BIOTECHNOLOGICAL IMPROVEMENT OF NEEM Priyanka Srivastavaa, Mithilesh Singha and Rakhi Chaturvedi*a *aDepartment of Biotechnology Indian Institute of Technology- Guwahati Guwahati - 781039, Assam, India *Corresponding Author *Department of Biotechnology Indian Institute of Technology- Guwahati Guwahati - 781039, Assam, India Tel: +91-361-2582211 (off)/ 2584211 (res) Fax: +91-361- 2582249; 2690762 Email: rakhi_chaturvedi@iitg.ernet.in rakhi_chaturvedi@yahoo.co.uk 2 BIOTECHNOLOGICAL IMPROVEMENT OF NEEM ABSTRACT Azadirachta indica A. Juss or Neem (Meliaceae) is a versatile tropical, evergreen tree,which has attained worldwide prominence in recent years due to its therapeutic and insecticidal properties. Its use in folklore has been documented since long where it has been utilized to cure and treat various ailments. The antifeedant and biopesticidal properties of the tree have further enhanced its global value. These diverse biological activities are attributed to several limonoids like Azadirachtin, present in various parts, particularly the seed kernels. However, these assets of neem have been underutilized due to limitations put forth by its long generation cycle and strict out-breeding nature that cause enormous variability in physical traits and chemical profile. Conventional breeding programmes for qualitative and quantitative improvements have been rendered inefficient due to prevalent heterozygosity and perennial nature of the tree. In this regard, plant tissue culture offers a lucrative alternative for quick propagation of plus neem trees. Till date, various explants ranging from nodes, leaves, zygotic embryos, endosperm, nucellus, cotyledons, hypocotyls, protoplasts, anthers and ovaries have been used for the micropropagation of this tree. Also, the in vitro raised cultures from many of the above stated explants have tested positive for presence of important metabolites like Azadirachtin. However, more strategic and intensive efforts are required to channelize laboratory outputs to a commercial scale. This chapter deals with the recent developments in tissue culture of neem and focuses on potential areas where 3 biotechnological intervention may play a key role in improvement of this important tree species. INTRODUCTION Mahogany’s botanical cousin, Neem or Azadirachta indica A. Juss. is an adaptable, tropical, evergreen tree of the family Meliaceae. Referred to as “Wonder Tree” by many, the plant is native to South and Southeast Asia and grows well in tropical and subtropical areas around the world. The history of Neem is inextricably linked to the history of Indian way of life. It is an integral part of Indian ayurveda since ages. This robust looking tree has numerous important medicinal, agrochemical and economic uses to its credit. Almost each and every part of this tree, particularly the leaves, bark and seeds, has multiple uses. Its deep root system is well adapted to retrieving water and nutrients from the soil profile and is very sensitive to water logging. It thrives best in hot, dry climates where shade temperatures often reach 50 degree celsius and annual rainfall ranges from 400 to 1,200 millimeters. Apart from this, neem tree is a renewable source of various useful products. Besides being a popular avenue tree, with a large crown, the wood of neem has been used as timber for house building, furniture and other domestic and agriculture tools. The wood of neem resembles teak wood in its strength, and is more resistant to shock, fungi, and insect attack. It is immune to termites and is durable even outdoors (Thengane et al., 1995). As far as commercial appeal is concerned, the major demand of neem is for its seed-oil, which contains several bioactive compounds. Azadirachtin, a highly oxidized limonoid (triterpenoid) present prominently in neem seed kernels, is mainly responsible for diverse 4 biological activities. It possesses insect repellent, antifeedant, larvicidal, growth inhibiting properties against a wide range of pests and, thus, has been well recognized as an environment friendly, biodegradable biopesticide. In neem, the reproductive phase normally begins after five years and the tree yields an average of 20 kg of fruits per year with maximum production reaching around 50 kg per year in a fully grown adult tree (>10 year old) (National Research Council, 1992). Of the fruit yield, only about 10% is attributed to seed kernels, and desired biological active compounds comprise only 10 grams per kilograms of kernel weight. Thus, an adult neem tree produces only about 20 grams of pesticidal compounds in a season (Schmutterer, 1990). Besides availability, the quality of seeds in terms of azadirachtin content and absence of aflatoxin is a major concern for the neem based industry. Fruits, in neem, are available from June-August which coincides with the rainy season in India. Thus, only about one-third of the fruits are collected due to operational problems and quality considerations (Jayaraj, 1993; Vyas and Mistry, 1996; Venkateswarlu and Mukhopadhyay, 1999). Keeping quality of the seeds is also poor due to high moisture (30-35%) and oil (40-45%) content. Neem trees show wide genetic variation in terms of tree size, morphology, fruit size, and fruit production within the natural population (Kumaran et al., 1993; Srivasuki et al., 1993; Ermel et al., 1984, 1987; Benge, 1989; Schmutterer, 1990; Ketker and Ketker, 1993; Ermel, 1995; Wewetzer, 1998). Considerable variability in the azadirachtin content of their seeds, irrespective of the habitat, is also observed (Ermel, 1995; Sidhu et al., 2003). Improvement in azadirachtin production can be achieved by clonal propagation of elite trees. Vegetative propagation of neem by the conventional methods is possible but 5 difficult (Dogra and Thapliyal, 1996). Therefore, it is normally grown from seeds, which yield a heterogeneous population due to strict cross-pollinating nature of the plant. Moreover, the seeds loose viability within two weeks (Mohan Ram and Nair, 1996). Plant cell and tissue culture would not only surmount these limitations but also hasten the production of clonal material for field planting. It has been suggested as an alternative means for year-round production of azadirachtin and other neem metabolites with the added potential of increasing yield by culture selection and manipulation, irrespective of the season (Allan et al. 1999; Van der Esch et al. 1993). Of late, numerous papers were published on in vitro plant regeneration from various somatic tissues of neem (Table 1). A large proportion of them deal with embryonal or seedling explants (Muralidharan and Mascarenhas, 1989; Nirmalakumari et al., 1993; Shrikhande et al., 1993; Sarker et al., 1997; Chaturvedi et al., 2004a; Rout, 2005; Singh et al., 2009 a,b). However, a few papers have reported shoot regeneration from endosperm tissues (Chaturvedi et al., 2003a) and anther/microspores (Chaturvedi et al., 2003b) of adult tree origin. To channelize the output obtained on laboratory scale more efforts are required in right direction to make the results more viable and of use to general public. This chapter deals with the recent developments in tissue culture of neem and focuses on potential areas where biotechnological intervention may play a key role in improvement of this important tree Reference: . PLANT TISSUE CULTURE AND APPLIED PLANT BIOTECHNOLOGY A uth ors : Kum ar, A . & R oy, S. Price : R s. 2200.00 Siz e : crow n s iz e Ed ition : 2011 ISBN : 9 78-81-79 10-363-0