Banana genomic library for Musa acuminata cv Giant Nain was constructed with DNA isolated from immature green leaves, using the EMBL3 vector (Stratagene) [14]. Approximately, 2 × 10⁶ primary plaques from the genomic library were plated, blotted and hybridized with ³²P labeled banana p31 cDNA [7]. Three successive hybridizations identified positive clones, from which DNA was isolated by restriction mapping. The genomic clone, Musa acuminata acidic chitinase class III was subcloned into plasmid Sport (Invitrogen) and sequenced by Sanger Method (GenBank: AY525367.1).

The 2.1 Kbp 5’ region of banana acidic chitinase class III (MaChIII) gene was obtained from genomic DNA clone. The NcoI site at –1739 bp from the start codon was removed by cutting with NcoI and filling the ends with Klenow enzyme. Then, a NcoI site was created at the start codon by PCR. The 2.1 Kbp BamHI-NcoI fragment containing the MaChIII promoter was fused to the GUS gene with the CaMV 35S 3’ region from pRTL2-GUS [15] and the expression cassette was inserted into pGPTV-Kan [16] to make pGPT-31G vector (Figure 1(A)).

Agrobacterium-mediated transformation of tomato cotyledons (variety Tanksley

TA234TM2R) was performed according to Frary [17], except those seeds were sterilized by soaking in 70% ethanol for 2 min before rinsing in sterile water and washing in a mixture of 10% bleach and 1% Tween-20 for 10 min. The seeds were rinsed three times in sterile distilled water before plating on half-strength MS medium [18] (half-strength MS: 50 mg·L⁻¹ myo-inositol, 2 mg·L⁻¹ thiamine HCl, 0.5 mg·L⁻¹ pyridoxine HCl, 0.5 mg·L⁻¹ nicotinic acid, 10 g·L⁻¹ sucrose and 8 g·L⁻¹ Difco bacto agar, pH 5.8). Plantlets were regenerated on medium containing kanamycin at 300 mg/L.

PCR MaChIII promoter fragment was amplified from transgenic tomato lines using total DNA as template and two internal primers for a 480 bp fragment of the promoter sequence (promoter region 1691 to 2152 pb): MaPromP31FW CCA AGA GGA TTT AAA TTT GGG C and MaPromP31RW CGG GGA CTT GTC GAA GTT TTC G under the following conditions: 5 min denaturation at 95˚C, followed by 25 cycles of amplification (95˚C for 30 s, 57˚C 30 s, 72˚C 30 s) in an Applied Biosystems 2720 Thermal Cycler.

We made fluorometric GUS assay following the method described by Jefferson [19]. Samples of leaves and fruits tissues were assayed. Transgenic tomatoes fruits were pick up at three ripening stages; green, brake and red (ripen). Fruits were divided in pericarp and placenta tissues. The volume equivalent to 20 μg of protein was incubated with 1 mM MUG buffer at 37˚C for 90 min. The enzymatic reaction was measured by spectrofluorometer Bio-Rad VersaFluor fluorometer (Bio-Rad Laboratories, Hercules, CA). Fluorescence was measured at an excitation wavelength of 365 nm and an emission wavelength of 450 nm. Proteins were extracted from different tissues and total protein concentration was determined according to Bradford assay and bovine serum albumin as standard. All the samples had three repetitions. To determine the pmol/min/mg value in each sample, a MU standard curve was constructed from the standard readings and absolute amount of MU (nM). Samples O.D. readings were applied to the equation, and a linear estimation was done to get protein activity/minute. The slope value (nM/min) was obtained for each sample, and conversions were applied to convert nM to nmolar, and then to nmol/min. Using the values obtained from the Bradford standard curve and several dilution factors, a value was obtained for each sample in units of pmol/min/mg.

Fruit sections were cut by hands and fixed in 0.3% formaldehyde in 10 mM MES, pH 5.6, 30 mM mannitol for 45 min at room temperature followed by several washes in 50 mM NaH₂ PO₄, pH 7.0. The samples were put in 1 mM X-Gluc (5-bromo-4-cloro-3-indolyl-β-glucuronic acid) solution and incubated at 37˚C overnight for blue color development [19].

The banana acidic chitinase promoter vector pGPT-31G was introduced into A. tumefaciens LBA4404 by electroporation. Transformants were selected on LB medium containing kanamycin and confirmed by PCR. Using pGPT-31G vector Tomatoes cotyledons were transformed using Agrobacterium system. Four transgenic plant lines were obtained; we chose the line GPT31G-2 for the next experiments. Transgenic line was grown under greenhouse conditions and grown over a period of six months. Tomato total DNA was obtained and used for PCR assays to verify the MaChIII promoter incorporation in the tomato genome (Figure 1(B)).

Samples from pericarp and placenta of transgenic tomato fruits of GPT31G-2 line at three stages of tomato fruit ripening; green, brake and ripen, were collected. We made fluorometric assay in these three tomato ripening tissues and leaves samples. Data was quantified, and then arrayed in column graphs plotting the pmol/min/mg values obtained for each sample. We can observe that the higher expression is specified in the pericarp and placental regions at tomato brake stage. Low values were obtained in pericarp and placenta tissues of green and ripen stages of tomato fruit ripening. Fluorometric leaves values are lower than obtained from fruit tissues (Figure 2(A)).

Figures 2(D)-(I) shows histochemical tomato GUS assays in the three tomato fruit ripening stages (green, break and ripe). In tomato green fruits the expression is confined to the vascular and funiculus tissues (Figure 2(D) & Figure 2(E)). In the tomato brake stage, the blue staining is hard in all pericarp, locular gel, vascular and placental tissues (Figure 2(F) & Figure 2(G)). This ripening stage is just before climacteric ethylene burst. During the ripen stage (red) after the climacteric period the expression was reduced to vascular and funiculus fruit tissues (Figure 2(H) & Figure 2(I)). This pattern of expression is like to the

acidic chitinase class III banana protein expression during fruit ripening [7]. The expression of the banana acidic chitinase class III promoter in a dicot model plant like tomato is interesting and suggests that this sequence gives organ specificity to the coding region and that is associated to fruit development too. The turn off the expression of the gene in presence of ethylene is similar, when climacteric ethylene production occurs during fruit ripening, the promoter turns off the expression of the gene in pericarp and placenta tissue. An interesting observation is the funiculus intense expression during three ripening stages, this result suggests that this acidic chitinase could be involved in seed development. Banana express abundantly this gene in the green pericarp like tomato, both fruits before of climacteric fruit stage.

The sequence of the 5’ flanking promoter DNA of the MaChIII was determined. The likely start codon is located at position 2153. The putative cis-element of this promoter was analyzed using PLANT Care program and Table 1. The TATA Box was found at position 1043. Potential regulatory cis elements associated with phytohormones, and stress related response were located within the MaChIII promoter, including 1 W-box, 5 light response, 4 stress, 3 Abscisic Acid, 1 Methyl JA and 4 for transcription factors related elements. The presence of these elements demonstrates that MaChIII may be involved in banana response to biotic and abiotic stresses and development more than pathogenic response.