A standard procedure for the micropropagation of the neem tree (Azadirachta indica A. Juss)

Micropropagated shoots were initiated from leaf explants of the neem tree, Azadirachta indica A. Juss. Regardless of their origin, shoots were successfully produced by culturing leaf explants on Murashige and Skoog medium containing benzylaminopurine (1 mg l^–1), kinetin (0.8 mg l^–1) and adenine sulphate (6 mg l^–1) in complete darkness. These shoots were further multiplied on Murashige and Skoog medium containing benzylaminopurine (0.1 mg l^–1), kinetin (0.08 g l^–l) and adenine sulphate (0.6 mg l^–1). Within 32 weeks, 80 shoots could be produced from a single leaf explant (10 mm×10 mm). Fifty-five percent of these shoots rooted on Murashige and Skoog medium containing indolebutyric acid (1 mg l^–1) and all of these grew on transfer to soil. Received: 5 May 1996 / Revision received: 23 August 1996 / Accepted: 5 October 1996     

Somatic embryogenesis and plant regeneration of neem (Azadirachta indica A. Juss.)

Somatic embryos were initiated with mature seeds of neem (Azadirachta indica A. Juss.) when cultured on Murashige and Skoog's medium supplemented with thidiazuron (TDZ). Regeneration occurred via somatic embryogenesis: direct embryo formation and through an intermediary callus phase. TDZ was very effective and induced somatic embryogenesis across a wide range of concentrations (1–50 μm). However, somatic embryogenesis was accompanied by callus formation at concentrations of 20 μm and above. Cell suspension cultures were established with the TDZ-induced callus and groups of large cell clumps were formed within 2–3 weeks. Plants were regenerated from both directly formed somatic embryos and somatic embryos derived from cell suspensions plated on semisolid medium devoid of growth regulators. Regenerated plantlets continued to grow after transfer to a greenhouse environment and were similar phenotypically to zygotic seedlings. This simple regeneration system may be beneficial for mass propagation of selected elite clones of neem. Received: 13 May 1997 / Revision received: 13 November 1997 / Accepted: 2 December 1997     

Statistical optimization of media for enhanced azadirachtin production from redifferentiated zygotic embryo cultures of neem (Azadirachta indica A. Juss.)

Callus cultures from zygotic embryos of neem (Azadirachta indica) were initiated and analyzed for azadirachtin production. Medium components were screened and optimized using the statistical techniques of Plackett–Burman and response surface methodology. The Plackett–Burman design, with five medium components (Murashige and Skoog major salts, sucrose, casein hydrolysate, indole-3-acetic acid, and N⁶-benzylaminopurine), was performed to screen the variables that significantly affected azadirachtin production. The three variables—Murashige and Skoog major salts, sucrose, and N⁶-benzylaminopurine—significantly affected azadirachtin production and were significant factors for optimization using response surface methodology. The experimental results were fitted to a second-order polynomial model with a correlation coefficient (R ²) of 0.9582. The optimal concentrations of variables for maximum azadirachtin production were full-strength Murashige and Skoog major salts, 5.68% sucrose, and 10.42 μM N⁶-benzylaminopurine. The maximum azadirachtin production by the predicted model was 5.13 mg/g dry weight, which was in agreement with the actual experimental value of 4.97 mg/g dry weight.     

The proteinaceous, gum polysaccharide from Azadirachta indica A. Juss

The gum exuded by Azadirachta indica trees contains 35% of proteinaceous material. The most abundant amino acid is aspartic acid; there are also considerable proportions of serine and threonine, and at least 2% of amino sugars. Attempts to deproteinise the polysaccharide material were unsuccessful. Fractionation of the gum exudate on agarose columns gave a protein-depleted fraction of high molecular weight and a protein-enriched fraction of lower molecular weight. Studies involving molecular-sieve chromatography showed a close correspondence in protein and carbohydrate content, and it appears that the proportion of proteinaceous component that is free or only loosely associated with carbohydrate is small. The carbohydrate component is much more complex than was indicated by earlier investigators. In addition to galactose and arabinose (major components), mannose, xylose, fucose, and rhamnose are also present. The uronic acid content (28%) is higher than previously believed, and a relatively high methoxyl content has been found. The major aldobiouronic acid, previously undetected, is 4-O-(4-O-methyl-- -glucopyranosyluronic acid)- -galactose, with 4-O-(- -glucopyranosyluronic acid)- -galactose also present. The exudate from Azadirachta indica is therefore a complex material having several features that are unusual in gum chemistry.     

Oxidative metabolism-related changes in cryogenically stored neem (Azadirachta indica A. Juss) seeds

The seeds of Azadirachta indica were successfully cryopreserved for 12 months with 45% survival following drying to 0.16gH(2)Og(-1) dry mass (DM). Highest survival (94-96%) was recorded during the first month of cryostorage. Subsequent cryopreservation up to 12 months resulted in decreasing germination. Post-thawing pre-heat treatment enhanced the recovery marginally in seeds cryopreserved from 3 to 12 months. Viability of cryostored seeds was negatively correlated with leachate conductivity and accumulation of thiobarbituric acid reactive substances (TBRS) estimated in cotyledons and axes. Leachate conductivity of imbibed seeds was low during the first month of cryostorage but increased gradually with the duration of cryostorage to a maximum after 12 months. TBRS accumulation was gradual throughout cryostorage. Relatively low amounts of active oxygen species (AOS) detected during the first month of cryostorage were closely associated with very high activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) in seeds (cotyledons and axes). Marked accumulation of AOS from 3 to 12 months was associated with decrease in antioxidant enzyme activity.     

Azadirachta indica A. Juss (neem) as a contraceptive: An evidence-based review on its pharmacological efficiency

BackgroundAzadirachta indica A. Juss. is an Indian medicinal plant with innumerable pharmacological properties. Studies have proven that the phytochemicals from neem possess remarkable contraceptive abilities with limited knowledge on its mechanism of action.PurposeThe present review aims to summarize the efficiency of A. indica treatment as a contraceptive.MethodsThe Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines were used. Published scientific articles on antifertility, antispermatogenic, antiovulation, hormone altering, contraceptive, and abortifacient activities of A. indica were collected from reputed Journals from 1980 to 2020 using electronic databases. Specific keywords search was completed to collect numerous articles with unique experiment design and significant results. This was followed by the selection of the requisite articles based on the criteria designed by the authors. Data extraction was based on the common research elements included in the articles.ResultsA total of 27 studies were considered for reviewing, which included key pharmacological investigations. In the beginning, authors evaluated a number of publications on the contraceptive properties of A. indica, in which it was revealed that most of the publications were made between 2005 and 2009. All the collected articles were categorised and reviewed as antifertility, antispermatogenic, antiovulation, hormone altering, contraceptive, and abortifacient. Authors also assessed studies based on the plant parts used for pharmacological evaluations including leaves, seeds, stem-bark, and flowers. The article was primarily divided into different sections based on the previous works of authors on phytochemistry and pharmacological review articles.ConclusionAlthough A. indica is not reported with the complete alleviation of reproductive system in both male and female animal models, studies have proven its efficacy as a contraceptive. Extracts and phytochemicals from neem neither reduced the libido nor retarded the growth of secondary sexual characters, thus indicating only a temporary and reversible contraceptive activity. However, there is a dearth for clinical studies to prove the efficacy of A. indica as a herbal contraceptive.     

Analysis of insecticidal Azadirachta indica A. Juss. fractions

As a result of chemical investigation on the ethanolic extract of fresh fruit coatings of Azadirachta indica A. Juss. (neem), twenty-seven compounds were identified in non-polar to less polar fractions which showed pesticidal activity determined by WHO method against Anopheles stephensi Liston. These identifications were basically made through GC-EIMS and were further supported by other spectroscopic techniques, including 13C NMR, UV and FTIR as well as retention indices. Thus sixteen n-alkanes, 1-16; three aromatics 2,6-bis-(1,1-dimethylethyl)-4-methyl phenol (17), 2-(phenylmethylene)-octanal (20), 1,2,4-trimethoxy-5-(1Z-propenyl)-benzene (27); three benzopyranoids 3,4-dihydro-4,4,5,8-tetramethylcoumarin (18), 3,4-dihydro-4,4,7,8-tetramethylcoumarin-6-ol (19), 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl-cyclopenta[g]-2-benzopyran (22); one sesquiterpene methyl-3,7,11-trimethyl-2E,6E,10-dodecatrienoate (21); three esters of fatty acids methyl 14-methyl-pentadecanoate (23), ethyl hexadecanoate (24), ethyl 9Z-octadecenoate (25) and one monoterpene 3,7-dimethyl-1-octen-7-ol (26) were identified. Except 6, 8, 24 and 25 all these compounds were identified for the first time from the pericarp and fifteen of these, 1-3, 7, 9, 10, 17-23, 26, 27, are hitherto unreported previously from any part of the tree. Although this tree is a rich source of various natural products, it is the first report of identification of mono- and sesquiterpenes 26 and 21 and a potent antioxidant, 17.     

Isolation and characterization of microsatellite markers in Indian neem (Azadirachta indica var. indica A. Juss) and cross-amplification in Thai neem (A. indica var. siamensis Valenton)

We developed eight polymorphic microsatellite loci in Indian neem (Azadirachta indica var. indica) and cross-amplified in closely related species Thai neem (A. indica var. siamensis). The number of alleles per locus in Indian neem and Thai neem ranged from 3 to 9 and 3 to 9, respectively. Average observed and expected heterozygosities in Indian neem and Thai neem were 0.63 and 0.70 and 0.61 and 0.65, respectively. Two loci exhibited significantly fewer heterozygotes than expected under Hardy–Weinberg equilibrium. These microsatellites may provide a useful tool for population genetics to establish conservation and management strategy.     

Effects of flask sealing and growth regulators on in vitro propagation of neem (Azadirachta indica A. Juss.)

In vitro propagation of neem (Azadirachta indica A. Juss.) may offer an efficient alternative to seed propagation of this species. For optimization of in vitro propagation, different basal salt formulations, growth regulators, and culture container sealants (polytetrafluoroethylene hydrophobic membranes [PTFE]) were evaluated. Nodal segments cultured on Murashige and Skoog (MS) medium showed the highest shoot formation per explant (1.67). Explants cultured in flasks containing MS medium with 0.5 mg L⁻¹ benzyladenine, 0.5 mg L⁻¹ kinetin, and 0.05 mg L⁻¹ naphthaleneacetic acid, and sealed with two PTFE membranes, produced the highest number of shoots (4.04). In contrast, explants cultured in flasks without membranes showed leaf chlorosis and senescence. For plant recovery, regenerants were acclimatized in a substrate of coconut fiber and eucalyptus bark (1:1) and showed 80% survival. Our results indicated that the use of flasks with vents was beneficial for in vitro propagation of this important plant.     

Field evaluation of the efficacy of neem oil (Azadirachta indica A. Juss) and Beauveria bassiana (Bals.) Vuill. in cotton production

Neem oil (Azadirachta indica A. Juss) alone and combined with the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (isolate Bb11) was applied to control cotton pests. The efficacy of these treatments was compared with that of synthetic insecticides applied either in a calendar‐based application or in the ‘Lutte Etagée Ciblée’ (LEC) strategy, consisting of using first calendar‐based (half‐dose) applications followed by threshold‐based treatments. The experiment was carried out in collaborative research with farmers in three cotton agro‐ecological zones differing in rainfall, pest prevalence, and farming practices. The neem oil and neem oil‐Bb11 treatments required 2 to 6 applications, while conventional and LEC received 6 to 8 applications. The percentage of damaged reproductive organs in plots treated with neem oil and neem oil plus Bb11 was higher than that recorded under the conventional and LEC strategy, with exception of the zone with the highest rainfall; this resulted in yields being 25% and 39% lower, respectively. Yields in the biopesticide plots were 26–42% higher and in the conventional and LEC plots 44–59% higher than those in the control plots that received only water. Overall, the LEC regime scored best, both in yield and profitability. The incidence of natural enemies was highest in the control and in the plots treated with biopesticides. Although the use of entomopathogen Bb11 and neem oil avoids many problems associated with the application of synthetic insecticides, their efficacy needs to be enhanced by improved formulation or by combining them with other pesticides.     

Desiccation-induced changes in lipid peroxidation, superoxide level and antioxidant enzymes activity in neem (Azadirachta indica A. Juss) seeds

The freshly harvested mature neem seeds (42.2 % seed moisture content) with 100 % viability deteriorate when naturally desiccated to below 10.9 %. The desiccation-induced loss of viability was closely associated with over accumulation of superoxide anion and lipid peroxidation products both in the embryonic axes and cotyledons. The levels of superoxide anion and lipid peroxidation products were higher in axes compared to cotyledons. Superoxide dismutase activity was not much affected, both in the axes and cotyledons of 100 % viable seeds during desiccation from 42.2 % to 10.9 % seed moisture content. Steep rise in its activity was observed during drying below lowest safe moisture content (LSMC). Activities of catalase and peroxidase exhibited substantially higher levels in the 100 % viable seeds dehydrated up to LSMC. Their activities declined sharply in seeds with water content below LSMC. Impairment of catalase and peroxidase activities possibly lead to enhanced accumulation of reactive oxygen species. The accumulation of superoxide anion, lipid peroxidation and differential expression of superoxide dismutase and catalse/peroxidase activities in response to desiccation (below LSMC) is discussed to explain the intermediate storage physiology of neem seeds.     

印楝植物内生真菌Phomopsis sp.的代谢产物研究

从印楝植物内生真菌Phomopsis sp．的菌丝体提取物中分离得到4个化合物,通过波谱技术分别鉴定为水苏碱（1）、甲基-β-D-葡萄糖苷（2）、过氧化麦角甾醇（3）、腺嘌呤核苷（4）,这些化合物均为首次从该属真菌中分离得到。     

Biological investigation and structure-activity relationship studies on azadirone from Azadirachta indica A. Juss

Azadirone 1, a limonoidal constituent of Azadirachta indica is found to possess potent cytotoxic activity against a panel of human cancer cell lines in our in vitro studies. In vitro screening of a number of semi-synthetic analogues of 1 revealed that the alpha,beta-unsaturated enone moiety or its equivalent conjugated system in A-ring, C-7 acetyloxy/chloroacetyloxy or keto group in B-ring and the furan moiety are responsible for the activity of 1 and its analogues. Compound 1 and two of the semi-synthetic analogues 10 and 13 were found to possess good in vivo antitumor activity in modified hollow fiber animal models.     

Side-effects of three neem (Azadirachta indica A. Juss) products on the predator Macrolophus caliginosus Wagner (Het., Miridae)

The side-effects of three neem formulations (Neem-Amin EC, Stardoor and B.P. 20/S) were tested on the mirid predator Macrolophus caliginosus Wagner in the laboratory. Direct toxicity tests on 1st instar nymphs exposed to fresh dry residues on glass plates at different doses demonstrated that all the products are harmful to the insects with LD₅₀ values much lower than the maximum recommended rate (1.217, 0.264, 1.083 mg a.i./l instead of 15, 31.5 and 80 mg a.i./l for Neem-Amin EC, Stardoor and B.P. 20/S, respectively). Moreover a reduction of fecundity of the surviving females was assessed with Neem-Amin EC and B.P. 20/S. Persistence tests were carried out on sharp pepper plants treated at the maximum recommended rate. High mortality was recorded when the insects were introduced onto the plants just after the treatment, but no significant differences compared with the controls were observed 5 days after the treatment and any consequence on the fecundity of surviving females was detected. Our experiments showed how azadirachtin can be noxious to M. caliginosus , but the short persistence makes this active ingredient a promising solution in integrated pest management programmes, when a lapse of time is guaranteed between the treatment and the introduction of the predator.     

Increased production of azadirachtin from an improved method of androgenic cultures of a medicinal tree Azadirachta indica A. Juss

Present report is the first direct evidence of azadirachtin production in androgenic haploid cultures of Azadirachta indica, a woody medicinal tree. Anther cultures at early-late-uninucleate stage of microspores were established on MS medium with BAP (5 µM), 2,4-D (1 µM) and NAA (1 µM) containing 12% sucrose. The calli, induced, were further multiplied on 2,4-D and Kinetin media. Shoots, differentiated on BAP (2.2 µM) + NAA (0.05 µM) medium, were elongated on MS + BAP (0.5 µM) and multiplied on MS + BAP (1 µM) + CH (250 mg/l). Thereafter, the shoots were rooted on ¼ MS + IBA (0.5 µM). Cytological analysis of the calli and regenerants have confirmed their haploid status with the chromosome number as 2n = x = 12. The haploid cell lines and leaves from in vitro grown plantlets were analyzed for azadirachtin by RP-HPLC and mass spectroscopy. Maximum azadirachtin (728.41 µg/g DW) was detected in calli supporting best shoot proliferation while least (49 µg/g DW) was observed in an undifferentiated line from maintenance medium. This study has brought us a step closer to develop genetically pure lines that could serve as new and attractive alternative ways of homogeneous controlled production of high value compounds, round the year, independent of geographical and climatic barrier.Key words: anther culture, Azadirachta indica, azadirachtin, haploid plants, RP-HPLC     

Studies on the Resin Glands of Azadirachta indica A. Juss. (Meliaceae)

The leafy shoots of Azadirachta indica have numerous, yellowresin glands, composed of a short stalk and sub-glandular tissuebeneath an epithelium-like 1-2-layered secretory tissue. Thefoot arises from the epidermis. The resin glands are derivedfrom a single papillate epidermal cell which functions as aresin gland initial. Histochemical localization using fluorescencemicroscopy confirmed the resinous nature of the secretion. Thegeneral structure of the glands shows similarity to extra-floralnectaries. Reports in the literature on the occurrence of extrafloralnectaries in Meliaceae should be confirmed by analysis of thesecretions if they are to be accepted. Azadirachta indica, resin glands, ontogeny, structure, secretion     

印楝内生真菌棘孢曲霉YM 311498的代谢产物研究

从印楝植物内生真菌棘孢曲霉（Aspergillus aculeatus）YM311498的发酵液提取物中分离得到4个化合物,通过波谱技术分别鉴定为大黄酚（1）、大黄素（2）、琥珀酸（3）、柠檬酸二甲酯（4）,以上化合物均为首次从该种真菌中分离得到。     

Enhanced production of azadirachtin by hairy root cultures of Azadirachta indica A. Juss by elicitation and media optimization

Azadirachtin is one of the most potent biopesticides so far developed from a plant sources. Influence of different culture media and elicitation on growth and production of azadirachtin by hairy root cultures of Azadirachta indica was studied. Out of the three media tested, namely Ohyama and Nitsch, Gamborg's and Murashige and Skoog's basal media, hairy roots cultured on Ohyama and Nitsch's basal medium produced maximum yield of azadirachtin (0.0166% dry weight, DW). Addition of biotic elicitor enhanced the production of azadirachtin by approximately 5-fold (0.074% DW), while signal compounds such as jasmonic acid and salicylic acid showed a approximately 6 (0.095% DW) and approximately 9-fold (0.14% DW) enhancement, respectively, in the production of azadirachtin as compared to control cultures on Ohyama and Nitsch medium. Extracts from hairy roots were found to be superior to those from the leaves for antifeedant activity against the larvae of Spodoptera litura.     

Neem,Azadirachta indica A. Juss. (meliaceae), and its potential for sustainable woodcarving in Kenya

The density of neem trees in parts of Malindi District in eastern Kenya was assessed to determine the sustainability of their use in the local woodcarving industry. Size and age structure of neem populations under different ownership were analyzed to establish the Annual Allowable Cut. Neem is an excellent carving wood and the available quantities along the Kenyan coast are sufficient to sustainably supply the industry of the whole country. However, because the resource comes from small farm holdings and the supply system to carving groups is not well established, further efforts are required to introduce a management regime that could be certified under the standards of the Forest Stewardship Council.     

Production of triploid plants from endosperm cultures of Phlox drummondii

Triploid plants of ornamental Phlox drummondii Hook. were raised from cultures of endosperm excised from immature fruits having zygotic embryo at early dicotyledonous stage. Endosperm tissue was firstly cultured with the embryo on the Murashige and Skoog’s (MS) medium supplemented with 5 μM 6-benzylaminopurine (BAP) + 10 μM α-napthaleneacetic acid (NAA) for 7 d and recultured after the embryo was removed. A friable callus appeared two weeks after removal of the embryo and it became compact callus mass in another three weeks. Upon transfer of this 5-week-old callus to the MS medium with 10 μM BAP + 2.5 μM indole-3-acetic acid (IAA), maximum percentage of green nodular shoot buds appeared from which regenerated dwarf shoots. Elongation of the dwarf shoots, however, required transfer of the individual dwarf shoots excised from the callus on the fresh medium and best results achieved on medium with low concentration of IAA (0.5 μM) in presence of 10 μM BAP. The shoots were then rooted in vitro and plants subsequently established in pots containing soil. Over 70 % of plants were triploid with a chromosome number of 2n=3x=21. Size of stem, leaves, flowers, pollen, and stomata of these triploid plants were higher and the plants were more vigorous as compared to naturally occurring diploid plants. In particular, flowers showed bright colour with enlarged central eye adding to their ornamental value.