papaya-1The mission of Plant Research Group, BIOTEC is to use gene transformation to increase the quality of economic crops. The fi rst example is to produce papaya resistant to Papaya ringspot virus (PRSV). PRSV is the serious problem for papaya production in Thailand (Figure 1). There are no resistant cultivars available to fi ght back this disease. The coat protein (CP) gene of PRSV was transformed into papaya cultivar Khak Nuan by particle bombardment. Few lines of transgenic papaya resistant to PRSV have been produced. The superior line was KN116/5 that was highly resistant to PRSV and produced high fruit yields with forty times higher than non-transgenic papaya infected with PRSV (Figure 2). Now, transgenic papaya line KN116/5 is in the process of biosafety assessment and breeding for homozygosity with high resistant performance under screen house condition.

Papaya-infected-with-PRSV
Figure 1 Papaya infected with PRSV with severe leaf distortion, ringspot on fruit and stunting
papaya1 papaya2
Figure 2 Transgenic papaya line KN116/5 showed resistance to PRSV as compared with non-transgenic papaya
(a). Transgenic papaya produced high yield of fruit with normal phenotype similar to non-transgenic
papaya (b).

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tomatoThe research on crop improvement has extended to tomato that has serious problem from disease caused by Tomato yellow leaf curl virus (TYLCV). In this case, another approach called trans-dominant resistance was used to produce transgenic tomato resistant to virus. This approach involves the inhibition of function of wild type gene by mutated gene product that acts as a competitive inhibitor. The candidate gene is Rep gene (replication-associated gene) of TYLCV responsible for replication of virus in tomato cells. The Rep gene was mutated, cloned into pCambia vector (Figure 3a) and then transformed into tomato cultivar VF134-1-2 by Agrobacterium-mediated transformation. Two transgenic lines DR#16-75 and DR#16-81 were resistant to TYLCV. These transgenic lines still contain the kanamycin-resistant gene used as marker gene for selection. Regarding to the public concern on biosafety of marker gene in transgenic plant, we are interested in production of transgenic tomato resistant to TYLCV and also free of marker gene. In this case, the mutated Rep gene was cloned into pMAT21 vector (Figure 3b). This vector contains ipt gene encoding for isopentenyl transferase used as marker gene instead of kanamycin-resistant gene, recombinase gene and the recognition sites for recombinase . The ipt gene is deleted from transformed plant cells by site-specifi c recombination mediated through the expression of recombinase gene. Three lines of transgenic tomatoes without marker gene were produced. One line-DR221 was self-pollinated and the progenies in R1 population were tested for resistance to TYLCV. Some of them were highly resistant and free of marker gene. In addition, they were physiologically normal (Figure 4).

map
Figure 3 Map of the mutated Rep gene cloned into pCAMBIA vector (a) and pMAT21 vector for marker-free
transgenic tomato (b)
tomato1
Figure 4 Marker-free transgenic tomato resistant to Tomato yellow leaf curl virus (TYLCV)

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orchid.The last mission is to extend the vase life of Dendrobium orchid. Ethylene plays a crucial role in flower senescence. To accomplish the mission, we use antisense technology to suppress gene expression of ethylene biosynthesis. Initially, two important genes for ethylene biosynthesis that are ACC oxidase (ACO) and ACC synthase (ACS) genes were identified from orchid flower cv. Pompadour (GeneBank accession no. EF487342 and EF488013, respectively). Then, both genes were cloned into plant expression vectorpCambia 1304 and pMAT21 in antisense orientation (Figure 5). All gene constructs were transformed into orchid cultivar Pompadour by Agrobacterium. Transformed orchid is in the process of development into mature plant.

Figure5-Map-of-ethylene
Figure 5 Map of ethylene-related genes (ACO or ACS genes) cloned into pCAMBIA1304 (a) and pMAT21 (b) in
the antisense orientation

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