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Rapid in vitro cloning of a 40-year-old tree of Azadirachta indica A. Juss. (Neem) employing nodal stem segments

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Abstract

An efficient in vitro process for rapid clonal propagation of a 40-year-old tree of Azadirachta indica employing nodal stem segments was developed. Season of collection and maturity of explants showed direct influence on bud-break. Nodal stem segments collected during the month of April gave best response. Maximum bud-break (78.6–81%) was obtained when middle order nodes (3rd or 4th node from apex) were taken. Amongst different cytokinins used, 6-benzylaminopurine (BAP) at the concentration of 1.11 μM was found most effective in inducing multiple shoots, whereas inorganic and organic constituents of the medium influenced growth and general condition of proliferating shoots. On an average 3.1 shoots per explant were regenerated in modified Murashige and Skoog medium supplemented with 1.11 μM BAP, 1.43 μM indole-3-acetic acid (IAA) and 81.43 μM adenine hemisulphate. Isolated shoots were rooted in presence of 2.46 μM indole-3-butryic acid (IBA). Root induction took place in 8–10 days with 100% rooting. The in vitro-raised plantlets were successfully transplanted in potted soil and finally grown under field conditions with 100% survival. The genetic fidelity of such in vitro-raised field-grown plants was ascertained by random amplified polymorphic DNA (RAPD) markers. Furthermore, the azadirachtin content of in vitro-cloned plants was found comparable to the mother tree proving their chemical stability also. The protocol developed holds good for in vitro cloning of mature elite neem trees.

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Abbreviations

BAP:

6-Benzylaminopurine

IAA:

Indole-3-acetic acid

IBA:

Indole-3-butyric acid

2iP:

N 6-(2-isopentenyl) adenine

MS medium:

Murashige and Skoog medium

NAA:

α-Naphthaleneacetic acid

PCR:

Polymerase chain reaction

PVP:

Polyvinylpyrollidone

RAPD:

Random amplified polymorphic DNA

Taq :

Thermus aquaticus

TDZ:

Thidiazuron or (N-phenyl-N′-(1,2,3-thiadiazol-yl) urea

Z:

Zeatin

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Acknowledgments

The authors thank the Director, National Botanical Research Institute (NBRI), Lucknow for the facilities provided. The first author also thanks the Council of Scientific and Industrial Research (CSIR), India, for awarding her Senior Research Fellowship. Thanks are also due to Dr. H. M. Behl and Dr. Vishal Kumar for providing facilities and guidance for work related to chemical analysis.

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Authors and Affiliations

  1. Tissue Culture Laboratory, National Botanical Research Institute, Lucknow, 226 001, India

    Kavita Arora, Meena Sharma & A. K. Sharma

  2. Plant Molecular Biology (Genomics) Division, National Botanical Research Institute, Lucknow, 226 001, India

    Jyoti Srivastava & S. A. Ranade

Authors
  1. Kavita Arora
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  2. Meena Sharma
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  3. Jyoti Srivastava
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  4. S. A. Ranade
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  5. A. K. Sharma
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Correspondence to A. K. Sharma.

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Arora, K., Sharma, M., Srivastava, J. et al. Rapid in vitro cloning of a 40-year-old tree of Azadirachta indica A. Juss. (Neem) employing nodal stem segments. Agroforest Syst 78, 53–63 (2010). https://doi.org/10.1007/s10457-009-9230-1

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