Callus culture of Azadirachta indica employing its stem

Azadirachtin,an insecticidal active ingredient present in neem tree i.e.Azadirachta indica A. Juss.seeds they varies considerably due to environmental factors also for genetic reasons. Furthermore, the tree does not grow in moderate climates and their germination ratio is very less. It seems appropriate, therefore, to employ tissue culture techniques for the production of Azadirachtin in order to obtain constant amounts of standardized quality. Here we try to create callus, an unspecialized, unorganized, growing and dividing mass of cells through which mass cultivation further initiate. The best media generally we use is MS Media along with different concentrations of hormones i.e. Auxins(BAP) and Cytokinin (Kinetin), employing Azadirachta indica stem.


INTRODUCTION
Neem or Azadirachta indica, is a versatile Indian tree having great importance. Neem is an untapped natural resource as like as its genetic potential is concerned. is one of the most valuable arid zone trees belonging to the family Meliaceae. A native of dry f of India and the subcontinent, Azadirachta indica widely cultivated in the arid and nutrient deficient regions of India and Africa. Besides being a popular avenue tree with a large crown and the wood of neem has been used as timber for furniture, house building, and other domestic as well as agricultural tools. The timber is reported to work well with hand and machine tools (Tewari 1992). Its wood resembles to teak wood in strength and is more resistant to shock, fungi and insect attack; it is also immune to termites and durable even outdoors (Thengane, 1995). Neem tree is known to increase the soil fertility. It has huge @ IJTSRD | Available Online @ www.ijtsrd.com | Volume -2 | Issue -3 | Mar-Apr 2018 Azadirachtin,an insecticidal active ingredient present in neem tree i.e.Azadirachta indica A. Juss.seeds and they varies considerably due to environmental factors also for genetic reasons. Furthermore, the tree does not grow in moderate climates and their germination ratio is very less. It seems appropriate, therefore, to the production of Azadirachtin in order to obtain constant amounts of standardized quality. Here we try to create callus, an unspecialized, unorganized, growing and dividing mass of cells through which mass cultivation further erally we use is MS Media along with different concentrations of hormones i.e. Auxins(BAP) and Cytokinin (Kinetin), employing

Callus, MS media,
, is a versatile Indian tree having great importance. Neem is an untapped natural resource as like as its genetic potential is concerned. It is one of the most valuable arid zone trees belonging to the family Meliaceae. A native of dry forest areas Azadirachta indica is widely cultivated in the arid and nutrient deficient regions of India and Africa. Besides being a popular avenue tree with a large crown and the wood of neem ture, house building, and other domestic as well as agricultural tools. The timber is reported to work well with hand and machine tools (Tewari 1992). Its wood resembles to teak wood in strength and is more resistant to shock, is also immune to termites and durable even outdoors (Thengane, 1995). Neem tree is known to increase the soil fertility. It has huge water holding capacity and the tree has a unique property of calcium mining which changes the acidic soil into neutral. The tree is resistant to high temperatures and drought and has been employed for afforestation of dry localities, reforesting bare ravines and checking soil erosion (Gill et al., 1996). Beside of that various parts of the neem tree, particularly leaves, bark and seeds have been traditionally used in India in ayurvedic medicines. The seed oil has been used as anti malarial, anthelminthic, vermifuge, antiseptic, and antimicrobial, it is also known to cure various skin disorders. Callus culture of Azadirachta indica employing its stem water holding capacity and the tree has a unique property of calcium mining which changes the acidic The tree is resistant to high temperatures and drought and has been employed for afforestation of dry localities, reforesting bare ravines and checking soil erosion (Gill et al., 1996). Beside of that various parts of the neem tree, particularly leaves, ark and seeds have been traditionally used in India in ayurvedic medicines. The seed oil has been used as anti malarial, anthelminthic, vermifuge, antiseptic, and antimicrobial, it is also known to cure various A. Juss is famous for its insecticidal properties and neem seed extracts show great potential as environmentally acceptable bioinsecticides for crop protection (Jacobson 1988; Mordue (Luntz) and Blackwell 1993; Schmutterer 1990). The first commercial neem insecticide, O, was registered by the Environmental Protection Agency (EPA) in 1985 for use on non-food crops (Jacobson 1988; Larson 1993) and from then the number of commercial and experimental neem insecticides has increased markedly with Azatin ne Technologies, USA) recently receiving EPA approval for use on food crops (Johnson et al. 1994). Neem having these properties due to the presence of several bioactive compounds; the most prominent one being Azadirachtin. Unfortunately, the atio is very less as compare to other In order to meet the economic demand of the neem tree, an efficient propagation technique is required through which we could get a large quantities and good quality of planting materials. Vegetative propagation of an adult neem tree by conventional In present context, the main objective of this experiment is to establish a procedure which can be used routinely to produce complete micro propagated plantlets from neem using stem i.e. Apical and lateral meristem explants of plants from mature trees.
The culture medium mostly used for the in vitro cultivation of Azadirachta indica is the Murishage and Skoog medium (Murishage and Skoog.1962).

Plant material
Simply cutting of axiallary or lateral stem of neem approximately size of 4-5 centimeters collected from campus of Adithya biotech lab, Raipur. After collection of stem along with the leaves immediately dipped into beaker having distilled water in it. Which helps to keep the plant away from any kind of infection. Page: 320

Nutrient Media
The cuttings were slightly trimmed at both ends to expose the fresh tissue and grown firstly on Murishage and Skoog (MS medium), whose composition are as follows and then secondly on MS medium with plant hormones Auxins and Cytokinin of different ratios.  1N NaOH and 1N HCl. The explants kept in the incubation chamber temperature must be 25±2 0 C and 16 hours in light. Later after 2 weeks for secondary culture explants were transferred into MS medium containing various ratio of Auxin and Cytokinin i.e. IBA: Kinetin, in different ratios. After 4-5 weeks plants were gradually moved away from lab and transferred to cocoa pit in a shed house at 25±2 0 C.After 2 and half months they were shifted to a area under natural conditions.

Sterilization process
Contamination of Azadirachta indica explants were a major trouble during incubation of culture. Specially in the month of June -August , monsoon season in India, where the chances of contamination increases around to 100% therefore normally researcher prefer the month of March-May.. Before inoculation the laminar airflow is cleaned by 70% methanol, each and every instrument like scalpel, forceps dipped in 70% methanol. We used new blade for scalpel regarding inoculation. We sterilize plastic sheets and media i.e. bottles containing MS media and test tubes in autoclave at 121 0 C at 15 psi for 20 minutes .After sterilization of media it was kept in inoculation chamber for 1-2 days to check whether any kind of infection is present or not. After 1-2 days we started the process of inoculation and on the day of inoculation once again sterilize the instruments use during the inoculation.

Callus induction
Azadirachta indica stems were used as explant material.During the process development of callus from the stem,a white color undifferentiated mass was developed.The callus initiated from stem grows rapidly within two weeks.After two weeks sufficient callus which was later sub culture on MS medium supplemented with lower dosage of Auxin and Cytokinin i.e IBA:Kinetin On this medium optimal callus growth was obtained and callus was maintained subsequently on this callus multiplication medium. Cultures were incubated in dark.

Hardening and Acclimatization of in vitro regenerated plantlets
After one week of transfer of in vitro raised plantlets to poly house. Bottles containing plantlets were the shifted from the pad section towards the fan section to provide growing conditions of low humidity (50 -60%) and high temperature (30±2°C). after 4 weeks, acclimatized plantlets were transferred to poly begs containing a mixture of soil+ sand+ FYM (Farm yard manure) (2:1:0.5). such plantlets were kept in the poly house for 3-4 weeks and then shifted to agro shade house then open environment.
Result and discussion:- Most of the sample 6 out of 10 contaminated by fungus, during rainy season it is very difficult to inoculate explant in India. Only out of 10 four sample survived, out of which one was control. New way of sterilization protocol required for inoculation of Azadirachta indica. Callus observed in the 3 samples and by further treatment with Cytokinin and Auxin, growth of shoot and root observed.Appropriate growth of shoot and leaves observed after treating with Kinetin . Later for rooting IBA at concerntration of 15µl/ 100 ml is used and growth of root observed.Further experiment can be continued by taking these two plant hormones, to initiate root and shoot from callus of Azadirachta indica.