Pinpointing towards improved transformation and regeneration of cassava ( Manihot esculenta Crantz)

Abstract

Friable embryogenic callus (FEC) of the cassava genotype 60444 was transformed by particle bombardment with DNA from the plasmid constructs pHB1 and pJIT100. Both plasmids contained the luciferase ( luc) marker gene under the control of the CaMV 35S promoter. In addition pJIT100 had the CaMV35S driven phosphinothricin acetyl transferase ( pat) gene, while pHB1 contained the cassava cDNA coding for the small subunit of ADP glucose pyrophosphorylase (AGPase B) in antisense orientation under the control of a double CaMV35S promoter. A total of 2 weeks after bombardment, luciferase (LUC) positive FEC units (spots) were isolated and subcultured separately for further proliferation. A total of 4 weeks later, those cultures having at least four positive LUC spots were subjected to three different selection regimes namely: stringent LUC selection, non stringent LUC selection and combined LUC/phosphinothricin (PPT) selection. A total of 16 weeks after bombardment, stringent LUC selection gave rise to cultures in which 92% of the FEC units were LUC positive. Within the same time period non stringently LUC selected cultures and LUC/PPT selection had only 1 and 41% of the units that were LUC positive, respectively. The number of LUC positive mature somatic embryos formed was similar to the percentage of LUC positive FEC units, within a culture, found with each selection method. Stringent LUC selection enabled transgenic plants to be produced in 28–36 weeks compared to 32–41 weeks for LUC/PPT selection and 53–78 weeks for non stringent LUC selection. This indicates that stringent selection is a more efficient method for obtaining transgenic cassava plants. Southern blot analysis of transgenic cassava plants revealed that they had between one to seven copies of the pHB1 and pJIT100 construct. The production of the first cassava plants carrying an agronomically important trait affecting starch biosynthesis is reported. Expression of the antisense AGPase B gene resulted in cassava plants within which the AGPase mRNA steady state levels was greatly decreased or even absent. The plants with no AGPase mRNA expression also had extremely low levels of starch, compared to control plants, as shown by iodine staining of in vitro thickened stems. In plants exhibiting the highest AGPase B antisense effect, starch formation was limited only to the epidermal layer of in vitro thickened stems.