In vitro regeneration of popular Kenyan dryland maize hybrids

Abstract

Immature embryos are widely used as explants in cereals, including maize. The objectives of this study are: 1) to evaluate the capacity immature maize embryos to form embryogenic calli, and 2) to evaluate the regeneration potential of selected maize hybrids. Immature embryos were isolated from four selected Kenyan dryland maize hybrids; Dryland Hybrid (DH) 02, Katumani (KAT), Pwani Hybrid (PH) 01 and PH 04 at different days post pollination. The embryos were cultured in N6 medium supplemented with proline, casein hydrolysate, silver nitrate and 2% sucrose containing different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), Calli started developing after 3-7 days of culture with best results obtained with 1.5-2.0 mg/L 2,4-D in all the hybrids. The calli were initiated in total darkness at 26 ± 20C for two weeks and then sub-cultured in a maintenance medium (N6) with conditions and supplements similar to those in calli initiation medium but devoid of silver nitrate White round somatic embryos started developing after 6-10 days of culture in maintenance medium with PH 01 showing the best response. The induction frequency of primary calli was over 70% for all hybrids tested. Embryogenic calli were sub-cultured in somatic embryo maturation medium composed of N6 medium supplemented with glycine, 1-naphthalene acetic acid (NAA) with 60g/L sucrose in total darkness. Mature somatic embryos were sub-cultured in shoot promotion medium consisting of Murashige and Skoog (MS) medium supplemented with glycine, 20g/1 sucrose with different concentration of 6-benzyl amino purine (BAP). Cultures were incubated under a photoperiodic regime of 16 hours of light and 8 hours of darkness. The highest number of shoots was obtained with hormone free MS medium. Root formation was promoted by sub-culturing the shoots in half strength MS medium supplemented with 20g/L sucrose with NAA concentration of 0.1 mg/L. Transfering regenerants to the pots containing sterilized vermiculite and sand in the ratio of 2:1 respectively acclimatized the planets before transplanting to soil in pots. The protocol developed in this study was used to regenerate twenty-five plants, which could easily be established in the field. The success in developing this protocol provides the basis for improvement of Kenyan maize genotypes tolerant to both biotic (e.g. diseases and insect pests) and abiotic (e.g. drought) stresses through genetic engineering.