In vitro Seed Germination and Micropropagation of Edible Bamboo Dendrocalamus giganteus Munro using Seeds
W. Sayanika Devi,
Successful propagation from seeds of Dendrocalamus giganteus Munro was achieved by in vitro methods. Incidence of sporadic flowering was recorded. The descriptions of inflorescence and floral morphology are in agreement with the prior taxonomic descriptions but a more detailed description and illustrations are presented in this communication. In vitro seed germination was enhanced in gibberellic acid (GA3) supplemented liquid Murashige and Skoog (MS) medium in the presence of light. Shoots multiplication was obtained directly from seeds inoculated on MS medium containing benzylaminopurine (BAP). In another method, callus was induced and proliferated on MS medium supplemented with 3 mg L-1 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg L-1 kinetin (Kn). De novo production of shoots took place from white, compact and nodular calli after transfer to MS medium containing 1 mg L-1 naphthalene acetic acid (NAA) and 0.5 mg L-1 Kn. Regenerated shoots were excised and induced to root on media with auxin. In vitro rooting percentage of shoots reached 86% with half-strength MS medium containing 5 mg L-1 indole-3-butyric acid (IBA). After the process of hardening and acclimatization, the plantlets established well when transferred to field during favourable season.
Received: January 15, 2012;
Accepted: February 03, 2012;
Published: May 25, 2012
Dendrocalamus giganteus Munro is a giant bamboo, native to southern
Myanmar and northwestern Thailand, which is cultivated in India, Sri Lanka,
Bangladesh and Southern China (Louppe et al., 2008).
It occurs naturally in humid areas at slightly higher altitudes (up to 1200
m). It can, also, be grown successfully at low altitudes on rich alluvial soils.
In Manipur (23°47'-25°41' NL; 92°58'-94°47' EL), it grows luxirantly
along the river banks on sandy and clay mixture of soils. The culms are erect,
14-25 m tall and 8-20 cm in diameter, grey-green and covered with white waxy
scurf when young. It is used extensively in making different kinds of decorative
pieces, house construction, water pipes, for making pulp etc. Besides, the young
tender shoots are considered as the most popular succulent vegetables found
in Manipur. The young succulent shoots are sold in the markets by vendors in
fresh, pickle, salad or fermented form. Furthermore, the ethnic communities
of Manipur prefer to consume the young shoots of this species as compared to
other edible bamboo species found in the region due to its delicious taste and
less irritant sensation in the mouth and throat. The irritability is caused
due to the presence of cyanogenic glycoside-taxiphyllin which can be removed
by cooking (Schwarzmaier, 1977; Ferreira
et al., 1995; Haque and Bradbury, 2002).
Bamboo shoots are rich in various nutrients, minerals, and also contain lignans
and phenolic acids which have anticancer, antibacterial, antifungal and antiviral
activity (Bhatt et al., 2005; David
and Nirmala, 2007). The demand for this bamboo species is increasing much
more than their availability, hence it is necessary to conserve this resource
for sustainable development. Bamboo is known for its monocarpic habit, i.e.,
flowering once before culm death (McClure, 1966). Bamboo
flowering is regarded as a bad omen in several states of India, especially where
the flowering incidence is accompanied by an increase in rodent population.
It is believed that flowering of bamboo brings disasters like famine and other
natural calamities, which has compelled the rural people to destroy the clumps
after or during blooming (Mohan-Ram and Hari Gopal, 1981;
John and Nadgauda, 2002). Conventional propagation of
bamboo through seeds is not reliable because the sporadic flowering that takes
place annually in isolated clump rarely set seeds (Ramayana
and Yakandawala, 1998) and so far, there is no report available on gregarious
flowering of Dendrocalamus giganteus. Furthermore, vegetative propagation
using culm cuttings and rhizomes is independable due to the bulky size of the
propagules for plantation purposes. The vegetatively propagated clones will
also have the same physiological state of their wild stock and will tend to
flower leading to the entire culm death. Hence, the present study was undertaken
to develop a method for in vitro propagation of Dendrocalamus giganteus
Munro, an important edible bamboo using seeds.
MATERIALS AND METHODS
Plant material: Surveys were carried out to record the incidence of flowering. During June, 2005, incidence of sporadic flowering of Dendrocalamus giganteus Munro was noticed in 3 locations along riverbanks of the Arapti river (24°44'N, 93°56'E), Manipur. All culms were flowered in the three flowering clumps. Inflorescences were collected and key morphological characters of fresh spikelets were recorded. Measurements were taken from at least five different fertile florets from each inflorescence. Seeds were harvested from flowering spikes and stored at 4°C for 4 months.
In vitro germination and direct shoot organogenesis from seeds: Seeds were dehusked and washed with 0.1% w/v Exalin (Merck, India) detergent solution for 10 min to remove foreign debris. The seeds were surface sterilized in 20% (v/v) of sodium hypochlorite (NaOCl) for 20 min, and rinsed five times with sterile distilled water. Single seeds were put into 100x10 mm test tubes containing MS culture medium supplemented with 2% sucrose (w/v) with or without 0.7% agar (v/w). Seeds incubated in liquid MS medium were supported with filter paper bridge. The experiments were repeated three times, each repetition comprising 25 seeds (tubes). All media were supplemented with different concentrations of PGRs i.e., BAP, Kn and GA3 (0.1, 0.2, 0.3, 0.5, 1.5 and 3.0 mg L-1) followed by incubation both in the dark and light. GA3 was sterilized using 0.2 μM millipore filters and added after autoclaving. After 2 weeks of incubation, percentage of seeds germination was recorded. Shoot proliferation from intact seeds were carried out in MS medium supplemented with various concentrations of BAP (1-10 mg L-1). Propagules of three shoots were excised and subcultured in MS medium containing different concentrations of BAP (1-10 mg L-1) for further multiplication. Data for multiplication rate were recorded after third subcultures.
Culture conditions: The pH of the medium was adjusted with 1 N NaOH
and 1 N HCl to 5.7±0 prior to the addition of 0.8% agar (Hi-Media). All
culture tubes containing media were autoclaved at 121°C for 20 min. Cultures
were maintained in a growth chamber at 25±2°C with a 16 h photoperiod
at 90-95 μmol m-2 sec-1 provided by cool white fluorescent
Callus induction, growth and maintenance: Modified MS (Murashige
and Skoog, 1962) medium containing B5 vitamins (Gamborg
et al., 1968) supplemented with various concentrations of 2, 4-D
(0.5, 1, 2, 3 and 5 mg L-1) either alone or in combination with BAP
or Kn (0.2, 0.5 and 1 mg L-1) was used for callus induction. Using
a dissecting microscope, fully matured aseptic seeds were selected based on
the completely filled endosperm and darked colored seeds without wrinkles on
the outer surface. Zygotic embryos were rescued from the seeds using scapel
with proper care and inoculated on the callus induction medium. Initially, the
cultures were maintained in the dark at 25±2°C till callus induction
and after 4 weeks, all cultures were maintained under 16 h photoperiod providing
an approximate irradiance of 15 μmol m-2 sec-1 at
the same temperature.
Root development and acclimatization of plantlets: In vitro raised shoots of about 1.5-2.5 cm were harvested for rooting experiment and cultured on half-strength MS medium supplemented with IBA or NAA at (1, 2, 3, 5 and 7 mg L-1). Rooting percentage and roots per propagules were counted after 4 weeks of culture in rooting medium. The rooted plantlets were washed with sterilized water to remove adhered agar and transferred to bottle containing sand and soil (1:1) for hardening. After 1 month, the plantlets were transplanted to pots containing sand:soil: FYM (farm yard manure) and kept under shade house with proper watering twice a day for 3 months.
Statistical analysis: All experiments were set up in a completely randomized
design. Data within each experiment was subjected to Analysis of Variance (ANOVA)
using Statistical package. If the data were found significant at p≤0.05,
Tukeys test was employed for comparison of treatment means.
RESULTS AND DISCUSSION
Description of inflorescence: Inflorescences were borne on leafless
branches between the leafy branches at a node (Fig. 1a) and
are up to 80 cm long. The branching pattern was similar to that of a vegetative
culm. Numerous floral buds were arranged with 1-7 pseudospikelets in capitate-like
clusters in the nodes (Fig. 1b). Spikelets were ovoid, laterally
compressed, 1-2x0.8-1.2 cm, reddish to dark purple, comprising 6-8 florets.
Florets were bisexual, 11-14 mm long, broad at the base; anthesis basipetal
(Fig. 1c); lemma broadly acuminate, concave, glabrous, pale
green in fresh specimens and overlapped with the palea (Fig. 1d);
palea subtending a bisexual floret, membranaceous, 2-keeled, ciliate on the
keels, 2-3 veined between them, pale green (Fig. 1e); Stamens
6; anthers 4-7 mm, linear, acute, pale green, basifixed, with linear dehiscence;
filaments thread-like, elongating to 10-12 mm during anthesis (Fig.
||Different floral parts of Dendrocalamus giganteus:
(a) Flowering clump, (b) Capitate arrangement of spikelets on nodes, (c)
A floret showing basipetal anthesis, (d) Lemma, (e) Palea, (f) Androecium,
(g) Stamen, (h) Pistil and (i) Caryopsis
Pollen released were yellow and powdery which were attractive and bees were
seen visiting the flower. Ovary was ovoid, 1-2 mm long and had style long ending
in 1-2 short purple stigma (Fig. 1h). Seeds caryopsis; cylindrical
to ovoid, 8-12x 4-6 mm, light brown, hairy, thin pericarp and filled with large
starchy endosperm (Fig. 1i). In this study, comprehensive
observations of floral morphology at several stages of development were documented,
in order to obtain seeds which would show genetic fidelity. The inflorescence
characters and floral morphology of D. giganteus as described above were
in agreement with descriptions given by Gamble (1896)
but more comprehensive. In the present report, the flowering did not produce
mass seeding but only few spikes set seeds which indicated sporadic flowering.
Such a flowering behavior was reported in Sri Lanka by Ramayana
and Yakandawala (1998).
In vitro seeds germination: Figure 2 illustrates
the effect of BAP, Kn and GA3 on in vitro seed germination.
In controls, about 20% seed germinated in the first 6 days and the rest germinated
evenly over the next 12 days. However, in the presence of optimum PGRs, between
50-85% seeds germinated during the first 6 days and reached almost the maximum
by the 12th day both in dark and light culture. (GA3) was found to
be better than BAP and Kn for promoting the germination of seeds. (GA3)
was found to be more effective at 0.5 mg L-1 in the presence of light.
In contrast, the cytokinins (BAP and Kn) increased germination at higher concentrations.
||Percentage in vitro germination of D. giganteus
seeds in MS medium supplemented with BAP, GA3 and Kn subjected
to light and dark. Values are Mean±SE of ten replicates. Columns
marked by a common letter do not differ significantly by Tukeys test
(p<0.05). Bars denote the standard error (SE)
||Effect of BAP concentration in MS medium on direct shoot organogenesis
from seeds of Dendrocalamus giganteus. Values are Mean±SE
of ten replicates. Columns marked by the same letter do not differ significantly
by Tukeys test (p<0.05). Bars denote the standard error (SE)
Our results agree with other studies showing that application of GA3
is an effective method to promote germination and to increase the germination
capability of the seeds of several other species (Perez-Garcia
and Duran, 1990; Upreti and Dhar, 1996; Khan
and Ungar, 1998; Hernandez-Verdugo et al., 2001;
Miransari and Smith, 2009; Prakash
et al., 2011). Contrary to the present observation regarding the
superiority of GA3, earlier report indicated that exogenous cytokinins
have various effects on seed germination in different species (Mahmoodzadeh
et al., 2010; Mirabdulbaghi et al., 2011).
The seedlings were transferred to MS medium without growth regulators for further
elongation and growth (Fig. 6a).
The shoots proliferated from seeds on MS medium supplemented with various concentration
of BAP within 4 weeks. Shoot proliferation per seed increased gradually with
the high levels of BAP (Fig. 3). The mean number of shoots
produced was 9.2 at 5 mg L-1 BAP (Fig. 6b). Although,
the highest mean number of shoots was obtained at 10 mg L-1 BAP,
the shoot length decreased insignificantly with the high levels of BAP. The
shoots developed were excised into 3-5 shoot clusters and subcultured in different
concentrations of BAP supplemented medium. The highest multiplication rate was
obtained at 3 mg L-1 BAP which showed no significant difference with
4 mg L-1 BAP (Fig. 4, 6c). After
the fifth subculture, the shoot multiplication rate declined drastically. The
present investigation indicated that the number of shoots produced was directly
correlated to BAP concentration.
||Effect of BAP concentration in MS medium on shoot multiplication
and shoot length of Dendrocalamus giganteus. Values are Mean±SE
of ten replicates. Columns marked by a common letter do not differ significantly
by Tukeys test (p<0.05). Bars denote the standard error (SE)
||Effect of Plant Growth Regulators (PGRs) on organogenic callus
induction from zygotic embryos of Dendrocalamus giganteus
|Values are means ± SE of ten replicates. Data scored
after 4 weeks in culture. Means followed by the same letter do not differ
significantly by Tukeys test (p≤0.05)
Cytokinin-induced direct shoot regeneration from intact seedlings had been
reported in Phaseolus species (Malik and Saxena,
1992), Arachis hypogaea (Saxena et al.,
1992), Murraya koenigii (Bhuyan et al.,
1997), Litchi chinensis (Das et al., 1999),
Dendrocalamus asper (Arya et al., 1999),
Cyclamen mirabile (Yamaner and Erdag, 2008).
Callus induction and adventitious shoots proliferation: Callus induction
from the zygotic embryos took place in modified MS medium (containing B5
vitamins) fortified with various concentrations of 2,4-D alone or in combination
with BAP or Kn at different concentrations after 2 weeks of culture (Table
1). The optimum concentration of 2,4-D for obtaining organogenic calli was
3 mg L-1 (Fig. 6d). When 2,4-D concentrations went
down from 3 to 0.5 mg L-1, callus induction percentage decreased
as well as non-organogenic texture were obtained. At the increased concentration
of 2,4-D, the callus obtained was brownish and hard.
||Optimum combinations of NAA with various concentrations of
BAP and Kn for shoot proliferation in callus cultures derived from zygotic
embryos of Dendrocalamus giganteus. Values are Mean±SE of
ten replicates. Columns marked by a common letter do not differ significantly
by Tukeys test (p≤0.05). Bars denote the standard error (SE)
The combination of 3 mg L-1 2,4-D and 0.5 mg L-1 Kn
produced maximum percentage of callus induction and organogenic calli proliferated
after 40 days culture (Fig. 6e). During organogenic callus
formation, calli with various textures was noticed and only the white, nodular
and compact responded for shoot regeneration. Our experiments proved that the
combined effect of 2,4-D and Kn showed best response for organogenic callus
induction. Similar to our results, in most of the dicot plants, a combination
of high amount of auxins (2,4-D or NAA) with low amount of cytokinins (BAP or
Kn) was widely used for the initiation of organogenic callus (Caboni
et al., 2000; Kumari et al., 2008;
Valizadeh et al., 2007; Siwach
et al., 2011; Sharma et al., 2012)
and including some bamboo species (Rout and Das, 1994).
Adventitious shoots were proliferated from the organogenic callus when subcultured
to modified MS medium supplemented with optimum levels of 1 mg L-1
NAA and various concentrations of BAP or Kn (0.5, 1, and 3 mg L-1).
After one month, maximum mean number of 12 shoots was regenerated from the callus
in the media supplemented with 1 mg L-1 NAA and 0.5 mg L-1
Kn (Fig. 5, 6f). Comparatively, BAP produced
a maximum of 4.3 shoots in 0.5 mg L-1 after 40 days culture. The
present investigation confirmed that the combination of NAA with cytokinins
promotes adventitious shoot proliferation from the callus cultures of Dendrocalamus
giganteus using zygotic embryos as an explants. Similar to our findings,
BAP or Kn was widely used for multiple shoot initiation from the callus cultures
(Huang et al., 1989; Martin,
2002; Satyavani et al., 2011).
||Seed germination and propagation of Dendrocalamus giganteus
using in vitro techniques: (a) Seedlings growth in MS medium,
(b) Direct shoots organogenesis on MS+5 mg L-1 BAP, (c) Shoot
multiplication on MS+3 mg L-1 BAP, (d) White, nodular and compact
calli obtained in MS+3 mg L-1 2,4-D, (e) Callus proliferation
in MS+3 mg L-1 2,4-D+0.5 mg L-1 Kn, (f) Clusters of
adventitious shoots regenerated from green and nodular organogenic calli,
(g) In vitro rooting of organogenic shoots, (h) Rooting of in
vitro microshoots and (i) Hardening
In some plant species, shoot multiplication and regeneration was efficiently
achieved by thidiazuron (TDZ) (Malik and Saxena, 1992;
Kumari et al., 2008; Kumar
and Kumari, 2010) and 2-isopentenyladenine (2iP) (Chang
and Chang 2000; Tonon et al., 2001).
In vitro rooting of shoots: Microshoots rooted on a PGR- free
½ strength MS medium following initial incubation for 30-40 days on medium
containing IBA. Initial exposure to IBA and subsequent withdrawn of auxin from
the medium was found very effective for overall root development. This rooting
procedure resulted best root growth within 7-10 days after transfer to auxin-free
medium. Among the various concentrations of IBA tested, significant rooting
was obtained at 5 mg L-1 producing 13 average root numbers with 86%
rooting in 40 days (Fig. 6h, Table 2).
||Effect of auxins on rooting of Dendrocalamus giganteus
on ½ strength MS medium
|Values are Mean±SE of ten replicates. Means followed
by the same letter do not differ significantly by Tukeys test (p≤0.05)
In the case of NAA also root induction was noticed but in the concentrations
of NAA tested rooting percentage was low as compared to IBA. The in vitro
raised shoots from embryogenic calli failed to root on a hormone-free MS medium.
Of the various auxin treatments, the best results were obtained with 1-3 mg
L-1 IBA supplemented ½ strength MS medium (Fig.
6g). Such similar stimulatory effect of IBA on in vitro root induction
from seedling has been reported in some bamboo species (Arya
et al., 1999; Bag et al., 2000) and
other important crops (Molla et al., 2004; Mirabdulbaghi
et al., 2011). On the contrary, the synergistic effect of NAA and
IBA proved to be beneficial for rapid root initiation of orchid via seed culture
(Nongdam and Chongtham, 2011). In the present case,
no auxin protector coumarin, additives and amino acids were required though
these have been reported to enhance in vitro root induction (Saxena,
1990; Agnihotri and Nandi, 2009).
Hardening and acclimatization: After following the process of hardening
and acclimatization (Fig. 6i), the newly transplanted plantlets
survived up to 80-90% exhibiting normal growth. As observed, these plantlets
established well and grew favorably during monsoon.
These reports present the evidence and importance of observing the incidence of sporadic flowering and behaviors for this important bamboo species, which will allow successful micropropagation using seeds. Furthermore, an efficient and simple protocol for in vitro seed germination and micropropagation through direct shoot organogenesis and indirect adventitious shoot multiplication from callus cultures have been described.
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