ABSTRACT
Aims: A procedure was developed for embryogenesis from embryo explants derived from mature seeds of freshly harvested Serenut 4T, Serenut 1R and Acholi-white groundnut cultivars representing the three broad groundnut botanical classifications. Methodology: This study explored the use of mature embryo axes as explants for somatic embryogenesis, and determined the factors that affect regeneration of three Ugandan groundnut cultivars. Freshly harvested mature seeds of the three groundnut cultivars were collected and the embryo explants were initiated on 3 media namely; Murashige and Skoog (MS) basal media with varying concentrations of the growth regulator 2,4-Dichlorophenoxy acetic acid (2,4-D); Chu N6 basal medium with vitamins (N6); and Callus Induction Medium (CIM). The shoot formation and elongation medium contained MS basal medium supplemented with indolebutyric acid (IBA) and 6- Benzylamminopurine (BAP) in isolation, and BAP in combination with a-naphthaleneacetic acid (NAA) and indoleacetic acid (IAA). For root induction, elongated shoots were transferred to MS medium supplemented with various combinations of NAA with IBA, BAP and a combination of IBA and Kinetin. Results and Conclusion: Different concentrations of 2,4-D elicited different callogenesis responses in the cultivars with Acholi white (Valencia botanical) and Serenut 4T (Spanish botanical) giving the optimal response at 5mg/l whereas Serenut 1R (Virginia botanical) showed best response at a concentration of 30 mg/l. N6 and CIM supported callogenesis in Acholi white (AW) and Serenut 4T only. In all cultivars, maximum root production was gained when using MS medium supplemented with NAA- 1 mg/l and IBA -2.0 mg/l. On the other hand, for Serenut 1R and Serenut 4T, BAP 2.5 mg/l; NAA 0.5 mg/l combination yielded higher shoot regeneration percentage whereas for AW BAP 3 mg/l; NAA 0.5 mg/l supported maximum shoot production.This is the first ever report of successful regeneration of the three groundnuts botanicals in Uganda. These results are likely to facilitate genetic transformation of three preferred Ugandan groundnut varieties.
ABSTRACT
Aims: To develop a sampling procedure for PCR-based screening of bacterial leaf spot (BLS)-causing xanthomonads without DNA extraction from infected tomato plants. Place and Duration of Study: University of Copenhagen, Denmark and Sokoine University of Agriculture, Morogoro, Tanzania between July 2008 and November 2010. Methodology: Flinders Technology Associates (FTA®) plant cards and Chromatography paper or Whatman® paper strips (WPS) were spotted with bacterial suspensions from 24- h-old cultures from reference strains of BLS-causing xanthomonads, or sap obtained by grinding or hand maceration of plant tissue, were used as templates in PCR reactions or isolation of live bacterial cells on Nutrient agar (NA) media. Samples were tested by PCR with Xan 7 genus/-specific Xanthomonas primers or in multiplex with 26S rRNA primers. Isolation of bacteria was done by streaking aliquots of 75 μl of a suspension from a disc (2-mm-punch by Harris Micro Punch®) in triplicate, removed from each of the FTA plant card and WPS onto NA media. Results: The FTA plant card spotted with pure cultures of reference strains of xanthomonads and sap from grinding or direct maceration of plant tissue resulted in more clear PCR bands (402 bp) and (594 bp of rRNA gene in multiplex) than the WPS samples. Sensitivity of detection by the FTA paper-based PCR was ≈ 5.0 x 102, while that of the WPS was > 1.0 x 103 CFU/ml. The WPS (but not the FTA) was proved to be useful for saving living bacteria cells for up to one week of storage at ambient temperatures. Conclusion: Both FTA plant card and WPS can be used for PCR detection of BLScausing xanthomonads in tomato. However, the FTA plant card is recommended as it produced clearer PCR products than WPS. WPS is recommended for experiments requiring isolation of live bacterial cells on NA media.