Transformation of tef (Eragrostis tef) by Agrobacterium through immature embryo regeneration system for inducing semi-dwarfism

Abstract

Successful application of genetic transformation for integration of a transgene is much dependent upon availability of an efficient in vitro plant regeneration procedure and detection of transgene insertion and expression. Isolated immature embryos (IEs) of E. tef cultivar DZ-01-196 were used for embryogenic callus formation and the callus was transformed with GA inactivating gene PcGA2ox under the control of a triple CaMV 35S promoter using Agrobacterium transformation procedure. Embryogenic callus was induced from immature embryos in a medium containing KBP minerals in the presence of 2,4- dichlorophenoxiyacetic acid. The embryogenic calli were further inoculated with Agrobacterium and the calli were grown in co-cultivation medium (CCM) followed by selection in KBP and regeneration (K4NB) media. Putatively transformed E. tef embryogenic calli were tolerant to treatment with the selectable marker kanamycin, while 75mg l-1 geneticin inhibited growth of non-transformed shoots derived from matured embryos completely after 12 days. A total of 55 plants were regenerated from all the embryogenic calli to fully viable plants setting seeds at maturity. Eight putatively transformed T0 plants were produced carrying the transgene in their genome which was detected by PCR. Sequence analysis confirmed amplified PCR products to have 97.2 and 99.8% sequence identity to PcGA2ox and nptII, respectively. However, detection of the transgene, PcGA2ox or nptII, in T1 plants was inconsistent although phenotypic analysis of T1 plants showed changes in pheno-morphic and agronomic characters such as plant height, number of internodes, tillering, panicle length, biomass, yield as well as GA content. Culm reduction was due to absence of elongation of the upper-most internodes. Panicle length in semi-dwarfed plants showed no relation with culm length. GA analysis showed plants with semi-dwarf phenotype to be associated with a low level of bioactive GA1 and its immediate precursors. Up to 3.7 fold increase in grain yield per plant was found in some semi-dwarfed plants.