Genetic Engineering For Improving The Storage Capacity Of Melon (Cucumis Melo L.)

Total RNA was extracted from mature fruits of Hetao melons (Cucumis melo L.cv Hetao). The cDNA fragments encoding 1-aminocyclopropane-l-carboxylic acid synthase (ACS), 1-aminocyclopropane-l-carboxylic acid oxidase (ACO), and polygalacturonase (PG) were amplified by reverse transcription polymerase chain reaction (RT-PCR). These cDNA fragments were cloned into pUC19 and transformed into the E.coli DH5 a strain. The recombinant plasmids were designated as pCMACS, pCMACO, and pCMPG, respectively. The analysis of the nucleotide sequence showed that the length of the cDNA clones are 627 bp (ACS), 545 bp (ACO), and 1183 bp (PG), respectively. Their nucleotide and deduced amino acid sequences shared high similarity to those of the melon ACS, ACO and PG reported previously.Cucumisin, an extracellular subtilisin-like serine protease, is highly expressed in the fruit of melon (Cucumis melo L.cv Hetao) and accumulates in its juice. Thepromoter region of the cucumisin gene from the nucleotide at the position of-310 to -1 relative to the transcriptional initiation site was amplified from the melon genomic DNA by PCR and cloned into the pUC19 vector. The sequence analysis showed that the nucleotide sequence is completely identical to the published sequence. The genomic fragment contains some signature motifs typically found in the fruit-specific promoters, such as TATA-box, CAAT-box, G-box, and I-box-like box. This cloned promoter region may be useful for genetic engineering of the melon fruit.In order to establish a simple and practical transformation technique with a high transformation rate in melon (Cucumis melo L.cv Hetao), the pollen-tube pathway transformation method has been developed. The melon cultivar, hetao melon, was used as recipient and the binary vector pPZP221 containing a GUS (B glucuronidase) gene was used as the donor DNA of transgene. The donor DNA solution was applied to the cut surface of stigma at 1, 2, 3, 4, 5, 6, 7, 8, 9, and10 h after hand-pollination, respectively. PCR results show that the transformation rate in the different transformation events was very high with the highest rate of 30% at the 7 h time point. Southern blot analysis confirmed that all of the T1 plants indeed contained the transgene.The cDNA clones of the ACC synthase and ACC oxidase genes from melon were constructed reversely into the plant binery vector pROKII. The melon (Cucumis melo L.cv Hetao) cultivar, Hetao was transformed with these constructs using the pollen-tube pathway transformation method. A large number oftransformed seeds were obtained. About 10 T6 strains with improved storage capacity and other desired traits have been selected from two thousands of T0 seeds. The PCR and Southern blot analyses showed that the transgene was inserted into the recipient's genome and inherited into the progeny of the primary transgenic plants. Northern analysis showed that the transgene was expressed in the fruits. The production of ethylene in the fruits of the transgenic plants was reduced approximately to 1% of that of the non-transgenic controls. In the field, non-transgenic melon fruits turned yellow upon ripening and yielded an aromatic flavor. The transgenic fruits remain green even at the late stages of ripening. The fruits from transgenic melon were stored for 2 months at room temperature without over-ripening or fungal attack. However, non-transgenic fruits have completely rotten and liquefied within two weeks of storage. The firmness of the fruits from transgenic melon was not changed during the storage period. The initial difference observed between the transgenic and non-transgenic fruits was the color of the rind. There was no noticeable difference between the transgenic and non-transgenic fruits in other physiological and biochemical parameters. The transgenic fruits turned yellow by the treatment of exogenous ethylene, and subsequently produced the aromatic flavor.