Regeneración de plantas a partir de cultivos de callos de <i>Vitex trifolia</i> (Lamiales: Lamiaceae): una planta medicinal potencial

Abstract

Regeneración de plantas a partir de cultivos de callos de Vitex trifolia (Lamiales: Lamiaceae): una planta medicinal potencial. Vitex trifolia is a shrub species with popular use as a medicinal plant, for which leaves, roots and flowers have been reported to heal different distresses. The increasing exploitation of these plants has endangered its conservation, and has importantly justified the use of biotechnological tools for their propagation. Our aim was to present an efficient protocol for plant regeneration through organogenesis; and simultaneously, to analyze the genetic homogeneity of the established clonal lines by Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Plantlet regeneration was achieved in callus cultures derived from stem, leaf and petiole explants of V. trifolia on a differently supplemented Murashige & Skoog medium, and incubated at 25±2ºC under a light intensity of 61µmol/m 2 s from cool white fluorescent lamps and a 16h photoperiod. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more rapidly from stem and petiole explants as compared to leaf explants on medium containing 11.10µM BAP in combination with 0.54µMNAA. Addition of 135.74-271.50µM adenine sulphate (Ads) and 0.72-1.44µM gibberellic acid (GA 3) to the culture medium increased the growth of shoot buds. The highest rate of shoot bud regeneration responses was obtained in stem explants using 11.10µM BAP in combination with 0.54µM NAA, 271.50µM Ads and 1.44µM GA 3. In vitro rooting of the differentiated shoots was achieved in media containing 1.23µM indole butyric acid (IBA) with 2% (w/v) sucrose. Regenerated plantlets were successfully established in soil with 86% survival under field condition. Randomly Amplified Polymorphic DNA and Inter Simple Sequence Repeat markers analyses have confirmed the genetic uniformity of the regenerated plantlets derived from the second up to fifth subcultures. This protocol may help in mass propagation and conservation of this important medicinal plant of great therapeutic potential.

Biotechnological advances in Vitex species, and future perspectives

“…86% of shoots rooted.∼90% survival of acclimatized plants in soil, sand and well decomposed manure (1:1:1). Genetic uniformity of regenerated plantlets of subculture 2–5 confirmed by RAPD and ISSR.[81] V . trifolia Shoot tips (5–8 mm long) from young shoots of 3-y-old treeMS + 5.0 μM TDZ (SIM).…”

“…Explants from in vitro germinated seedlings (hypocotyls, cotyledons, roots and shoot tips) can be employed [22], although seed-derived material is genetically dissimilar and is thus not suitable for clonal tissue culture experiments. There is one report on somatic embryogenesis but none on direct organogenesis (adventitious shoot regeneration) in the Vitex genus, although there are reports on callus-mediated organogenesis in V. trifolia [15], [35], [81], V. negundo [103], [26], [79], V. rotundifolia [72] and V. leucoxylon [25]. The chances of somaclonal variation are greater in the case of callus-mediated organogenesis or somatic embryogenesis [52], [62], although the latter can provide a useful platform for genetic transformation studies.…”

“…Ahmad and Anis [5] isolated DNA from micropropagated and field-grown plants (plant parts and other conditions were not reported) by the Doyle and Doyle [36] method and used random amplified polymorphic DNA (RAPD) markers (10 GC-rich decamer primers, OPA1-10) to verify the genetic fidelity of two-year-old micropropagated V. negundo plants versus the mother plant (control), and detected no genetic variation. Samantaray et al Samantaray et al [81] isolated DNA from fresh leaves of micropropagated and a field-grown mother plant by the cetyltrimethyl ammonium bromide (CTAB) method [20] with minor modifications; 1% polyvinylpyrrolidone (PVP) was added to remove polyphenols and RAPD and inter simple sequence repeats (ISSR) were used to test the genetic fidelity of V. trifolia ; they found no genetic variation relative to the mother plant . Ahmad et al Ahmad et al [8] also carried out RAPD analysis of V. trifolia plantlets developed from axillary shoots and extracted DNA from young leaves using the [36] protocol.…”

“…Ahmad et al Ahmad et al [8] also carried out RAPD analysis of V. trifolia plantlets developed from axillary shoots and extracted DNA from young leaves using the [36] protocol. No phenotypic variation was observed in micropropagated plants of V. trifolia [81], [8] and V. negundo [5]. A wider selection of molecular markers could assist in the discrimination of adulterants from pure sources, and to screen out somaclonal variants derived from bioreactors, synthetic seeds, or cryopreservation.…”

“…trifolia Shoot tips → RTW 10 min → 5% Tween-20 15 min → DW → 0.5% NaOCl 5 min → DDW → 0.01% HgCl 2 10 min → 0.1% HgCl 2 5 min → Bavistin® 30 min → DDW 1×[64] V . trifolia 5–7 cm long shoots → 0.1% HgCl 2 20 min → rinse 4–5 × in SDW[81]AA, ascorbic acid; CA, citric acid; DDW, double distilled water; DW, distilled water; EtOH, ethyl alcohol (ethanol); HgCl 2 , mercuric chloride; NaOCl, sodium hypochlorite; NR, not reported; RTW, running tap water; s, second(s); SDW, sterilized (by autoclaving) distilled water; SDDW, sterilized (by autoclaving) double distilled water; SW, sterile water.…”