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Agrobacterium-mediated cassava transformation for the Asian-elite variety KU50

ABSTRACT: Agrobacterium-mediated cassava transformation via friable embryogenic callus (FEC) has allowed the robust production of transgenic cassava. So far, the transformation has been performed mostly for 60444 (the model cassava variety for transformation) and African varieties due to the high ability of callus induction and good regeneration capacity from embryogenic tissues. It is important to develop the transformation methods for elite cassava varieties in Asian area which is one of main cassava production area in the world, however, the suitable transformation method for Asian elite variety via FEC has not been reported. Here, we developed the transformation method in Kasetsart 50 (KU50) which has the highest planting area in Thailand and Vietnam. In cassava transformation, the preparation of FEC is considered as a key step. The ability of FEC induction from KU50 was improved efficiently by the use of media with reduced nutrients and excess vitamin B1, even if the FEC-inducible efficiency was 5 times lower compared with cv. 60444. The optimal concentration of NAA for regeneration from FEC to cotyledon was 1.0 mg/L and the optimal concentration of BAP for shoot formation from cotyledon was 0.4 mg/L. The transformation efficiency was estimated as 45% for 60444 and 22% for KU50. Transcriptome analysis indicated that KU50 FEC is out of balance between cell wall production and assembly, because the expression of the genes related to loosing cell wall was upregulated in the FEC from KU50 compared with 60444. We hope that the developed technology will contribute to molecular breeding of useful cassava plants in Asia by the simultaneous use of genome-editing technology.

ORGANISM(S): Manihot esculenta

PROVIDER: GSE169685 | GEO | 2022/03/26

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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Agrobacterium-mediated cassava transformation for the Asian-elite variety KU50

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