“ transgene carrying progeny will lead to contamination of the original wild rice populations, and even to the extinction of endangered wild rice populations in local ecosystems “
LU Baorong**, SONG Zhiping and CHEN Jiakuan
(Ministry of Education Key Laboratory for Biodiversity and Ecological Engineering, Institute of Biodiversity Science, Fudan University, Shanghai, 200433)
Abstract: Alien transgene escape from genetically engineered rice to non-transgenic varieties or close wild relatives (including weedy rice) may lead to unpredictable ecological risks. However, for transgene escape to occur three conditions need to be met: (i) spatially, transgenic rice and its non-transgenic counterparts or wild relatives should have sympatric distributions; (ii) temporally, the flowering time of transgenic rice and the non-transgenic varieties or wild relatives should overlap; and (iii) biologically, transgenic rice and its wild relative species should have a sufficiently close relationship, that their interspecific hybrids can have normal generative reproduction. This paper presents research data on the geographic distribution, flowering habits, interspecific hybridization, and gene flow of cultivated rice (Oryza sativa) and its closely related wild relatives containing the AA genome. The objective is to estimate the possibility of transgene escape to non-transgenic rice varieties and wild relatives of rice, which may result in unpredictable ecological risks.
Key words: oryza species, transgene escape, biosafety, ecological risk, introgression, gene flow
The tremendous achievements associated with transgenic biotechnology and its rapid application in agriculture has been based on substantial crop genetic improvements. As a consequence, a great number of genetically modified crops (GMC) have been released into the environment and have entered commercial markets [1-4]. Undoubtedly, the so-called “gene revolution” offers new possibilities for global food security, but at the same time has also
* Supported by the National Natural Science Foundation of China for Distinguished Young Scholars
(Grant No. 30125029)
** To whom correspondence should be addressed. E-mail: email@example.com
brought serious problems of biosafety . The biosafety issues raised in relation to transgenic biotechnology and its products have been most extensively debated worldwide in recent years, and have become the “bottle-neck” to further development of transgenic biotechnology and wider application of transgenic products [5-10]. Are transgenic products safe in terms of impacts on human health and the environment? Can transgenes escape and persist in the environment through outcrossing? Will transgene escape cause significant ecological risks? All these questions relating to the biosafety of transgenic crops need to be addressed scientifically. It is also important to educate publics about biotechnology and biosafety, as well as the current achievements on biosafety research. Effective strategies to minimize transgene escape and its ecological risks, can only be made when a better understanding of the pathways and consequences of transgene escape is achieved, which will in turn lead to more safe and efficient use of transgenic crops. The present paper discusses whether or not release of genetically modified crops into the environment will result in transgene escape and consequently lead to ecological risks, using rice as an example.
1 Transgenic rice and possible ecological risks following transgene escape
Rice is one of the most important world’s cereal crops, providing staple food for nearly one half of the global population . More than 90% of rice is grown and consumed in Asia where about 55% of the world’s population lives , reflecting the importance of rice in Asian people’s daily life. Rice is one of the earliest of the world’s crop species to which transgenic biotechnology has been effectively used for genetic improvement [13, 14]. Although no transgenic rice varieties have yet been officially approved for extensive commercial cultivation anywhere in the world, genes conferring traits such as high protein content, disease and insect resistance, virus resistance, herbicide resistance, and salt tolerance, have been successfully transferred into different rice varieties through transgenic techniques [13 ～ 16]. Transgenic biotechnology has rapidly developed and been extensively applied in rice breeding in China. To date, transgenic rice varieties resistant to three major diseases and insects, namely, rice stem borer (using Bt and CpTI genes), rice hopper, and rice bacterial blight (using the Xa21 gene isolated from an African wild rice, Oryza longistaminata), have been developed and released into the environment for testing . In addition, some herbicide resistant and salt tolerant transgenic rice varieties have also been produced in China . We are confident that, as an important world cereal crop, transgenic rice varieties will be released into environment for commercial production in the near future, after authorized food and environmental biosafety assessments have been made by the concerned Chinese agencies.
Transgene escape refers to a gene or a group of genes introduced to a rice variety by genetic engineering moving to non-transgenic rice varieties or to wild relative species (including weedy rice) through gene flow. Cross-pollination between transgenic and non-transgenic rice varieties or wild relatives is the major pathway for transgene escape, which may ultimately causes possible ecological risks. Normally, for transgene escape to happen the following requirements need to be met: (i) spatially, transgenic rice and its non-transgenic varieties or wild rice relatives should be sympatrically distributed, i.e. grow in the same vicinity; (ii) temporally, the flowering time (including flowering duration within a year and flowering time within a day) of transgenic rice and its non-transgenic varieties or wild relatives should overlap; and (iii) biologically, transgenic rice and its wild relative species should have a sufficiently close relationship, also resulting interspecific hybrids should be able to reproduce normally. It is therefore necessary to know geographic distribution patterns and flowering habits of cultivated and wild rices, and to understand genetic relationships and actual gene flow frequencies between the cultivated and wild rice species. This will facilitate the effective prediction of transgene escape and its potential ecological risks, and the development of strategies to minimize the escape of alien transgenes.
When alien transgenes escape to non-transgenic rice varieties and disseminate within rice populations, the purity of non-transgenic varieties will have been affected by transgenic contamination. This will largely affect the strategic deployment of transgenic and non-transgenic rice varieties in a given agricultural system. When normal rice varieties are mixed with individuals of transgenic rice, international trade in rice might be affected because of the contamination. This will influence rice exports particularly to countries where biosafety control is rigid and transgenic products are tightly restricted. The exporting of rice varieties contaminated by transgenes might also cause some legal difficulties. On the other hand, when alien transgenes escape to and express normally in wild relatives and weedy species of rice, the transgenes will persist and disseminate within the wild or weedy populations through sexual reproduction and/or vegetative propagation. If the transgenes are encoding for traits, such as high protein content, special vitamins, and better grain quality, which are not associated with the ecological fitness of the wild or weedy species and not related by natural selection to the survival of rice plants, the ecological risks caused by escape of these genes will be minimum. However, if the transgenes are responsible for resistance to biotic and abiotic stresses (such as drought and salt tolerance, and herbicide resistance), and the genes significantly enhance ecological fitness of wild and weedy rice species, escape of these genes will probably cause ecological problems. If several of such fitness enhancing genes are stacked in the same individual wild or weedy rice species, the ecological consequences might become more significant through formation of aggressive weeds that escape human control and cause unpredictable damage to local ecosystems. On the other hand, when transgenes escape to wild rice populations through outcrossing, the persistence and rapid spread of the resulting hybrids and their transgene carrying progeny will lead to contamination of the original wild rice populations, and even to the extinction of endangered wild rice populations in local ecosystems [17,18]. This will make in situ conservation of wild rice germplasm more difficult. In addition, perennial hybrids of cultivated and wild rices carrying transgenes may serve as a bridge to spread their transgenes through outcrossing to other wild grass species, such as Leersia and Hygroryza, causing even more significant ecological risks.
2 The close wild relatives of cultivated rice and their geographic distribution
Cultivated rice is classified in the genus Oryza L. of the tribe Oryzeae in the grass family (Poaceae). The genus Oryza includes two cultivated species and over 20 wild species widely distributed in the pan-tropics and subtropics . The Asian cultivated rice O. sativa had its origin in South and Southeast Asia, and is grown worldwide in the tropics, subtropics and some temperate regions, whereas the African cultivated rice O. glaberrima was domesticated in western Africa and is now cultivated only in local agricultural ecosystems in West Africa. The cultivated rice that we discuss here in this paper is only referred to as O. sativa.
Species in the genus Oryza included ten different genome types, i.e. the AA, BB, CC, BBCC, CCDD, EE, FF, GG, JJHH, and JJKK genomes [19～21]. Species containing different genomes have significant reproductive barriers. Therefore, genetically they are distantly related and spontaneous hybridization between species with different genomes is extremely difficult. Cultivated rice contains the AA genome and is relatively easy to cross with its close relative species (including weedy rice) also containing the AA genome. Theoretically speaking, transgene escape from transgenic rice varieties will only occur to species with the AA genome. Therefore, this paper only describes geographic distribution of the AA genome Oryza species.
There are eight diploid (2n=2x=24) Oryza species containing the AA genome. Apart from the two cultivated rice species, the perennial common wild rice O. rufipogon and annual common wild rice, O. nivara from Asia, the perennial O. longistaminata and annual O. barthii from Africa, the perennial O. glumaepatula from Latin America, and the annual O. meridionalis from northern Australia and New Guinea are all comprised of the AA genome [11, 12]. Weedy rice usually has its origin in crop-to-wild rice hybridization or degenerated individuals of cultivated rice, is mostly found in the rice field alongside cultivated rice, but also occurs in the vicinity of rice fields, in ditches, or in sympatric regions of cultivated and wild rices . It is shown in Figure 1 that the AA genome wild Oryza species is distributed across a significantly wide geographic region, and cultivated rice is grown sympatrically with these wild rice species in many areas, particularly in Southeast and South Asia, Central Africa, and Latin America. It is concluded from the geographic distribution data that spatially transgenes from cultivated rice have a great potential to escape to its wild relative species.
3 Flowering habits of cultivated and wild rice species
Flowering habits of cultivated rice grown in different parts of the world vary considerably depending on local cultivation time and seasons, and differences between varietal types (such as photoperiod and thermal sensitivity). The flowering and pollinating time of different wild rice species or different populations of the same species also varies significantly across different geographic regions. In general, the flowering habit of wild rice species is characterized by a protracted flowering period. In other words, different individuals within the same population, and different tillers and spikelets of the same individuals, will flower at a considerably different time. For example, the Asian perennial common wild rice usually starts its flowering at the beginning of September and terminates its flowering in December or towards the end of January in the next year. Obviously, the comparison of flowering habits becomes meaningful only when specific wild species and cultivated rice varieties from the same location are selected for flowering studies under the same conditions. Therefore, we selected the perennial common wild rice, O. rufipogon, and two cultivated rice varieties, a late-maturing local variety and an improved variety (Minghui-63) at a field site in Chaling of Hunan Province, to compare their flowering habit. The results given in Table 1 show that both the flowering period in a year and flowering time in a day had considerable overlap for the perennial common wild rice and the two rice varieties. Our additional experimental data demonstrated that pollen grains of the perennial common wild rice and a cultivated rice variety could be actively alive in the air for more than 60 minutes . In summary, it is essentially possible that cross-pollination between the O. rufipogon and cultivated rice will occur, if the two species have sympatric distribution and are grown near to each other.
4 Biosystematic relationships of cultivated and wild rice species
Biosystematic relationships of the AA genome Oryza species can be estimated from the following aspects: (i) crossability between cultivated rice and its wild relatives; (ii) meiotic chromosome pairing in the F1 interspecific hybrids; and (iii) fertility of the F1 hybrids. If cultivated rice has relatively high crossability with its wild relatives, normal meiosis forms in the F1 hybrids, where chromosomes from different parental species will pair and genetic recombination will take place, observed in F1 hybrids, and if the F1 hybrids have comparatively high fertility, the transgenes will easily escape to wild relative species through cross pollination and persist in environment. The transgenes will also spread out through reproductive procedures or through vegetative propagation if the hybrids and their progeny are perennial.
4.1 Crossability between cultivated and wild rice species
Research on crossability between cultivated and wild rice species has occasionally been reported [25～27]. We also conducted interspecific experiments between eight AA genome Oryza species under greenhouse conditions [28, 29], and the results of crossability are summarized in Figure 2. The results from interspecific hybridization show that most of the AA genome wild rice species have relatively high compatibility with the cultivated rice, although with comparatively large variations among species (Figure 2). Only the Australian O. meridionalis has crossability of less than 5% with the cultivated rice.
4.2 Meiotic chromosome pairing in the F1 hybrids between cultivated and wild rice species
The chromosome pairing ability at metaphase-I in meiosis of the F1 hybrids reflects the genetic relationships of the parental species. The high frequency of meiotic pairing indicates a close genetic relationship between their two parents. In this case, exchange of genetic materials between the parents will occur during meiosis through genetic recombination. Extensive studies showed that very high frequency of meiotic pairing was found in F1 hybrids between cultivated rice and its AA genome wild relatives [25, 27, 30]. Our cytogenetic studies also indicated that nearly all chromosomes from parental species formed 12 ring bivalents in meiosis of the hybrids [31, 32].
4.3 Spikelet fertility of the F1 hybrids between cultivated rice and its wild relatives
A great variation in spikelet fertility of F1 hybrids between cultivated rice and its AA genome wild relatives was reported [25, 26]. Results from our spikelet fertility investigation in F1 hybrids between cultivated and wild AA genome rice species show a similar rate of panicle fertility [29, 30, 33]. Our data summarized in Figure 3 indicate that spikelet fertility of the F1 hybrids from all available combinations is relatively high under bagged self-pollination conditions, particularly for the hybrids of Asian cultivated rice and its ancestral wild species O. rufipogon and O. nivara, and the African cultivated rice and its ancestor O. barthii, with spikelet fertility over 11%.
5. Gene flow between cultivated rice and O. rufipogon
It is clear that cultivated rice and O. rufipogon have sympatric distribution and overlap in flowering in many Asian countries and regions, and genetically the two species have close relationships and low reproductive isolation. As a consequence, introgression between the two species occurs frequently in nature [26, 34, 35]. Although crossability between the two species obtained under artificial hybridization is considerably high, our knowledge on gene flow between the two species is limited. Information on gene flow frequency between the two species becomes essential for the assessment of transgene escape from transgenic rice varieties to wild relatives. In order to obtain actual data for the maximum gene flow frequency between cultivated rice and O. rufipogon under natural conditions, we designed an experiment where gene flow between a cultivated rice variety, Minghui-63 and O. rufipogon were examined under controlled conditions. Four different experimental designs were made with 12 treatments for gene flow detection, in which the cultivated Minghui-63 was planted to encompass the wild O. rufipogon, or be surrounded by the wild rice species, Minghui-63 and O. rufipogon were planted alternatively in rows, and O. rufipogon was planted at the downwind direction of Minghui-63. The simple sequence repeat (SSR) was used as molecular markers to determine cross-pollination rates between Minghui-63 and O. rufipogon under different designs. The results demonstrated that the maximum frequency of gene flow from Minghui-63 to O. rufipogon reached nearly 3% in natural habitats of Chaling in Hunan Province 1), indicating clearly that gene flow between cultivated rice and the widely distributed perennial wild common rice O. rufipogon occurs considerably under natural conditions.
6 Ecological risks of transgenic rice and its environmental release
Transgene escape to the environment and its ecological impacts are problematic issues that should receive serious and long-term attention by the public, scientists and government agencies, because it is difficult monitor ecological problems caused by the transgene escape within a limited period. However, if alien, engineered genes escape and persist in the environment they may cause considerable damage in ecosystems, which could not be recovered from in a short period. Bad examples can be found in the cases of invasive species, such as Alternanthera philoxeroides, Solidago canadensis, Mikania micrantha, and Euptorium adenothorum that have extensively invaded agricultural ecosystems, grasslands, wetlands, and forest ecosystems, causing tremendous damages to our agriculture, animal husbandry and forestry industry, and have resulted in great economic losses. However, we did not pay adequate attention to invasive species till recently when disasters linked to these species become an extraordinary headache. The price paid has been too high. Most alien genes carried by genetically modified agricultural products are not from crops. Instead, they are from other organisms or microorganisms, even from an artificially synthesized origin. These genes may completely alter the natural habit of crop species and significantly change wild relatives of the
1) Unpublished data from the doctoral dissertation thesis of Song Zhiping (2001)
crop species when transgene escape happens. If we do not pay attention to this matter and scientifically study, assess, and manage transgenic crops, we might encounter similar ecological disasters as have happened with invasive species.
Transgene escape to the environment and its environmental impacts have become increasingly challenging biosafety issues worldwide. However, this problem could be controlled or minimized using scientific methods, if we have a better understanding of the mechanisms and conditions for transgene escape through outcrossing. A previous report based on different field experimental designs already proved that gene flow from a transgenic herbicide resistant rice variety to its non-transgenic counterpart was about 0.05%-0.53% . Results from our own studies also confirmed that cultivated rice and its wild relatives O. rufipogon have sympatric distribution and overlapping flowering times, which meets the spatial and temporal conditions for transgene escape from cultivated rice to its wild relatives. Our experimental data further show that most of the AA genome wild Oryza species have relatively close biosystematic relationships and high crossability with cultivated rice, particularly the ancestors of cultivated rice (O. rufipogon and O. nivara) and weedy rice (O. spontanea), and that they do not have significant reproductive isolation with the cultivated rice, and spontaneous introgression with the cultivated rice occurs with considerable frequency in the field. It was reported by Japanese scientists that outcrossing rates between O. rufipogon (including weedy rice) and cultivated rice could be as high as 50% in nature, although with a considerable variation . Data obtained from our experiment further showed that the frequency of gene flow from cultivated rice (Minghhui-63) to O. rufipogon in Chaling of Hunan Province could reach as high as 3%. All experimental data from previous reports and our studies support the indisputable fact that transgene escape will occur if transgenic rice varieties are grown in the vicinity of the wild relative species, and if no effective isolation measures are taken.
It is therefore a very important biosafety strategy to establish an effective buffering isolation zone between transgenic rice and non-transgenic rice varieties or its closely related wild species of rice, to avoid or significantly minimize transgene escape to the environment, given the fact that the spatial, temporal, and biological conditions for rice transgene escape are satisfied in many rice producing countries or regions. It is general knowledge that pollen flow is the principal pathway for transgene escape because pollen can act as a vehicle to disseminate transferred alien genes in nature. We also studied pollen flow of cultivated rice —a typical wind pollinating species, and our results showed that dispersal range of rice pollen grains increases with the increase of wind speed (Figure 4). The maximum distance of rice pollen flow can be as far as 110 m at downwind direction when the wind speed reached 10 m per second (equal to grade 5 wind power). This occurs frequently during the rice flowering period in southern China, the major region for Chinese rice production and wild rice O. rufipogon distribution 1). Therefore, to effectively minimize transgene escape to environment, it is recommended to have a buffering isolation zone wider than 110 m or use tall crops such as sugarcane as an effective buffer objective between transgenic rice and its wild relatives, to limit the distant dispersal of transgenic pollen.
It is generally recognized that a better understanding of species biosystematic relationships, pollen flow, and gene flow will facilitate efficient prediction of transgene escape and its potential ecological risks, as well as appropriate management of ecological risks. However, more scientific questions regarding biosafety issues relating to transgenic agricultural products need to be properly addressed and thoroughly studied. These questions include whether transgenic hybrids and their progeny have better ecological fitness than parental species if introgression between transgenic rice and its wild relative species does occur? Whether transgenes can be normally expressed in the interspecific hybrids and their progeny? Whether transgenes will change genetic structures and population dynamics of the natural wild rice populations? Will the wild rice hybrids carrying transgenes become a “genetic bridge” that further passes transgenes to other wild plant species? What kind of ecological consequences and risks will the individuals and populations carrying alien transgenes cause? What is the effective method for assessing and managing the biosafety risks related to environmental change? The appropriate answers to all these scientific questions will assist us to effectively assess and manage the potential ecological risks resulting from rice transgene escape, which will also promote the possibility of safe utilization of transgenic rice varieties.
1) Song et al. submitted to Biodiversity and Conservation in 2002
Mr. J. Keeley of the University of Sussex, UK, kindly corrected English of this paper.
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PS: Pease see the attached articles on Biodiversity study of rice. I shall send you the reprint of the rice gene flow paper published in Progress in Natural Science (Ref: Lu, B.-R., Song, Z. P. and Chen, J. K., 2003. Can transgenic rice cause ecological risks through transgene escape? Natural Science 13: 17-24.)
Bao-Rong Lu Ph. D.
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