The Cre/ lox site-specific recombination controls the excision of a target DNA segment by recombination between two loxsites flanking it, mediated by the Cre recombinase. We have studied the functional expression of the Cre/ lox system to excise a transgene from the rice genome. We developed transgenic plants carrying the target gene, hygromycin phosphotransferase ( hpt) flanked by two lox sites and transgenic plants harboring the Cre gene. Each lox plant was crossed with each Cre plant reciprocally. In the Cre /lox hybrid plants, the Cre recombinase mediates recombination between two lox sites, resulting in excision of the hpt gene. The recombination event could be detected because it places the CaMV 35S promoter of the hpt gene adjacent to a promoterless gusA gene; as a result the gusA gene is activated and its expression could be visualized. In 73 Cre /lox hybrid plants from various crosses of T0 transgenic plants, 19 expressed GUS, and in 132 Cre /lox hybrid plants from crosses of T2 transgenic plants, 77 showed GUS expression. Molecular data proved the excision event occurred in all the GUS(+) plants. Recombination occurred with high efficiency at the early germinal stage, or randomly during somatic development stages.

As an important step toward free access and, thus, impact of GoldenRice, a freedom-to-operate situation has been achieved for developing countries for the technology involved. Specifically, to carry the invention beyond its initial "proof-ofconcept" status in a Japonica rice (Oryza sativa) cultivar, we report here on two transformed elite Indica varieties (IR64 and MTL250) plus one Japonica variety Taipei 309. Indica varieties are predominantly consumed in the areas with vitamin A deficiency. To conform with regulatory constraints, we changed the vector backbone, investigated the absence of beyondborder transfer, and relied on Agrobacterium tumefaciens-mediated transformation to obtain defined integration patterns. To avoid an antibiotic selection system, we now rely exclusively on phosphomannose isomerase as the selectable marker. Single integrations were given a preference to minimize potential epigenetic effects in subsequent generations. These novel lines, now in the T 3 generation, are highly valuable because they are expected to more readily receive approval for follow-up studies such as nutritional and risk assessments and for breeding approaches leading to locally adapted variety development.We reported previously on a genetically modified rice line, frequently termed GoldenRice, engineered to synthesize and accumulate pro-vitamin A (␤-carotene) in the endosperm (Ye et al., 2000). Since then, the concept of genetic engineering-based nutritional enhancement of rice to contribute to a sustained reduction of vitamin A deficiency (VAD) has evoked strong expectations to develop in the public sector and to deliver a safe product in a short period of time. However, to carry the project from the scientific discovery to impact, i.e. to actually provide grain to subsistence farmers and urban poor in developing countries free of charge for the technology used, a large number of obstacles, not only scientific in nature, need to be dealt with. These are contractual and regulatory aspects.Contractual issues have largely been solved to date, including a "freedom-to-operate" intellectual property situation for GoldenRice, the inventors, and their licensees. Free licenses have been granted by our license collaborators for patents used in this research designed to benefit resource-poor farmers in developing countries. The next step to be taken is to find regulatory acceptance in these countries, the prerequisite for most of the tasks ahead such as to allow grain production enabling feeding trials and to begin with diversified variety development.GoldenRice as published (Ye et al., 2000) demonstrates the feasibility of the scientific approach but does not yet represent a product. Its development was based on the finding that the precursor geranylgeranyldiphosphate, which wild-type rice endosperm is capable of synthesizing, can be used by the subsequent enzyme phytoene synthase (PSY) when the latter is supplemented by transformation (Burkhardt et al., 1997). Providing the full supplement of all necessary biosynthetic genes ...

To increase the beta-carotene (provitamin A) content and thus the nutritional value of Golden Rice, the optimization of the enzymes employed, phytoene synthase (PSY) and the Erwinia uredovora carotene desaturase (CrtI), must be considered. CrtI was chosen for this study because this bacterial enzyme, unlike phytoene synthase, was expressed at barely detectable levels in the endosperm of the Golden Rice events investigated. The low protein amounts observed may be caused by either weak cauliflower mosaic virus 35S promoter activity in the endosperm or by inappropriate codon usage. The protein level of CrtI was increased to explore its potential for enhancing the flux of metabolites through the pathway. For this purpose, a synthetic CrtI gene with a codon usage matching that of rice storage proteins was generated. Rice plants were transformed to express the synthetic gene under the control of the endosperm-specific glutelin B1 promoter. In addition, transgenic plants expressing the original bacterial gene were generated, but the endosperm-specific glutelin B1 promoter was employed instead of the cauliflower mosaic virus 35S promoter. Independent of codon optimization, the use of the endosperm-specific promoter resulted in a large increase in bacterial desaturase production in the T(1) rice grains. However, this did not lead to a significant increase in the carotenoid content, suggesting that the bacterial enzyme is sufficiently active in rice endosperm even at very low levels and is not rate-limiting. The endosperm-specific expression of CrtI did not affect the carotenoid pattern in the leaves, which was observed upon its constitutive expression. Therefore, tissue-specific expression of CrtI represents the better option.

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