The purpose of this study was to evaluate the effectiveness of using RNA interference in down regulating the expression of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Eksotika papaya. One-month old embryogenic calli were separately transformed with Agrobacterium strain LBA 4404 harbouring the three different RNAi pOpOff2 constructs bearing the 1-aminocyclopropane-1-carboxylic acid oxidase gene. A total of 176 putative transformed lines were produced from 15,000 calli transformed, selected, then regenerated on medium supplemented with kanamycin. Integration and expression of the targeted gene in putatively transformed lines were verified by PCR and real-time RT-PCR. Confined field evaluation of a total of 31 putative transgenic lines planted showed a knockdown expression of the targeted ACO1 and ACO2 genes in 13 lines, which required more than 8 days to achieve the full yellow colour (Index 6). Fruits harvested from lines pRNAiACO2 L2-9 and pRNAiACO1 L2 exhibited about 20 and 14 days extended post-harvest shelf life to reach Index 6, respectively. The total soluble solids contents of the fruits ranged from 11 to 14° Brix, a range similar to fruits from non-transformed, wild type seed-derived plants.

Biofortification is the process of improving the bioavailability of essential nutrients in food crops either through conventional breeding or modern biotechnology techniques. Rice is one of the most demanding staple foods worldwide. Most global population live on a diet based on rice as the main carbohydrate source that serve as suitable target for biofortification. In general, polished grain or white rice contains nutritionally insufficient concentration of iron (Fe) to meet the daily requirements in diets. Therefore, iron biofortification in rice offers an inexpensive and sustainable solution to mitigate iron deficiency. However, understanding on the mechanism and genes involved in iron uptake in rice is a prerequisite for successful iron biofortification. In this chapter, the overview of iron uptake strategies in plants and as well as different iron-biofortified approaches used in rice will be outlined. Then, the challenges and future prospects of rice iron biofortification to improve global human health will also be discussed.

The Malaysian Eksotika papaya (Carica papaya L.) has poor keeping quality due to its fast ripening attribute, which leads to post-harvest losses. This study is aimed at extending the shelf life of this perishable Eksotika fruit using antisense technology. A total of 6,000 Eksotika somatic embryogenic calli was transformed with the antisense 1-aminocyclopropane-1-carboxylic acid oxidase 2 gene (ACO2) construct, and 46 PCR-positive putative transformants were obtained. Gene expression analysis using real-time PCR on the 46 regenerated putative transgenic lines revealed that 42 showed down-regulation of the ACO2 gene with two- to five-fold differences among the lines. Out of 22 independently selected transformed lines grown under net house conditions, 16 harbored a single copy of the transgene. Physical stature of the transgenic plants was not significantly different from that of the non-transformed seed-derived papaya plants. Physiological evaluations of the transgenic fruits showed a 15-day delay in ripening compared with 4 days of the non-transformed seed-derived papaya fruits. The total soluble solid (TSS) of the transgenic fruits was comparable to that of the non-transformed seed-derived fruits with similar 11-15°Brix, implying the transgenes did not affect the TSS content. The transgenic fruits remained firm for additional 4 to 8 days at room temperature (25 ± 2°C) after achieving the full maturity index (index 6), whereas the non-transformed seed-derived fruits lost their firmness after 2 days. The outcomes of this study revealed that reduction of ethylene was successful in the Eksotika papaya by manipulating the ACO2 gene using the antisense technique.