Indirect in vitro Regeneration of the Medicinal Plant, Aspilia africana, and Histological Assessment at Different Developmental Stages

Abstract

The medicinal plant, <i>Aspilia africana</i>, has been traditionally used in several African countries to treat many diseases such as tuberculosis, cough, inflammation, malaria, osteoporosis, and diabetes. In this study, we developed a protocol for <i>in vitro</i> propagation of <i>A. africana</i> using indirect shoot organogenesis from leaf and root explants of <i>in vitro</i>-grown seedlings and assessed the tissues at different developmental stages. The highest callus induction (91.9 ± 2.96%) from leaf explants was in the Murashige and Skoog (MS) medium augmented with 1.0 mg/L 6-Benzylaminopurine (BAP) and 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) while from root explants, the highest callus induction (92.6 ± 2.80%) was in the same plant tissue culture medium augmented with 0.5 mg/L BAP and 1.0 mg/L 2,4-D. The best shoot regeneration capacity from leaf-derived calli (i.e., 80.0 ± 6.23% regeneration percentage and 12.0 ± 6.23 shoots per callus) was obtained in medium augmented with 1.0 mg/L BAP and 0.05 mg/L α-Naphthaleneacetic acid (NAA); the best regeneration capacity for root-derived calli (i.e., 86.7 ± 6.24% shoot regeneration percentage and 14.7 ± 1.11 shoots per callus) was obtained in the MS medium augmented with 1.0 mg/L BAP, 0.05 mg/L NAA, and 0.1 mg/L Thidiazuron (TDZ). Regenerated plantlets developed a robust root system in 1/2 MS medium augmented with 0.1 mg/L NAA and had a survival rate of 93.6% at acclimatization. The <i>in vitro</i> regenerated stem tissue was fully differentiated, while the young leaf tissue consisted of largely unorganized and poorly differentiated cells with large intercellular airspaces typical of <i>in vitro</i> leaf tissues. Our study established a protocol for the indirect regeneration of <i>A. africana</i> and offers a basis for its domestication, large-scale multiplication, and germplasm preservation. To the best of our knowledge, this is the first study to develop an indirect regeneration protocol for <i>A. africana</i> and conduct anatomical assessment through the different stages of development from callus to a fully developed plantlet.