Formation of friable embryogenic callus in cassava is enhanced under conditions of reduced nitrate, potassium and phosphate

Abstract

Agrobacterium-mediated transformation is an important research tool for the genetic improvement of cassava. The induction of friable embryogenic callus (FEC) is considered as a key step in cassava transformation. In the present study, the media composition was optimized for enhancing the FEC induction, and the effect of the optimized medium on gene expression was evaluated. In relative comparison to MS medium, results demonstrated that using a medium with reducing nutrition (a 10-fold less concentration of nitrogen, potassium, and phosphate), the increased amount of vitamin B1 (10 mg/L) and the use of picrolam led to reprogram non-FEC to FEC. Gene expression analyses revealed that FEC on modified media increased the expression of genes related to the regulation of polysaccharide biosynthesis and breakdown of cell wall components in comparison to FEC on normal CIM media, whereas the gene expression associated with energy flux was not dramatically altered. It is hypothesized that we reprogram non-FEC to FEC under low nitrogen, potassium and phosphate and high vitamin B1. These findings were more effective in inducing FEC formation than the previous protocol. It might contribute to development of an efficient transformation strategy in cassava.

Cassava breeding and agronomy in Asia: 50 years of history and future directions

“…At ILCMB, efforts to establish genomeediting technologies in farmer-preferred varieties are underway. The application of this technology in cassava has indicated the importance of establishing a sustainable induction system of friable embryogenic callus (FEC) in the preferred varieties including KU50 (Utsumi et al 2017), even though regeneration of whole cassava plants from transformed FEC was already reported in the mid-1990's (Li et al 1996). Recently, an in planta genomeediting procedure was established for wheat cultivars using shoot apical meristems (SAMs) that are target cells for biolistic delivery (Hamada et al 2018).…”

Effect of nitrogen sources and 2, 4-D treatment on indirect regeneration of ginger (Zingiber officinale Rosc.) using leaf base explants

“…Maximum number of friable white calli were observed in MS Macronutrients containing 1216 mg/L KNO 3 and 1056 mg/L NH 4 NO 3 ( Table 1). The effect of nitrogen on callus production is studied on different plants like Sorghum (Elkonin et al 2000), Wheat (He et al 1989) and Cassava (Utsumi et al 2017). MS3 (Table 1) medium containing 2.0 mg/L 2,4-D in combination with 0.5 mg/L NAA found to be the best for maximum callus induction (92%) ( Table 2).…”

“…Nitrogen is an essential macronutrient for the synthesis of nucleic acids, proteins and secondary metabolites (Wada et al 2015). In tissue culture, Optimum level of nitrogen is essential for callus production, shoot and root multiplication and embryogenesis (Shanjani 2003;Utsumi et al 2017). Ammonium and nitrate are the two major sources of nitrogen in MS medium (Murashige and Skoog 1962).…”

Genetic Transformation of Recalcitrant Cassava by Embryo Selection and Increased Hormone Levels

“…However, the main obstacle to translational research in cassava remains the production of cassava tissues suitable for genetic transformation and the regeneration of transgenic lines. The FEC induction and multiplication steps appear to be particularly problematic and often require protocols with substantial modifications to achieve the production of FEC amenable for genetic transformation and regeneration of transgenic plantlets [17,19,21,22].…”