PINPOINTING TOWARDS IMPROVED TRANSFORMATION AND REGENERATION OF CASSAVA (MANIHOT-ESCULENTA CRANTZ)

Friable embryogenic callus (FEC) of the cassava genotype 60444 was tra nsformed 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 h ad 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 unde r the controled a double CaMV35S promoter. A total of 2 weeks after bo mbardment, luciferase (LUC) positive FEC units (spots) were isolated a nd subcultured separately for further proliferation. A total of 4 week s later, those cultures having at least four positive LUC spots were s ubjected to three different selection regimes namely: stringent LUC se lection, 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 LU C positive. Within the same time period non stringently LUC selected c ultures and LUC/PPT selection had only 1 and 41% of the units that wer e LUC positive, respectively. The number of LUC positive mature somati c embryos formed was similar to the percentage of LUC positive FEC uni ts, within a culture, found with each selection method. Stringent LUC selection enabled transgenic plants to be produced in 28-36 weeks comp ared to 32-41 weeks for LUC/PPT selection and 53-78 weeks for non stri ngent LUC selection. This indicates that stringent selection is a more efficient method for obtaining transgenic cassava plants. Southern bl ot analysis of transgenic cassava plants revealed that they had betwee n one to seven copies of the pHB1 and pJIT100 construct. The productio n of the first cassava plants carrying an agronomically important trai t affecting starch biosynthesis is reported. Expression of the antisen se AGPase B gene resulted in cassava plants within which the AGPase mR NA steady state levels was greatly decreased or even absent. The plant s with no AGPase mRNA expression also had extremely low levels of star ch, compared to control plants, as shown by iodine staining of In vitr o thickened stems. In plants exhibiting the highest AGPase B antisense effect, starch formation was limited only to the epidermal layer of i n vitro thickened stems. (C) 1998 Elsevier Science Ireland Ltd. All ri ghts reserved.