MICROPROPAGATION AND PLOIDY STABILITY OF Lippia lacunosa Mart. & Schauer: AN ENDANGERED BRAZILIAN MEDICINAL PLANT

Abstract

Lippia lacunosa is a Brazilian savanna plant that belongs to the Verbenaceae family. It has been used in folk medicine as a treatment for different diseases. This species represents an endangered Brazilian medicinal plant, and this is the first report documenting a reliable protocol for the in vitro propagation and regeneration of L. lacunosa. Axenic explants were cultivated in MS medium containing different concentrations of naphthalene acetic acid (NAA) to induce root growth. The mean shoot length and the number of roots were highest with 0.06 mg·L-1 NAA. The highest number of buds in shoot regeneration was induced with 2 mg·L-1 6-benzylaminopurine (BA). To obtain a long-term culture, the dwarf shoots were elongated on MS media containing 0.5 mg·L-1 BA alternated with MS containing 2 mg·L-1 BA every 40 days. In the present protocol, the long-term shoots retained the ability to root even after long periods of BA treatment. In addition, we evaluated the nuclear DNA content and ploidy levels, including the occurrence of endopolyploidy, in long-term micropropagated plant leaves using flow cytometry analysis. The plants propagated in vitro over several years possessed nuclear DNA contents ranging from 2.940 to 3.095 pg, and no differences in DNA content were found among in vitro plants or between these plants and the control (L. lacunosa from a greenhouse with a DNA content of 3.08 pg). The flow cytometry analysis also demonstrated that there was no polyploidization. The present study will be useful for biotechnological approaches and provides the first estimate of the nuclear DNA content of this species using flow cytometry.

High-Frequency Plant Regeneration, Genetic Uniformity, and Flow Cytometric Analysis of Regenerants in Rutachalepensis L.

“…In the present investigation, no major differences between fluorescence peak derived from nuclei of Ruta chalepensis plantlets and ex vitro plants were found. The findings of flow cytometric analysis are consistent with those obtained from earlier research on Mentha arvensis [69], Puya berteroniana [112], Solanum lycopersicum [113], Cucumis melo [114], Bacopa monnieri [34], Curcuma zedoaria [72], and Juglans regia [115].…”

“…All RAPD and DAMD-generated bands were monomorphic and identical, confirming the complete absence of somaclonal variations in regenerated plantlets. It has been shown that the use of RAPD and DAMD molecular markers is efficient in determining the genetic stability of regenerated plants in a variety of medicinal plant species, including Withania somnifera [108], Henckelia incana [109], Avicennia marina [110], Ruta graveolens [18], Curcuma zedoaria [72], Artemisia vulgaris [100], Anarrhinum pubescens [111], and Anthurium andraeanum [106]. In recent years, it has been shown that a flow cytometry-based approach for evaluating in vitro raised plantlets is an excellent technique for assessing the clonal integrity and ploidy status in micropropagated plants [34,71,72,112,113].…”

“…It has been shown that the use of RAPD and DAMD molecular markers is efficient in determining the genetic stability of regenerated plants in a variety of medicinal plant species, including Withania somnifera [108], Henckelia incana [109], Avicennia marina [110], Ruta graveolens [18], Curcuma zedoaria [72], Artemisia vulgaris [100], Anarrhinum pubescens [111], and Anthurium andraeanum [106]. In recent years, it has been shown that a flow cytometry-based approach for evaluating in vitro raised plantlets is an excellent technique for assessing the clonal integrity and ploidy status in micropropagated plants [34,71,72,112,113]. In the present investigation, no major differences between fluorescence peak derived from nuclei of Ruta chalepensis plantlets and ex vitro plants were found.…”

“…Homogeneity of in vitro propagated plants can also be ascertained using flow cytometry, which offers a quick, accurate, and simple method for assessing the ploidy level, genome size, cell cycle, and DNA content within plant nucleus homogenates [60,68]. This technique has been successfully employed to determine the nuclear DNA content, genome size, and ploidy level in Mentha arvensis [69], Pongamia pinnata [70], Nardostachys jatamansi [71], Bacopa monnieri [34], Curcuma zedoaria [72], and Lippia lacunosa [73].…”

In vitro propagation from nodal segments of Lippia origanoides (chemotype A)

“…Although, the in vitro micropropagation technique has been efficient and described for some species of the genus Lippia (CASTELLANOS-HERNÁNDEZ et al, 2013;GUPTA et al 2001;JOSÉ et al 2019;JULIANI et al 1999;PEIXOTO et al 2006;RESENDE et al 2015), currently, there is only a single report on the effects of different PGRs (benzylaminopurine, indole acetic acid and kinetin) on in vitro development of L. origanoides (CASTILHO et al 2019). It is important to point out that this approach does not describe a useful methodology for ex vitro acclimatization.…”