Formation of Friable Embryogenic Callus is Enhanced under Nitrate, Potassium and Phosphate Limited Condition in Cassava.

Agrobacterium-mediated transformation is an important research tool and a practical tool for an improvement of cassava cultivar. The induction of friable embryogenic callus (FEC) is considered as a one of key step in cassava transformation. In this article, we optimized a media composition for improving the frequency of FEC induction and investigated about the physiological mechanism of FEC induction under optimized media condition using the transcriptome analysis. These results showed that changing to 10-folds lower media in comparison to MS media on nitrogen, potassium and phosphate contents and the use of excess vitamin B1 (changing from 0.1 mg/L to 10 mg/L) in addition to use of picrolam were necessary for leading to the effective induction to FEC and delaying the growth of non-FEC. An increase of transcripts concerning lipid metabolism, cell membrane and cell cuticle and a decrease of transcripts related to cell-wall modification process under long-term subculture were observed by microarray analysis, expecting that the nitrogen, potassium and phosphate limited condition and use of excess vitamin B1 provide the suitable condition for FEC induction through inhibition of excess cell proliferation and maintenance of fine FEC through formation of protective barriers in cell membranes. Our results will benefit as a one of significant techniques for cassava transformation, providing the more convenience transformation process using cassava genotypes and landrace adapted to the various field environments The friable embryogenic callus (FECs) were prepared in modified MS basal medium with 12mg/L piloram, 2% sucrose and 1.5% agarose (CIM-NPK) at 28ºC for 6 months (dark condition). The FECs were transferred to CIM-NPK or normal MS basal media with 12mg/L piloram, 2% sucrose and 1.5% agarose (CIM). The FECs were incubated for 1 month at 28ºC (dark condition). Total RNA was prepared from the incubated FECs and used for the microarray hybridization. Four replicative hybridization experiments were carried out using the independent biological samples.