An Optimized Protocol for in Vitro Regeneration of Tropical Maize Inbred Lines through Cell Suspension and Semi-Protoplast Cultures
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Date
2023
Authors
Moemi, Billiah
Masanga, Joel
Runo, Steven
Journal Title
Journal ISSN
Volume Title
Publisher
AJB
Abstract
Maize being an important staple food crop is widely consumed in Kenya but its production remains low
due to biotic and abiotic challenges that have not been addressed sufficiently through conventional
breeding. This study sought to optimize the establishment of cell suspension and protoplast cultures
for 10 tropical inbred maize genotypes in order to identify genotypes that can readily establish in liquid
cultures and those whose cell suspensions and protoplasts are capable of in vitro regeneration. Callus
were induced using immature zygotic embryos on Murashige and Skoog (MS) medium supplemented
with 3 mg/L 2, 4-D, 5 mg/L dicamba or 5 mg/L picloram. Dicamba and picloram induced friable calli that
readily dispersed into cell suspensions in liquid MS medium supplemented with 0.1 g/L asparagine and
either 0.4 or 0.8 g/L proline. Cell growth was determined by packed cell volume (PCV) every seven days.
The highest PCV (240 µl/ml) was recorded in genotype EO4 followed by CML 216 (188 µ/ml). Optimal
growth was observed in cells maintained in MS Amended with 0.4 or 0.8 g/L of proline in combination
with 0.1 g/L asparagine compared to medium without proline. It was also observed that cells cultured in
media with reduced Ammonium Nitrate (12 fold reduction) recorded higher PCV values than controls.
Protoplasts were generated from the resulting cells using 2% cellulase and 0.5% pectolyase in an
enzyme digestion cocktail containing Mannitol and Calcium Chloride (MaCa) and washed in MS with
vitamins containing Mannitol (MSMa). Only cell clusters of genotype EO4 gave rise to plants with a
regeneration frequency of 42.51%. In conclusion, success was achieved in callus initiation, formation of
cell suspension cultures and their eventual regeneration into whole plants for selected tropical maize
genotypes.
Description
Article
Keywords
Cell suspensions, callus, somatic embryos, packed cell volume, protoplasts
Citation
Moemi, B., Masanga, J., & Runo, S. (2023). An optimized protocol for in vitro regeneration of tropical maize inbred lines through cell suspension and semi-protoplast cultures.