The effects of plant growth regulators on accumulation of pinocembrin and cardamonin of adventitious root suspension cultures of boesenbergia rotunda (L.) mansf. / Siti Zulaiha Ahamad Azhar

Boesenbergia rotunda is an important medicinal plant. The rhizome requires a long
period of time to produce in a large scale for this plant. In vitro culture is the best
method as a continuous source of supply of disease free planting material for
commercial utilization because it offers an opportunity to exploit the cell, tissue, organ
or entire organism by growing them in vitro. Secondary metabolites of B. rotunda
possess many biological and pharmacological activities such as antioxidant,
antibacterial, antitumor, anti-inflammatory and anti-cancer activities. Pinocembrin has
shown cytotoxicity against certain cancer cell lines whereas cardamonin was reported
to have strong inhibition of HIV-1 protease activity. There were a few studies on
secondary metabolites of B. rotunda in cell culture and callus culture. However, this
plant has low content of secondary metabolites in cell suspension cultures and callus
culture. This study was conducted to establish the protocol for adventitious root
induction from shoot meristem of B. rotunda and to determine the effects of PGRs on
the biomass and accumulation of pinocembrin and cardamonin in adventitious root
suspension cultures of B. rotunda. Surface sterilization with 60% clorox for 30
minutes, followed by 20% clorox for 15 minutes was the best treatment to establish
the aseptic explants with mean value of 4.667 ± 0.724. For the adventitious root
induction, half strength of Murashige and Skoog (MS) medium was supplemented
with various concentrations of auxins; 1-naphthaleneacetic acid (NAA), indole-3-
butyric acid (IBA), and 2,4-dichlorophenoxyacetic acid (2,4-D) at a range of 0.5
mg/L-3.0 mg/L either alone or in combination of cytokinins; kinetin (Kn) and
thidiazuron (TDZ) at a range of 0.1 to 0.5 mg/L. The most effective treatment for
adventitious root induction was supplemented with 0.5 mg/L NAA which produced
the highest number and length of roots with a mean value of 7.667 ± 2.082 and 1.733
± 0.416 respectively. The most effective combination of auxin and cytokinin for
adventitious root induction was 0.5 mg/L NAA+ 0.1 mg/L Kn that produced the
highest number and length of roots with a mean value of 3.400 ± 2.408 and 1.330 ±
1.021 respectively. The best treatment for the biomass production of adventitious root
of B. rotunda was 0.5 mg/L NAA + 0.1 mg/L TDZ with a mean value of 3.684 ±
1.233 g fresh weight and also produced the highest number of shoots with a mean
value of 3.167 ± 1.169. Histological examinations of adventitious root of B. rotunda
revealed the presence of important root anatomic structures including epidermis,
endodermis, pericycle, phloem, cortex, late metaxylem and protoxylem pole. HPLC
analysis showed that both pinocembrin and cardamonin were present in the extract of
adventitious root suspension cultures of B. rotunda. The highest compound contents
were 13.09 ± 3.92 mg/g for pinocembrin whereas 16.95 ± 4.01 mg/g for cardamonin
when the adventitious root were respectively cultured into 0.5 mg/L NAA + 0.3 mg/L
TDZ and 0.5 mg/L NAA + 0.1 mg/L TDZ. The result shows that the PGRs (NAA and
TDZ) is a good approach for enhancing the secondary metabolite content in
adventitious root cultures of B. rotunda. Further research using bioreactor, elicitors,
application of hairy root culture, histological analysis at cellular level and
pharmacological properties should be done as B. rotunda is an important aromatic and
medicinal plant.