STUDY ON COMPLETING A REGENERATION SYSTEM AND CONSTRUCTING A SYNTHETIC AGPOPT VECTOR FOR TRANSFORMATION INTO CASSAVA (MANIHOT ESCULENTA CRANTZ).
Abstract
Cassava is a high value crop for food security and industry. Improving cassava using biotechnology is providing important scientific and practical values. The longterm objective of this study is to create AGPopt transgenic cassava plants for enhancing the starch synthesis. A plant regeneration protocol of the KM140 and KM94 cassava varieties via somatic embryos from shoots and young leaf lobes was optimized on MS supplemented with 12 mg/l picloram for induction of somatic embryogenesis, and germinated on MS supplemented with 0.3 mg/l BAP. The AGPopt gene is synthetized using data from glgC protein of E. coliK12 (code AKD93525.1) with 1527 bps, added with the G336D mutation, their genetic codes optimized in compliance with plants, the 5’ head attached with the 171 bps fragment encoding for the signal peptide (57 amino acids), the 3’ end attached the c-myc (11 amino acids), and the KDEL (4 amino acids) sequence. The synthetic AGPopt has been constructed into the pBI121 vector for transformation. Using tobacco as the model plant, the transformation of AGPopt vector has created 32 transgenic tobacco lines which are proved to be positive by PCR; also positive by Southern blot; positive for AGPase protein expression by Western blot; increasing leaf AGPase activity (from 121 to 153%), and also starch content (from 140.05 to 168.99%). This has successfully proved that the AGPopt vector has been used for transformation into KM94 cassava via somatic embryos and resulted in 6 plant lines, 5 of which have been proved to be positive for the AGPopt gene. Further analyses of the transgenic cassava lines have been implemented.
