A PROCEDURE FOR STERILISING LEAF EXPLANTS COLLECTED FROM WILD NEEM ( AZADIRACHTA INDICA ) TREES

Abstract :Experiments conductccl to find suitable sterilisation procedures for ncem explnnts collcctcd from trees growing in the wilcl showed tha t they could not be sterilised by thc method for leaves collected from the trees reared in [.he laboratory (10% NaOCl for 10 mjn.) but required more rigorous sterilisntion methocls. Leaf cxplants collectecl from the trees growing in the wild could not bc stcrj.lisec1 using different conccntrntions ofNaOCl (lo%, 160/0 20% & 25%), HgCI, (0.1%, 0.5(Zb, ST 1%) fbr different time intervals (5, 10, 15, 20 ancl 30 mins) but could be sterilisecl by combining HgCI, ancl NaOCl (0.1% /o:[gCl, for 5 rnin fbllowed by 1.0'8, NaOCI for 10 min). Leaf damage was observed when 70% ethanol was u.sed a s a PI-e-treatment.


INTRODUCTION
Azadirachtin, which is extractcd from neem secds, shows insect antifcedant as well as growth regulatory activity against a wide range of insect pests.'It has been shown that azadirachtin is biodegradable without having residual effects to the environment2 and therefore, can be used as an ellvironmentally friendly insecticide in crop protection programmes.
However, the low quantities of azadirachtin present in neem seeds and the expense of purification makes the pure compound very expensive (15,000 U.S. $ per kg).Therefore, the chemical synthesis2 of azadirachtin and production using plant cell c u l t ~r e techniques '<-%have been attempted.
Maximisation of product synthesis is desirable when plant cell culture techniques are applied to produce valuable secondary metabolites.Strategies for producing high yielding cell cultures include, establishment of cell lines from selected high yielding trees, followed by clonal multiplication and optimisation of culture conditioi~s.~Therefore, it is very important to establish cell lincs from high producing wild growing neem trees (referred as wild neem ) when cell culture techniques are used to produce azadirachtin.Kearney et ul., (1994)" reported successful callus initiation from neem 1.eaf explants which was subsequently shown to be successful irrespective of the country of the origin of the plant materials by Eeswara et uL., (1997)."Even though, the medium was successful in initiating calli, high rate of contamination of cultures were seen when leaf explants collected :from wild growing neem were sterilised by the method (i.e.10% NaOCl for 10 min) used for laboratory grown materi.als.Therefore, investigations were carried out to find techniques suitab1.e for sterilisation of neem lezif explants from wild trees.

METHODS AND MATERIALS
Leaf explants coll.ectedfrom a wild neem tree at University experimen.ta1station at Dodaugolla, which is 15 km away from the Uni.versity of Peradeniya were used for the experiments.Immature 'leaves (slightly reddish colour) were picked directly from the tree and sprayed with water before packing in polythene bags which were placed in a water bucket during the transport (1 hour) from the experimental station to the tissue culture laboratory a t University of Peradeniya.Tlle leaves were thoroughly washed with tap water followed by mild liquid soap (Teepol C.I.C, Sri Lanka) and sterilised distilled water (pre-treatment) as soon as they were taken to the laboratory, prior to receiving further specific treatments.After this stage all the treatments (experiments 1 to 6, Table 1) were done und.er clean environmental con.ditions in a laminar flow cabinet.Treated leaf explants were thoroughly washed with sterile distilled water in between each treatment in each experiment and prior to being incubated a t 25 'c, i n thc dark on Murashige and Sl-oog medium containing 1 m g 1: ' BAP and 4 mg 1-' IBA (Maintenance Medium).:'Each treatment was replicated 30 times.In addition, leaf explants treated under differen.tsterilisation regimes were kept in Petri dishes containing glucose Agar and n.utrientAgar (Oxoid, UK) to observe any micl-obial contamination.Thc number of cultures without contamination was recorded.Results were subjected to Chi-squared analysis after categorisation into cultures, wh.ich contaminated and those, which were not.The experiment 6 was conducted to replace the Bleach with standard chemicals (NaOC1, Sigma, UK).
Pre-treated leaf explants were first sterilised with 0.1% HgCl, for 5 mill and thereafter, leaves were surface sterilised with three different concentrations of NaOCl(Sigma UK) for 10 minutes (Table 1).Leaf explants sterilised with 10% commercially available NaOCl (Bleach, Care product) in addition to 0.1% HgClz treatment were used a s the control (Table 1).
The succcss of stcrilisation procedure established based on the results of experiments 1 to 6 was further investigated using neem I ear cxplants collected from the 15 wild neem trees from four locations (Dambulla, Ethalawa, Maha-Illuppallama and Kalawewa) in two agro-ecological zones (dry and intermcdiate) in Sri Lanka.Leaves were picked directly from trees, and transported (4 hours) to University of Pcradeniya as described for the leaves from tree a t Dodangolla farm.Thereafter,  pre-treated leaves were surface sterilised with 0.1% HgC1, for 5 minutes followed by 10% NaOCl for 10 minutes and were cultured on Maintenance Medium.

RESULTS AND DISCUSSION
Immature leaves were selected since young tissues are often found to be free of detectable internal population of viroids, viruses, mycoplasms, bacteria and f u i ~g i .~ I t was found that sterilisation of neem leaf explants collected from wild growing trees was unsuccessful (100% contamination) when they were treated with 10% Bleach alone or following exposure to 70% ethanol for 3 min (Table 1).Furthermore, leaf explants treated with concentrations of Bleach of up to 25 % for up to 30 minutes also showed 100% contamination (Table 1).After these treatments leaf explants cultured on Glucose and Nutrient Agar showed very high levels of both bacterial and fungal contamination.
Very few leaf explants treated with 0.1% HgC1, were observed to be free of contamination and the results were not significant a t the 5% probability level (Table 1).Exposure to a variety of concentrations of HgC1, for different time intervals, also produced few culturcs without contamination (Table 1) but results werc not significantly different Leaf cxplants treated with HgC1, and culturcd on Glucose Agar and Nutrient Agar showed more fungal than bacterial contamination.Thesc results clearly showed that sterilisation of wild neem Icaf explants could not be achieved followii~g the proccdure for laboratory-grown materials:'.Taking explants from other field-grown plants has previously been shown to reduce the efficiency of the subsequent disinfection process.*Furthermore, wild neem leaf' explants could not be surface sterilised using either NaOCl or HgCI, alone.
The effects of different concentrations of HgCI, combined with 10% Bleach solution on the sterilisation of wild neem leaf explants are shown in Fig 1 .In this experiment very low levels of contamination were observed where lcaf explants were treated with 70% ethanol prior to being subjected to IlgC1, and 10% Bleach treatments.However, leaf explants treated with 70% ethanol became brown in colour and callus production from uncontaminated leaf explants was very low (Fig. 1).Chi-squared analysis showed a highly significant effect of ethanol on production of brownish leaf explants (p<0.001).The use of ethanol as a pre-treatment may cause the penetration of NaOCl and HgC1,into internal tissues and consequently damage the leaf explants.Further statistical analysis conducted only on those leaf explants, which had not been treated with 70% ethanol, showed significant differences (p<0.05)This was due to the high contamination % observed in treatment 5J, in which leaf discs had only been treated with 10 "/o Bleach.There were no significant differences a t 5% level for the other four treatments (5F, 5G, 5H & 51).Therefore, treatment 5F containing the lowest concentration of HgC1, (0.1% HgCl, for 5 min followed by 10% Bleach for 10 min) was used for further work.The results of t h e experiment, which was conducted to compare the commercially available Bleach with s t a n d a r d chemicals a r e shown in Table 1.Chi-squared analysis showed significant differences (p<0.01) between 10% Bleach and all the other treatments except for 5% NaOCI.There was no significant difference ( ~1 0 .0 5 ) between 10% NaOCl and 15% NaOC1.At the end of the sixth week period the amount of callus produced was measured and one-way analysis of variance sl-rowed no significant differences for the 10% Bleach and 10% NaOCl (Sigma, UK) treatments.
The surface sterilisation procedure described (0.1% HgCl, for 5 min followed by 10% NaOCl (Sigma, UK) for 10 min) was effective in 85% of explants but this was found to be acceptable at practical level.Leaf explants collected from 15 wild neem trees from four locations (Dambulla, &thalawa, Maha-Illuppallama and Kalawewa) i n two agro-ecological zones (Dry zone and Intermediate zone), could be successfully sterilised by applying this method."Callus was successfully initiated using the maintenance mehum, in accord with the earlier results", irrespective of differences i n stock plant origin.Callus was brown i n colour and contained hard, partially differentiated, callus clumps, which became white and friable with continuous subculturing.The present study showed t h a t by combining two disinfectants successful surface sterilisation could be achieved.Disinfection with HgC1, was reported to be practically useful when the presence of fungal contaminants made disinfection by hypochlorite ineffective.Vhus, HgCI, may kill the microorganisms, which cannot be killed by NaOCl and vice versa.