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In VitroCulture Manipulation on Pruatjan for Secondary

Metabolite Production

Ika Roostika1, Ragapadmi Purnamaningsih1, Ireng Darwati2, and Ika Mariska1

1Indonesian Center for Agricultural Biotechnology Research and Development, Jl. Tentara Pelajar 3A, Bogor 16111

2Indonesian Medicinal and Aromatic Plants Research Institute, Jl . Tentara Pelajar 3, Bogor 16111

ABSTRAK

Manipulasi Kultur In Vitro pada Tanaman Purwoceng

untuk Produksi Metabolit Sekunder.Ika Roostika, Raga-padmi Purnamaningsih, Ireng Darwati, dan Ika Mariska.

Purwoceng (Pimpinella pruatjan Molk. atau Pimpinellaalpina KDS.) adalah tanaman obat langka yang dapat diman-faatkan sebagai bahan obat afrodisik, diuretik, dan tonik.

Kulturin vitrotidak hanya dapat digunakan untuk konservasidan perbanyakan tanaman, melainkan dapat juga diterap-kan untuk produksi metabolit sekunder. Melalui teknik ini,produksi metabolit sekunder tidak bergantung kepada sum-ber tanaman di lapang. Penelitian ini dilakukan dengan tuju-an untuk meningkatkan kadar stigmasterol melalui kulturinvitrodengan menggunakan prekursor asam mevalonat. Pe-nelitian dibagi menjadi dua tahap, yaitu induksi kalus danmanipulasi kulturin vitrountuk meningkatkan kadar stigma-sterol. Pada tahap induksi kalus, terdapat 16 perlakuan yangmerupakan kombinasi perlakuan 2,4-D dan piklorammasing-masing pada taraf 0,5; 1,0; 1,5; dan 2,0 ppm. Untukmeningkatkan kadar stigmasterol, digunakan asam mevalo-nat pada taraf 0, 250, 500, dan 750 ppm dengan masa inku-basi selama 4 dan 6 minggu. Kandungan stigmasterol diana-lisis menggunakan GC-MS. Hasil penelitian menunjukkanbahwa media P2 (DKW + 2,4-D 0,5 ppm + pikloram 1,0ppm) adalah media terbaik untuk induksi kalus. Eksplan da-un lebih baik daripada eksplan petiol. Hasil analisis GC-MSmenunjukkan bahwa kandungan stigmasterol tertinggi(0,0356 ppm) diperoleh dari kalus dengan masa inkubasi 4minggu pada media dengan penambahan asam mevalonat250 ppm. Peningkatan taraf asam mevalonat tidak mampumeningkatkan kandungan stigmasterol. Kadar tersebut miripdengan kandungan stigmasterol pada planlet dari GunungPutri (0,0365 ppm) dan Dieng (0,0414 ppm). Dibandingkandengan kadarnya dalam akar tanaman dari lapang, kan-dungan tersebut sekitar 10-100 kali lipat lebih tinggi.

Key words:Kultur in vitro, metabolit sekunder, PimpinellapruatjanMolk.

BACKGROUND

Indonesia has a megabiodiversity of plant geneticresources, the second largest in the world after Brazil,including those of the medicinal plants. Since medicin-al herb industries in the country were mostly obtainedtheir raw materials from nature without efforts to in-tensively cultivate and they use the material excessive-

Hak Cipta 2007, BB-Biogen

ly of the natural capacity, medicinal plant species are,therefore, the most eroded plants among agriculturalcrop species. Until l992, there were at least 30 medi-cinal plant species are categorized as eroded, includ-ing the commercial plant of Pruatjan (Pimpinella prua-tjan Molk. or Pimpinella alpina KDS.). This plant canbe used as an ingredient for diuretic, aphrodisiac, and

body fit enhancers or tonics. The use of Puatjan as anaphrodisiac compound has even been patented by theUniversity of Diponegoro, Semarang, Central Java(http://www.laksamana.net/vnews.cfm?ncat=34news_id=6397).

Based on the erosion levels, many medicinalplants in Indonesia were categorized as extinct,endangered, rare, and indeterminate (Rifaiet al.l992).Pruatjan is categorized as one of the endangered spe-cies. Recently, Rahardjo (2003) and Syahid et al.(2004) reported that Pruatjan plant was currentlygrown by farmers only in a small area at Sekunangvillage, Dieng Plateau, Central Java. It is quite difficult,therefore, to fullfil enough supply of raw materials ofPruatjan plant for production of medicines. To solvethis limitation, an alternative technology needs to bedeveloped.

In vitro culture is not only applicable for plantconservations and propagations but also for secondarymetabolite manipulations since this technique doesnot dependent on plant sources from the field. Sec-ondary metabolites, such as terpenoid, glycoside (ster-oid, phenol), and alkaloid are products from plantmorphogenetic processes. Thein vitroculture offers abetter way for secondary metabolite productions, es-

pecially medicinal compounds, than production fromintact plants (Wetter and Constable 1991). This tech-nique has been applied in production of secondarymetabolite from several plants. Some metaboliteswere accumulated in cultured cells at a higher levelthan those in the native plants, particularly when op-timization of the cultural conditions was optimized. Forexamples, ginsenosides of Panax ginseng, rosmarinicacid of Colleus blumei, shikonin ofLithospermum ery-throrhizon, diosgenin of Dioscorea, ubiquinone-10 ofNicotiana tabacumwere accumulated in much higherlevels in cultured cells than those in the intact plants

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(Misawa 1994). It is expected, therefore, that in vitromanipulation can also be done in the production ofsecondary metabolites of pruatjan.

Under in vitroculture, the addition of precursorsor elicitor into a medium could increase the secondarymetabolite contents (Ravishandar and Grewal 1991).The precursor used in the culture may dependent onstructures of secondary metabolites to be produced. Apruatjan plant produces stigmasterol as the secondarymetabolite. This compound is formed through themevalonic biosynthesis pathway (Vikery and Vikery1981). Wikinsonet al.(1994) reported that the additionof mevalonic acid on cell suspension of celery cultureincreased sitosterol content, higher than control. Inaccordance to this, the use of mevalonic acid may alsoincrease concentration of stigmasterol in the pruatjanculture, which is dependent on the concentration andexposure time of the precursor. Therefor, the objectiveof the study was to develop an in vitro technique forthe production of stigmasterol from pruatjan throughinvitro culture by using mevalonic acid as a precursor.

MATERIALS AND METHODS

The research was conducted at theTissue CultureLaboratory of Biology Cell and Tissue Culture Division,Indonesian Center for Agricultural Biotechnology andGenetic Resources Research and Development from

February 2004 to December 2005. The pruatjan plantmaterials used in the in vitrocultures were collectedfrom Gunung Putri, Bogor, West Java. The cultureswere proliferated on a DKW (Driver and Kuniyaki)basal medium with additions of 1 ppm BA (benzyl-adenine), 0.2 ppm thidiazuron, and 100 ppm arginine.The cultures were subcultured every two-months andincubated at 9oC, 1000 lux, with a 12-hour photoperiod.This research was divided into two experiments, i.e.,induction of callus formation andin vitromanipulationof the stigmasterol content.

Induction of Callus Formation

The explants used in the study were petioles andleaves of in vitro culture of pruatjan. These explantswere cut into squares of 0.25 cm2 and subsequentlyplaced in the DKW basal medium. Sixteen treatmentswere used for callus inducing compounds consistingof combinations of 2,4-D (2,4-dichlorophenoxyaceticacid) and picloram with concentrations of 0.5, 1.0, 1.5,and 2.0 ppm, respectively. The parameters observedwere initiation time of callus formation, percentage ofcallus formation, as well as fresh weight and dryweight of the calli.

In VitroManipulation to Increase

Stigmasterol Content

The explants used in this study were were calli of

pruatjan. The calli were maintained in the best mediaformulation for callus formation that obtained from theprevious experiment. After one month of culturing, thecalli were transferred into a fresh medium. Mevalonicacid was added to the fresh medium to increase thestigmasterol production. Concentrations of the me-valonic acid used were 0, 250, 500, and 750 ppm, res-pectively. The cultures were then incubated at roomculture of about 20oC for 4 and 6 weeks. The param-eter observed was the stigmasterol content, whichwas detected by the GC-MS.

The calli were harvested after certain periods of

incubation time (4 and 6 weeks). Samples of approxi-mately 1.0 g were freeze-dried overnight. One gram ofdried samples were ground and soaked in a-100 mlmethanol (p.a). This was then filtered by a Whatmanpaper no. 41 followed by silica gel (60GF254). Thefiltrate was then injected to the GC-MS apparatus. Theplantlets (in vitroaccessions) that were collected fromDieng, Central Java and Gunung Putri, West Java, werealso analyzed by the GC-MS, to com-pare thesecondary metabolite contents.

RESULT AND DISCUSSION

Induction of Callus Formation

A large amount of plant materials were needed toproduce secondary metabolite underin vitroculture. Acallus is an undifferentiated cell with high level of celldivision. Therefore, callus is used in thein vitroculture,since it can proliferate quickly and continuously. Inorder to obtain a large amount of calli, the applicationof a high activity auxin is needed, such as 2,4-D, 2,3,5-T, dicamba, and picloram. Results of the callus induc-tion experiment indicated that callus initiations couldbe induced from pruatjan leaves and petioles. Gener-ally, the growth response of the leaf was more variable

than response of the petiole. Moreover, the callus initi-ation from petioles looked more uniform. According toRamulu (1985), leaves and roots were possible to havepolysomic cells, while according to Gunawan (1988),explants from stems, roots, and leaves commonly pro-duced heterogenous cells. Figure 1 showed that theinitiation times of calli formations from the petioleswas 9 days after planting, earlier than that from theleaves, 13 days after planting. It is assumed that thepetiole cells were looser than those of the leaf cells.Gunawan (1988) reported that a callus resulted fromparenchyma cells showed a loose binding.


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2007 ROOSTIKA ET AL.: In VitroCulture Manipulation on Pruatjan 57

The percentage of calli formations from petiolesranged from 87.5 to 100%, which was higher thanthose from the leaves (62.5-93.8%) (Table 1). Growth

of calli from the leaf explants, however, were fasterthan those from the petioles. This was indicated by theleaf explants that produced heavier fresh weight anddry weight calli (Figure 2 and 3). These figures alsoshowed that the treatments that gave higher levels ofcalli fresh weights did not always produced higherlevel of dry weights. Among all treatments, the leafexplant that was grown on the P2 medium (2,4-D 0.5ppm + 1.0 ppm picloram) gave the best result. Thismedium gave the highest percentage of calli freshweight and dry weight. The P2 medium was thereforeselected as the most suitable formulation for the in-duction of callus formation in the secondary metab-

olite production.

In VitroCulture Manipulation to

Increase Stigmasterol

Pruatjan plant produced a stigmasterol, which isused as an aphrodisiac compound. According to Tau-fiqurrachman and Wibowo (2005), stigmasterol mightbe altered into testosterone that functions in sexualactivity. Therefore the stigmasterol content in the invitrocultures was measured. The use of phenylalanineas a precursor in callus cultures of pruatjan has beenreported (Fauzi et al. 2005). However, the content of

secondary metabolite in the culture was lower thanthat in the leaves extract. In this experiment, me-valonic acid was used as a precursor in the culture.

Mevalonic acid is an intermediate compound inthe biosynthesis of stigmasterol, therefore the applica-tion of this precursor is expected to increase the stig-masterol content of the pruatjan culture. Results of thetrial showed that when the pruatjan calli were sub-cultured on the medium containing mevalonic acid,they were still growing even when the highest concen-tration of mevalonic acid (750 ppm) was added. How-ever, the growth of the four weeks old cultures de-creased with the increasing level of mevalonic acidcontents at 500 and 750 ppm. As reported by Wikinsonet al.(1994), the excessive mevalonic addition causedaccumulation intermediate compound of xycloartenolin cells which might inhibit the biosynthesis of thefollowing compound such as sitosterol and stigma-sterol. The best growth of the culture was obtained ona medium that contain 250 ppm mevalonic acid. Un-fortunately, most of the cultures were contaminatedwith microbes, so that it needs the other explantssources to check thesstigmasterol content from 6weeks old cultures. The condition of the sources wasrather vitrified so that the growth of the 6 week-old

P1 = 0.5 ppm 2,4-D + 0.5 ppm picloram, P2 = 0.5 ppm 2,4-D + 1.0ppm picloram, P3 = 0.5 ppm 2,4-D + 1.5 ppm picloram, P4 = 0.5ppm 2,4-D + 2.0 ppm picloram, P5 = 1.0 ppm 2,4-D + 0.5 ppmpicloram, P6 = 1.0 ppm 2,4-D + 1.0 ppm picloram, P7 = 1.0 ppm2,4-D + 1.5 ppm picloram, P8 = 1.0 ppm 2,4-D + 2.0 ppm picloram,P9 = 1.5 ppm 2,4-D + 0.5 ppm picloram, P10 = 1.5 ppm 2,4-D + 1.0ppm picloram, P11 = 1.5 ppm 2,4-D + 1.5 ppm picloram, P12 = 1.5ppm 2,4-D + 2.0 ppm picloram, P13 = 2.0 ppm 2,4-D + 0.5 ppmpicloram, P14 = 2.0 ppm 2,4-D + 1.0 ppm picloram, P15 = 2.0 ppm2,4-D + 1.5 ppm picloram, P16 = 2.0 ppm 2,4-D + 2.0 ppm picloram.

Figure 1. Time (days) of calli initiations from leaf (A) and petiole

explants (B) grown on different formulation of media.

Table 1. Effect of different media formulations on percentage of calliformations of pruatjan.

Calli formations (%) from:Medium formulation

Leaf petioles Leaves

P1P2P3P4P5P6P7P8P9P10P11P12P13P14P15P16

10001000

91.714.4100095.87.21000

93.311.595.87.210001000

91.714.410001000100087.512.51000

62.507535.47535.4

81.38.868.88.881.38.8

75093.88.881.326.562.517.793.88.887.517.793.88.8

7535.462.517.7

7535.4


Callus initiation from leaf explants

0

5

10

15

2025

30

35

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16

Treatment

Da

ys

A

Callus initiation from petiole explants

0

5

10

15

20

25

30

35


Treatment

Days

B


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cultures were not very good. The dry materials were

cultures were not very good. The dry materials werecultures were not very good. The dry materials were

only about 5% (Table 2).The results showed that stigmasterol was pro-

duced in the callus culture of pruatjan, either on me-dium with or without mevalonic acid. This result sug-gested that stigmasterol can be producedin vitrofromundifferentiated tissues (callus). Table 2 also showsthat stigmasterol content from four weeks old calliwere higher than from six weeks old calli. This level ofstigmasterol content in the culture was dependent onthe level of dry material of the callus. The stigmasterolcontent was low or undetected when the amount drymaterial of the culture was also low (Table 2). Thisresult suggests that the amount of callus biomass wasthe most important variable in the production of prua-tjan secondary metabolites (stigmasterol). A similar re-sult was reported by Hiraoka et al.(2004) on culturesof Corydalis ambigua,when the callus fresh weight in-creased gradually until the end of incubation period, itsdry weight increased only in the first nine days andremained constant thereafter.

The highest stigmasterol content (0.0356 ppm)was obtained from a four week-old cultures on mediacontaining 250 ppm of mevalonic acid. This contentwas almost similar to the stigmaterol content of prua-tjan plantlets that was collected from Gunung Putri,

Bogor (0.0365 ppm) and Dieng, Central Java (0.0414ppm). When the content was compared with that ofroot from a 9 month-old intact plant (Rahardjo andDarwati 2005) it was about ten to a hundred timeshigher.

This results proved thatin vitro technique can beapplied to produce secondary metabolite of pruatjan.However, this technique needs to be optimized byusing good plant materials, other suitable mediaformulations, and optimum level of mevalonic acid inthe media, as well as the use of other precursors.Siregar et al. (2005) reported that the combination of

P1 = 0.5 ppm 2,4-D + 0.5 ppm picloram, P2 = 0.5 ppm 2,4-D + 1.0ppm picloram, P3 = 0.5 ppm 2,4-D + 1.5 ppm picloram, P4 = 0.5ppm 2,4-D + 2.0 ppm picloram, P5 = 1.0 ppm 2,4-D + 0.5 ppmpicloram, P6 = 1.0 ppm 2,4-D + 1.0 ppm Picloram, P7 = 1.0 ppm2,4-D + 1.5 ppm picloram, P8 = 1.0 ppm 2,4-D + 2.0 ppm picloram,P9 = 1.5 ppm 2,4-D + 0.5 ppm picloram, P10 = 1.5 ppm 2,4-D + 1.0ppm picloram, P11 = 1.5 ppm 2,4-D + 1.5 ppm picloram, P12 = 1.5ppm 2,4-D + 2.0 ppm picloram, P13 = 2.0 ppm 2,4-D + 0.5 ppmpicloram, P14 = 2.0 ppm 2,4-D + 1.0 ppm picloram, P15 = 2.0 ppm2,4-D + 1.5 ppm picloram, P16 = 2.0 ppm 2,4-D + 2.0 ppm picloram.

Figure 2. Fresh weights of calli from leaf (A) and petiole explants(B) grown on different formulation of media.


Figure 3. Dry weight of calli from leaf (A) and petiole explants(B) grown on different media formulations.

Table 2. Stigmasterol content in pruatjan cultures on a mediumcontaining different levels of mevalonic acid.

MediumCulture fresh

weight (g)

Culture dry

weight (g)

Dry material

(%)

Stigmasterol

(ppm)R4 Mev0R4 Mev250R4 Mev500R4 Mev750

4.57634.12713.96653.2275

0.41970.38500.18150.1524

9.049.334.584.72

0.0059190.0356240.0016440.013154

R6 Mev0R6 Mev250R6 Mev500R6 Mev750

4.19534.62994.34952.5334

0.22800.29790.26160.2180

5.436.436.014.66

0.0017540.0112900.001315

not detected

Data in each column indicating the total amount of four replications.R = age of the cultures in weeks, and Mev = mevalonic acid in ppm.

Fresh weight of calli from leaf explants

00.10.20.30.40.50.60.7

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12P13

P14 P15 P16

Treatment

Weigh

t(g)

A

Fresh weight of calli from petiole explants

00.10.20.30.40.50.60.7


Treatment

Weight(g)

B

Dry weight of calli from leaf explants

0

0.01

0.02

0.03

0.04

0.05

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10P11P12P13P14 P15 P16Treatment

Weight(g) A

Dry weight of calli from petiole explants

00.010.020.030.040.05


Weight(g)

Treatment

B


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2007 ROOSTIKA ET AL.: In VitroCulture Manipulation on Pruatjan 59

2,4D and NAA could produce good quality of callusand induced cell suspension with high rate of growthand high level of alkaloid in Eurycoma longifolia

cultures.

CONCLUSIONS

A secondary metabolite stigmasterol was pro-duced from in vitroculture of pruatjan. The P2 (DKW+ 0.5 ppm 2,4-D + 1.0 ppm picloram) was the mostsuitable medium for the induction of callus formation.Leaf explants was better than petiole explants for thesecondary metabolite production. Results from theGC-MS analysis showed that the highest level ofstigmasterol production was obtained from a four-weeks old of callus culture that was applied with 250

ppm mevalonic acid. This content was similar to thatproduced in plantlets collected from Gunung Putri,West Java (0.0365 ppm) and Dieng, Central Java,which was 0.0414 ppm. As compared to that producedin roots of a 9-month old intact plant, this content wasabout ten to a hundred times higher.

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