Abstract: Ochlandra wightii (Munro) Fisch., an endemic reed bamboo in the Southern Western Ghats has been multiplied using tissue culture techniques. In vitro production of O. wightii plantlets has been achieved by using isolated embryos from mature seeds and nodal segments from in vitro seedlings. Better germination of isolated embryos (85%) was noticed in BAP (6-benzyl amino purine) (0.5 mg L-1) with 1-2 shoots in 30 days. Half-strength MS medium supplemented with BAP (0.5 mg L-1) and TDZ (Thidiazuron) (0.5 mg L-1) enhanced the rate of shoot multiplication to an average of 9.8 shoots in 60 days. Simultaneous development of roots along with shoots was also noticed. Rhizome induction was achieved from 65% of seedling cultures when treated with KN (Kinetin) (1 mg L-1) and higher concentration of sucrose (4%) in 35 days. Multiple shoots were also induced from nodal explants obtained from in vitro seedlings. Average 3.6 shoots were achieved in 40 days when treated with BAP (2 mg L-1) and KN (0.5 mg L-1). Twenty percent of the nodal cultures developed 1-2 roots during shoot development and the rest were rooted in half-strength MS solid medium supplemented with IBA (indole-3-butyric acid) (0.5 mg L-1). Plantlets with healthy roots were hardened and transplanted to the clay pots. They showed 80% survival after 6 months. However, the plantlets with in vitro rhizomes showed 100% survival. Development of new culms and rhizomes were noticed after 3 months of greenhouse growth. Around 880 plantlets could be generated in 9 months from 10 embryos isolated from mature seeds using this protocol.
INTRODUCTION
Ochlandra wightii (Munro) Fisch. belongs to the family Poaceae is an endemic reed bamboo of the Southern Western Ghats. Its distribution is restricted to the hilly tracts of the extreme Southern regions of Kerala (Kumar, 2002). The genus, Ochlandra comprises 11 species, of which ten are endemic to the Western Ghats and one to Sri Lanka (Koshy and Mathew, 2009). The plant consists of rhizomes, culms and culm branches. Rhizomes are continuously grown horizontally and new culms are produced from the lateral buds. Culms are erect and slender up to 5 m in length and 3-4 cm girth. Leaves are oblong-lanceolate, glabrous and the ligule is long and papery. The species is monocarpic which flowers only once in their life span and dies. The flowering cycle of this species is presumed as 15-17 years (Jijeesh and Seethalakshmi, 2011).
Bamboos and reeds have received great attention as a forest produce due to its multifarious uses. The culms of O. wightii are widely used in pulp, paper, plyboard and cottage industries. It is also used as a material for fencing, for making flute stake for plantations etc. Leaves are used as fodder and thatching substitutes (Seethalakshmi and Kumar, 1998). The rapidly increasing bamboo-based industries have resulted in severe loss of forest stocks which demands adequate re-plantation of elite bamboo species to strengthen the raw material stock (Mudoi and Borthakur, 2009).
Vegetative propagation in reeds is very difficult due to limited availability of offsets from established culms and continuous harvesting cause damage to the parent culms. Moreover, the offsets are bulky and heavy to dig out and hence it is labour intensive and difficult to transport. Vegetative propagation of reeds by culm cuttings was standardized by Seethalakshmi et al. (1990). Propagation through seeds is undesirable due to the rapid loss of viability, infection of seeds by pests and diseases during storage and the uncertain flowering cycle. Conventional propagation techniques are insufficient and inefficient for large scale propagation and to satisfy the growing demand of planting materials, in vitro propagation is inevitable (Gielis, 1999) . These bottlenecks are also applicable to O. wightii for large scale production of planting materials. Numerous reports on tissue culture multiplication of bamboo have been published by several authors (Alexander and Rao, 1968; Huang and Murashige, 1983). However, reports on in vitro regeneration of Ochlandra spp. are very limited (Philip, 1998). Thus, potential techniques need to be developed to scale up sapling production of this endemic species to meet the requirements of afforestation programmes. The present study elucidated the in vitro multiplication of O. wightii from embryos isolated from mature seeds.
MATERIALS AND METHODS
Embryo culture: Flowering populations of O. wightii were located in Ponmudi, Palode forest range, Kerala. As the species is closely allied to O. travancorica, proper taxonomic identification was done and matched with live collections in the bambusetum, TBGRI. Mature seeds of O. wightii harvested were washed thoroughly in running tap water and treated with 1% commercial bleach (Ranbaxy, S.A.S. Nagar) and 0.2% labolin (Qualigens, Mumbai) for 30 min. They were rinsed in sterile distilled water and flame sterilized using ethanol. The embryos were carefully isolated by dissecting the seeds and inoculated onto half strength MS (Murashige and Skoog, 1962) solid (0.7% agar) medium supplemented with 2% sucrose and Plant Growth Regulators (PGR) such as BAP and KN at various concentrations and combinations (0.1-1.0 mg L-1). pH of the media was adjusted to 5.7 prior to autoclaving at 121°C for 20 min. All cultures were incubated at 24±2°C and under a photoperiod of 16/8 h provided by cool white fluorescent tubes.
The isolated embryos mostly developed single shoots with one or two leaves and roots in 30 days of incubation. The shoots were decapitated and long roots were trimmed before transferring to liquid basal medium supplemented with TDZ, BAP and KN (Table 1). Data regarding the number of shoots and roots and length of shoots were recorded after 30 days of subculture. The embryo culture is followed by axillary shoot proliferation and multiplication.
In vitro nodal culture: In a separate study, nodal explants of 1.0 - 1.5 cm size isolated from in vitro seedlings were inoculated onto half strength MS solid (0.7% agar) medium supplemented with various concentrations and combinations of BAP and KN (Table 2). After 40 days, they were subcultured for rooting.
In vitro rhizome production: Decapitated in vitro seedlings were cultured in half-strength MS liquid medium fortified with different concentrations of sucrose (0, 2%, 3, 4, 5 and 6%) for rhizome production.
| Table 1: | Effect of cytokinins on in vitro propagation of O. wightii embryos |
| *Basal medium: Half-strength MS+2% sucrose and pH 5.7, **Means±SE of 8 replicates after 60 days of culture, Means followed by the same letter do not differ statistically at p = 0.05 according to DMRT | |
| Table 2: | Shoot production from nodal explants obtained from in vitro seedlings of O. wightii |
| *Basal medium: Half-strength MS+2% sucrose and pH 5.7, **Means±SE of 8 replicates after 40 days of incubation, Means followed by the same letter do not differ statistically at p = 0.05 according to DMRT | |
Effect of cytokinins like BAP and KN on rhizome production was also tested. The rate of in vitro rhizome production was recorded after 30 days.
Rooting and field establishment: Bunches of 2-4 shoots isolated from nodal cultures were subjected to in vitro rooting in basal MS medium containing IBA (0.5 mg L-1). Well rooted plantlets were washed thoroughly in tap water and treated with 3% commercial fungicide (Indofil M45) before planting in a mixture of river sand and coarse charcoal (3:1). The plantlets were planted (5-10 clumps) in clay pots and kept in semi-shade (50%) and high humid (80-85%) mist house for hardening. After 4-5 week, the plantlets were transplanted to small plastic cups or poly bags and the survival rate was noticed.
Statistical analysis: Each of the growth regulator treatments contains a minimum of 8 replicas. Mean and standard error values were calculated on parameters like shoot number, shoot length and root number. One way analysis of variance was performed (p<0.05) and the means were compared using Duncan’s Multiple Range Test (DMRT).
RESULTS AND DISCUSSION
Embryo culture: Embryos from mature seeds were used to initiate in vitro culture. Germination of isolated embryos was noticed after 10-12 days of culture in half strength MS solid medium supplemented with cytokinins (BAP and KN 0.1-1.0 mg L-1). But the rate of germination was varied according to the type and concentration of cytokinins. BAP (0.5 mg L-1) supplemented cultures showed the highest rate of germination (85%). All other treatments showed less response of average 60-80% germination (data not included). Normal seedlings with 1-2 shoots having leaves and roots developed in all the cytokinin treatments. However, medium devoid of growth regulators delayed the germination of isolated embryos to 20 days and showed only 60% response. Low rate of contamination and rapid induction of morphogenic responses have forced to use embryos as the most suitable explants in bamboo tissue culture (Woods et al., 1992; Yeh and Chang, 1987; Rout and Das, 1994). Further, multiple shoot induction from mature explants is a difficult task in bamboo tissue culture (Ravikumar et al., 1998). However, in vivo nodal segments (Ramanayake et al., 2006), inflorescence (Lin et al., 2005), nodes from in vitro raised seedlings (Rout and Das, 1994), shoot apices (Huang et al., 1989) and leaf explants (Hassan and Debergh, 1987) have also been successfully tested in different bamboos. Embryos have been utilized for in vitro propagation of difficult to propagate woody plants. Isolated embryos without endosperm showed shoot multiplication in Taxus media when treated with BAP or 2,4-D (Liao et al., 2006). In Taxus baccata, BAP and NAA enhanced the rate of production from embryo explant (Abbasin et al., 2010).
After 30 days of germination, the seedlings were decapitated and the roots were completely removed and subcultured for further multiplication in 25 mL liquid medium supplemented with cytokinins (Table 1). The suitability of liquid medium for shoot multiplication in Bambusa tulda (Saxena, 1990) and Dendrocalamus strictus (Nadgir et al., 1984) has been reported. Incorporation of agar detrimentally affects the shoot multiplication rate. Saxena (1990) avoided the necessity of a shaker to provide aeration to the shoots of B. tulda by reducing the quantity of liquid medium. This method was successfully adopted in the present study in O. wightii.
Significant variation in morphogenic response was observed in different cytokinin treatments. The roles of cytokinins on in vitro bud break and shoot multiplication is well known. The present study also showed the importance of cytokinin with preferential morphogenic responses. Among the cytokinins tested, KN seems to be least effective whereas TDZ and BAP showed better response. The potential effect of BAP over other cytokinins on shoot multiplication has been reported in several bamboos such as D. asper (Arya et al., 1999), Bambusa bambos (Kapoor and Rao, 2006), B. vulgaris (Ramanayake et al., 2006) and Guadua angustifolia (Jimenez et al., 2006). In O. wightii combined action of two cytokinins resulted in enhanced shoot multiplication response. Half-strength MS medium supplemented with BAP (0.5 mg L-1) and TDZ (0.5 mg L-1) recorded the rate of multiplication to a maximum of 9.8 shoots in 60 days after subculture. Further increase in BAP or TDZ concentrations did not show favourable response on shoot production. Synergistic effect of PGRs showed improved morphogenic responses in vitro. Thidiazuron, a substituted phenylurea (1-phenyl-3-(1, 2, 3-thidiazo-5-yl)-urea) has been identified for its cytokinin-like activity in various explants cultured in vitro (Mok et al., 1982). Huetteman and Preece (1993) reported TDZ as the most potential cytokinin-like substance for micropropagating woody plants. Similar to our observation, addition of TDZ along with BAP enhanced the multiplication over 50% in Amomum hypoleucum (Bejoy et al., 2010). In Bambusa wamin (Arshad et al., 2005), BAP in combination with KN was found ideal for proliferation of mature nodal explants. Similar synergistic effect of two cytokinins on in vitro regeneration has been reported from many species such as Tamarindus indica (Farooq and Farooq, 2003), Eugenia singampattiana (Pavendan and Rajasekaran, 2011), Cinnamomum camphora (Soulange et al., 2007), Boesenbergia pulcherrima, (Anish et al., 2008) whereas, cytokinin and auxin enhanced multiplication was reported from Taxus baccata (Abbasin et al., 2010) and Curcuma haritha (Bejoy et al., 2006). The length of shoots was taken as a measure of growth in various cytokinin treatments. In the present study, shoot length was significantly less in presence of BAP and KN whereas TDZ (0.5 mg L-1) showed the highest shoot length (Table 1). Simultaneous development of shoots and roots were also observed in 85-95% of isolated embryos in O. wightii. Similar results were reported in other bamboos (Shirgurkar et al., 1996; Huetteman and Preece, 1993). However, the rooting response was varied according to the growth regulator treatments (Table 1). Among the different treatments, TDZ supplemented cultures showed better response of 3.3 roots per explant. Murch and Saxena (2001) have reported the enhanced effect of TDZ on rooting in Pelargonium x hortum.
In vitro rhizome production: Modifications in the in vitro culture media can lead to the development of storage and perennating structures like tubers, bulbils, rhizomes, corms etc. In vitro rhizomes not only help in early establishment of plants in the field but also culm production (Kapoor and Rao, 2006). The decapitated in vitro seedlings of O. wightii were cultured onto half-strength MS liquid medium supplemented with various concentrations of sucrose for the induction of in vitro rhizomes. Most of the cultures showed 2-4 rhizomes developed from the base of the proliferating shoot which grew horizontally in the medium and later developed into shoots (Fig. 1b). The rate of rhizome production was varied along with the growth regulator and sucrose concentration (Fig. 2).
| Fig. 1(a-d): | In vitro propagation and rhizome induction on Ochlandra wightii, (a) Multiple shoot induction on ½ MS+BAP (2 mg L-1) and KN (0.5 mg L-1) from node harvested from in vitro seedlings, (b) In vitro rhizome formation on ½ MS+KIN (1 mg L-1)+Sucrose (4%), (c) Deflasked plantlets after 30 days in rooting medium, (d) Ex vitro plants of O. wightii after hardening |
This event occurred spontaneously in D. strictus during the rooting phase (Shirgurkar et al., 1996). In the present study, basal medium without growth regulator and sucrose was found ineffective in rhizome induction. Kinetin proved to be more effective on rhizome development when 3-5% sucrose was used whereas; BAP (1 mg L-1) was less effective and 20 percent of cultures developed rhizomes. Availability of higher levels of sucrose has also influenced rhizome development in O. wightii. Four percent sucrose was found ideal for rhizome production in both the cytokinins tested. Half strength MS with KN (1 mg L-1) and 4% sucrose showed the highest rate (65%) of in vitro rhizome production in 30 days. Further decrease or increase in sucrose concentration did not show positive response (Fig. 2). In vitro rhizome formation has also been reported in B. bambos var. gigantea with the addition of 5% sucrose along with growth regulators and recorded 100% rhizome formation within four weeks (Kapoor and Rao, 2006). Apart from growth regulators, increased sucrose levels in the medium also have a significant role in the induction of storage organs in vitro. Bamboo rhizomes mainly function as storage of starch and fibres (Hsiung et al., 1980). Reports revealed that enhanced sucrose concentrations foster the development of storage organs in Curcuma longa (Shirgurkar et al., 2001), C. aromatic (Nayak, 2000), Bunium persicum (Grewal, 1996). However, microtubers were achieved on half MS medium supplemented with BAP (2 mg L-1) and NAA (0.1 mg L-1) under normal sucrose (3%) regime (Yamaner and Erday, 2008).
In vitro nodal culture: The use of nodal explants from in vitro cultures has certain advantages viz., availability of explants for raising axenic cultures, irrespective of season. Once an aseptic culture system has been developed, it can be utilized for the continuous production of plantlets. In O. wightii nodal explants (1.0-1.5 cm) isolated from in vitro seedlings were inoculated onto basal medium supplemented with various concentrations and combinations of BAP and KN (Table 2). Initial axillary bud proliferation was observed in 15 days and subsequently more shoots from the base (Fig. 1a). A combination of BAP (2 mg L-1) and KN (0.5 mg L-1) treated cultures showed most number of shoots (3.6) in 40 days. Individual use of the cytokinins showed bud proliferation but rarely multiple shoot development. Though shoot growth was moderate in all the treatments, KN (0.5 mg L-1) exhibited best growth response and obtained 50 mm shoots in 40 days.
| Fig. 2: | Effect of cytokinins and sucrose concentration on in vitro rhizome production in O. wightii seedlings after 30 days of incubation, Basal medium: ½ MS liquid medium |
Incidence of callusing or in vitro rhizome formation was not observed in any of the treatments. Rout and Das (1994) used in vitro nodal explant for callus induction and subsequent somatic embryogenesis in three species of bamboo. Around 880 plantlets could be generated from ten embryos isolated from mature seeds using this protocol (embryo culture followed by axillary bud proliferation and multiplication) in 9 months. In D. hamiltonii, in vitro shoots raised from adult nodal cultures exhibited profuse multiplication (20 fold) when treated with 2 mg L-1 BAP and 0.2 mg L-1 NAA (Agnihotri and Nandi, 2009).
Rooting and field establishment: Cultures established from embryos showed simultaneous development of roots along with shoots. Similarly, in cultures having in vitro rhizomes, roots were developed from rhizome nodes. In these cases a separate rooting phase is not necessary. Similar results were reported in D. strictus where in vitro shoots rooted in BA containing multiplication medium, in the absence of Auxin (Shirgurkar et al., 1996). Twenty percent of the nodal cultures of O. wightii developed 1-2 roots during shoot development and the rest were rooted separately. Small clumps of 2-4 shoots obtained from in vitro nodal explants were treated in half-strength MS solid medium supplemented with IBA (0.5 mg L-1). Under this treatment 70% cultures developed 2-4 healthy roots in 30 days of incubation. IBA is widely used as rooting hormone in several bamboo species like B. vulgaris (Ramanayake et al., 2006), B. wamin (Arshad et al., 2005) and D. hookeri (Ramanayake et al., 2008). Agnihotri and Nandi (2009) reported the improved rooting of D. hamiltonii microshoots when treated with 20 mg L-1 IBA for 10 days followed by hormone-free medium. In yellow bamboo, shoots from adult culms showed only 40% rhizogenic response with 3 mg L-1 IBA, while pretreatment with TDZ (0.5 mg L-1) and incubation under continuous light increased the rootability of neo-formed shoots to 100% (Ramanayake et al., 2006).
Plantlets with healthy roots were deflasked (Fig. 1c) and treated with a commercial fungicide (3%) for 5 min. before planting in community pots containing river sand and coarse charcoal (3:1). The plantlets were kept in semi-shade (50%) and high humid (80-85%) greenhouse conditions for 3 months for initial establishment. Development of new culms and rhizomes were noticed during hardening. The hardened plantlets were then repotted in plastic cups and recorded 80% survival in 6 months (Fig. 1d). However, in vitro seedlings with rhizomes showed 100% survival.
In conclusion, the study clearly demonstrates an efficient in vitro protocol for the propagation of O. wightii through embryo culture and subsequent axillary shoot proliferation. In vitro propagation technique allows the mass production of plantlets within a short period and is less labour intensive. Thus, it offers distinct advantageous over conventional propagation methods to scale up sapling production of this endemic species. The present investigation has the potential of generating many thousands of plants per year to recover cleared natural stands or to start new plantations of O. wightii.
ACKNOWLEDGMENTS
This study was undertaken with the support of Western Ghats Cell, Planning and Economic Affairs Department, Govt. of Kerala. The authors acknowledge Director TBGRI for encouragement. The authors also wish to thank Mr. J. Jose and Mrs. Manjuladavi for lab assistance.