Regeneration Potential and Transient Expression of B-glucuronidase Mediated via Agrobacterium tumefaciens in Immature Embryos of Kenya Maize Genotypes

Abstract

Production of maize is constrained by both abiotic and biotic stress factors in the field and by post-harvest pest problems; the most important being the larger grain borer, Prostephanus truncatus (Horn) and maize weevil, Sitophilus zeamais. Developing insect resistance crop varieties through conventional plant breeding is elusive, expensive and time consuming due to the limited genetic variation within the maize genotypes as well as difficulty in maintaining quantitatively controlled traits such as insect resistant. However, by using genetic engineering tools, modified novel genes (e.g. from Bt or plant proteins) can be introduced into maize to produce transgenic maize that confer resistance to these insects pests. The present study investigated regeneration potential of Kenyan maize genotypes as a prerequisite to genetic transformation. Twelve parental inbred lines and their respective single cross hybrids were planted in Kiboko and Kabete and evaluated for callus induction, somatic embryo formation and subsequent plant regeneration. Embryos were excised from surface sterilized kernels harvested at different physiological stages namely 10, 15, 18, 21 and 24 days after pollination (DAP). They were used as explants to initiate callus on N6 induction media with varying levels of 2,4-D (020mg/L) supplemented with 2.87g/L proline, 0.1 g/L casein hydrolysate, 2g/L glycine, 30g/L sucrose and 3g/L gelrite. The pH was adjusted to 5.8 before autoclaving. The induction frequency of primary calli at 2mg/L 2, 4 D was genotype dependent. Callus induction ranged from 80-90% for hybrids and 50-80% inbred lines. Following two biweekly subculture, the embryogenic calli formation was initiated. Three types of calli were initiated: Type I accounted for 17.6%, type II 52.9% while the remaining 29.4% made up type 0. Using this system, somatic embryo competence was demonstrated in 6 inbred lines and 4 hybrids. However, plant regeneration was only achieved in 4 inbred lines and 3 hybrids. The frequency of shoot formation ranged between 4-40%. The development of this efficient and reproducible regeneration system sets a basis for genetic transformation via Agrobacterium tumefaciens. Six Agrobacterium strains carrying two types of plasmids (pBECK2000.4) and (pCAMBIA2301) were used to introduce Gus A (Gus) reporter gene encoding -(3-glucuronidase to maize embryogenic tissues. The expression of gus activity on transformed embryogenic tissues was evaluated by histochemical staining with X-Gluc. Gus staining revealed variation in both intensity and pattern of blue staining in embryos transformed with the same plasmid and bacterial strain. EHA105(pCAMBIA2301) AGL1(pBECK) revealed high infectivity across various genotypes. Fifteen days (15 DAP) was optimal embryo stage for gus expression while incubation for 24 hrs was appropriate for co cultivation stage. There was no gus activity in bacterial strains that were devoid of plasmid.