African Journal of Biotechnology Vol. 12(15), pp. 1811-1819, 10 April, 2013 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB12.2478 ISSN 1684–5315 ©2013 Academic Journals Full Length Research Paper Agrobacterium mediated transformation of banana (Musa sp.) cv. Sukali Ndiizi (ABB) with a modified Carica papaya cystatin (CpCYS) gene Namuddu, A.1, Kiggundu, A.1*, Mukasa, S. B.2, Kurnet, K.4, Karamura, E.3 and Tushemereirwe, W.1 1 National Agricultural Research Organization, P.O. Box 7065, Kampala. 2 Crop Science Department, Makerere University Kampala, P.O. Box 7062, Kampala. 3 Bioversity International, P.O. Box 24384, Kampala-Uganda. 4 Forestry and Agricultural Biotechnology Institute, Department of Plant Science, University of Pretoria, 74 Lunnon Road, Hillcrest, Pretoria, 0002. South Africa. Accepted 31 December, 2012 Conventional banana breeding for pest and disease resistance is a very difficult and slow process due to the limited sources of resistance, sterility of cultivated banana varieties, high polyploidy levels, long cropping cycle and the lack of rapid screening methods. Molecular breeding using the transgenic approach with candidate genes such as cystatins offers an alternative method to banana improvement. Cystatin proteins inhibit the activity of cysteine proteases responsible for the breakdown of dietary proteins in the gut of many pests including nematodes resulting in protein deficiency. In this study, the papaya cystatin gene was introduced into the banana genome. Embryogenic cell suspension (ECS) cultures of the banana cultivar Sukali Ndiizi (ABB) were used as explants material for the successful transformation of banana. The Carica papaya cystatin gene (CpCYS-Mut89) previously modified to improve its inhibitory potential against banana pests was introduced into this cultivar using Agrobacterium tumefaciens, strain LBA4404 and the gus reporter gene was used to observe successful transformation process. We report the successful protocol for routine transformation of this cultivar, which was completed in six months with plant regeneration observed at a frequency of 23%. An additional four months was required to multiply the regenerant lines in order to have at least 20 plants per line for downstream challenging studies. Putatively transgenic plants were analyzed by PCR using hpt and CpCYS-Mut89 specific primers to confirm the presence of transgenes. Out of 28 selected lines, 27 were positive for both hpt and CpCYS-Mut89 transgenes giving 96.4% transformation efficiency. Five lines were then selected on the basis of putative PCR positives and a Southern blot analysis gave hybridization signals with 1 to 4 copy number integration patterns characteristic of Agrobacterium mediated transformation. These results confirm stable gene integration in East African banana cultivar cv. Sukali Ndiizi (genome group ABB) through an efficient Agrobacterium-mediated transformation protocol described for routine use in future improvement of this crop with genes of economic importance. Key words: Cystatins, banana, Agrobacterium mediated transformation, southern blot. INTRODUCTION Banana is one of the most important staple food crops in most important staple crops contributing about 30% of the tropics and a source of income to millions of poor the total food consumption and 14% total crop value subsistence farmers. It is the developing world's fourth (Kalyebara et al., 2005). About 24% of the agricultural most important food crop after rice, wheat and maize households are engaged in banana production. Banana (Bioversity International, 2006). In Uganda, it is one of the being a year round fruiting crop ensures food security at 1812 Afr. J. Biotechnol. household level, providing food to more than 70% of (0°25’N, 32°32’E) is 13 km north of Kampala city and 1,195 m Uganda’s population on a regular basis. Banana is above sea level. The climate is tropical with bimodal rainfall. Embryogenic cell suspensions (ECSs) of banana cv. Sukali Ndiizi primarily grown for subsistence needs and any surplus (AAB) developed in the tissue culture laboratory at National for sale to local markets. Agricultural Research Laboratories were used. ECS were sub- Two major pests affect banana productivity through cultured and maintained as previously described (Cote et al., 1996). damage to the stem and root system. These are the The Agrobacterium strain LBA4404 was used. The transformation banana weevil (Cosmopolites sordidus) (Gold et al., vector was designed using pCAMBIA1300 binary vector obtained 2001) and parasitic nematodes of various species from CAMBIA. The papaya cystatin gene (CpCYS-Mut89), a donation from the laboratory at University of Pretoria was cloned (Speijer et al., 1998). Banana weevil and nematodes are within the multiple cloning sites (MCS) under the double enhancer heavily responsible for the decline and disappearance of version of Cauliflower Mosaic Virus (CaMV) 35S promoter and highland bananas from their traditional growing zones in CaMV 35S terminator. The pCAMBIA vector back-bone sequence East Africa (Gold et al., 1999). Several migratory also constituted kanamycin resistance gene (npt11) for bacterial endoparasitic nematodes including, Pratylenchus selection and hygromycin B phosphotransferase (hpt) resistance goodeyi, Radopholus similis and Helicotylenchus gene as the plant selectable marker gene. The hpt gene was driven by the CaMV 35S promoter (Figure 1). In addition, ECS were multicinctus have been reported to attack bananas in transformed with the binary vector pGreen constituting the gusA Uganda leading to serious crop losses (Gold et al., 1994). gene under the control of the maize poly-ubiquitin promoter as the R. similis, the burrowing nematode is reported to be the positive control. Successful transformation was monitored by most damaging, with a life cycle of 20 to 25 days. R. transient histochemical Gus assay. similis feeds and reproduces within living corm and root tissue, affecting the root cortex leading to necrosis before Agrobacterium- mediated transformation of ECSs of the death (Sarah et al., 1996). Tissue damage impairs water banana cv. Sukali Ndiizi and nutrient uptake and causes poor plant anchorage leading to plant toppling, crop losses and reduced bunch Embryogenic cell suspensions (ECSs) from male flowers of banana weight (Speijer et al., 1998). cultivar Sukali Ndizi were transformed using the Centrifugation Conventional banana breeding for pest and disease Assisted Agrobacterium-mediated Transformation system (CAAT) as previously reported by Khanna et al.(2004). To increase the resistance is a difficult and slow process due to the competence of ECSs and therefore transformation efficiency, they limited sources of resistance, sterility of cultivated banana were refreshed with new media five days prior to transformation. varieties, high polyploidy levels, long cropping cycle and Prior to transformation, a settled cell volume (SCV) of 1.25 ml ECSs the lack of rapid screening methods. Biotechnology tools in 50 ml of liquid MA2 were cultured for five days to induce fast cell such as genetic engineering can offer potentially effective division and subsequently increase their competence for and sustainable solution to the problem of controlling Agrobacterium infection. Liquid MA2 consisted of standard MS salts and vitamins (Murashige and Skoog, 1962), 4.1 µM biotin, 4.5 µM banana parasitic nematodes and banana weevils by 2, 4-D, 680 µM glutamine, 100 mg/l malt extract, 20 mg/L ascorbic transferring important genes into the banana genome acid, 45 g/L sucrose and the pH was adjusted to 5.3 before without altering the original characteristics of a given autoclaving. A 0.5 ml five day-old aliquot settled cell volume of cultivar. Agrobacterium- mediated transformation is one banana ECSs were re-suspended in 10 ml activated Agrobacterium of the methods of gene transfer that has been successful suspension, adjusted to 0.6 (OD600nm). The mixture of ECS and Agrobacterium were incubated in the dark at 22°C for 5 days. in the production of transgenic banana of other Infected ECS were then washed in liquid MA2 containing a broad internationally important cultivars (Khanna et al., 2004; spectrum antibiotic cefotaxime and then transferred onto selective Ghosh et al., 2009). It is a well-known fact that banana as embryo formation media (MA3) comprising 3.2 g/L SH salts, in many monocotyledonous plants cultivar or variety standard MS vitamins, 4.1 µM biotin, 100 mg/L malt extract, 680 recalcitrance to tissue culture and Agro-bacterium µM glutamine, 230 mg/L proline, 100 mg/L myo-inositol, 60 mg/L mediated genetic transformation. Therefore the objective citric acid, 40 mg/L ascorbic acid, 10 g/L PVP 10, 400 mg/L L- cysteine, 1.1 µM NAA, 0.2 µM zeatin, 0.5 µM kinetin, 0.7 µM 2-ip, of this work was to develop an efficient agrobacterium 45 g/L sucrose, 10 g/L lactose and 2.3 g/L phytagel). mediated transformation protocol for routine genetic improvement of this popular east African Cultivar based on recently developed embryogenic cell suspension Histochemical Gus assay cultures. Five days after co-cultivation of ECS with the A. tumefaciens harbouring pGreen binary vector with gusA gene encoding the β- glucuronidase (GUS) enzyme, transformed ECS of the banana cv. MATERIALS AND METHODS Sukali Ndiizi were assayed for transient expression of the gusA reporter gene. Three samples of 0.02 ml SCV were incubated in a The study was carried out at the National Agricultural Research Gus assay substrate constituting 100 mM sodium phosphate (pH Laboratories Kawanda, one of the research institutes of the 7.0), 50 mM ascorbate, 0.1% tritonX-100, 0.4 mM potassium National Agricultural Research Organization, in the tissue culture ferricyanide, 0.5 mM potassium ferrocyanide and 1 mM 5-bromo-4- laboratory, biotechnology laboratory and green house. Kawanda chloro-3-indolyl-β-D-glucuronic acid (X-Gluc) according to Jefferson (1987). Sterile sheets of filter paper were laid in sterile 15 cm diameter Petri dishes and 1 ml of X-Gluc staining solution was placed at the center of each sterile filter paper. Transformed cells *Corresponding author. E-mail: akiggundu@kari.go.ug. plated on 50 µM nylon mesh, were transferred onto the wet filter Namuddu et al. 1813 Figure 1. Schematic representation of the T-DNA for pCAM35S-CysMut89. papers and stained with additional 200 µL of X-Gluc staining approximately 5 cm in height were weaned and placed in a high solution. The Petri dishes were sealed with cling film and incubated humidity chamber at 27°C in the biosafety level II green house. at 37°C overnight for expression of β-glucuronidase (gusA) gene. After a month, the shade was removed and plants transferred to The numbers of blue foci were then observed under a stereo pots. The ECS transformed with the gusA gene went through the microscope and photographed using a digital camera. same selection and regeneration process but with 50 μg/μl Histochemical GUS staining of leaves, stem and roots of geneticin for plant selection. greenhouse transgenic plants two months after weaning (~30 cm tall) was also performed for detection of stable gus expression in mature tissues. To prevent interference of visual analysis and DNA isolation and PCR analysis photographing from chlorophyll and other pigments, the stained materials were soaked and washed in a solution containing acetic: Genomic DNA of papaya cystatin transformed and non ethanol (1:3). transformed, two month old green house banana plants was extracted from cigar leaf tissue using the modified cetyltrimethylammonium bromide (CTAB) protocol (Gawel and Selection and regeneration of transgenic banana plants Jarret, 1991). DNA was analyzed using PCR to detect presence of the hygromycin B phosphotransferase (hpt) selectable marker and Infected ECS were washed with liquid MA2 medium containing a the papaya cystatin gene (CpCYS-Mut89). The PCR reaction broad spectrum antibiotic cefotaxime (300 μg/μL) and drained. contained 1.0 mM MgCl2 for CpCYS-Mut89 primers and 1.5 mM Transformed ECS were then transferred to selective semisolid MA3 MgCl2 for hpt primers, 0.4 μM of each of the primer pairs, 1× PCR medium with 300 μg/μL cefotaxime to kill off bacteria and 50 μg/μL buffer, 0.24 mM dNTPs and 0.02Unit Taq/reaction (25 μl). The hygromycin to select transformed cells. Transformants maintained primers CYS_For (5’-GGA ATT GTG ATC GGT GGT TT-3’), selective semisolid MA3 medium with subsequent subculture every CYS_Rev (5’- CGA GCT AAT GAG CAT CAC CA-3’), HYG-F (5’- two weeks until embryos were obtained. After three months of CTA TCG GCG AGT ACT TCT ACA CAG-3’) and HYG-R (5’-CCC incubation in the dark at 26°C, grayish-white masses of cell clusters ATG TGT ATC ACT GGC AAA C-3’) were designed using an online appeared. The cell clusters were individually transferred onto primer designing software (Primer3, version 0.4.0, selective semisolid RD1 media consisting of (standard MS salts and http://frodo.wi.mit.edu) as forward (For) and reverse (Rev) for vitamins (Murashige and Skoog, 1962), 20 mg/L ascorbic acid, 100 CpCYS-Mut89 gene, and as forward (F) and reverse (R) for hpt mg/L myo-inositol, 30 g/L sucrose, pH 5.3 and 2.3 g/L phytagel) gene. The reaction mixture was subjected to an initial denaturation with 300 μg/μL cefotaxime and 50 μg/μL hygromycin for further step of 94°C for 3 min followed by 35 cycles of 94°C for 30 s; embryo initiation, until good embryos were obtained. The embryos annealing temperatures of 60°C for hpt and 61°C for CpCYS-Mut89 were maintained on RD1 for for months. Single embryos were then for 1 min; 72°C for 1 min and a final extension step of 72°C for 8 transferred onto selective semisolid MA4 media constituting min. standard MS salts supplemented with Morel vitamins (Morel and Wetmore, 1951), 0.22 µM 6-benzylaminopurine (6-BAP), 1.14 µM IAA, 30 g sucrose, pH 5.8 and 2.3 g phytagel (which is embryo Southern blotting analysis germination media according to Namanya et al. (2004)) with 300 μg/μL cefotaxime and 50 μg/μL hygromycin. After one month, the Genomic DNA putatively transformed lines was restricted with 100 selection pressure was removed by transferring the regenerated units of EcoRI (New England Biolabs). EcoRI restriction enzyme shoots onto anti-biotic free semisolid proliferation media containing has one cleavage site in the binary vector. The restricted DNA was standard MS salts and vitamins, 20 mg/L ascorbic acid, 30 g/L resolved on 0.8% (w/v) agarose gel and blotted onto the positively sucrose, 5 mg/L 6-BAP, pH 5.8 and 2.3 g/L phytagel for shoot charged nylon membrane (Boehringer Mannheim) by the method of multiplication, with subculture of 2 weeks interval. The banana Southern (1975). The blots were hybridized with DIG labeled shoots were then transferred onto proliferation media with half CpCYS-Mut89 probes generated using PCR DIG probe synthesis strength of 6-BAP after obtaining at least four clones per plantlet to kit (Roche). Hybridization and detection of the probe was done maintain single shoots. After ten months, 57 rooted lines of using the DIG luminescent detection kit for nucleic acids (Roche 1814 Afr. J. Biotechnol. Figure 2. Transformation and regeneration of transgenic bananas. A. White clusters of Ndiizi cells with black cells in the background show that the black cells were non-transformed and thus died due to the toxicity of the hygromycin antibiotic in the selection MA3 media. B. Mature embryos on selection embryo initiation media (RD1) 2 months after transferring the white clusters to RD1 with some embryos germinating into shoots on selection MA4 1 month after transferring them onto MA4. D. Multiple shoots regenerated on non-selection proliferation media E. Transgenic lines in the green house. Diagnostics) according to manufacturer’s instructions. The selective MA3, whereas untransformed cells showed hybridization signal was detected by autoradiography. necrosis (Figure 2A). The cell clusters were then transferred onto selective semisolid RD1 media with subsequent sub culturing every two weeks to allow RESULTS embryo initiation. Embryos were observed on selective RD1 after two months (Figure 2B). Mature antibiotic Agrobacterium mediated transformation of the resistant embryos were then transferred onto selective banana cv. Sukali Ndiizi semisolid MA4 media and embryos were observed germinating into shoots after one month (Figure 2C). The Readily available ECSs of the banana cultivar Ndiizi were 57 hygromycin resistant shoots obtained were multiplied transformed with Agrobacterium confirmed to carry the on proliferation media without any selection and about 3 binary vector with CpCYS-Mut89 gene. Distinct whitish to 8 shoots per plant were obtained in two months (Figure cell clusters were observed three months after transfer to 2D). All plants regenerated from one cell clump were Namuddu et al. 1815 Figure 3. Histochemical assay for transient and stable expression of the gusA gene in Agrobacterium- transformed ECSs and o mature tissues from 2 month old greenhouse grown plantlets of banana cv. Sukali Ndiizi after overnight incubation at 37 C. Gus expression in gus transformed embryogenic cells (A), leaf tissues (B); root tissues (C). Figure 4. Schematic representation of the transformation of banana cultivar Sukali Ndiizi, selection and regeneration process indicating the time intervals between stages and number of cell clusters, embryos and shoots obtained, before transfer to a biosafety level-II greenhouse. considered to be clones and counted as one line. A month old greenhouse grown transgenic plants and summary of the whole transformation, selection and histochemically stained. The dark blue spots show that regeneration process is represented in the schematic Ndizi cells and mature tissues took up the reporter gene diagram (Figure 4). (Figure 3). Positive results from the histochemical gus expression assay indicate that there was successful Histochemical GUS assay on the transformed ECSs insertion of the target gene into banana using the gus of the banana cv. Sukali Ndiizi expressing cells and mature tissues as an indicator. The samples of transformed ECSs were histochemically Molecular analysis of transgenic plants stained for the expression of the gusA gene. Samples of leaves, stems and roots were also collected from two The presence and integration of the transgene in the 1816 Afr. J. Biotechnol. Figure 5. A, PCR analysis of the putatively transformed banana plants amplifying a 300 bp internal fragment of the cystatin g e n e. B, PCR analysis of the putatively transformed banana plants amplifying a 500 bp internal fragment of the hygromycin gene. M, DNA sizing marker (Hyperladder1); P, plasmid; Lanes 1-57, putatively transformed plants. genome of transgenic plants was confirmed by PCR and initial explants is a key in producing transgenic plants Southern blot analysis. PCR was carried out with hpt and (Ghosh et al., 2009). The histochemical Gus assay was CpCYS-Mut89 specific primers and amplified products of performed to assess the success of insertion of the gusA about 500 bp (Figure 5A) and 300 bp corresponding to an gene into the embryogenic cells using Agrobacterium- internal fragment of the hpt and CpCYS-Mut89 gene mediated transformation. High expression of gusA gene were obtained (Figure 5B). Amplified products were in ECSs indicates competence of the embryogenic cells observed in 27 out of the 28 selected transformed lines. to Agrobacterium-mediated transfer. Positive results were Line 15 neither amplified for hpt primers nor CpCYS- also obtained from histochemical staining of leaves, Mut89 specific primers, indicating that it was an escape. stems and roots indicating stable gus expression in No amplified product was observed in case of the non- mature tissues. High transient and stable gus expression transformed plant (negative control). Southern blot in ECSs and mature tissues of banana has also been analysis of 5 randomly selected transgenic banana plants observed by Khanna et al. (2004) in Cavendish and Lady of individual events digested with EcoRI and hybridized finger and Huang et al. (2007) in Musa acuminate cv. with CpCYS-Mut89 probe confirmed integration of Mas respectively. CpCYS-Mut89 gene in the banana genome. Different Agrobacterium-mediated transformation has been banding patterns were obtained indicating independent successfully used to obtain transgenic banana plants transgenic events. The non-transformed plant did not using different initial explants. May et al. (1995) used show any signal (Figure 6). apical meristems of the cultivar Grand Naine as initial explants to obtain transgenic plants. Intercalary meristematic tissues of East African highland bananas DISCUSSION have also been used as initial explants for transformation (Tripathi et al., 2008). In this study, embryogenic cell An efficient transformation system using appropriate suspensions (ECSs) of the banana cv. Sukali Ndiizi were Namuddu et al. 1817 Figure 6. Southern blot analysis of five randomly selected transgenic banana lines. transformed using Agrobacterium strain LBA4404. This infected cells recover from infection after-effects like method is less tedious and faster for the cultivars for phenolics before being transferred to selection media. which formation of ECS is possible because repeated After three subcultures Agrobacterium contamination was rounds of selection are not necessary (Ghosh et al., completely eliminated and cells proliferated more 2009). Other methods of transformation as vigorously. After three weeks of Agrobacterium-mediated electroporation (Sagi et al., 1994), and biolistics have transformation, ECS turned brown due to necrosis and been used for banana transformation (Becker et al., massive death of non-transformed embryogenic cells on 2000). However, Agrobacterium-mediated transformation selective MA3 media also occurred. One month later, is the most preferred due to the high transformation numerous whitish cell clumps appeared on the surface frequencies (Khanna et al., 2004) with fewer transgene with dead cells in the background. Similar results were copy numbers and significantly higher transient and obtained by Arinaitwe (2008) and Ghosh et al. (2009). stable gene expression compared to biolistics (Arinaitwe, One of the critical steps after transformation is the 2008). selection procedure used for the recovery of transgenic Agrobacterium was pre-induced with a surfactant, shoots (Joersbo, 2001). This is because embryogenic cell pluronic F68 (Cheng et al., 1997) to aid in the colonies obtained during the selection procedure must transformation of banana cells (Khanna et al., 2004). The survive the stress induced by the combination of media co-cultivation process was done at 22°C for 5 days as and antibiotics used to kill both Agrobacterium and compared to 3 days by Khanna et al. (2004) because untransformed plant cells (Arinaitwe, 2008). In this study, overgrowth occurs at higher temperature leading to the hygromycin B phosphotransferase (hpt) was used as a death of banana cells. Acetosyringone was included in selectable marker and hygromycin antibiotic (50 μg/μl) pre-induction media (TMA1) and co-cultivation media to was used as a selection agent for stable integration. enhance transfer of the T-DNA, a compound that Hygromycin is a preferred selection for easily scoreable enhances chemotactic movement and attachment of phenotype and faster inhibition response of explants Agrobacterium tumefaciens to wounded tissues of (Sreeramanan et al., 2006). Sreeramanan et al. (2006) banana cultivars (Hernández et al., 1999). Heat shocking further reported that hygromycin inhibited growth of non- of banana cells prior to transformation was done to transformed banana buds at lower concentrations. minimize cell death response resulting in recovery of a Distinct cell clusters were observed on selective MA3 large number transformed plants following Agrobacterium after 3 months and distinct embryos were observed on infection. Centrifugation was done to increase contact of RD1 after 2 months. Embryos were able to germinate on Agrobacterium with host cells (Khanna et al., 2004) MA4 after 1 month. Embryos from a cell clump were hence increased transient gene expression. Post co- assumed to have resulted from the same transformation cultivation wash of ECSs in liquid recovery media event and all plants from such a cluster were counted as removed Agrobacterium colonies sticking to ECSs and one line. Although, Agrobacterium-mediated transforma- 1818 Afr. J. Biotechnol. tion is a good transformation system, not all the nematodes, and hence the need to generate transgenic transformed ECSs developed into embryos and not all bananas with novel genes such as cystatins to confer embryos developed into shoots. This is because of the elevated resistance against the banana weevil and differences in embryogenic competences among the banana parasitic nematodes. In this study, ECSs of the ECSs. Variation in embryogenicity depends on banana cultivar Sukali Ndiizi were successively accumulation of auxin in embryogenesis competent transformed with the modified papaya cystatin (CpCYS- protodermal and subprotodermal explants cells as a Mut89) using the Agrobacterium- mediated result of auxin treatment, expression of embryogenesis transformation method. When transgenic plants are controlling gene especially the genes related to auxin obtained, the earliest essential step is molecular signaling as LEC2 (Kurezyńska et al., 2007; Ledwoń and characterization. Many primary transformants must be Gaj, 2009), cell division and cytological changes in pre- analyzed due to the random insertion of new DNA into embryogenic cells including formation of thick cell wall the plant genome, often leading to a generation of plants and cutin deposition, and symplasmic isolation of with multiple transgene copies integrated into one or embryogenic cells (Kurezyńska et al., 2007). more chromosomal locations. The number of copies of The PCR technique was used to confirm successful the exogenous gene inserted into the genome is one of introduction of the transgene into the banana genome. the reasons for reported transgenic silence (Weng et al., However, presence of residual Agrobacterium in 2004). Most plants with one or two integration events transgenic plants leads to false positives making yield high-level expression of the exogenous gene, conventional PCR unconvincing unless verified by stable whereas lower and unstable transgene expression and integration of the transgene in the plant genome (Chen et transgene silencing has been associated with high al., 2006; Ghosh et al., 2009). To minimize this problem, numbers of copies (Flavell, 1994; Kooter et al., 1999; Iyer putatively transformed plants were maintained on et al., 2000). In this study, a total of 57 transgenic lines antibiotic free media for four months and then transferred were generated. PCR analysis of the selected transgenic to greenhouse and maintained in the green house for at lines of Sukali Ndiizi shows 96.4% presence of the least two months before samples were collected for PCR CpCYS-Mut89 gene. Southern blot analysis of the analysis. putative positives confirmed stable integration of the Stable integration of the papaya cystatin gene was CpCYS-Mut89 gene into the banana genome. The further confirmed by Southern blot analysis. The mobility transgenic lines show one to four integration patterns, of the bands differed in most transgenic lines, verifying which are characteristic of Agrobacterium- mediated that the plants were the result of independent transformation. The integration patterns were also transformation events which are probably randomly generally random, implying different transformation located on the genome. Simple integration patterns have events in the generated lines. been reported in banana with different genes using Other cystatins like the rice cystatin (Oryzacystatin-I) Agrobacterium-mediated transformation (Khanna et al., have been integrated into many plant species to provide 2004; Arinaitwe, 2008; Ghosh et al., 2009). Lines 37 and protection against plant parasitic nematodes and 56 shows similar integration profiles indicating that they coleopterans. These include potato (Atkinson, 1996), rice were most likely clones from one transformation event (Vain et al., 1998), Cavendish banana (Atkinson et al., and hence derived from a single transformed 2004) for nematodes and potato (Lecardonnel et al., embryogenic cell. Southern blot analysis with the 1999) for coleopterans. In all these cases, increased modified papaya cystatin (CpCYS-Mut89) probes gave protection to nematode and coleopteran damage was hybridization patterns in the transformed and not the observed. In the current study, we introduced a cystatin untransformed control suggesting that there is no from Carica papaya (Song et al., 1995) that was later existence of highly complementary papaya cystatin modified to improve its inhibition capacity by Kiggundu sequences in banana or the probe design was efficient (2008) into the banana cultivar Sukali Ndiizi in an enough not to pick them up. The negative signals in the approach to protect banana against the banana weevil untransformed controls confirm that the observed signals and banana parasitic nematodes. truly indicate the integration of the transgene. This was further supported by the positive signal from the vector construct pCAMCYSMUT89, implying that the probes ACKNOWLEDGEMENTS correctly detected the integration events. This work was supported by funding from Bioversity International through the National Banana Research Programmme of the National Agricultural Research Conclusion Organization, Uganda. 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