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Evaluation of genetic fidelity of in vitro raised plants of Dendrocalamus asper (Schult. & Schult. F.) Backer ex K. Heyne using DNA-based markers

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Abstract

Dendrocalamus asper, an edible bamboo is valued for its tender edible shoots in the food industry. However, overexploitation of natural stands of D. asper coupled with minimal conservation and reforestation efforts has led to its rapid depletion in nature. Therefore protocol for rapid multiplication of D. asper via direct regeneration using nodal segments from mature clumps was standardized and more than 25,000 plants were transferred to the field (Singh et al. 2012a). However, genetic fidelity of these in vitro raised plants needs to be authenticated for commercial scale application of the developed micropropagation protocol. PCR-based molecular markers have emerged as simple, fast, reliable and labor-effective tools for testing the genetic fidelity of in vitro raised plants. This study report the genetic fidelity analysis of in vitro raised plants of D. asper for the first time using arbitrary (Random Amplified Polymorphic DNA, RAPD), semi-arbitrary (Inter-Simple Sequence Repeat, ISSR; Amplified Fragment Length Polymorphism, AFLP), and sequence-based (Simple Sequence Repeat, SSR) markers. Bulked DNA samples of 20 in vitro raised shoots (collected after every three subculture cycles starting from 3rd to 30th passage) and field transferred plantlets were compared with the mother plant DNA using 90 primer combinations (25 each of RAPD, ISSR, SSR, and 15 AFLP) and scorable bands were produced by 78 (22 RAPD, 24 ISSR, 21 SSR, and 11 AFLP) primers. A total of 146 distinct and scorable bands were produced by 22 RAPD primers with an average of 6.6 bands per primer while the number of bands for ISSR primers varied from 3 (ISSR-4 and 9) to 13 (ISSR-17), with an average of 7.1 bands per primer. Similarly, SSR markers also showed wide variation in number of bands, ranging from 2 (RM 261) to 12 (RM 44, 140, and 224) with an average of 7.8 bands. AFLP primer combinations could generate 35–72 bands with an average of 48.7 bands per primer pair. Amplification of monomorphic bands with all primer combinations authenticated the true to type nature of the in vitro raised plants of D. asper which underwent up to 30 subculture passages over a period of approximately 2 years thereby supporting the commercial utilization of the developed micropropagation protocol.

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Abbreviations

AdS:

Adenine sulfate

AFLP:

Amplified fragment length polymorphism

BAP:

6-Benzyl-amino-purine

CTAB:

Cetyl trimethyl ammonium bromide

dNTPs:

Deoxyribonucleotide triphosphates

FYM:

Farm yard manure

IBA:

Indole-3-butyric acid

ISSR:

Inter-simple sequence repeat

MS:

Murashige and Skoog’s medium

NAA:

α-Naphthalene acetic acid

PCR:

Polymerase chain reaction

RAPD:

Random amplified polymorphic DNA

RFLP:

Restriction fragment length polymorphism

SSR:

Simple sequence repeat

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Acknowledgments

Our sincere thanks to 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 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.

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Author notes

  1. A. K. Dhawan

    Present address: Central University of Punjab, Bhatinda, 151001, Punjab, India

  2. Rajwant K. Kalia

    Present address: Central Arid Zone Research Institute (CAZRI), Jodhpur, 342003, Rajasthan, India

Authors and Affiliations

  1. Centre for Plant Biotechnology, CCS HAU New Campus, Hisar, 125004, Haryana, India

    Sharbati R. Singh, Rohtas Singh, A. K. Dhawan & Rajwant K. Kalia

  2. Department of Biotechnology, Kurukshetra University, Kurukshetra, 136119, India

    Sharbati R. Singh & Sunita Dalal

Authors
  1. Sharbati R. Singh
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  2. Sunita Dalal
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  3. Rohtas Singh
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  4. A. K. Dhawan
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Correspondence to Rohtas Singh.

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Communicated by B. Borkowska.

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Singh, S.R., Dalal, S., Singh, R. et al. Evaluation of genetic fidelity of in vitro raised plants of Dendrocalamus asper (Schult. & Schult. F.) Backer ex K. Heyne using DNA-based markers. Acta Physiol Plant 35, 419–430 (2013). https://doi.org/10.1007/s11738-012-1084-x

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  • DOI: https://doi.org/10.1007/s11738-012-1084-x

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