Deepti Bhadrawale?Jay Prakash Mishra?Yogeshwar Mishra

Abbreviations

BA 6-Benzyladenine

HgCl2Mercuric Chloride

Kn Kinetin(N6-Furfuryladenine)

IAA Indole-3 acetic acid

MS Murashige and Skoog(1962)

Introduction

Bambusa tulda Roxb.or Indian timber bamboo is considered as one of the most useful bamboo species in India.It is normally found in Bihar,Assam,Meghalaya,Mizoram,Nagaland,Tripura and exhibits an inter-mast flowering cycle of 30–60 years(Uppin 1980).In India it is distributed naturally in the North and North-Eastern parts of the country and cultivated in Orissa,Arunachal Pradesh,Uttar Pradesh,Karnataka and Bengal.It is a tall,sturdy and quick growing bamboo suitable for the production of high quality paper and furniture.In northeast India,its culms are used for manufacture of many handicraft items(Chakraborty 1988),house construction(Tewari 1992)and manufacturing of high quality paper(Upreti and Sundriyal 2001).The succulent shoots are rich in phytosterols and the fermented shoots used for the production of sterol drugs(Srivastava 1990).Due to prolonged flowering cycle,the commercial level propagation of B.tulda through seeds is not always possible.It appears to have an intermast period of about 48 years(Mohan Ram and Harigopal 1981).Similarly,clonal propagation en masse is also not routinely feasible,for a micropropagation protocol developed only for seedling explants by Saxena(1990).However,this method of in vitro regeneration possesses a higher risk of genetic instability(somaclonal variation),thereby impeded the purpose of micropropagation.The unknown genetic background and the heterogeneity in seedling populations are the major constraints in seed-based micropropagation protocols.To overcome these constraints,we developed micropropagation protocol for in vitro regeneration system for B.tulda employing nodal explants from field grown culms with twofold shoot multiplication rate(Mishra et al.2008).

In vitro season specific morphogenetic response have been reported in many bamboo species viz.,Dendrocalamus giganteous Munro(Ramanayake and Yakandawala 1997),Bambusa balcoa Roxb(Das and Pal 2005)and Gigantochola atroviolaceae Widjaja(Bisht et al.2010).Sterilizing agents have also a definite effect on shoot bud differentiation from nodal segments in various bamboo species including Bambusa nutans Wall.(Yadav et al.2008)and Bambusa balcooa Roxb(Mudoi and Borthakur 2009).Therefore,in the present communication we described the role of season and concentrations of mercuric chloride on culture establishment of B.tulda.Besides,the report deals with augmentation of shoot multiplication rate for the production of clonal plants through axillary branch proliferation from field-grown clumps of B.tulda.

Materials and methods

Culture establishment

The young side branches of current year growth of B.tulda were collected from NWFP nursery of the institute(Fig.1a)during four seasons,i.e.summer(April–May),rain(July–August),autumn(October–November)in 2014 and winter(January–February)in 2015.The axillary branches(Fig.1b)were selected as source of explants and nodal segments of 2.0–3.0 cm length were excised from these shoots and prepared as explants(Fig.1c).Culmsheaths were removed and then washed for 10 min with 0.1 × diluted aqueous solution of Dettol?(1:10 v/v)(Avalon Cosmetics Pvt.Ltd.,Sirmour,India).Subsequently,these explants were continuously washed with distilled water so as to remove foam followed by treatment with 0.2% aqueous solution of Bavistin,a systemic carbendazim fungicide(BASF India Ltd,Mumbai,India)and surface sterilization with(1)70%ethyl alcohol for 20 s and(2)three doses viz.,0.05,0.1 and 0.2%each of aqueous mercuric chloride and sodium hypochlorite solution separately for 10 min under aseptic condition.The explants were thoroughly rinsed with sterile distilled water to remove traces of sterilizing agents.

The sterilized explants were implanted on MS(Murashige and Skoog 1962)semi-solid medium supplemented with 10 μM BA+0.1 μM IAA in test tube.The axillary bud break(Fig.1d)started from third week and the axillary shoots for in vitro multiplication became available only after fifth week and were subsequently maintained through ten subculture cycles each of 15 days on liquid MS medium supplemented with 10 μM BA+0.1 μM IAA for the present investigation.

In vitro shoot multiplication and rooting

A propagule(bunch of three axillary shoots)was excised and inoculated on MS liquid medium supplemented with 100 μM glutamine+0.1 μM IAA and different concentrations of BA(0,5.0,10.0 and 15.0 μM)alone or in combinations with Kn(0,5.0 and 10.0 μM).The rate of shoot multiplication was determined after 15 days of incubation.For rooting we tried our own procedure(Mishra et al.2008)implanting shoot clusters of 2 shoot(＞2.0 cm length)harvested after 10th subculture cycle on MS liquid medium supplemented with 40.0 μM coumarin.

Culture condition and statistical analysis

The inorganic salts were obtained from Qualigens Pvt.Ltd.,India and phytohormones and B vitamins from Sigma Chemicals Pvt.Ltd.,India.The medium was solidified with 0.8%(w/v)agar and fortified with 3%sucrose(Loba Chemie Ltd.,India).The pH of the medium was adjusted to 5.8 before autoclaving for 15 min at 1.06 kg cm-2(121°C). Each explant was cultured in a 2.5 cm×15.0 cm glass tube(Borosil,India)containing 10 ml sterilized semi-solid medium for culture initiation and 150 ml conical flasks containing 25 ml liquid medium for in vitro shoot multiplication,physically supported with‘M’shape Sonar?filter paper(Axiva Sichem Pvt.Ltd.,New Delhi,India)bridges.The cultures were incubated at(25 ± 2)°C for 16 h with fluorescent light(approx.45 μmol m-2s-1).Each experiment had three replicates each of ten single propagules and repeated twice.The data recorded for culture establishment and in vitro shoot multiplication were subjected to two-way analysis of variance(ANOVA)and for ascertaining level of significance‘F’test least significant differences(LSD)was used.If the data were found significant at p ≤ 0.05,Duncan’s Multiple Range Test(DMRT)was employed for comparison of treatment means(Gomez and Gomez 1984).

Fig.1 Micropropagation of B.tulda:a Mother clump;b Current year axillary branches selected for preparation of explants;c Nodal segment collected form explants prepared for inoculation;d Sprouting ofnodalexplanton MS semisolid medium with 10 μM BA+0.1 μM IAA;eMultiplicationonMS liquidmedium supplemented with 5.0 μM BA+5.0 μM Kn;f Rooting on MS liquid medium supplemented with 40 μM coumarin;f Transfer of in vitro raised plantlets to culture room for in vitro hardening and h Transfer of in vitro raised plantlets to polythene bags

Hardening and transplantation

The in vitro raised plantlets were removed from the flasks and dipped for 30 s in 0.2%(w/v)bavistin solution and washed with tap water and transferred to root trainers comprising 25 cells each of 150 cc(Neevedita Plastic Industries,Nagpur,India),filled with a mixture of autoclaved soilrite:compost(1:1).The planted root trainers shifted to plastic tray filled with half strength of iron free MS salts covered with perforated transparent polythene sheet and maintained at(25 ± 2)°C under 16 h photoperiod for in vitro hardening for 2–3 weeks followed by 2 weeks of ex vitro hardening.

Results

Culture establishment

We used various concentrations of sodium hypochlorite and mercuric chloride for sterilization of B.tulda explants.The explants treated with various concentrations of sodium hypochlorite in all seasons exhibited heavy bacterial and fungal contamination(data not shown).It appears that the release of chlorine from sodium hypochlorite was too inadequate to compete with the growth of pathogens on B.tulda.As a result,mercuric chloride emerged to be more effective than sodium hypochlorite for sterilization of B.tulda explants.

All three concentrations of mercuric chloride used for sterilization of explants did not show significant variation for aseptic culture establishment and bud break(Table 1).However,various seasons and their interaction with mercuric chloride had significantly pronounced effect on aseptic culture establishment and bud break.Thus,rains had significantly maximum aseptic culture establishmentand winter-summer exhibited the highest bud break.The autumn recorded significantly lowest aseptic culture establishment and bud break(Table 1).Interestingly,ratio of bud break to aseptic culture establishment declined from 0.68 in 0.05%mercuric chloride to 0.59 in 0.2%mercuric chloride but did not alter in other seasons presumably due to more succulent nature,and consequent intolerance to high concentrations of mercuric chloride of explants in rains than in other seasons.Thus,aseptic culture establishment and bud break depends upon physiological status of the collected explants and amount of contaminations during various seasons of the year.Our investigation suggests that administration for 10 min of 0.05 and 0.1%mercuric chloride to explants collected in winter and summer seasons,respectively facilitates optimum culture establishment and bud break.On the other hand,administration of 0.1–0.2%mercuric chloride in rainy season enhanced aseptic culture establishment but inhibited bud breaks due to toxicity to explants(Table 1).

Table 1 Effect of season,mercuric chloride,concentration and their interactions on aseptic culture establishment and bud break in B.tulda.Data are mean of two experiments

Shoot regeneration

A three-fold shoot multiplication was achieved till the 5th subculture cycle on MS liquid medium+10 μM BA+0.1 μM IAA.Thereafter,the multiplication rate significantly declined and shoots turned brown and eventually dead.To overcome this problem the medium was supplemented with 100 μM glutamine and a large number of cultures could be saved after 5th subculture cycle.For in vitro shoot multiplication the shoots were divided into propagules of 2–3 shoots and cultured on MS medium containing different combinations of BA and Kn.BA and Kn at any concentration could not sustain shoot multiplication whereas the synergistic effect of BA+Kn signif icantly enhanced the shoot multiplication rate and showed significant variation in two way analysis.The combination of 5.0 μM BA and 5.0 μM Kn in MS medium was judged as the optimum dose for maximum shoot multiplication of 4.75 fold and overall shoot growth and development(Table 2;Fig.1e).Since this interaction proved to be most effective,regular subculturing for maintenance of shootcultures was done in this media with additional supplementation of 100 μM glutamine+0.1 μM IAA at periodic interval of 3–4 weeks.

Table 2 Effect of BA and Kn alone and their interaction on shoot regeneration in B.tulda.Data are mean of two experiments

Rooting

In the present communication we affirm our previous results(Mishra et al.2008)and obtained 98%rooting in the medium with 40 μM coumarin in the species(Fig.1f).The pattern of root induction and root number was found to be similar to our earlier investigation.

Hardening and transplantation

The in vitro raised plantlets were hardened and acclimatized prior to transplantation in the field.The plantlets were first transferred to culture room for 2–3 weeks for in vitro hardening(Fig.1g)and then transferred to semi controlled conditions of shadehouse for 2 weeks.The healthy plantlets showed 98%acclimatization after 6 months transfer to polythene bags(Fig.1h).

Discussion

Culture establishment

The magnitude of contaminations and physiological status of donar plants play significant role in aseptic culture establishment and bud induction of the explants collected during various seasons of the year(Ramanayake and Yakandawala 1997;Yadav et al.2008).We used various concentrations of sodium hypochlorite and mercuric chloride for sterilization of B.tulda explants.In contrast to earlier investigations(Woods et al.1991;Ogita 2005),the explants treated with various concentrations of sodium hypochlorite in all seasons exhibited heavy bacterial and fungal contamination.It appears that the release of chlorine from sodium hypochlorite was too little to compete with the growth of pathogens on B.tulda.As a result,mercuric chloride emerged to be more effective than sodium hypochlorite for sterilization of B.tulda explants.Similar observation was also been recorded by Moutia and Dookun(1999)in bud cultures of sugarcane.Therefore,we describe the data obtained with mercuric chloride.All three concentrations of mercuric chloride used for sterilization of explants did not show significant variation for aseptic culture establishment and bud break and corroborate with the findings of several researchers(Saxena and Bhojwani 1991;Arya et al.2001;Mishra et al.2001)who routinely used 0.1%mercuric chloride as an effective sterilizing treatment for different bamboo species.However,various seasons had significantly pronounced effect on aseptic culture establishment and bud break.Thus,rains had significantly maximum aseptic culture establishment and winter-summer exhibited the highest bud break.The autumn recorded significantly lowest aseptic culture establishment and bud break.In fact,the autumn season denotes fluctuations of climatic conditions favouring pathogen growth at the expense of poor growth adjustment of B.tulda.As winter dormancy sets in,the growth of both B.tulda culms and pathogens slows down,resulting in better aseptic culture establishment.Also in this season,explants accumulate considerable reserved food material but remain dormant on donor culms.On detachment,the explants come out of dormancy and utilize reserved food for their bud break.In tropical region like central India summer starts with the continuation of active growth of bamboos(Agnihotri and Ansari 2000;Kumar et al.2005)that competes with pathogens,resulting in excellent culture establishment and bud break.The rainy season sustaining the highest culture establishment possibly relates to fast growth of culms overriding pathogen growth,for increasing concentrations of mercuric chloride in rains signif icantly facilitated aseptic culture establishment.This indicates that pathogenesis was more in rains than other seasons,which exhibited very little change in aseptic culture establishment with increasing concentrations of mercuric chloride.

Interestingly,ratio of bud break to aseptic culture establishment declined from 0.68 in 0.05% mercuric chloride to 0.59 in 0.2%mercuric chloride but did not alter in other seasons presumably due to more succulent nature,and consequent intolerance to high concentrations of mercuric chloride of explants in rains than in other seasons.Thus,aseptic culture establishment and bud break depends upon physiological status of the collected explants and amount of contaminations during various seasons of the year.Our investigation suggests that administration for 10 min of 0.05 and 0.1%mercuric chloride to explants collected in winter and summer seasons,respectively facilitates optimum culture establishment and bud break.On the other hand,administration of 0.1–0.2%mercuric chloride in rainy season enhanced aseptic culture establishment but inhibited bud breaks due to toxicity to explants.Correlation between rainfall and bud-break frequency has also been reported in many bamboo species such as Bambusa balcooa Roxb(Das and Pal 2005),Pseudoxytenanthera stocksii Munro(Sanjaya et al.2005)and Bambusa balcooa Roxb(Negi and Saxena 2011).which they attributed to the strong maternal effect,which prevented rejuvenation and survival of shoots,despite phytohormone amendments or change in physical conditions.In bamboos,glutamine has been recognized to be effective as a source of reduced nitrogen for in vitro culture of Phyllostachys nigra Munro var.Henonis(Ogita 2005)and Pseudoxytenanthera stocksii Munro(Sanjaya et al.2005).As compared to our previous findings(Mishra et al.2008),significant increase in shoot multiplication rate by 2.5 times has been achieved on MS liquid medium with 5 μM BA+5 μM Kn with additional supplementation of 100 μM glutamine+0.1 μM IAA.The result is in concurrence with the findings of Arshad et al.(2005)who employed combination of BA and Kn for higher multiplication rate in Bambusa wamin Branids ex Camus.Higher concentration of BA resulted in stunted and vitrified shoot growth inappropriate for subculturing.Similarly,Arya and Arya(1997)in Dendrocalamus asper(Schult and Schult)Backer ex Heyne and Bag et al.(2000)in Thanacolamus spathiflorus Munro also reported higher concentration of BA produced thin,small leafy shoots which adversely affected the rate of shoot multiplication.

Rooting

Being a natural source of phenol coumarin reported to augment plant growth(Pollock et al.1954)and affect IAA biosynthesis and increases the endogenous free IAA level during the induction phase of rooting to initiate more roots(Tartoura et al.2004).Working with the same species,Sharma and Sarma(2013)reported maximum rooting success to the tune of 95%employing 5 mg/l NAA.However,as per current catalogue of Sigma Aldrich(2016)the rate of NAA is just double as compared to coumarin.Therefore,for making the procedure cost effective using 40 μM coumarin is cheap and economical.

Hardening and transplantation

The in vitro raised plantlets were hardened and acclimatized prior to transplantation in the poly bags.The plantlets were first washed with bavistin to avoid any bacterial attack and then transferred to culture room for 2–3 weeks for in vitro hardening and then transferred to semi controlled conditions of shadehouse for 2 weeks for ex vitro hardening.The healthy plantlets showed 100%acclimatization after 6 months of transfer to polythene bags.

Shoot regeneration

In accordance to the present observation,Saxena and Bhojwani(1993)has also reported severe decline in shoot multiplication rate in D.longispathus Kurz(Kurz)Wendl.,

Conclusion

We developed an efficient and improvised micropropagation system for field grown culms of Bambusa tulda Roxb,which is very difficult to root species.The study exhibited the role of season and concentration of HgCl2on aseptic culture establishment and bud break.The summer and 0.1%HgCl2had emerged as the best season and concentration which resulted in 73%aseptic culture establishmentand bud break.Besides,we enhanced the existing shoot multiplication rate by 2.5 times in the present study by supplementing MS liquid medium with 100 μM glutamine+0.1 μM IAA+5.0 μM BA+5.0 μM Kn,which ensured a stable 4.75 fold shoot multiplication rate at a subculture cycle of 15 days.Forty micro molar coumarin induced≥98%in vitro adventitious roots after 1 month of inoculation.The in vitro regenerated plantlets exhibited 98%survival during hardening and thus,manifesting the efficacy of this procedure for B.tulda.

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