In Vitro Regeneration, Virus Elimination and Transformation of Selected Cassava Landraces in Tanzania

Abstract

Cassava is among the most produced and consumed crops in Africa with an annual production of 176 million metric tons. Lack of clean planting materials of farmer-preferred cassava varieties is one of the main constraints to increased cassava productivity. This study aimed at evaluating in vitro regeneration, virus elimination and transformation protocols for high quality farmer preferred cassava landraces in Tanzania. Cassava cuttings, Kibandameno and Paja la mzee collected from Coast area of Tanzania were grown in two media, conventional Murashige and Skoog medium and an alternative medium for six weeks. Four parameters were measured namely; plant height, number of leaves, number of nodes and number of roots. The cost of production was calculated for both media and compared. The use of the alternative medium led to a cost reduction of 93% over the conventional Murashige and Skoog medium. The efficacy of chemotherapy and thermotherapy in viral elimination was also determined, where cassava plants infected with East African cassava mosaic virus were subjected to chemical treatment in medium supplemented with Ribavirin and Salicylic acid at different concentrations. In another separate experiment, infected plants were subjected to thermotherapy. DNA was extracted from plant leaves after 42 days of treatment and Polymerase chain reaction amplification was performed using East African Cassava Mosaic Virus (EACMV) specific primers. The most effective concentrations were 20 mg/l of ribavirin, and 30 mg/l of salicylic acid which resulted in 85.0% and 88.9% virus-free plantlets, respectively. Thermotherapy at 35°C resulted in 71.4% virus-free plantlets. The regenerable cassava varieties were assessed for transformability using Agrobacterium tumefaciens with the reporter gene. The established FEC from two cassava varieties TMS60444 and Albert were infected with Agrobacterium LBA4404 harboring the vector Pc062 having the reporter gene β-glucuronidase (GUS). Transformants were selected and taken through regeneration. Transformability was confirmed by GUS histochemical assay and later by PCR using specific primers. The highest transformation efficiency of 86% and 83% was observed for TMS60444 and Albert, respectively. The high-throughput cost-effective platform for mass production of cassava plantlets, viral cleaning and genetic modification protocols evaluated will greatly enhance availability of clean cassava planting materials to farmers at an affordable cost and conservation of valuable germplasm.