Effects of abiotic factors on cell biomass and rosmarinic acid production in cell suspension cultures of Orthosiphon Stamineus benth

doi: 10.9755/ejfa.2015.04.018

O. stamineus is generally propagated vegetatively by cuttings, but rooting diffi culties inhibits fulfi lling the market demands (Lee and Chan, 2004).Biotechnological technique which is a good alternative has the privilege of screening in cells rather than intact plants (Akin-Idowu et al., 2009).In vitro propagation protocols for O. stamineus has been established (Lee and Chan, 2004;Lim et al., 2006).The optimization of medium for callus induction of O. stamineus was also established by Lee and Chan (2004).However, there are no studies so far classifying the differences among fast, intermediate and slow growing cell lines of O. stamineus.
The objectives of the present study were to evaluate the effects of medium pH, photoperiod, temperature and sucrose concentrations on cell biomass and RA production of three different selected growing cell lines (fast, intermediate and slow) of this species.

Callus induction
Two in vitro plant lines of O. stamineus, maintained in Plant Tissue and Cell Culture Laboratory, School of Biological Sciences, Universiti Sains Malaysia, Penang were used for the callus induction.The leaves w ere dissected (0.5mm × 0.5mm) and weighed and inoculated on MS (Murashige and Skoog, 1962) medium supplemented with 1 mg/L NAA and 1 mg/L 2,4-D (Lee and Chan, 2004).Ten (10) replicates were used in this study.Three (3) leaf explants were cultured in each vessel and they were maintained at temperature 25 ± 2ºC under continuous illumination using cold white fl uorescent tubes at intensity of 32.5 μEm-² s-¹ for six weeks.Induced callus from leaves were weighed and the subculture procedure was continued every three weeks for the subsequent study.

Categorization and selection of callus lines
From each line, total of 0.5 g callus was inoculated on MS medium supplemented with 1 mg/L NAA and 1 mg/L 2,4-D.All the cultures were maintained at 25 ± 2ºC under continuous illumination at intensity of 32.5 μEm-² s-¹.The fresh callus weight was recorded after three weeks.This procedure was repeated nine times.Growth index was calculated for each subculture cycle using the following formula: Growth index (GI) = final weight-initial weight /initial weight After nine subculture cycles, callus lines were categorized as fast (GI > 7), intermediate (5≤ GI ≥7) and slow growing (GI < 5), on the basis of their growth index.To determine the harvesting day for each cell line, fresh callus mass of 0.75 g from one representative of each growing cell line (fast, intermediate and slow) was weighed and inoculated into 100 mL Erlenmeyer fl asks containing 20 mL liquid cell proliferation medium (MS medium, supplemented with 1 mg/L 2,4-D and 1 mg/L NAA).Every three days, fi ve random samples from each growing cell line (fast, intermediate and slow) were collected and the fresh cell biomass was determined after cells fi ltered through Whatman filter papers (No.1) using Buncher funnel connected to a vacuum pump.Fresh cells were air-dried until the constant weight was achieved.The growth pattern of each cell line was determined based on the fresh and dried cell masses.

Study of abiotic factors
The infl uence of abiotic factors was studied on cell biomass and rosmarinic acid production of the three cell lines of O. stamineus.Cell proliferation medium (MS medium plus 1 mg/L NAA and 1 mg/L 2,4-D) was experimented by using two sucrose concentrations (30 g/L and 45 g/L).Effect of pre autoclaved medium pH in different levels (5, 5.50, 5.65, 5.70, 5.75, 5.80, 6 and 6.50) was observed.In this study medium was prepared and adjusted to different pH levels (EUTECH-510) before autoclaving.Photoperiod impact was studied on the three cell lines.In this study, each cell line was grown under different conditions: Continues illumination, total darkness (flasks were covered by aluminium foil), the fi rst half growth cycle was illumination followed by darkness for the second half and the fi rst half growth cycle was darkness followed by illumination for the second half.The cells were also incubated at different temperatures (20, 23, 26 and 29˚C).In all experiments, each cell line was studied individually hence, tests carried out using completely randomized design (CRD) and the data were analysed using One-Way ANOVA.The signifi cant differences of means were compared using Tukey test at p ≤ 0.05.

Rosmarinic acid (RA) detection using ultra performance liquid chromatography (UPLC) system
In order to detect rosmarinic acid, total of 0.5 g dried cells of each cell line (fast, intermediate and slow), derived from sucrose, medium pH, photoperiod and temperature experiment was grinded into powder and soaked in 10 ml methanol and put on a rotary shaker (120 rpm) for 24 hours.It was then fi ltered by using fi lter paper No.1.After the methanol was evaporated, the remained extract was diluted with methanol HPLC grade using Millipore fi lter (0.20μm).The Quantitative estimation of RA was carried out using UPLC system (Waters UPLC TM ) equipped with C18 2.1×50 mm (1.7 M) with a fl ow rate of 0.25 ml/min.The PDA detector wavelength was set to 210 -400 nm.For RA detection two mobile phases were used, mobile phase A consisted of 10% acetonitril, 1% acetic acid and 90% distilled water and mobile phase B, consisted of 45% acetonitril and 55% distilled water (Lim et al., 2006).RA content was determined by comparing sample curves with the RA standard curve (Aldrich Chem.Co.).Rosmarinic acid standard was serial diluted (1000-3500 ppm) and it was injected before each sample run.Using rosmarinic acid standard curves in different concentrations provided us a formula to compute the quantity of rosmarinic acid found in the samples.

Callus induction
Two callus lines (ZFIN and ZIIN) were induced and added to eight other previously provided callus lines (MK, SA, BF, ZI, ZZ, ZZNEW, II and ZF) for a more accurate selection study.In this procedure the excised leaf pieces enlarged and swollen when cultured on the callus induction medium after seven days.The callus formation fi rst took place at the cutting edges of the leaf pieces.The callus cells gradually grew over the leaf pieces.The friable callus was established after nine subculture cycles.Callus lines were categorized into three main groups, on the basis of growth index, the fast, intermediate and slow growing lines.At the end of nine subculture cycles, callus lines were categorized.The induced callus line, ZIIN, was eliminated after 6 th subcultures due to abnormal growth.Four callus lines were categorized as fast growing lines, four lines as intermediate growing lines and only one was categorized as slow growing line (Table 1).

Growth pattern of the three cell lines
Growth pattern of the three selected cell lines (line MK for slow, line ZF for intermediate and line SA for fast growing) showed that there was a short lag phase (three days after inoculation) followed by a long logarithmic phase that took 12 days in the fast growing cell line and 15 days in the slow and intermediate growing cell lines.The fast growing cell line reached the stationary phase in 15 days and completed a growth cycle whereas the intermediate and slow growing cell lines reached the stationary phase in 18 days.Fast growing cell line had the longest stationary phase.This cell line produced more aggregated cells while the slow and intermediate growing lines produced more fi ne and separated cells.Comparing the dried and fresh cell biomass showed that cells contained mainly water.Growth pattern results indicated that regardless of differences among the three cell lines, the slow and intermediate growing cell lines of O. stamineus were more similar (Fig. 1).

Effect of sucrose, medium pH, photoperiod and temperature on cell growth and rosmarinic acid production
Cells cultured in the cell proliferation medium (MS plus 1 mg/L 2,4-D and 1 mg/L NAA) supplemented with 30 g/L sucrose produced signifi cantly higher fresh cell biomass in the intermediate and fast growing cell lines.Fast growing line produced 3.36 g of cell mass when cultured in cell proliferation medium supplemented with 30 g/L sucrose while in cell proliferation medium with 45 g/L sucrose the cell biomass production was decreased by almost 50 percent (1.31 g).The same trend was observed in the intermediate growing cell line while the growth of the slow growing cell line was not affected by the amount of sucrose presented in the cell proliferation medium (Table 2).
With regards to rosmarinic acid (RA) production, only the slow growing cell line favoured RA production when cultured in cell proliferation medium supplemented with 45 g/L sucrose.However, the intermediate cell line showed higher amount of RA when cell proliferation medium was   supplemented with 30 g/L sucrose (Fig. 2).This result was in contrast with previous studies on O. stamineus done by Lim et al. (2006), in which it was reported that medium with 45 g/l sucrose led to both high amount of RA and fresh cell biomass production.Similarly the addition of 45 g/L sucrose in the culture medium was found to be effective for cell biomass and pigment production of Melastoma malabathricum (Koey et al., 2010).However Akalezi et al. (1999) reported the highest cell biomass of Panax gensing was produced in medium with 30 g/L sucrose.A study on Taxus chinensis cell cultures showed that the addition of only 20 g/L sucrose could support the highest fresh cell biomass and taxane production (Wang et al., 1999).Ilieva and Pavolov (1997) reported the Lavandula vera cells produced not only more cell biomass but also higher RA content when cells cultured in culture medium supplemented with 45 g/L sucrose.In Perilla frutescens, using lower sucrose concentrations led to pigment production in the cells, but the supplement of higher concentrations (60 and 50 g/L sucrose) induced higher fresh cell biomass (Zhong and Yoshida, 1995).
The present study showed that the medium pH affected both cell biomass and rosmarinic acid production of the three cell lines of O. stamineus.In terms of biomass production, the slow growing cell line produced the highest fresh cell biomass when the medium pH was adjusted to 5.65, 5.70, 5.75 or 5.80.The most alkaline media (pH 6 and 6.5) supported the lowest amount of fresh cell biomass production.This cell line also produced low cell biomass in very acidic medium pH (5).In terms of rosmarinic acid production, the slow growing cell line produced the highest amount of RA in medium pH 5.70 (44.76 mg/g dried cell) and 5.75 (40.39 mg/g dried cell).The intermediate growing cell line produced high fresh cell biomass when the medium pH was adjusted to 5 and 5.65.This cell line also had the least biomass production in the most alkaline media (PH 6 and 6.5).In this cell line, medium pH 5.65 (6.22 mg/g dried cell) and 5.80 (5.76 mg/g dried cell) resulted in the highest RA production.RA was not detected in the most alkaline media (pH 6, 6.5) and also the most acidic medium (pH 5) resulted in trace amount of RA production.The fast growing cell line responded differently to medium pH.The least fresh cell biomass production was observed in the most acidic medium (pH 5 and 5.5).High fresh cell biomass was produced in medium pH of 5.65, 5.70 also 6 and 6.5.Fast growing cell line was the only cell line that could produce high fresh cell mass at very alkaline medium pH (6 and 6.5).In terms of RA production in the fast growing cell line, medium pH 5.70 (25.93 mg/g dried cell), 5.75 (18.55 mg/g dried cell) and 5.80 (18.68 mg/g dried cell) resulted in signifi cantly high RA production.In the most alkaline medium (pH 6.5) RA was not produced, other medium pH levels also resulted in very little amount of RA production (Fig. 3).
The results of the present study on O. stamineus were in harmony with the study on Centella asiatica cells in which the optimal medium pH for both triterpens and cell biomass production was at the range of 5.5 -5.6 (Ling, 2004).In another study on Eurycoma longifolia callus cultures it was also reported that the highest amount of 9-methoxycanthin-6-one produced when the medium pH adjusted to 5.5 (Rosli et al., 2009).Luthfi (2004) also reported the highest alkaloids and cell biomass production of Eurycoma longifolia cell cultures achieved when cells cultured in medium pH 5.75.Anthocyanin accumulations on Melastoma malabthricum affected by medium pH 5.25 and 6.25, however it did not infl uence cell biomass production (Chan et al., 2010).
Photoperiod effected cell biomass and rosmarinic acid production of the three cell lines differently.The slow growing cell line was able to produce high cell biomass either under continuous illumination or total darkness, or when it was incubated under darkness for the 1 st half cycle followed by illumination for the 2 nd half cycle.The highest amount of RA was produced when this cell line was incubated under either darkness for the 1 st half cycle followed by illumination for the second half (45.82 mg/g dried cell) or illumination for the 1 st half followed by darkness for the 2 nd half (47.25 mg/g dried cell).The intermediate growing cell line produced the highest fresh cell biomass when incubated at either the fi rst half cycle was illumination followed by darkness or the fi rst half cycle was darkness followed by illumination (L/D or D/L).The amount of cells produced was not signifi cantly different when cultured at continuous illumination or total darkness The intermediate growing cell line produced the highest amount of RA when incubated at either continuous illumination (4.56 mg/g dried cell) or when the fi rst half cycle was illumination followed by darkness in the second half (4.52 mg/g dried cell) (Fig. 4).In the fast growing cell line, a complete cycle of total darkness caused the least fresh cell biomass production, whereas cells cultured in three other conditions (illumination, L/D and D/L) did not produce signifi cantly different fresh cell biomass.In this cell line, the highest RA production was observed when either cell incubated at total darkness (15.80 mg/g dried cell) or continuous illumination (12.36 mg/g dried cell).In this cell line the lowest RA content was produced when cells incubated at illumination in the fi rst half cycle followed by darkness in the second half cycle (6.21mg/g dried cell) (Fig. 4).
Production of some secondary metabolites like anthocyanin is highly dependent on illumination as Koay ( 2008) reported that in cell suspension cultures of Melastoma malabathricum although the cell growth was promoted in total darkness but anthocyanin production was highly decreased however under continuous illumination high anthocyanin was produced from the cell cultures.In Vitis vinifera cell cultures, 2.7 fold increased in anthocyanin production was reported when incubated at continuous illumination, this condition on the other hand showed negative results in terms of cell biomass production (Zhang et al., 2002).Jenkins et al (1995) described the importance of illumination as the photoreceptors that control the expression of specifi c genes involved in cell growth, development and secondary metabolite production.
Temperature affected fresh cell biomass and rosmarinic acid production of the three growing cell lines.The slow growing cell line produced the highest amount of fresh cell biomass when incubated at 26˚C.This cell line did not produce any signifi cantly different amount of fresh cell biomass when incubated at 20, 23 and 29˚C.In terms of rosmarinic acid production, slow growing line produced high amount of RA (23.02 mg/g dried cell) when incubated at 29˚C, while incubation at other temperatures the amount of RA produced was not significantly different.The intermediate growing cell line produced more cell biomass when incubated at 20˚C.This cell line also did not produce signifi cantly different cell biomass when incubated at other temperatures.The intermediate growing cell line produced the highest amount of RA (15.88 mg/g dried cell) when incubated at 26˚C followed with 29˚C (7.54 mg/g dried cell).The fast growing cell line produced more cell biomass when incubated at 23˚C or 29˚C.This cell line did not produce signifi cantly different cell biomass when incubated at 20˚C and 26˚C.Fast growing line produced high amount of RA (19.65 mg/g dried cell) when incubated at 20˚C.In this cell line an obvious declining trend in terms of RA production, was observed at higher temperatures (Fig. 5).These results indicated that different cell lines produced different amount of RA at different temperatures.Physiology and metabolism of cell cultures infl uenced by temperature effected cell growth and secondary metabolite production.Temperature in the range of 17 -25ºC known as the optimal temperature range for most callus and cell cultures.However, it could be different depending on the plant species (Rao and Ravishankar, 2002).The present study on O. stamineus cell lines, indicated that some stress factor like a different range of temperature was required for cell lines to trigger the enzymes needed for secondary metabolite production however only in the slow growing cell line at the normal temperature those enzymes were produced and subsequently the rosmarinic acid was produced.It was reported that in cell suspension cultures of Daucus carota, the highest fresh cell biomass and secondary metabolite was produced when maintained at 25ºC (Narayan et al., 2005).In strawberry cell cultures, 15ºC was the optimal temperature that increased anthocyanin production by 35 folds (Zhang et al., 1997).

CONCLUSIONS
The fresh cell biomass and rosmarinic acid production in cell suspension cultures of O. stamineuse was affected by different physical factors like medium pH, photoperiod and carbohydrate source although in a diverse range among the three different cell lines.The study on sucrose concentrations proved that medium supplemented with 3% sucrose was more suitable for cell biomass and RA production however adding more sucrose to the medium acted as an elicitor and improved the RA production in the slow growing line.Similar to most other cell lines, cells grown in medium with normal pH range (5.65-5.80)were more productive in terms of cell biomass and RA.
In terms of photoperiod and temperature O. stamineuse the three cell lines showed inconsistency for cell biomass and RA production.Signifi cant differences observed among the three selected cell lines indicated the importance of selection and categorization in the study of cell lines.

Fig 1 .
Fig 1.Growth pattern according to fresh cell weight (FW) and dried cell weight (DW) of the (a) slow, (b) intermediate, and (c) fast growing cell lines of Orthosiphon stamineus.c

Fig 2 .Fig 3 .
Fig 2. Effect of sucrose supplemented into the cell proliferation medium on RA production of the three cell lines of O. stamineus.Mean values followed by the same alphabets (in each cell line) were not signifi cantly different (Student t-test at p ≤ 0.05).

Fig 4 .
Fig 4. Effect of photoperiod on rosmarinic acid and cell biomass production of the three cell lines of O. stamineus (I= illumination, D= darkness, I/D= illumination/darkness, D/L= darkness/illumination).Means within each group (each line) having the same letter are not signifi cantly different (Tukey test, p ≤ 0.05).

Fig 5 .
Fig 5. Effect of temperature on rosmarinic acid and biomass production of the slow, intermediate and fast growing cell lines of Orthosiphon stamineus.The mean values followed by the same alphabets for each parameter (biomass or RA) were not signifi cantly different (Tukey test, p ≤ 0.05).