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

ABSTRACT: 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

ORGANISM(S): Manihot esculenta

PROVIDER: GSE89971 | GEO | 2019/10/29

REPOSITORIES: GEO

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Project description:Friable embryogenic callus (FEC) has been considered as the most suitable materials for efficient genetic transformation of cassava. Due to heavy genotype dependence on FEC induction and being amenable to somaclonal variation, the production and maintaining of reliable FEC is a limitation factor for cassava genetic transformation. Identification of key elements involving in biological processes from somatic embryos (SEs) to the FEC at different stages will provide critical insights for FEC improvement. Cytological observation showed the dramatic change of subcellular structures among SEs, fresh FEC (FFEC) and old FEC (OFEC). Decrease of sucrose and increase of fructose and glucose were detected in OFEC. A total of 6871 differentially expressed genes (DEGs) were identified from SEs, FFEC and OFEC by RNA-seq. Analysis of the DEGs showed that FEC induction was accompanied by the process of dedifferentiation, whereas the epigenetics modification occurred during the continuous subculturing process. The cell structure was reconstructed, mainly including the GO terms of "cell periphery" and "external encapsulating structure"; in parallel, the internal mechanisms were changed correspondingly, including the metabolisms of process (glycolysis) and compounds(alanine, aspartate, and glutamate). The significant reduction of genomic DNA methylation in OFEC indicates altered gene expression via chromatin modification. These results indicate that the induction and long-term subculture of FEC is a complicated biological process involving changes of genome modification, gene expression and subcellular reconstruction. The findings will be useful for improving FEC induction and maintenance from farmer-preferred cassava cultivars that are recalcitrant to genetic transformation, hence improving cassava through genetic engineering. Two-week-old SEs cultured on GD, FFEC emerging from SEs, and 9-month-old OFEC were used for analysis. Each sample was mixtured from six indepedent replicates and were collected for RNA extraction respectively.

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

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