In Vitro Propagation OfBuah Merab (Pandanu Conoidells Lam) Through Lateral Bud Proliferation

Maria Imelda Aida Wnlansari and umarniez

I Research Centreor Biotechnology LIPI Cibinong 2Research Centreol Biology LIP Cibinong

maria_tahardiyahoocom

ABSTRACT

Pal1danus collodeus Lam or Buah mcrah of the Pandanaceae i middot native to Ea t Tndonesia particularly Papua and North Maluku Traditionally the fruits are used for health promolion and mai ntenance as well as for curing several illnesses Recent ly it has been reported that the fm its are potential fo r cancer medication As a result Lhere has been an overexploitation of the plants from the ir habitats In order to anticipate their po iblt disappearance due 10 overexploilation in the wild an efficient and eITective technology for the mass propagation con cnalion and ull ivalion orlbe e plant shou ld be developed Generally buah merah is propagated vegetat ively by If ho IS and Le m cuttings or genera tively by seeds Micropropagalion ha many advantages over the conventional method because tbe technique a llows mass cI nal and pa thogen-free production o f plan ts at high rate of multiplicati on all year round In this re earch Ue eITects of O I-02 mgll lhidiazuron (TDZ) 05- 10 b nzyl amino purine (BAP) and 025-05 mgl Kinetin (KN) n shooL bud induction and pro lifera tion )r p LOI(Jideu were investigated 1I ing noda l ections or laLera l bud or P conoideus on modj fied Murashige and

koog medium Sho ts were rooted on MS medilUTI without plant growth reguJator (PGR) The result howed that lateral bud of Pondanu start d to initiate growth after 4-7 days in culture The be t medium for sho t proliferation was MS conta ining either O - mgll BAP with 01 mgl TDZ or I mgl BAP with 05 mgI KN giving a multiplication rate of I65 shootlets per shootbud explant after 8 weeks Rooting ofsho Is was succc sfull y conducted on MS medium withou t PGR Acclimatization of rooted plantlets wa achieved on a mixed med ium of ocopcat and soi l (1 I )

Keywords Bliah merah (Pandal7l11 middotol()idells) lateral buds B P TDZ K

TffROO CflON Recently it bas been reported that the fruits of buah mera h ar pot ntial for cancer

Pandanus conoiJelis Lam or Suah merah med ication (Budi amp Paimin 2002 Redaksi of the Pandanaceac is nati ve to Ea t Indonesia Trubus 2005) A a result Lhere has been an namely Papua and orth Malltku The plant can over explo itation ofthis poten tial medicinal plant b found from lowland to highland growing be t from it habitats In order to anticipate their at 1200- [500 m above ealevel generally in the pas ible disappearance due t ovcrexploitation open air or lightly in haded area (Fig I a) In in the wild an efficient and effective technology Papua there are about 30 different kind of buah for the mass propagation conservation and merah vhich can be divided IOto 3 group based cultivation of these plant should be developed on iLS colour namely red (Fig I b) yellow and Buah merah is generally propagated brown TraditionaJly the fruits are us d for health egetatively by otTshoots and I m cuttings or promotion and maintenance as well a lor curing generatively by eedltgt (Fig I c) but their viability several Wne es The active compound of the wer very low (Bueli amp Paimin 2002) fruit are beta carotene alpha toeoferrol oleic Micropropagation has many advantages over the

acid linoleic acid and decanoic acid (Yahya amp conventional propagation method since lhc

Wiryallto 2005) technique allows mass clonal and pathogen-free production ofplants at a high rate ofmulliplication all year round (George amp Sberrington1984)

nnale Bogorienses n ~ Vol 12 o I 200R 19

Figure l In vitro propagat ion of buah merah (Pandanlls conodells) (a) Mature plants in Uleir wild habilat in Papua (b) The fruit of buah merah (c) Infertile seeds

However the application of thi technique ha not yet been reported on this plant

Cytokinin j a group of plant growth regulator (PGR) which has an important role in cell division breaking dormancy apical dominance stimulates growth of dormant bud and influence shoot growth (Mok et aI 2000) In plant ti ue c ulture cytokinin such as thidiazuron (TDZ) benzylaminopurin (BAP) and kinetin (KN) ar generally very effective in supporting th formation and proLifi ration of in vitro hoot (George amp Sherrington 1984)

In this research nodal s ctions or lateral buds of P conoiJeus were used a explant with the aim of proliferat ing high quality shoots and regenerating plantlets The effects ofthidiazuron (TDZ) benzyl amino purine (BAP) and Kinetin (KN) on shoot bud induction and pro liferation were inve ligated

MATER) L AND METHODS

Preparation and inoculation oexpanls Young plants ofP conoideult abou t I-month

old collected from Papua Indonesia wer grown in the glasshouse of Zhej iang Unjversity PR China Sho of about 15 cm in height were collected from the g lasshouse The shoots wer cut into plusmn 5 cm length and weTe then washed in running tap water immersed in 70 ethanol for 30 seconds then rinsed several lime with distilled water

Under a laminar air flow cabinet the ShOOlS were further sterilized in 01 HgCI

2 fOT 12 min

and then rinsed three time with steril distilled water The shoots were transferred to a sterile

Petri-di h containing a filter paper after lheir leaves were removed the shoots were cut into internode segments with lateral buds of about I em in length for inoculation on the prepared medium

All cuJ ture were incubated in a growth room at 25degC and under a 16-h photoperiod with light intensity 000 1I110Im21 ec provided by cool whjte inflorescent tube Subculture to the me medium was done every 4 weeks

Preparation omedium Aba al medium of Mura hige and koog

(MS) (1962) containing 3 sucrose and 25 gil phytagel wa used in the experiment The pH of the medium was adjusted to 58 by the add it ion of I N KOH or 0 I N HC before autoclaving at 121 0 C for 15 min at 1 atm

Treatments Shoot proliferation was indu ced by the

addition of BAP (0 05 and 10 mg I) in ombination with TDZ (0 0 1 and 02 rugI) and

BAP (0 05 and 10 mgll) in combination with KN (0 025 and 05 mgI) into the m dium Totally there were 18 treatm nts with 2 replicates

Roofing and Acclimatization Rooting of shoots was induced in MS

medium without the addition ofPGR A hundred rooted plantlets were removed from cu lture rin ed in tap water to remove the agar medium and planted in plastic pot) containing soil and coco peat medi u m (I I) The polled plant were watered to satura t ion a nd cove red wi th

Annales Bogorien e n Vol 12 No I 2008 40

tran parent pIa tic bag for abou t _ weeks and placed in the greenhouse

Observations Ob ervati n wa done after 4 and 8 weeks

on the number of hoot produced by each treatment The data were statistically analyzed u ing least ignificant difference (LSD) test

RE ULT A DDI 10

NodaJ section of lateral buds of Pandanus grown on MS basal media with everal combination of TDZ and BAP or BAP and KN (Table I) started to grow after 4-7 days in culture (Fig 2 a) The buds began to swell then developcd into new shoot bud However in a medium without plant growth regulator new h ot buds appeared atler 4 weeks Similar result were also bOWD in hoot culture of lndian pandan (Pandanus amatlIifolius) which could be continu u Iy mult iplied u ing the arne combination of plant growth regulators namely BAP and KN (Thinunaraju et ai 2005)

In thi experim nt all media produced shoots but the highest number of shoots was obtained on treatment contailling 2 combination ofplant growth regulators BAP and TDZ or BAP and KN The addition of2 cytokinins produced highe t number of adventive hoot compared with if only TDZ or BAP added When KN wa added into the medium without any other plant growth rcglLlator only 1-2 shoots were fomled after 4 weeks (Table I)

TDZ and BAP are the mo t effecti e cytokinins in inducing shoot prol iferation in vitro in many plant species compared with other cytokinins (George amp S herrrington 1 984) In this inve ligation the highest number of in vitro sh ot ofbuah merah (75 shoots after 4 week and 165 boots after w eks) was attained on MS medium

contailling 05 mgI BAP and 0 1 mgI TDZ (Fig 2 b) The addition ofBAP in combination with KN also showed the highest proliferation of advenli ve shoots (7 shoots after 4 weeks and 16 hoot afler 8 weeks) It appear that the

combination of these two PGR has a synergic effect in proliferating adventive shoots The arne phenomenon could al 0 be seen on Arachis stenosperma and A vilosa where the addition of TDZ at low concentration (10 JlM) in combination with BAP and lAA could increase

Annale Bogorien es n s Vol 12 No 1 200

the regeneration percentage of in vitro shoots of Arachis spp (Laxmi amp Giri 2003)

In low concentration TDZ induces high r il1 vitro shoots proliferation than other cytokinin in many plant species such as on Lenti l (Khawar e f aI 2004) whereas BAP have be n reported to be effective in stimulating shoot prolipound ration in everaJ plants like Sida cordiolia (Sivanesan amp

Jeong 2007) and sungkai (Peronema canescen ) (Imelda I al 1999) In buah mcrab BAP alone at I mgI or 05 mgl could stimulate ad entivc shoots even though it wa les than when it was added in combination with other cytokinin KN is a less effective cytokinin compared with BAP or TDZ in inducing shoot formation (Table I) The additi n of KN a a ingle plant growth regulator showed a lack in it ab ility in proliferating in vitro shools KN at 025 or 05 mgI can only produce 1-2 shoots within 4 weeks but interaction between BAP and KN significantly influence shoot bud induction and proliferation of P conoideus imilarobservations have been reported in other plant such a Chiorophytllln borivilianuffl (Purohit el aI 1994) and Hibiscus cannabinus (Herath et ai 2004)

However pr longed cullure containing 0022 mM BAP resulted in vitrified plantlets A higher percentage of normal plantlets of P amQlyllijolius was recorded with decrea ing concentrations ofBAP for four c nsecutive ubshycultural pa age (0022 mM 00 II mM0005 mM 0002 mM) before transferring to rooti ng medium (Gangopadhyay 2004)

Rooting of in vitro hoots i generally done on a medium containing root-promoting p lant growth regulator such a lBA or NAA Gangopadbyay (2004) used solid and liquid MS media supplemented with 00 I mM fBA and 0002 mM KN for root induclion in Pandanus amaJyllioliu Howe er in certain plant species like buah merah roots can be directly formed without the addition of auxin (Fig2 c d) The same condition has also been fOlmd in in vilro shoot of banana (Tmelda L991) and Amorphophalu muelleri (Imelda ef aI 2006) In vitro shoots of P ammyllifolius can aJso be rooted in a half-strength MS solid med ium without PGR (Thirnmaraju el ai 2005)

Acclimatization is an adaptation process for in vitro pLantlets from the in vitro condition in which both temperature or humidity was under control to the ex vitro green house condition that

41

tran parent pIa tic bag for abou t _ weeks and placed in the greenhouse

Observations Ob ervati n wa done after 4 and 8 weeks

on the number of hoot produced by each treatment The data were statistically analyzed u ing least ignificant difference (LSD) test

RE ULT A DDI 10

NodaJ section of lateral buds of Pandanus grown on MS basal media with everal combination of TDZ and BAP or BAP and KN (Table I) started to grow after 4-7 days in culture (Fig 2 a) The buds began to swell then developcd into new shoot bud However in a medium without plant growth regulator new h ot buds appeared atler 4 weeks Similar result were also bOWD in hoot culture of lndian pandan (Pandanus amatlIifolius) which could be continu u Iy mult iplied u ing the arne combination of plant growth regulators namely BAP and KN (Thinunaraju et ai 2005)

In thi experim nt all media produced shoots but the highest number of shoots was obtained on treatment contailling 2 combination ofplant growth regulators BAP and TDZ or BAP and KN The addition of2 cytokinins produced highe t number of adventive hoot compared with if only TDZ or BAP added When KN wa added into the medium without any other plant growth rcglLlator only 1-2 shoots were fomled after 4 weeks (Table I)

TDZ and BAP are the mo t effecti e cytokinins in inducing shoot prol iferation in vitro in many plant species compared with other cytokinins (George amp S herrrington 1 984) In this inve ligation the highest number of in vitro sh ot ofbuah merah (75 shoots after 4 week and 165 boots after w eks) was attained on MS medium

contailling 05 mgI BAP and 0 1 mgI TDZ (Fig 2 b) The addition ofBAP in combination with KN also showed the highest proliferation of advenli ve shoots (7 shoots after 4 weeks and 16 hoot afler 8 weeks) It appear that the

combination of these two PGR has a synergic effect in proliferating adventive shoots The arne phenomenon could al 0 be seen on Arachis stenosperma and A vilosa where the addition of TDZ at low concentration (10 JlM) in combination with BAP and lAA could increase

Annale Bogorien es n s Vol 12 No 1 200

the regeneration percentage of in vitro shoots of Arachis spp (Laxmi amp Giri 2003)

In low concentration TDZ induces high r il1 vitro shoots proliferation than other cytokinin in many plant species such as on Lenti l (Khawar e f aI 2004) whereas BAP have be n reported to be effective in stimulating shoot prolipound ration in everaJ plants like Sida cordiolia (Sivanesan amp

Jeong 2007) and sungkai (Peronema canescen ) (Imelda I al 1999) In buah mcrab BAP alone at I mgI or 05 mgl could stimulate ad entivc shoots even though it wa les than when it was added in combination with other cytokinin KN is a less effective cytokinin compared with BAP or TDZ in inducing shoot formation (Table I) The additi n of KN a a ingle plant growth regulator showed a lack in it ab ility in proliferating in vitro shools KN at 025 or 05 mgI can only produce 1-2 shoots within 4 weeks but interaction between BAP and KN significantly influence shoot bud induction and proliferation of P conoideus imilarobservations have been reported in other plant such a Chiorophytllln borivilianuffl (Purohit el aI 1994) and Hibiscus cannabinus (Herath et ai 2004)

However pr longed cullure containing 0022 mM BAP resulted in vitrified plantlets A higher percentage of normal plantlets of P amQlyllijolius was recorded with decrea ing concentrations ofBAP for four c nsecutive ubshycultural pa age (0022 mM 00 II mM0005 mM 0002 mM) before transferring to rooti ng medium (Gangopadhyay 2004)

Rooting of in vitro hoots i generally done on a medium containing root-promoting p lant growth regulator such a lBA or NAA Gangopadbyay (2004) used solid and liquid MS media supplemented with 00 I mM fBA and 0002 mM KN for root induclion in Pandanus amaJyllioliu Howe er in certain plant species like buah merah roots can be directly formed without the addition of auxin (Fig2 c d) The same condition has also been fOlmd in in vilro shoot of banana (Tmelda L991) and Amorphophalu muelleri (Imelda ef aI 2006) In vitro shoots of P ammyllifolius can aJso be rooted in a half-strength MS solid med ium without PGR (Thirnmaraju el ai 2005)

Acclimatization is an adaptation process for in vitro pLantlets from the in vitro condition in which both temperature or humidity was under control to the ex vitro green house condition that

41

are more fluctuative This process is an important was still difficult due to their physiologal tep for acclimatization orall regenerated plants morphologica l and anatomical abnormalit ies

produced by ti ue culture since in vitro plant induced during the in vilfO culture (Ziv 1994) are very sensitive to temperature and humidity In this research the accljmatization ofbuah merah fluctuation in the greenhouse and easily attacked plantlet on oi 1 and cocopeat medium was by diseases Acclimatization ofsome tree plants resulting in 90 of 100 planlets survived (Fig 2

e)

Table 1 The effect of plan growth regulators on shoot bud initialion of buah merah

Treatment No

PGR (mgll) Ntmber of shoots BAP mz KN 4 weeks weeks

1 0 0 - 10 plusmn 029 a 20 plusmn 033 a 2 0 0 1 - 40 plusmn 026 b 50 plusmn 000 e 3 deg 02 - 50 plusmn 029 be 8 0 plusmn 029 de 4 05 0 - 4 0 plusmn0 17 b 70 + 050 d 5 05 01 - 75 plusmn 029 f 1 65 plusmn 1 00 i 6 0 5 0 2 - 60 0 fIJ coo 160 058i 7 1O 0 - 50 050 be 125 100 f 8 1 0 0 1 - 65 100 def 140 058 gh 9 1 0 0 2 - 60 plusmn1 00 cde 155 058 hi

10 deg deg 10 029 a 20 033 a 11 deg - 0 25 10 050 a 30 029 b 12 deg - 05 2 o 050 a 4 degplusmn 029 be 13 05 - deg 40+ 050 b 70 029 d 14 0 6 - 025 60 050 be 80 0 29 de 15 06 - 0 5 55 1DO cd 9 5 0 29 e 16 10 deg 50 100 be 125 058 fg 17

1 deg - 0 26 60 1 00 ede 140 058 gn 18 1 0 - On 70 100 ef 165 087 i

Note Mean within olumn followed by the same feller are not ignificantly different according to LSD test at 5 level

a

Figure 2 111 vitro propagation of buah merah (Pandanus cOlloideus ) (a) Shoots appear fTom lateral buds (b) Shoots proliferation in MS medium+O5 mgI BAP+Ol mgI TDZ (c) Plantlets in rooting medium (MS without PGR) (cI) Rooted plantlets beD re acclimatization (e) Young plant from ti sue culture in pots containing oil and cocopeat medium

Annales Bogorien es n Vol 12 No 1 2008 42

CONCLU 10 Khawar KM Sancak C Uranbey S amp Ozean (2004) Efiect ofthidiazuron on shoot regeneration from

Buah me rah (P conoidells) ca n be propagated througb lateral bud proliferation The best medium for shoot prohferation wa MS + 05 mglBAP + 01 mgl TDZ or MS 1 rugl BAP +O5mgl1 KN giving a mullipUcation rate of 165 shootletsexplant after 8 weeks Rooting of shoots was ucce sfully conducted on MS medjum without PGR Acclimatization of rooted plantle gave a 90 survival rate

ACKyenOWLEDGEMENTS

We would like to acknowledge Prof Fu Chengxin who provided me with the opportuniLy to iniliate this research at the College of Life Science ZheJiang Univer ity (ZU) under the Collaborative Project between LIPI Indonesia and ZU PR China

REFERENCE

Budi 1M amp Paim in FR (2002) Buah Merah Pcnebar Swadaya Depok 75 pp

angopadhyay G Bandyopadhyay T Modak BK Wongpornchai S amp Mukherjee KK (2004) Micropropaga tion of Ind ia n pandan (Pandanus tlmary llifolius Roxb) a rich source of 2-acetyl - I-pyrro line urrent cienc 87 ( 11) 1589-1592

George EF amp Sh e rrington PD (1984) Plant propagation by tis ue culture Handbook and Directory ofCommercial Laboratories Eastem Pre s Reading UK p 284-310

Hcralh SP Suzuki T amp Halori K (2004) Multiple hoot regeneration from young shoots ofkenaf (Hibiscus cannabinus) Plant Cell Ti Organ Cult 77 49-53

I meda M ( 1991) Penerapan teknologi in iumiddoto dalam peoyediaan bibi t plsang Prosiding eminar Bioleknoiogi Perkebllnan dan Lokakarya Biopolimer untuk Industri PAU Bioteknologi IPB Bogor 71-79

Imelda M etyowati T amp luleha (1999) Penyediaan bibit s ungka i (Peronema canescens Jack) melalu i pro li feras i tunas adven lif Jurnal Bioteknologi Pertanian 3 (2) 53 -57

Imelda M Wul an a r i A amp P erba YS (200 7) Mikropropagas i tanaman ile -iles (Amorphophalus mlle eri Blum e) Berita Biologi (4) 27 1-277

Annales Bogorienses n s Vol 12 No1 2008

Cibinong 16911

Kabupaten Bogor Indonesia

E-mail annaJes_hogorienesyahoocom

Title page contains

different exp lant of len til (Lens culinaris Medik) via organogenesis Tu rk J Bo t 28(2004) 42 1-426

Laksmi GV amp Giri CC (2003) Plant Regeneration via organogenesis from hoot base derived callus of Arachis s tenosperma and A villomiddota Current ceienee 85 (II) 1624-1629

Mok MC Ma n in RC amp Mok DW (2000) ytokinins B iosynthesis metabolism and

perception InVitro Cellular and Developmental BiologymiddotPlanl36 (2) 102- 107

Mun im A Andradjati R amp SlIsilowati H (2006) Uj i hambatan tumorigenesis sari bllah merah (Pandanus conoideu Lam) terhadap tikllS pUlil1 bel ina yang diindllksi 7 12 dimetilbenz (a) antra en (DMBA) Majalah Ilmu Kefarmasian m(3) 152-1 6 1

Murasruge T amp koog F (1962) A revised mediwn for rapid growth and bioassays with tobacco tissue cultures Phy io l Plant 15 473-497

Puro hi t D Da e A amp Kukda G (1994) Micropropagat ion of safed mlls li (Ch lorophy tllfll borivilianum) a rare Indian medicinal herb Planl Cell Tis Organ ull 3993-96

Redak i T rubu (2005 ) Panduan Praktisi Bua h Merah Bukti Empiris dan Ilm iah Penebar Swadaya Depok 104 pp

Sivane an I amp Jeong BR (200 7) Direct hoo t regeneration from nodal explant of Sida cordifo lia Linn In Vitro ell ular and Developmental Biology-PlanI43(5) 436-441

Thimmaraj u R Bhagyalakshmi N Narayan M Venkata balam L amp Ravi hankar GA (2005) In vitro cu lture of Palldanus amaryllifolius and enhancement of 2-acetyl-l shypyrrolinc the major flavouri ng compound of aromatic rice by precursor fecding ofL-proline

cienee of Food and Agriculture 85 ( 15) 2527 -2534

Yahya HM amp Wir anta BTW (2005) Kbasiat amp manfaat buah merah si emas da ri Papua P

gro Media Pustaka Depok 84 pp Z iv M (1 994) In vitro acclima tization In J Aitken shy

C hri tie T Koza i amp MAL Smi th (Eds ) Automat ion and Environmenta l contro l in P lan t Tissue Cu lt ure K luwer Acad mle Publisher Dordrecht 493-51 6

A concise and information title written in a sentence format The name(s) of the author(s) The affiliation(s) and addresses) of the author(s) The e-mail addre of the communicating author

4 3

CONCLU 10 Khawar KM Sancak C Uranbey S amp Ozean (2004) Efiect ofthidiazuron on shoot regeneration from

Buah me rah (P conoidells) ca n be propagated througb lateral bud proliferation The best medium for shoot prohferation wa MS + 05 mglBAP + 01 mgl TDZ or MS 1 rugl BAP +O5mgl1 KN giving a mullipUcation rate of 165 shootletsexplant after 8 weeks Rooting of shoots was ucce sfully conducted on MS medjum without PGR Acclimatization of rooted plantle gave a 90 survival rate

ACKyenOWLEDGEMENTS

We would like to acknowledge Prof Fu Chengxin who provided me with the opportuniLy to iniliate this research at the College of Life Science ZheJiang Univer ity (ZU) under the Collaborative Project between LIPI Indonesia and ZU PR China

REFERENCE

Budi 1M amp Paim in FR (2002) Buah Merah Pcnebar Swadaya Depok 75 pp

angopadhyay G Bandyopadhyay T Modak BK Wongpornchai S amp Mukherjee KK (2004) Micropropaga tion of Ind ia n pandan (Pandanus tlmary llifolius Roxb) a rich source of 2-acetyl - I-pyrro line urrent cienc 87 ( 11) 1589-1592

George EF amp Sh e rrington PD (1984) Plant propagation by tis ue culture Handbook and Directory ofCommercial Laboratories Eastem Pre s Reading UK p 284-310

Hcralh SP Suzuki T amp Halori K (2004) Multiple hoot regeneration from young shoots ofkenaf (Hibiscus cannabinus) Plant Cell Ti Organ Cult 77 49-53

I meda M ( 1991) Penerapan teknologi in iumiddoto dalam peoyediaan bibi t plsang Prosiding eminar Bioleknoiogi Perkebllnan dan Lokakarya Biopolimer untuk Industri PAU Bioteknologi IPB Bogor 71-79

Imelda M etyowati T amp luleha (1999) Penyediaan bibit s ungka i (Peronema canescens Jack) melalu i pro li feras i tunas adven lif Jurnal Bioteknologi Pertanian 3 (2) 53 -57

Imelda M Wul an a r i A amp P erba YS (200 7) Mikropropagas i tanaman ile -iles (Amorphophalus mlle eri Blum e) Berita Biologi (4) 27 1-277

Annales Bogorienses n s Vol 12 No1 2008

Cibinong 16911

Kabupaten Bogor Indonesia

E-mail annaJes_hogorienesyahoocom

Title page contains

different exp lant of len til (Lens culinaris Medik) via organogenesis Tu rk J Bo t 28(2004) 42 1-426

Laksmi GV amp Giri CC (2003) Plant Regeneration via organogenesis from hoot base derived callus of Arachis s tenosperma and A villomiddota Current ceienee 85 (II) 1624-1629

Mok MC Ma n in RC amp Mok DW (2000) ytokinins B iosynthesis metabolism and

perception InVitro Cellular and Developmental BiologymiddotPlanl36 (2) 102- 107

Mun im A Andradjati R amp SlIsilowati H (2006) Uj i hambatan tumorigenesis sari bllah merah (Pandanus conoideu Lam) terhadap tikllS pUlil1 bel ina yang diindllksi 7 12 dimetilbenz (a) antra en (DMBA) Majalah Ilmu Kefarmasian m(3) 152-1 6 1

Murasruge T amp koog F (1962) A revised mediwn for rapid growth and bioassays with tobacco tissue cultures Phy io l Plant 15 473-497

Puro hi t D Da e A amp Kukda G (1994) Micropropagat ion of safed mlls li (Ch lorophy tllfll borivilianum) a rare Indian medicinal herb Planl Cell Tis Organ ull 3993-96

Redak i T rubu (2005 ) Panduan Praktisi Bua h Merah Bukti Empiris dan Ilm iah Penebar Swadaya Depok 104 pp

Sivane an I amp Jeong BR (200 7) Direct hoo t regeneration from nodal explant of Sida cordifo lia Linn In Vitro ell ular and Developmental Biology-PlanI43(5) 436-441

Thimmaraj u R Bhagyalakshmi N Narayan M Venkata balam L amp Ravi hankar GA (2005) In vitro cu lture of Palldanus amaryllifolius and enhancement of 2-acetyl-l shypyrrolinc the major flavouri ng compound of aromatic rice by precursor fecding ofL-proline

cienee of Food and Agriculture 85 ( 15) 2527 -2534

Yahya HM amp Wir anta BTW (2005) Kbasiat amp manfaat buah merah si emas da ri Papua P

gro Media Pustaka Depok 84 pp Z iv M (1 994) In vitro acclima tization In J Aitken shy

C hri tie T Koza i amp MAL Smi th (Eds ) Automat ion and Environmenta l contro l in P lan t Tissue Cu lt ure K luwer Acad mle Publisher Dordrecht 493-51 6

A concise and information title written in a sentence format The name(s) of the author(s) The affiliation(s) and addresses) of the author(s) The e-mail addre of the communicating author

4 3