Molecular Assessment of Clonal Fidelity in In Vitro-Regenerated Heinsia crinita (African bush apple) Micro Shoots Using ISSR and SCoT markers
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Abstract
The African bush apple Heinsia crinita (Wennberg) G. Taylor is a valuable orphan shrub with important nutritional and medicinal uses, especially in southern Nigeria. To support germplasm conservation and biotechnology-driven improvement, we previously developed the first in vitro regeneration system for the crop using stem and hypocotyl explants. In this study, genetic stability was assessed in ten in vitro regenerants, comprising five stem-derived and five hypocotyl-derived microshoots, using 10 Inter-Simple Sequence Repeat (ISSR) and 3 Start Codon-Targeted (SCoT) primers selected for clear and reproducible amplification. Using ISSR primers, 38 reproducible bands (250 ~ 2200 bp) were amplified, and microshoots regenerated from stem and hypocotyl explants showed 5.26% and 0.00% loci polymorphism, respectively. The SCoT primers produced 6 reproducible bands (150–2100 bp) and showed 0.00% polymorphism across both explant types. The polymorphism assessment was based on the presence or absence of clear, reproducible bands across the regenerants relative to the mother plant. Similarity coefficients ranged from 0.955 to 1.000, and UPGMA clustering grouped the regenerants into one major cluster with two minor sub-groupings. The mother plant clustered with all hypocotyl-derived microshoots and most stem-derived microshoots, indicating a high degree of apparent genetic similarity. Overall, hypocotyl-derived regenerants showed slightly greater stability in this dataset, however further validation using additional markers as well as larger sample sizes, would strengthen the recommendation
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