Advantage of Basillus thurengiensis (Bt) gene engineering in Plant Biotechnology
By:Ali Parsaeimehr
Introduction:
Basillus thurengiensis (Bt) is an aerobic, motile , gram-positive, endospore-forming bacillus initially isolated in Japan by Ishiwata and formally described by Berliner in 1915 (20). The insecticidal ,tivity of Bt used commercially thus far comes from included in crystals formed during sporulation, although “vegetative insecticidal proteins” (Vips) from before sporulation (24) are also being developed. The crystals of different strains of most Bts contain varying to different groups of insects. More than 100 Bt toxin genes have been cloned and sequenced, providing an array of proteins which can be expressed in plant or in foliar applications of Bt products (10).
Insecticidal crystal proteins (Cry) of Bacillus thuringiensis (Bt) have been used since long to control insect pest (Tabashnik 1997).In recent years transgenic cultivar expressing the cry gen have been shown to defend insect attack in a wide variety of crops (Cheng et al 1998; Xiang et al 2000).
Insecticidal products containing subspecies of bacterium B.thuringiensis were first commercialized in France in the late 1930s with the product Sporeine.Bt was first introduced into tobacco plants in 1987 (21). However , much more effective plants that use synthetic genes modeled on those from Bt but designed to be more compatible with plant [removed]18 ) were introduced a few years later.of the $US 8.1 billion spent annually on all insectides world wide, it has been estimated that nearly $2/7 billion could be substituted with Bt biotechnology products (14).Insect targeted for control by Bt plant are primarily Lepidoptera through the production of Cry1Ab, Cry1Ac, and Cry9c proteins, and with some experiment this proteins don’t have any harm for mankind.
Genetically transformed plant incorporating genes from the soil bacterium Bacillus thuringiensis (Bt) were developed in the 1980` s. Tabacco, cotton and tomatoes were among the first crop plants transformed to produce insecticidal protein to protect the plant from insect attack and consequent yield losses. Bt cotton varietes were developed and field tested by the early 1990`s.
Agrobacterium tumefaciens is a Mediated Gene Transfer this Bacterial plant pathogen found in the soil that results in tumorous growths and/or roots to develop in infected plants (“Agrobacterium tumefaciens” 2001) ,This infection is known as Crown Gall Disease (Deacon 2002) The bacteria transfers a tumor-inducing (Ti) plasmid located in a section of its DNA (known as T-DNA) into the nucleus of an infected plant cell this bacteria could altered from the bacterial genes of Bacillus thuringiensis (Cry1Ab, Cry1Ac,…) by insertion of the cauliflower mosaic virus (CaMV) promoter and enhancers, the leader sequence. An antibiotic resistance marker nptII, also with the CaMV promoter and the NOS transcription terminator from Agrobacterium tumefacians nopaline synthetase gene could insert into the cotton chromosome along with the Bt-Cry1Ac gene which could expression toxin crystal proteins that has affect on pests of Cotton and the modified Cry1Ac gene has been produced by the modifying amino acid sequence of the wild- type Cry1Ac protein which produced in Bacillus thuringiensis (22, 25) .
2-Review of Literature and Relevant Topics:
In an experiment at the University of Georgia,Zygotic hypocotyls of canola (Brassica napus L.) varieties of cv Oscar, cv Westar, and the breeding line UGA188-20B were transformed with a truncated synthetic Bacillus thuringiensis insecticidal crystal protein gene (Bt cry1Ac) under the control of the cauliflower mosaic virus 35S promoter using Agrobacterium tumefaciens-Mediated transformation. Fifty-seven independently transformed lines were produced, containing 1 to 12 copies of the transgenes. A range of cry expressors was produced from 0 to 0.4% Cry as a percentage of total extractable protein (Xiang Y et al,2000).
Helicoverpa armigera is a primary pest in Iran and it could raise it damage’s to 75%. Gharosy et.al transferred Bt cry1Ac insecticidal crystal protein gene under the control of the cauliflower mosaic virus 35S promoter using Agrobacterium tumefaciens-Mediated strains; LBA4404 and plasmid pBII21 to hypocotyls explants of Coker,Varamin and Sahil varieties of cotton .PCR analysis of putative transgenic plant showed the integration of the 1/66 Kb especial band of cry1Ac gene in the produce transgenic plant by variety of Varamin (Gharosy et.al,2004).
Conventional breeding methods have not been very successful in producing pest-resistant genotypes of pigeon pea, due to the limited genetic variation in cultivated germplasm. Kiran K. Sharma et.al developed an efficient method to produce transgenic plants of pigeon pea by incorporating the cry1Ab gene of Bacillus thuringiensis through Agrobacterium tumefaciens-mediated genetic transformation. The novel tissue culture protocol have been the direct regeneration of adventitious shoot buds in the axillary bud region of in vitro germinating seedlings by suppressing the axillary and primary shoot buds on a medium containing a high concentration of N 6-benzyladenine (22.0?M). The tissue with potential to produce adventitious shoot buds explanted used for co-cultivation with A. tumefaciens carrying the synthetic cry1Ab on a binary vector and driven by a CaMV 35S promoter. Following this protocol, over 75 independently transformed transgenic events of pigeon pea were produced and advanced to T2 generation. Amongst the recovered primary putative transformation events, 60% showed positive gene integration based on initial polymerase chain reaction (PCR) screening. PCR analysis of the progenies from independent transform ants followed gene inheritance in a Mendelian ratio and 65% of the transformants showed the presence of single-copy inserts of the introduced genes. Reverse transcription-polymerase chain reaction analysis showed that the transcripts of the introduced genes were normally transcribed and resulted in the expression of Cry1Ab protein in the tested T2 generation plants. Interestingly, the content of Cry1Ab protein as a percent of total soluble protein varied in different tissues of the whole plant, showing the highest expression in flowers (0.1%) and least in the leaves (0.025%) as estimated by enzyme-linked immunosorbent assay. The transgenic plants produced in this study offer immense potential for the improvement of this important legume of the semi-arid tropics for resistance to insect pests (Kiran K. Sharma et.al ,2005).
In the other experiment Guo, X. et.al transfered, Three constructs harbouring novel Bacillus thuringiensis genes (Cry1C, Cry2A, Cry9C) and bar gene into four upland cotton cultivars Ekangmian, Emian, Coker and YZ via Agrobacterium-mediated transformation. With the bar gene as a selectable marker, about 84.8 % of resistant calli have been confirmed positive by polymerase chain reaction (PCR) tests, and totally 50 transgenic plants were regenerated. The insertions were verified by means of Southern blotting. Bioassay showed 80 % of the transgenic plantlets generated resistance to both herbicide and insect (Guo, X. et.al ,2007 ).
In an experiment Si-Jun Zheng et al use Agrobacterium-mediated genetic transformation for applied to produce beet armyworm (Spodoptera exigua Hübner) resistant tropical shallots (Allium cepa L. group Aggregatum). A cry1Ca or a H04 hybrid gene from Bacillus thuringiensis, driven by the chrysanthemum ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (Rubisco SSU) promoter, along with the hygromycin phosphotransferase gene (hpt) driven by the CaMV 35S promoter, was employed for genetic transformation. An average transformation frequency of 3.68% was obtained from two shallot cultivars, Tropix and Kuning. After transfer of the in vitro plants to the greenhouse 69% of the cry1Ca and 39% of the H04 transgenic shallots survived the first half year. After one year of cultivation in the greenhouse the remaining cry1Ca and H04 transgenic plants grew vigorously and had a normal bulb formation, although the cry1Ca transgenic plants (and controls) had darker green leaves compared to their H04 counterparts. Standard PCR, adaptor ligation PCR and Southern analyses confirmed the integration of T-DNA into the shallot genome. Northern blot and ELISA analyses revealed expression of the cry1Ca or H04 gene in the transgenic plants. The amount of Cry1Ca expressed in transgenic plants was higher than the expression levels of H04 (0.39 vs. 0.16% of the total soluble leaf proteins, respectively). There was a good correlation between protein expression and beet armyworm resistance. Cry1Ca or H04 gene expression of at least 0.22 or 0.08% of the total soluble protein in shallot leaves was sufficient to give a complete resistance against beet armyworm. This confirms earlier observations that the H04 toxin is more toxic to S. exigua than the Cry1Ca toxin. The results from this study suggest that the cry1Ca and H04 transgenic shallots developed could be used for introducing resistance to beet armyworm in (sub) tropical shallot (Si-Jun Zheng et al ,2005 ).
In another experiment transgenic Japanese lawngrass plants containing a synthetic cryIA(b) gene from Bacillus thuringiensis under the control of a maize ubiquitin promoter were developed by Agrobacterium-mediated transformation. A total of 1540 embryogenic calluses derived from dormancy-removal mature seeds were co-cultured with the disarmed strain EHA105 harbouring the binary vector pKUB. Three days after co-culture with EHA105 in the dark at 21°C, transient ?-glucuronidase (GUS) expression frequency was 74.2%. After selection with 100 mg/l hygromycin B, a total of over 50 independent resistant cell clones and 25 regenerated plants were obtained. The integration and expression of the cryIA(b) gene into the genome was confirmed in 22 regenerated plants by the GUS histochemical assay, PCR amplification, Southern blotting and Western blotting analysis, with a transformation efficiency of 1.4%. The entire process from callus induction of mature seeds to production of transgenic plantlets was 80–100 days. T1 progeny segregation analysis of these transgenic lines demonstrated that 59.1% of the transgenic events were inherited in a typical Mendelian fashion (Lei Zhang,2007).
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1.PhD student at the field of plant Biotechnology at the National Academy of Science of Republic of Armenia
Ali Parsaeimehr
