Protocol development for micropropagation of Phyllanthus niruri linn. (dukung anak) via in vitro culture

Phyllanthus niruri, commonly known as Dukung Anak or Gale of Stone, is an herbaceous plant belonging to the family Phyllanthaceae that has commonly been distributed in tropical countries, including Malaysia. Even though this plant is considered a problematic weed for farmers, it has been used traditionally to treat various ailments such as diabetes, flu, and cough. Overexploitation in Malaysia, on the other hand, has resulted in wide destruction of this plant from its natural habitat. Besides, there is no information on the production of P. niruri in Malaysia. This study has provided a solution to this problem through in vitro technology approach. Specifically, this study's objectives were to study the growth efficiency of P. niruri with different surface sterilants, to study in vitro growth responses of P. niruri towards different growth factors, and to determine the factors affecting in vitro rooting and acclimatization of P. niruri. P. niruri explant's nodal segment was prepared for surface sterilization using Clorox® and nanosilver with 10, 20, 30 (% and ppm, respectively). The aseptic culture was established with the lowest percentage of contamination, 4.44% using 30% Clorox®. The explant from the previous experiment was multiplied by different plant growth factors, which were different types of basal media and its strength, different cytokinin and its concentrations, different carbon sources and its strength, and different initial pH of basal media. Murashige and Skoog (MS) media and Gamborg B5 (B5) with the strength of half, full and double were used in the first experiment. Full-strength MS media produced a decent number of shoots with 2.33 shoots, 3.11 cm length of shoots and 27.91 number of leaves. In a subsequent experiment, MS media was supplemented with different types of cytokinin, which were 6-benzylaminopurine (BAP), kinetin (Kn), zeatin (Zn), and 2-isopentenyl adenine (2iP) with different concentrations of 2.5, 5.0, 7.5, and 10 μM. The best treatment was obtained from MS media without cytokinin (control), which produced 5.00 number of shoots, 3.68 cm length of shoot, and 27.33 number of leaves. In the next experiment, basal media were manipulated with different carbon sources like sucrose, fructose, and glucose at different concentrations of 20, 25, 30, 35, and 40 g/L. The result showed the explant in media enriched with 30 g/L of sucrose has the highest number of shoots with an average of 3.2 shoots and shoot elongation of 2.27 cm. Lastly, the initial pH media was tested at 5.2, 5.4, 5.6, 5.8, 6, and 6.2. The highest number of shoots was recorded at a pH level of 5.6 (2.93). In the last experiment, the explant was used for root induction using different auxins at different concentrations. There were indole-3-butyric acid (IBA), 1-naphthalene acetic acid (NAA), and indole-3-acetic acid (IAA) at 1.25, 2.5, and 5 μM, respectively. Media with 2.5 μM IBA produced the highest number of the shoot and the most extended length of the root with 17.92 shoots and 1.29 cm root length. Lastly, the explant was acclimatized ex vitro in different potting media; peat moss: cocopeat, peat moss: vermiculite, peat moss: perlite at a ratio of 1:1 w/w and mixed of all media peat moss: cocopeat: vermiculite: perlite at a ratio of 1:1:1:1 w/w. The plantlet in potting media consisting of peat moss: cocopeat (1:1 ratio) showed the best performance with 88% plant survival. In conclusion, protocol for micropropagation of P. niruri has been developed in this study.

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