Application of laser microbeam cell surgery and Agrobacterium-mediated gene transformation systems in melon (Cucumis melo L).

In the current investigation, the application of laser microbeam cell surgery as a transformation system in melon has been studied and compared with transformation via Agrobacterium tumefaciens as well as a combine of the two systems. All these transformation treatments have been carried out on the hypocotyl proximal zone of melon cv. Shahd El-Dokki using the plasmid pISV2456 that harboring gus-intron and bar genes. The treated explants were regenerated on shoot regeneration medium supplemented with 250 μg/l bialaphos. Results showed that there was no significant difference of shoot regeneration between the laser and Agrobacterium treatments, however, the regeneration percentage varied among the treatments, the laser microbeam treatment showed highest percentage than the Agrobacterium and combined methods, (76, 72 and 68 % respectively). The transformation systems were evaluated by detecting the expression of the gus gene using histochemical assay, while the integration of the both genes (gus and bar) were confirmed by the PCR assays, indicating that the two genes were successfully transferred to the plants in the three methods. Seven shoots out of 30 tested shoots were PCR-positive, representing 23.33%, in both Agrobacterium and laser treatments, while, only 6 out of 30 tested shoots were PCR-positive, representing, 20%. This study demonstrates using laser tool as a new transformation method in melon cv Shahd El-DOkk, indicating that laser technique could be considered a competitive transformation method with the Agrobacterium-system. INTRODUCTION Melon is an important vegetable crop that is widely cultivated in SouthEast Asia, China, East Africa and throughout the tropical and subtropical regions (Yadav et al., 1996). Plant biotechnology has the potential to genetically transform plants in order to transfer novel characteristics (Guis et al., 2000). Twenty-five years ago, the concept of using A. tumefaciens as a vector to create transgenic plants was viewed as a prospect and a "wish. " Today, many agronomically and horticulturally important species are routinely transformed using this bacterium, and the list of species that is susceptible to Agrobacterium mediated transformation seems to grow daily 46 Khames et al., Pak. J. Biotechnol. using Agrobacterium and gun-particle bombardment methods, several transgenes, which provide different phenoltypic characteristics, has been transferred to the Cucumis melo explants. These have included different marker and reporter genes such as, npt II and gus (Fang and Grumet, 1990 and Valles and Lasa, 1994); bar and gus genes (El-Dossoky et al., 2006) and dhfr and the gus (Dong et al., 1991); virus resistance genes as ZYMV-cp gene (Fang and Grumet, 1993; Hosny, 1996 and 2003.) and ACC oxidase gene (Nunez-Palenius et al., 2005). On the other hand, the production of transgenic melon plants expressing the CMV-cp has been reported using the microprojectile-mediated gene transfer by Gonsalves et al., (1994). Recently, the leaser microbeam has been studied as a new approach in the genetic manipulation of higher plants, Badr et al., (2004 and 2005) reported that a highly focused laser beam could be used as an “optical” microbeam to produce tiny submicrometer self-healing holes momentarily made in the cell membrane facilitating the exogenous DNA uptake into the cultured cells. The laser microbeam has been utilized as a new approach in the genetic manipulation of plant cells has been reported for introducing a foreign DNA into a higher plant which leads to plant cell perforations that might facilitate transformation experiments (Weber et al., 1988 and 1990). They introduced bisbenzimid labeled DNA of the plasmid pBR322 in the cell wall of the pollen grains of Brassica napus L. cv. Lindora, this using 0.4 M sorbitol. They also reported that the cell wall of the plasmolyzed cell was perforated with a laser pulse, while a second pulse directed at the plasma membrane punctured a hole, which closed again. Moreover, Sanford (1993) demonstrated the feasibility of puncturing the cell wall of individual Vinca minor pollen grains without killing them by using the laser microbeam. Guo et al., (1995) used laser microbeam under osmotic condition to establish an effective system for introducing foreign plasmid DNA's (pBI121, pB221, pACI 1-D and pRq6) into cells of embryonic calli of Oryza sativa L. cv. Japonica. In addition, Buer et al., (1998) reported a detailed protocol for precisely inserting microscopic objects into the periplasmic region of plant callus cells using laser microsurgery. The bacterium A. rhizogenes and the plant Ginkgo biloba were used as the model system for developing the optical tweezers and scalpel techniques using a single laser. Results showed that more than 95% survival after plasmolyzing G. biloba cells, ablating a 2-4 μm hole through the cell wall using a pulsed UV laser beam, trapping and translating bacteria into the periplasmic region using a pulsed infrared laser beam, and then deplasmolyzed the cells. Insertion of bacteria is also described. A homemade UV excimer laser with two dimensional translation stages, a suitable computer program and a proper optical device has been modified by Badr et al., (2004) to introduce the plasmid pAB6 DNA that harboring the gus and bar genes into the embryogenic calli of the Egyptian wheat (Triticum aestivum L) cv. Giza 164. Osmotic treatment was performed Vol. 6. (1-2) 2009 Application of laser microbeam cell. 47 with 0.4 M manitol that mixed with the plasmid DNA. For irradiating the calli with laser microbeam focused for puncturing holes ~ 0.5 μm in the cell wall allowing DNA uptake. Results showed that this modified procedure of laser-mediated can be a successfully transformation system in wheat. Thereafter, in 2005 Badr et al., produced transgenic wheat cv. Giza 164 plants using the same homemade setup of the UV excimer laser. The obtained plants behaved bialaphos-postive in a selection and rooting stage, indicating the success of the laser system for introducing foreign DNA. This study deal with the application of laser microbeam cell surgery (LMCS) for gene transfer in melon cv Shahd El-Dokki and compare it with the common transformation system Agrobacterium. MATERIALS AND METHODS Seeds: Seeds of the melon Shahd ElDokki cultivar were obtained from the Horticulture Research Institute, ARC, Giza, Egypt and used in all regeneration and transformation experiments. Plasmids: The plasmid pISV2678 (Unpublished data) harbors the gus– intron under the control of 35S promoter and nos terminator as well as the bar fused the AMV Leader gene under the control of nos promoter, pAg7 terminator (Becker et al., 1992) was used to optimize the transformation system in melon via Agrobacterium mediated transformation (Horch et al., 1985). The plasmid was kindly provided by Dr. P. Ratet, Institut des Sciences Vegetales (ISV), Centre National de la Recherche Scientifique (CNRS), Gif-Sur-Yvette, France. Primers: Two sets of specific oligonucleotide for bar and gus genes were synthesized at AGERI, ARC, Giza, Egypt and used to detect the presence of the two genes in putatively transgenic plants. Tissue culture and regeneration condition: In order to study the laser microbeam cell surgery system as a new transformation method in melon, following treatments have been carried out on the hypocotyl proximal zone explants of melon cv. Shahd El-Dokki with the DNA of the plasmid pISV2678 that harboring gus-intron and bar genes, i.e., the laser microbeam cell surgery system; Agrobacterium tumefaciens and a combine of laser microbeam cell surgery and Agrobacterium tumefaciens treatment. All treated explants were regenerated and rooted as described by El-Dessoky et al., (2006). Agrobacterium-mediated transformation: The Agrobacterium mediated transformation was carried out on melon with strain LBA4404 carrying the plasmid pISV2678 as described by El-Dessoky et al., 2006. The hypocotyl explants were soaked in the Agrobacterium culture for 10-15 min. Laser microbeam cell surgery application: The proximal zone of hypocotyl as explants of melon were placed under osmotic condition by incubating the explants on MSBA1 that composed of MS (Murashige and Skoog 1962) medium and 1.0 mg/l 6benzylaminopurine (BAP) supplemented with 0.4 manitol for 4 hours at 25C at dark. Subsequently, these explants were then exposed to the leaser microbeam irradiation under 48 Khames et al., Pak. J. Biotechnol.