Transient Expression of Red Fluorescent Protein Gene from Discosoma sp. in Calli and Immature Embryos of Different Maize (Zea mays L.) Genotypes

To demonstrate the feasibility of the transformation and expression of a foreign gene, transient expression of red fluorescent protein (RFP) gene, DsRed2, was investigated in maize embryogenic calli and 5-d old pre-cultured maize embryos using Particle Inflow Gun technology. The study involved two phases - (1) looking into the response of maize genotypes to culture media and subculture cycles and (2) determination of the optimal bombardment parameters affecting the efficiency of the transient expression of RFP gene. Ten maize genotypes were used to test for their response to induction of callus using two kinds of media, NC6 Dicamba (NC6DIC), a synthetic auxin and silver nitrate. Embryogenic calli were seen in cultures grown in NC6DIC with silver nitrate and best observed in the third subculture cycle. Out of 10 genotypes used, five responded favorably to NC6DIC with silver nitrate, namely; IPB Pi17, CML 161, IPB Var 4, CML 482 and IPB P51. Using nested RCBD, results showed that the production of calli is dependent on genotypes, media and/or subculture cycles. Previously incubated embryogenic calli and pre-cultured embryos were subjected to bombardment. The bombardment phase followed the 4-hr and 16-hr post-bombardment incubation in an osmoticum containing NC6 medium supplemented with 0.2 M mannitol and 0.2 M sorbitol and the gene construct coated with 0.7 mu m tungsten microparticles. The percent RFP transient expression (%TE) in both bombarded pre-cultured embyos and calli observed after 24 hr was highest in genotype IPB P51 at 400kPA. At acceleration pressure of 800kPA, %TE in IPB 51 was relatively lower than that of IPB Var 4 which exhibited the highest %TE of 70%. The number of fluorescent loci was not significantly different in embryos and maize calli bombarded at 400 and 800 kPA at 10 cm and at 25 '' Hg vacuum pressure. The results of the study are of potential use in stable transformation in the future.