Phyllanthus niruri (P. niruri) or Dukung Anak is a herbal plant in the Phyllanthaceae family that has been used traditionally to treat various ailments such as diabetes, jaundice, flu and cough. P. niruri contains numerous medicinal benefits such as anti-tumor and anti-carcinogenic properties and a remedy for hepatitis B viral infection. Due to its beneficial properties, P. niruri is overharvested and wild plants become scarce. This study was conducted to develop an appropriate in vitro culture protocol for the mass production of P. niruri. An aseptic culture of P. niruri was established followed by multiplication of explants using different types of basal medium and its strength and plant growth regulators manipulation. This study also established the induction of in vitro rooting utilizing various types and concentrations of auxin. Treatment of Clorox® with 30% concentration showed the lowest percentage (%) of contamination, 4.44% in P. niruri culture. Nodal segments of P. niruri were successfully induced in full-strength of Murashige and Skoog (MS) basal media with 2.33 number of shoots, 3.11 cm length of shoot and 27.91 number of leaves. In addition, explants in full-strength MS media without any additional cytokinin were recorded as the optimum results for all parameters including the number of shoots (5.0 shoots), the length of shoots (3.68 cm) and the number of leaves (27.33 leaves). Treatment of 2.5 µM indole-3-butyric acid (IBA) showed the highest number of roots (17.92 roots) and root length (1.29 cm). Rooted explants were transferred for acclimatization, and the plantlet showed over 80% of survival rate. In conclusion, plantlets of P. niruri were successfully induced and multiplied via in vitro culture, which could be a step closer to its commercialization.
1. Introduction
Over the past several years, medicinal plants have gained greater attention and recognition in Malaysia, particularly as demand for alternative medicine and natural health products increases in 2019
[1,2][1][2]. A total of 351 hectares of Malaysian land has yielded 1317 metric tonnes of herbs in 2007. Since then, herb production grows to 2800 metric tonnes from 578 hectares of land cultivated with different herb species
[3]. In addition, the Malaysia Economic Transformation Program, through the National Key Economic Areas for the agriculture sector, has identified numerous high potential herbs that can be commercially utilized as a new economic growth source in the herbal industry. One of these herbs is
Phyllanthus niruri (
P. niruri), identified locally as Dukung Anak
[4].
P. niruri (Phyllanthaceae) has traditionally been used to treat several ailments such as jaundice, kidney stone, flu, fever, and diabetes
[5,6,7][5][6][7]. Several researchers discovered that
P. niruri possesses anti-tumor, anti-oxidant, anti-carcinogenic, and hepatoprotective properties
[8,9,10][8][9][10]. In addition, certain phenolic compounds such as gallic acid, epicatechin, gallocatechin, epigallocatechin, epicatechin 3-O-gallate, epigallocatechin 3-O-gallate have gained significant interest from the researcher
[11]. Furthermore, a novel compound named niruriside was isolated from methanol extract of
P. niruri that can inhibit the binding of human immunodeficiency virus Rev protein (HIV REV)
[10]. These secondary metabolites have a wide range of therapeutic activities that have essential pharmacological effects on humans
[12]. Hence, many products were made from this medicinal plant have been successfully commercialized.
The main issue for the commercialization of herbal-based products is the uniformity and consistency of the planting materials
[13]. In addition, the growing demand for medicinal plants would undoubtedly decrease the sustainable supply of raw materials in the future
[14]. Overharvesting and unsustainable agriculture practices lead to many consequences, such as a limited supply of herbs
[15]. Moreover, to extract elevated amounts of secondary metabolites, large quantities of raw materials are needed
[16].
Plant micropropagation may serve as an alternative solution to ensure the sustainable supply of plant materials. It is an excellent technique to produce plants in a large amount in a short time
[17]. Micropropagation is the in vitro aseptic culture of cells, tissues, or even organs of plants grown under a controlled environment and nutrient media for growth and multiplication. This technology can be used to eliminate diseases, for secondary metabolite production, plant improvement, and conservation of threatened and endangered species
[18,19,20,21][18][19][20][21]. A study conducted by Padma and Ilyas
[22] resulted in a maximum number of
P. niruri shoots (15.28 ± 0.96) on MS medium supplemented with BAP (0.5 mg/L). In addition, a maximum number of
P. niruri shoots (3.16 ± 0.16) was achieved from nodal explants inoculated on MS medium supplemented with BAP (3.0 mg/L)
[23].
Appropriate selection of chemical sterilization is essential in the first stage of plant micropropagation. All microorganisms that potentially could contaminate the culture should be removed and eliminated
[24]. This step will establish an aseptic culture to be carried out in the following phase of micropropagation. On the other hand, numerous factors affect the explant’s development in the growth and multiplication phase
[25,26][25][26]. For instance, the source of plant material, kind of explant, the type of basal media and its strength and plant growth regulators and its concentration
[27].
Culture medium supplemented with plant growth regulators; cytokinin and auxin were used in many plants to propagate via in vitro techniques
[28,29][28][29]. Auxin and cytokinin play a crucial role in many aspects of plant development and growth
[30]. The interplay of auxin and cytokinin is particularly critical for controlling a few developmental processes, such as the production and maintenance of meristems, which are necessary for the establishment of the entire plant body
[31]. Cytokinins play a central role in the regeneration of multiple shoots in many medicinal plant species
[32,33][32][33] and sometimes in combination with auxins. Cytokinins trigger cell division and influence differentiation
[34], while auxins are a major signal for apical dominance
[35] and are responsible for elongation in phototropism and gravitropism
[36]. In vitro plantlets will then be transferred to potting media during acclimatization, the final stage. In this stage, the survival rate of plantlets is also influenced by the type of potting media
[37].
Due to its high medicinal values and properties,
P. niruri has captured people’s attention worldwide. The plant was harvested with no sanitary consideration taken on the collected samples. The explant obtained from wild or greenhouse is typically contaminated with microorganisms such as bacteria and fungi
[38]. In vitro contamination of plant cultures could be induced by either internal explant tissue or the presence of microorganisms on the surface of the explant
[39]. The microorganism would kill plants eventually, whether because of their overgrowth or the release of toxic substances into the basal medium. Hence, in the surface sterilization stage, the effectiveness of removing all microorganisms with minimal damage to the plant cell is essential. Chemical sterilants could be used to reduce the microbial contaminant and increase the percentage of survival at the same time
[40].
2. Results
2.1. Surface Sterilization
2. Surface Sterilization
Nodal segments of
P. niruri were cultured on MS medium without any plant growth regulators following the surface sterilization procedure. There were significant differences between different chemical sterilants and their concentrations on the percentage of contamination of explant of
P. niruri. Different concentrations of nanosilver showed no significant effect on their percentage of contamination (
Table 1). For Clorox
®, there was no significant difference between 10% and 20% Clorox
® on their contamination percentage. The contamination percentage on explants had no significant difference between 20% and 30% Clorox
®. However, 10% and 30% Clorox
® showed a significant difference in their percentage of contamination, whereby 10% Clorox
® showed 130% higher contamination than 30% Clorox
®.
Table 1. Effect of different chemical sterilants and concentrations towards the percentage of contamination (%) of P. niruri on week 2 of incubation.
Treatment/Chemical Sterilants |
Concentration |
Percentage of Contamination (%) |
Clorox® (%) |
12.00 ± 2.28 a |