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Micropropagation and validation of genetic homogeneity of Alhagi maurorum using SCoT, ISSR and RAPD markers

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Abstract

A reliable and reproducible protocol for in vitro regeneration has been developed for Alhagi maurorum, a rare and medicinally important plant of family fabaceae. MS medium with BAP (2.0 mg l−1) proved to be the best for shoot bud induction from nodal segments. The rate of shoot multiplication was found to be influenced by a number of factors, viz., media composition, plant growth regulator’s type and concentration, successive transfer of mother explant for different passage, culture vessels and gelling agents. Modified MS medium (modified having nitrates reduced to half) solidified with 0.14 % gelrite and containing BAP (0.5 mg l−1), IAA (0.1 mg l−1) and additives was found optimum for shoot multiplication which gave rise to maximum number of shoots (33.5 ± 3.43 per culture vessel). The in vitro regenerated shoots were rooted under both in vitro (on half strength MS salts with 1.0 mg l−1 IBA + 100 mg l−1 activated charcoal) as well as ex vitro (on sterile soilrite by treating shoot base with 250 mg l−1 each of IBA and NOA for 4 min in green house) conditions. Thereafter, the in vitro and ex vitro rooted plantlets were hardened under green house conditions with 70 and 90 % survival rate, respectively. Start codon targeted (SCoT), inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers were used to validate the genetic homogeneity of seven tissue cultured plantlets growing in green house condition with mother plant. The amplification products were monomorphic across all the seven micropropagated plants as well as mother plant produced by all SCoT, ISSR and RAPD primers applied. The monomorphic banding pattern in micropropagated plants and the mother plant confirms the genetic homogeneity of the in vitro raised plants and demonstrates the reliability of our in vitro propagation system for A. maurorum. To the best of our knowledge, this is the first report on micropropagation and genetic homogeneity assessment of A. maurorum, which can be applied for large scale multiplication of elite genotypes of A. maurorum.

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Abbreviations

AC:

Activated charcoal

BAP:

6-benzylaminopurine

IAA:

Indole-3-acetic acid

IBA:

Indole-3-butyric acid

ISSR:

Inter simple sequence repeats

Kn:

Kinetin

MMS:

Modified Murashige and Skoog

NAA:

Naphthalene acetic acid

NOA:

Naphthoxy acetic acid

PFD:

Photon flux density

PGR:

Plant growth regulator

RAPD:

Random amplified polymorphic DNA

RH:

Relative humidity

SCoT:

Start codon targeted

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Acknowledgments

We are thankful to Council of Scientific and Industrial Research (CSIR), New Delhi for financial assistance in the form of Junior Research Fellowship (JRF) to authors T.A. and Senior Research Fellowship (SRF) to A.K.P., respectively. We are also thankful to Mr. Ramesh Dagla for his assistance in collecting the plant material.

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Authors and Affiliations

  1. Biotechnology Unit, Department of Botany, Jai Narain Vyas University, Jodhpur, 342001, Rajasthan, India

    Tanvi Agarwal, Ashok Kumar Patel & N. S. Shekhawat

  2. Department of Botany and Biotechnology, L.M. College of Science and Technology (Autonomous), Jodhpur, 342001, Rajasthan, India

    Amit K. Gupta

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  1. Tanvi Agarwal
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  2. Amit K. Gupta
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  3. Ashok Kumar Patel
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  4. N. S. Shekhawat
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Correspondence to Amit K. Gupta.

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Agarwal, T., Gupta, A.K., Patel, A.K. et al. Micropropagation and validation of genetic homogeneity of Alhagi maurorum using SCoT, ISSR and RAPD markers. Plant Cell Tiss Organ Cult 120, 313–323 (2015). https://doi.org/10.1007/s11240-014-0608-z

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