Cytokinin influence on in vitro shoot induction and genetic stability assessment of Dendrocalamus latiflorus Munro: a commercially important bamboo in Manipur, North-East India

, , , ,

Research Articles | Published:

Print ISSN : 0970-4078.
Online ISSN : 2229-4473.
Pub Email:
Doi: 10.1007/s42535-022-00392-5
First Page: 1085
Last Page: 1095
Views: 414

Keywords: DNA markers, Somaclonal variation, Monomorphism, Cytokinins, Genetic stability


In vitro propagation of Dendrocalamus latiflorus is another alternative method to natural propagation which is limited by poor seed production and irregular and prolonged flowering cycles. The effect of BAP (6-benzylaminopurine) and KN (kinetin) was tested for bud break, shoot initiation, and multiplication during in vitro stage. Successful bud break and higher shoot formation were observed at the elevated level of KN (8.0 mg/L) or BAP (8.0 mg/L). Multiple marker systems were employed to determine genetic variation that could appear at the shooting stage due to high cytokinin concentration. A high level of monomorphism was detected among the in vitro clones with inter simple sequence repeats (ISSR) and start codon targeted polymorphism (SCoT) markers detecting 98.4% and 98% monomorphism, respectively. The inter-primer binding site (iPBS) and CAAT box-derived polymorphism (CBDP) markers also recorded 100% monomorphism. Nei’s genetic distance from the pooled ISSR, SCoT, CBDP and iPBS data showed close genetic distance values ranging from 0.000 to 0.006, disclosing high genetic identity among the genotypes. The Unweighted Pair Group Method with Arithmetic Mean (UPGMA) based dendrogram generated from combined marker data revealed a closed genetic association of the mother plant (MP) and in vitro clones (R1-R7). Principal coordinate analysis (PCoA) further confirmed the grouping of the genotypes as per the dendrogram cluster. The high monomorphism (99.1%) and close clustering patterns between the genotypes indicated the maintenance of high genetic stability at the shooting stage despite elevated levels of BAP and KN in the medium.

DNA markers, Somaclonal variation, Monomorphism, Cytokinins, Genetic stability

*Get Access

(*Only SPR Members can get full access. Click Here to Apply and get access)



Agnihotri RK, Nandi SK (2009) In vitro shoot cut: a high frequency multiplication and rooting method in the bamboo Dendrocalamus hamiltonii. Biotech 8:259–263

Amom T, Nongdam P (2017) The use of molecular marker methods in plants: a review. Int J Cur Res Rev 9:1–7

Amom T, Tikendra L, Apana N, Goutam M, Sonia P, Koijam AS, Potshangbam AM, Rahaman H, Nongdam P (2020) Efficiency of RAPD, ISSR, iPBS, SCoT and phytochemical markers in the genetic relationship study of five native and economical important bamboos of North-East India. Phytochemistry.

Anand M, Brar J, Sood A (2013) In vitro propagation of an edible bamboo Bambusa bambos and assessment of clonal fidelity through molecular markers. J Med Bioeng 2:257–261.

Arya S, Sharma S (1998) Micropropagation technology of Bambusa bambos through shoot proliferation. Indian Forester 124(9):725–731

Banerjee M, Gantait S, Pramanik BR (2011) A two step method for accelerated mass propagation of Dendrocalamus asper and their evaluation in field. Physiol Mol Biol Plants 17:387.

Bisht P, Pant M, Kant A (2010) In vitro propagation of Gigantochloa atroviolaceae Widjaja through nodal explants. J Am Sci 6:1019–1026

Biswas P, Nandy S, Dey A, Tikendra L, Nongdam P (2022) Molecular markers in assessing genetic clonal fidelity for in vitro propagated endangered medicinal plants. In: Kumar A, Choudhury B, Dayanandan S, Khan ML (eds) Molecular genetics and genomics tools in biodiversity conservation. Springer, Singapore.

Bornet B, Branchard M (2001) Non-anchored inter simple sequence repeat (ISSR) markers: reproducible and specific tools for genome fingerprinting. Plant Mol Biol Rep 19:209–215

Cichorz S, Gośka M, Mańkowski DR (2018) Miscanthus × giganteus: regeneration system with assessment of genetic and epigenetic stability in long-term in vitro culture. Ind Crop Prod 116:150–161

Collard BC, Mackill DJ (2009) Start codon targeted (SCoT) polymorphism: a simple, novel DNA marker technique for generating gene-targeted markers in plants. Plan Mol Biol Rep 27:86–93

Damasco OP, Graham GC, Henry RJ, Adkins SW, Smiths MK, Godwin ID (1996) Random amplified polymorphic DNA (RAPD) detection of dwarf off-types in micropropagated Cavendish (Musa spp. AAA) bananas. Plant Cell Rep 16(1):118–123

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

Devi WS, Sharma GJ (2009a) In vitro propagation of Arundinaria callosa Munro—an edible bamboo from nodal explants of mature plants. TOPSJ 3:35–39

Devi SW, Sharma GJ (2009b) In vitro shoot proliferation and enhancement of rooting in Melocanna babusoides Trin. from nodal explants. J Bamboo Ratt 8:43–52

Dey A, Nandy S, Nongdam P, Tikendra L, Mukherjee A, Mukherjee S, Pandey DK (2020) Methyl jasmonate and salicylic acid elicit indole alkaloid production and modulate antioxidant defence and biocidal properties in Rauvolfia serpentina Benth. ex Kurz. in vitro cultures. S Afr J Bot 135:1–17.

Dey A, Nongdam P, Nandy S, Mukherjee S, Mukherjee A, Tikendra L, Hazra AK, Pandey DK (2021) Polyamine elicited aristolochic acid production in in vitro clonally fidel Aristolochia indica L.: an ISSR and RAPD markers and HPTLC based study. S Afr J Bot 140:326–335

Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus (madison).

FAO (2020) Global forest resources assessment 2020. Rome, Italy.

Gao X, Yang D, Cao D et al (2010) In vitro micropropagation of freesia hybrida and the assessment of genetic and epigenetic stability in regenerated plantlets. J Plant Growth Regul 29:257–267.

George PS, Ravishankar GA, Venkataraman LV (1993) Clonal multiplication of Gardenia jasminoides Ellis through axillary bud culture. Plant Cell Rep 13:59–62.

Godbole S, Sood A, Thakur R, Sharma M, Ahuja PS (2002) Somatic embryogenesis and its conversion into plantlets in a multipurpose bamboo, Dendrocalamus hamiltonii Nees et Arn. Ex Munro Curr Sci 83(7):885–889

Goyal AK, Pradhan S, Basistha BC, Sen A (2015) Micropropagation and assessment of genetic fidelity of Dendrocalamus strictus (Roxb.) nees using RAPD and ISSR markers. 3 Biotech 5:473–482

Haisel D, Hofman P, Vágner M, Lipavská H, Tichá I, Schäfer C, Čapková V (2001) Ex vitro phenotype stability is affected by in vitro cultivation. Biol Plant 44:321–324.

Hsu YH, Annamalai AP, Lin CS, Chen YY, Chang WC, Lin NS (2000) A sensitive method for detecting bamboo mosaic virus (BaMV) and establishment of BaMV-free meristem tip cultures. Plant Path 49:101–107

Kalendar R, Antonius K, Smýkal P, Schulman AH (2010) iPBS: a universal method for DNA fingerprinting and retrotransposon isolation. Theor Appl Genet 121:1419–1430

Khare SR, Kharate PS, Sahu RK, Jha Z (2021) The rapid in-vitro micropropagation of bamboo (Dendrocalamus strictus) and its genetic fidelity testing using ISSR markers. Environ Conserv J 22(3):69–77.

Krishna H, Alizadeh M, Singh D, Udayvir S, Chauhan N, Eftekhari M, Sadh RK (2016) Somaclonal variations and their applications in horticultural crops improvement. 3 Biotech 6:54.

Lakshmanan V, Venkataramareddy SR, Neelwarne B (2007) Molecular analysis of genetic stability in long-term micropropagated shoots of banana using RAPD and ISSR markers. Electron J Biotechnol.

Liu M, Qiao G, Jiang J, Yang H, Xie L, Xie J, Zhuo R (2012) Transcriptome sequencing and de novo analysis for Ma bamboo (Dendrocalamus latiflorus Munro) using the illumine platform. PLoS ONE 7(10):e46766.

Mamgain J, Mujib A, Ejaz B, Gulzar B, Malik MQ, Syeed R (2022) Flow cytometry and stsrt codon targeted (SCoT) genetic fidelity assessment of regenerated plantlets in Tylophora indica (Burm.f.) Merrill. Plant Cell Tissue Organ Cult 28:1–12.

Mok DW, Mok MC (2001) Cytokinin metabolism and action. Annu Rev Plant Physiol Plant Mol Biol 52:89–118.

Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol Plant 15:473–497

Mustafa AA, Derise MR, Yong WTL, Rodrigues KF (2021) A concise review of Dendrocalamus asper and related bamboos: germplasm conservation, propagation and molecular biology. Plants 10(9):1897.

Nadgauda RS, Parasharami VA, Mascarenhas AF (1990) Precocious flowering and seeding behaviour of tissue cultured bamboos. Nature 344:335-336G

Nadha HK, Rahul R, Sharma RK, Anand M, Sood A (2011) Evaluation of clonal fidelity of in vitro raised plants of Guadua angustfolia Kunth using DNA-based markers. J Med Plant Res 5(23):5636–6564

Negi D, Saxena S (2011) In vitro propagation of Bambusa nutans Wall. Ex Munro through axillary shoot proliferation. Plant Biotech Res 5:35–43

Nongdam P, Tikendra L (2014) The nutritional facts of bamboo shoots and their usage as important traditional foods of Northeast India. Int Scholarly Res Notices.

Nongdam P (2015) Traditional fermented bamboo shoot foods of North-East India and their characteristic natural microbial flora. In: 10th World Bamboo Congress, Korea. Available at:,%20Potshangbam.pdf

Peakall R, Smouse PE (2012) GenAlEx 6.5: genetic analysis in excel. Population genetic software for teaching and research—an update. Bioinformatics 28:2537–2539.

Premlata T, Sharma V, Bisht MS, Nirmala C (2020) Edible bamboo resources of Manipur: consumption pattern of young shoots, processing techniques and their commercial status in the local market. Indian J Tradit Knowl 19(1):73–82

Purayil FT, Kurup SS, Alkhaili MMS, Tawfik NH, Dhaheri SM, Cheruth AJ, Dhaheri SA, Subramaniam S (2018) ISSR-assisted analysis of clonal fidelity supported with SEM and histology using in vitro propagated plants of Moringa peregrine (Forssk.) iori—an endangered desert tree. S Afr J Bot 114:163–170

Rajkumari M, Gupta A (2013) A note on the bamboo diversity and utilization from Manipur in N.E. India. Pleione 7(2):449–455

Ramanayake SMSD, Meemaduma VN, Weerawardene TE (2006) In vitro shoot proliferation and enhancement of rooting for the large-scale propagation of yellow bamboo (Bambusa vulgaris ‘Striata’). Sci Hortic 110:109–113.

Rodrigues PHV, Tulmann Neto A, Cassieri Neto P, Mendes BMJ (1998) Influence of the number of subcultures on somaclonal variation in micropropagated Nanico {Musa spp., AAA group). Acta Hort 490:469–473

Rout GR, Senapati SK, Aparajita S, Palai SK (2009) Studies on genetic identification and genetic fidelity of banana cultivars using ISSR marker. Plant Omics J 2:250–258

Sandhu M, Wani S, Jiménez V (2018) In vitro propagation of bamboo species through axillary shoot proliferation: a review. Plant Cell Tissue Organ Cult.

Schaller GE, Street IH, Kieber JJ (2014) Cytokinin and the cell cycle. Curr Opin Plant Biol 21:7–15.

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

Singh SR, Dalai S, Singh R, Dhawan AK, Kalia RK (2012) Seasonal influences on in vitro bud break in Dendrocalamus hamiltonii Arn. ex Munro nodal explants and effect of culture microenvironment on large scale shoot multiplication and plantlet regeneration. Indian J Plant Physiol 17:9–21

Singh SR, Dalai S, Singh R, Dhawan AK, Kalia RK (2013) Ascertaining clonal fidelity of micropropagated plants of Dendrocalamus hamiltonii Nees et Arn.ex Munro using molecular markers. In Vitro Cell Dev Biol-Plant 49:572–583

Singh AK, Rana MK, Singh S, Kumar S, Kumar R, Singh R (2014) CAAT box-derived polymorphism (CBDP): a novel promoter-targeted molecular marker for plants. J Plant Biochem Biotechnol 23:175–183.

Singh MK, Ibrahim M, Bordoloi S, Meena D, Kakoti S (2018) Bamboo diversity, distribution and utility in forest fringe villages of Manipur (India). Pharma Innov 7(4):503–507

Smith MK (1988) A review of factors influencing the genetic stability of micropropagated bananas. Fruits 43:219–223

Suwal MM, Lamichhane J, Gauchan DP (2021) Assessment of genetic stability of micropropagated Bambusa balcooa Roxb. using RAPD marker. Plant Tissue Cult Biotech 31(1):89–95

Tikendra L, Amom T, Nongdam P (2019) Molecular genetic homogeneity assessment of micropropagated Dendrobium moschatum Sw.—a rare medicinal orchid, using RAPD and ISSR markers. Plant Gene.

Tikendra L, Choudhary R, Sanayaima Devi R, Dey A, Potshangbam AM, Nongdam P (2021a) Micropropagation of bamboos and clonal fidelity assessment using molecular markers. In: Ahmad Z, Ding Y, Shahzad A (eds) Biotechnological advances in bamboo. Springer, Singapore.

Tikendra L, Potshangbam AM, Dey A, Dev TR, Sahoo MR, Nongdam P (2021b) RAPD, ISSR, and SCoT markers based genetic stability assessment of micropropagated Dendrobium fimbriatum Lindl. var. oculatum Hk. f.—an important endangered orchid. Physiol Mol Biol Plants 27:341–357.

Tikendra L, Apana N, Potshangbam AM, Amom T, Choudhary R, Sanayaima R, Dey A, Nongdam P (2021c) Dendrobium sp.: in vitro propagation of genetically stable plants and ethno-medicinal uses. In: Mérillon JM, Kodja H (eds) Orchids phytochemistry, biology and horticulture, reference series in phytochemistry. Springer Nature, Switzerland.

Tikendra L, Sushma O, Amom T, Devi NA, Paonam S, Bidyananda N et al (2022) Genetic clonal fidelity assessment of rhizome-derived micropropagated Acorus calamus L.—a medicinally important plant by random amplified polymorphic DNA and inter-simple sequence repeat markers. Phcog Mag 18:207–215

Waikhom SD, Louis B (2014) An effective protocol for micropropagation of edible bamboo species (Bambusa tulda and Melocanna baccifera) through nodal culture. Sci World J.

Wu FH, Kan DP, Lee SB, Daniell H, Lee YW, Lin CC, Lin NS, Lin CS (2009) Complete nucleotide sequence of Dendrocalamus latiflorus and Bambusa oldhamii chloroplast genomes. Tree Physiol 29(6):847–856

Yew CK, Balakrishnan B, Sundasekaran J, Subramaniam S (2010) The effect of cytokinins on in vitro shoot length and multiplication of Hymenocallis littoralis. J Med Plant Res.

Zhang N, Fang W, Shi Y, Liu Q, Yang H, Gui R, Lin X (2010) Somatic embryogenesis and organogenesis in Dendrocalamus hamiltonii. Plant Cell Tissue Organ Cult 103:325–332



The authors would like to thank the SERB, Department of Science and Technology (DST), New Delhi, India, for providing financial assistance for the research work.

Author Information

Tikendra Leimapokpam
Department of Biotechnology, Manipur University, Canchipur, India

Dey Abhijit
Department of Life Sciences, Presidency University, Kolkata, India

Jamir Imlitoshi
Department of Biotechnology, Nagaland University, Dimapur, India

Sahoo Manas Ranjan
ICAR-Indian Institute of Horticultural Research (ICAR-IIHR), Bhubaneswar, India

Nongdam Potshangbam
Department of Biotechnology, Manipur University, Canchipur, India