Agrobacterium tumefaciens transformation genetic transformation of banana

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1 84 Agrobacterium tumefaciens transformation genetic transformation of banana EFFECTS OF VARIOUS VIRULENT STRAINS OF Agrobacterium tumefaciens ON GENETIC TRANSFORMATION OF BANANA (Musa sp.) CULTIVAR WILLIAMS Esuola CO *1, 2, Tripathi L 1, and Fawole I 3 1 International Institute of Tropical Agriculture, P.M.B 5320, Oyo-Road, Ibadan, Nigeria 2 Botany Department, University of Ibadan, Ibadan, Oyo State, Nigeria 3 Crop Protection and Environmental Biology Department, University of Ibadan, Ibadan, Oyo State, Nigeria * Correspondence: bolajiius@yahoo.com Abstract Investigations were conducted to select the appropriate Agrobacterium tumefaciens strains for efficient transformation of the banana cultivar Williams. Three A. tumefaciens strains (EHA105, C58, and GV2260) were used for the study. The ability of these strains to transfer β-glucuronidase (GUS) gene into banana tissues was assessed through GUS histochemical assay. Results obtained demonstrated that GUS activity was more enhanced in banana meristem pieces that were co-cultivated with EHA105 strain than in tissues that were co-cultivated with the other two strains. The least transient GUS expression was recorded in banana tissues that were co-cultivated with C58. The effect of the strains on efficient regeneration also indicated that EHA105 is more efficient than the other two strains. Therefore, Agrobacterium tumefaciens strain EHA105 appears to be the appropriate strain for efficient transformation of banana. Keywords: Agrobacterium tumefaciens, β-glucuronidase, banana cultivar Williams Introduction Bananas and plantains (Musa spp. L.) are major staple food, supplying up to 25% of the carbohydrates for approximately 70 million people in the humid zone of Sub- Saharan Africa (Faostat, 2007; Swennen, 1990). Banana production is continuously threatened by numerous pests and diseases (Jones 2000). The decrease in production of plantain can be as much as 50% (Mobambo et al. 1993). Black sigatoka (Mycosphaerella fijiensis), wilt (Fusarium oxysporium f.sp.cumbense), vascular bacterial wilts, viruses [Banana bunchy top virus (BBTV), Banana streak virus (BSV)] and nematodes cause significant crop losses worldwide (Carlier et al., 2000; Ploetz and Pegg, 1999; Lockhart and Olszewski, 1993). Although most of these diseases can be controlled by the application of chemicals (pesticides, fungicides, etc), this option places a heavy economic cost on the farmer and may not be feasible for the resource poor, subsistence farmers in sub-saharan Africa. Moreover, the adverse environmental effect of chemical control on the ecosystem is undesirable. Breeding for disease-resistant banana cultivars using conventional methods remain a difficult endeavour because of the various ploidy levels, slow propagation, long-time span from one generation to the next, and a large area for field - testing. Genetic engineering may offer an alternative solution to solve these problems. For that purpose, a wide set of target genes is currently available which can be introduced into banana and plantain for valuable agronomic traits such as pest and disease resistance, fruit maturation and storage (Swennen, 1990). There have been a number of reports on the genetic transformation of banana via microprojectile bombardment (Becker et al., 2000; Sagi et al., 1995). At present time the gene transfer by Agrobacterium is the method of choice for the genetic transformation of most plant species. It is perceived to have several advantages over other forms of transformation such as microprojectile bombardment, including the ability to transfer large segments of DNA with minimal rearrangement and with fewer copies of inserted genes at higher

2 Esoula et al. 85 efficiencies with lower cost (Lindsey, 1992; Raineri et al., 1990; Bytebier et al., 1987; Schafer et al., 1987)). Agrobacterium mediated gene transfer into banana has been reported only for few cultivars, for example, Grand Nain and Rasthali (Tripathi et al., 2008; Khanna et al., 2004; Ganapathi et al., 2001; May et al., 1995). Bosque-Perez et al. (2000) reported the efficiency of transformation using three different Agrobacterium tumefaciens strains with the banana cultivar Grand Nain; however, there are currently no published reports on the cultivar Williams. This work aims at studying the effect of three virulent strains of Agrobacterium tumefaciens (EHA105, C58 and GV2260) on genetic transformation of cultivar Williams. Materials and Methods Plant Materials The suckers of the cultivar Williams were collected from the screen house at IITA- Ibadan. Plantlets were regenerated through micropropagation using apical shoot tips as described by Tripathi et al. (2003). The apical shoot tips of banana cv. Williams were cultured on MS medim (Murashige and Skoog, 1962) supplemented with Benzylaminopurine (BAP 5 mg l-1). The ph was adjusted to 5.8 and the medium was solidified with 0.2% gelrite after which it was dispensed into culture tubes and autoclaved at 121 o C for 15 minutes. The culture tubes were sealed using parafilms and incubated in the culture room at the temperature of 28 o C with fluorescent tubes providing light for four weeks. Established cultures were routinely sub- cultured on fresh semi-solid medium every three-four weeks by subdividing the shoot clusters with a scalpel for further proliferation of shoots. For the elongation of shoots, the individual shoot was transferred to medium supplemented with BAP (3 mg l-1) and IAA (0.3 mg l-1). Strains of Agrobacterium tumefaciens and plasmid Agrobacterium tumefaciens strains EHA105, C58, and GV2260 obtained from IITA were used in the transformation experiments. The plasmid pcambia1201 containing the gusa gene encoding β- glucuronidase with an intron as reporter marker and hpt encoding hygromycin phosphotransferase as selection marker was acquired from CAMBIA, Australia. A schematic representation of the plasmid vector is shown in Fig 1. The gus intron reporter gene results in expression of GUS activity in plant tissues but not in A. tumefaciens cells (Vancanneyt et al., 1990). Transformation of Agrobacterium tumefaciens strains with pcambia 1201 The pcambia 1201 plasmid DNA was isolated from E. coli strain containing this plasmid according to Sambrook et al. (1989). The plasmid DNA recovered from E.coli cells was purified by precipitation with polyethylene glycol (PEG) according to the method described by Sambrook et al. (1989) and confirmed on agarose gel before use for transformation with the A. tumefaciens strains. A. tumefaciens strains were transformed with the plasmid pcambia1201 according to the modified method of Gynheung (1987). Isolation of plasmid DNA from transformed Agrobacterium cells was done according to Sambrook et al. (1989) to confirm successful integration of the plasmid DNA into transformed bacterium cells.

3 86 Agrobacterium tumefaciens transformation genetic transformation of banana Figure 1: Schematic representation of T-DNA region of binary vector pcambia Agroinfection of explants and regeneration of plantlets A single colony of each of the Agrobacterium strains (EHA105, GV2260 and C58 separately) carrying the plasmid 1201 DNA was inoculated in 25 ml LB broth (1% tryptone, 0.5% yeast extract and 1% Sodium chloride) with appropriate antibiotics and grown at 28 o C for 48 hours. About 1ml of inoculum from the 48 hours grown culture was used to inoculate 25 ml of LB and the culture was grown at 28 o C till Optical Density (O.D600 nm) reached 0.8. The bacteria cells were harvested at 5,000 rotations per minute for 10 minutes at 4 o C and pellet was resuspended in MS Liquid containing 100 µm Acetosyringone. The apical shoot tips (approximately 2 mm) were isolated from the in vitro grown shoots. The shoot tips were bisected longitudinally and arranged in the centre of the plate containing MS + BAP medium.the plates were incubated in dark at 28oC overnight. Explants were micro-wounded prior to co-cultivation with A. tumefaciens strains through microprojectile bombardment using naked Gold particles (1µm, BioRad). The fifty micro-wounded explants were added to the bacterial suspension and vacuum infiltrated at 25 inches of Mercury (inhg) for 30 seconds. Co-cultivation was done for 30 minutes with gentle shaking. The tissue pieces were then blotted on sterile tissue paper and transferred to co-cultivation medium containing MS Agar, 5 mg l-1 BAP and 100 µm Acetosyringone and incubated in the dark at 28oC for three days. Co-cultivated explants were then transferred to medium containing cefotaxime (200 mg l-1) to kill Agrobacterium and incubated in the culture room at the temperature of 28 o C with 16 hours photoperiod with fluorescent tube providing light. Some of the explants were randomly picked after two days and tested for GUS histochemical activity following the method of Jefferson (1987). The remaining explants were transferred to the selective medium (regeneration medium containing 25 mg l-1 hygromycin and 200 mg l-1 cefotaxime). The cultures were transferred to fresh selection medium every two weeks. The putatively transformed shoots regenerated on selection medium were transferred to rooting medium. Histochemical GUS assay The GUS histochemical assay for transient gene expression was performed 5 days after co-cultivation according to the modified procedure of Jefferson (1987). The explants were washed in 70% ethanol followed by incubation in fixation solution (0.3% v/v formaldehyde, 10 mm MES, ph 5.6, 0.3 M mannitol) for minutes at room temperature. The explants were vacuum infiltrated for 2 minutes for proper fixation and extensively washed with 50 mm phosphate buffer (ph 7.0). The fixed explants were incubated with the substrate

4 Esoula et al. 87 solution (1 mm X-gluc, 50 mm sodiumphosphate (ph 7.0), 5 mm potassium ferricyanide, 5 mm ferrocyanide, 10 mm EDTA, 50 mm Ascorbic acid), vacuum infiltrated for 2 minutes and incubated at -24 hours. Chlorophyll was removed by immersing the explants in solution of methanol and glacial acetic acid (3:1) for 3 to 4 hours followed by dehydration in a series of ethanol (50, 70, and 95%). Results and Discussion Transformation of Agrobacterium tumefaciens strains with pcambia 1201 The pcambia1201 plasmid DNA was successfully transferred into the three strains of Agrobacterium tumefaciens EHA105, C58 and GV2260 and colonies were grown on selective medium. The plasmid DNA extracted from the transformed Agrobacterium tumefaciens strains was checked on agarose gel for confirmation. The expected size bands, which correspond to that of the pcambia1201 (13 Kb) was observed (Fig. 2). Marker EHA105 C58 GV2260 GV2206 Kb Figure 2: The three strains of Agrobacterium tumefaciens containing Plasmid 1201 DNA after transformation Evaluation of Agro-infected Explants for Result of the transient GUS expression transient GUS assay shown by the blue colouration of the Histochemical Gus assay was conducted transformed meristem pieces indicates that on explants that were co-cultivated with genes could be introduced into meristematic cells of the three different strains of tissues on banana via Agrobacterium Agrobacterium tumefaciens (EHA105, C58 mediated transformation. Ganapathi et al. and GV2260 respectively), harbouring the (2001) also reported a successful plasmid pcambia The results transformation of embryogenic cells of demonstrated that transformation of explants banana cultivar Rasthali co-cultivated with with EHA105 produced higher number of Agrobacterium tumefaciens EHA 105 strain. blue spots (94%) than with C58 (44%) or The blue staining was indicative of the GV2260 (22%) [Figs 3 and 4]. presence of GUS noted over the entire surface of the tissues while non-

5 88 Agrobacterium tumefaciens transformation genetic transformation of banana transformed control cells did not stain blue. Comparison of the Agrobacterium tumefaciens strains for genetic transformation of banana cultivar Williams suggests that EHA105 is more efficient in the transformation of banana meristem tip pieces than GV2260, while the latter strain is more efficient than C58 (Figs 3 and 4). Probably EHA105 is more compatible with banana tissues. Figure 3: Response of explants of banana cultivar William to - -Glucuronidase assays Figure 4: Response of transformed Explants with EHA105 strain to GUS assay (A-D). Blue colouration indicates GUS positive as compared with the control Explant in plate (E). Regeneration of Explants The recovery of stable transformants mainly depends on the regenerative competence of the target tissues and their recovery after co-cultivation. The regeneration of explants after co-cultivation with various Agrobacterium strains was compared. The explants co-cultivated with EHA105 regenerated at an efficiency of about 70% as compared to both GV2260 and C58 (60% each) [Fig. 5]. Hiei et al. (1997) on their work on rice reported that the optimization of transient activity is a waste of time if experiments are conducted on non-regenerable tissues. Hence an ideal system of Agrobacterium mediated transformation must have a high rate of T-DNA transformation into plant cells with a strong regeneration potential.

6 Esoula et al. 89 The test conducted on efficient regeneration of cultivar Williams meristem pieces after transformation with the three Agrobacterium tumefaciens strains separately showed that EHA 105 is more efficient than the other two strains. This can be explained by the fact that EHA 105 is a supervirulent strain. Bosque-Perez et al. (2000) reported similarly that EHA105 is more efficient in the transformation of Grand Nain using meristem pieces. Tripathi et al. (2005) have also demonstrated that A. tumefaciens strain EHA105 is the best strain for transformation of apical shoot tips of the plantain cultivar Agbagba. Therefore, EHA105 can be suggested for use in Agrobacterium mediated transformation of plant regenerable tissues as compared to C58 and GV2260. Figure 5: Regeneration percentage of explants of bananas cultivar Williams Conclusion The result of this study confirms that Agrobacterium mediated transformation can be used with Agrobacterium - based vectors for foreign gene transfer into bananas and plantains. Bananas and plantains have enormous economic and nutritional value. The availability of transformation systems that can complement conventional breeding programmes is thus, of great importance for the improvement of Musa spp. The genetic technologies should allow the incorporation into existing Musa cultivars desired traits including resistance to fungal and viral diseases. Acknowledgements Practical assistance of P. Ogunsanya is acknowledged. This research was supported by IITA. References Bosque-perez N, May GD, Arntzen CJ Applicability of an Agrobacterium based system for the Transformation of Musa spp. with diverse genomic constitution and ploidy level. In: Craenen K, Ortiz R, Karamura EB, Vuylsteke DR (Eds.). Proceedings of the first International Conference on Banana and Plantain for Africa. Acta Horticulturae 540, Bytebier B, Deboeck F, De Grere H, Van Montagee M, Hernlsteens JP T- DNA organization in tumor cultures and transgenic plants of the monocotyledon Asparagus officinalis. Proceedings of the National Academy of Sciences, USA. 84, Carlier J, Fouré E, Gaul F, Jones DR, Lepoivre P, Mourichon X, Pasberg- Gauhl C, Romero RA Fungal

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