The aim of this study was to establish an effective protocol for callus induction from the seed explants of Solanum dubium and to investigate the callus extract ability in milk clotting activity. The effects of growth regulator, basal media strength and sucrose were studied using different concentrations (0.5, 1.0, 1.5, and 2.0 mg/L) of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthalene acetic acid (NAA) and 2,4-dichlorophenoxy-acetic acid (2,4-D) alone or in combination with 0.5 mg/L of 6-benzylamino purine (BAP). For milk clotting activity, about 50 or 100 μL extracts of seed callus was mixed with 2 mL 50% milk held at 55°C for 5 and 10 min until milk clotting occurred. The results showed that NAA alone or in combination with BAP gave a higher callusing percentage (80 to100%) compared to the other plant growth regulators at the same concentrations. When an auxin was supplied in combination with BAP, a significant increase in callusing percentage or degree of callusing was observed. The time required for callus to be developed was shortened and the quality of the induced callus improved. An increase in callus growth in low sucrose (10 g/L) concentration was found be comparable (88%) to high sucrose concentration (30 g/L; 60%). Crude extracts obtained from S. dubium callus were shown to exhibit milk clotting activity.
The cockle, Anadara granosa, was experimentally exposed to low (0.1 mg/L) and sublethal (1.0 mg/L) doses of copper (Cu) for a period of 24 hrs. Significant increase in Cu concentrations in whole tissues and hepatopancreas compared to control animals were observed. In order to study the effect of copper exposure at molecular levels, a subtractive cDNA library was constructed from the hepatopancreas of cockles exposed to 1.0 mg/L Cu. Screening of the subtractive cDNA library using reverse northern analysis resulted in several differentially expressed genes, including one that codes for metallothionein (MT). The complete coding sequence of the MT gene (designated as AnaMT2) reveals an open reading frame of 234 bp in length that encodes a 77 amino acid polypeptide as revealed by the deduced amino acid composition. Although showing similarities with other molluscan MTs, AnaMT2 can be distinguished by its lower glycine and higher asparagine and proline content. Expression analysis of the AnaMT2 by northern analysis indicated higher mRNA level in cockle exposed to 1.0 mg/L Cu and was undetectable in those treated with 0.1 mg/L. This suggests that AnaMT2 represents a primarily inducible MT not highly expressed under basal conditions.