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. 2021 Jul 29:12:704896.
doi: 10.3389/fpls.2021.704896. eCollection 2021.

An in vitro Propagation of Aspilia africana (Pers.) C. D. Adams, and Evaluation of Its Anatomy and Physiology of Acclimatized Plants

Denis Okello  1   2 , Sungyu Yang  1 , Richard Komakech  1   3 , Endang Rahmat  1 , Yuseong Chung  1 , Roggers Gang  1   2   4 , Yong-Goo Kim  1   5 , Francis Omujal  3 , Youngmin Kang  1   2
Affiliations

An in vitro Propagation of Aspilia africana (Pers.) C. D. Adams, and Evaluation of Its Anatomy and Physiology of Acclimatized Plants

Denis Okello et al. Front Plant Sci. .

Abstract

Aspilia africana (Pers.) C. D. Adams is an important medicinal plant, that has been used as traditional medicine in many African countries for the treatment of various health problems, including inflammatory conditions, osteoporosis, tuberculosis, cough, measles, diabetes, diarrhea, malaria, and wounds. We developed an efficient and reproducible protocol for in vitro regeneration of A. africana from nodes. We assessed the effects of plant tissue culture media on A. africana growth, cytokinins for in vitro shoot regeneration and proliferation, and auxins for the rooting of regenerated shoots. Furthermore, chlorophyll content, photosynthetic rates, anatomy (leaves, stems, and roots), and Fourier transform near-infrared (FT-NIR) spectra (leaves, stems, and roots) of the in vitro regenerated and maternal A. africana plants were compared. Murashige and Skoog media, containing vitamins fortified with benzylaminopurine (BA, 1.0 mg/l), regenerated the highest number of shoots (13.0 ± 0.424) from A. africana nodal segments. 1-naphthaleneacetic acid (NAA, 0.1 mg/l) produced up to 13.10 ± 0.873 roots, 136.35 ± 4.316 mm length, and was the most efficient for rooting. During acclimatization, the in vitro regenerated A. africana plants had a survival rate of 95.7%, displaying normal morphology and growth features. In vitro regenerated and mother A. africana plants had similar chlorophyll contents, photosynthetic rates, stem and root anatomies, and FT-NIR spectra of the leaf, stem, and roots. The established regeneration protocol could be used for large-scale multiplication of the plant within a short time, thus substantially contributing to its rapid propagation and germplasm preservation, in addition to providing a basis for the domestication of this useful, high-value medicinal plant.

Keywords: Aspilia africana; FT-NIR; anatomy; in vitro propagation; micropropagation; nodal segments; physiology.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Summary of in vitro Propagation of A. africana. (A1) Seeds of A. africana. (A2) Four-month-old A. africana maternal plant. (B1) Nodal segments inoculated in MS medium supplemented with BA (1.0 mg/l). (B2) Morphology of inoculated nodal segment showing the shoot bud. (C) Proliferated shoots in same medium [in (B)] after 3 weeks. (D) Proliferated shoots in same medium as in (B) after 6 weeks. (E) Fully developed roots in MS medium supplemented with NAA (0.1 mg/l) at 4 weeks. (F) Plantlets removed from medium and roots washed. (G) Regenerated A. africana plantlets transferred to soil and covered with polythene bags to maintain moisture (H). acclimatized potted plants.
FIGURE 2
FIGURE 2
Chlorophyll content measurements of A africana in vitro regenerated and maternal plant leaves. (A) Chlorophyll content of leaf being measured using SPAD instrument. (B) Different points/regions on a leaf considered for the average chlorophyll content of each leaf. (C) Chlorophyll contents of in vitro generated A africana plants compared to that of the maternal plants over a period of 8 weeks.
FIGURE 3
FIGURE 3
Comparison of mean Fv/Fm ratio of in vitro regenerated and maternal A. africana plants over a period of 8 weeks.
FIGURE 4
FIGURE 4
(A) Comparison of FT-NIR spectral lines of samples from different parts of in vitro regenerated and maternal A. africana plants. (B) Clustering Dendrogram for the different samples of in vitro regenerated and maternal A. africana plants analyzed from FT-NIR. In vitro regenerated A. africana plant samples analyzed: IL-leaf, ISt-stem, and IR-root. Maternal A. africana samples analyzed: ML-leaf, MSt-stem, and MR-root.
FIGURE 5
FIGURE 5
Transverse sections of A. africana in vitro regenerated and maternal plant tissues. (A) In vitro plant root. (B) Maternal plant root. (C) In vitro plant stem. (D) Maternal plant stem. (E) In vitro plant leaf. (F) Maternal plant leaf. cor, cortex; xy, xylem; ph, phloem; ep, epidermis; col, collenchyma; sc, secretory duct; pa, palisade parenchyma; sp, sponge parenchyma; tr, trichome. Scale bars = 100 pm.
FIGURE 6
FIGURE 6
Plant tissue culture media effects on shoot growth of A. africana plants from shoot tip explants. (A) Effect on shoot lengths. (B) Effect on leaf numbers. (C) Effect on fresh weights. (D) Effect on overall growth indices. Same letter are not significantly different by Tukey’ s test and p = 0.05.
FIGURE 7
FIGURE 7
Effects of different cytokinins on in vitro shoot regeneration of A. africana from nodal explants. (A) Effects on percentage regeneration. (B) Effects on of shoot numbers formed per nodal explants. Same letters are not significantly different by Tukey’s test and p = 0.05.
FIGURE 8
FIGURE 8
Effects of auxins on in vitro rooting of regenerated A. africana shoots. (A) Effects on percentage rooting. (B) Effects on number of roots formed per shoot. (C) Effects on root lengths. Same letters are not significantly different by Tukey’s test and p = 0.05.
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