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  bacillus thuringiensis et lutte biologique?
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biouided
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Féminin Bélier (21mar-19avr)
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MessagePosté le: Mar 16 Déc - 21:16 (2008) Répondre en citant

bonjour tout le monde. je demande votre aide pour me dirigez vers des informations ou une souce où je peut trouver une méthodologie bien détaillée en français décrivant le protocole de la trangénèse d'une plante par la bactéris bacillus thuringiensis dés son isolement et culture et la manipulation génétique jusqu'a l'introduction du gène cry dans la plante d'intéret . svp aidez moi et merci d'avence .


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MessagePosté le: Mar 16 Déc - 21:16 (2008)

PublicitéSupprimer les publicités ?
sidali
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Masculin Verseau (20jan-19fev)
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MessagePosté le: Mar 16 Déc - 21:45 (2008) Répondre en citant

essaie ça http://www.gnis-pedagogie.org/pages/classbio/chap3/36.htm


sidali
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Masculin Verseau (20jan-19fev)
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MessagePosté le: Mar 16 Déc - 21:47 (2008) Répondre en citant

http://www.senat.fr/rap/r97-440/r97-44070.html


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MessagePosté le: Mar 16 Déc - 21:48 (2008) Répondre en citant

cherche ici : www.sciencedirect.com et si jamais tu trouve un article la bas tu me file le lien par message privé et je ferai de mon mieux pr le télécharger 

_________________
Il faut que le disciple de la sagesse ait le coeur grand et courageux


sidali
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Masculin Verseau (20jan-19fev)
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MessagePosté le: Mar 16 Déc - 21:58 (2008) Répondre en citant

http://www.techno-science.net


biouided
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Féminin Bélier (21mar-19avr)
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MessagePosté le: Jeu 18 Déc - 20:29 (2008) Répondre en citant

1. Introduction
Significant advances in tissue culture and gene delivery techniques have allowed the incorporation of beneficial genes for specific agronomic traits into diverse crop plants. Genetic engineering of rice enables breeders to design new varieties by the introduction of desired alien genes into existing commercial lines. In the past few years considerable progress has been made in optimizing and refining genetic transformation techniques in rice. Direct DNA uptake into rice explants was reported for japonica and a few indica varieties [1, 2, 3, 4, 5 and 6]. These procedures, however, have resulted in the integration of multiple copies of transgenes into host genomes, rearrangement of transgenes, sterility of transgenic plants, besides their non-Mendelian inheritance [5, 7 and 8]. To obviate these problems, efforts have been made to transform rice through Agrobacterium-mediated method. The Agrobacterium-mediated transformation was found very effective mainly because of single copy integration of T-DNA region, high fertility of transgenics and transmission of transgenes in a Mendelian fashion. Hence, this technique has become the most preferred option to transform rice varieties. Hiei et al. [9] reported the unequivocal evidence of Agrobacterium-based transformation in japonica rice. Later, the host range was extended to a few javanica and indica rice varieties [10, 11 and 12]. However, the efficiency of Agrobacterium-based transformation in rice is modulated by genotype, choice of tissue and choice of vector besides culture conditions [13]. In rice, different binary vectors have been used for achieving genetic transformation. Hiei et al. [9], Rashid et al. [12], and Cheng et al. [14] used pIG121Hm which is a derivative of the most commonly used vectors, viz. pBI121 [15] and pBIN19 [16].


In cereal crops, maize and wheat, the Agrobacterium-mediated transformation was achieved using super-binary vectors [17 and 18]. Super-binary vectors, such as pTOK233, were decidedly more efficient in transforming the recalcitrant genotypes of rice [9]. These vectors carry the vir genes in the plasmid comprising T-DNA region, in addition to the native vir genes present in the acceptor vector, thus making them super-virulent. In this study, we have used the pSB super-binary vectors to transform different indica rice varieties. The pSB1 is a disarmed Ti acceptor vector of the Agrobacterium strain LBA4404, and pSB11 is an intermediate vector carrying the T-DNA region. All such vectors carry a 15.2 kb Kpn 1 fragment derived from pTiBo542, and this fragment is present in the pSB1 acceptor vector. In triparental mating, genetic recombination occurs between a 2.7 kb homologous region of the acceptor and intermediate vectors [19].


Based on the success of super-binary vectors with japonica rice varieties, we attempted to extend the host range of super-binary vectors to elite indica rice varieties. The present investigation deals with the optimization of Agrobacterium-mediated gene transfer in different indica varieties. High yielding and popular rice varieties, susceptible to stem borers and sap-sucking insects, have been employed in this study. The snowdrop lectin (GNA) gene was found highly effective against sap-sucking insects [20], while Bt genes proved lethal to the stem borers [14 and 21]. In the present study, we have demonstrated the stable integration of bar, gna and cry genes into genomes of different rice lines, and their faithful transmission to subsequent generations in a normal Mendelian fashion.


2. Materials and methods
Genetic transformation experiments were carried out using three leading cultivars (cvs) of rice, viz. Chaitanya, Phalguna and Swarna, besides three parents, viz. IR58025A (CMS line), IR58025B (maintainer line) and Vajram (restorer) used in hybrid rice programmes.


2.1. Construction of Agrobacterium vectors for transformation studies
Three Ti plasmid based vectors were constructed and used for transformation studies. The plasmid constructs contained the common selectable marker gene bar driven by CaMV 35S promoter, and the Bacillus thuringiensis synthetic crystal protein genes, cry1Ab/cry1Ac, driven by maize ubiquitin promoter, and the snowdrop lectin gene, gna, driven by phloem-specific rice-sucrose synthase promoter (RSs1). Expression cassettes of bar (CaMV 35S-bar-nos) [22], cry1Ab (maize ubi-cry1Ab-nos), cry1Ac (maize ubi-cry1Ac-nos) [23], and gna (RSs-gna-nos) [24] were cloned at the multiple cloning site of the intermediate vector pSB11 [19] obtained from Japan Tobacco Inc., Japan. The recombinant clones were introduced into Agrobacterium strain LBA4404 by triparental mating [25] and the resulting co-integrate vectors were designated as pSB111ubi-cry1Ab-35S-bar, pSB111-ubi-cry1Ac-35S-bar, and pSB111-RSs1-gna-35S-bar (Fig. 1). Initial transformation experiments were carried out using the vector pTOK233 [9] which has the reporter gene int-gusA along with the selectable marker gene hygromycin phosphotransferase (hpt).


.2. Genetic transformation studies using pTOK 233, pSB111-ubi-cry1Ab/cry1Ac-35S-bar and pSB111-RSs-gna-35S-bar
Mature rice seeds were surface-sterilized in 0.1% HgCl2 and placed on the Murashige and Skoog [26] medium (3MN62) ( Table 1) for callus induction. After 3 weeks of incubation, the scutellar-derived calli were used for transformation experiments. Agrobacterium cultures were initiated by inoculating a single colony of the bacterium into 5 ml of ABG liquid medium containing 50 mg/l spectinomycin at 29 °C for about 24 h. The bacterial culture was pelleted at 2500 rpm and resuspended in 10 ml of PIM II medium [10] supplemented with 100 mM acetosyringone (AS) and incubated for 16 h at 29 °C. Before co-cultivation, the calli (2–3 mm) were treated with MS basal medium containing 100 mM AS for 30 min at 29 °C. Later, the calli were transferred into the Agrobacterium culture and left on the shaker at 150 rpm for about 30 min. These calli were placed on co-cultivation medium (CCM) (Table 1), and 20 μl of Agrobacterium culture was used for infecting each callus. The infected calli were incubated for 72 h at 29 °C in the dark, and washed in MS basal medium with 100 mg/l cefotaxime and 250 mg/l carbenicillin before transferring to the MS medium (3MN62) (Table 1) containing 100 mg/l cefotaxime and 250 mg/l carbenicillin for 2 weeks. The proliferated calli were placed on First stage selection media containing 8 mg/l phosphinothricin (Table 1) and cultured for 3 weeks. Care was taken to see that the calli were no more than 2–3 mm in size and calli of the same origin were grouped together. The actively growing calli were selected and placed on Second stage selection media containing 10 mg/l phosphinothricin (Table 1). After 3 weeks of incubation on this medium, the surviving calli were transferred to proliferation medium (3MN62) ( Table 1) for 2 weeks, and later transferred to the regeneration medium containing BAP (2 mg/l) and NAA (0.5 mg/l) (Table 1). Subsequently, the regenerated plants were transferred to the rooting medium ( Table 1). The rooted plants were transferred to Hoagland’s solution for hardening of the roots, and were established in the glass house in pots containing a mixture of vermiculite and soil.


2.3. Transient assays for gusA gene expression
Histochemical GUS assay was carried out according to Jefferson et al. [27]. Ten days after eliminating the bacteria, the co-cultivated calli were fixed in the assay buffer containing 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc), and were incubated overnight at 37 °C.


2.4. Analysis of putative transformants using herbicide (Basta) test
The leaves of about 30–40-days old putatively transformed plants along with the untransformed controls were dipped in 0.25% ‘Basta’ solution and the damage was recorded after 4 days.


2.5. Molecular analysis of putative transformants
Genomic DNA was isolated from Basta resistant and untransformed control plants by the CTAB method [28]. PCR analyses were carried out using the primers 5′- CGG ATC CAT GGC TAA GGC AAG TCT CCT C-3′, 5′-CGG TAC CTC ATT ACT TTG CCG TCA CAA G-3′; 5′-CTA CCA TGA GCC CAG AAC G-3′, 5′-TCA GAT CTC GGT GAC GGG-3′; 5′-GTT CGC AGT CCA GAA CTA CCA AG-3′, 5′-TGG GTG ATT TGA GAG GAA GGA-3′; and 5′-GTT CGC AGT CCA GAA CTA CCA AG-3′, 5′-AGT TTC CCT TCA CTG CAG GGA-3′, respectively, for detection of gna-, bar-, cry1Ab- and cry1Ac-coding sequences in primary transformants [20]. The DNA from the untransformed plants was used as negative control and the intermediate vector was used as positive control. The primers were expected to amplify about 560, 480, 930 and 980 bp regions, respectively.


2.6. Southern blot analyses of T0 plants
Southern analyses were done using approximately 15 μg each of undigested and digested genomic DNA from the PCR positive plants according to Sambrook and Russel [29]. Genomic DNA was digested with EcoRI, BamHI and HindIII, separately, in the case of the Bt transformants and with HindIII and Sal I, independently, for the gna transformants. The digested DNA was resolved on 0.8% agarose gel and transferred to positively charged nylon membranes as per the manufacturer’s instructions (Amersham). The coding sequences of bar, cry and gna were radiolabeled with [α-32P]dCTP using random primer labeling beads (Amersham) and were used as probes.


2.7. Northern blot analyses of T0 plants
Total RNA was isolated from Southern positive plants along with untransformed control plants according to Sambrook and Russel [29]. Approximately, 12 μg of the total RNA was run on 1.4% denaturing agarose gel containing formaldehyde and then transferred to positively charged nylon membrane (Amersham). The coding sequences of cry and gna were used as radiolabeled probes.


2.8. Inheritance of transgenes in T1 generation
Selfed seeds from the primary transformants (T0) were collected and germinated. The T1 progeny plants were maintained in the glass house under controlled conditions. Forty-days old T1 plants were tested for tolerance to Basta. PCR and Southern blot analyses of DNA from these plants were carried out as described earlier.


2.9. Insect bioassays on transgenic rice plants expressing Cry1Ac and GNA
Insect bioassays were carried out with five neonate larvae of yellow stem borer-YSB (Scirpophaga incertulas) (Walker) per cut stem of primary transformants of cry1Ac/Ab along with untransformed control plants, using the no choice method [30]. Ten such replications were used for each of the transformant. The stems were dissected after 4 days and screened for feeding damage in the pith and presence of dead larvae. The T1 and T2 progenies of rice plants expressing GNA were used in insect bioassays against brown planthopper (BPH; Nilaparvata lugens) (Stal), green leafhopper (GLH; Nephotettix virescens) (Distant) and whitebacked planthopper (WBPH; Sogatella furcifera). Twenty third instar nymphs of BPH, GLH and WBPH were independently released on each plant confined in a mylar cage and were allowed to feed for 10 days, in 15 replications. After 10 days of infestation with BPH/GLH/WBPH, the survived plants were scored as resistant while the dead were recorded as susceptible. The resistance exhibited by transgenic plants was scored based on a scale of 0–9 used in the international rice-testing programme. Data were recorded on the nymphal survival up to 3 weeks after infestation


sidali
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Masculin Verseau (20jan-19fev)
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MessagePosté le: Ven 19 Déc - 11:38 (2008) Répondre en citant

voici une traduction biouided:



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