Molecular identification and elimination of endophytic contamination using antibiotics from in vitro culture of Vitex peduncularis Wall. Ex Schauer
摘要
Vitex peduncularis Wall. Ex Schauer, a medicinal tree in Jharkhand, is rich in flavonoids, iridoids, terpenoids, and essential oils. Its low germination rate, due to the hard seed coating, in vitro micropropagation presents a promising alternative for its propagation. During the establishment of the in vitro culture of V. peduncularis, nodal explants were surface sterilized using 0.1% HgCl2 for 8 min, resulting in a maximum survival rate of 76.19 ± 0.95% with minimal contamination (19.04 ± 1.90%). Despite this, endophytic bacterial contamination caused explant death after the first subculture. To address this contamination, antibiotics (gentamicin, streptocycline, kanamycin, and neomycin) were incorporated into Murashige and Skoog (MS) medium at concentrations of 250–1000 ppm. Among these antibiotics, 500 ppm of kanamycin exhibited the highest efficacy in inhibiting contamination, followed by streptocycline. The endophytic bacteria isolated from the inoculated nodal explants were identified as Pseudoxanthomonas japonensis by 16S rRNA. Following the bacterial identification, nodal explants were cultured on a bud induction medium without antibiotics. MS medium supplemented with 5 µM BAP and 2.5 µM kinetin increased bud break response (94.28 ± 1.64%), promoting shoot proliferation and lateral bud outgrowth. Furthermore, BAP (5 µM), kinetin (2.5 µM), NAA (0.5 µM) glutamine (100 µM), and phloroglucinol (792.95 µM) gave the highest number of shoots (2.58 ± 0.01) and shoot length (4.47 ± 0.01 cm). The synergistic effects of cytokinin and phloroglucinol significantly enhanced V. peduncularis growth. This study highlights antibiotics in preventing contamination and improving micropropagation strategies for the sustainable development and conservation of V. peduncularis.