Abstract
Routine production of large numbers of transgenic plants is required to fully exploit advances in cassava biotechnology and support development of improved germplasm for deployment to farmers. This article describes an improved, high-efficiency transformation protocol for recalcitrant cassava cultivar TME14 preferred in Africa. Factors that favor production of friable embryogenic calli (FEC) were found to be use of DKW medium, crushing of organized embryogenic structures (OES) through 1–2 mm sized metal wire mesh, washing of crushed OES tissues and short exposure of tyrosine to somatic embryos; and transformation efficiency was enhanced by use of low Agrobacterium density during co-cultivation, co-centrifugation of FEC with Agrobacterium, germination of paramomycin resistant somatic embryos on medium containing BAP with gradual increase in concentration and variations of the frequency of subculture of cotyledonary-stage embryos on shoot elongation medium. By applying the optimized parameters, FEC were produced for cassava cultivar TME14 and transformed using Agrobacterium strain LBA4404 harboring the binary vector pCAMBIA2301. About 70–80 independent transgenic lines per ml settled cell volume (SCV) of FEC were regenerated on selective medium. Histochemical GUS assays confirmed the expression of gusA gene in transformed calli, somatic embryos and transgenic plants. The presence and integration of the gusA gene were confirmed by PCR and Southern blot analysis, respectively. RT-PCR analysis of transgenic plants confirmed the expression of gusA gene. This protocol demonstrates significantly enhanced transformation efficiency over existing cassava transformation protocols and could become a powerful tool for functional genomics and transferring new traits into cassava.
Highlights
Genetic transformation technology has become a high throughput platform for cultivar improvement in several crops as well as for studying gene function in plants
The comparative potential of somatic embryogenesis was evaluated based on both the frequency of organized embryogenic structures (OES) production for each basal medium [Frequency of OES = ∗ 100], and scoring of the amount of somatic embryos (SE) obtained per OES cluster on 0–5 scale, where 0 = no Somatic embryos (SE) obtained, 1 = very low SE, up to 10% of the OES cluster, 2 = low SE, 11–25% of the OES, 3 = medium SE, 26–50% of the OES, 4 = high SE, 51–75% of the OES, and 5 = very high SE with mostly structures embryogenic on entire OES cluster
Cassava remains popular staple food crop among resource poor farmers mostly due to its resilience and capacity to grow on marginal lands, and its flexible harvest period, which can be as long as 30 months after planting
Summary
Genetic transformation technology has become a high throughput platform for cultivar improvement in several crops as well as for studying gene function in plants. Several protocols using FEC as target tissues and particle bombardment or Agrobacteriummediated transformation procedures have been reported (Taylor et al, 2012; Zainuddin et al, 2012; Nyaboga et al, 2013). Establishing such embryogenic tissues is cultivar dependent and requires optimization of FEC production for each particular cultivar making it a prerequisite for agronomic improvement in that particular genetic background. The development of efficient high-throughput genetic transformation capabilities for popular cassava cultivars is required
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