Abstract: This investigation highlights for the first time the application of 24-epibrassinolide in the micropropagation of orchids. Successful initiation of protocorm-like bodies and in vitro regeneration of Cymbidium elegans was achieved using shoot tip sections and 24-epibrassinolide supplemented Mitra et al. (1976) basal medium. The highest percentage of explants (91.0%) producing PLBs (24.0±2.1) was recorded on 4.0 μM 24-epibrassinolide supplemented basal medium. All the newly formed protocorm-like-bodies survived and after nearly 12 weeks, small bud-like structures formed healthy shoots. Shoots produced roots when cultured on basal medium supplemented with 2.0 μM triacontanol. The well-rooted shoots were transferred to pots containing charcoal chips, coconut husk and broken tiles (2:2:1) and 100% survival rate was achieved. Our results for the first time demonstrate that 24-epibrassinolide provided compelling evidence that they can be effectively used in the micropropagation of orchids.
INTRODUCTION
Brassinosteroids are widely distributed in plants and form a unique class of endogenous plant growth regulators with pleiotropic effects, which play an essential role in growth and developmental processes (Sasse, 1997; Clouse and Sasse, 1998). Since the first discovery of brassinosteroids from rape (Brassica napus L.) pollen by Grove et al. (1979) as a plant growth regulator with remarkable biological activity, 40 BRs and 4 BR conjugates have been characterized from 44 plant species including 37 angiosperms (9 monocots and 28 dicots), 5 gymnosperms, 1 algae and 1 pteridophyte (Fujioka, 1999). In several bioassays, they have been reported to affect cell elongation, division and differentiation of plant cells and splitting of internode (Mandava, 1988; Sakurai and Fuijoka, 1993, 1997; Clouse et al., 1996; Clouse and Sasse, 1998; Fujioka et al., 1998; Altmann, 1999; Khripach et al., 2000). BRs have also been shown to enhance trachery element differentiation, act synergistically with cytokinins in the growth of cell cultures, stimulate hyperpolarisation, promote ethylene biosynthesis, control microtubule orientation, alter mechanical properties of cell walls and modulate environment stress signals as well as other developmental processes (Mayumi and Shibaoka, 1995; Dhaubhadel et al., 1999).
Some workers have demonstrated their role in plant cell cultures. Embryogenic callus induction and growth of Coffee, lettuce and potato was improved by the use of Spirostane analogues of brassinosteroids in the culture medium as a cytokinin substitute or complement (Lu et al., 2003; Oh and Clouse, 1998; Nakajima et al., 1996; Nunez et al., 2004). Successful initiation of embryogenic tissue in conifers and cotton, organogenesis in sweet pepper and cauliflower was established using 24-epibrassinolide (Pullman et al., 2003; Wang et al., 1992; Franck-Duchenne et al., 1998; Sasaki, 2002). Ronsch et al. (1993) reported an improvement in the rooting efficiency and survival of Norway spruce seedlings using 22S, 23S-homobrassinolide. In various bioassays, brassinolide has been shown to be more active than, or synergistic with, auxins such as IAA or NAA (Brosa, 1999). Oh and Clouse (1998) demonstrated that brassinolide increased the rate of cell division in isolated leaf protoplasts of Petunia hybrida. Hu et al. (2000) suggested that 24-epibrassinolide may promote cell division through Cyc D3, a D-type plant cyclin gene through which cytokinin activates cell division. They also showed that 24-epibrassinolide can substitute cytokinin in culturing Arabidopsis callus and suspension cells. Work with Chinese cabbage protoplasts has shown that 24-epibrassinolide promoted cell division in presence of 2, 4-D and kinetin (Nakajima et al., 1996). However, very few reports are available with respect to the effect of brassinosteroids in the micropropagation and tissue culture.
Cymbidium elegans Lindl, an endangered orchid species is of grate horticultural value. This is an unusual species in which inflorescences carries a dense bunch of beautiful funnel-shaped buttercup yellow blossoms. It grows epiphytically and litophytically at elevations of 1500-2500 m (Kumar et al., 1994). Many attractive hybrids of Cymbidium orchid have become commercially important in cut flower and potted plant industries. Cymbidium was the first orchid to be propagated using shoot-tip culture (Wimber, 1963; Morel, 1964). Indiscriminate collection and severe habitat loss are the two potent factors responsible for its depletion in India (Kataki, 1977). Its large-scale multiplication in botanic gardens to meet trade demand has been recommended. Tissue culture techniques have been widely used for the in vitro mass multiplication of several commercially important orchids (Rao, 1977; Lakshmanan et al., 1995; Ichihashi, 1998; Kanjilal et al., 1999; Malabadi et al., 2004, 2005). In vitro regeneration of Cymbidiums has been reported by Wimber (1963), Morel (1964), Sagawa et al. (1966), Ueda and Torikata (1968), Steward and Mapes (1971), Wang (1988), Begum et al. (1994), Ichihashi (1997), Chang and Chang (1998), Phukan and Mao (1999), Nayak et al. (1997, 2002) and Huan et al. (2004). In vitro regeneration of Cymbidium elegans was achieved using axenic seeds germination (Sharma et al., 1991; Sharma and Tandon, 1990). In this study we report for the first time an efficient multiplication method for Cymbidium elegans through shoot tip thin section culture by using 24-epibrassinolide (24-epiBL) and higher percentage of rooting by the use of triacontanol (TRIA). Our results for the first time demonstrate that 24-epibrassinolide provided compelling evidence that they can be effectively used in the micropropagation of orchids.
MATERIALS AND METHODS
Twenty-five plants of Cymbidium elegans (Lindl.) collected from the Khasi and Jaintia Hills, Shillong, Meghalaya state, India were established in pots and grown under greenhouse conditions at Department of Botany, Karnatak University, Dharwad, India. Shoot tips (0.5-0.8 cm) harvested from mother plants were carefully washed in distilled water. They were surface decontaminated sequentially with 0.1% streptomycin (20 sec), 70% (v/v) ethanol (50 sec) and 0.1% (w/v) HgCl2 (2 min) and thoroughly rinsed with sterilized double distilled water. Transverse- thin sections of 1-5 mm thick were cut from shoot tips and these sections were cultured on Mitra et al. (1976) basal medium with 3.0% sucrose (Hi media, Mumbai), 0.7% agar (Sigma), 0.5 g L-1 meso inositol, 1.0 g L-1 casein hydrosylate, 0.5 g L-1 L-glutamine, 250 mg L-1 peptone, 0.2 g L-1 p-aminobenzoic acid and 0.1 g L-1 biotin. The 24-epibrassinolide was purchased from CID Tech. Research Inc., Mississauga, Ontario, Canada. The medium was supplemented with a range of 24-epibrassinolide (24-epiBL) concentrations (0.5, 1, 2, 3, 4, 5, 6, 7 10, 15 and 20 μM) without any other growth hormones in 25x145 mm glass culture tubes (Borosil) containing 15 mL of the medium under cool white fluorescent light (100 μmol m-2 s-1) at 25±3°C with a relative humidity of 55-60%. The pH of the media was adjusted to 5.8 with NaOH or HCl before agar was added. Media without 24-epibrassinolide is served as control. The media were then sterilized by autoclaving at 121°C at 1.04 kg cm-2 for 15 min. L-glutamine, biotin, p-aminobenzoic acid and 24-epibrassinolide (24-epiBL) were filter sterilized and added to the media after autoclaving when the medium had cooled to below 50°C.
The cultures were maintained for 6-10 weeks for the initiation of PLB’s or proliferating shoot buds. The freshly initiated PLB’s transferred to Mitra et al. (1976) basal medium containing 4.0 μM 24-epiBL. Healthy shoots with 2-3 leaves developed within 10-12 weeks. They were subcultured on the same medium for another 2 weeks for further shoot development. All experiments contained only 25 cultures for one replicate. Four replicates (100 cultures) were maintained for one set of experiment and experiments were repeated three times (100x3 = 300). Data presented in the tables were arcsine transformed before being analyzed for significance using ANOVA and the differences contrasted using a Duncan’s multiple range test. All statistical analyses were performed at the 5% level using the SPSS statistical software package.
The well-developed shoots were further transferred on fresh Mitra et al. (1976) basal medium supplemented with various concentrations of auxins (IAA, NAA, IBA) for rooting. All the shoot buds failed to produce rooting with IAA and IBA. Furthermore, poor rooting was observed with NAA. Therefore, the other growth regulators such as triacontanol (TRIA) were studied for the first time for the rooting efficiency in C. elegans. The well-developed shoot buds were transferred on fresh basal medium of Mitra et al. (1976) supplemented with various concentrations of TRIA (1, 2, 3, 4 and 5 μM) (Table 2). The shoots with well developed roots on 4.0 μM TRIA supplemented basal medium were washed thoroughly under running tap water and transplanted into 15 cm diameter pots in a potting mixture of charcoal chips, coconut husks and broken tiles (2:2:1). Three to four plants were planted in each pot and the plants were watered daily and fertilized at weekly intervals with a foliar spray of a mixture of commercial DAP (Di Ammonium phosphate) and NPK (Nitrogen 20: phosphorous 10: Potassium 10) (Malabadi et al., 2004, 2005). Five months later, the rooted plants were individually transferred to 15 cm pots.
RESULTS AND DISCUSSION
This study demonstrated that 24-epiBL has a high potential to induce proliferating shoot buds or PLBs with callusing from thin shoot tip sections of Cymbidium elegans. The highest percentage of explants (91.0%) producing PLBs (24.0±2.1) was recorded on 4.0 μM 24-epiBL in a period of 14 weeks (Table 1) and (Fig. 1A). These PLBs or proliferating shoot buds formed the maximum number of healthy shoots (17.0±1.23). Lower (0.5-1.0 μM) or higher concentrations (6.0-20.0 μM) of 24-epiBL resulted in the browning of explants and failed to produce PLBs (Table 1). Initiation of PLBs or proliferating shoot buds increases/decreases with increase in the concentrations of 24-epiBL from 2.0-5.0 μM (Table 1). However, the percentage of PLBs decreasing with increase in the concentrations of 24-epiBL from 4.0-5.0 μM. Explants cultured on basal medium (Mitra et al., 1976) supplemented with lower concentrations of 24-epiBL (3.0-4.0 μM) showed luxuriant growth of PLBs or proliferating shoot buds. Hence the effective range of 24-epiBL for the initiation of PLBs in C. elegans is 3.0-4.0 μM (Table 1). The thin shoot tip sections remained green and developed small bud-like structures when cultured on 4.0 μM of 24-epiBL supplemented basal medium of Mitra et al. (1976) within 9 weeks. They were further subcultured on the same medium and maintained for another 10-12 weeks. After nearly 12 weeks, small bud-like structures formed healthy shoots (Fig. 1A, B and C).
Embryogenic callus induction and growth of coffee and potato was improved
by the use of spirostane analogues of brassinosteroids in the culture
medium as a cytokinin substitute or complement (Garcia, 2000; More et
al., 2001). Two spirostane analogues of brassinosteroids (BB6 and
MH5) were tested for callus induction and plant regeneration in lettuce.
Results indicated that both BB6 and MH5 enhanced both callus formation
and shoot regeneration from cotyledons in lettuce (Nunez et al.,
2004). In case of rice seeds, the application of brassinosteroids reduced
the impact of salt stress on growth, prevented photosynthesis pigment
loss and increased nitrate reductase activity (Anuradha and Rao, 2003).
Brassinosteroids are of ubiquitous occurrence in plants and elicit a wide
spectrum of physiological responses (Gupta et al., 2004). In this
study, we have tested the influence of 24-epiBL alone without any combination
with other plant growth regulator to test its role on in vitro
regeneration of plantlets in orchids. Till today no other reports are
available on the effect of brassinosteroids on in vitro regeneration
of orchids. Embryogenic tissue were induced from longitudinally bisected
segments of protocorm-like bodies of Cymbidium Twilight Moon Day
Light, a hybrid orchid on modified Vacin and Went medium (1949) supplemented
with 2-4D in combination with 0.01 mg L-1 TDZ (Huan et al.,
2004).
| Table 1: | Effect of various concentrations of 24-epiBL on the initiation of PLBs or proliferating shoot buds in Cymbidium elegans |
| Data scored after 14 weeks and represent the mean±SE of at least three different experiments. In each column, the values with different letter(s) are significantly different (p<0.05) | |
| Fig. 1: | In vitro multiplication of C. elegans using 24-epiBr. (A) Initiation of PLBs from thin sections of shoot tip on the basal medium of Mitra et al. (1976) supplemented with 4.0 μM 24-epibrassinolide. (B) Formation of healthy shoot from PLBs. (C) Healthy shoots with well-developed leaves ready for rooting |
Whereas, in vitro regeneration of Cymbidium elegans was achieved using 24-epiBL and survival rate of seedlings was 100%. The other advantages of using 24-epiBL were seedlings are very healthy and more resistant than rest of the regeneration protocols at least in Cymbidium elegans. In case of Oryza sativa, an increase in the soluble protein content was noticed by 24-epiBL application and considerably alleviated oxidative damage that occurred under NaCl-stressed conditions and improved seedling growth in part under salt stress in sensitive IR-28 seedlings (Ozdemir et al., 2004). Our results are in conformity with the literature.
The shoots regenerated on 4.0 μM 24-epiBL supplemented basal medium
were tested for rooting efficiency with different concentrations of auxins
such as IAA, IBA and NAA. Shoots failed to produce roots at all the different
concentrations of auxins (IAA, IBA). There was a poor rooting with different
concentrations of NAA (data not shown) since this paper presents only
optimum results. Therefore, the other growth regulators such as triacontanol
(TRIA) were studied for the first time for the rooting efficiency in C.
elegans. The highest percentage of rooting efficiency (95.0±6.0)
was recorded at 2.0 μM of TRIA within a time period of 4 weeks (Table
2). On the other hand the lowest percentage of rooting (4.0±0.65)
was observed at 4.0 μM of TRIA. Shoots failed to produce roots at
higher concentrations (5.0 to 10.0 μM) of TRIA (Table
2). A sudden decrease in the rooting efficiency of shoots was noticed
when the concentrations of TRIA was increased from 2.0 to 4.0 μM
(Table 2). This also confirmed our previous results
of higher rooting efficiency of an endangered orchid, Dendrobium nobile
at a concentration of 2.0 μg L-1 TRIA supplemented
with Mitra et al. (1976) basal medium (Malabadi et al.,
2005). According to Tantos et al. (1999), successful rooting was
observed at 2.0 μg L-1 TRIA in M. officinalis.
Similarly Fraternale et al. (2003) reported that 2.0-5.0 μg
L-1 TRIA was optimum for efficient rooting in T. mastichina.
Lower concentrations of TRIA may be biologically effective because of
the sensitivity of whole explants to extremely low doses of TRIA (Biernbaum
et al., 1998; Malabadi et al., 2005).
| Table 2: | Effect of different concentrations of TRIA on rooting of shoots regenerated at 4.0 μM 24-epiBL supplemented Mitra et al. (1976) basal medium |
| Data scored after 4 weeks and represent the mean±SE of at least three different experiments. In each column, the values with different letter(s) are significantly different (p<0.05) | |
The well rooted shoots were washed thoroughly under running tap water and transplanted to 15 cm community pots in a potting mixture of charcoal chips, coconut husk and broken tiles (2:2:1) for hardening.
In conclusion this study reports for the first time the optimized parameters for the successful initiation of PLBs and regeneration of plantlets by using 24-epiBL from shoot tip sections and efficient rooting by the incorporation of TRIA in the basal medium in Cymbidium elegans. This protocol is very simple, significant for the large-scale propagation to meet commercial demands in a short period and conservation of this valuable orchid species.
ACKNOWLEDGMENT
We are grateful to the Head, Department of Botany, Karnatak University, Dharwad, India for providing necessary facilities for this work. RBM also wishes to thank lab mates for every help during this investigation. KSCST is warmly acknowledged for the financial support for this project.