Meena Bakshi?Chandrakant Tiwari?Sajad Razvi

Conservation of an important montane bamboo Thamnocalamus falconeri,Hook.f.ex Munro through axillary bud proliferation

Meena Bakshi?Chandrakant Tiwari?Sajad Razvi

Thamnocalamus falconeri,Hook.f.ex Munro., an important bamboo species belonging to the family Poaceae,locally known as Ringal,occurs in the hills of Uttarakhand,India.This species has been traditionally exploited by localcommunities to supporttheirlivelihoods. Increasing needs of the hill villages impose unsustainable pressure on natural stands of Ringal in the Uttarakhand hills and forests have been degraded.The long history of excessive cutting of Ringal from natural forests and the lack of replanting threaten villager livelihoods.Replanting is required to conserve the species.We propose a protocol for generation of planting material through axillary bud proliferation for multiplication and conservation of this species.We collected offsets/rhizomes from a naturalstand of T.falconeri in the Chopta Mandal areas(Chamoli district,India).These were planted atsites ofvaried elevation and fresh single nodal segments were collected from them as explants.Differentsterilization treatments were assessed to combatcontamination.Among these,treatmentof 0.1% HgCl2followed by 5%NaOCl,proved best.Among two cytokinin treatments,viz.BAP and Kinetin,singly or in combination,BAP alone(5 mg L-1)proved superior and resulted in 100%bud break.BAP-supplemented MS media yielded maximum vigorous shootformation(90%) and maximum number of shoots(8.9).Subculturing of shoots on the same medium with similar BAP treatment (5 mg L-1BAP)enabled continuous production of healthyshoots at similar frequency.Maximum rooting(100%) was recorded on half-strength MS medium supplemented with 5 mg L-1IBA.Micropropagated plants were hardened and acclimatized in soil mixture(2:1:1)and then transplanted to field sites(Magra,Uttarakhand,1,834 m). Eightto ten months after field transplantation we recorded 100%survival of transplanted material.This micropropagation protocol could be used successfully for raising a stock of genetically homogenous plant materialin bulk for field plantations and for conservation of the species.

Thamnocalamus falconeriMicropropagationMS mediumAxillary bud proliferationHormones

Introduction

Bamboo is one of the most ubiquitous,multi-use plant species in the tropical and sub-tropical regions of the world.Itis importantin socialforestry programs due to its rapid growth and shortrotation cycle.Biologicalproperties and ecological functions of bamboo make it an important economic resource for a wide range of commercial uses, for alleviating environmental problems by erosion control and sequestration of carbon dioxide(Zhou et al.2005).

Thamnocalamus falconeri Hook.f.ex Munro,often known as Himalayacalamus falconeri(Munro)Keng f. (commonly Dev Ringal)is a dwarf bamboo species of Uttarakhand.The species is used for a variety of purposes by local communities,including basket making,mats, hookah pipes,handicrafts,fencing,flooring and roofing of houses,and italso provides income to artisans.Due to the diverse utility of the species,excessive harvesting prevails in the natural stands.Augmentation of the species throughplantations is imperative in orderto conserve and Maintain resources of Ringal.Some replanting has been undertaken in the hills of Uttarakhand where large volumes of planting material are needed.Sources of the planting material needed for restoration of natural stands are as yet unavailable.

Collection of seed from bamboo species is complicated by their long flowering cycles.The flowering cycle of T. falconeri is 28–33 years.Janzen(1976)reported that it flowered in England during 1902–1908,1929–1932,and 1964–1967.In 2002,T.falconeri flowered throughout Uttarakhand and,after seeding,whole clumps died(Naithani et al.2003).

Vegetative propagation through culm cuttings,branch cuttings or rhizomes/offsets is difficultdue to the presence of small numbers of propagules,season-specificity,and poor rooting capacity of culm and branch cuttings.In contrast,in vitro propagation techniques allow mass proliferation within relatively short time periods and might offer reliable approaches to the generation of planting material in bulk.We describe here an efficient in vitro protocol for the mass production of planting material of T. falconeri through in vitro axillary shoot proliferation,the first report for this species.Bulk generation of planting material would notonly overcome the acute shortages but would also conserve the species through reduced pressure on natural forests.

Materials and methods

Explant source

Offsets of T.falconeri were cut from natural stands, wrapped in peatmoss and wetsacks,and then transported from the Chopta-Mandal region of Uttarakhand,India to planting sites.Offsets were planted during July in the field at Khirsu(Pauri 1,900 m),Magra(1,834 m),and in the shade of trees at the Forest Research Institute,Dehradun, India.The growing shoots were later collected during different seasons from these three sources.Nodalsegments (2.5–3.0 cm.)of shoots were removed using sharp secateurs and then washed thoroughly under running tap water to remove dustparticles.This was followed by washing in liquid detergent(labolein)solution with 5–10 drops per 100 mL of Teepol(Glaxo India Ltd,Mumbai,India)for 5 min and then pre-sterilizing in a mixture of1%Bavistin and 1%Blitox fungicide solutions for 15 min.The treated explants were then washed three times with sterile water before sterilizing in 0.1%(w/v)HgCl2with continuous shaking for10 min followed by 5%NaOClfor20 min.To remove any traces of sterilant,explants were rinsed vigorously in sterile water at least three times.Prior to inoculation,proximal and distal ends of explants were trimmed to reduce them to 15–20 mm in length.

Culture condition

Liquid Murashige and Skoog(MS)basal medium(consisting of salts,vitamins and 3%sucrose)was used for inoculation.One of three plant growth regulators(PGRs), viz.6-benzylaminopurine(BAP),6-furfurylaminopurine (Kn),indole-3-butyric acid(IBA)and naphthalene acetic acid(NAA)was added atvarious concentrations to the MS medium before the pH of the medium was adjusted to 5.6. Media were autoclaved at 1.06 kg cm-2and 121C for 15 min.Cultures at all growth stages were incubated in artificial conditions:(25±2)C,60%RH and a 16-h photoperiod(using white fluorescent tubes)under photosynthetic photon flux density of 40 l mol m-2s-1.

Axillary bud initiation and multiple shoot proliferation

The explants were inoculated in liquid MS medium at various concentrations(1–6 mg L-1)of BAP and Kn separately or in combination using paper bridges in tissue culture tubes.A minimum of 24 replicates was maintained for each treatment.After 1 week,buds were induced in incubated cultures.After 15 days,elongated axillary buds were separated and cultured formultiple shootproliferation on the same medium.For further shoot multiplication,six levels of BAP(1–6 mg L-1)supplemented with 2 mg L-1indole-3-butyric acid(IBA)were tested.The control was devoid of PGRs.

Data recording

Data were recorded with respectto multiple bud induction (number of multiple buds,percentage of cultures responding).Only those explantswhich formed 3 ormore budswere scored for multiple buds and average number of buds induced was calculated by subtracting existing axillary buds at the time of inoculation.For axillary shoot proliferation, datawererecorded with respectto numberofaxillary shoots/ explants,percentage conversion of buds into shoots(total numberofshoots/totalnumberofbuds 9 100),and length of induced shoots.The treatmentyielding bestresults,viz.BAP 5 mg L-1,was chosen for further multiplication.Multiplication rate was recorded with respectto gain in number of shoots per subculture cycle(increase in number of shoots is number of shoots induced—number of shoots cultured)and dividing the gain by the numberofshootsin a propagule.The shootsobtained from these concentrations were subcultured for further multiplication on a medium of the same composition.

Subculturing

Subculturing was done at30-day intervals.A clump of 3–4 shoots was subcultured on the same medium and the effect of subsequent subculturing was observed as the multiplication of shoots.

Root induction and elongation

For rooting,cultured shoots were excised in clumps of 3–4 and cultured on half-strength MS liquid basal medium supplemented with severalconcentrations(1–6 mg L-1)of IAA,IBA and NAA.MS medium without PGR was used as the control.The efficiency of auxins was compared in terms ofpercentage response to rootinduction and number and length of roots.For all treatments 24 replicates were maintained.The plantlets with well-developed roots were advanced for acclimatization.

Acclimatization

A simple acclimatization process was established to make the most of the survival rate.Prior to the core part of acclimatization,the well-rooted plantlets were kept in an incubation room in plastic Thermocol pots filled with sterilized vermiculite(coarse grain)under artificial conditions where they grew for 1 week.Rooted plantlets were transferred to a mist chamber with a transparent cover of polythene sheet for 20 days,with good aeration and high humidity.The partially acclimatized plantlets were shifted to a shade house in earthen pots containing soil mixture(soil,sand and farm yard manure in 2:1:1 ratio).Over the next 6 months these plants developed profuse root systems.

Multiplication in vivo and transfer to the field

The plantlets in earthen pots were separated via macroproliferation technique in which all three essential entities, viz.rhizome,root and a culm,were maintained and then planted in polybags containing the same soilmixture.The propagules were kept under controlled conditions of temperature and humidity(under Green House condition)for 30 days.Subsequently,on formation of new leaves,they were transferred to natural light conditions.

Field planting

Six months after transplanting to polybags,the acclimatized plants were shifted to nursery at Magra,Mussorriee (1834 m),Uttarakhand during July–August(the monsoon season).

Experimental design and statistical analysis

The experimentaldesign was completely randomized.Each treatment included 24 replicates.The experiment was repeated thrice to ensure reproducibility ofthe results.Data were subjected to analysis of variance(ANOVA)and significant differences between treatments were tested using Duncan’s multiple range test at 5%significance level using SPSS(Version 16).

Results

In vitro shoot induction

The effects of BAP and Kn alone or in combinations on shoot induction and proliferation in a two-way factorial experimentare summarized in Table 1.Maximum(100%) bud break was achieved with BAP 5 mg L-1.Treatmentof BAP and Kn individually and in combination had statistically significanteffects on the rate of shootproliferation.In all treatments,shoot induction increased up to BAP concentration of 5 mg L-1,beyond which there was a sharp decline.Rates of shoot multiplication varied significantly by BAP concentration.Maximum shoot induction(90%) was recorded for 5 mg L-1BAP alone and for 5 mg L-1BAP?1 mg L-1or 2 mgL-1Kinetin,both of which yielded significantly more shoots than did other BAP treatments over the trial period of 3 weeks.

The effect of Kn on shoot induction showed a similar trend butnumbers of shoots were much lower than for BAP treatment alone.The effect on shoot induction of lower concentration of kinetin in combination with BAP was insignificant as compared to treatment with BAP alone. Maximum shoot number(8.9±0.70)was recorded when MS media was supplemented with 5 mg L-1BAP alone or with 5 mg L-1BAP combined with 1 or 2 mg L-1Kinetin.Maximum shoot length(3.18±0.24)was recorded with 5 mg L-1BAP?2 mg L-1of Kinetin.The interactive effect of BAP and Kn on mean shoot number and mean shoot length was insignificant.Based on its performance,Ms medium supplemented with BAP(5 mg L-1) was used for further multiplication of shoots(Fig.1a,b).

Shootmultiplication

Effects of BAP on shoot multiplication in T.falconeri in a two-way factorial experiment are summarized in Table 2. Shoot multiplication varied by BAP concentration.The maximum rate of shoot multiplication(6.55±0.25)was recorded for medium supplemented with 5.0 mg L-1of BAP,and this mean was significantly greater than meansfor shoot multiplications obtained with other BAP treatments over the eight-week period(Fig.1c,d).

The longest shoots(5.01±0.11)were obtained on the same medium,i.e.,5.0 mg L-1of BAP,and were significantly longer than those of other treatments.A significant reduction in mean shoot length was observed upon the addition of BAP beyond 5 mg L-1(Table 2).

In vitro rooting

For in vitro rooting,auxins IAA,NAA and IBA were supplemented at various concentrations(1–6 mg L-1)to half-strength MS liquid medium.Root induction varied significantly between treatments.Root initiation started after 2 weeks of shoot culturing.Rooting was achieved only in medium supplemented with IBA(Table 3).Significant differences were observed between concentrations of IBA alone,with maximum rooting(100%)achieved using IBA 5 mg L-1supplemented cultures while 40 and 60%rootinduction was observed at4 and 6 mg L-1IBA, respectively.

Mean root number(6.3±0.67),mean root length (4.67±0.32),and maximum rooting resulted from treatment using IBA at 5 mg L-1and means for all three parameters were significantly higher than those of other treatments.Auxins IAA and NAA failed to induce rooting. A well developed root system resulted after 6 weeks of culture(Fig.1d).

Hardening and acclimatization

Table 2 Effect of different concentrations of BAP on rate of multiplication and length of shoots(cm)from propagules*of T.falconeri cultured on MS media.(Observations recorded after 8 weeks of culture on MS media)

Plantlets with welldeveloped rootand shootsystems were transferred to a 500 ml glass jars containing sterilized vermiculite presoaked with half strength MS nutrient medium(Sucrose-free).The jars were initially kept for 2 weeks in a culture room.More than 90%of the plantlets survived when subjected to 2 weeks hardening in the culture room.These plantlets were shifted to plastic pots containing soil,sand,and farmyard manure at 2:1:1 ratio and were then placed in a mist chamber for 20 days and subsequently shifted to an open shade house for acclimatization to the externalenvironment(Fig.1e).The plantlets were then transplanted in polybags containing an identical soil mixture and kept under a shade house.Over the next 6 months,these plantlets showed well developed rhizome and root systems.Gradually the plantlets were exposed to naturalconditions survivalofthe transplanted materialwas 100%.

Discussion

Our aim was to develop an efficient micropropagation protocol for T.falconeri by the axillary bud proliferation method using vegetative tissue of field-grown bamboo clumps.Many researchers have reported on micropropagation of bamboos,e.g.,Thamnocalamus spathiflorus(Bag et al.2000),Bambusa arundinacea(Mehta et al.1982),B. glaucescens(Shirin and Rana 2007),B.multiplex(Huang et al.1989),D.strictus(Nadgir et al.1984;Rao et al. 1985),Phyllostachys attrea(Huang et al.1989),Sasa pygmaea(Huang et al.1989)and Sinocalamus latiflora (Yeh and Chang 1986).Since micropropagation through callus culture causes loss of genetic fidelity,axillary bud proliferation is a more reliable procedure than callus culture forclonalpropagation.The protocoldescribed here for T.falconeri by axillary branching is suitable for true-totype clonal shoot multiplication.

Liquid cultures were also assessed in this study.Incorporation of agarresulted in delayed growth and declines in rates of shoot multiplication.Similar responses in liquid and semi-solid media was reported for D.strictus(Nadgir et al.1984).Slower growth or poor shoot multiplication rates on semi-solid medium could be due to the fact that solubilized agarforms a gelthatcan bind waterand adsorb compounds,resulting in reduced uptake of water,nutrient ions,hormones and other constituents of the medium (Debergh 1983).

Since BAP and kinetin remain stable athigh temperature and are autoclavable,these were selected for this study. Moreover,greater efficiency of BAP over other cytokinins is evident from the findings of Zaerr and Mapes 1982 and Thomas and Blakesley 1987.Nadgauda et al.(1990) obtained in vitro shoots in Dendrocalamus brandisii and Bambusa arundinacea on MS medium supplemented with BA and coconut milk.Chambers et al.(1991)obtained maximum numbers of shoots from intact cultured epicotyl tissue of seedlings of Dendrocalamus hamiltonii on medium containing BA.Similarly,many other workers also recommended the use of BA for bamboo shoot multiplication(Das and Rout 1991;Prutpongse andGavinlertvatana 1992;Ansari et al.1996;Arya and Arya 1997;Huang and Huang 1995;Yasodha et al.1997;Arya and Sharma 1998).

Table 3 Effectofdifferentconcentrations of Auxins viz.IAA,NAA,IBA and Coumarine on percentrootinduction,number ofroots perculture and length of roots(cm)of T.falconeri cultured on 1/2 MS media.(Observations recorded after 4 weeks of inoculation)

This study documented the efficiency of BAP over Kinetin(Kn)for bud break,shoot proliferation and multiplication.Low proliferation on Kn-supplemented medium was consonant with the work of Nadgir et al.(1984)who reported better multiplication in nodal segments collected from mature plants of Dendrocalamus strictus,Bambusa arundinacea and Bambusa vulgaris.Das and Rout(1991) also reported reduced shoot multiplication on Kn-supplemented medium in Dendrocalamus giganteus and Dendrocalamus strictus.The interactive effect of BA and Kn was insignificant on shoot induction in T.falconeri.Maximum shoot induction(90%)was obtained on medium supplemented with 5 mg L-1of BAP alone or in combination with very low concentration of Kn.A similar trend resulted for mean number of shoots per explant,whereas maximum mean length of shoots(3.18±0.24)was recorded for 5 mg L-1BAP?2 mg L-1Kn and 5 mg L-1of BAP(3.14±0.24).The maximum shoot multiplication rate(6.55-fold)was recorded for 5 mg L-1of BAP and was significantly higher than for other treatments.

The ability of plant tissues to form adventitious roots depends on the interaction of many different endogenous and exogenous factors.The role of auxins in root development is well established and was reviewed by Torrey (1976).The clumps of at least 4 proliferated shoots were transferred to auxin-supplemented half-strength MS medium,where profuse rooting was observed with simultaneous shoot elongation and proliferation,probably due to the‘‘cytokinin carryover effect’’in the shoots.

In tissue culture,roots are mostly induced in the presence of a suitable auxin in the medium.In T.falconeri, only few auxin treatments stimulated adventitious root formation with large numbers of long roots.Of the various auxin treatments,IBA was the most suitable auxin,yielding 100%rooting.IBA has been used for rooting of many bamboo species,like Dendrocalamus strictus(Nadgir etal. 1984),Dendrocalamus brandisii and Bambusa arundinacea(Nadgauda etal.1990),Dendrocalamus giganteus,D. strictus(Das and Rout1991),Thamnocalamus spathiflorus (Bag et al.2000)and Bambusa balcooa(Das and Pal 2005).Among six different concentration of IBA, 5 mg L-1of IBA proved best for root induction(100%) and for root growth.

We recorded survival success of 90%when propagules were subjected to 2 weeks of hardening in a culture room and acclimatization in a net house.Further,hardened plantlets showed 100%survival after transplantation in field conditions,whereas 70–80%survival was reported by Nadgir et al.(1984)for propagules of D.strictus.

Conclusion

Dev Ringal is the most preferred ringal species in the hills of Uttarakhand and its abundance is declining due to natural calamities,habitat fragmentation,forest fires and excessive harvesting coupled with gregarious flowering.To meet the livelihood demands of villagers,large scale planting is required.This species flowers at an interval of 28–33 years and has seed viability of just 6–8 months so seeds cannot be kept in storage for long periods.Considering the limitation of seed supply,we developed anin vitro protocol for production of plantlets using axillary shoot proliferation from field-grown mature culms.This not only enables multiplication of the species en masse but also conserves its genetic resources.The other advantage of the technique is that the plantlets generated through this method will be genetically stable,true-to-type plantlets as there is no intervention of callus.

AcknowledgmentsOur profound thanks to Forest Research Institute(FRI),Dehradun,India for providing the facilities.Authors are also thankful to the staff of Plant Physiology Discipline,FRI,Dehradun for providing necessary assistance.

Ansari SA,Kumar S,Palanisamy K(1996)Peroxidase activity in relation to in vitro rhizogenesis and precocious flowering in Bambusa arundinacea.Curr Sci 71:358–359

Arya ID,Arya S(1997)In vitro culture and establishment of exotic bamboo Dendrocalamus asper.Indian J Exp Biol35:1252–1255

Arya S,Sharma S(1998)Micropropagation technology of Bambusa bambos through shoot proliferation.Indian For 124:725–731

Bag N,Chandra S,Palni LMS,Nandi SK(2000)Micro propagation of Dev-ringal[Thamnocalamus spathiflorus(Trin.)Munro]—a temperate bamboo,and comparison between in vitro propagated plants and seedlings.Plant Sci 156:125–135

Chambers SM,Heuch JHR,Pirrie A(1991)Micropropagation and in vitro flowering of bamboo Dendrocalamus hamiltonii Munro. Plant Cell Tissue Organ Cult 27:45–48

Das M,PalA(2005)In vitro regeneration of Bambusa balcooa Roxb: factors affecting changes of morphogenetic competence in the axillary buds.Plant Cell Tissue Organ Cult 81:109–112

Das P,Rout GR(1991)Mass multiplication and flowering of bamboo in vitro.Orissa J Hortic 19:118–121

Debergh P(1983)Effects of agar brand and concentration on the tissue culture medium.Physiol Plant 59:270–276

Huang LC,Huang BL(1995)Loss of the species distinguishing trait among regenerated Bambusa ventricosa.Plant Cell Tissue Organ Cult 42:109–111

Huang LC,Huang BL,ChertWL(1989)Tissue culture investigations of bamboo-IV Organogenesis leading to adventitious shoots and plants in excised shootapices.Environ Exp Bot 29:307–315

Janzen DH(1976)Why bamboos wait so long to flower.Annu Rev Ecol Syst 7:347–391

Mehta U,Rao IVR,Ram HY.1982.In Proceedings of the fifth international congress on plant tissue culture(ed.Fujiwara A.), Tokyo,pp 109–110

Nadgauda RS,Parsharami VA,Mascarenhas AF(1990)Precocious flowering and seedling behaviour in tissue cultured bamboos. Nature 344:335–336

Nadgir AL,Phadke CH,Gupta PK,Parasharmi VA,Nair S, Mascarenhas AF(1984)Rapid multiplication of bamboo by tissue culture.Silvae Genet 33:219–233

Naithani HB,Pal M,Lepcha STS(2003)Gregarious flowering of Thamnocalamus spathiflorus and T.falconeri,bamboos fromUttaranchal India.Indian For 129:517–526

Prutpongse P,Gavinlertvatana P(1992)In vitro micropropagation of 54 species from 15 Genera of Bamboo.Hortic Sci 27:453–454

Rao IU,Rao VR,Narang V(1985)Somatic embryogenesis and regeneration of plants in the plants in Dendrocalamus strictus. Plant Cell Rep 4:191–194

Shirin F,Rana PK(2007)In vitro plantlet regeneration from nodal explants of field-grown culms in Bambusa glaucescens Willd. Plant Biotechnol Rep 1:141–147

Thomas TH,Blakesley D(1987)Practical and potential uses of cytokinins in agriculture and horticulture.Br PlantGrowth Regul Group Monogr 14:69–83

Torrey JG(1976)Root hormones and plant growth.Annu Rev Plant Physiol 27:435–459

Yasodha R,Sumathi R,Malliga P,Gurumurthi G(1997)Genetic enhancement and mass production of quality propagules of Bambusa nutans and Dendrocalamus membranaceus.Indian For 4:303–306

Yeh ML,Chang WC(1986)Plant regeneration through somatic embryogenesis in callus cultures of green bamboo(Bambusa oldahamii Munro).Theor Appl Genet 73:161–163

Zaerr JB,Mapes MO(1982)Action of growth regulators.In:Bonga JM,Durzan D(eds)Tissue Culture in Forestry.Martinus Nijhoff/Dr W.Jun,The Hague,pp 231–255

Zhou B,Fu M,Xie J,Yang X,Li Z(2005)Ecological functions of bamboo forest:research and Application.J For Res 16:143–147

7 December 2012/Accepted:20 April 2014/Published online:20 January 2015

The online version is available at http://www.springerlink.com

Corresponding editor:Zhu Hong

Plant Physiology Discipline,Botany Division,Forest Research Institute,Dehradun 248006,India

e-mail:tiwari.chandrakant08@gmail.com