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Tropical and Subtropical Agroecosystems
E-ISSN: 1870-0462
ccastro@uady.mx
Universidad Autónoma de Yucatán
México
Chandrasekhara Reddy, M.; Bramhachari, P. V.; Rama Murthy, K. Sri
OPTIMIZED PLANT TISSUE CULTURE PROTOCOL FOR IN VITRO MORPHOGENESIS OF AN
ENDANGERED MEDICINAL HERB Ceropegia ensifolia Bedd.
Tropical and Subtropical Agroecosystems, vol. 18, núm. 1, enero-abril, 2015, pp. 95-101
Universidad Autónoma de Yucatán
Mérida, Yucatán, México
Available in: http://www.redalyc.org/articulo.oa?id=93938025012
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Tropical and Subtropical Agroecosystems, 18 (2015): 95 - 101
OPTIMIZED PLANT TISSUE CULTURE PROTOCOL FOR IN VITRO
MORPHOGENESIS OF AN ENDANGERED MEDICINAL HERB Ceropegia
ensifolia Bedd.
[PROTOCOLO ÓPTIMO DE CULTIVO DE TEJIDOS IN VITRO PARA
MORFOGÉNESIS DE LA HERBACEA MEDICINAL AMENAZADA Ceropegia
ensifolia Bedd.]
M. Chandrasekhara Reddy 1&2, P. V. Bramhachari 2 and K. Sri Rama Murthy 1*
1Department of Biotechnology, Montessori Mahila Kalasala, Vijayawada,
Andhra Pradesh - 520 010, India
2
Department of Biotechnology, Krishna University, Machilipatnam -521 001,
Andhra Pradesh, India
*Corresponding author
SUMMARY RESUMEN
In the present study effect of different concentrations Se estudió diferentes concentraciones de sucrose y
of sucrose and Thidiazuron (TDZ) on in vitro thidiazuron (TDZ) en la morfogenésis in vitro de C.
morphogenesis of C. ensifolia was investigated and ensifolia y se desarrolló un protocolo rápido de
rapid micropropagation protocol was developed from micropropagación in vitro a partir de explantes
in vitro derived nodal explants. Among the sucrose nodales. Entre las concentraciones de sucrosa
concentrations tested, medium concentration (3%) of evaluadas, la concentración media (3%) indujo el
sucrose induced maximum number of healthy shoots mayor número de rebrotes saludables (6.50.68). Los
(6.50.68). The shoots formed in this concentration rebrotes obtenidos a esta concentración fueron
are normal and elongated rapidly. Whereas at higher normales y elongaron rápidamente. A
concentrations delayed shoot induction with stout concentraciones mayores se retrasó la inducción del
shoots stunted in their growth was observed. Among rebrote y se observó brotes robustos atrofiados en su
different concentrations of TDZ tested healthy shoots crecimiento. Entre las concentraciones de TDZ se
with well developed leaves were formed in lower observaron rebrotes saludables con hojas bien
concentration i.e. 0.2 mg/l. the number of shoots desarrolladas a concentraciones bajas (0.2 mg/l)
formed in this concentration was limited to 3.60.18 donde el número de rebrotes fue limitado a 3.60.18
shoots/explant. The shoots raised in vitro were best rebrotes por explante. Los rebrotes cultivados in vitro
rooted on MS medium supplemented with 0.1 mg/l α- enraizaron mejor en medio suplementado con 0.1
Naphthaleneacetic acid (NAA) in combination with mg/l de ácido α-Naphthaleneacetico (NAA) en
0.05 mg/l 6-Benzyladenine (BAP). The maximum combinación con 0.05 mg/l 6-Benzyladenine (BAP).
root induction (84%) with mean number of roots of La máxima inducción de rebrotes (84%) con un
6.70.26 and with mean root length 3.50.28 cm was promedio de 6.70.26 longitud media de raíz de
observed after six weeks of inoculation. Complete 3.50.28 cm fue observado 6 semanas posteriores a la
plantlets developed in vitro were acclimatized inoculación. Plantuelas completamente desarrolladas
successfully with 85% survival in field conditions. in vitro fueron exitosamente aclimatizadas con un
85% de sobrevivencia en condiciones de campo.
Keywords: Ceropegia ensifolia; Thidizuron (TDZ);
sucrose; multiple shoots. Palabras clave: Ceropegia ensifolia; thidizuron
(TDZ); sucrosa; rebrotes múltiples.
INTRODUCTION According to the International Union of Conservation
of Nature (IUCN), it is anticipated that the current
It is a well-known fact that worldwide thousands of species extinction rate is between 1000 and 10,000
plant species are endangered and facing extinction times higher than it would naturally be. Biodiversity
with the current trend of their exploitation and conservation is of global concern which requires a
destruction (Poi et al., 2010; Wyse et al., 2009). holistic approach. Recognizing the need, the
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Chandrasekara Reddy et al., 2015
biodiversity convention was adopted by United carbohydrates in culture media to maintain the
Nations in 1992 (Sharma and Sharma, 2013). osmotic potential and serve as energy and carbon
Convention on Biodiversity seeks to conserve the sources for developmental processes (Chae, 2013). A
endangered plants at their genetic, species and variety of carbon sources viz. glucose; maltose,
ecosystem levels. Nonetheless it is now recognized sucrose, dextrose etc are used in culture media
that ex situ techniques can be efficiently used to depending upon genotypes, species and specific
complement in situ methods, and they represent stages of growth (Bozena and Szczerba, 1991).
conservation of highly endangered and rare species However, sucrose is extensively used as a major
(Ramsay et al., 2000). Recently, a combination of ex transport-sugar in the phloem sap of many plants
situ and in situ conservation methods of a critically (Lemoine, 2000). In micropropagation systems,
endangered plant (Ceropegia fantastica Sedgw.) has morphogenetic potential of plant tissues can greatly
been demonstrated (Chandore et al., 2010). be manipulated by varying concentration of sucrose
Nowadays in vitro multiplication has become an (Yaseen et al., 2013). In addition to that TDZ (N-
advanced tool for protecting medicinal plants, phenyl-N’-1,2,3-thidiazol-5-ylurea) is a substituted
particularly rare and endangered species (Khan et al., phenyl urea compound which was developed for
2012). mechanized harvesting of cotton bolls and emerged as
a highly effective bioregulant of morphogenesis in
The species of Ceropegia as a whole are under threat, plan tissue culture (Guo et al., 2011). Unlike
owing to either destructive collection or habitat conventional phytohormones, it was proved that TDZ
degradation. They are not only genetically depleted individually contented the requirements of various
but also are scarcely available. These species are of regenerative responses of many different plant species
economic importance (Jagtap and Singh, 1999) due to (Sanikhani et al., 2006). However, in previous studies
their starchy edible tubers with medicinal value it has shown that TDZ exhibited both auxin and
(Murthy and Kondamudi, 2011). The tubers of this cytokinin like effects, although, it is chemically,
genus contain sugars, starch, fats, albuminoids, gums different from auxins and cytokinins (Murthy et al.,
and crude fibers which are useful as nutritive tonic 1998). It exhibits strong cytokinin like activity and
(Kirtikar and Basu, 1935). The active compound of promotes the proliferation of axillary shoots (Faisal et
tuberous roots is the alkaloid cerpegin which is active al., 2005). In addition to that TDZ also releases the
against diarrhea and dysentery inflammation of gums lateral bud dormancy and stimulates adventitious
and delirious fevers of parturition (Nadkarni, 1976). organ regeneration (Mroginski et al., 2004). These
Recently this genus has attracted the attention of interesting findings on TDZ raised many tantalizing
several biologists for conservation, since many of the questions for researching in plant morphogenesis. The
Ceropegia species are listed in RED data book of purpose of the present work was to investigate the
Indian medicinal plants (Srinivasarao et al., 2010). effects of sucrose and TDZ on morphogenesis of C.
Furthermore, the application of advanced tissue ensifolia using nodes with axillary buds as explants.
culture protocols is of economic use for the industrial So for the reports are not available on in vitro
production and consequently, conservation of micropropagation for the regeneration of C.ensifolia.
Ceropegia species. However, for effective
reintroduction of these plants, in vitro morphogenesis MATERIALS AND METHODS
protocols must be established along with the habitat
conservation and other appropriate ex vitro strategies. In the present study nodal segments derived from the
Ceropegia ensifolia Bedd. (Asclepiadaceae) is an in vitro grown plantlets were used as explants to
endemic, perennial, tuberous twining herb distributed study the effect of different concentrations of sucrose
originally isolated from Peermadu Ghat of Kerala. and TDZ on in vitro morphogenesis of C. ensifolia. In
This species is now extended its distribution in Tamil order to study the effect of sucrose on morphogenesis
Nadu, i.e. at high wavy mountains in Theni district of of C. ensifolia, single nodal segments were separated
Western Ghats, India. In recent days the population of (1 cm long) and cultured on Murashige and
this species is very rare due to habitat degradation. Skoog (MS) media containing varying concentrations
Conventional propagation of this plant species is of sucrose (1 to 5%) along with BAP (3.0 mg/l)
apparently difficult, owing to low fruit and seed respectively. The pH of the media was adjusted to 5.6
setting, less viability of seeds, poor seed germination to 5.8. Then the agar (0.8%) was added to the media
and delayed rooting of seedlings. Therefore we o
and autoclaved for 20 min at 121 C and15 lbs
propose an in vitro propagation method as an pressure. The cultivation was performed at 25±2°C
alternative technique for propagation of this with optimized room conditions for 6 weeks under
endangered species and reestablishment of this plant fluorescent lightening (Philips) of 3000 lux (16/8,
population in natural habitat. D/N cycle) at the top of the culture vessels. The
plantlets grown in vitro were harvested from all
In vitro plant cells, tissues and organ cultures are not replicate vessels of a treatment and evaluated their
fully autotrophic, establishing a need for morphogenesis status. The parameters considered
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Tropical and Subtropical Agroecosystems, 18 (2015): 95 - 101
were a) percentage of shooting response, b) number on shoot initiation and proliferation. Different
of shoots per explants, c) shoot height (cm). concentrations of sucrose showed varied responses
during plant morphogenesis of C. ensifolia. The
In order to study the influence of TDZ on shoot optimum response that is healthy shoot formation was
proliferation of C. ensifolia, nodal segments was observed when 3.0 mg/l sucrose was added to the
inoculated on MS basal media augmented with TDZ medium (Table – 1). The explants inoculated in this
at different concentrations (0.1 to 3.0 mg/l). The medium showed fast sign of shoot sprouting when
explants derived from sprouted shoots were compared with the explants inoculated on other
transferred periodically to the fresh medium and in media. The shoots formed in this medium are good to
each subculture the transfer of explants were carried observe with maximum number of shoots (6.50.68)
out for every six weeks. The frequency of explants and with maximum shoot length (8.40.25 cm) (Fig –
sprouting, number of shoots formed per explants and 1B). These cultures survived for a longer duration in
shoot length after six weeks of culture were recorded culture conditions. The shoots which are formed in
as observations to know the optimum concentration the lower concentrations of sucrose are with linear
of TDZ for effective shoot proliferation in C. stems with pale yellowish green leaves (Fig – 1A).
ensifolia. Initially in the lower concentration (1.0 mg/l) the fast
sign of shoot sprouting was observed, which
Attempts were made to root the cloned shoots under developed into thin linear shoots very rapidly and
in vitro conditions by using various combinations of latter these shoots under gone weathering and failed
NAA, BAP and Kinetin (KN). The healthy shoots to survive for longer durations. The explants taken
were harvested and cultured on 0.6% agar gelled MS from these shoots were failed to sprout in further
medium supplemented with 0.1% NAA in subcultures. Whereas at higher concentrations of
combination with different concentrations of BAP sucrose the initial shoot sprouting was delayed for
and KN. Plantlets with well developed shoots and longer durations (nearly 2 weeks) and the shoots
roots were taken out from the culture vials and were formed in these concentrations are stout and stunted
cleaned with distilled water to remove traces of agar. in their growth (Fig – 1C). Browning of the medium
Roots of each plantlet were treated with 0.5% was also observed due to leaching of phenolics from
fungicide (carbendazim 50% WP) solution before the base in to the medium. Swelling of the base of
planting into plastic cups contained sterile soil, sand explants and tuber formation was also noticed when
and vermicompost in different ratios. The pots were higher concentrations of sucrose present in the
covered with polythene covers; these hardened medium (Fig – 1F). The level of sucrose in tissue
plantlets were irrigated with sterilized 1/4 MS salt culture can affect the growth and development of the
solution devoid of sucrose for initial 2 to 3 days and plants. To address this problem, several researchers
◦
were maintained in culture room at 25 ± 1 C with showed that sucrose at 3% level was optimum for
photoperiod of 16 h light. Subsequently after 4 to 6 healthy shoot regeneration (Vinterhalter and
weeks the covers were pored to allow free gaseous Vinterhalter, 1999: Adjei, 2001, Gauchan, 2012).
exchange with the outer environment and then
removed to expose the plantlets to ex vitro conditions. In C. ensifolia lower concentrations of TDZ played a
Primary hardened plants after two weeks were prominent role in shoot induction. The shoots that are
transferred to earthen pots and irrigated with tap formed at lower concentration of TDZ showed
water and observed for their further growth. excellent results in their morphogenesis especially
with healthy stem and broad dark green elongated
Each experiment was repeated three times and twenty leaves which resembled the plants of the natural
replicates were maintained. The data regarding environment. The maximum shoot sprouting (90)
number of shoots formed, shoot length, number of percentage with mean number of shoots (3.60.18)
roots formed and root length was recorded and mean shoot length (10.10.81 cm) was observed
periodically (six weeks) for shoot multiplication and on MS medium supplemented with 0.2 mg/l TDZ
rooting respectively. Raw data were subjected to (Table–2, Fig–1D). However, with the increase in
analysis of variance (ANOVA) carried out by the concentration of TDZ, decrease in shoot sprouting
SPSS 20 (SPSS Inc. Chicago, IL, USA). The values percentage and shoot number and length was
with P≤ 0.05 were considered to be statistically observed. The shoots that are formed at higher
significant. concentrations of TDZ showed reduction in leaf size
(Fig–1E). Even though the shoots contain reduced
RESULTS AND DISCUSSION leaf size, the nodal explants derived from these shoots
are good in their shoot sprouting and produced
The carbon sources (sucrose) serves as energy and healthy shoots in further subcultures. Some studies
osmotic agents to support the growth of plant tissues. have previously reported the significant role of TDZ
In the present study different concentrations of in shoot proliferation like cytokinins in a number of
sucrose were added in the media to know their effect
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