Preliminary results on development of transgenic tomato plants tolerant to Tomato yellow leaf curl virus (original) (raw)

Tomato yellow leaf curl virus (TYLCV), a whitefly transmitted geminivirus, is the major limiting factor for tomato production in many countries in the Middle East, Europe, Africa and was reported in North and South America. Development of resistant varieties represents the most economical management measure. DNA sequences of the six major strains of TYLCV reported in the Middle East and Europe were aligned and sequences with more than 21 conserved nucleotides were determined. Three primer pairs were designed and used in PCR assays to amplify three overlapping regions of 240, 355 and 505bps. The amplicons were cloned in pHELLSGATE 2 using BP and LR clonase. This protocol allows the insertion, in one step, of the genes of interest in sense and antisense orientation with an intron in between. Once introduced into the plant it will be transcribed into dsRNA leading to post-transcriptional gene silencing of homologous genes. Colonies of Agrobacterium strain LBA 4404 were transformed with pHELLSGATE vectors carrying each of the three constructs and used to inoculate cotyledons of the tomato variety MP1. The produced transgenic tomato seedlings with the 505 bp construct developed TYLCV like symptoms upon inoculation but recovered rapidly thereafter. However, TYLCV concentration in the recovered tissue, as determined by semi-quantitative PCR, does not seem to have been reduced considerably. This construct includes partial pV1, pC2, and pC3, indicating that part of this region may play a significant role in development of TYLCV symptoms.