Factors influencing somatic embryogenesis, regeneration, and Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14

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.

Faithful transmission and expression of transgenes via somatic embryos of transgenic cassava plants at the sixth cycle of vegetative propagation

“…In addition, we observed that the frequency of somatic embryos and the number of somatic embryos per explant in primary, secondary and cyclic somatic embryogeneses were higher in immature leaf lobes than axillary buds, which confirms the report of Rossin andRey (2011), Opabode et al (2013) and Opabode et al (2014) in non-transgenic cassava plants. The difference in the frequency of somatic embryos and the number of somatic embryos per explant could be due to the larger surface area of immature leaf lobes than axillary buds, as the frequency of regenerating cotyledonary-stage embryos and the number of shoots per cotyledon have been reported to be affected by the size and section of cotyledonary-stage embryos used in the studies (Nyaboga et al, 2015).…”

The Uses of Somatic Embryogenesis for Genetic Transformation

“…Genetic transformation based on somatic embryogenesis is still limited to those species showing high embryogenic capacity. However, since embryogenic responses are dependent on the genotype and on the environmental factors, more efforts should be focused to find the right in vitro culture conditions to overcome recalcitrance as it was described for citrus species (Dutt and Grosser 2010) and cassava (Nyaboga et al 2015). Alternatively, embryogenic capacity in recalcitrant plants can be improved by traditional genetic techniques (Kita et al 2007) or by the genetic manipulation of specific master genes involved in the control of embryogenesis as reported for Norway spruce (Picea abies) (Belmonte et al 2007).…”

“…Genetic improvement and functional genomics of cassava have been limited for low-efficiency transformation protocols. Nyaboga et al (2015) investigated the influence of factors that favor plant regeneration and genetic transformation of the recalcitrant cultivar TME14 preferred in Africa. They observed that production of friable embryogenic callus from axillary buds and immature leaf lobes of aseptic plantlets was promoted using DKW medium (Driver and Kuniyuki 1984), crushing of organized embryogenic structures (OES) through a metal mesh (1-2 mm pore size), washing of crushed OES tissues and exposure of somatic embryos to tyrosine (12 mg L −1 ) in the presence of 12 mg L −1 picloram.…”

Pengaruh Sitokinin, Jenis Eksplan, dan Genotipe terhadap Embriogenesis Somatik Kakao

“…Formation of cacao somatic embryos and planlets induced by kinetin. Somatic embryos at heart and early cotyledonary stages of (A) Cimanggu 3 accession, (B Górecki, Michalczyk, & Michalczyk, 2010;Zuyasna, Hafsah, Fajri, Syahputra, & Ramadhan, 2012;Nyaboga, Njiru, & Tripathi, 2015). Perbedaan respons embriogenik dari eksplan mahkota bunga dan staminoid pada kakao juga telah dilaporkan sebelumnya oleh Avivi et al (2010); Ajijah et al (2014); dan Ajijah et al (2016).…”