Determination of the Best Culture Medium and Plant Growth Regulators for Micropropagation of Neem Tree (Azadirachta indica A.Juss)

Document Type : Research paper

Authors

1 Department of Arid Land and Desert Management, Faculty of Natural Resources and Desert Studies, Yazd University, Yazd, Iran

2 Department of Environmental Science, Faculty of Natural Resources and Desert Studies, Yazd University, Yazd, Iran

Abstract

Azadirachta indica is a tree with high medicinal value that is conventionally propagated by seed while exhibiting heterozygosity. The aim of this research was to determine the best culture media and plant growth regulators for the micropropagation of Neem. Lateral and terminal buds of A. indica were sterilized with 0.15% and 0.2% mercuric chloride for 10, 11, 12, and 13 min, followed by washing with sterilized double-distilled water three times. At the proliferation and elongation stage, WPM and MS media were tested with different concentrations of BAP either alone or in combination with 0.01 mg L-1 IBA. LS and MS media containing four different levels of IBA (0, 0.5, 1.0, and 2.0 mg L-1) were used for the rooting stage. Pulsing technique with different IBA concentrations was investigated at the rooting stage. Hardening of rooted plantlets was done in potting soil containing peat and perlite (2:1), at 23-24 °C prior to transfer into the natural environment. Maximum survival percentage (70.83%) with minimum browning (10.42%) was achieved by sterilizing the explant with 0.15% of mercuric chloride at all times. The longest shoots (3.66 cm) were observed in the media containing BAP (0.5 mg L-1). Furthermore, the highest number of leaves (14.2 leaves per plant) was recorded in MS medium. Additionally, the MS media containing BAP hormone alone at 0.7 mg L-1 produced the highest number of shoots (3.6 shoots per treatment). LS medium supplemented with IBA (4.0 mg L-1) using the pulsing technique gave the best result at the rooting stage.

Keywords


Arora K, Sharma M, Srivastava J, Ranade SA, Sharma AK. 2011. In vitro cloning ofAzadirachta indica from root explants. Biologia Plantarum 55(1), 164-168.
 
Benelli G, Buttazzoni L, Canale A, D'Andrea A, Del Serrone P, Delrio G, Foxi C, Mariani S, Savini G, Vadivalagan C, Murugan K, Toniolo C, Nicoletti M, Serafini M. 2017. Bluetongue outbreaks: Looking for effective control strategies against Culicoides vectors. Research in Veterinary Science 115, 263-270.
 
Bennett I, McComb J, Tonkin C, McDavid D. 1994. Alternating cytokinins in multiplication media stimulates in vitro shoot growth and rooting of Eucalyptus globulus Labill. Annals of Botany 74(1), 53-58.
 
Cha-um S, Mosaleeyanon K, Kirdmanee C, Supaibulwatana K. 2003. A more efficient transplanting system for Thai neem (Azadirachta siamensis Val.,) by reducing relative humidity. ScienceAsia 29, 189-196.
 
Chaturvedi R, Razdan MK, Bhojwani SS. 2004. In vitro clonal propagation of an adult tree of neem (Azadirachta indica A. Juss.) by forced axillary branching. Plant Science 166(1), 501-506.
 
Chaudhary S, Kanwar RK, Sehgal A, Cahill DM, Barrow CJ, Sehgal R, Kanwar JR. 2017. Progress on Azadirachta indica Based Biopesticides in Replacing Synthetic Toxic Pesticides. Frontiers in plant science 8, 610-610.
 
Gautam VK, Nanda K, Gupta SC. 1993. Development of shoots and roots in anther-derived callus of Azadirachta indica A. Juss.—a medicinal tree. Plant cell, tissue and organ culture 34(1), 13-18.
 
Houllou LM, Souza RAd, Santos ECPd, Silva JJPd, Barbosa MR, Sauvé JPG, Harand W. 2015. Clonal propagation of neem (Azadirachta indica A. Juss.) via direct and indirect in vitro regeneration. Revista Árvore 39(3), 439-445.
 
Islam R, Hoque A, Khalekuzzaman M, Joarder O. 1997. Micropropagation of Azadirachta indica A.Juss. from explants of mature plants. Plant Tissue Culture 7, 41-46.
 
Joshi M. 1996. In vitro propagation of Azadirachta indica A. Juss (neem) by shoot proliferation. Indian Journal of Experimental Biology 34, 480-485.
 
Kumar R, Jakhar M. 2018. Factor Affecting Tissue Culture Experiments Of Woody Plant: A Review. Journal of Cell & Tissue Research. 18(2), 6467-6471.
 
Kuravadi NA, Yenagi V, Rangiah K, Mahesh H, Rajamani A, Shirke MD, Russiachand H, Loganathan RM, Lingu CS, Siddappa S. 2015. Comprehensive analyses of genomes, transcriptomes and metabolites of neem tree. PeerJ 3, e1066.
 
Pandey V, Pati A. 2017. Limitation of Improvement in Germination by Osmopriming of Differentially Aged Non-Orthodox Neem (Azadirachta indica) Seeds. Biochemistry and Physiology 6(217), 2.
 
Patel SM, Venkata KCN, Bhattacharyya P, Sethi G, Bishayee A. 2016. Potential of neem (Azadirachta indica L.) for prevention and treatment of oncologic diseases. In: Seminars in Cancer Biology 40, 100-115.
 
Phukan H, Kumar R, Mitra PK. 2017. Plant Regeneration by Somatic Embryogenesis in Azadirachta indica A. Juss.(Neem). International Research Journal of Engineering and Technology 4, 3212-3217.
 
Quraishi A, Koche V, Sharma P, Mishra S. 2004. In vitro clonal propagation of neem (Azadirachta indica). Plant cell, tissue and organ culture 78(3), 281-284.
 
Rout GR. 2005. In vitro somatic embryogenesis in callus cultures of Azadirachta indica A. Juss.—a multipurpose tree. Journal of Forest Research 10(4), 263-267.
Rupani R, Chavez A 2018. Medicinal plants with traditional use: Ethnobotany in the Indian subcontinent. Clinics in Dermatology, 36(3), 306-309.
 
Shrikhande M, Thengane S, Mascarenhas A.1993. Somatic embryogenesis and plant regeneration in Azadirachta indica A. Juss. In Vitro Cellular & Developmental Biology-Plant 29(1), 38-42.
 
Singh M, Chaturvedi R. 2009. An efficient protocol for cyclic somatic embryogenesis in Neem (Azadirachta indica A. Juss.). In: Proceedings of World Academy of Science Engineering and Technology656-658.
 
Srinidhi HV, Gill RIS, Sidhu DS. 2008. Micropropagation of Adult and Juvenile Neem (Azadirachta indica A. Juss). Journal of Crop Improvement 21(2), 221-232.
Suresh KS, Suresh P, Kudre TG. 2019. Prospective ecofuel feedstocks for sustainable production. In:  Advances in Eco-Fuels for a Sustainable Environment. India, Elsevier.89-117.
 
Venkateswarlu B, Katyal J, Choudhuri J, Mukhopadhyay K. 1998. Micro-propagation of plus neem (Azadirachta indica A. Juss) and evaluation of field transferred plants. Indian forester 124(7), 537-543.