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Jouf University Science and Engineering Journal 2018; 5(1): 29- 33 Published online June 
EFFECT OF PLANT GROWTH REGULATORS IN SHOOT REGENERATION OF PHALERIA MACROCARPA IN VITRO
ROSILAH AB AZIZ1, 2, NUR ATIQAH JAMARI2, IZZATUL ZULIANA ZOLKEFLI2

Phaleria macrocarpa (Mahkota Dewa) is a medicinal plant species widely used by old folks for its medicinal values. Mass propagation for this valuable plant is necessary to fulfill its high demand without disturbing the rainforest biodiversity. In this study, in vitro shoot regeneration from seed of P. macrocarpa was established. The explant was surface sterilized with 20% Clorox and aseptic seed was sub-cultured onto Murashige and Skoog (MS) media supplemented with different concentrations of 6-benzyloaminopurine (BAP) with or without combination of 0.1 mg/L of α-naphthalene acetic acid (NAA). The appearance of bacteria was first seen after two days followed by fungi after 1 week of culture. The percentage of seed clean culture was 18.57%, followed by bacteria contamination (41.43%) and fungi contamination (40%). Hypocotyl was first observed in all treatments after 2 weeks in culture. Among all treatments, explant cultured on MS0 gave the highest length of shoot (3.57±1.86). As a conclusion, all treatments successfully induced P. macrocarpa shoot.
Keywords: In vitro culture, Murashige and Skoog (MS) media, 6-benzyloaminopurine (BAP), α-naphthalene acetic acid (NAA), Phaleria macrocarpa.
         1. INTRODUCTION
Phaleria macrocarpa is an Indonesian plant of family Thymelaceae. This medicinal plant is indigenous to Indonesia and Malaysia and is commonly known as God's crown, Mahkota Dewa or Pau (1). P. macrocarpa fruit consist of pericarp, mesocarp and seed. Recent study showed that different parts of P. macrocarpa fruit contain bioactive compounds such as flavonoids, alkaloids, polyphenols and saponins (1). Phenolics are secondary metabolites which are synthesized by plants and are utilized as UV, wounding and infection protectant in plants (2). This compound has been related to have several biological activities such as antioxidant, antimutagenic, anticarcinogenic, anti- inflammatory and antimicrobial activities in human (3). The main constituents such as flavonoids, glycosides, saponin glycosides, phenolic compounds, steroids, tannins, and terpenoids that present in P. macrocarpa seeds contain bioactivities that include antibacterial, analgesic, antifungal, anti-inflammatory agents and cytotoxicity (4). P. macrocarpa has been widely used by the traditional folk from Indonesia and Malaysia to biotreat cancer, diabetes, common cold, viral infections, allergy problem, asthma, cardiovascular, high blood pressure, acne and insect bites (5). This plant is also believed to have potential to treat hypertension, diabetes, cancer and diuretic conditions (2) and also used to treat various skin diseases including acne (5). Therefore, due to these facts, the exploration of new alternative medicines derived from this plant is required (2).

Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant (totipotency) (6). This technique is capable to regenerate and propagate plants from single cells, tissues and organs that is keep in controlled and sterile environmental conditions (7). The technique is widely used for genetic improvement of crops, rapid multiplication of plants, obtaining disease-free clones and also for preserving valuable germplasm (8). This valuable technique is widely recognized for its contribution in improvement of the crop.
P. macrocarpa has already known as the medicine plant that gives many benefits to the health. For its potential, the demand for this medicine plant has greatly increase. Therefore, there is need for the continuous supply of raw materials in order to fulfill the increasing demand. Thus, in order to produce quality medicine product, the production of plant source must be carried out by using various strategies that include plant tissue culture technique. In this study, in vitro shoot regeneration of P. macrocarpa is established.
2. MATERIALS AND METHODS
2.1 Preparation of explant
The fruits of P. macrocarpa was obtained from Forest Research Institute of Malaysia (FRIM) and wet markets in Kuala Pilah, Malaysia. The outer layer of the fruits was cleaned with wet tissues and was put under running tap water for 15 minutes before being transferred into Scott bottle.
2.2 Surface sterilization of fruits
Once in the laminar air flow, the fruits were soaked in sterile distilled water with few drops of Tween 20 followed by washing with sterile distilled water for few times. The fruits were then agitated in 20% of Clorox for 10 min before rinsing several times with sterile distilled water and were air dried for 10 min.
2.3 Inoculation onto MS regeneration medium
Seed was dissected from the fruits under aseptic conditions and transferred onto MS regeneration medium supplemented with various concentrations of BAP with or without NAA
(Table 2). All cultures were maintained at 25±2 􏱍C with illumination powered by cool-white florescent light (16-h photoperiod). Cultures were transferred onto fresh media every 4 weeks. Observation was made every day for shoot regeneration and contamination. Cultures that were contaminated with bacteria or fungi is recorded and discarded by autoclaving.
3. RESULTS AND DISCUSSION
3.1 Percentage of seed clean culture after surface sterilisation
Surface sterilisation is most critical part in plant tissue culture. This step is crucial to produce clean culture that is necessary to proceed for the next step of producing shoot multiplication and rooting of plantlets. In this study, the percentage of clean cultures and contamination by bacteria and fungi were recorded after 4 weeks in culture (Table 1). It was observed that the surface sterilised techniques used in this study for P. macrocarpa produced 18.57% clean cultures, and cultures contaminated by bacteria were 41.43% followed by fungi (40%). In most cases, bacteria are the frequent contaminants (9). High contamination rates was due to the fact that explant from outdoor source may contains pathogens that affect the viability and the growth of in vitro shoot (9).
 Bacteria was first seen after two days of culture and fungi appeared after 1 week of culture. The bacteria appeared as creamish white (10) (Figure 1B) while fungi appeared as white and wooly colonies (11) (Figure 1C). Bacterial and fungal contamination is always a major problem in plant tissue culture and is well documented (12). Their presence obstructs successful in vitro propagation and establishment of aseptic cultures. The nutrient media that is used to cultivate plant tissue is a good source of nutrient for microbial growth. With their presence, in vitro plant tissues will have compete for nutrient for its growth (13).

In addition, the presence of microbes will result in increased plant culture mortality, variable culture growth, necrosis in plant tissue, reduced shoot proliferation and reduced rooting (14).
Contamination occurred in in vitro plant can be caused by exogenous and endogenous microbes. The endogenous microbes are embedded in the plant tissues (15) and the exogenous microbes may due to the unsterilized tool used for dissection and inoculation, and also improper handling of the operator. Contamination may also cause by insufficient sterilisation procedures of explants and unautoclaved of growth medium. Insufficient flaming of tools may also cause the spreading of bacterial contamination in plant cultures (16). Therefore, to reduce contamination rate, proper handling and precaution is important. Surface sterilisation technique used in this study can be improve by increasing the percentage of Clorox and also increasing the agitation time.
3.2 The effect of BAP and NAA towards the in vitro shoot regeneration
The aseptic P. macrocarpa seeds grown in regeneration medium started to germinate after two weeks in culture. The first sign of germination was the emergence of hypocotyls (Figure 1D), followed by development of leaves on the subsequent week. The respond of explants was recorded after 5 weeks in culture (Figure 2). Cultures responded well on all tested medium (Table 2). Of all medium, T1(MS0) produced the highest length of shoot (3.57 ± 1.86), followed by T6 (MS with 0.5 mg/L BAP + 0.1 mg/L NAA; 1.78 ± 0.92) and T5 (MS with 0.25 mg/L BAP + 0.1 mg/L NAA; 1.73 ± 0.90). The least respond was observed in T2 (MS with 0.25 mg/L BAP; 0.27 ± 0.27).
Note: Means in a column with different small letters are significantly different at p≤0.05 by Duncan New Multiple Range Test. Each treatment consists of 10 replicates
 'Plant growth' regulators is very important for shoot regeneration. To test the effect of plant growth regulators in shoot regeneration of P. macrocarpa, MS supplemented with cytokinin (BAP) alone or combination with auxin (NAA) were used. In this study, it was shown that P. macrocarpa produced optimum shoot length in MS without plant growth regulators. Previous study reported the used of MS supplemented with BAP alone at 0.25 mg/L for shoot multiplication of P. macrocarpa (17). The success of shoot regeneration using MS without growth regulators has been described by several studies including Allium ampeloprasum (18), Momordica charantia (19) and Cicer arietinum (20). The used of BAP alone for shoot regeneration for in vitro plants is also well documented in many plant species e.g. Zingiber officinale (21); Oroxylum indicum (22) Chlorophytum borivilianum (23) and Begonia pavonina (24).

Many studies also reported the success of in vitro plant regeneration using combination of cytokinins and auxins (25, 26, 27, 28, 29). In this study, combination of BAP and NAA produced increased number of leaves (Figure 2). Unfortunately, the combination of BAP and NAA does not produced maximum shoot height for P. macrocarpa. The combination of hormones however, produced sizeable plantlets that suitable for further subculturing. It is also noted that P. macrocarpa grew healthily with stable morphological characteristics and no hyperhydric shoots was observed in all treatments.
4. CONCLUSION
P. macrocarpa is an important medicinal species which is needed to be conserved and cultivated. The in vitro techniques reported in this study provides powerful tool for mass multiplication of this plant species. Obtaining clean in vitro culture is prerequisite for the success in plant tissue culture. It is recommended to explore other surface disinfectants such as ethanol, hydrogen peroxide, bromine water, mercuric chloride, silver nitrate, and antibiotics to produce efficient surface sterilization method of P. macrocarpa. It is also concluded that MS without plant growth regulators is sufficient for shoot regeneration. However, it is recommended for further investigation of optimum combination of auxins and cytokinins for high frequency shoot multiplication and rooting of explants.
5. ACKNOWLEDGEMENT
The authors would like to give special thanks to staff members at the Tissue Culture Unit
(MARA University of Technology, Negeri Sembilan) for their technical assistance.