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An efficient protocol for in vitro regeneration and conservation of Shirui lily (Lilium mackliniae Sealy): a lab-to-land approach to save the rare endangered Asiatic lily species

  • Manas Ranjan Sahoo
  • Mayengbam Premi Devi
  • Madhumita Dasgupta
  • Narendra Prakash
  • Shishom Vanao Ngachan
Protocols/Methods
  • 14 Downloads

Abstract

An efficient protocol for direct and indirect shoot regeneration and proliferation from bulb scales of Shirui lily (Lilium mackliniae Sealy), an endangered Asiatic lily species endemic to the Shirui hill peak, Manipur, India, has been developed. Bulb scales were isolated from mature bulbs and cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of 6-benzylaminopurine (BAP), kinetin (KIN), or thidiazuron (TDZ). For direct shoot regeneration from bulb scale explants, 0.5 mg L−1 BAP yielded the highest shoot induction (3.5 shoots per scale; a 96.7% response). For indirect de novo organogenesis, optimum callus induction was achieved with 2.0 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D), and shoot organogenesis was higher (16.2) when subcultured onto 0.5 mg L−1 BAP medium. Multiple shoot regeneration and pseudo-bulb formation protocols were assessed; the highest shoot proliferation (10.1) occurred with 0.5 mg L−1 BAP and 1.0 mg L−1 gibberellic acid (GA3). Rooting response was 96% with 0.5 mg L−1 1-naphthalene acetic acid (NAA), with multiple roots per shootlet. Plantlet survival was increased to 92.5% during the hardening-off process by using hydroponics with Hoagland’s solution in a mist chamber. Clonal fidelity was assessed through random amplified polymorphic DNA (RAPD) analysis comparing the mother plant and regenerated plantlets. After confirming genetic uniformity, the pseudo-bulblets with four to six leaves and three to four roots were successfully established at the Shirui hills peak. This in vitro regeneration and ex vitro conservation approach could be helpful to save this rare endangered species in a sustainable way.

Keywords

Bulb scale Callus Endangered Lilium mackliniae Sealy Micropropagation RAPD 

Notes

Acknowledgments

The authors are thankful to the Director and Joint Director of ICAR Research Complex for NEH Region for the infrastructure facilities. The “Shirui village authority” is gratefully recognized for providing the necessary plant materials for the present study. The authors also express gratitude for the support of “Shirui youth club,” Mr. Ajit Kumar Singh, SMS (Plant Protection), and Dr. Y. Ramakrishna, Head, Farm Science Centre, Ukhrul, for the necessary cooperation in awareness and transplantation of the in vitro plantlets at the Shirui hill peak.

Authors’ contribution

MRS and MD participated in designing the experiment, analyzing the data, and drafting the manuscript. MPD and MD executed the experiments. All authors read and approved the final manuscript.

Funding information

The authors wish to acknowledge the financial support of the Indian Council of Agricultural Research (ICAR), Government of India, under institutional program, and the Department of Science and Technology (DST), Government of India, for internship to MPD for carrying out this work.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11627_2018_9916_Fig5_ESM.png (409 kb)
Figure S1

Random amplified polymorphic DNA (RAPD) analysis using (a) OPA; (b) OPC; and (c) OPW primers showed no variability among Lilium mackliniae plants (lanes SL1; SL2; and SL3 represent mother plant collected from Shirui hills; plantlets derived directly from bulb scale; and plantlets derived through callus culture, respectively).100 bp lane with molecular ladder. (PNG 409 kb)

11627_2018_9916_MOESM1_ESM.tif (397 kb)
High resolution image (TIF 397 kb)

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Copyright information

© The Society for In Vitro Biology 2018

Authors and Affiliations

  1. 1.ICAR Research Complex for North Eastern Hill RegionManipur CentreImphalIndia
  2. 2.University of TennesseeKnoxvilleUSA

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