A question that worries many people when dealing with genetically modified foodstuff is whether unintended changes have taken place in the final product. For many, this hard-to-define eventuality has been the major stepping stone to the acceptance of transgenic produce, but without a concrete fear there is of course no target to look for. Detailed molecular analyses have failed to find new allergens showing up as a consequence of having introduced a new gene into a plant, and determination of the expression levels of ten-thousands of genes have also shown that the only changes encountered are related to the introduced genes and those involved in related metabolic pathways. Gene expression levels vary probably less after the introduction of a foreign gene than after the traditional crossing of two varieties, where thousands of regulatory factors and pathways interact in unexpected ways.

Genetically modified plants destined for field release are some of the most in detail examined organisms worldwide, and this is not different for Golden Rice, in fact it is one of the reasons why it has not reached the people who need it yet. Many test have been performed on Golden Rice and many are yet to be performed. A number of tests require kilogram amounts of seed; these tests have been unnecessarily delayed by the difficulties in being able to carry out field trials with Golden Rice, not enough seed can be produced when grown in the glasshouse.

In this section we will report on various tests conducted on Golden Rice and also on pending analyses. From the nutritional point of view the most important question is that of bioavailability—terms like this one will explained in the corresponding sections, but see also the FAQs section. Not less important are retention studies, taste and cooking tests, agronomic performance, storability of the seed and other tests.

Bioavailability relates to the fraction of carotene that the body can extract out of a given foodstuff and make available for utilisation; it is determined to a great extent by the food matrix, however, within a plant tissue carotenoids can be stored in several ways:

• Protein-bound, stoichiometrically (in light-harvesting complexes, in green vegetables); also in lobsters.
• Protein-associated; by a protein that organises a proteolipid-carotenoid complex with a protein called fibrillin and homologs thereof, as in red pepper.
• Free in membranes; in chromoplasts, like in daffodil (probably etioplasts and amyloplasts).
• In plastoglobules, frequently met in flowers, algae (probably the most bioavailable form).
• In crystals, as in tomato and carrot (this is probably the least available form).
• Additional determinants may act as promoters or inhibitors of resorption.

This again means that a person can in principle obtain more utilisable ß-carotene from an orange-fleshed sweet potato which has ca. 18 µg/g ß-carotene than from carrots that have around 60 µg/g.

In a given human population there are also genetic factors that determine bioavailability of carotenoids. The expression/modification of the ß-carotene receptor determines availability and transport into the blood serum (and probably also into cells).

Bioconversion is fraction of available ß-carotene converted to the active form, ie retinol or vitamin A.

Bioefficacy is the capability of an individual —governed by individual internal factors— to convert ß-carotene (provitamin A) into retinal, retinol (vitamin A) and retinyl esters. It is expressed as the amount of ß-carotene required to yield one retinol activity equivalent (1 RAE).

The best way to determine bioavailability is by feeding human subjects and measuring the levels of retinol in the blood. For an excellent review on the determination of bioavailability and bioconversion see Yeum and Russell (2002).

• Yeum K-J and Russell RM (2002) Carotenoid bioavailability and bioconversion. Annu Rev Nutr 22:483-504.
• Tanumihardjo SA (2002) Factors influencing the conversion of carotenoids to retinol: bioavailability to bioconversion to bioefficacy. Int J Vitam Nutr Res 72:40-45.

Golden Rice has a pleasant yellow colour, very similar to rice prepared with saffron or curcuma, which are traditional condiments in many parts of the world. Naturally, the taste of an increased level of carotenoids is not that of condiments. At the concentration levels encountered in the present and future generations of carotenoids it is expected that these will not affect the taste of rice at all, but that must be demonstrated first by expert taste panels.

Taste trials not only involve an elaborate sensory analysis using its own descriptive language but also gas chromatographic and mass spectrometric identifcation of volatiles. For more details you may consult the cited liteture below. These trials have also been delayed by the reduced availability of Golden Rice seed and the ethical and safety requirements involved in the testing of genetically modified foodstuff using human subjects.

• Champagne ET, Bett KL, Vinyard BT, McClung AM, Barton FE, Moldenhauer K, Linscombe S, McKenzie KS (1999) Correlation between cooked rice texture and rapid visco-analyser measurements. Cereal Chem 76:764-771.
• Goodwin HL, Jr, Koop LA, Rister ME, Miller RK, Maca JV, Chambers E, Hollingsworth M, Bett K, Webb BD, McClung AM (1996) Developing a common language for the US rice industry: Linkages among breeders, producers, processors and consumers. TAMRC Consumer Product Market Research CP2-96. Texas A&M: College Station, TX.
• Lyon BG, Champagne ET, Vinyard BT, Windham WR, Barton FE, Webb BD, McClung AM, Moldenhauer KA, McKenzie KS, Kohlwey DE (1999) Effects of degree of milling, drying condition, and final moisture content on sensory texture of cooked rice. Cereal Chem 76:56-62.
• Meilgaard M, Civille GV, Carr BT (1999) Sensory Evaluation Techniques. CRC Press: Boca Raton, FL.
• Surles RL, Weng N, Simon PW, Tanumihardjo SA (2004) Carotenoid profiles and consumer sensory evaluation of specialty carrots (Daucus carota, L) of various colors. J Agric Food Chem 52:3417-3421.

It would be very hard to convince a farmer to adopt Golden Rice if varietal productivity ran counter to its improved nutritional quality. This is especially understandable in developing countries where population density is high and the areas of production owned by single farmers are small. Moreover, consumers in those countries should not and would not be able to pay an premium for the Golden Trait. Carotenoids are isoprenoid derivatives as are vitamin E, chlorophyll and the phytohormones absiscic acid and gibberellic acid; diversions of the pathway could have consequences on the amounts of these compounds produced and hence on plant architecture or photosynthetic activity. Further, carotenoids are photosensitive compounds involved in light protection. For these reasons it is very important to determine whether the expression of the introduced genes has any deleterious effect on the agronomic performance of the rice plants.

The first Golden Rice field trial carried out in 2004 in Louisiana, gave us the first opportunity to determine its agronomic performance under real field conditions. Previous experiments in the glasshouse had not given any indication of reduced or altered performance, but varying conditions typically encountered in the field could have led to unexpected outcomes. Such varying conditions not encountered in the glasshouse are related to temperature, humidity, light intensity, and other biotic and abiotic interactions.

In the field trial, the Golden Rice plants stood side by side together with control plants of the same variety as well as azygotic siblings (genetically modified plants where the introduced trait had been crossed out). The reassuring result was that Golden Rice was no different from its progenitors and other controls in a number of agronomic parameters measured (plant height, days to flowering, seed set, 100-seed weight, total biomass, etc).

The question to be answered here is, how much β-carotene is left intact after various typical ways of preparing rice, eg boiling or frying. It has been determined that, for most vegetables, the best way to make ß-carotene bioavailable is by cooking and adding some oil. Carotenoids are highly soluble in oil, therefore addition of oil or butter to boiled vegetables makes it easier for them to be taken up by the intestine. In general, carotenoids are quite heat stable, hence most of it—80% or more—remains utilisable after most typical cooking procedures applied to various vegetable sources. The losses are generally compensated by the increased bioavailability.

The first Golden Rice version (SGR1) produced an average of 1.6 µg β-carotene per gram seed dry weight. First field trial results indicate that it may be possible to obtain higher ß-carotene production levels—ca. three to four times higher—under field conditions. This could be related to higher irradiation intensities in the open, but this is speculative at present. Because of their chemical nature—several conjugated double bonds—carotenoids are susceptible to light and oxidation. The effects of light and air after harvest can be studied now that the first field trials have begun. From these studies it will be possible to make recommendations as to how and how long to store Golden Rice without losing its beneficial nutritive effects.