) through nodal culture - hindawi publishing...

Research ArticleAn Effective Protocol for Micropropagation ofEdible Bamboo Species (Bambusa tulda andMelocanna baccifera) through Nodal Culture

Sayanika Devi Waikhom12 and Bengyella Louis134

1 Institute of Bioresources and Sustainable Development Takyelpat Imphal Manipur 795001 India2 Centre of Advanced Study in Life Sciences Manipur University Imphal Manipur 795003 India3 Department of Biotechnology The University of Burdwan Golapbag More West Bengal 713104 India4Department of Biochemistry University of Yaounde I BP 812 Yaounde Cameroon

Correspondence should be addressed to Sayanika Devi Waikhom sayanikawaikhomgmailcom

Received 3 March 2014 Revised 1 May 2014 Accepted 7 May 2014 Published 21 May 2014

Academic Editor Amparo Latorre

Copyright copy 2014 S D Waikhom and B LouisThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

High demand for edible bamboo shoots of Bambusa tulda andMelocanna baccifera inmany Asian ethnic groups has led to the needfor developing intensive bamboo farming To achieve this in vitro regeneration of bamboo plantlets is needed due to the long andirregular bamboo flowering cycle and scarcity of bamboo seeds An effective protocol for plantlets regeneration in B tulda andMbaccifera from nodal explants following validation of the species using the sequence of trnL-F intergenic spacer region is describedEffective axillary bud breaking was achieved at 3mgL of 6-benzylaminopurine (BAP) in MS medium Importantly combining2mgL of kinetin (Kn) with 3mgL of BAP produced a synergistic effect for shootmultiplication inB tulda andM baccifera Underoptimized conditions in half-strengthMSmedium supplemented with 3mgL of indole-3-butyric acid (IBA) 10mgL of coumarinand 3 sucrose profuse production of dark-brown rhizome in B tulda and abundant rooting (8167 119875 lt 005 119865 = 1546) forMbaccifera within 30 days were achieved The established protocol and the validation of the reported species at the molecular levelwill be of help to stakeholders in edible bamboo trade to conserve gene-pool and increase productivity

1 Introduction

The demand for bamboo is on the rise in Asian countriesfor their utility in handicraft industry construction papermaking fishery and human consumption [1 2] Ediblebamboos have been identified and grouped based on the totalcyanogen content [3] Such highly demanded edible bamboowith substantial nutritional attributes are Dendrocalamushamiltonii [4ndash6] Chimonobambusa callosa Bambusa tuldaandMelocanna baccifera [3]M baccifera is sparsely found inthe valleys of North Eastern states of India such as ManipurSikkim Arunachal Pradesh and Mizoram [7] The marketprices of fermented bamboo shoots for B tulda and otherspecies in India are at the range of Rs 40 (asympUS $066) to Rs50 (asympUS $083) per kg [8] and they cost more than US $120

when canned or fried Due to intense shifting cultivation andthe use of fire for clearing the forest M baccifera is nowrestricted to the hilly regions and facing extinction

There is no in vitro protocol for B tulda reportingrhizogenesis herein described and additionally there is noreport for micropropagation of M baccifera Thus there isneed to develop a new protocol for rhizome induction whichforms buds that develops into new culms or shoots easilyShoot regeneration and rooting are key steps for in vitromicropropagation Recent advances in tissue culture exploredthe combined effects of silicon and sodium chloride topromote adventitious shoot regeneration in Ajuga multiflora[10] Nonetheless protocols based on zygotic embryos (ieexplant from seed tissue) for woody bamboo plantlet regener-ation have been developed [11ndash15] Firstly the limitation with

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 345794 8 pageshttpdxdoiorg1011552014345794

2 The Scientific World Journal

zygotic embryos protocols in bamboo plantlets regenerationis that a given protocol is species specific and consequentlydoes not often apply to other genera Secondly the long andirregular bamboo flowering cycle [16 17] as well as low seedviability and scarcity in bamboo seeds [18 19] often limitszygotic embryo protocols for bamboo plantlet regenerationTherefore new accurate protocols are required for successfulpropagation of several species since nodal explants are readilyavailable

B tulda and M baccifera are sympodial species whichproduce nutritive shoots [3 20] Protocols for plantletsregeneration were developed for B tulda through seeds [11]and nodal explants [21] without achieving rhizogenesis Tilldate there is no protocol for plantlets regeneration for Mbaccifera Plantlets regeneration protocols in bamboo gen-erally suffer from poor taxonomic identification and clonalfidelity for the reported species Because of these setbacksprotocols reported on several bamboo species are frequentlynot reproducible In this study we first used trnL-F intergenicspacer to accurately ascertain the taxonomic placement of thestudied species Secondly we developed an effective protocolfor in vitro shoot proliferation from the nodal buds andoptimized rooting of B tulda andM baccifera

2 Material and Methods

21 Plant Material and Phylogenetic Placement Bambusatulda andMelocanna baccifera were identified by the author-ities of the Botanical Survey of India (BSI) Kolkata Thelateral branches of the authenticated specimen were collectedfrom culms of B tulda and M baccifera which lies 23∘471015840ndash25∘411015840 NL and 92∘581015840ndash94∘471015840 EL from the Forest Departmentof Imphal District Manipur India during the month ofJuly 2011 Genomic DNA was isolated as described [22] Thequantity and quality of DNA were checked on a nanodropspectrophotometer (BioSpec-nano Shimadzu Japan) and08 wv agarose gel electrophoresis respectively The trnL-F intergenic spacer was amplified as previously described[23] and sequenced Sequences were analysed and assignedto molecular species based on 98ndash100 sequence similarityin the GenBank as accessions JX507132 (Bambusa tulda)and JX507133 (Melocanna baccifera) respectively Sequenceswere aligned usingMuscle program [24] and best substitutionmodel was determined based on Akaike Information Crite-rion corrected (AICc) and Bayesian Information Criterion(BIC) Phylogenetic analysis was performed using the maxi-mum likelihood method in MEGA6 software [25]

22 Explants and Surface Sterilization The leaf sheaths cov-ering the nodal segments containing axillary buds (15ndash2 cm)were carefully removed and wiped with 70 (vv) ethanolusing sterilized cotton The explants were surface sterilizedin 01 (vv) solution ofmercuric chloride (HgCl

2) for 15min

andwashed 4 times with eachwashing step lasting 5minThesterilized nodal segments were cultured in liquid Murashige-Skoog (MS) medium supplemented with 100mgL myo-inositol (Sigma Saint Louis MO USA) and 30 gL sucroseIt is worth noting that the pH of the medium was adjusted to

57plusmn01with 1NNaOHor 1NHCl before autoclaving at 121∘Cand 11768 kPa for 20min For bud breaking the MS mediumwas supplemented with 6-benzylaminopurine (BAP) at 10levels (1 2 3 4 5 6 7 8 9 and 10mgL)

23 Shoots Multiplication Proliferated axillary shoots of 3ndash5 shoot clusters were excised and transferred to MS mediumsupplemented either with (1) BAP at 5 levels (1 2 3 4 and5mgL) (2) 6mgL kinetin (Kn) singly or (3) combination ofKn at levels (1 2 and 4mgL) with optimum concentrationof 3mgL of BAP It is worth mentioning that the choice of6mgL of Kn at one level was based on preliminary findingwhere best effect was observed (data not shown) Shootmultiplication was performed using 3ndash5 axillary proliferatedshoots excised from the clusters of shoots Multiplied shootsin liquid cultures were supported by sterile filter paperbridges Regularly the shoots were transferred at intervalof 10 days under sterile conditions to fresh medium inorder to prevent phenolic oxidation that can trigger theshoots to become yellowish The multiplied shoots on eachculture medium were counted after 45 days to evaluate themultiplication rate of the nodal explants

24 Rooting Process We used 5 levels (1 2 3 4 and 5)of sucrose in half-strength MS medium to determine theoptimum concentration that induces rooting Furthermorerooting was achieved by aseptically transferring multipliedshoots to a half-strength MS medium prepared with 3sucrose (ie optimum concentration) and supplementedwith different combinations of growth regulators as followsFor B tulda we used (1) 3mgL of indole-3-butyric acid(IBA) (2) 3mgL of IBA with 10mgL of coumarin and(3) 3mgL of IBA 3mgL of indole-3-acetic acid (IAA)and 10mgL of coumarin For M baccifera we used (1)3mgL of IBA (2) 3mgL of IBA with 10mgL of coumarinand (3) 3mgL of IBA 005mgL of BAP and 10mgL ofcoumarin All cultures in the experiment were incubated ina growth chamber at 25 plusmn 1∘C with a 16 h photoperiod atlight intensity of 45120583molm2sec photosynthetic photon flux(PPF) provided by cool white fluorescent tubes (TLD CoolWhite 40W Phillips India) Rooting percentage and averagenumber of days for rootingwere recorded Following rootingMSmediumwas removed and the plantlets were hardened ina bottle jar of 76mm height times 60mm diameter and 143mLcap containing autoclaved soil The soil was made of 4 1( ww) rice-straw-vermin compose and sand The plantletswere cultured for 25 days Acclimatization was achieved at30 plusmn 2

∘C and at 84 humidity in the greenhouse

25 Clonal Fidelity Test We checked clonal fidelity by se-quencing the trnL-F intergenic spacer of in vitro raisedplantlets and compared the sequences with the motherexplants

26 Statistical Analysis The experiments were performed inreplicates for a total of 3 biological repeats carried out atseparate times All data on shoot regeneration and in vitrorooting were analysed using one-way ANOVA associated

The Scientific World Journal 3

JX507134 Schizostachyum dullooaDQ137362 Schizostachyum funghomiiEU434083 Neohouzeaua fimbriataHQ292331 Schizostachyum zollingeriJX507133 Melocanna bacciferaEF137536 Melocanna bacciferaJX564905 Dendrocalamus manipureanusJX507132 Bambusa tuldaJX564906 Bambusa vulgarisHM448971 Dendrocalamus dumosusEU434058 Dendrocalamus giganteusJX507131 Bambusa oliveriana

KC013282 Chimonobambusa callosaKC013288 Bambusa tuldoides

KC013285 Bambusa cacharensis100

65

100

01

JX564907 Bambusa sp

JX564906 Bambusa sp

Figure 1 Molecular phylogenetic analysis by maximum likelihood method based on the T93 substitution model [9] AIC is 155128 BICis 176370 the highest log likelihood is minus74159 and bootstrap values ge50 from 1000 iterations are shown The taxa we developed in vitroplantlets regeneration based on nodal explants are highlighted in blue

with Tukeyrsquos post hoc test at 119875 le 005 Statistical analysis wasperformed in IBM SPSS statistical software v190

3 Results and Discussion

31 Phylogenetic Placement of Species Based on trnL-Fintergenic spacer sequence similarity test between B tuldaM baccifera and closely related species the overall meandistance between taxa was 054 A total of 167 patternswere found out of a total of 1040 sites and 832 sites werewithout polymorphism (8000) The maximum likelihood(ML) inference authenticated the taxonomic placement ofBambusa tulda andMelocanna baccifera with respect to theirclosely related taxa (Figure 1) The ML inference confirmedthe studied species were different Failure to accuratelyidentify bamboo species prior to propagation can lead tomisleading reports on developed protocols Motivated byrecurring failure to achieve plantlets regeneration for Btulda based on previously reported nodal protocol [21] wesuspected the problem could probably be due to factors likespecies identity the physiological state of mother explant thequality of growth regulators used and so forth As reviewedin Mudoi et al [16] poorly identified nodal clones somaticclones or the mother explant compromises the fidelity ofprotocols Considering the species specificity of protocolsin in vitro bamboo plantlets regeneration we suggest thatprotocols should be validated at theDNA level for the identityof the explants so as to limit the number of factors that mightaffect reproducibility There was no significant difference inthe sequences of trnL-F intergenic spacer between motherexplants and in vitro raised plantlets (data not shown) Thusvalidating clonal fidelity is based on the developed protocol

32 Culture Initiation and In Vitro Axillary Bud BreakThe explants of B tulda and M baccifera were establishedsuccessfully on the modified MS medium with low rateof contamination Previous work on micropropagation of

Table 1 Effect of BAP Kn and Kn + BAP in MS medium on shootmultiplication of Bambusa tulda

Growthhormones

Concentration(mgL)

Average shootsproduced per

explant

Multiplicationrate per explant

BAP

1 833aplusmn 182 250

abplusmn 067

2 1400abcplusmn 180 433

abplusmn 067

3 1683cplusmn 048 533

bplusmn 021

4 1650bcplusmn 092 517

abplusmn 031

5 1183abcplusmn 194 350

abplusmn 067

Kn 6 750aplusmn 231 217

aplusmn 083

Kn + BAP1 + 3 1383

abcplusmn 281 417

abplusmn 101

2 + 3 1767cplusmn 056 550

bplusmn 022

4 + 3 1367abcplusmn 232 417

abplusmn 087

Mean of 10 replicates plusmn SE scored after 4 weeks in culture medium Withineach column values followed by the same letter are not significantly differentaccording to Tukeyrsquos test at 119875 lt 005

B tulda from nodal explants had encountered problemsin culture initiation due to fungal contamination [21] Inthis protocol we minimized the level of contamination byavoiding the use of water for cleaning the lateral branchesat the initial step of surface sterilization Additionally it isshown that supplementingMSmediumwithNaCl and siliconsignificantly enhances the activities of antioxidant enzymes[10] and equally curbs contaminations encountered in tissueculture Axillary proliferation took place after 15 days of budbreak for B tulda and M baccifera The highest frequencyof bud break was observed at the optimum concentrationof 3mgL BAP for B tulda and M baccifera This washallmarked by the production of 1ndash3 shoots as a result ofaxillary bud break for B tulda andM baccifera (Figure 2)

As shown (Figure 2) above or below 3mgL of BAPa low rate of bud break was recorded In vitro bud breakwas low in the basal medium for B tulda and M baccifera


0

10

20

30

40

50

60

70

80

BAP concentration (mgL)

Bud

brea

k (

)

Control 1 2 3 4 5 6 7 10

P le 005

y = minus25082x2 + 27218x

R2 = minus0161

B tulda

Bud break ()Poly (bud break ())

(a)

y = minus244942x2 + 26148x

P le 005

0

10

20

30

40

50

60

70

80

Bud

brea

k (

)

BAP concentration (mgL)Control 1 2 3 4 5 6 7 10


R2 = 01621

Bud break for M baccifera ()

(b)

Figure 2 Effectiveness of BAP on bud break inMSmedium revealed 3mgL is the optimum concentration (a) Bambusa tulda (b)Melocannabaccifera

Although we tested other cytokinin such as Kn only BAPefficiently promoted bud break at all tested concentrations Inprevious studies BAP was successfully used in bud breakingin plant species like Arundinaria callosa B vulgaris andMelocanna bambusoides [26ndash28] and in Origanum species[29 30] Previous studies on B tulda did not explore theeffect of BAP on axillary bud break [21] Herein rapid budbreaking of axillary explants dormancy for M baccifera andB tulda was achieved at 3mgL of BAP in 10 and 15 daysrespectively Details of the bud break (Figures 2 3(a) and4(a)) shoot multiplication (Figures 3(b) and 4(b)) and invitro rooting (Figures 3(c) and 4(c)) are depicted for B tuldaandM baccifera

33 Effects of BAP and Kn on Shoot Multiplication In thisstudy it was observed that increase in concentration ofBAP (2ndash4mgL) enhanced the rate of shoot multiplicationNonetheless above 5mgL of BAP sharp decline in the shootmultiplication rate was observed (Figures 3(b) and 4(b))BAP has been observed to be the most effective plant growthregulator for woody bamboo shoot in vitro multiplication[28 31 32] In our study the highest shoot multiplication rateper explant was 550 (119875 lt 005 119865 = 318) in B tulda underthe influence of 3mgL BAP and 2mgL Kn (Table 1) Alsothe maximum shoot multiplication rate per explant was 583(119875 lt 005119865 = 385) inM baccifera under the effect of 3mgLBAP and 2mgL Kn (Table 2) Comparatively a single doseeffect of Kn on B tulda and M baccifera did not increasethe rate of shoot multiplication Interestingly the combinedeffect of BAP and Kn increased shoot multiplication rate aswell as the quality of shoots in B tulda and M baccifera(Figures 3(b) and 4(b)) In this regard the effective combinedconcentration was 2mgL of Kn and 3mgL of BAP whichproduced 1767 and 1817 of average shoots per explant in Btulda andM baccifera respectively

Table 2 Effect of BAP Kn and Kn + BAP in MS medium on shootmultiplication ofMelocanna baccifera

Growthhormones

Concentration(mgL)


explant


BAP

1 850aplusmn 180 267

aplusmn 061

2 1417abplusmn 196 450

abplusmn 067

3 1700bplusmn 044 533

bplusmn 021

4 1667bplusmn 095 50

bplusmn 044

5 1200abplusmn 214 367

abplusmn 076

Kn 6 1583bplusmn 128 483

abplusmn 048

Kn + BAP1 + 3 1717

bplusmn 087 550

bplusmn 034

2 + 3 1817bplusmn 031 583

bplusmn 017

4 + 3 1583bplusmn 114 483

abplusmn 048


To achieve optimal shoot multiplication other authorshave combined synthetic auxin (such as 1-naphthalene aceticacid NAA) natural auxin (such as IBA) with cytokinin(such as BAP) in culture medium [4 27 33] In somecases combination of BAP with Kn enhanced the rate ofshoot multiplication [14 34] In the present investigationcombination of BAP and Kn produced a synergistic effectleading to an increase in shoot multiplication as well asthe texture of shoots which turned deep green in both Btulda and M baccifera (Figures 3(b) and 4(b)) In contrastMishra et al [21] achieved shoots multiplication of B tuldaby combining BAP IAA and glutamine

34 Rooting Process and Rhizome Formation The shootclusters of 1-2 cm transferred to a half-strength MS medium


(a) (b) (c) (d)

Figure 3 Micropropagation of Bambusa tulda (a) Node with axillary bud breaks in liquid MS medium (b) Shoot multiplication in 2mgLKn + 3mgL BAP (c) Formed rhizome (tagged with arrows) produced multiple profuse whitish roots in 05 MS medium supplemented with3mgL IBA + 3mgL IAA + 10mgL coumarin + 3 sucrose (d) Plantlets established in the soil under greenhouse conditions

(a) (b) (c) (d)

Figure 4 Micropropagation of M baccifera (a) Node with axillary bud breaks in liquid MS medium (b) Shoot multiplication in 2mgLKn + 3mgL BAP (c) Rooting in 05 MS medium supplemented with 3mgL IBA + 10mgL coumarin + 005mgL BAP + 3 sucrose (d)Plantlets established in the soil under greenhouse conditions

fortified with different plant growth regulators were success-fully rooted As shown by the regression analysis (Figure 5)varied concentrations of sucrose in combination with 3mgLIBA+ 10mgL of coumarin influenced the rooting percentagein B tulda and M baccifera (Figure 5 Table 3) Optimumrooting was obtained with 3 of sucrose Primarily at 3sucrose we obtained increased rooting in B tulda and Mbaccifera It was observed that 3mgL IBA + 3 sucroseproduced moderate rooting of 4500 at an average rateof 76 days in B tulda Adding 10mgL of coumarin inIBA supplementedmedium increased rooting efficiency from4500 to 7700 (Table 3) Interestingly rhizome formationoccurred in the culture medium supplemented with 3mgLof IBA + 10mgL of coumarin + 3 sucrose for B tuldaImportantly the highest rate of roots regenerated from thenodes of rhizome stood at 8670 within 25 days in MSmedium supplemented with 3mgL of IBA + 3mgL of IAA+10mgL of coumarin + 3 sucrose B tulda produced dark-brown rhizome from which profuse whitish roots emerged

(Figure 3(c)) Rhizogenesis is hard to achieve in in vitromicropropagation of woody bamboo Nonetheless similarrhizome morphology was described in Dendrocalamus asperand D membranaceus in the presence of 3 sucrose in MSmedium [35] It is worth noting that a unique dose of 3mgLof IBA + 3 sucrose produced low level rooting (5500 119875 lt005 119865 = 235) forM baccifera (Table 3) To achieve optimalrooting we combined 3mgL IBA + 10mgL coumarin +005mgL BAP + 3 sucrose This combination produced8167 rooting within 30 days (Table 3) No rhizome for-mation occurred in all the growth regulators combinationsfor M baccifera culture (Table 3) In this study we exploitedthe potentials of sucrose BAP coumarin and IBA to rootB tulda and M baccifera Previous findings demonstratedthat induction of in vitro rooting from shoot clusters inbamboos required auxins treatment alone or combined eitherwith BAP or coumarin [14 21 36ndash38] As shown in thisstudy coumarin was required in combination with IBAand BAP for promoting root induction in B tulda and M


Table 3 Optimized rooting conditions in 05 strength MS medium in the presence of multiple growth hormones at 3mgL sucrose

Plant growth regulators Concentration (mgL) rooting Mean daysBambusa tulda

IBA 3 4500aplusmn 494 7600

abplusmn 403

IBA + coumarin 3 + 10 7700bplusmn 70 5167

aplusmn 783

IBA + IAA + coumarin 3 + 3 + 10 8667bplusmn 333 2500

abplusmn 483

Melocanna bacciferaIBA 3 5500

aplusmn 756 4083

abplusmn 271


abplusmn 342

IBA + BAP + coumarin 3 + 005 + 10 8167bplusmn 654 3000

abplusmn 207

Mean of 10 replicates plusmn SE scored after 4 weeks in culture medium Within each column values followed by the same letter are not significantly differentaccording to Tukeyrsquos test at 119875 lt 005

0102030405060708090

100

1 2 3 4 5

Root

ing

()

Sucrose in MS medium ()

y = minus78683x2 + 524x

R2 = 07368

P le 005

(a)

y = minus7778x2 + 52452x

R2 = 08705

P le 005

0102030405060708090

100

Root

ing

()

1 2 3 4 5Sucrose in MS medium ()

(b)

Figure 5 Effect of sucrose on rooting inMSmedium revealed 3 is the optimum concentration (a) Bambusa tulda (b)Melocanna baccifera

baccifera Coumarin is a phenolic compound which may actsynergistically with IBA to enhance endogenous liberationof IAA in explant during rooting process [39] The freedendogenous IAA in turn favours roots initiation as earlierdescribed [39] Plantlets were successfully acclimatized (Fig-ures 3(d) and 4(d)) under greenhouse conditions at a survivalrate of 8181 and 7031 for B tulda and M bacciferarespectively

4 Conclusion

In these established protocols 3mgL BAP was found to beeffective for bud break and shoot multiplication of B tuldaandM baccifera Rhizogenesis in bamboo micropropagationis difficult to achieve and has not been reported previouslyin B tulda protocols [11 21] The developed protocol suc-cessfully produced rhizome in half-strength MS mediumsupplemented with 3mgL IBA 10mgL coumarin and 3sucrose for B tulda for the first time This study providesan effective protocol for micropropagation of B tulda andM baccifera from the nodal segments of the field grownculm This protocol shall be of help to stakeholders inedible bamboo trade to conserve gene pool and increaseproductivity

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was jointly supported by the Academy ofSciences for Developing World (TWAS) Trieste Italy andthe Department of Biotechnology Government of India(Program no 3240223450)

References

[1] J M O Scurlock D C Dayton and B Hames ldquoBamboo anoverlooked biomass resourcerdquo Biomass and Bioenergy vol 19no 4 pp 229ndash244 2000

[2] L M Bal P Singhal S Satya S N Naik and A Kar ldquoBambooshoot preservation for enhancing its business potential andlocal economy a reviewrdquo Critical Reviews in Food Science andNutrition vol 52 no 9 pp 804ndash814 2012

[3] S D Waikhom B Louis C K Sharma et al ldquoGrappling thehigh altitude for safe edible bamboo shoots with rich nutritionalattributes and escaping cyanogenic toxicityrdquo BioMed ResearchInternational vol 2013 Article ID 289285 11 pages 2013


[4] R K Agnihotri and S K Nandi ldquoIn vitro shoot cut a highfrequency multiplication and rooting method in the bambooDendrocalamus hamiltoniirdquoBiotechnology vol 8 no 2 pp 259ndash263 2009

[5] S Sangita W Shivani G Mahesh and S Anil ldquoNutritionalcharacterization of shoots and other edible products of anedible bamboomdashDendrocalamus hamiltoniirdquo Current Researchin Nutrition and Food Science vol 1 pp 169ndash176 2013

[6] S D Waikhom S Ghosh N C Talukdar and S S MandildquoAssessment of genetic diversity of landraces of Dendrocalamushamiltonii using AFLP markers and association with biochem-ical traitsrdquo Genetic and Molecular Research vol 11 no 3 pp2107ndash2121 2012

[7] H B Naithani ldquoDiversity of bamboo species in North EastIndiardquo in Ecology Diversity and Conservation of Plants andEcosystems in India H N Pandey and S K Barik Eds pp 312ndash322 New Dehli India 2006

[8] P K Singh S P Devi K K Devi D S Ningombam andP Athokpam ldquoBambusa tulda Roxb in Manipur State Indiaexploring the local values and commercial implicationsrdquo Notu-lae Scientia Biologicae vol 2 no 2 pp 35ndash40 2010

[9] K Tamura andMNei ldquoEstimation of the number of nucleotidesubstitutions in the control region of mitochondrial DNA inhumans and chimpanzeesrdquo Molecular Biology and Evolutionvol 10 no 3 pp 512ndash526 1993

[10] I Sivanesan and B R Jeong ldquoSilicon promotes adventitiousshoot regeneration and enhances salinity tolerance of AjugamultifloraBunge by altering activity of antioxidant enzymerdquoTheScientific World Journal vol 2014 Article ID 521703 10 pages2014

[11] S Saxena ldquoIn vitro propagation of the bamboo (Bambusa tuldaRoxb) through shoot proliferationrdquo Plant Cell Reports vol 9no 8 pp 431ndash434 1990

[12] S Arya and S Sharma ldquoMicropropagation technology ofBambusa bambos through shoot proliferationrdquo Indian Forestervol 124 no 9 pp 725ndash731 1998

[13] P Chowdhury M Das S R Sikdar and A Pal ldquoInfluence ofthe physiological age and position of the nodal explants onmicropropagation of field-grown Dendrocalamus strictus neesrdquoPlant Cell Biotechnology and Molecular Biology vol 5 no 1-2pp 45ndash50 2004

[14] F Shirin and P K Rana ldquoIn vitro plantlet regeneration fromnodal explants of field grown culms in Bambusa glaucescensWildrdquo Plant Biotechnology Reports vol 1 pp 141ndash147 2007

[15] D Negi and S Saxena ldquoIn vitro propagation of Bambusa nutansWall ex Munro through axillary shoot proliferationrdquo PlantBiotechnology Reports vol 5 no 1 pp 35ndash43 2011

[16] K M Mudoi P S Siddhartha G Adrita G Animesh BDebashisha and B Mina ldquoMicropropagation of importantbamboos a reviewrdquoAfrican Journal of Biotechnology vol 12 no20 pp 2770ndash2785 2013

[17] W S Devi B Louis and G J Sharma ldquoIn vitro seed germina-tion and micropropagation of edible Dendrocalamus giganteusmunro using seedsrdquoBiotechnology vol 11 no 2 pp 74ndash80 2012

[18] D H Janzen ldquoWhy bamboos wait so long to flowerrdquo AnnualReview of Ecology and Systematics vol 7 pp 347ndash391 1976

[19] S D Waikhom L Bengyella R Pranab M S Wakambam KB Pardeep and C T Narayan ldquoScanning electron microscopyof pollen structure throws light on resolving Bambusa-Dendrocalamus complex bamboo flowering evidencerdquo PlantSystematics and Evolution vol 299 article 10 2013

[20] C-S Lin C J Liang H W Hsaio M J Lin and W CChang ldquoIn vitro flowering of green and albino Dendrocalamuslatiflorusrdquo New Forests vol 34 no 2 pp 177ndash186 2007

[21] Y Mishra P K Patel S Yadav F Shirin and S A Ansari ldquoAmicropropagation system for cloning of Bambusa tulda RoxbrdquoScientia Horticulturae vol 115 no 3 pp 315ndash318 2008

[22] S Aras A Duran and G Yenilmez ldquoIsolation of DNA forRAPD analysis from dry leaf material of some Hesperis Lspecimensrdquo Plant Molecular Biology Reporter vol 21 no 4 pp461ndash462 2003

[23] P Taberlet L Gielly G Pautou and J Bouvet ldquoUniversalprimers for amplification of three non-coding regions of chloro-plast DNArdquo PlantMolecular Biology vol 17 no 5 pp 1105ndash11091991

[24] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[25] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 pp 2725ndash2729 2013

[26] W S Devi and G J Sharma ldquoIn vitro propagation of Arundi-naria callosa Munro-an edible bamboo from nodal explants ofmature plantsrdquoTheOpen Plant Science Journal vol 3 pp 35ndash392009

[27] S M S D Ramanayake V N Meemaduma and T EWeerawardene ldquoIn vitro shoot proliferation and enhancementof rooting for the large-scale propagation of yellow bamboo(Bambusa vulgaris ldquoStriatardquo)rdquo Scientia Horticulturae vol 110no 1 pp 109ndash113 2006

[28] W S Devi and G J Sharma ldquoIn vitro proliferation andenhancement of rooting inMelocanna bambusoides Trin fromnodal explantsrdquo Journal of Bamboo and Rattan vol 8 no 1-2pp 43ndash52 2009

[29] E A Oluk and A Cakir ldquoMicropropagation of Origanumsipyleum L an endemic medicinal herb of Turkeyrdquo AfricanJournal of Biotechnology vol 8 no 21 pp 5769ndash5772 2009

[30] M U Yildirim ldquoMicropropagation of Origanum acutidens(HANDMAZZ) IETSWAART using stem node explantsrdquoTheScientific World Journal vol 2013 Article ID 276464 3 pages2013

[31] S Islam and M M Rahman ldquoMicro-cloning in commerciallyimportant six bamboo species for mass propagation and at alarge scalerdquo Plant Tissue Culture and Biotechnology vol 15 no2 pp 103ndash111 2005

[32] R Yasodha S Kamala S P A Kumar P D Kumar and KKalaiarasi ldquoEffect of glucose on in vitro rooting ofmature plantsof Bambusa nutansrdquo Scientia Horticulturae vol 116 no 1 pp113ndash116 2008

[33] I D Arya P K Rana R Satsangi F S Muzaffar S Sharma andS Arya ldquoRapid and mass multiplication of bamboos throughtissue culture techniquesrdquo in Role of Plant Tissue Culture inBiodiversity Conservation and Economic Development S KNandi LM S Palni andA Kumar Eds pp 29ndash39 GyanodayaPrakashan Nainital India 2002

[34] M Das and A Pal ldquoIn vitro regeneration of Bambusa balcooaRoxb factors affecting changes of morphogenetic competencein the axillary budsrdquo Plant Cell Tissue and Organ Culture vol81 no 1 pp 109ndash112 2005

[35] C Nirmala A H Ali and T Badal ldquoDe novo organogenesisin the form of rhizome in Dendrocalamus asper and D mem-branaceusrdquo Current Science vol 100 no 4 pp 468ndash470 2011


[36] S Arya P K Rana R Sharma and I D Arya ldquoTissue culturetechnology for rapidmultiplication ofDendrocalamus giganteusMunrordquo Indian Forester vol 132 no 3 pp 345ndash357 2006

[37] A Ndiaye M S Diallo D Niang and Y K Gassama-Dia ldquoInvitro regeneration of adult trees of Bambusa vulgarisrdquo AfricanJournal of Biotechnology vol 5 no 13 pp 1245ndash1248 2006

[38] S M S D Ramanayake K M M N Maddegoda M CVitharana and G D G Chaturani ldquoRoot induction in threespecies of bamboo with different rooting abilitiesrdquo ScientiaHorticulturae vol 118 no 3 pp 270ndash273 2008

[39] K Tartoura A Da Rocha and S Youssef ldquoSynergistic interac-tion between coumarin 12-benzopyrone and indole-3-butyricacid in stimulating adventitious root formation in Vigna radiata(L) Wilczek cuttings I Endogenous free and conjugated IAAand basic isoperoxidasesrdquo Plant Growth Regulation vol 42 no3 pp 253ndash262 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Zoology


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Volume 2014

Stem CellsInternational



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Microbiology


zygotic embryos protocols in bamboo plantlets regenerationis that a given protocol is species specific and consequentlydoes not often apply to other genera Secondly the long andirregular bamboo flowering cycle [16 17] as well as low seedviability and scarcity in bamboo seeds [18 19] often limitszygotic embryo protocols for bamboo plantlet regenerationTherefore new accurate protocols are required for successfulpropagation of several species since nodal explants are readilyavailable

B tulda and M baccifera are sympodial species whichproduce nutritive shoots [3 20] Protocols for plantletsregeneration were developed for B tulda through seeds [11]and nodal explants [21] without achieving rhizogenesis Tilldate there is no protocol for plantlets regeneration for Mbaccifera Plantlets regeneration protocols in bamboo gen-erally suffer from poor taxonomic identification and clonalfidelity for the reported species Because of these setbacksprotocols reported on several bamboo species are frequentlynot reproducible In this study we first used trnL-F intergenicspacer to accurately ascertain the taxonomic placement of thestudied species Secondly we developed an effective protocolfor in vitro shoot proliferation from the nodal buds andoptimized rooting of B tulda andM baccifera

2 Material and Methods

21 Plant Material and Phylogenetic Placement Bambusatulda andMelocanna baccifera were identified by the author-ities of the Botanical Survey of India (BSI) Kolkata Thelateral branches of the authenticated specimen were collectedfrom culms of B tulda and M baccifera which lies 23∘471015840ndash25∘411015840 NL and 92∘581015840ndash94∘471015840 EL from the Forest Departmentof Imphal District Manipur India during the month ofJuly 2011 Genomic DNA was isolated as described [22] Thequantity and quality of DNA were checked on a nanodropspectrophotometer (BioSpec-nano Shimadzu Japan) and08 wv agarose gel electrophoresis respectively The trnL-F intergenic spacer was amplified as previously described[23] and sequenced Sequences were analysed and assignedto molecular species based on 98ndash100 sequence similarityin the GenBank as accessions JX507132 (Bambusa tulda)and JX507133 (Melocanna baccifera) respectively Sequenceswere aligned usingMuscle program [24] and best substitutionmodel was determined based on Akaike Information Crite-rion corrected (AICc) and Bayesian Information Criterion(BIC) Phylogenetic analysis was performed using the maxi-mum likelihood method in MEGA6 software [25]

22 Explants and Surface Sterilization The leaf sheaths cov-ering the nodal segments containing axillary buds (15ndash2 cm)were carefully removed and wiped with 70 (vv) ethanolusing sterilized cotton The explants were surface sterilizedin 01 (vv) solution ofmercuric chloride (HgCl

2) for 15min

andwashed 4 times with eachwashing step lasting 5minThesterilized nodal segments were cultured in liquid Murashige-Skoog (MS) medium supplemented with 100mgL myo-inositol (Sigma Saint Louis MO USA) and 30 gL sucroseIt is worth noting that the pH of the medium was adjusted to

57plusmn01with 1NNaOHor 1NHCl before autoclaving at 121∘Cand 11768 kPa for 20min For bud breaking the MS mediumwas supplemented with 6-benzylaminopurine (BAP) at 10levels (1 2 3 4 5 6 7 8 9 and 10mgL)

23 Shoots Multiplication Proliferated axillary shoots of 3ndash5 shoot clusters were excised and transferred to MS mediumsupplemented either with (1) BAP at 5 levels (1 2 3 4 and5mgL) (2) 6mgL kinetin (Kn) singly or (3) combination ofKn at levels (1 2 and 4mgL) with optimum concentrationof 3mgL of BAP It is worth mentioning that the choice of6mgL of Kn at one level was based on preliminary findingwhere best effect was observed (data not shown) Shootmultiplication was performed using 3ndash5 axillary proliferatedshoots excised from the clusters of shoots Multiplied shootsin liquid cultures were supported by sterile filter paperbridges Regularly the shoots were transferred at intervalof 10 days under sterile conditions to fresh medium inorder to prevent phenolic oxidation that can trigger theshoots to become yellowish The multiplied shoots on eachculture medium were counted after 45 days to evaluate themultiplication rate of the nodal explants

24 Rooting Process We used 5 levels (1 2 3 4 and 5)of sucrose in half-strength MS medium to determine theoptimum concentration that induces rooting Furthermorerooting was achieved by aseptically transferring multipliedshoots to a half-strength MS medium prepared with 3sucrose (ie optimum concentration) and supplementedwith different combinations of growth regulators as followsFor B tulda we used (1) 3mgL of indole-3-butyric acid(IBA) (2) 3mgL of IBA with 10mgL of coumarin and(3) 3mgL of IBA 3mgL of indole-3-acetic acid (IAA)and 10mgL of coumarin For M baccifera we used (1)3mgL of IBA (2) 3mgL of IBA with 10mgL of coumarinand (3) 3mgL of IBA 005mgL of BAP and 10mgL ofcoumarin All cultures in the experiment were incubated ina growth chamber at 25 plusmn 1∘C with a 16 h photoperiod atlight intensity of 45120583molm2sec photosynthetic photon flux(PPF) provided by cool white fluorescent tubes (TLD CoolWhite 40W Phillips India) Rooting percentage and averagenumber of days for rootingwere recorded Following rootingMSmediumwas removed and the plantlets were hardened ina bottle jar of 76mm height times 60mm diameter and 143mLcap containing autoclaved soil The soil was made of 4 1( ww) rice-straw-vermin compose and sand The plantletswere cultured for 25 days Acclimatization was achieved at30 plusmn 2

∘C and at 84 humidity in the greenhouse

25 Clonal Fidelity Test We checked clonal fidelity by se-quencing the trnL-F intergenic spacer of in vitro raisedplantlets and compared the sequences with the motherexplants

26 Statistical Analysis The experiments were performed inreplicates for a total of 3 biological repeats carried out atseparate times All data on shoot regeneration and in vitrorooting were analysed using one-way ANOVA associated





65

100

01

JX564907 Bambusa sp

JX564906 Bambusa sp







Growthhormones

Concentration(mgL)


explant


BAP

1 833aplusmn 182 250

abplusmn 067

2 1400abcplusmn 180 433

abplusmn 067

3 1683cplusmn 048 533

bplusmn 021

4 1650bcplusmn 092 517

abplusmn 031

5 1183abcplusmn 194 350

abplusmn 067

Kn 6 750aplusmn 231 217

aplusmn 083

Kn + BAP1 + 3 1383

abcplusmn 281 417

abplusmn 101

2 + 3 1767cplusmn 056 550

bplusmn 022

4 + 3 1367abcplusmn 232 417

abplusmn 087





0

10

20

30

40

50

60

70

80


Bud

brea

k (

)

Control 1 2 3 4 5 6 7 10

P le 005

y = minus25082x2 + 27218x

R2 = minus0161

B tulda


(a)

y = minus244942x2 + 26148x

P le 005

0

10

20

30

40

50

60

70

80

Bud

brea

k (

)



R2 = 01621


(b)





Growthhormones

Concentration(mgL)


explant


BAP

1 850aplusmn 180 267

aplusmn 061

2 1417abplusmn 196 450

abplusmn 067

3 1700bplusmn 044 533

bplusmn 021

4 1667bplusmn 095 50

bplusmn 044

5 1200abplusmn 214 367

abplusmn 076

Kn 6 1583bplusmn 128 483

abplusmn 048

Kn + BAP1 + 3 1717

bplusmn 087 550

bplusmn 034

2 + 3 1817bplusmn 031 583

bplusmn 017

4 + 3 1583bplusmn 114 483

abplusmn 048





(a) (b) (c) (d)


(a) (b) (c) (d)







IBA 3 4500aplusmn 494 7600

abplusmn 403


aplusmn 783


abplusmn 483


aplusmn 756 4083

abplusmn 271


abplusmn 342


abplusmn 207


0102030405060708090

100

1 2 3 4 5

Root

ing

()


y = minus78683x2 + 524x

R2 = 07368

P le 005

(a)

y = minus7778x2 + 52452x

R2 = 08705

P le 005

0102030405060708090

100

Root

ing

()


(b)



4 Conclusion




Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





65

100

01

JX564907 Bambusa sp

JX564906 Bambusa sp







Growthhormones

Concentration(mgL)


explant


BAP

1 833aplusmn 182 250

abplusmn 067

2 1400abcplusmn 180 433

abplusmn 067

3 1683cplusmn 048 533

bplusmn 021

4 1650bcplusmn 092 517

abplusmn 031

5 1183abcplusmn 194 350

abplusmn 067

Kn 6 750aplusmn 231 217

aplusmn 083

Kn + BAP1 + 3 1383

abcplusmn 281 417

abplusmn 101

2 + 3 1767cplusmn 056 550

bplusmn 022

4 + 3 1367abcplusmn 232 417

abplusmn 087





0

10

20

30

40

50

60

70

80


Bud

brea

k (

)

Control 1 2 3 4 5 6 7 10

P le 005

y = minus25082x2 + 27218x

R2 = minus0161

B tulda


(a)

y = minus244942x2 + 26148x

P le 005

0

10

20

30

40

50

60

70

80

Bud

brea

k (

)



R2 = 01621


(b)





Growthhormones

Concentration(mgL)


explant


BAP

1 850aplusmn 180 267

aplusmn 061

2 1417abplusmn 196 450

abplusmn 067

3 1700bplusmn 044 533

bplusmn 021

4 1667bplusmn 095 50

bplusmn 044

5 1200abplusmn 214 367

abplusmn 076

Kn 6 1583bplusmn 128 483

abplusmn 048

Kn + BAP1 + 3 1717

bplusmn 087 550

bplusmn 034

2 + 3 1817bplusmn 031 583

bplusmn 017

4 + 3 1583bplusmn 114 483

abplusmn 048





(a) (b) (c) (d)


(a) (b) (c) (d)







IBA 3 4500aplusmn 494 7600

abplusmn 403


aplusmn 783


abplusmn 483


aplusmn 756 4083

abplusmn 271


abplusmn 342


abplusmn 207


0102030405060708090

100

1 2 3 4 5

Root

ing

()


y = minus78683x2 + 524x

R2 = 07368

P le 005

(a)

y = minus7778x2 + 52452x

R2 = 08705

P le 005

0102030405060708090

100

Root

ing

()


(b)



4 Conclusion




Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

10

20

30

40

50

60

70

80


Bud

brea

k (

)

Control 1 2 3 4 5 6 7 10

P le 005

y = minus25082x2 + 27218x

R2 = minus0161

B tulda


(a)

y = minus244942x2 + 26148x

P le 005

0

10

20

30

40

50

60

70

80

Bud

brea

k (

)



R2 = 01621


(b)





Growthhormones

Concentration(mgL)


explant


BAP

1 850aplusmn 180 267

aplusmn 061

2 1417abplusmn 196 450

abplusmn 067

3 1700bplusmn 044 533

bplusmn 021

4 1667bplusmn 095 50

bplusmn 044

5 1200abplusmn 214 367

abplusmn 076

Kn 6 1583bplusmn 128 483

abplusmn 048

Kn + BAP1 + 3 1717

bplusmn 087 550

bplusmn 034

2 + 3 1817bplusmn 031 583

bplusmn 017

4 + 3 1583bplusmn 114 483

abplusmn 048





(a) (b) (c) (d)


(a) (b) (c) (d)







IBA 3 4500aplusmn 494 7600

abplusmn 403


aplusmn 783


abplusmn 483


aplusmn 756 4083

abplusmn 271


abplusmn 342


abplusmn 207


0102030405060708090

100

1 2 3 4 5

Root

ing

()


y = minus78683x2 + 524x

R2 = 07368

P le 005

(a)

y = minus7778x2 + 52452x

R2 = 08705

P le 005

0102030405060708090

100

Root

ing

()


(b)



4 Conclusion




Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c) (d)


(a) (b) (c) (d)







IBA 3 4500aplusmn 494 7600

abplusmn 403


aplusmn 783


abplusmn 483


aplusmn 756 4083

abplusmn 271


abplusmn 342


abplusmn 207


0102030405060708090

100

1 2 3 4 5

Root

ing

()


y = minus78683x2 + 524x

R2 = 07368

P le 005

(a)

y = minus7778x2 + 52452x

R2 = 08705

P le 005

0102030405060708090

100

Root

ing

()


(b)



4 Conclusion




Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




IBA 3 4500aplusmn 494 7600

abplusmn 403


aplusmn 783


abplusmn 483


aplusmn 756 4083

abplusmn 271


abplusmn 342


abplusmn 207


0102030405060708090

100

1 2 3 4 5

Root

ing

()


y = minus78683x2 + 524x

R2 = 07368

P le 005

(a)

y = minus7778x2 + 52452x

R2 = 08705

P le 005

0102030405060708090

100

Root

ing

()


(b)



4 Conclusion




Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

) through nodal culture - hindawi publishing...

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