A prerequisite for successful in vitro culture is the establishment of an aseptic technique, thus the experiment was to investigate suitable sterilization regimes for the leaf explants of Gnetum africanum, an endangered green leafy vegetable. Three sterilization regimes were tested to establish the best regime using three to four days old leaves. The surface sterilized explants were later aseptically introduced onto the surfaces of sterile Murashige and Skoog agar media, incubated at 25°C for three weeks in the growth chamber. 100% sterility was observed from the regime which was significantly different (P<0.05) from the other two regimes thus the best regime adopted for further experiments was; washing in two drops of Tween 20/100 mls of sterile water, soaking in 70% ethanol for 2 min and later in 1% sodium hypochlorite for 20 min. Fungal contaminants responsible for in vitro contaminations was also investigated and possible isolates were identified as Asperigilus niger (28.71%); A. flavus (26.73%); Rhyziopus spp. (24.75%) and Mucor Spp (19.81%) respectively.

ABSTRACT. To better understand the reproductive transformation mechanism of Daphnia carinata, a Doublesex (Dsx) gene was cloned based on rapid amplification of cDNA ends (RACE), and was designated DapcaDsx2. Next, we compared similarities and assumed homology based on deduced amino acid sequences. It showed 97.52, 87.94, and 85.11% identity to orthologous genes in D. magna, D. pulex, and D. galeata respectively. Phylogenetic analysis revealed that DapcaDsx2 clustered in the same class, and was evolutionarily more distant to sequences from other species. qRT-PCR showed that DapcaDsx2 was most abundantly expressed during sexual reproduction (P < 0.05). Using digoxigenin-labeled RNA probes, we studied DapcaDsx2 expression in parthenogenetic and sexual females by whole-mount in situ hybridization. The results revealed that DapcaDsx2 was mainly Cloning and expression of Doublesex in D. carinata expressed in the second antennae and several sites of the ventral carapace, whereas higher expression levels were found in sexual than in parthenogenetic females. This suggests that the DapcaDsx2 gene is involved in switching modes of reproduction and in sexual differentiation.