Title of Invention

A MANNOSE BINDING LECTIN FROM LEAVES OF ALLIUM SATIVAM EFFECTIVE AGAINST WHITEFLY, AND PROCESS FOR ITS PREPARATION

Abstract

Heretofore pest control was usually done by application of chemical pesticides, such as, for instance, pyrethroid and pyrethrin derivatives, organophosphates, chlorinated hydrocarbons, nicotin and nicotinic acid derivatives and particularly in India, neem extracts. But such conventional pesticides did not produce desired effect on certain pests like whitefly and /or cotton aphid as the pesticides when applied often did not or could not reach the target pests because of their peculiar perch. The present invention aims at overcoming the above defects of the the conventional pesticides and protect crop loss by affecting the target pests, whitefly and cotton aphids with the help of mannose binding insecticidal lectins from leaves of Allium sativum (garlic). The lectin has undernoted protein sequence marnlltnge glyagqsldv eqykfimqdd cnlvlyeyst piwasntgvt gkngcravmq rdgnfwydv ngrpvwasns vrgngnyilv Iqkdrnwiy gsdiwstgtl lehrvptsfy. The above lectin has been expressed in rice, mustard and chickpea. The transgenic plants have shown pronounced antagonistic effects against green leaf hopper of rice, mustard aphid and chickpea aphids. The present invention also relates to a process for isolation of pure lectin from leaves of Allium sativum which is a rather cost effective one. Figures 1 and 2 of the drawings illustrate the invention

Full Text

The present invention relates to a mannose binding insectisidal ectin from leaves of Allium sativum, effective against whitefly (Bemisia tabaci) and cotton aphid (Aphis gossipi) also process for preparing the same. More particularly, this invention pertains to isolation of mannose binding insectisidal lectin from vegetative leaf tissue, purification thereof and a novel bioassay procedure to demonstrate its insecticidal effect on the sap sucking homopteran insects, Bemisia tabaci, commonly known as whitefly, affecting a large number of important crop plants and Aphis gossipi, these days becoming a very important pest of cotton. Background of invention Bemisia tabaci, commonly known as whitelly, has been known for nearly a century as a tobacco pest and afterwards has become one of the most notable pests of the world agriculture affecting many economically important plants. In addition to the direct feeding damage, the insect vectors a number of devastating plant viruses (e.g.yellow mosaic virus), causing destructive diseases and by the excretion of honeydew, reduces the quality of harvested products. Likewise, Aphis craccivora (chickpea aphid), Aphis gossipi (cotton aphid) and green leafhopper of rice are other important sucking pests which cause enormous damage on respective crop plants. However, developing pest resistance through transgenic approach has revolutionized crop protection scenario in the last two decades. The process of creating resistant cultivars begins with identifying the active compound showing toxic or antifeedant activity against target pests. Control agents reported thus far are u nable t o c ontrol the homopteran pests. On the other hand, there are some mannose binding plant lectins, which have been proved to be antagonistic to different homopteran insects. These lectins are carbohydrate-binding proteins of non-immune origin. Although these lectins are primarily storage proteins due to their inherent carbohydrate binding property, they are sometimes utilised in nature as defense component by binding to the cell membrane of the attacking pathogen, thereby destabilizing its metabolism. Keeping this in mind mannose-binding lectins from leaves and bulbs of Allium sativum (garlic) were purified and tested on red cotton bug, mustard aphids (Bandyopadhyay et al 2001 and Roy et al 2002), chickpea aphids and green leafhopper (Majumder et al 2004). Many lectins are reported to be toxic to mammals, e.g. Phaseolus vulgaris lectin remaining undigested exhibits adverse effects on rats. Hence, lectins which show detrimental effect on growth and fecundity of insects, need to be checked for their non-toxicity to non-target organisms or consumers, and also investigated, whether they are allergenic or not, before introducing them in plants for development of resistance against pests/insects. Their efficacy on growth and survival of the target pests, whitefly (Bemisia tabaci), chickpea aphid (Aphis craccivora) and cotton aphid (Aphis gossipi) green leaf hopper of rice (Nephottetix sp.) were monitored. Finally the lectin (ASAL) gene was expressed in mustard, tobacco, chickpea and rice. All transgenic plant types exhibited detrimental effect on survival and fecundity of mustard aphid, peach potato aphid, chickpea aphid and green leafhopper of rice respectively. Extraction and purification of Allium sativum leaf lectin was performed by following steps which was followed by whitefly and cotton aphid bioassay in artificial diet. • Homogenisation of leaves in diaminopropane (DAP) • Filtration of the extracts followed by centrifugation • Addition of CaCl2 to the supernatant followed by centrifugation • Addition of NaCl to the supernatant followed by centrifugation • Dialysis of the supernatant against phosphate buffered saline (PBS pH 7.4) • Binding of the suspension to a mannose agarose column followed by eliminating the unbound proteins and eluting the lectin with DAP • Conducting the ion exchange chromatography followed by elution of the pure lectin with 20 mM TrisCl pH 5.0. Whitefly and cotton aphid bioassays were set up with different doses of purified lectin in artificial diet and the LC 50 values have been calculated. The present invention attempts to overcome the difficulties above by providing a pure mannose binding Allium sativum leaf agglutinin, herein referred to as ASAL in this specification for the sake of brevity and convenience, on a large scale which is characterized by their non-toxicity to non-target consumer and/organisms and also their non-allergenicity. The principal objective of this invention is to provide a mannose binding lectin from leaves of Allium sativum, effective against whitelly ( Bemisia tabaci) and cotton aphid (Aphis gossipi) among others. A further objective of this invention is to provide a mannose binding lectin of native molecular weight of around 25 kDa, gives a spot in 2-D PAGE denoting approximate pI of 5.25 and is able to agglutinate rabbit erythrocytes. A still further objective of the present invention is to provide a mannose binding lectin which is stable at room temperature, digested by pepsin and whose LD 50 values against whitefly and cotton aphid in artificial diet are 8.5µ g/ml and 8.49 µg/ml, respectively. Another objective of this invention is to provide a mannose binding lectin which when expressed in mustard, tobacco and rice, the transgenic plants themselves show antagonistic action against mustard aphid, peach potato aphid and green leafhopper of rice in in vivo condition. Yet another objective of this invention is to provide a process for extraction and purification of insecticidal mannose binding lectin from leaves of Allium sativum . The foregoing objects are fulfilled by the subject invention which relates to a mannose binding insectisidal lectin from plant parts of Allium sativum particularly effective against whitefly ( Bemisia tabaci) and cotton aphid ( Aphis gossipi), having protein sequence as given below. ASAL protein sequence: The nucleotide sequence of the present protein and blast analysis with the related sequences are shown above. The present application also provides a process for extraction and purification of mannose binding insectisidal lectin from Allium sativum leaf as defined above, which comprises (i) collecting leaves of aforementioned plant, cleaning them by washing with distilled water, drying them and homogenizing the dried leaves in 20mM diaminopropane ; (ii) filtering the extracts followed by addition of 0.02M PMSF and incubation at room temperature ; (iii) adding 20mM Calcium chloride to the supernatant liquid after centrifuging the same and incubation overnight; (iv) addition NaCl to a final concentration of 200mM and incubating overnight at 4°C; (v) dialyzing the supernatant against phosphate buffered saline (PBS) maintained at around pH 7.4; (vi) binding the suspension from step (v) to a mannose agarose column and eliminating the unbound proteins by standard technique(s); (vii) eluting the bound lectin from the said column with diaminopropane (DAP) and (viii) conducting ion exchange chromatography followed by elution of the pure lectin with 20mM Tris-Cl at around pH5.0 and collecting the eluant containing pure lectin. Description of the invention Four mannose-binding lectins have been isolated from the plant species of Colocasia esculantum, Diffenbachia sequina of family Araceae and Allium sativum and Allium cepa (Alliaceae family) for determination of their efficacy against homopteran pests (Bandyopadhyay etal, 2001 and Roy et al 2002). They have been characterized as the members of the monocot mannose binding lectin superfamily based on their molecular weight, sequence similarity and mannose specificity. The crude extract of leaves homogenized in Tris buffer was initially loaded on mannose agarose column, which strongly supports the mannose specific binding property of the lectin. Subsequently the lectin was purified through DEAE-Sephacel column, analysed in gel. For purifying ASAL on a large scale the initial extraction and the steps of column purification were modified and optimized into a novel procedure, which is described below in detail in the following sections. After purification, determination of the molecular weight of the purified protein, non- denaturing gel running and comparative analysis with known molecular weight standard through HPLC were carried out. For the verification of the purity of the lectin, agglutination reaction with rabbit erythrocytes was conducted. Bioassay had been set against homopteran pests, whitefly in artificial diet supplemented with different concentrations of pure lectin. Insect mortality had been recorded at every 24 hours. The particular lectin showed detrimental effect on growth and development of the tested insects. LD 50 value of the lectin against target insects, Bemisia tabaci (whitefly) and Aphis gossipi (cotton aphid) had been determined by statistical analysis. For determining the allergenecity of the lectin, enzymatic assay with pepsin in simulated gastrointestinal fluid had been conducted and subsequent profile of the protein in SDS-PAGE was analysed. The coding sequence of the lectin had been isolated previously and cloned initially in pUC18 vector and the sequence has been submitted and accepted on 12th January by the GenBank of National Centre for Biotechnology Information (NCBI) US National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894, USA (Accession no. AY 866499). The ASAL gene has further been cloned in plant vector and through Agrobacterium mediated transformation was introduced in mustard, tobacco and IR64, the elite cultivar of rice. The transgenic plants showed resistance against Lipaphis erysimi , Myzus persicae, Aphis craccivora and Nephottetix sp., respectively. The above process can be elaborated by means of the following example which is given by way of illustration and not by way of limitation. Example: Purification of ASAL on a large scale: For purification of ASAL in large scale, a novel extraction protocol has been devised as described below. Fresh tender leaves of garlic were taken and homogenized in an extraction buffer of 20 mM diaminopropane (DAP) at 2mI per gram of fresh tissue. The homogenate was filtered through cheesecloth. To the filtrate 0.02 M PMSF (dissolved in DMSO) was added and allowed to stay at room temperature. The suspension was centrifuged at 12,000 rpm for 15 minute and the supernatant was collected. 20 mM CaCl2 was added and incubated for overnight at 4°C. The mixture was centrifuged for 12,000 rpm for 20 minutes. To the supernatant NaCl was added to a final concentration of 200mM and kept at 4°C, overnight. Next morning the suspension was centrifuged at 12000 rpm for 15 minutes. The supernatant liquid was dialysed against 20 mM phosphate buffered saline (PBS pH 7.4). The suspension was centrifuged again at 12000 rpm for 20 mins. The clear s upernatant liquid was then used for affinity chromatic purification. Affinity Chromatography Mannose agarose column was equilibrated with 20 mM PBS (pH 7.4). 100 ml of above supernatant was loaded onto the matrix and the flow through was collected. The flow through was re-loaded and the final flow through was washed with 20 mM PBS pH 7.4. The protein fractions were eluted against 20 mM DAP (unbuffered) and collected in fraction collector (BioRad model 2110) in 2 ml fractions. The elution profile was monitored in a Spectrophotometer (Beckman DU-70) at 280 nm wavelength. The fractions were pooled and dialysed against 20mM TrisC1 (pH 7.5). The dialysed sample was then concentrated and analysed in polyacrylamide gel and stained with coomassie brilliant blue and visualised on light box. Ion-Exchange Chromatography To the affinity eluted lectin fraction, solid α-D-mannose was added to a final concentration of 1M. This suspension was incubated at 4°C overnight and subsequently centrifuged at 15,000 rpm for 15 minutes. The clear supernatant was then loaded onto a freshly regenerated DEAE-Sephacel matrix, pre-equilibrated with 20 mM TrisC1, pH 7.5. This step was repeated twice or thrice to ensure complete binding, and the unbound proteins were eliminated by washing the matrix with the same equilibration buffer. Thorough washing was resumed with the same buffer (20 mM TrisCl) but at pH 5.0. The OD of the wash fractions were monitored spectrophotometrically at 280 nm and the major peak was collected and pooled. The purified protein was analysed in SDS-PAGE. Characterisation of the ASAL a) Determination of molecular weight The pure protein was analysed in HPLC TSK4000-SW (LKB, Bromma) gel permeation chromatography for purity. The HPLC analysis was done in an analytical scale with lug of the pure ASAL with Carbonic anhydrase as a standard. 20 mM TrisCl (pH 7.5) was used as a running buffer with a flow rate of 1 ml/min maintained with a LKB 2150 HPLC pump. The elution was monitored with a UV cord S-II online monitor at 220 nm and recorded on a LKB 2210 dual channel recorder. b) Non denaturing PAGE Analysis The pure protein fraction was analysed through non-denaturing PAGE. The gel used for analysis had discontinuous buffer system, 10% T, 0.8%C with Carbonic anhydrase as a standard. The gel was stained after electrophoresis with Coomassie Brilliant Blue. c) SDS-PAGE Analysis The pure protein was analysed through SDS-PAGE. The gel used for analysis had discontinuous buffer system, 15% T, 0.8% C with broad range denatured proteins (Bangalore Genei) as standard. The gel was stained after electrophoresis with Coomassie Brilliant Blue. d) 2-D PAGE Analysis To determine the isoelectric focusing point of purified fraction of Annona lectin, two- dimensional gel electrophoresis was done in the Bio-Rad mini protean II Isoelectric Focusing (IEF) module with carrier ampholytes as the focusing agents. Ampholytes purchased from Pharmacia and both the ranges of 3-10 and 5-8 were used. The protocol followed was taken from the Mini Protean II user's manual. The second dimension was run in a 15%SDS-PAGE. e) Agglutination Assay The mannose specific lectins have a typical characteristic feature of agglutinating activity with rabbit erythrocytes. Keeping these in mind an agglutination assay was set up in microtitre plates. 1 ml of rabbit blood was drawn into a hypodermic syringe, pre-filled with 1 ml of physiological saline solution (0.9% NaCl). The two solutions were mixed quickly and poured into a 2 ml microfuge tube and pulse centrifuged for a few seconds to sediment down the erythrocytes. Washing steps with the saline (resuspension and centrifugation) were repeated until the supernatant was clear and colorless. The sedimented erythrocytes were resuspended in 0.9% saline solution to make the erythrocyte concentration 20% (v/v). 20µl of this 20% erythrocyte suspension was dispensed into the wells of a microtitre plate. Pure lectin was dispensed into the wells of the plate (pre-filled with the erythrocytes) in different concentrations (0.1, 0.5, 1-10 µg/ml with 1 µg increment). The total volume of each well was made up to 100 µl with 0.9% saline. The plate was incubated for 1 hour at room temperature and the agglutination was monitored manually over a light box. 1) Monitoring of the stability of the purified lectin The stability assay of ASAL in simulating gastro intestinal fluid (SGF) of mammals was followed according to the protocols described by Fu et al (2002) with some modification. SGF was prepared by dissolving 10mg/ml pepsin in 0.3M NaCl at pH 1.2. ASAL was dissolved into 0.3M NaCl pH 7.5 at a concentration of 5mg/ml and for each digestion lµ1 of lectin solution were added to 7µ1 of SGF in 0.5ml micro centrifuge tube and incubated at 37 C. The ratio of test lectins versus pepsin was 14:1. At the interval of 0, 1, 5, 15, 30, 60 and 120 minutes, 1µl of 4N NaOH was added to the mixture to stop the reaction. 1.25µl of 5X SDS-PAGE sample buffer was then added to the mixture and heated in a boiling water bath for 5 minutes. Samples were then loaded onto SDS-PAGE (15% T, 1.8% C) and run. The PAGE was then stained with coomassie brilliant blue (CBB). Bioassay on white fly with purified lectin Transparent high quality plastic cup (3.2 cm diameter and 3.7 am high) with one open surface was taken as bioassay chamber. On the side wall of the chamber a tiny hole was made manually with hot needle, sufficient for whitefly entry. 20 early adult whitefly were put into the chamber through the hole by gentle aspirator in each experimental set up in three replicates. Each of the experiment was repeated at least five times. The hole was plugged with cotton to prevent insects escaping from the chamber. The open surface of the chamber was covered with a parafilm stretched four times of its original dimensions. A micro hole was made on stretched paraflim by pointed needle in sterile condition. A 200 µ1 of synthetic diet (15% sucrose and 2.5% yeast extract) mixture, supplemented with different concentrations of ASAL (2,5,10,20 µg/ml) was put on the paraflim layer, and covered with another paraflim layer to form a diet pouch. In control plate, only 15% sucrose and 2.5% yeast extract (autoclaved) were used as diet. Data on insect mortality were collected at 12-hour interval for 72 hours. In-vitro insect Bioassay on Aphis gossipi Polycarbonate Petri plates were made perforated for air passage at the bottom. Second instar nymphs of Aphis gossypii were released into the Petri plates in the multiple sets. The stretched Para film membrane was used to cover the upper edges of the plates. A synthetic diet mixture (total volume 200 µl) supplemented with different concentrations of ASAL (5, 10, 20ug/'ml) was dispensed on the stretched Para film membrane. Another layer of stretched Para film was put on it to make a diet pouch. In the control plates, 20 mM TrisCl, pH 7.4, was used in the artificial diet instead of ASAL. Three replications for each set were used. Data on number of nymphs survived were collected at a 12 h interval unto 60 h. The LC50 value of ASAL was determined according to Probit analysis for each insects. Cloning of the gene sequence coding for ASAL Total RNA was prepared from the frozen seed in liquid nitrogen. PolyA rich RNA was purified by using polyA mRNA isolation kit (Promega). The first strand cDNA had been synthesized using the Superscript II RNAse H reverse transcriptase (Gibco BRL). The first strand cDNA was used as template for RT PCR. The primers were designed from available ASAL sequence. Interestingly the amplification of approximately 360 bp band was obtained. The amplified band was digested with Bam HI and cloned in pUC 18 vector. The positive white clones were selected in Xgal / Ampicillin plate. Plasmid from a bacterial culture, grown from a single colony was isolated and digested with Bam HI that generated band of expected size in 1 % Agarose gel. Nucleotide sequencing of the positive clones Positive clones were selected and nucleotide sequencing was carried out by using standard techniques, which has been submitted in NCBI, the GenBank accession number of which has been allotted as AY866499. Blast analysis of the present lectin sequence with already available similar sequences. The present sequence, referred as ASAL shows 98% identity with other mannose binding lectin isolated from Allium sativum. Development of transgenic mustard, tobacco and rice plants with the chimeric ASAL gene. Well formed, light brown, healthy seeds of uniform dimension were taken as source material for explant preparation. Establishment of efficient and fast regeneration protocol for chickpea for transforming chickpea with ASAL In earlier study it was shown that ASAL affects the survival of Aphis craccivora (Majumder et al 2004), the homopteran insect which affects one the most important pulse crop, chickpea. While expressing ASAL in chickpea to develop resistance against A. raccivora, it appeared to be difficult due to non availability of suitable regeneration/transformation protocol for chickpea. Hence, an efficient, quick and reproducible regeneration protocol was developed which is amenable for A grobacterium mediated transformation experiments (Chakraborti et al. 2006). Seeds were surface sterilised in 0.05% HgCl2 with 0.1% Tween 20, washed repeatedly with autoclaved double distilled water and kept for another 20-24 hours for germination. After removing the seed coat, radicle portions were discarded and a longitudinal dissection along the plemule region of the embryo axes were carried out. The dissected single cotyledon with half embryo was chosen as explant. The explants were incubated in MS (Murashige and Skoog 1962) major salts, 4XMS minor salts, B5 (Gamborg et. al, 1968) vitamins, 10mM MES buffer alongwith 3%(w/v) sucrose, and 0.8% bacto-agar (Difco) supplemented with BAP 1.5 mg- 1 and NAA 0.04mgl-l for multiple shoot regeneration. The regenerated shoots were further incubated in elongation media containing IAA 0.2 mgl 1 where efficint elongation took place. Thereafter the elongate shoots were grafted on root stock prepared as mentioned below. Surface sterilized properly germinated seeds were cultured in Hoagland (Hoagland and Anion, 1950) agar media (2 seeds per Magenta box containing 50 ml medium) in above mentioned environmental condition. After 5-7 days of germination the shoot at first node was cut and a 2 mm incision made with a sterile scalpel. Healthy shoot from elongated shoot stock as described above was taken, a V-shaped cut made at the bottom and grafted on incised rootstock supported by a sterile Teflon ring (Morton et. al, 2001) and incubated in same condition for 4-5 days till the healing of the tissue of the grafted region was over. After 4-5 days of grafting the plantlets were taken out from the culture vessel, the remaining pieces of agar was removed carefully and transferred to plastic pots containing autoclaved synthetic soil (soilrite) and sprayed with ¼ strength of Hoagland Solution and covered with another transparent plastic pot. After 5 days the plantlets were transferred to 25-30 cm diameter pots containing soil, sand and organic manure (6:3:1). Transformation of mustard, tobacco and rice plants with ASAL gene cassette The hypocotyls segment of germinating seedling of mustard, tobacco leaf disc, single cotyledon with half embryo axis of germinating seedling of chickpea and scutellar derived embryonic callus of IR64 rice lines were used as respective explants for Agrobacterium mediated transformation following earlier reported protocol. The transformed plants were selected in presence of antibiotic (hygromycin in case of mustard, tobacco and rice and kanamycin in case of chickpea). The selected transgenic lines and their T1 progenies were analysed through Southern, northern and western blot techniques (Dutta et al 2005a, Dutta et al 2005b and Saha et al 2006). Functional assay of the transgenic mustard, tobacco, chickpea and rice plants In planta bioassay on mustard aphid, peach potato aphid, chickpea aphid and green leafhopper of rice were carried out in replicates on high ASAL expressing transgenic mustard, tobacco, chickpea and rice plants. The subject invention will further be illustrated by means of the drawings accompanying the Provisional Specification as well as this Specification As already indicated in the Provisional Specification- Fig. 1 shows purification and characterisation of ASAL. A. SDS-PAGE analysis of the purified ASAL. Lane M: Protein molecular weight Marker; lane 1: sample extracted from garlic leaf; lane 2: affinity purified sample; lane 3: purified protein showing ~12kDa single monomeric band. B. Two-Dimensional PAGE analysis of purified ASAL. The single spot showing the separation of ASAL at pH of ~5.25. Fig. 2 shows agglutination activity of pure ASAL. Well A: Tris buffer, Well B: 1 µg/ml ASAL in Well C: Colocasia tuber lectin fraction Fig. 3 depicts SDS-PAGE analysis of the ASAL digestion with pepsin at different time points. Lane M: Protein molecular weight marker, Lane 1-7: Digestion profile at different time point (0, 1, 5, 15, 30, 60, 120 minutes), Lane 8: Undigested ASAL, Lane 9: Pepsin Fig. 4 shows white fly mortality at different concentrations of ASAL in artificial diet. The following figures are described in relation to the present complete specification. Fig. 1 of the drawings accompanying this specification depicts in -vitro bioassay of Aphis gossypii ( cotton aphid) by ASAL. The graph shows the percentage of survival of cotton aphid on control diet and diet supplemented with 5,10and 20 µg/mal of ASAL, respectively. After lapse of 60 hours, around 80% insect survived on control diet, whereas only around 10% survived after feeding on diet supplemented with ASAL. Fig. 2 of the drawings accompanying this specification the following pictures are shown wherein A-is the explant; B- shows regeneration of multiple shoots from an explant after 15 days of incubation in a medium comprising 1.5mg/l of 6-benzyladenine and 0.04mg/lof α-naphthalene acetic acid; C-shows elongation of multiple shoots generated from an explant after 7 days of incubation in a medium containing 0.2mg/lof Indole-3-acetic acid; D- depicts elongated shoots separated from shoot stock; E-shows grafted shoot on rootstock, arrow indicating the junction point; F- shows a plantlet in soilrite during hardening with a teflon ring, and G- illustrates an established plant. Results: Purification and characterization of ASAL: In 15% SDS-PAGE, the affinity purified fraction resolved into two bands, 12 kDa monomer of ASAL band and higher mol. wt. contaminating band. Further analytical experiments necessitated the purification of the ASAL protein in large amount and of high quality. The contaminating bands were e liminatcd b y u sing D EAE-Sephacel column(Fig 1A of the drawings accompanying the provisional specification). The purified lectin fraction was analysed in non-denaturing PAGE and HPLC and found to have a native molecular weight of ~25kDa. The Purified ASAL also gave a spot in 2-D PAGE at an approximate pI of 5.25 (Fig 1B of the drawings accompanying the provisional specification). Fig 2 (the drawings accompanying the provisional specification) shows agglutination activity of pure ASAL in the well B at a concentration of 1 µg/ml with respect to the positive Colocasia tuber lectin fraction in well C. The well A was treated with no lectin as negative control. Inhibition of agglutination was monitored by adding ASAL pre-incubated with 1-100 mM of α-D-mannose that further determine its mannose specificity. Purified ASAL was found to be degraded in simulated gastric fluid with time as evidenced in SDS-PAGE profile of the pepsin treated and untreated lectins (Fig 3 the drawings accompanying the provisional specification). The process of enzymatic degradation is slow in first 30 minutes but it degraded completely between 60 to 120 minutes. Monitoring of the insecticidal activity of ASAL against whitefly From the insect bioassay experiments, it was evident that 20µg/ml concentration of ASAL were optimal, when supplemented in artificial diet for insect mortality. Close to 91% of the total population mortality was evident within 72 h in diet supplemented with 20ug/ml ASAL (Fig 4). The condition of the remaining 9% insects had been found to be weak Determination of LC 50 value of ASAL against whitefly LC50 value of ASAL for white fly was calculated to be 8.5µg/ml by Probit analysis using Abbott's formula. Monitoring of the insecticidal activity of ASAL against cotton aphid From the Figure 1 of this specification it became evident that 10 µg/ml ASAL was able to control the survival of cotton aphid which reached to less than 10% at 60 hrs. Determination of LC 50 value of ASAL against cotton aphid LC50 value of ASAL for cotton aphid was calculated to be 8.49 µg/ml by Probit analysis using Abbott's formula. Blast analysis of the present lectin sequence with already available similar sequences (in this case ASAL). The sequence of present ASAL coding gene represented hereinabove was subjected to blast analysis with earlier reported other related sequences by previous authors exhibited 98% sequence identity. Thus, through the present study an effective and fast regeneration protocol has been developed for facilitating deployment of agronomically important components through genetic transformation in chickpea improvement programme. Monitoring of the efficacy of transgenic plants on respective insect survival An in planta bioassay with peach potato aphid revealed that the percentage of insect survival decreased significantly to 16%-20% on transgenic tobacco T0 and T1 progeny plants, whilst approximately 75% of insects survived on untransformed tobacco plants after 144 h of incubation. Likewise, mustard aphids when tested on ASAL expressing transgenic mustard plants showed 89% of insect mortality. Fecundity of the same aphids was also noted to reduce by 60-64%. The survival and fecundity of chickpea aphid was reduced to 18.5 and 20.5%, respectively. Figure 2 of this specification demonstrated the steps of chickpea whole plant regeneration. Similarly, survival and fecundity green leafhopper on transgenic rice plant were reduced to 40.5% and 29.5% respectively. Conclusion: In the present report we are describing an efficient, rapid and modified method of ASAL purification, which has been, optimized for large-scale preparation of 25kDa ASAL lectin from Allium sativum leaf. The lectin being mannose specific had been preliminarily purified from crude leaf extract homogenized in DAP, through mannose agarose column. Finally purified through Sephacel column and eluted by 20mM TrisCl (pH 5.0). The native molecular weight of 25 kDa has been determined in non-denaturing Polyacrylamide gel and also by fractionating in HPLC gel filtration column and comparing the profile of known molecular weight protein. One single band of ~12 kDa resolved in SDS- PAGE determines its dimeric nature. Like other mannose-binding plant lectin, ASAL purified through this modified protocol agglutinates rabbit erythrocytes spontaneously. The agglutinin inhibition assay with various carbohydrate determined that agglutination inhibition takes place in presence of mannose. The western blot assay of pure ASAL earned out using anti ASAL antibody authenticated the purity of the lectin. The lectin is stable at room temperature and digested by pepsin in the simulated gastrointestinal fluid. Therefore it can be concluded that ASAL is well digested in the mammalian intestinal environment that substantially excludes the possible allergenic property. The pure ASAL is highly effective to reduce the survival rate of potentially damaging homopteran sucking pest, such as whitefly and cotton aphid. Transgenic mustard, tobacco, chickpea and rice plant types were also found to be effective to control the survivability of the respective sucking pests at significant level. Important features of the mannose binding lectin of this invention: • Native molecular weight of mannose binding ASAL purified through the new protocol is ~25kDa. • It is a dimeric protein, each monomer is of ~12 kDa. • The expected pI of ASAL is 5.25 as shown in 2D- PAGE. • It is able to agglutinate rabbit erythrocytes. • Purified ASAL is effective to confer mortality of whitefly and cotton aphid. . The LD 50 values of ASAL against white fly in artificial diet are 8.5µg/ml . The LD 50 values of ASAL against cotton aphid in artificial diet are 8.49µg/ml It is stable at room temperature, and digested by pepsin. • The blast analysis of ASAL sequence showed 98% identity with previously reported • ASAL sequence by Smeet et al (1997). • ASAL expressed in mustard, tobacco, chickpea and elite Indian cultivar of rice, IR- 64 shows antagonistic properties against mustard aphid, peach potato aphid, chickpea aphid and green leafhopper of rice, respectively. • The plant source being common and easily accessible, lectin isolation and purification is considered to be cost effective, particularly due to its stability. Thus, ASAL being a mannose specific non allergenic plant lectin qualifies itself as a member of "mannose binding lectin super family" and provides itself as an important control agent against the newly emerging important pest, whitefly (Bemesia tabaci) and cotton aphid (Aphis gossypii). Transgenic T1 ASAL plants of mustard, tobacco, chickpea and rice have shown detrimental effects on mustard aphid, peach potato aphid, chickpea aphid and green leafhopper of rice, respectively. While the invention has been described in detail and with reference to specific embodiment thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without deviating or departing from the spirit and scope of this invention. Thus, the disclosure contained herein includes within its ambit the obvious equivalents and substitutes as well. Having described the invention in detail with particular reference to the illustrative Examples and drawings accompanying the Provisional Specification, it will now be more specifically defined by means of claims defined hereafter. We claim : 1. A mannose binding insectisidal lectin from leaves of Allium sativum, effective against whitefly ( Bemesia tabaci) and cotton aphid (Aphis gossypii), having protein sequence as given below: marnlltnge glyagqsldv eqykfimqdd cnlvlyeyst piwasntgvt gkngcravmq rdgnfvvydv ngrpvwasns vrgngnyilv lqkdrnvviy gsdiwstgtl Iehrvptsfy 2. A mannose-binding lectin (ASAL), isolated from leaves of Allium sativum as claimed in Claim1 having molecular weight of around 25kDa and giving a spot in 2-D PAGE at a pI of 5.25,which is able to agglutinate rabbit erythrocytes. 3. A mannose-binding lectin as claimed in Claim 2, which undergoes enzymatic degradation in gastric fluid between 60 and 120 minutes 4. A mannose-binding lectin as claimed in Claim 3, which is stable at room temperature and has LD 50 values against whitefly and cotton aphid in artificial diet as 8.5 and 8.49 µg/ml, respectively. 5. A mannose-binding lectin as claimed in Claims 1 and 4, which when expressed in mustard, tobacco, chickpea and rice shows detrimental effects on survivability of mustard aphid, peach potato aphid, chickpea aphid and green leafhopper of rice. 6. A mannose-binding lectin, ASAL effective against whitefly (Bemisia tabaci) and cotton aphid ( Aphis gossypii), substantially as hereinbefore described with particular reference to the illustrative Examples and accompanying drawings. 7. A process for extraction and purification of a mannose binding insecticidal lectin from leaves of Allium sativum as claimed in Claims 1 to 6, which comprises (i) collecting leaves of aforementioned plant, cleaning them by washing with distilled water, drying them and homogenizing the dried leaves in 20mM diaminopropane (DAP); (ii) filtering the extracts followed by addition of 0.02M PMSF and incubation at room temperature; (iii) adding 20mM calcium chloride to the supernatant liquid after centrifuging the same and incubating overnight; (iv) adding NaCl to a final concentration of 200mM and incubating overnight at 4° C; (v) dialyzing the supernatant against phosphate buffered saline ( PBS) maintained at around pH7.4; (vi) binding the suspension from step (v) to a mannose agarose column and eliminating the unbound proteins by standard technique(s); (vii) eluting the bound lectin from the said column with diaminoprpane(DAP) and (viii) conducting ion exchange chromatography followed by elution of the pure lectin with 20mM Tris-C1 at around pH5.0 and collecting the eluent containing lectin 8. A process for extraction and purification of insecticidal lectin from leaves of Allium sativum, substantially as hereinbefore described with particular reference to the illustrative Example and accompanying drawings. Heretofore pest control was usually done by application of chemical pesticides, such as, for instance, pyrethroid and pyrethrin derivatives, organophosphates, chlorinated hydrocarbons, nicotin and nicotinic acid derivatives and particularly in India, neem extracts. But such conventional pesticides did not produce desired effect on certain pests like whitefly and /or cotton aphid as the pesticides when applied often did not or could not reach the target pests because of their peculiar perch. The present invention aims at overcoming the above defects of the the conventional pesticides and protect crop loss by affecting the target pests, whitefly and cotton aphids with the help of mannose binding insecticidal lectins from leaves of Allium sativum (garlic). The lectin has undernoted protein sequence marnlltnge glyagqsldv eqykfimqdd cnlvlyeyst piwasntgvt gkngcravmq rdgnfwydv ngrpvwasns vrgngnyilv Iqkdrnwiy gsdiwstgtl lehrvptsfy. The above lectin has been expressed in rice, mustard and chickpea. The transgenic plants have shown pronounced antagonistic effects against green leaf hopper of rice, mustard aphid and chickpea aphids. The present invention also relates to a process for isolation of pure lectin from leaves of Allium sativum which is a rather cost effective one. Figures 1 and 2 of the drawings illustrate the invention

Documents:

889-KOL-2005-CORRESPONDENCE.pdf

889-KOL-2005-FORM 27-1.1.pdf

889-KOL-2005-FORM 27.pdf

889-KOL-2005-FORM-27.pdf

889-kol-2005-granted-abstract.pdf

889-kol-2005-granted-claims.pdf

889-kol-2005-granted-correspondence.pdf

889-kol-2005-granted-description (complete).pdf

889-kol-2005-granted-drawings.pdf

889-kol-2005-granted-examination report.pdf

889-kol-2005-granted-form 1.pdf

889-kol-2005-granted-form 18.pdf

889-kol-2005-granted-form 2.pdf

889-kol-2005-granted-form 3.pdf

889-kol-2005-granted-form 5.pdf

889-kol-2005-granted-form 9.pdf

889-kol-2005-granted-pa.pdf

889-kol-2005-granted-reply to examination report.pdf

889-kol-2005-granted-specification.pdf