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Indian Journal of Biotechnology
Vol 5, April 2006, pp 223-228
Effect of different factors on non-symbiotic seed germination,
formation of protocorm-like bodies and plantlet morphology of
Cleisostoma racemiferum (Lindl.) Garay
Temjensangba and Chitta Ranjan Deb*
Department of Botany, Nagaland University, Headquarters: Lumami, Mokokchung 798 601 (University Branch Office), India
Received 31 August 2004; revised 3 May 2005; accepted 2 July 2005
Cleisostoma racemiferum, an epiphytic orchid of primary forest under threat in their natural habitat, was studied.
Immature seeds of different developmental stages [(8-20 week after pollination (WAP)] were cultured on Knudson ‘C’,
Mitra et al and MS media supplemented with sucrose (0-3%, w/v), coconut water (CW, 0-20%, v/v) and NAA (0-30 μM) +
BA (0-8 μM) singly or in combination. After 7 week of culture, first sign of germination was recorded as nodular swelling
of seeds. Amongst the three different basal media tested, better germination was supported by MS medium, followed by
Mitra et al and Knudson ‘C’ media containing sucrose (3%) and NAA (10.0 μM ) + BA (8.0 μM). Of various developmental
stages of the seed, better germination was obtained from green pods of 16 week after pollination. Younger seeds did not
show any sign of germination, while mature seeds exhibited delayed and deformed germination. Though incorporation of
CW in the medium did not show much influence on seed germination, but 15% (v/v) CW in the initiation medium enhanced
the early differentiation of protocorm-like bodies (PLBs) into plantlets. Within 14-16 week of culture on germination
medium, the PLBs started releasing the first set of leaflets. The advanced stage PLBs were converted into rooted plantlets on
MS medium containing IAA (10.0 μM) + Kn (9.0 μM). Although the medium containing NAA (10.0 μM) + BA (8.0 μM)
resulted in multiple shoot buds, but the leaves were thin and etiolated. Further, the medium enriched with NAA (10.0 μM) +
Kn (9.0
μ
M) resulted in stunted growth of plantlets, while presence of IAA (10.0 μM) + BA (8.0 μM) resulted in plantlets
with poor roots.
Keywords: Cleisostoma racemiferum, green pod culture, plant growth regulators, seed age
IPC Code: Int. Cl.8 A01H4/00
Introduction
Orchid seeds are microscopic and non-
endospermous with undifferentiated embryos. They
are produced in large numbers and their germination
in nature depends upon a suitable association with
mycorrhizal fungus, which provides an essential
physico-chemical stimulus for initiating germination1.
The orchids are propagated through vegetative means
as well as seeds. However, the rate of vegetative
propagation is very slow and the seed germination in
nature is very poor, i.e. 0.2-0.3%2. In vitro
germination of seeds is an important part in the orchid
multiplication and conservation programmes since the
‘dust seeds’ are tiny and contain few food reserves.
Knudson3 demonstrated the possibility of by-passing
the fungal requirement of orchid seeds during in vitro
germination and since then non-symbiotic seed
germination has been accepted as an important tool
for propagating orchids4. The non-symbiotic seed
germination potential of fertilized ovules has been
positively tested in several commercially viable
and/or threatened orchid taxa5,6. However, non-
symbiotic seed germination of orchids is greatly
influenced by several factors, like seed age, different
nutrient media with adjuvants and plant growth
regulators3,5-8. Moreover, none of these basal nutrient
media with different adjuvants fulfills the
requirements of the entire orchidaceous group.
Cleisostoma racemiferum (Lindl.) Garay is an
epiphytic orchid and native of primary forests. The
species is under threat due to removal of their natural
habitat for ‘Slash and Burn’ cultivation, unplanned
developmental activities, etc. In this communication,
we describe the effect of different factors like green
pod age, culture media and plant growth regulators on
non-symbiotic seed germination, formation of
protocorm-like bodies (PLBs) and plantlet
morphology of C. racemiferum.
______________
*Author for correspondence:
Tel: 91-369-2268221; Fax: 91-369-2268204
E-mail: debchitta@rediffmail.com
INDIAN
J
BIOTECHNOL. APRIL
2006
Materials and Methods
In
Vitro
Seed Germination
,
C.
racem$erum green pods/capsules were harvested
at 2 week interval starting from 8 week after
pollination (WAP) up to 20 WAP. The pods were
surface sterilized with 0.5% (w/v) mercuric chloride
for 5 min and rinsed 3-4 times with sterilized distilled
water. Thereafter, the pods were flamed on gas burner
and seeds were scooped out by making longitudinal
slit and inoculated on different media, viz. Knudson
'c",
MS"
and Mitra et al". These media were
fortified with coconut water (CW; 0-20%, v/v) and
sucrose (0-3%, w/v) and then supplemented with
different plant growth regulators, like NAA (0, 10.0,
20.0 and 30.0 pM) and BA (0, 8.0, 16.0 and 24.0
CLM)
singly or in combination.
PLBs
and Multiple Shoot Formation
The PLBs developed from germinating seeds were
transferred on optimum culture conditions and
maintained for 2 more passages at 4 week interval.
The advanced stage PLBs (with first set of leaflets)
were transferred on both
MS
and Mitra et a1 media
containing sucrose (3%, w/v), CW (lo%, vfv) and the
following plant growth regulator for plantlet
regeneration: (i) IAA (0-20.0
CLM)
and
Kn
(0-18.0
pM),
(ii)
IAA (0-20.0
CLM)
and BA (0-16.0
C1M),
(iii)
NAA (0-20.0
CLM)
and BA (0-16.0 pM), and (iv)
NAA (0-20.0
CuZ/I)
and Kn (0-18.0
CLM),
single or in
combination.
Before autoclaving at 121°C and 1.05 kg cm-2 for
20 min, 0.8% agar was incorporated as gelling agent
and
pH
of the media was adjusted to 5.6 using 0.1
N
NaOH and HCI. Cultures were maintained at 25e°C
under cool white fluorescent light at 40 pmole m-2 s-'
light intensity and 12/12 h photo cycle. For seed
germination, 10 culture vials were maintained for
each treatment. The cultures were sub cultured at
4
week intervals both for seed germination and plantlet
regeneration, and data were recorded at 1 week
interval. Cultures on regeneration media were
monitored for plantlet formation and morphology. All
the experiments were repeated thrice and the
experimental design was completely randomized.
Results and Discussions
In
Vitro
Seed Germination
After
7
week of inoculation, the first sign of
germination was observed as yellowish nodular
swelling of seeds (Fig. la), which subsequently
converted into PLBs. Amongst three different basal
nutrient media studied in the present investigation on
C.
racemiferum, better germination was supported by
MS medium followed by Mitra et a1 and Knudson
'C'. The developmental stage of green pods was
found to be the most crucial factor for both nodular
swelling of seeds and PLBs formation from the
cultured seeds. Seeds from the pods of 16 WAP
initiated germination after 52, 62 and 68 d after
initiation of culture on MS, Mitra et
al,
and Knudson
'C' media, respectively; while for PLBs formation,
another additional 40, 53 and 56 d were required on
the respective media (Table 1). Green pods of age >16
WAP required longer duration and, in some cases, did
not show any sign of germination. The seed sown at
relatively early stage of development (c14 WAP) did
not germinate at all; though, in some cases, showed
nodular swelling, but did not form PLBs and
degenerated subsequently. It may be due to the fact
that embryos have not been reached proper stage of
Fig. I-Different stages of seed germination and plantlets'formation in
C.
rucenu-jerum:
a. Nodular swelling of seeds in culture,
b.
First
set of leaflets are formed in PLBs,
&
c. Rooted plantlets and repetitive PLBs are formed in culture
TEMJENSANGBA & DEB: IN VITRO CULTURE OF C. RACEMIFERUM FROM IMMATURE SEED
225
maturation as reported in Vanda coerulea12,
Cymbidium iridioides and Cy. lowianum8. The
importance of time interval between pollination and
fertilization has also been stressed13. The relative time
taken by ovules after pollination for successful
Table 1—Effect of green pod age and basal nutrient media* on
asymbiotic seed germination and PLBs formation of C.
racemiferum
Basal nutrient Age of Time taken to respond (d)
media capsule
(WAP)
Nodulation PLBs formation
Knudson ‘C’9 8 - -
10 - -
12 90 -
14 84 60 (degenerated)
16 68 56
18 75 70
20 70 -
Mitra et al11 8 - -
10 - -
12 80 degenerated
14 63 42
16 50 53
18 58 60
20 60 degenerated
MS10 8 - -
10 - -
12 120 68
14 70 47
16 52 40
18 55 40
20 70 60
* Media containing sucrose (3%, w/v), CW (15%, v/v) and
NAA (10.0 μM) + BA (8.0 μM) in combination.
germination seems to vary with species14. Other
workers also reported the effect of green pod age on
in vitro germination of orchid seeds8,15-17. In
Dactylorhiza hatagirea17, seeds of 16 WAP exhibited
better germination over younger and older seeds but
in case of Cy. macrorhizon better response was
recorded from seeds of 12 WAP and seeds from
mature pods (20 WAP) did not germinate15. While
Jamir et al8, reported better germination from 120
days old pods in Cy. iridioides.
Previous studies revealed that no single nutrient
medium is universally suitable for asymbiotic seed
germination of all or most orchid taxa. For example,
Mitra et al medium was found suitable over other
nutrient media for Cy. macrorhizon15 and Goodyera
biflora16; Knudson ‘C’ medium for Cy. elegans,
Coelogyne punctulata5 and D. hatagirea17; VW
medium6 for V. coerulea; Nitsch medium8 for Cy.
iridioides; and Knudson ‘C’, VW and MS media for
Aerides rosea18. In the present study, however, MS
medium was found most suitable over Mitra et al and
Knudson ‘C’ media for asymbiotic seed germination
of C. racemiferum (Tables 1 & 2).
Besides seed age and basal nutrient media, other
factors, like organic carbon concentration, CW and
plant growth regulators, have also shown marked
effect on asymbiotic seed germination and subsequent
differentiation in C. racemiferum. The sucrose
concentration (3%) supported better germination
(~90%) on MS10 medium; while on Mitra et a1
medium, 2% sucrose concentration found to be
superior over other concentration and exhibited 80%
germination (Table 2). At other concentrations,
Table 2—Effect of sucrose concentration in different media* on asymbiotic seed germination of C. racemiferum
Basal media Sucrose conc. Germination rate Type of response
(%) (%) (±SE)**
Knudson ‘C’9 0 0 No germination
1 40 (± 3.0) Germinated seeds degenerated subsequently
2 40 (± 2.0) Very few germinated seeds converted into PLBS
3 60 (± 2.0) Germinated seeds formed PLBs and browning of PLBs observed in some cases
Mitra et al11 0 0 No germination
1 45 (± 1.0) Very few germinated seeds converted into PLBS
2 80 (± 2.0) Germinated seeds formed healthy PLBs
3 50 (±2.0) Conversion of PLBs was comparatively poor
MS10 0 0 No germination
1 50 (± 2.0) Germinated seeds mostly degenerated and very few converted into PLBs
2 60 (± 3.0) PLBs were not healthy
3 90 (± 2.0) Germinated seeds formed healthy PLBs
* Media containing CW (15%, v/v) and NAA (10.0 μM) + BA (8.0 μM) in combination
**Standard error
INDIAN
J
BIOTECHNOL,
APRIL
2006
--
germination was poor. Sharma and
and on'
reported
the effect of different organic carbon sources on
in
vitro seed and found 2-3% sucrose,
D-fructose and D-glucose suitable for seed
germination of
Cy.
elegans and Co. punctulatas.
Incorporation of
CW
in the medium did not show
much influence on seed germination in
C.
racemiferum. It, however, enhanced the early
incorporated at 15% (v/v) concentration in the
germination medium (data not presented). Devi et a16
and Vij
et
all9 also reported the promotory effect of
F
differentiation of PLBs into plantlets when
-
CW
on seed germination. Amongst the different
'
concentrations and combinations of plant growth
regulators studied for asymbiotic seed germination of
i
C.
racemiferum, a combined treatment of NAA (10.0
,
pkf)
+
BA (8.0
CLM)
exhibited better PLBs formation,
followed by single treatment of both NAA (10.0
CLM)
and BA (8.0
CLM)
(data not presented). Devi et a16
reported the promontory effect of NAA (0.1-0.5 mg
L")
+
Kn
(1-2
mg L-I) in
V.
coerulea, while Sinha et
a1" reported inhibitory effect of NAA, BA and
Kn
in
the medium. In the present study, the first set of
leaflets started appearing in 14-16 week of cultures on
germination medium (Fig. lb).
I
PLBs
and
Multiple Shoot Formation
On regeneration media, the advanced stage PLBs
started converting into young rooted plantlets and
repetitive PLBs within 3-4 weeks (Fig. lc). Table
3
shows the effect of plant growth regulators on PLBs
formation and regeneration of plantlets. Amongst the
two media tested, it was noted that
MS
medium
I
supported the optimum growth and differentiation
I
into rooted plantlets. While, out of different plant
growth regulator combinations, IAA (10.0
CLM)
+
Kn
(9.0 was found to be optimum, where well rooted
plantlets developed
on
both the media. ~h~ number
of
Fig. 2-Effect of culture media and plant growth regulators' on
multiple shoot formatien and plantlet morphology of
C.
shoot formation
was
higher
On
(figures
are
the optimum regulator
medium containing NAA (10.0
CLM)
+
BA (8.0
combinations only).
*
mi:
on Mitra
et
al"
medium and
rns:
on
but leaves were thin and etiolated (Fig. 2a). The
medium containing NAA (10.0
CLM)
+
Kn
(9.0
pA4)
resulted in stunted growth of plantlets; very few shoot
buds and callusing were observed at the base, though
roots were very long (Fig. 2b). In case of IAA (10.0
pM)
+
BA (8.0
pM)
combination, multiple shoot with
stunted growth and poor roots (Fig. 2c) were
obtained, while IAA (10.0
CLM)
+
Kn
(9.0
CLM)
resulted in well rooted plantlets (Fig. 2d) with
moderate numbers of developed multiple shoots.
MS"
medium (fighres in subscript indicate the concentrations of
plant growth regulators in
CuM)
The protocol described here reveals the different
factors controlling immature seed germination of
C.
racemiferum and could also be used for clonal mass
multiplication of this threatened species facing rapid
denudation. However, further work on in vitro mass
multiplication and short- to medium-term
conservation of the species is in progress.
TEMJENSANGBA & DEB: IN VITRO CULTURE OF C. RACEMIFERUM FROM IMMATURE SEED
227
Acknowledgement
Authors are thankful to the Department of
Biotechnology, Ministry of Science and Technology,
Government of India, New Delhi for financial
assistance through a research grant to Dr Chitta
Ranjan Deb.
References
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Table 3—Effect of different plant growth regulators* on PLBs development and plantlet regeneration of C. racemiferum
Conc. of growth Time taken to Type of response
regulators (μM) respond (d)
NAA10.0 40 Plantlets were short, stout with broad leaves
NAA20.0 44 Plantlets slightly elongated and leaf long and linear
NAA30.0 42 As above
IAA10.0 36 Growth retarded, stunted and etiolated
IAA20.0 40 Growth healthy, leaf broad and long but no multiple shoot
IAA30.0 38 Slightly stunted growth and leaf linear and elongated
BA8.0 43 Growth healthy with few PLBs from the explants
BA16.0 43 As above
BA24.0 42 Moderate plant growth but no multiple shoot buds
Kn9.0 41 Healthy growth with broad leaf
Kn18.0 40 As above
Kn27.0 40 Plant growth retarded and slightly etiolated
NAA10.0 + BA8.0 38 Multiple shoots but leaves are thin and etiolated
NAA20.0 + BA16.0 40 Plant growth normal, leaf thin and short
NAA30.0 + BA24.0 40 Plantlets thin, elongated and etiolated
IAA10.0 + BA8.0 42 Multiple plantlets, leaf long but poor rooting
IAA20.0 + BA16.0 40 Plantlets growth slightly retarded with poor or no root
IAA30.0 + BA24.0 45 As above
IAA10.0 + Kn9.0 43 Well rooted multiple plantlets with repetitive PLBs
IAA20.0 + Kn18.0 42 Growth slightly stunted, leaf small and linear
IAA30.0 + Kn27.0 40 Plant growth stunted and etiolated dies after sometime
NAA10.0 + Kn9.0 40 Well differentiated rooted plantlets with stunted growth
NAA20.0 + Kn18.0 47 Well developed plantlets but growth slightly retarded and stunted
NAA30.0 +Kn27.0 45 Well differentiated plantlets but retarded and stunted growth, and etiolated
*In MS medium containing sucrose (3%, w/v) and CW (15%,v/v)
INDIAN J BIOTECHNOL, APRIL 2006
228
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