
vitro culture was between 16-18 days post anthesis. However, the physiological state of the
embryo of different genotypes at the same age varied. This indicates that panicle harvesting
decisions for in vitro culture should be based on the embryo size rather than age. Strong
genotype by medium type influenced in vitro culture responses rather than the genetic control only. The genotypes MUC007/I94 and MUC007/I93 had a short regeneration time on MS
basal medium than on L3 medium. However. L3 medium was the best medium for most of
the sorghum genotypes included in the study. Among the genotypes advanced for
Agrobacterium mediated transformation, MUC007/I93 and MUC007/I24 were susceptible to
Agrobacterium infection and no significant sorghum genotype by Agrobacterium strain was
found. Generally, EHA 105 Agrobacterium infected sorghum explants exhibited highest
frequency of putatively transformed material with the high level ofgz/5 plus gene expression.
Overall, this study has demonstrated that indeed Ugandan local sorghum genotypes can be
amenable to in vitro processes. The model Ugandan sorghum genotypes for in vitro culture
are the genotypes MUC007/I93 and MUC007/I94. Genotypes MUC007/I93 and
MUC007/124 had highest transformation frequencies and thus can be used for further stable
transformation studies. The high recalcitrance of sorghum, however, implies that candidate
genotypes amenable to in vitro processes can be used as systems to introgress foreign genes
into non recalcitrant but commercially viable genotypes.