Journal

Journal of Applied Horticulture, 21(1): 20-24, 2019 Appl

In vitro cormlet production- an efficient means for conservation

in Ensete superbum (Roxb.) Cheesman

T.G. Ponni* and Ashalatha S. Nair

Department of Botany, University of Kerala, Kariavattom, Thiruvananthapuram. *E-mail: [email protected]

Abstract
Ensete superbum from the family Musaceae is commonly known as Kallu vazha (wild/ rock/cliff banana). The species holds a precise
position in the field of medicine for its anti-hyperglycemic, anti-diuretic and spermicidal potential as well as ornamental value in
botanical gardens. Due to deforestation, habitat fragmentation, indiscriminate harvesting for commercial gain, absence of suckers,
and recalcitrant nature of seeds; this species is facing a drastic reduction in its propagation. The present study developed a protocol
for the production of cormlets from explants isolated from inflorescence. The explants were cultured on MS media supplemented with
4mg L-1 BAP and 1.5 mg L-1 KIN and an average of six to ten cormlets were produced/ explants within eight weeks. Shoot induction
occurred from the cormlets on MS medium with 3mg L-1 IBA and 1.5 mg L-1 BAP. Cormlets inoculated on MS medium supplemented
with 1000 mg L-1 glutamine for a period of four weeks enhanced the size of cormlets which in turn increased the number of shoots.
An average of ten multiple shoots were obtained on MS medium supplemented with 5 mg L-1 BAP. Maximum rooting was obtained
on half strength MS medium with 3 mg L-1 IBA, 0.1 mg L-1 BAP and 1% activated charcoal. The plantlets were transferred to Knop’s
solution for acclimatization. Rooted plants were hardened successfully in cocopeat along with sand in 1:1 combination and transferred
to soil with 98% survival rate.
Key words: Ensete superbum, conservation, micropropagation, cormlets, glutamine, Knop’s solution.

Introduction 2014). It is commonly known as ‘false banana’ since unlike

commercial bananas, its pseudo stem and corm are edible
Ensete superbum (Roxb.) Cheesman, a threatened medicinal
plant, commonly known as rock, wild, or cliff banana, shows rather than ripe fruits and possess high starch compared to
dissimilarities with Musa both morphologically as well as potato (Birmeta et al., 2004). Sethiya et al. (2016) reported
genetically even though residing under the same family musaceae. that the methanolic extract of its pseudostem showed highest
The genus Ensete which comprises of nine species having a concentration of phenolics, flavonoids and had significant anti-
chromosome number 2n= 18 (Cheesman 1947, Simmonds 1960, oxidant activity. TLC fingerprint is also used in characterization
1962). It is a monocarpic, non- stoloniferous perennialshrub of plant extract for standardization (Sethiya et al., 2016).
usually propagated by seeds. It is endemic to the Western Ghats, Unregulated harvesting of seeds, seedlings from its natural
the Aravalli range and North-eastern hills of India and Northern habitat and nonscientific leaf harvesting drastically retarded
Thailand (Vasundharan et al., 2010). Ensete possess a swollen flowering and fruiting which in turn reduced its population (Bhise
base called corm, a pseudo stem, very large broad leaves with et al., 2015). Hence it is considered as rare, relict, endangered,
prominent midribs, inflorescence that resembles a lotus flower, threatened and conservation concern species (Vasundharan et
all contribute to its ornamental value in botanical garden.
al., 2011).Vasundharan et al. (2015) reported the need of further
Its endosperm is used as remedy for different ailments such as initiatives from the authorities to empower Good Agriculture and
diabetes, kidney stone and leucorrhoea. Presence of non-steroidal Collection Practices (GACP) for medicinal plant recommended
phytosterole-4-hydroxyl-3-methyl-hex-5-enyl is reported to lower by world health organization and to study the present population
cholesterol when it is used as a food additive (Vasundharan et al., status of the species by IUCN’s guidelines and recommendations
2013, Kachroo et al., 2011). The ethanolic seed extract possess to conserve this Wild Banana species struggling for survival.
appreciable spermicidal potential and can be used as an effective
contraceptive (Sarwar et al., 2014). It was reported that aqueous A few reports on clonal propagation and somatic embryogenesis
seed extract of Ensete can inhibit the growth of calcium hydrogen were reported for this species (Mathew et al., 1996, 2000, 2002).
phosphate dehydrate, thereby preventing urinary stone formation Somatic embryogenesis from male floral apices of Ensete was
(Diana et al., 2013).The plant or plant product was used as a published by Kulkarni et al. (2002). Negash et al. (2001) reported
prescribed medicine by indigenous practitioners for nineteen the in-vitro conservation of Ensete ventricosum shoot tips for
etiological symptoms (Vasundharan et al., 2015). six months. The present paper reports in-vitro production of
Its unripe fruits and flowers were used as vegetables and was cormlets, which is the key organ for multiple shoot induction in
reported that its antioxidant activity was found higher than Musa species, as a tool for micropropagation and conservation
commercial banana after thermal processing (Sasipriya et al., of this species.
Journal of Applied Horticulture (www.horticultureresearch.net)
 In vitro cormlet production- an efficient means for conservation in Ensete superbum 21

Materials and methods Regenerated shoots were transferred for shoot multiplication on
MS medium supplemented with BAP at concentration of 3, 4, 5
Plant materials: Inflorescence (apical meristem with male floral or 6 mg L-1 alone or in combination with IBA (at concentration of
buds) having 22 cm length and 30-32cm diameter (Fig.1A), 0.5, 1, 1.5 or 2 mg L-1), respectively. Observations were recorded
were collected from three to four years old wild plants growing at one week interval for a month.
at Sethathodu, Pathanamthitta district, Kerala, India (9.34oN,
76.99oE), during of November 2014. The outer bracts were Shoots, after attaining a height of 4-5 cm were transferred
removed to attain an inflorescence with 6cm length and 3 cm to rooting medium consisting of half strength MS medium
diameter, washed first in in 10% labolene (Qualigens, India) for supplemented with 3 mg L-1 IBA along with 0.1 mg L-1 BAP
15 minutes and then in running tap water for 30 minutes. It was with or without 1% wv-1 activated charcoal. After four weeks of
surface sterilized by 0.1% HgCl2 for ten minutes in laminar air culture, shoots with roots were washed thoroughly to remove any
flow hood, rinsed with sterilized double distilled water for five trace of medium and were shifted to a Knop salt solution without
any sucrose (Prasad et al., 1975).
Plantlets with healthy shoots and roots were taken from Knop
solution and washed thoroughly with a pinch of bavistin in sterile
water. They were transferred to plastic pots with coco peat (Home
Green Agri Private Limited) and sand in 1:1 combination and
were covered with polythene bags to maintain high humidity and
were watered on a daily basis. Polythene bags were perforated
gradually for acclimatization and were transferred to greenhouse
conditions.
All the data were analyzed by Analysis of Variance (ANOVA)
Fig. 1. (A) Inflorescence explant. (B)3.5 cm represents explant with followed by Duncan’s multiple range tests using SPSS version 24.
apical meristem. Fig.1 (B Inset) represents explants of approximately
1cm in size
times, five minutes for each wash. Both ends were trimmed to get
Results and discussion
a final size of 3.5 cm length and 1.5 cm diameter, and were divided Explants inoculated on MS medium supplemented with BAP
longitudinally into two pieces. Explants of approximately 1cm alone or in combination with KIN showed, darkening of the
in size were isolated from the basal region aseptically in such a cut surface, bulging and velvety texture on the surface within
way that each piece consisted of basal apical meristem (Fig.1B). two to three days after inoculation. The explants showed the
development of a basipetal bud like structure which later swollen
The explants were inoculated on MS medium (Murashige and into globular structures called cormlets within four weeks. In the
Skoog, 1962) supplemented with 6-benzylaminopurine (BAP) medium with lower concentration (1, 2 mg L-1 BAP) of cytokinin,
at concentration of 1, 2, 3, 4 or 5 mg L-1 alone or in combination the explants initiated green callus. An average of four cormlets
with l, 6-furfuryl amino purine (KIN) at concentration of 0.5, per explants were obtained on MS medium supplemented with
1, 1.5 or 2 mg L-1 respectively. The pH of the medium was 4mg L-1 BAP within four weeks of culture (Table 1).The explants
adjusted to 5.8, solidified with 0.7% agar (wv-1) and sterilized inoculated on MS medium supplemented with 4 mg L-1 BAP and
by autoclaving at 121ºC at 15 lbs. pressure for 15 minutes. The 1.5 mg L-1 KIN developed maximum number of cormlets per
explants were inoculated horizontally on the medium. Cultures explants (Fig. 2). About 90 % of explants showed four to eight
were maintained under 16 hours photoperiod at 25±1ºC and cormlets production, after eight weeks of culture (Fig. 3A).
60 - 80% relative humidity. The light intensity of 2500 lux was
At the time of shoot regeneration, black surface of the cormlets
provided by means of white fluorescent light. The response was
developed white spotted appearance, which later produced
observed after four weeks of culturing and number of cormlets
numerous clumps of shoot buds, developing white and brownish
per explants were recorded.
mass of tissues within two weeks. An average of 19 shoot buds/
Cormlets obtained were sub cultured on MS medium supplemented Table 1. Effect of different concentrations of BAP on MS medium in
with IBA at concentration of 1, 2, 3, or 4 mg L-1; along with 1.5 cormlet induction. Mean number of cormlets per explant after four and
mg L-1 BAP to induce shoot regeneration. The average number eight weeks of culture
of shoot buds, length of longest fleshy leaves, length of longest Concentration of No: of cormlets / No: of cormlets /
BAP explant after four explant after eight
leaf sheaths and number of normal shoots were recorded at two mg L-1 weeks weeks
weeks interval up to 12 weeks. .
1 1.20±0.374d 2.00±0.548 d
The cormlets were inoculated on MS medium supplemented
2 2.00±0.316cd 3.60±0..400c
with 3 mg L-1 IBA, 1.5 mg L-1 BAP and 500, 1000 or 1500 mg
3 3.20±0.374 b
5.20±0.374 b
L-1glutamine. Average number of shoot buds, length of longest
fleshy leaves, length of longest leaf sheaths and number of normal 4 4.40±0.245a 7.40±0.510 a
shoots were recorded at two weeks interval up to 12th week. 5 2.80±0.374bc 5.20±0.374 b
Photographs of free hand sections of the cormlets were taken
Values represent means ± SE of 10 replicates, those representing different
using image analyzer (Leica DM 2000) to analyze the mode of letters in appropriate column are significantly different (ANOVA,
shoot regeneration. P<0.05).

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22 In vitro cormlet production- an efficient means for conservation in Ensete superbum

Fig. 2. Effect of different concentrations of BAP and KIN in different combinations on cormlet induction after
four weeks of culture

Fig. 3. (A) Explant with eight to ten cormlets in 4 mg L-1BAP along with 1.5 mg L-1KIN.(B) Clumps of bud like white structures open out after
six weeks resulted in thick, fleshy, white leafy shoot, later get transformed to cream, pale green ( without glutamine). (C) One with glutamine.
The path of regeneration from cormlets was exactly the same in both cormlets with and without glutamine. (D) Green fleshy leaves emerging
from cormlets. (E) Leaf sheath and normal leaves developing after 12 weeks of culture. Bars:1 cm (A, B, C, D, E).
cormlets were observed after another two weeks on MS medium size of cormlet without glutamine was observed to be 1.14 cm in
supplemented with 3 mg L-1 IBA and 1.5 mg L-1 BAP. These diameter while one with glutamine showed 2.70 cm in diameter.
clumps of buds developed to fleshy, white leafy shoot, which It was reported early that the role of proline and glutamine in
then further turned to cream, pale green and finally green in improving callus induction and subsequent shooting in rice
colour. Bud developmentfrom cormlets without glutamine (Fig. (Pawar et al., 2015). Green et al. (1990) reported that the nature
3B) and one with glutamine (Fig. 3C) showed similar path of of nitrogen source can significantly affect the in-vitro responses
developments. When it attained an average height of 2cm (Fig. of the explant. Organic nitrogen sources like amino acids proved
3D), normal shoots and leaves appeared within the clumps (Fig. to be beneficial for cell proliferation, increasing biomass of the
3E). On an average, 13 shoots/cormlet developed after six weeks explants as well as regeneration rate and helped to maintain high
of culture. Maximum number of shoot development was observed growth rate for a longer period than inorganic source like nitrates
from explants inoculated on MS medium supplemented with 3 and ammonium (Gamborg et al., 1968, Franklin et al., 1991,
mg L-1 IBA and 1.5 mg L-1 BAP (Table 2). Vasudevan et al., 2004).
The cormlets inoculated on MS medium supplemented with 3mg Shoot regeneration from cormlets inoculated on MS medium with
L-1 IBA along with 1.5 mg L-1 BAP and 1000 mg L-1glutamine glutamine showed higher number of shootbuds, sprouted shoot
showed larger cormlets. After four weeks of culture, an average buds and shoots (Table 2).The path of regeneration of shoots from
Table 2. Effect of different concentrations of IBA (1, 2, 3, 4 mg L-1) and cormlets was exactly the same both with or without glutamine
glutamine (500, 1000, 1500 mg L-1) along with 1.5 mg L-1 BAP on MS treatment. Histlogical study showed the presence of shoot
medium for shoot regeneration from cormlets.Mean number of shoot meristems, emerging fleshy leaves within the cormlets (Fig. 4A, B
buds, sprouts and shoots were represented at fourth, and twelfth week and C). In the present study an average of 13 shoots developed per
of culture respectively.
cormlet, and an addition of glutamine increased the cormlet size
Concentration Glutamine No. of shoot No. of shoots
of mg L-1 buds fourth twelfth
IBA mg L-1 week week
1 - 5.40± 0.92d 1.80±0.37d
2 - 13.40±0.92b 9.00±0.70b
3 - 19.80±1.35a 13.60±1.50a
4 - 9.00±1.00c 5.00±0.70c
3 500 22.60±1.20 b 17.00± 1.41b
3 1000 28.60±0.92a 21.20± 0.58a Fig. 4. (A) Longitudinal section of in vitro regenerated corm showed
an apical meristem enclosed with fleshy leaves on either side which
3 1500 17.40±1.28c 11.40± 1.03c later gives rise to normal shoots, (B) elongated leaf sheath emerging
Values represent means ± SE of 10 replicates, those representing from corm. (C) Lower portion of leaf sheath (upper arrow), inner to
different letters in appropriate column are significantly different that internal apical meristem (lower arrow) for normal shoot appeared.
(ANOVA, P<0.05). Bars: 500 µm (A, B, C).
Journal of Applied Horticulture (www.horticultureresearch.net)
 In vitro cormlet production- an efficient means for conservation in Ensete superbum 23

and produced an increased number of 21 shoots/cormlets. Similar Table 4. Effect of activated charcoal and Knop’s Solution on rooting.
results were reported by Aboshama (2011) where addition of 500 3 mg L-1 IBA along with 0.1 mg L-1BAP in half strength MS medium
for root induction. NR- number of roots, LR-Length of the longest root
mg L-1 glutamine to shoot regeneration medium showed highest was represented in cm. Results were recorded after fourth and eighth
number of shoot induction in Buddelia globosa and by Hamasaki week of culture.
et al. (2005) where 8 mM glutamine along with shoot induction Concentration Activated Fourth week Transferred to
medium indirectly increased endogenous IAA and iP levels of PGHs IBA 3 charcoal Knop’s Solution
mg L-1 + 1% wv-1 eighth week
resulting in increased explant competence for organogenesis in BPA 0.1 Number Length Number Length of
pineapple leaves. mg L-1 of of longest of longest
roots root roots root
The clumps of shoots obtained above were separated and grouped _ _ 1.00 ± 1.16± 2.40± 3.14±
into ten, each with two plantlets and transferred to fresh medium 0.31b 0.33b 0.24c 0.15c
with varying concentrations of BAP viz., 3, 4, 5 or 6 mg L-1 for _ + 1.60 ± 1.80± 4.20± 4.14±
shoot multiplication (Table 3). Higher concentration of BAP 0.51ab 0.46b 0.37b 0.18b
showed increase in shoots as well as leaf length. MS medium + _ 1.20 ± 1.10± 3.40± 3.76±
0.37b 0.33b 0.24bc 0.25bc
supplemented with 5 mg L-1 BAP, showed significantly increased + + 3.00 ± 3.54± 6.40± 6.24±
shoot as well as leaf numbers after four weeks of culture (Fig. 5A). 0.31a 0.20a 0.51a 0.34a
When leafy shoot alone was transferred to shoot development Values represent means ± SE of 10 replicates, those representing different
medium, no response was observed, whereas the same with a letters in appropriate column are significantly different (ANOVA,
little portion of corm showed normal shoot development.The rate P<0.05).
of shoot multiplication was lower in IBA and BAP combination 98% survival rate and weremaintained in greenhouse (Fig. 5C).
rather than BAP alone (Table 3). Hence in the present study
Table 3. Effect of different concentration of BAP either alone or in
Most of the earlier reports on micropropagation of Ensete, used
combination with IBA on MS medium for shoot multiplication.Mean shoot tips from corm tissue as explants (Afza et al., 1996, Negash
number of shoots, leaves and length of shoot (cm) after four weeks of et al., 2000, Diro et al., 2004). The source of shoot tip explant was
culture.
either greenhouse grown plants or in-vitro germinated seedlings
Concentration Number Number Length of
(mg L-1) of of shoots (Negash et al., 2000). Availability of such explants was not easy
BAP IBA shoots leaves (cm) since the species population is decreasing. Hence present study
3 - 5.80±0.37c 12.20±0.97b 3.14±0.09b used inflorescence tip as explants.
4 - 7.20±0.37b 16.20±1.46a 3.30±0.15b
5 - 9.00±0.44 a
19.00±1.00 a
4.02±0.16a Earlier report on clonal propagation of Ensete through shoot tip
6 - 2.40±0.51d 7.60±1.50c 3.90±0.18a cultures showed an average of 1-4 cormlets per explant on MS
5 0.5 0.50±0.298c 1.75±1.03c 1.45±0.83b medium with 1.5 mg L-1 BAP and 1mg L-1 KIN, within four weeks
5 1.0 4.00±0.548 b 13.80±0.917b 3.540±0.248 a of culture (Mathew et al., 1996). However inflorescence explant
5 1.5 6.60±0.510 a 18.60±1.030 a 3.660±.2657 a showed higher yield of cormlets with in a shorter period of four
5 2.0 1.00±0.447 c 3.20±1.463 c 1.760±0.719 b
Values represent means ± SE of 10 replicates, those representing different
weeks. Even though high phenolic exudation retards responses,
letter in appropriate column are significantly different (ANOVA, darken the medium, showed degradation and necrosis from the
P<0.05). distal end of the explant, it helped in reducing contamination,
a higher cytokinin concentration was found to be better for development of both corms as well as healthy green leafy shoots.
maximum shoot multiplication. After attaining a height of 3-4 Similar to corm development, leafy shoots development was
cm, shoots were transferred to rooting medium. also basipetal. Earlier report on E. ventricosum showed normal
shoot development required a little portion of corm (Birmeta et
The shoots developed roots on half strength MS medium with 3
al., 2004).
mg L-1 IBA, 0.1 mg L-1 BAP and 1% wv-1activated charcoal (Fig.
5B). Averages of three roots with 3.54cm length were obtained In the present study a single inflorescence may yield approximately
after four weeks of culture. Then the plantlets were transferred more than 60 explants and contamination rate was also less or
to Knop’s solution. The plantlets adapted well in Knop solution completely absent. An average of 21 shoots developed per cormlet
and increased root number as well as root length. A maximum of indicating the potential of this method for conservation of this
six to eight roots/shoot having a length of 6-10 cm were observed species.
with new leaves within four weeks (Table 4). Rooted plants were
transferred to cocopeat and sand in 1:1 combination and showed Traditional vegetative propagation methods already published for
this species (Karlsson et al., 2015), reportedly took about nine
months for sucker induction at the expense of 63 corms of two
year old plants, buried under soil. A similar vegetative propagation
method was reported by Simmonds (1966) in E. superbum, where,
before flowering, hollowed corms were stuffed with soil and dung.
Both found to be time consuming. The present study introduced
an efficient micropropagation method using apical meristem in
the inflorescence as explants, without harming its natural means
Fig. 5. (A) Multiple shoots obtained in MS medium with 5 mg L-1BAP
(B) Rooting in half strength MS with high auxin to cytokine (IBA 3 mg of propagation or its habitat, thereby successfully contributing
L-1 , BPA 0.1 mg L-1) along with activated charcoal.(C) Hardened plants to the conservation of wild gene pool and therapeutic potentials
in green house. Bars: 1cm (A, B), 4 cm (C). of this rare, threatened rock banana - E. superbum.
Journal of Applied Horticulture (www.horticultureresearch.net)
24 In vitro cormlet production- an efficient means for conservation in Ensete superbum

Acknowledgements Mathew, M.M. and V.J. Philip, 2002. Somatic embryogenesis versus
zygotic embryogenesis in Ensete superbum. Plant Cell, Tissue and
We are grateful to Dr: Suhara Beevy, Associate Professor and Organ Culture, 72: 267-275.
Head of the Department, Department of Botany, University Mathew, M.M., R. Manuel and V.J. Philip, 2000. Callus Regeneration
of Kerala for continuous encouragement and support. We also And Somatic embryogenesis in Ensete superbum (Roxb. Cheesman).
Indian Journal of Plant Physiology, 5(4): 392-396.
thank Kerala University authority for providing instrumentation
facilities and University Junior Research Fellowship for the Murashige, T. and F. Skoog, 1962. A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiology Plant, 15:
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Negash, A., K. Puite, J. Schaart, B. Visser and F. Krens, 2000. In vitro
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