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Generation of transgenic oriental melon resistant to Zucchini yellow mosaic virus by an improved cotyledon-cutting method

Wu, Hui-Wen, Yu, Tsong-Ann, Raja, Joseph A. J., Wang, Hui-Chin, Yeh, Shyi-Dong
Plant cell reports 2009 v.28 no.7 pp. 1053-1064
Agrobacterium, Cucumis melo, Northern blotting, Southern blotting, Zucchini yellow mosaic virus, coat proteins, cotyledons, cultivars, gene silencing, greenhouses, hosts, immunity, melons, transgenes, viruses, East Asia
Production of melon (Cucumis melo L.) worldwide is often limited by the potyvirus, Zucchini yellow mosaic virus (ZYMV). In order to engineer melon lines resistant to ZYMV, a construct containing the translatable coat protein (CP) sequence coupled with the 3' non-translatable region of the virus was generated and used to transform an elite cultivar of oriental melon (Silver light) mediated by Agrobacterium using an improved cotyledon-cutting method. Removal of 1-mm portion from the proximal end of cotyledons greatly increased the frequency of transgenic regenerants by significantly decreasing the incidence of false positive and aberrant transformants. Results of greenhouse evaluation of transgenic lines by mechanical challenge with ZYMV identified transgenic lines exhibiting different levels of resistance or complete immunity to ZYMV. Southern hybridization of transgenic lines revealed random insertion of the transgene in host genome, with insert numbers differing among transformants. Northern hybridization revealed great variations in the levels of accumulation of the transgene transcripts among transgenic lines, and evidenced an inverse correlation of the levels of accumulation of transgene transcript to the degrees of virus resistance, indicating post-transcriptional gene silencing (PTGS)-mediated transgenic resistance. These transgenic melon lines with high degrees of resistance to ZYMV have great potential for the control of ZYMV in East Asia.