(Pandanu Conoidells Lam) Through Lateral Bud Proliferation

In Vitro Propagation OfBuah Merab (Pandanu Conoidells Lam) Through Lateral Bud Proliferation Maria Imelda '., Aida Wnlansari ' and umarnie z
Centre/or Biotechnology. LIPI, Cibinong
2Research Centre/ol" Biology. LIP!. Cibinong
*[email protected]
I Research
ABSTRACT
Pal1danus collo/deus Lam or 'B uah mcrah' of th e Pa ndanaceae i · native to Ea t Tndonesia , particularly
Papua and North Maluku. Traditionally the fruits are used for health promolion and mai nte nance 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 w ild, 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 vegetatively
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 ll ows 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 U,e eITects of O. I-0.2 mgll lhidi.azuron (TDZ), 0.5- 1.0
b nzyl amino purine (BAP) and 0.25-0.5 mg/l Kin eti.n (KN) n shooL bud induction and pro lifera tion )r p
L"OI/(Jideu... were investigated 1I ing noda l 'ections or laLera l b ud
P conoideus on modj fied Murashige and
koog medium. Sho ts were rooted on MS medilUTI w ithout plant growth reguJator (PGR) . T he 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 w ith 0.1 mg/l TDZ or I mg/l BAP with 0.5 mg/I KN.
giving a multiplication rate of I6.5 s hootlets 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 ' ac hieved on a mixed med ium
of ocopcat and soi l ( 1: I ).
or
Keywords: Bliah merah (Pandal7l1.1 ,·ol/()idells). lateral buds, B P. TDZ. K
T'ffROO CflON
Pandanus conoiJelis Lam or 'Suah merah'
of the Pandanaceac is nati ve to Ea t Indonesia
namely Papua and orth Malltku. The plant can
b found from lowland to highland, growing be t
at 1200- [500 m above ealevel, generally in the
open air or lightly in haded area (Fig. I a). In
Papua, there are about 30 different kind of buah
merah vhich can be divided IOto 3 group based
on iLS colour namely red (Fig. I b). yellow and
brown. TraditionaJly. the fruits are us d for health
promotion and maintenance as well a lor curing
several Wne es. The active compound of the
fruit are beta carotene, alpha toeoferrol, oleic
acid, linoleic acid and decanoic acid (Yahya &
Wiryallto. 2005).
nnale Bogorienses n
~.
Vol. 12 o. I. 200R
Recently, it bas been reported that the fruits
of buah mera h ar pot ntial for ca ncer
med ication (Budi & Paimin, 2002; Redaksi
Trubus, 2005). A a result, Lhere has been an
over explo itation of this poten tial medicinal plant
from it habitats. In order to anticipate their
pas ible disappearance due t ovcrexploitation
in the wild, an efficient and effective technology
for the mass propagation, conservation and
cultivation of these plant should be developed.
Buah merah is generally propagated
egetatively by otTshoots and I m cuttings or
generatively by eed<> (Fig. I c). but their viability
wer very low (Bueli & Paimin, 2002) .
Micropropagation has many advantages over the
conventional propagation method , since lhc
technique allows mass clonal and pathogen-free
production ofplants at a high rate ofmulliplication
all year round (George & Sberrington.1984).
.19
Figure l. In vitro propagat ion of buah merah (Pandanlls cono/dells) . (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), benzylam inopurin (BAP), and
kinetin (KN) ar generally very effective in
supporting th formation and proLifi ration of in
vitro hoot (George & 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 o/exp/anls
Young plants of P. conoideu..<; abou t I-month
old collected from Papua, Indonesia wer grown
in the glass house of Zhej iang Unjversity, PR
China. Sho
of about 15 cm in height were
collected from the g lasshouse. The shoots wer
cut into ± 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 0.1 % HgCI 2 fOT 12 min
and then rinsed three time with steril distilled
water. The shoots were transferred to a sterile
40
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 25°C and under a 16-h photoperiod with ligh t
intensity 000 ,.1I110I!m21 ec provided by cool whjte
inflorescent tube . Subculture to the me medium
was done every 4 weeks.
Preparation o/medium
Aba al medium of Mura hige and koog
(MS) (1962) containing 3% sucrose and 2.5 gil
phytagel wa used in the experiment. The pH of
the medium was adjusted to 5.8 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, 0.5 and 1.0 mg/ I) in
ombination with TDZ (0, 0. 1, and 0.2 rug/I) and
BAP (0, 0.5, and 1.0 mgll) in combination with
KN (0, 0.25, and 0.5 mg/I) 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 med ium
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 n d cove red wi th
Annales Bogorien e n. . Vol. 12 No. I, 2008
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 prod uced 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 amat}lIifolius) 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 proliferation in vitro
in many plant species compared with other
cytokini.ns (George & S herrrington, 1984) .In this
inve ligation, the highest number of in vitro sh ot
ofbuah merah (7.5 shoots after 4 week and 16.5
boots after w eks) was attained on MS medium
contailling 0,5 mg/I BAP and 0, 1 mg/I 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. vil/osa where the addition of
TDZ at low concentration (1.0 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 & Giri, 2003).
In low concentration TDZ induces hi gh 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 proli£ ration in
everaJ plants like Sida cordi/olia (Sivanesan &
Jeong. 2007) and sungkai (Peronema canescen )
(Imelda I al., 1999). In buah mcrab, BAP alone
at I mg/I or 0.5 mg/l 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 0.25 or 0.5
mg/I 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. Chiorophytlll'n
borivilianuffl (Purohit el aI, 1994) and Hibiscus
cannabinus (Herath et ai, 2004).
However, pr longed cullure containing
0.022 mM BAP resulted in vitrified plan tlets. A
higher percentage of normal plantlets of P
amQlyllijolius was recorded with decrea ing
concentrations ofBAP for fo ur c nsecutive ub­
cultural pa age (0.022 mM. 0.0 II mM.0.005
mM, 0.002 mM) before transferring to rooti ng
medium (Gangopadhyay, 2004).
Rooting of in vitro hoots i generally done
on a medium containing root-promoti ng p lant
growth regulator such a lBA or NAA .
Gangopadbyay (2004) used solid and li quid MS
media supplemented with 0.0 I mM fBA and
0.002 mM KN for root induclion in Pandanus
amaJylli/oliu . Howe er, in certain plant species
like buah merah, roots can be directly formed
without the addition of auxin (Fig.2 c, d). The
same condition has also been fOlmd in in vilro
shoot of banana (Tmelda, L991), and
Amorphophal/u 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. T his process is an important
tep for acclimatization orall regenerated plants
produced by ti ue culture since in vitro plant
are very sensitive to temperature and humidity
fluctuation in the greenhouse and easily attacked
by diseases. Acclimatization of some tree plants
was still difficult due to their physiologal
morphologica l and anatomical abnormalit ies
induced during the in vilf'O culture (Ziv, 1994).
In this research, the accljmatization ofbuah merah
plantlet on oi 1 and cocopeat medium was
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
1
2
PGR (mgll)
BAP
0
mz
0
-
0
0..1
-
°
0.2
-
0
-
0 .1
02
-
3
4
5
6
7
8
9
10
11
12
13
'14
15
16
0.5
0 .5
0 .5
1O
10
10
°°
°
05
0 .6
0.6
10
°
17
18
1
10
KN
-
0
-
0 .1
0 .2
-
.
-
.
-
-
°
°
°
On
0 .25
0.5
0.25
0 .5
0 .26
Ntmber of shoots
:;} weeks
4 weeks
1,0 ± 0,29 a
2,0 ± 0,33 a
4,0 ± 0.26 b
5,0 ± 0,00 e
5.0 ± 029 be
8 0 ± 0,29 de
4 0 ±0 17 b
70 + 050 d
75 ± 029 f
'1 65 ± 1 00 i
6,0 :!: 0 fIJ coo
160 :!: 058i
12,5:!: 1,00 f
5,0 :!: 0,50 be
6,5 :!: 1,00 def
14,0 :!: 0,58 gh
60 ±1 ,00 cde
15,5 :!: 0,58 hi
1,0 :!: 0,29 a
2,0:!: 0,33 a
1,0:!: 0,50 a
3,0:!: 0,29 b
2 o :!: 050 a
4 ± 029 be
40+ 050 b
70:!: 029 d
6,0 :!: 0,50 be
8,0:!: 0, 29 de
5,5 :!: 1.. DO cd
9.. 5 :!: 0 ,29 e
5,0 :!: 1,00 be
12,5 :!: 0,58 fg
6,0:!: 1, 00 ede
14,0 ! 0,58 gn
7,0 ! 1,00 ef
16,5! 0,87 i
°
Note : Mean within olumn followed by the same felle r are not ignificantly different according
at 5% level.
to
LSD test
a
Figure 2. 111 vitro propagation of buah merah (Pandanus cOlloideus ) (a). Shoots appear fTom lateral buds. (b).
Shoots proliferation in MS mediu m+O.5 mg/I BAP+O.l mg/I 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
42
Annales Bogori en es n. . Vol. 12 No. 1, 2008
CONCLU 10.
Buah me rah (P conoidells) ca n be
propagated througb lateral bud proliferation . The
best medium for shoot prohferation wa MS +
0.5 mg/lBAP + 0.1 mg/l TDZ or MS 1 rug/l
BAP +O.5mgl1 KN giving a mullipUcation rate
of 16.5 shootlets/explant after 8 weeks. Rooting
of shoots was ucce sfully conducted on MS
medjum without PGR. Acclimatization of rooted
plantle gave a 90 % survival rate.
ACK.."¥OWLEDGEMENTS
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.
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Cibinong 16911
Kabupaten Bogor
Indonesia
E-mail: annaJes_ [email protected]
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