Micropropagation and validation of genetic and biochemical fidelity among regenerants of Nothapodytes nimmoniana (Graham) Mabb. employing ISSR markers and HPLC.

Lokesh Prakash,Lokesh Prakash,Sudipta Kumar Mohanty,Sushil Kumar Middha,Mallappa Kumara Swamy

doi:10.1007/s13205-016-0490-y

Lokesh Prakash, Lokesh Prakash + Show 3 more

Open Access

https://doi.org/10.1007/s13205-016-0490-y

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Journal: 3 Biotech	Publication Date: Aug 16, 2016
Citations: 28	License type: CC BY 4.0

Affiliation: Universiti Putra Malaysia

Abstract

An in vitro protocol has been established for clonal propagation of Nothapodytes nimmoniana which is an important source of Camptothecin (CPT). Elite source was identified based on the chemical potency to accumulate the optimum level of CPT. Different types and concentrations of plant growth regulators were used to study their effect on inducing multiple shoots from the explants regenerated from embryos of N. nimmoniana. Of these, a combination of N6-benzyladenine (0.2 mg L−1) and Indole-3-butyric acid (IBA) (0.1 mg L−1) proved optimum for differentiating multiple shoots in 90.6 % of the cultures with an average of 10.24 shoots per explant obtained within 8 weeks of inoculation. Nearly, 92 % of the excised in vitro shoots rooted on half strength Murashige and Skoog (MS) medium containing 0.05 % activated charcoal, supplemented with 1-naphthaleneacetic acid and IBA at 0.1 mg L−1 each. The micropropagated plants were evaluated for their genetic fidelity by employing inter simple sequence repeats (ISSR) markers. Ten individuals, randomly chosen from a population of 145 regenerants, were compared with the donor plant. The regenerated plants were also evaluated for their chemical potency using high-performance liquid chromatography (HPLC) analysis of CPT content. The true-to-type nature of the micropropagated plants was confirmed based on their monomorphic banding profiles with that of the mother plants using ISSR markers. Besides, HPLC evaluation of the CPT content confirmed the existence of chemical uniformity among the regenerated plants and the elite mother plant.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-016-0490-y) contains supplementary material, which is available to authorized users.

Highlights

Nothapodytes nimmoniana (Graham) Mabb. belonging to the family, Icacinaceae is a medicinally valuable tree
An in vitro protocol has been established for clonal propagation of Nothapodytes nimmoniana which is an important source of Camptothecin (CPT)
A combination of N6-benzyladenine (0.2 mg L-1) and Indole-3-butyric acid (IBA) (0.1 mg L-1) proved optimum for differentiating multiple shoots in 90.6 % of the cultures with an average of 10.24 shoots per explant obtained within 8 weeks of inoculation

Summary

Introduction

Nothapodytes nimmoniana (Graham) Mabb. belonging to the family, Icacinaceae is a medicinally valuable tree. In addition to the difficulty in the synthesis of CPT and its derivatives, the natural source becomes extinct due to the problems of drastic weather and excessive trade. Due to this fact, C. acuminata was recommended for protection by World Conservation Monitoring Centre in 2006. Though some work on in vitro regeneration of N. nimmoniana has been published using different explants, none of them has established protocols pertaining to the genetic and biochemical fidelity of the mature regenerants. Analyzing the CPT content of N. nimmoniana mature regenerants and the elite mother plant will further confirm the biochemical fidelity. The genetic and biochemical fidelity among the micropropagated plants was established by ISSR and HPLC analysis

Methods

Results

Conclusion