Standarizing in Planta Agrobacterium Tumefacience Mediated Genetic Transformation Protocol to Develop New Events by Transforming G. Hirsutum Cotton based on Cry1Ac-Cry1Ec Genes
American Journal of Life Sciences
Volume 2, Issue 4, August 2014, Pages: 190-199
Received: Jul. 13, 2014;
Accepted: Jul. 22, 2014;
Published: Aug. 10, 2014
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Yanal A. Alkuddsi, Agricultural Research Station, University of Agricultural Sciences, Dharwad- 580005, Karnataka, India
Shreekanth S. Patil, Agricultural Research Station, University of Agricultural Sciences, Dharwad- 580005, Karnataka, India
S. M. Manjula, Agricultural Research Station, University of Agricultural Sciences, Dharwad- 580005, Karnataka, India
K. J. Pranesh, Agricultural Research Station, University of Agricultural Sciences, Dharwad- 580005, Karnataka, India
B. C. Patil, Agricultural Research Station, University of Agricultural Sciences, Dharwad- 580005, Karnataka, India
Cotton breeding for insect resistance has been limited by a lack of sufficient genetic variation in the existing germplasms. Therefore, genetic engineering provides the possibility of creating varieties carrying new properties coming even from heterologous source. Exogenous pesticidal transgenes can be introduced into plants. Agrobacterium mediated plant transformation offers advantages like reducing copy number of the transgene and little co-suppression. Inter specific hybrids are known to be more susceptible to biotic stress. It is hence important to develop Bt version for inter specific hybrid. Presently, the Bt gene commercialized are owned by private sector. It is necessary to develop public sector’s Bt event and commercialize them. UAS Dharwad is involved in developing public sector Bt cotton genotypes. One variety, RCR4 (Gossypium hirsutum, L.) was used in the present investigation. Cry1Ac-Cry1Ec genes are to control Helicoverpa armigera and Spodoptera litura. The seedlings in pots were co-cultivated with solid Agrobacterium culture after cutting the meristematic tip with sharp knife. The number of seedlings co-cultivated, number of seedlings established and the number of seedlings showing transformed status are presented in this study. PCR was performed to confirm the presence of the transgene in the plants that were selected to be advanced further. The results showed that non of plants had trangenes Cry1Ac- Cry1Ec as detected through PCR amplification. In planta genetic transformation was carried out and the plants were tested in T0 generation by means of PCR amplification for the genes Cry1Ac- Cry1Ec. The results obtained were not amplified the Cry1Ac- Cry1Ec. Hence the transformation of the genes was not up to mark and the plants of T1 generation are also not confirmed.
Yanal A. Alkuddsi,
Shreekanth S. Patil,
S. M. Manjula,
K. J. Pranesh,
B. C. Patil,
Standarizing in Planta Agrobacterium Tumefacience Mediated Genetic Transformation Protocol to Develop New Events by Transforming G. Hirsutum Cotton based on Cry1Ac-Cry1Ec Genes, American Journal of Life Sciences.
Vol. 2, No. 4,
2014, pp. 190-199.
Aslam, M., Hrzog, C. A. and Chalfant, R. B., 2000, Different cotton strains screened for resistance to Heliothis spp. (Lepidoptera : Noctuidae) in the field. Pak. J. Biol. Sci., 3 (8) : 1290-1291.
Bevan, M., Barnes, W. M. and Chilton, M. D., 1983a, Structures and transcription of nopaline synthase gene region of T-DNA. Nucleic Acids Res., 11 : 369-385.
Bosch, D., Schipper, B., Van Der Klei, H., De Maagd, R. A. And Stiekema, W. J., 1994, Recombinant Bacillus thuringiensis crystal proteins with new properties : possibilities for resistance management. Bio/Tech., 12 : 915–918.
Broer, I., Droge-Laser, W., Barkers, R. F., Neumann, K., Klipper, W. and Puhler., 1995, Identification of Agrobacterium tumefaciens C58 T-DNA genes c and f and their impact on crown gall tumor formation. Plant Mol. Biol., 27 : 41-57.
Chambers, J. A., Jelen, A., Gilbert, M. P., Jany, C. S., Johnson, T. B. and Gawron-Burke, C., 1991, Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai. J Bacteriol., 173 : 3966–3976.
Dhaliwal, H. S., Kawai, M. and Uchimiya, H., 1998. Genetic engineering for abiotic stress tolerance in plants. Plant Biotech., 15 : 1-10.
Enriquez-Obregon, G. A., Vazquez-Padron, R. I., Prietsosansonov, D. L., Delariva, G. A. and Selman-Housein, G., 1998, Herbicide resistant sugarcane (Saccharum officinarum L.) plants by Agrobacterium mediated transformation. Planta., 6 : 20-27.
Firoozabady, E., Deboer, D. L., Merlo, D. J., Halk, E. L., Amerson, L. N., Rashka, A. E. and Murray, E. E., 1987, Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants. Plant Mol. Biol., 10 : 105-116.
Gill, S. S., Cowles, E. A. and Pietrantonio, P. V., 1992, The mode of action of Bacillus thuringiensis endotoxins. Ann Rev Entomol., 37 : 615–636.
Graves, A. C. F., and S. L. Goldman, 1986. The transformation of Zea mays seedlings with Agrobacterium tumefaciens, Plant Mol. Biol., 7 : 43-50.
Hansen, G., Shillito, R. D. and Chilton, M. D., 1997, T- strand integration in maize protoplants after co delivery of a T-DNA substrate and virulence genes. Proc. of the Nation. Acad. of Sci. of USA., 94 : 11726-11730.
Herrnstadf, C., Soanres, G. G., Wilcox, E. R. and Edward, D. L., 1986, A new strain of Bacillus thuringiensis with activity against coleopteran insects. Biotech., 4 : 305-308.
Hoftey, H. and Whiteley, H. R., 1989, Insecticidal crystal protein of Bacillus thuringiensis. Microbiol Rev., 53 : 242-255.
Hone´e, G., Van Der Salm, T, and Visser, B., 1988, Nucleotide sequence of crystal protein gene isolated from B. thuringiensis subspecies entomocidus 60. 5 coding for a toxin highly active against. Spodoptera species Nucleic Acids Res., 16 : 6240.
Kalman, S., Kiehne, K. L., Libs, J. L. and Yamamoto, T., 1993, Cloning of a novel cryIC-type gene from a strain of Bacillus thuringiensis subsp. galleriae. Appl Environ Microbiol., 59 : 1131–1137.
Katageri, I. S., Vamadevaiah, H. M., Udikeri, S. S., Khadi, B. M. and Kumar, P. A., 2007, Genetic transformation of an elite Indian genotype of cotton (Gossypium hirsutum L.) for insect resistance. Current Sci., 93 (12) : 1843-1847.
Konez, C., Nemeth, K., Redei, G. P. and Scel, J., 1994, Homologous recombination and gene silencing in plants. Plant Cell Rep., 22 : 167-174.
Krattiger, K. and Anatole, F., 1997, Insect resistance in crops : a case study of Bacillus thuringiensis (Bt) and its transfer to developing countries. ISAAA Briefs. pp. 42.
Krieg, A., Huger, A., Langenbuch, G. and Schneter, W., 1983, Bacillus thuringiensis var. tenebrionis : a new pathoype effective against larvae of Coleoptera. J. Appl. Entomol., 96 : 500-508.
Kumaraswamy, G. K, 2005, Expression of cry1Ac from native Bacillus thuringiensis D1 in Escherichia coli, M. Sc. (Agri) Thesis, Univ. Agricl. Sci., Dharwad.
Lambert, B., Buysse, L., Decock, C., Jansens, S., Piens, C. and Saey, B., 1996, A Bacillus thuringiensis insecticidal crystal protein with a high activity against members of the family noctuidae. Appl Environ Microbiol., 62 : 80–86.
Lycett, G. W. and Grierson, D., 1990. Genetic engineering of crop plants. Butter worth, London.
Lyer, V. N., Klee, H. J. and Nester, E. W., 1982, Units of genetic expression in the virulence region of a plant tumor inducing plasmid of Agrobacterium tumefaciens. Mol. Gen. Genet., 188 : 418-424.
Maizer, M., Pannetie, C., Tourneur, J., Jouanin, L. and Giband, M., 1997, Expression of Bt toxin genes in plant cells. Biotech. Annu. Rev., 3 : 313-347.
Masson, L., Moar, W. J., Van Frankenhuyzen, K., Bosse, M. and Brousseau, R., 1992, Insecticidal properties of a Crystal protein gene product isolated from Bacillus thuringiensis subsp. kenyae. Appl Environ Microbiol., 58 (2) : 642–646.
Perlak, F. J., Oppenhuizen, M., Gustafson, K., Voth, R., Sivasupramaniam, S. and Heering, D., 2001, Development and commercial use of Bollgard cotton in the USA – early promises versus today’s reality. Plant J., 27 : 489–501.
Rohini, V. K., and K. Sankara Rao, 2001. Transformation of peanut (Arachis hypogaea L.) with tobacco chitinase gene : variable response of transformants to leaf spot disease, Plant Sci., 160 (5) : 883-892.
Rohini, V. K. and K. Sankara Rao, 2000a. Transformation of peanut (Arachis hypogeae L.) : a non-tissue culture based approach for generating transgenic plants, Plant Sci., 150 : 41-49.
Rohini, V. K., and K. Sankara Rao, 2000b. Embryo transformation, a practical approach for realizing transgenic plants of safflower (Carthamus tinctorius L.), Ann. Bot. 86 : 1043-1049.
Sawant, S. V., Kiran, K., Singh, P. K. and Tuli, R., 2001, Sequence architecture downstream of the initiator codon enhances gene expression and protein stability in plants. Plant Physiol., 126 : 1630–1636.
Selvapandiyan, A., Reddy, V. S., Kumar, P. A., Tewari, K. K. and Bhatnagar, R. K., 1998, Transformation of Nicotiana tabacum with a native cry1Ia5 gene confers complete protection against Heliothis armigera. Molec. Breed., 4 : 473–478.
Singh, R. K. and Chaudhary, B. D., 1996, Biometrical methods in quantitative genetic analysis. Kalyani publishers, Ludhiana, pp. 191-200.
Stachel, S. E. and Nester, E. W., 1986, The genetic and transcriptional organization of the vir region of the A6T plasmid of Agrobacterium tumefaciens. Embo. J., 4 : 891-898.
Umbeck, P., Johnson, G., Barton, K. and Swain, W., 1987, Genetically transformed cotton (Gossypium hirsutum L.) plants. Bio/Tech., 5 : 263-266.
Visser, B., Munsterman, E., Stoker, A. and Dirkse, W. G., 1990, A novel Bacillus thuringiensis gene encoding a Spodoptera exigua – specific crystal protein. J Bacteriol., 172 : 6783– 6788.
Von Tersch, M. A., Robbins, H. L., Jany, C. S. and Johnson, T. B., 1991, Insecticidal toxins from Bacillus thuringiensis subsp. kenyae : gene cloning and characterization and comparison with B. thuringiensis subsp. kurstaki CryIAI toxins. Appl Environ Microbiol., 57 : 349–358.