Evaluation of a morphological marker selection and excision system to generate marker-free transgenic cassava plants

Abstract

The efficacy of the ipt-type Multi-Auto-Transformation (MAT) vector system to transform the extensively grown cassava cultivar "KU50" was evaluated. This system utilizes the isopentenyltransferase (ipt) gene as morphological marker for visual selection of transgenic lines. The extreme shooty phenotype (ESP) of transgenic lines is lost due to the removal of ipt gene mediated by the yeast Rint/RS system. As a result, phenotypically normal shoots, considered marker-free transgenic plants, could be obtained. When transforming KU50 cassava cultivar with two different ipt-type MAT vectors, transformation frequency at 19-21% was observed. Among the total number of ESP explants, 32-38% regained normal extended shoot phenotype and 88-96% of which were confirmed to represent the marker-free transgenic plants. This is the first demonstration of the efficacy of Rint/RS system in promoting excision of ipt marker gene in cassava specie, with the consequent rapid production of marker-free transgenic plants. The high efficiency of this system should facilitate pyramiding a number of transgenes by repeated transformation without having to undergo through laborious, expensive and time-consuming processes of sexual crossing and seed production. The generation of marker-free, thus environmentally safe, genetically modified cassava clones should also ease the public concerns regarding the use of transgenic cassava in both food and nonfood industries.

Cassava Genetic Transformation and its Application in BreedingF

“…In 2005, Jørgensen et al constructed several RNAi and antisense vectors to interfere with the expression of CYP79D1 and AYP79D2, and transformed the somatic cotyledons of cassava MCol22 using an Agrobacterium ‐mediated method. Saelim et al (2009) reported the production of transgenic cassava via shoot organogenesis of KU50 somatic cotyledons, using a multi‐auto‐transformation (MAT) vector system of isopentenyl transferase (ipt) type to obtain marker‐free transgenic plants with conversion efficiency up to 19–21%. Transgenic cassava with senescence‐inducible expression of the ipt was also developed using Agrobacterium ‐inoculated TMS60444 somatic cotyledons via shoot organogenesis (Zhang et al 2010).…”

Genetic potential of cassava biodiversity in Bangka Island, Indonesia

“…Tubers are potential to be developed as the main ingredient for local food industry in Bangka. Saelim et al (2008) stated that genetically modified cassava is environmental friendly plant which derives product in both food and nonfood industries.…”

Involvement of miR160/miR393 and their targets in cassava responses to anthracnose disease

“…The HN and HB60 plantlets were grown in vitro on CBM medium supplemented with 2% sucrose and 2 M CuSO 4 , solidified with 0.6% plant agar pH 5.8 (Saelim et al, 2009). The plantlets were grown at 26 ± 1 • C with a 16-h photoperiod (fluorescent tubes, 200 M m −2 s −1 ) and subcultured at 8-week intervals.…”