Abstract
Dendrocalamus hamiltonii is a giant, evergreen, clumping, multipurpose bamboo with strong culms which are mainly used for construction, handicrafts and fuel. The tender shoots are also used as food. Overexploitation of existing natural stocks coupled with harvesting of culms before seed formation, a long flowering cycle, irregular and poor seed production, short seed viability, seed sterility, limited availability of offsets and rhizomes and seasonal dependence are some of the major bottlenecks in conventional propagation of this species. Therefore, alternative methods like micropropagation can fill the gap in demand and supply of true-to-type planting material. Recently, our micropropagation protocol for rapid multiplication of D. hamiltonii through axillary bud proliferation using nodal explants from mature culms was standardized, and more than 3,000 plants were transferred to the field. However, somaclonal variations are known to appear in the in vitro-derived clones due to culture-induced stresses. Therefore, the present investigation was conducted to ascertain the effect of the length of in vitro culture age on clonal fidelity of regenerated plants using random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. The genomic DNA samples (i.e. mother plant, in vitro-raised shoots from the 3rd to 30th passage, and in vitro-raised plants transferred to the field) were subjected to PCR amplification using 90 primer combinations (25 each of RAPD, ISSR and SSR, and 15 AFLP primer combinations) of which 76 (23 RAPD, 24 ISSR, 21 SSR and 8 AFLP) markers showed amplified DNA fragments. The 23 RAPD primers produced 162 distinct amplified DNA fragments from 2 (OPE-5) to 16 (OPE-16) fragments per primer, while 24 ISSR primers produced 181 distinct amplified DNA fragments with an average of 7.5 fragments per primer. The number of bands generated by SSR primers varied from 3 (RM-7 and RM-240) to 14 (RM-44), and the eight combinations of AFLP primers produced 369 distinct and scorable amplified DNA fragments with an average of 46.1 fragments per primer. Appearance of monomorphic bands with all the tested primer combinations confirmed the true-to-type nature of the in vitro clones of D. hamiltonii and hence the suitability of the developed micropropagation protocol for commercial-scale plant production.
Similar content being viewed by others
References
Agnihotri RK, Mishra J, Nandi SK (2009) Improved in vitro shoot multiplication and rooting of Dendrocalamus hamiltonii Nees et Arn. Ex Munro: production of genetically uniform plants and field evaluation. Acta Physiol Plant 31:961–967
Agnihotri RK, Nandi SK (2009) In vitro shoot cut: a high frequency multiplication and rooting method in bamboo Dendrocalamus hamiltonii. Biotechnol 8:259–263
Bisht P, Pant M, Kant A (2010) In vitro propagation of Gigantochloa atroviolaceae Widjaja through nodal explants. J Am Sci 6:1019–1026
Chambers SM, Heuch JHR, Pirrie A (1991) Micropropagation and in vitro flowering of the bamboo Dendrocalamus hamiltonii Munro. Plant Cell Tiss Organ Cult 27:45–48
Chauhan SK, Sharma PK, Moorthi TV (1992) Studies on wooing wastelands for environmental protection and economical biomass productivity with bamboos Dendrocalamus hamiltonii in Western Himalayas—some projections. Indian J Forest 15:20–130
Cuesta C, Ordas RJ, Rodriguez A, Fernandez B (2010) PCR-based molecular markers for assessment of somaclonal variation in Pinus pinea clones micropropagated in vitro. Biol Plant 54:435–442
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 Org Cult 81:109–112
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 83:885–889
Goto S, Thakur RC, Ishii K (1998) Determination of genetic stability in long-term micropropagated shoots of Pinus thunbergii Parl. using RAPD markers. Plant Cell Rep 18:193–197
Huang LC, Huang BL (1995) Loss of the species distinguishing trait among regenerated Bambusa ventricosa McClure plants. Plant Cell Tiss Org Cult 42:109–111
Jin W-M, Dong J, Wang Y-H, Mao H-L, Xiao Z, Chen M-X (2009) Genetic fidelity of regenerated adventitious shoots in grapes through organogenesis. Mol Plant Breed 2:375–379 (in Chinese)
Joshi P, Dhawan V (2007) Assessment of genetic fidelity of micropropagated Swertia chirayita plantlets by ISSR marker assay. Biol Plant 51:22–26
Kabir MF, Bhattacharjee DK, Sattar MA (1993) Use of bamboo skin in the cottage industry. BIC-India Bulletin 3:49
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 10:106–113
Leroy XJ, Leon K, Charles G, Branchard M (2000) Cauliflower somatic embryogenesis and analysis of regenerant stability by ISSRs. Plant Cell Rep 19:1102–1107
Lodhi MA, Ye GN, Weeden NF, Reisch BI (1994) A simple and efficient method of DNA extraction from grapevine cultivars and Vitis species. Plant Mol Biol Rep 12:6–13
Martin M, Sarmento D, Oliveira MM (2004) Genetic stability of micropropagated almond plantlets as assessed by RAPD and ISSR markers. Plant Cell Rep 23:492–496
Marulanda ML, Lopez AM, Claroz JL (2007) Analyzing the genetic diversity of Guadua spp. in Colombia using rice and sugarcane microsatellites. Crop Breed Appl Biotechnol 7:43–51
McGregor CE, Lambert CA, Greyling MM, Louw JH, Warnich L (2000) A comparative assessment of DNA fingerprinting techniques (RAPD, ISSR, AFLP and SSR) in tetraploid potato (Solanum tuberosum L.) germplasm. Euphytica 113:135–144
Mehta R, Sharma V, Sood A, Sharma M, Sharma RK (2011) Induction of somatic embryogenesis and analysis of genetic fidelity of in vitro-derived plantlets of Bambusa nutans Wall., using AFLP markers. Eur J Forest Res 130:729–736
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol Plant 15:473–97
Nadha HK, Kumar R, Sharma RK, Anand M, Sood A (2011) Evaluation of clonal fidelity of in vitro raised plants of Guadua andustifolia Kunth using DNA-based markers. J Med Plants Res 5:563–5641
Negi D, Saxena S (2010) Ascertaining clonal fidelity of tissue culture raised plants of Bambusa balcooa Roxb. using inter simple sequence repeat markers. New Forests 40:1–8
Negi D, Saxena S (2011) In vitro propagation of Bambusa nutans Wall. ex Munro through axillary shoot proliferation. Plant Biotechnol Rep 5:35–43
Panda MK, Mohanty S, Subudhi E, Acharya L, Nayak S (2007) Assessment of genetic stability of micropropagated plants of Curcuma longa L. by cytophotometry and RAPD analyses. Int J Integr Biol 1:189–195
Pandey RN, Singh SP, Rastogi J, Sharma ML, Singh RK (2012) Early assessment of genetic fidelity in sugarcane (Saccharum officinarum) plantlets regenerated through direct organogenesis with RAPD and SSR markers. Aust J Crop Sci 6:618–624
Peschke VM, Phillips RL (1992) Genetic implications of somaclonal variation in plants. Adv Genet 30:41–75
Phillips RL, Kaeppler SM, Olhoft P (1994) Genetic instability of plant tissue cultures: breakdown of normal controls. Proc Natl Acad Sci USA 91:5222–5226
Ramanayake SMSD, Yakandawala K, Nilmini Deepika PKD, Ikbal MCM (1995) Studies on micropropagation of Dendrocalamus gigateus and Bambusa vulgaris var. striata. In: Bamboo, people and the environment, vol. 1. Propagation and management. INBAR Tech Rep 8:75−85
Rani V, Raina SN (2000) Genetic fidelity of organized meristem derived micropropagated plants: a critical reappraisal. In Vitro Cell Dev Biol Plant 36:319–330
Rao KS, Ramakrishnan PS (1988) Architectural plasticity of two bamboo species, Neohouzeua dullooa Camus and Dendrocalamus hamiltonii Nees in successional environment in North-East India. Proc Indian Acad Sci (Plant Sci) 98:121–133
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
Rohlf FJ (2000) NTSYS: Numerical Taxonomy and Multivariate Analysis System. Version 2.0 Exeter Software, Setauket, New York, USA
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
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Saxena S, Dhawan V (1994) “Micropropagation research in South Asia”. Constraints to production of bamboo and rattan. INBAR Tech Rep 5:101–113
Singh SR, Dalal S, Singh R, Dhawan AK, Kalia RK (2012a) 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, Dalal S, Singh R, Dhawan AK, Kalia RK (2012b) Micropropagation of Dendrocalamus asper Schult. & Schult. F. Backer ex k. Heyne: an exotic edible bamboo. J Plant Biochem Biotechnol 21:220–228
Sood A, Ahuja PS, Sharma M, Sharma OP, Godbole S (2002) In vitro protocols and field performance of elites of an important bamboo Dendrocalamus hamiltonii Nees et Arn. Ex Munro. Plant Cell Tiss Org Cult 71:55–63
Uppin SF (1980) High yielding bamboos for artificial regeneration. Myforest 16:55–62
Venkatachalam L, Sreedhar RV, Bhagyalakshmi N (2007) Micropropagation in banana using high levels of cytokinins does not involve any genetic changes as revealed by RAPD and ISSR markers. Plant Growth Regul 51:193–205
Vos P, Hogers R, Bleeker M, Reijans M, Vandelee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995) AFLP—a new technique for DNA-fingerprinting. Nucleic Acids Res 23:4407–4414
Yadav S, Patel P, Shirin F, Mishra Y, Ansari SA (2008) In vitro clonal propagation of 10-year-old clumps of Bambusa nutans. J Bamboo Rattan 7:201–210
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
Acknowledgments
Our sincere thanks to the Director (Technical) CPB and Chairman, Department of Bio- and Nanotechnology, GJUST, for providing the laboratory facilities. The Forest Department, Yamuna Nagar (Haryana), is acknowledged for maintenance of tissue culture-raised plants in the field. The financial support extended to SRS and RS by the Department of Biotechnology, Ministry of Science and Technology, Govt. of India, New Delhi, under project no. BT/PR/5261/AGR/16/459/2004 and BT/PR11091/NDB/51/179/2008 is also gratefully acknowledged.
Author information
Authors and Affiliations
Corresponding author
Additional information
Editor: J. Forster
Rights and permissions
About this article
Cite this article
Singh, S.R., Dalal, S., Singh, R. et al. 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 (2013). https://doi.org/10.1007/s11627-013-9520-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11627-013-9520-1