Azad Takestan University, Iran Email: firstname.lastname@example.org
Although scientists often generically refer to the procedure by which plants are genetically engineered as “transformation”, it really entails three separate processes. The first, transformation, involves stable integration of DNA into the chromosome of an individual plant cell. The second step, regeneration, is the process by which individual transformed cells are coaxed, through hormonal and cultural manipulations, to develop into a whole plant. The third step , controlling Agrobacterium after trensformation. Generally,in Iran limitation of Agrobacterium is the most limiting of the three processes ( the usal antibiotics is very expensive and not available.
Oilseed rape (Brassica napus L.) is one of the leading crops which benefit from the application of genetic engineering through recombinant DNA technology in Iran too and we have optimized gene transformation in Brassica napus. In this research after testing 40 antibiotics combinations we have recognized the best media with Cefatoxim ( 200 mg/l) and Vancumycine ( 150 mg/l) for limitation the Agrobacterium growth after gene treansformation ( Gus Reporter).
Brassica napus- Transformation- Antibiotic
Plant tissue culture technology has been successfully used for the commercial production of pathogen-free plants (Debergh and Maene 1981), and to conserve the germplasm of rare and endangered species (Fay 1992). Techniques such as meristem culture (Hu and Wang 1983) and hot-water treatment of explants before in vitro culture (Hol and van der Linde 1992, Langens-Gerrits et al. 1998) have been used to produce plants free from pathogens. The most common method used for the transformation of B. napus is via Agrobacterium mediated gene transfer. Both Agrobacterium tumefaciens and Agrobacterium rhizogenes have been used for genetic transformation. A. tumefaciens mediated transformation has been attempted using a variety of explants such as hypocotyl (De Block et al., 1989; Radke et al 1988; Schroder et al 1994 ), inflorescence stalks (Boulter et al 1990), microspores (Huang 1992), thin cell layer (Ovesna et al 1993), and stem internodes (Fry et al., 1987). Other methods such as electroporation (Chapel and Glimelius 1990; Bergman and Glimelius 1993), direct DNA uptake using PEG (Golz et al,1990; Mukhopadhyay et al., 1991), microinjection (Jonesvilleneuve et al., 1995) and microprojectile bombardment (Chen and Beverdorf 1994) have also been used for transformation. Gene transformation in to Barassica napus by Agrobacterium is one of the successfully methods, but One of the major problems for gene transformation into Brassica napus is controlling the Agrobacterium after gene trasfermation. Carbenicylin is one of the important antibiotics that must used in the growth of media and is very effective for limitation of bacteria growth is very expensive and is not available so in this research we have tested different antibiotics with various doses for recognizing the best combination.
Seven commercial genotypes (Maluka, Hyola 308, Global, PF ,NSA,SLM, westar and falcon) of Brassica napus L., were used. Seeds of these varieties were stored at 40C until used. Seeds were surface-sterilised in
8 % (w / v ) sodium hypochlorite by vigorous shaking for 25 minutes. The seeds were placed on to germination medium after rinsing five times in sterile distilled water. Tissue culture media including germination medium used in the study were essentially the same as described by Murshig and skoog. Hypocotyl sections (07-1 cm long section ) and cotyledons with 3-5 mm long petioles were cut from germinated seedlings and dipped in an Agrobacterium tumefaciens C58 (pGV3101) containing pBI121 plasmid( with Gus Reporter gene) suspension for 30 seconds. After infection, the explants were placed immediately onto basal media. The explants were co-cultivated with Agrobacterium for two days and transferred to callus induction medium for one week with 500 mg/l carbenicillin to inhibit bacterial growth. Since the carbenicillin was not avaiable and its costs was high so we have tested 40 combinations of cheap antibiotics. In this method, dark green transgenic shoots with thick, odd-shaped leaves are induced after 14-21 days on supplemented media, followed by recovery of normal shoot morphology on a shoot elongation media with 4.5 mgl-1 BAP. Well-developed leafy green shoots were transferred to a common root-stimulating media, and planted into soil when roots had formed. The occasional shoot which formed callus on the rooting media was transferred directly to soil after removal of the callus. glucuronidase (GUS) activity were used to confirm transformation of plantlets and to determine the frequency of transgene rearrangements. In most cases, transformation efficiencies were calculated conservatively by only including plants which grew to produce seed in the greenhouse. ( Greenhouse condition : Mercury lights were used to supplement natural lighting and provide a 16 hour day-length. Greenhouse temperature was not effectively controlled. However, daytime temperatures were around 21 ± 5°C).
For introducing the best antibiotics and the best controling Agrobacterium we have tested 40 differents antibiotics. One of the major limitation in gene transformation in to Brassica napus in Iran was finding a cheap and available antibiotics after gene transformation. Data analysis ( SPSS soft ware ) have showed that there are significant difference between anti biotics combination and their doses. After mean cpmpration ( Dunkan ) we have introduced the best media . Combination of Cefatoxim ( 200 mg/l) and Vancumycine ( 150 mg/l) showed the best results for limitation the Agrobacterium growth. By meaning compration ( LSD ) cotyledone showed the high regeneratin than hypocotyle and we could obtain 23% transformed complete plants from Westar cotyledon explants. In the another research, peduncles from flowers and cotyledonary petioles have been used for transformation of other Brassica crops such as cabbage, cauliflower and kale (Christey and Sinclair 1992; Bhalla and Smith 1998; Metz et al 1995) but no study has been conducted to compare using hypocotyl, cotyledon and root explants of oilseed rape in order to maximize the recovery of viable transformed shoots from genetic transformation experiments.
Bahalla , P. L and N. Smith, 1991, Agrobacterium tumefacies mediated transformation of cluliflower . Brassica Oleracea Var. botrytis. Molec Breeding, Vol. 4: 531-541.
Bamberg J. B., Del Rio, A., Singsit, C and B . Radcliffeedward, 2002, RAPD analysis of genetic diversity in solanum populations to predict the need for fine screening. Tektran.
Belarmino, M. M ., Abe, T and T. Sasahara, 1994, Plant regeneration from stem and petiol protoplasts of sweet potato and wild relative . Plant Cell Tiss.Org.Cult,Vol,
37: 145- 150.
Carodoza, V and C . N. Stewar, 2003, Increased Agrobacterium mediated transformation and rooting efficiencies in Canola ( Brassica napus. L ) from hypocotyls segment explant, Plant Cell Rep . Vol , 21:599- 604.
Carputo, D., Cardi, T., Ferraiolo,G and L. Frusciante , 1995 , Tissue culture response in various wild and cultivated Solanum germplasm accessions for exploitation in potato breeding. Plant Cell Tiss. Org. Cult. Vol, 41: 151-158.
Centeno, M. L., A. Rodriguez., I. Feito and B. Femandez ,1996, Relationship between endogenous auxin and cytokinin levels and morphogenic responses in Actinidia deliciosa tissue cultures. Plan Cell Rep. Vol, 16: 58- 62.
Charles, G., Rosignol, L and M. Rosignol, 1992, Environmental effects on potato plants in vitro . J. Plant Physiol. Vol, 139: 708- 713.
Debergh, P.C. and L.J. Maene. 1981. A scheme for commercial propagation of ornamental plants by tissue culture. Scientia Horticulturae 14: 335-345.
De Block,M ., De Brouwer, D and P. Tenning , 1989, Transformation of Brassica napus and Brassica Oleracea using Agrobacterium tumefacies and the expression of the bar and neo genes in the transgenic plants. Plant Phisiology. Vol , 91: 694- 701.
Dietert, M.F.,Barron,S.A and O.C.Yoder , 1982,Effects of genotype on in vitro culture in genus Brassica.Plant Sci.Letters,Vol.26:233-240.
Eapen , S., Tivarekar and L. Goerge, 1998, Thidiazuron -induced shoot regeneration in pigeonpea (Cajanus cajan L.). Plant Cell Tissue and Organ Culture. Vol, 53: 217- 220.
Escatland , Z and A. R. Langille, 1998, Photoperiod , temprature, gibberellin and antigiberellin affect tuberization of potato stem segments in vitro. Hortscience. Vol,
33: 701- 703.
Fay, M.F. 1992. Conservation of rare and endangered plants using in vitro methods. In: Vitro Cellular and Developmental Biology. pp. 1-4. 28pp.
Fry, J., Barnason, A and R. Horsch, 1987, Transformation of Brassica napus with Agrobacterium tumefacies based vectors. Biological Sciences . Vol, 6:321- 325.
Gruber, M., Auser, P and G, Rakow, 2001, Plant Transformation in yellow – seeded Brassica napus Breeding germplasm.Saskatoo Research Center , SK., S7No X2, Canada.
Hol, G.M.G.M. and P.C.G. van der Linde. 1992. Reduction of contamination in bulb-explant cultures of Narcissus by a hot-water treatment of parent bulbs. Plant Cell, Tissue and Organ Culture 31: 75-79.
Hu, C.Y. and P.J. Wang. 1983. Meristem, shoot tip, and bud cultures. In: Evans, D.A., Sharp, W.R., Ammirato, P.V. and Y. Yamada (Eds.). Handbook of Plant Cell Culture: Techniques for Propagation and Breeding. Vol. 1. Macmillan Publishing Co., New York, pp. 177-227.
Jalali- Javaran, M, Morris, K and V. Buchanan – Wollustrow, 2001 , Transformation of Brassica napus with antisenseglutamine synthetase gene. Experimental Botany . pp,48-49.
Menze, A and C. Mollers , 2001, Transformation of different Brassica napus cultivars with three different strains of Agrobacterium rhizogenes . Institue fur P flanzeb au and p flanzen zuchtung in France.
Metz, T. D., Dixit , R and E. D. Earle, 1995 , Agrobacterium tumefaciens – medited transformation of broccoli (Brassica Oleracea Var . italica) and eabbage . (B.Oleracea Var Capitate ). Plant Cell Reports. Vol,15: 287- 292.
Muhaammad , R. K., Hamid , R., Muhammad , A and C. Zubeda, 2003, Hight frequency shoot regeneration and Agrobacterium mediated DNA transfer in canola (Brassica napus). Plant Cell, Tissue and Organ Culture . Vol, 75: 223- 231.
Murashinge, T and F. C .Skoog, 1962, A revised medium for rapid growth and bioassays with tissue culture. Physiologia Plantarum. Vol, 15, 473- 497.
Radlke , S., Andrews , B., Molony, M., Kridi ,Y and V, Knauf, 2000, Transformation of Brassica napus using Agrobacterium tumefaciens : development regulated expression of reintroduced napin gene .Theor .Appl.Genet Vol . 75: 685- 694.
Takasaki, T., Hatakeyama, KOjima ,M., Watanube, K and K. Hinata , 1997 , Factors influencing Agrobacterium- mediated transformation of Brassica napus. Breeding Science ,Vol. 47:127- 134.
Tang, G. X., Zhou,W. J., Li , H. Z., Mao, B. Z., H and K. Yoneyama , 2003, Medium, Explant and genotype factors in fluencing shoot regeneration in Oilseed Brassica.Cpp.