Agrobacterium mediated transformation of Tunisian Cucumis melo cv. maazoun

Transgenic Cucumis melo cv. Maazoun containing the neomycin phosphotransferase II (NPT II) chimeric gene conferring resistance to kanamycin were obtained from cotyledons explants inoculated with Agrobacterium tumefaciens (GV3101) that contained the binary vector plasmid pADI. Transformed shoots were obtained on Murashige and Skoog medium supplemented with 1.50 mgl IAA, 0.10mgl BAP, 0.01 mgl NAA and 6 mgl kinetin. Transformants were selected by using only 100 mgl of kanamycin and 4 days of pre-culture. Putative transformants were confirmed for transgene insertion through polymerase chain reaction (PCR) analysis. From the inoculated explants, 6.66% produced transgenic shoots.


INTRODUCTION
In Tunisia, Cucumis melo is cultured over an area of 9629.39 hectares with a production of 140645 tons per year (DG/PDIA, 2004).However cultures are subject to several viral (e.g.Cucumber Mosaic Virus, CMV) and fungal diseases (e.g.Sphaerotheca fuliginea, Fusarium oxysporum var.Melonis) that severely limit yield and for which adequate levels of native resistance are not available (Trionfetti et al., 2002;Kuzuya et al., 2003;Vengadesan et al., 2005).Genes resistance against these pathogens exists essentially in wild species (Pitrat and Risser, 1992).However, their introduction via conventional breeding techniques (artificial hybridizations) is very low, expensive and limited because of the interspecific and intergeneric reproductive barriers (Debeaujon and Branchard, 1992;Guis et al., 2000).Plant biotechnology strategies, including genetic transformation could help overcome these problems.
the first time by Fang and Gourmet (1990).A few several procedures for C. melo transformation have been established (Vallès and Lasa, 1994;Dias et al., 2002;Akasaka et al., 2004) for genes encoding for kanamycin resistance (Chee, 1990;Sarmento et al., 1992) and coat protein gene conferring virus resistance.Subsequently, transgenic melon plants with useful traits such as long shelf-life of fruit (Chee and Slightom, 1991;Ayub et al., 1996;Vengadesan et al., 2005) and halo-tolerance (Bordas et al., 1997) have been produced.However, melon genetic transformation using Agrobacterium strains is at present limited to few varieties.The restriction of the use of this technique was mainly due to the low number of plant regenerated and the high frequency of chimeric plants (Ray et al., 1993;Simandjuntak et al., 1996).
Beji (charentais) and Maazoun (Eastern type) C. melo cultivars are widely cultivated in Tunisia and very appreciated by the consumer (Jebberi, 1988).However, the two cultivars are subject to climatic stresses and pathogens.Conventional breeding methods, using wild rustic melon species in order to transfer desirable agronomic genes into the cultivar, are limited because of the intra and interspecific barriers.The present work aims to opti- mize a genetic transformation system permitting introduction of desirable genes into C. melo cv.Maazoun.

Mature seeds of Maazoun cultivar were provided by INRAT (1998).
Seeds were surface sterilized with 5% calcium hypochlorite for 10 min and rinsed three times with sterile distilled water.Seeds were aseptically cultured in Petri dishes (8 x 8 x 2 cm) containing 25 ml of MS medium (Murashige and Skoog, 1962) solidified with 0.8% agar.Cotyledons were taken from ten days old seedlings.They were cut perpendicularly to the longitudinal axis into fragments (7/5 mm). 1 mm wide along the edges was removed.

Media and in vitro culture conditions
Explant organogenesis potentialities were tested on the MS basal medium solidified with 0.7% agar and added with NAA (0.01 mgl -1 ), IAA (1.5 mgl -1 ), BAP (0.10mgl -1 ) and kinetin (6 mgl -1 ).The pH of medium was adjusted to 5.8 before autoclaving at 100 KPa for 20 min.Explants were placed horizontally in Petri dishes containing each 25 ml of medium.Cultures were incubated at 22 ± 1°C under 16 h photoperiod (cool white fluorescent lamps, 5 Mm -2 s -1 ).For each experiment, 30 explants were used.Callus induction medium consisted of MS basal medium supplemented with 1.5 mgl -1 IAA and 6 mgl -1 kinetin.In the regeneration medium the above growth regulators were replaced by 0.01 mgl -1 NAA and 0.1 mgl -1 BAP.

Bacteria strain and plasmid vector
Genetic transformation was performed using the Agrobacterium strain GV3101 containing the binary plasmid pADI (Figure 1) that carries a neomycin phosphotransferase II (NPT II) gene under the control of CaMV promoter (Hernould et al., Unpublished).

Infection, co-cultivation and selection
After four days of pre-culture, explants were inoculated with an overnight culture of Agrobacterium diluted to OD600 nm = 0.8 (10 7 -10 8 bacteria ml -1 ) for 20 min.Infected explants were blotted dry using sterile Whatman paper to remove excess bacteria then cocultivated for three days at 28°C in the dark on callus induction medium.Bacteria were then removed by washing with liquid MS medium containing 300 mgl -1 Claventin (Duchhefa, Nederland), explants were transferred onto selective callus induction medium containing 100 mgl -1 kanamycin and 300 mgl -1 Claventin, and subcultured every week.After four weeks of incubation, green kanamycin resistant calluses were transferred to the regeneration medium containing 100 mgl -1 kanamycin and 300 mgl -1 Claventin.Resistant organogenic calluses were obtained through several subcultures under the same selective pressure.Putative transgenic plants were then tested using PCR analysis.

DNA preparation and polymerase chain reaction (PCR) analysis
Polymerase chain reaction was used to detect specific DNA sequences of the NPT II gene transferred.Genomic DNA was isolated from putative transformed callus (0. was performed.The PCR reaction containing 100 ng of total melon DNA was incubated in a final volume of 100 µl with 0.08U µl -1 Taq polymerase, 0.50 pmol µl -1 for each primer and 200 µM de dNTP.Reaction cycles (35 cycles) consisted of 30 s at 94°C for denaturation, 30 s at 52°C for annealing and 30 s at 72°C for extension.The forward and reverse primers for NPT II gene were 5'-CTTGGGTGGAGAGGCTAT-3' and 5'-AGAACTCGTCAAGAAGGC-3', respectively, amplifying a fragment of 750 bp.PCR products were analysed by 1.5% agarose gel electrophoresis.DNA was visualized under UV and photographed.

RESULTS
Calli formation started after a week for all explants (97%) from the proximal end.Calli were smooth, compact and greenish.Shoots began to differentiate after thirteen days.The induction of adventitious buds reached 79% on MS added with 0.01 mgl -1 NAA combined with 0.1 mgl -1 BAP.

Selection and regeneration of transgenic plants
Cotyledon explants co-cultivated with bacteria were washed with liquid MS medium containing 300 mgl -1 Claventin, blotted on sterile Whatman paper and then placed on the selection medium solidified with 0.7% agar.

PCR analysis
Gene transfer mediated by Agrobacterium inoculation to cotyledonary explants was tried by transient NPT II expression.The integration of the transgene into the plant genome was confirmed by PCR analysis.650 bp corresponding to the amplified fragment of NPT II gene was detected (Figure 3).The amplified products were observed in all the organogenic callus tested, confirming the presence of transgenes and non-calli escapes.

DISCUSSION
The genetic transformation via A. tumefaciens is commonly used for the genes transfer to the vegetable cells.Many varieties transformed for agronomic genes were obtained.However, the efficiency of this technique can be influenced by many factors including the species or the variety to be transformed, the bacteria used, the explant and the presence of inductive virulence genes substances, particularly the acetosyringone (Wenck et al, 1999;Galperin et al., 2003).The success of the genetic trans-formation requires combination of a transformation vector and a regeneration technique.This combination is coupled with efficient techniques of selection and confirmation of the transformation.this paper, we have shown that the potentiality of C. melo for genetic transformation depends on regeneration.The melon regeneration using cotyledon as an explant is efficient in terms of regenerated plants and transformation level.To confirm the integration of the foreign DNA into the melon genome, PCR amplification of the NPT II gene was conducted and yielded the correct size of the band (650 bp) corresponding to the NPT II gene in the vectors.Genetic manipulations for disease and pest resistance, as well as abiotic factors require the use of rapid and non-species specific transformation and regeneration system.The use of appropriate constructs, after screening and biosafety concerns have been conducted which allow production constraints of nematodes, fungi, bacteria and virus infection in a sustainable and environmentally friendly manner.It may also be possible to incorporate other characteristics such as drought tolerance, thus increasing the area that can support the growth of melon and contributing significantly to food security and poverty alleviation.

Figure 1 .
Figure 1.Schematic representation of the T-DNA binary plasmid pADI.This plasmid carries the NPT II gene, conferring resistance to kanamycin, under the control of the 35S-CaMV promoter and 35S-CaMV terminator.