Neem (Azadirachta Indica A. Juss) callus induction and its larvaecidal activity against Anopheles mosquito.
The aim of this study was to establish an effective protocol for callus induction from the leaf explant of the neem (Azadirachta indica A.Juss) and to investigate its larvaecidal activity against the larvae of Anopheles mosquito. The effects of growth regulator, basal media strength, sucrose and crude aqueous extracts were investigated. Growth regulator effect was studied by using four different concentrations (0.5, 1.0, 1.5, and 2.0 mg/L) of indolyl- 3-acetic acid (IAA), indolyl- 3- butyric acid (IBA), naphthalene acetic acid (NAA) and 2,4-dichlorophenoxy--acetic acid (2,4-D) alone or in combination with 0.5 mg/l of 6-bezyladenine (BAP). IBA alone or in combination with BAP produced the highest callusing percentage and the best callusing degree compared to other auxin at the same concentrations. Auxin when supplied in combination with BAP did not induce any significant increases in callusing percentage or degree of callusing. However, the times required for callus to be developed was shortened and the quality of the induced callus was improved. Increase in callus growth on medium with low salt and sucrose concentration was greater than on medium supplemented with high salt and sucrose concentration.
The effect of crude aqueous extracts obtained from the neem leaf callus and neem fresh leaf against the larvae of Anopheles mosquito was investigated separately. Exposure of the larvae to 20 ml extracts of leaf callus and fresh leaf for 24 hours led to 100 and 63% mortality rate, respectively.
Keywords: larvaecidal activity, Growth regulator, Aqueous extracts,
The neem (Azadirachta indica A. Juss) is an evergreen, fast growing tree grows in subtropical parts of Africa and Asia and is renowned for its insecticidal properties. Neem tree has been reported to contain several biologically active constituents such as azadirachtin , meliantriol , salanin , as well as nimbin and nimbidin .
Azadirachtin is particularly well-known for its effectiveness as a defensive compound against insects, acting as a strong anti-feedant and causing growth disruption in many insect species [5, 6]. Due to its favorable properties such as low toxicity against non-target organisms, low persistence in chromatognature and systemic action , azadirachtin and its natural or synthetic analogues are highly promising candidates for the development of novel naturally derived insecticides. In fact, a variety of extracts obtained from seed, bark and leaf of the neem tree have been successfully investigated as environmentally acceptable bioinsecticides used in crop protection  and control of mosquito's larvae . However, since these insecticides are based on extracts obtained from intact neem organs, the concentrations of azadirachtin and related triterpenoids are rather low and may vary due to fluctuating contents in the natural sources, or susceptibility of the compounds to environmental influences (heat, light) .
Plant cells/tissue cultures offer an opportunity for continuous production of plant metabolites, and represent an attractive production source, since it is scalable according to specific needs . Moreover, it is well known that, in vitro culture is able to provide secondary metabolite, some times even in quantities that allow economically feasible production [11, 12, 13]. Furthermore, tissue culture technique like callus culture could provide an alternative supply of compounds for use in medicine, stimulating the production or inducing the biosynthesis of novel compounds not found in the intact plant [14, 15]. In recognition of all these important properties of tissue culture, the present study is an attempt to develop an efficient protocol for callus induction from the leaf explant of the neem and investigate its crude extract larvaecidal activity against the larvae of anopheles mosquito. The choice of crude extract was to ensure adaptability by the local populace and low-cost intervention strategy
Mosquitoes constitute a major public health menace as vectors of serious human diseases . Anopheles mosquito is the principal vector of malaria in Sudan, according to the Federal Ministry of Health National Malaria Control Programme 2003, this parasitic disease infects 7.5 million patients each year and 35,000 die from this disease, which accounts for up to 20% of hospital deaths .
Materials and methods
Fresh leaves obtained from ten months-old neem (Azadirachta indica A. juss) tree located at the nursery of the Commission of Biotechnology and Genetic Engineering were used as source of explants through out this experiment.
Surface sterilization of explants
Leaf explants were brought to the laboratory then washed and cleaned carefully with continuous running tap water for 15 minutes to remove all the surface dust. Next, the leaves were disinfected by rinsing in sterilized distilled water with 15 mg ascorbic acid, 100 mg citric acid and one gram of activated charcoal and shaked continuously for 10 minutes, and then the leaves were allowed to soak in a 70% ethanol solution for one minute. Finally, the leaf explants were soaked in 10 % Clorox with two drops of Tween 20 for 15 minutes followed by three times rinsing with sterile distilled water.
The medium used for callus culture was MS  medium, supplemented with 30 g/L sucrose, 100 mg /L myo-inositol, 0.4 mg /L thiamine-HCl and 8 g/L agar. All media used in this experiment were adjusted to pH 5.8, and then distributed in 25x 150 mm culture tubes covered with plastic Bellco kaptus before autoclaving at 121[degrees]C for 15 minutes under pressure of 15 PSI. The media were left to cool as slant in the culture room until use.
Inoculation of explants material
Under laminar flow hood, the leaves were cut into three pieces using a scalpel and the leaf discs were placed adaxial side down into culture tube containing 15 ml of MS media. The culture tubes were then placed in incubator at 25 [degrees]C under 16-hours light of 1000 lux using fluorescent lamps. The resulting callus was transferred to fresh media every 4 weeks.
Effects of growth regulator on callus growth
This experiment was designated with four different concentrations (0.5, 1.0, 1.5 and 2.o mg/L) of indolyl- 3- acetic acid (IAA), indolyl- 3- butyric acid (IBA), naphthalene acetic acid (NAA) and 2,4-dichlorophenoxy--acetic acid (2,4-D) alone or in combination with 0.5 mg/l of 6-bezyladenine.
MS basal medium (MS macro + MS micro) supplemented with 8g/l agar was used throughout the experiment. All the experiment treatments were replicated at least 10 times, and data for callusing percentage and degree of callusing were recorded after 8 weeks of culture.
Effects of sucrose and basal medium strength on callus growth
This experiment was designed with sucrose concentration of 10, 20 and 30 gm/l in half and full strength of MS basal medium supplied with 1.0 mg/L of IBA in combination with 0.5 mg/l of 6-bezyladenine.
Extractions and larvaecidal assays
The freeze-dried powdered callus and fresh leaf (obtained from the same explants source) of neem were extracted with MeOH at room temperature. The MeOH extract was evaporated and 3.0 g dissolved in 50 ml distilled water was used. Mosquito larvae were collected in a plastic bucket from a pool of stagnant water. The bucket containing the larvae was kept in netted enclosure in the Laboratory at room temperature (30 [degrees]C [+ or -] 2). Three sets of five Petri dishes were laden with moist filter paper and placed on a laboratory bench. Using a metal loop, 60 mosquito larvae were transferred from the plastic bucket into each of the twelve Petri dishes. The sets of Petri dishes were labeled a, b, c, d and e. In the first set, 5 mls of the leaf callus extract were added into dish (a), 10 mls in (b), 15 mls in (c) and 20 mls in (d). In Petri dish (e), 20 ml of distilled water was added as control. In the second sets of Petri dishes, leaf extracts were added in the same manner as for callus in the first set. The effects of the four extracts were monitored by counting the number of dead larvae at one-hour intervals for the first 12 hours, then at 6 hours interval for the next 12 hours. Three replicates of the experiments were conducted and the mean were taken. The data obtained was statistically assessed to determine variance and standard error of the mean.
Effects of growth regulator, sucrose and basal media strength on callus growth
The neem callus growth was remarkably affected by growth regulator type and concentration. While there was a little extension growth of the explants, no callus growth was observed during the culture period in the basal medium without growth regulator (Control). However, callus developed on the surface of the leaf explant cultured on MS basal medium supplemented with varying concentrations of either IBA, NAA, IAA or 2, 4-D alone or in combination with BAP (Table 1). Leaf explants enlarged and developed callus at the cut surface in all media within 1-3 weeks of inoculation depending on auxin type and concentration. Of the various auxins tested IBA at different concentrations induced rapid, maximum callusing percentage, healthiest invariably greener and more granular callus from leaf explant compared to IAA, NAA and 2, 4-D when supplied at the same concentrations. Within the different concentrations of IBA (0.5-2mg/l), MS basal medium supplemented with 1mgl\L promoted rapid growth, produced the highest callusing percentage (92.6%) and the best callusing degree (more than 50%). However, IBA at higher concentration (2mg/l) induced the lowest callusing percentage (80.0%) and compact calli.
Auxin at different concentrations when supplied in combination with BAP (Table 1) did not induced any significant increases in callusing percentage or degree of callusing when compared to auxin alone. However, the times required for callus to be developed on surface of leaf explant was shortened to only one week and the appearance of the induced callus was improved and become a more viable and look healthier even at higher auxin concentrations. Similar to the result we obtained with using auxin alone, IBA at different concentrations when added in combination with BAP induced rapid and maximum callusing percentage from leaf explant compared to IAA, NAA and 2, 4-D when supplied at the same concentrations. MS basal medium containing 1mgl\L of IBA in combination with 0.5 mgl\L of BAP promoted rapid growth, produced the highest callusing percentage (95.0%) and the best callusing degree (more than 50%) (Table 1).
The effects of three levels of sucrose concentrations and two MS basal medium strength on callus induction is shown in table 2. The callus induction decreased by increasing sucrose concentration. When the basal media was supplied with 10g/l of sucrose the percentage of leaves callused, the degree of callusing and induced callus appearance were significantly better than at 20 or 30g sucrose in both half and full MS basal medium strengths. Callusing response and callus appearance were better in half strength MS basal medium than full strength only for 20 and 30 g/l levels of sucrose concentrations tested.
The effects of crude aqueous extracts obtained from the callus and fresh leaves of the neem on the mortality of the larvae of Anopheles sp. mosquito are presented in Table 3. When 20 ml of the callus extract was applied, all the larvae (100%) died within 24 h. However, for the same period, only 63% of the larvae died when leaf extract used. Decreasing the amount of the callus extracts to 15, 10 and 5 ml, reduced the mortality rate to 97, 90 and 82%, respectively, after 24 h. (Table 3 A). On the other hand, decreasing the amount of the leaf extracts to 15, 10 and 5 ml gave mortality rate of 53, 50 and 40% respectively, after the same time (Table 3 B).
The cultural conditions for neem callus induction and growth were established. We have shown that the presence of auxin alone or in combination with the cytokinine (BAP) in the medium was capable of inducing callus. However, the callusing percentage, degree of callusing and callus appearance were dependent on auxin type and concentration.
The leaf explant induced the best callus when cultured on half strength MS basal medium supplemented with 1.0 mg/L of IBA, 0.5 mg/L of BAP and 10g/l of sucrose. However, it failed to produce callus in media without auxin, this declared that, in vitro callus formation is attributed to the presence of growth regulators in the medium. Generally cytokinine and auxin are known to promote callus formation in tissue culture [19, 20, 21]. Moreover, similar findings on using IBA in combination with BAP for neem callus induction were already reported . Furthermore, in agreement with our result, in a other study on neem callus induction it was found that low sucrose concentration in the medium favors more callus induction compared to high concentration .
The use of neem as a pesticide is well known [24, 25] and extracts obtained from seed, bark and leaf of the neem tree have been successfully investigated as environmentally acceptable bioinsecticides for use in crop protection [5, 8] and control of mosquito's larvae . All these extracts were obtained from intact neem plant organ. Here in this study we used extract obtained from in vitro produced undifferentiated cells. Therefore, the higher larvaecidal activity recorded might be attributed to the higher quantity of azadirachtin in the in vitro induced callus compared to that of the intact leaf. It is well known that in vitro cultures are able to produce secondary metabolites, sometimes even in quantities that allow economically feasible production [11, 12, 13]. Further more, previous studies [14, 15] have reported that callus culture could provide an alternative supply of secondary metabolite for use in medicine, stimulating the production or inducing the biosynthesis of novel compounds not found in vivo. This is because in callus cultures cells are undifferentiated, which means the genes that are in control of secondary metabolite production may be turned off or not under specific control .
In conclusion, the results of this study shows that extracts derived from neem callus have demonstrable larvaecidal activities, therefore further work is needed to isolate the secondary metabolites from the extracts studied in order to test specific larvaecidal activity. Moreover, large scale neem callus system production for the commercial production of secondary metabolites is a promising system and deserves further investigation.
This work was supported by Commission for Biotechnology and Genetic Engineering, (CBGE), National Centre for Research (NCR), Khartoum, Sudan. We are grateful for comments on this paper from Professor Rashied Modawi, Biology Department, School of life Sciences, Faculty of Science and technology, Al Neelain University, Khartoum Sudan
 Naganishi, K., 1975, "Structure of the insect antifeedant azadirachtin," In: Recent advances in phytochemistry, VC Runeckles (ed.), Plenum, New York, N.Y.Vol.5 pp. 283-298.
 Lavie D, Jain M K., and Shpan-Gabrielith, S R., 1965, "A locust Phagorepellent two Melia species," Chem. Commun. 910-1911.
 Warthen, J D., Jr -Uebel, E C., Dutky, S R., Lusby, W R., and Finegold, H., 1978, "an adult housefly feeding deterrent from neem seeds," UD Agric., Agric. Res. Result RR-NE 2.
 Shin-foon, C., 1984, "The active principles and insecticidal properties of some chine's plant, with special Reference to Melicaceae," pp 255-262 in Schmutterer and Ascher, (see under above conference reports).
 Mordue (Luntz), A J., and Blackwell, A., 1993, "Azadirachtin--an update," J. Insect Physiol. 39:903-924.
 Mordue (Luntz), A J., Simmonds, M S J., Ley ,S V., Blaney, W M., Mordue ,W.,Nasiruddin M, Nisbet AJ (1998) Actions of azadirachtin, a plant allelochemical, against insects. Pestic. Sci. 54: 277-284.
 Otmar, S., Andrew, P., Jarvis, S/, Andrew, V D E., Germina, G., and Neil, J O., 2000, "Rapid and sensitive analysis of azadirachtin and related triterpenoids from neem (Azadirachta indica) by high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry" Journal of Chromatography A, 886, pp. 89-97.
 Jacobson, M., 1988 "Focus on photochemical pesticides" Volume-1. The neem tree. CRC Press, Inc. Boca Raton, Florida. U.S.A.
 Aliero, B L., 2003, "Larvaecidal effects of aqueous extracts of Azadirachta indica (neem) on the larvae of Anopheles mosquito" African Journal of Biotechnology., 2 (9), pp. 325-327.
 Smith, M A., and Pepin, M., 1999, "Stimulation of bioactive flavonoid production in suspension and bioreactor-based cell cultures" In: Altman A, Ziv M, Izhar S., editors. Plant Biotechnology and In Vitro Biology in the 21st Century. Dordrecht: Kluwer Academic Publishers, pp. 333-336.
 Fujita, Y., 1988, "Industrial production of shikonin and berberine" in: Bock, G. and Marsh, J. [Eds.] Applications of plant cell and tissue culture. John Wiley and Sons Ltd., Chichester, UK. pp. 228-235.
 Fujita, Y., Hara, Y., Ogino, T., and Suga, C., 1981, "Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon" Plant Cell Rep., 1, pp. 59-60.
 Hara, Y., Morimoto, T., and Fujita, Y., 1987 "Production of shikonin derivatives by cell suspension cultures" Plant Cell Rep., pp. 6:8-11.
 Furmanowa, M., Glowniak, K., "Syklowska-Baranek K, Zgorka G, Jozefczyk A. (1997) Effect of picloram and methyl jasmonate on growth and taxane accumulation in callus culture of Taxus x media var. Hatfieldii" Plant Cell Tissue Organ Cult. 49, pp. 75-79.
 Zhao, J., Hu, Q., Guo, Y Q., and Zhu, W H., 2001, "Effects of stress factors, bioregulators, and synthetic precursors on indole alkaloid production in compact callus clusters cultures of Catharanthus roseus" App Microbiol Biotechnol, 55, pp. 693-8.
 El Hag, E A., Nadi, A H., and Zaitoon, A A., 1999, "Toxic and growth retarding effects of three plant extracts on Culex pipiens larvae (Diptera Culicidae)" Phytother. Res., 13, pp.388-392.
 RBM in Sudan progress report., 2002, "Achievements, constraints and challenges"[http://www.emro.who.int/rbm/SUDProgReport02.pdf] Republic of the Sudan Federal Ministry of Health National Malaria Control Programme 2003.
 Murashige, T., and Skoog, T F., 1962, "A revised medium for rapid growth and bioassays with tobacco tissue cultures" Physiolgia Plantarum, 15, pp. 473- 479.
 Skoog, F., and Armstrong, D J., 1970, "Cytokinin" Annu. Rev. Plant Physiol, 21, pp. 359-384.
 Letham, D S., 1974 "Regulators of cell division in plant tissues. XX. The cytokinines of coconut milk" Physiol. Plant, 32, pp. 66-70.
 Akiyoshi, D E., Morris, R O., Hinz, R., Mischke, B S., Kosuge, T., Garfinkel, D J., Gordon M P, and Nester, E W., 1983, "Cytokinin/ auxin balance in crown gall tumors is regulated by specific loci in the T-DNA". Proc. Natl. Acad. Sci. USA., 80, pp. 407-411.
 Murthy, B N S., and Saxena, P K., 1998, "Somatic embryogenesis and plant regeneration of neem (Azadirachta indica A. Juss.)" Plant Cell Rep, 17, pp. 469-475.
 Wewetzer, A., 1998, "Callus cultures of Azadirachta indica and its potential for the production of azadirachtin" Phytoparasitica 26 (1), pp. 47 -52.
 Ascher, K R S., 1993, "Nonconventional insecticidal effects of pesticides available from the neem tree, Azadirachta indica. Arch" Insect Biochem. Physiol., 22, pp.433-449.
 Schmutterer, H., and Ascher, K R S., 1987, "Natural Pesticides from the Neem Tree and Other Tropical Plants" Proc. 3rd Int. Neem Conf. (Nairobi, Kenya).
 Wink, M., 1986, "Storage of quinolizidine alkaloids in epidermal tissues" Z. Naturforschung 41 c, pp. 375-380.
Mutasim Mohamed Khalafalla (1) *, Eisa Ibrahim El Gaali (1), Fatima Misbah Abbas (1) and Hind Ahmed Ali (2)
(1) Commission for Biotechnology and Genetic engineering, National Center for Research P. O. Box 2404Khartoum, Sudan
(2) Faculty of Sciences & Technology, Al-Neelain University, Khartoum, Sudan E-mail: firstname.lastname@example.org
Table 1: Effects of phytohormones on callusing percentage and degree of callusing in leaf explant culture of neem Phytohormones (mg/l) No. of % of explant Degree of explants callused (a) Callusing (b) cultured Auxin Cytoki- nine IBA 0.0 BAP 0.0 30 0 -- 0.5 0 30 86.5 [+ or -] 1.12 ++ 1.0 0 30 92.6 [+ or -] 2.10 +++ 1.5 0 30 89.0 [+ or -] 1.94 +++ 2.0 0 30 80.0 [+ or -] 2.47 ++ 0.5 30 89.5 [+ or -] 2.03 +++ 1.0 30 95.0 [+ or -] 1.49 +++ 1.5 30 81.5 [+ or -] 2.69 ++ 2.0 30 68.5 [+ or -] 1.98 ++ IAA 0.5 0 30 75.5 [+ or -] 1.50 ++ 1.0 0 30 72.5 [+ or -] 1.71 ++ 1.5 0 30 72.0 [+ or -] 2.00 ++ 2.0 0 30 59.0 [+ or -] 1.00 + 0.5 30 51.0 [+ or -] 3.86 ++ 1.0 30 43.0 [+ or -] 1.33 ++ 1.5 30 38.5 [+ or -] 1.98 ++ 2.0 30 34.0 [+ or -] 3.23 + NAA 0.5 0 30 79.0 [+ or -] 1.33 ++ 1.0 0 30 72.0 [+ or -] 0.83 ++ 1.5 0 30 61.0 [+ or -] 2.89 ++ 2.0 0 30 60.0 [+ or -] 2.47 ++ 0.5 30 53.0 [+ or -] 0.76 ++ 1.0 30 47.0 [+ or -] 0.82 ++ 1.5 30 34.0 [+ or -] 1.25 ++ 2.0 30 22.5 [+ or -] 0.83 ++ 2, 4-D 0.5 0 30 38.0 [+ or -] 3.50 + 1.0 0 30 33.0 [+ or -] 4.03 + 1.5 0 30 25.0 [+ or -] 3.00 + 2.0 0 30 12.0 [+ or -] 2.25 + 0.5 30 39.0 [+ or -] 1.63 + 1.0 30 18.5 [+ or -] 1.30 + 1.5 30 19.5 [+ or -] 1.38 + 2.0 30 12.5 [+ or -] 0.83 + (a) [+ or -] Standard error. (b) Area of explant surface covered by callus is directly related to the number of plus signs. + less than 25% area covered, ++ 25-50% area covered, +++ more than 50% area covered Table 2: Effect of MS basal media strength and sucrose concentration on callusing Basal media Sucrose No. of % of explant Degree strength (a) (g) explant callused (b) of call- cultured using (c) Half MS 10 30 87.0 [+ or -] 2.60 +++ 20 30 63.0 [+ or -] 5.64 ++ 30 30 57.0 [+ or -] 6.55 + Full MS 10 30 86.0 [+ or -] 3.37 +++ 20 30 38.0 [+ or -] 4.64 ++ 30 30 29.0 [+ or -] 2.39 + (a) MS supplemented with 1.0 mg/ IBA and 0.5 mg/l BAP. (b) [+ or -] Standard error (c) Area of explant surface covered by callus is directly related to the number of plus signs. + less than 25% area covered, ++ 25-50% area covered, +++ more than 50% area covered percentage and degree of callusing in leaf explant culture of neem Table 3: Mortality rate of neem leaf callus (A) and neem leaf (B) extracts on anopheles mosquito larvae A Extract Concentration (ml) Time (hour) 1 2 3 4 5 6 7 8 9 5 2 3 3 3 5 3 5 4 3 10 3 2 2 3 2 3 4 3 4 15 4 3 3 2 4 3 3 4 3 20 4 4 3 3 2 3 3 4 4 Control 0 0 0 0 0 0 0 0 0 A Extract Concentration (ml) Time (hour) 10 11 12 18 24 Total % Mean SE [+ (%) or -] 5 3 3 3 4 5 49 82 4 0 10 5 3 4 6 10 54 90 4 1 15 4 3 4 8 10 58 97 4 1 20 3 3 5 8 11 60 100 4 1 Control 0 0 0 0 0 0 0 0 0 B Extract Concentration Time (hour) (ml) 1 2 3 4 5 6 7 8 9 5 0 0 0 3 2 2 1 1 2 10 1 1 2 3 3 3 2 2 1 15 1 2 2 3 3 3 2 2 2 20 3 3 3 3 3 3 3 2 3 Control 0 0 0 0 0 0 0 0 0 B Extract Concentration Time (hour) (ml) 10 11 12 18 24 Total % Mean SE [+ (%) or -] 5 3 2 2 3 3 24 40 2 0 10 3 2 3 2 2 30 50 2 0 15 3 1 2 3 3 32 53 2 0 20 3 1 2 3 3 38 63 3 0 Control 0 0 0 0 0 0 0 0 0
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|Author:||Khalafalla, Mutasim Mohamed; El Gaali, Eisa Ibrahim; Abbas, Fatima Misbah; Ali, Hind Ahmed|
|Publication:||International Journal of Biotechnology & Biochemistry|
|Date:||Jan 1, 2007|
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