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29. Agrobacterium- and particle bombardment-mediated transformation of a wide range of banana cultivars - Arinaitwe, G.*, S. Remy, H. Strosse, R. Swennen, L. Sági

* Permanent address: Makerere University, Faculty of Agriculture and Forestry, Department of Crop Science P.O. Box 7062, Kampala, Uganda.

Laboratory of Tropical Crop Improvement,
Catholic University of Leuven,
Kasteelpark Arenberg 13,
B-3001 Leuven,


Genetic transformation of banana (Musa spp.) by particle bombardment and Agrobacterium is established only in a few laboratories worldwide. In general, transformation frequencies are reported to be cultivar-dependent. Thus there is a need to adapt and optimise transformation protocols for any particular type of banana. In this study, the two transformation methods were compared and the effect of physical parameters on transformation frequency was investigated in four banana cultivars, 'Grand Nain' (AAA), 'Three Hand Planty' (AAB), 'Obino l'Ewai' (AAB), and 'Orishele' (AAB). DNA transfer frequency was measured by monitoring expression of b-glucuronidase and the green fluorescent protein gene. The results indicate major differences between the efficiency of the two transformation systems. Significantly higher transient and stable gene expression, in most banana cultivars, were obtained with the Agrobacterium-based method. The effect of the volume of cell suspensions and the length of infection time on transformation frequency were optimised. The cultivars were categorised on the basis of their competence for transformation and their capacity for regeneration. Molecular and biochemical analysis will be performed to confirm integration and expression of the transgenes in the different cultivars.


Banana (Musa spp.), the fourth most important crop in the world [1], is a staple food for over 600 million people in the tropical and subtropical areas. An annual production of 85.5 million metric tons was reported in 1998 [2]. However, in major banana growing regions, production is severely threatened by pests and diseases. The production decline, in most areas, is attributed to the Sigatoka complex caused by the fungal pathogens Mycosphaerella fijiensis (Morelet) Deighton (black Sigatoka) and M. musicola Leach ex Mulder (yellow Sigatoka), nematodes, and weevils [3]. Breeding for resistance to these pathogens is limited by the crop's long life cycle, and triploidy and sterility in most commercial banana cultivars [4].

There has been success in the establishment of regenerable embryogenic cell suspension (ECS) cultures of banana [5-7], by which genetic transformation could become a viable solution to the above constraints, without altering locally accepted post-harvest characteristics. There have been a few reports on genetic transformation of bananas, using different methods and cultivars [8-12]. However, in each of the reports only one or two banana cultivars have been transformed using one gene transfer method, either particle bombardment or cocultivation with Agrobacterium. Therefore, in this paper, we describe and compare the two transformation systems for the generation of transgenic plants from ECS of four different cultivars. The effects of selected parameters (volume of ECS, infection time) on the frequency of Agrobacterium transformation in the plantain 'Three Hand Planty' and in the commercial dessert banana cultivar 'Grand Nain', respectively, have also been studied.


2.1. Plant materials

Embryogenic cell suspensions of the banana cultivars 'Grand Nain' (AAA), 'Three Hand Planty' (AAB), 'Obino l'Ewai' (AAB), and 'Orishele' (AAB) were initiated from in vitro proliferating meristems and maintained as described [5]; they were subcultured for 6 days before transformation unless indicated otherwise. Embryogenic cell suspensions of 'Obino l'Ewai' were maintained in basal MS medium [13] supplemented with 5 mM 2,4-D, 100 mg/l malt extract and 45 g/l sugar (M2 medium). For the other cultivars half-strength MS medium containing 5 mM 2,4-D and 1 mM zeatin (ZZ medium) was used. Cells were maintained on a rotary shaker (70 r.p.m.) at 26 ± 2°C under fluorescent light, and subcultured at an interval of 2 weeks.

2.2. Agrobacterium-mediated transformation (A-MT)

2.2.1. Agrobacterium and plasmids

Agrobacterium tumefaciens EHA101 harbouring pFAJ3000 and AGLO harbouring pBINUbi-sgfpS65T were used for transformations. The plasmid pFAJ3000 contains both a reporter and a selectable marker gene. The reporter gene is an intron-containing gusA gene under the control of the CaMV 35S promoter and neo, a selectable marker gene conferring resistance to aminoglycoside antibiotics. Plasmid pBINUbi-sgfpS65T contains the green fluorescent protein (GFP) gene under the control of the maize ubiquitin promoter and the neo selectable marker gene. Agrobacterium was plated on selective semi-solid YM medium supplemented with appropriate antibiotics, and incubated at 28°C for 40 h. Single colonies were cultured in selective liquid YEP medium and incubated at 210 r.p.m. and 28°C to an OD600 of 1.2 for approximately 24 h.

2.2.2. Transformation of embryogenic cell suspensions

ECSs in liquid ZZ (for 'Grand Nain', 'Three Hand Planty', and 'Orishele') and M2 (for 'Obino l'Ewai') medium were used for transformation. ECSs (200 ml unless indicated otherwise) of 33.3% settled cell volume were infected with A. tumefaciens cells adjusted to an OD600 of 0.4 in liquid ZZ or M2 medium containing acetosyringone (AS). One millilitre of diluted bacterial suspension was mixed with ECS in 24-well titre plates and the plates were incubated for 6 h (unless indicated otherwise) at 25 r.p.m. in the dark. The ECS were then transferred to 10 ml semi-solid AS-containing ZZ or M2 medium in 5 cm Petri dishes and incubated for 6 days.

2.3. Particle bombardment-mediated transformation (P-MT)

2.3.1. Plasmid constructs

Plasmid DNA was purified from E. coli by the QIAGEN midi-prep procedure. Plasmids pAct1Fneo and pMy-GUS (containing a banana streak virus promoter [14]) were used for co-transformation. The two plasmids were equally and uniformly mixed before being coated on tungsten particles.

2.3.2. DNA preparation and particle bombardment

The preparation of plasmid DNA, precipitation of plasmid DNA onto tungsten particles, and particle bombardment were carried out as described previously [8]. The efficiency of plasmid DNA precipitation onto tungsten particles was monitored by viewing Hoechst 33258-stained coated particles under a UV microscope. Then 8 µl of plasmid DNA-coated particles were pipetted onto 200 µm stainless steel mesh and accelerated in a home-made particle gun by helium at a pressure of 8 bars.

2.4. Transient gene expression assays

Six days after Agrobacterium-cocultivation or 2 days after particle bombardment, the transformed samples were tested histochemically for GUS expression. Cells were immersed in the assay buffer in the presence of the reporter enzyme substrate X-Gluc for 3 h at 37°C as described [15], and the number of blue foci was counted. Non-transformed ECSs were used as a negative control. GFP expression was detected under a Leica äMZFL III stereomicroscope equipped with a GFP plant fluorescence filter.

2.5. Selection and regeneration of transformants

After 6 days of cocultivation with Agrobacterium, the cells were transferred to semi-solid ZZ or M2 medium supplemented with geneticin (50 mg/l) and timentin (200 mg/l), and incubated in the dark at 25 ± 2°C for 2 months, with subcultures every 2 weeks. Finally, transformants were regenerated through successive culture steps in the presence of geneticin and timentin. After particle bombardment, embryogenic cells were cultured on non-selective semi-solid ZZ medium for 7 days and later on ZZ medium supplemented with 50 mg/l geneticin with a two-week subculture interval. After 2 months, cell aggregates were selected and transferred to selective proliferation and regeneration medium as described [4, 8].


3.1. Comparison of cultivars and transformation methods

Microparticles coated with plasmids pAct1Fneo and pMy-GUS or vir-induced agrobacteria carrying the plasmids pFAJ3000 or pBINUbi-sgfpS65T were used to transform ECSs of the four banana cultivars mentioned. Co-cultivation and selection of ECSs was done on semi-solid ZZ medium. This medium stimulates cell division, which is required for efficient incorporation of foreign DNA into the host plant genome as reported in Agrobacterium-mediated transformation [16-19] as well as for direct gene transfer [20]. Two days after bombardment or after 6 days of co-cultivation, histochemical GUS analysis revealed transient gene expression in transformed banana cells (Figure 1).

Figure 1 Transient GUS expression in 50 mg fresh weight cells of ECSs of 'Grand Nain' (G.N), 'Three Hand Planty' (THP), 'Obino l'Ewai' (OE), and 'Orishele' (ORI), transformed with pMy-Gus by particle bombardment (P-MT) or with pFAJ3000 by Agrobacterium (A-MT). Mean ± SE of at least four replicates

Transient GUS expression (TGE) appeared to be cultivar-dependent in both transformation systems, and ranged between an average of 240 blue foci per plate ('Orishele' after cocultivation with A. tumefaciens) and 1600 blue foci per plate ('Grand Nain' with A. tumefaciens). In 'Three Hand Planty', TGE counts did not differ significantly in the two transformation systems, whereas higher TGE was observed for 'Obino l'Ewai' and 'Orishele' after particle bombardment. In contrast, in 'Grand Nain', Agrobacterium transformation generated the highest TGE overall (Figure 1).

At the level of stable transformation, the number of colonies at the end of the selection process was significantly higher with the Agrobacterium method for all banana cultivars (Figure 2.). The same result was obtained by Pérez Hernández [21], who observed up to a tenfold increase in the number of selected colonies after Agrobacterium transformation, compared to 2 to 20 colonies per plate after particle bombardment [8].

Figure 2. Number of independent colonies after a 2 months selection in 'Grand Nain' (G.N), 'Three Hand Planty' (THP), 'Obino l'Ewai' (OE), and 'Orishele' (ORI), transformed by particle bombardment (P-MT) or Agrobacterium (A-MT). Mean ± SE of ten replications each consisting of 50 mg fresh weights cells

Visualisation of GFP fluorescence in ECSs transformed with the vector pBINUbi-sgfpS65T was performed using a GFP stereomicroscope. The number of GFP fluorescent spots and selected colonies after the selection process are shown in Table 1.

Table 1. Transient expression of GFP (TGFPE) 6 days after Agrobacterium transformation and number of colonies after 2 months of selection, in 'Grand Nain' (GN), 'Three Hand Planty' (THP), 'Obino l'Ewai' (OE), and 'Orishele' (ORI)a

Banana cultivar


No. of independent colonies


996.3 ± 315

8.6 ± 5.4


556.8 ± 108

17.6 ± 8.2


545.9 ± 185

175.8 ± 23.7


334.8 ± 88.5

12.3 ± 4.8

a Mean ± SD of 10 replicates both for transient GFP expression and the number of independent selected colonies.

There was a difference in the expression of the two reporter genes used. Transient GUS expression (Figure 1) was higher than transient GFP expression (Table 1). This was probably due to differences in the efficiency of the two A. tumefaciens strains used (EHA101 and AGLO, respectively), which may be resolved by transforming both plasmids (pFAJ3000 and pBINUbi-sgfpS65T) in the same Agrobacterium strain. Nevertheless, the order of the cultivars according to the frequency of transient gene expression was exactly the same with both reporter systems.

Based on the transient expression level of reporter genes (parameter for competence for transformation) and the number of selected colonies after 2 months in selection medium (indicator for regeneration capacity), the ECS lines of the four banana cultivars were categorised as in Table 2.

Table 2. Transformation competence of ECS lines of four banana cultivars after 2 months of selection

Transformation competence

High regeneration

Low regeneration


'Grand Nain'


'Obino l'Ewai'

'Three Hand Planty'



3.2. Effect of the volume of ECS and infection time on Agrobacterium-mediated transformation frequency

Prior to co-cultivation, variable volumes of ECS (in microlitres) were plated (Figure 3) and uniformly spread over a 50 mm nylon mesh. After co-cultivation, transient GFP expression was used to assess transformation frequency in each treatment. The results indicate that T-DNA transfer is more efficient when small volumes of ECS (50-200 ml) are plated during co-cultivation. The highest number of GFP spots was observed at 100 ml. This dropped sharply when the volume of ECS was increased to 300 ml or 600 ml. This effect could be caused by decreased attachment and access of agrobacteria to individual embryogenic cells or cell clusters. This appears to be confirmed by the increased GFP spot counts in certain areas of the 300 ml and 600 ml samples, where more uniform spreading was achieved during plating. Whether the increased transient transformation frequency also results in a higher number of stable transformants will soon be determined when the selection process is completed.

Figure 3. Effect of ECS volume on transformation frequency of the plantain 'Three Hand Planty' after co-cultivation with Agrobacterium strain AGLO harbouring pBINUbi-sgfpS65T. Mean ± SE of two independent experiments.

The effect of infection time on transformation frequency was investigated in 'Grand Nain'. The number of blue foci did not differ significantly between 4 h and 6 h of infection. However, after 6 h or more of infection, the number of blue foci increased to over 1500, and it became difficult to quantify accurately (Table 3). This increased transient expression rate suggests that T-DNA transfer could be increased with a prolonged infection period. This investigation will be extended to other cultivars to confirm the observed trend of transient GUS expression.

Table 3. Transient GUS expression (TGE) in 'Grand Nain' suspension cells infected for different times with Agrobacterium tumefaciens EHA (pFAJ3000)

Infection time (h)








No. of blue foci

794 ± 87.9

881 ± 91.8





a Mean ± SD of blue foci in at least five replicates

In most transformation systems, an increase in foreign DNA integration into the host genomic DNA is the main target. It was important, therefore, to observe the above transformed ECSs on selective medium. A large number of colonies survived on selective medium, at geneticin concentrations of both 50 mg/l and 100 mg/l. The GUS test after increased selection pressure indicated 100% of colonies were positive, though with fully blue and partly blue stained colonies (Table 4). The proportion of fully expressing colonies increased in parallel with increasing infection time up to 12 h. At 14 h of infection the blue coloration became partial because of the necrosis caused by the prolonged contact with Agrobacterium.

Table 4. Effect of infection time on stable GUS expression (SGE) in 'Grand Nain' cell colonies after three months of selection

Infection time (h)









3.7 ± 1.5

10 ± 6.1

13 ± 5.3

13 ± 7.2

18.7 ± 1.5

2.3 ± 1.5

Partly blue

16.3 ± 1.5

10 ± 6.1

7 ± 4.7

7 ± 6.4

2.3 ± 1.5

18.7 ± 1.0

a Mean ± SD of blue foci in at least three replicates of 20 colonies each. Transformed cells were maintained on selective ZZ (50 mg/l geneticin) for 2 months, 1 month on selective ZZ (100 mg/l geneticin), and 10 days in liquid selective ZZ (100 mg/l geneticin).


(a) Agrobacterium transformation proved to be efficient in a wide range of banana cultivars,

(b) Two reporter genes (gfp and gusA) were highly expressed in all these cultivars,

(c) The effect of two parameters (volume of ECS and infection time) on transformation frequency was optimised for two cultivars.


The study was conducted at the Laboratory of Tropical Crop Improvement, Catholic University of Leuven (KUL) as part of a collaborative biotechnology project between the Government of Uganda and KUL, co-ordinated by the International Network for the Improvement of Bananas and Plantain (INIBAP).


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